Methods of Treating Cancer with Kinase Inhibitors

Abstract

An aspect of the present disclosure relates to a method for treating a subject with cancer. A particular aspect relates to treating a subject with lung cancer by administering a kinase inhibitor to a subject that is determined to have a specific class of EGFR mutation from the analysis of tumor DNA from the subject. A further aspect relates to a method for treating a subject with lung cancer by detecting a specific class of EGFR mutation in tumor DNA from the subject and administering an effective amount of a kinase inhibitor to the subject. Also disclosed is a method for stratifying and prognosing a subject based on EGFR mutation classification.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 143,710, filed January 29, 2021, and U.S. Provisional Patent Application No. 63 / 244,169, filed September 14, 2021, which are incorporated by reference herein in their entireties.

[0002] This invention was made with Government support under R01CA247975 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] I. Field of Disclosure Aspects of the present disclosure relate generally to at least the fields of cancer biology, molecular biology, and medicine. [Background technology]

[0004] II. Background Epidermal growth factor receptor (EGFR) mutations are established driver mutations in non-small cell lung cancer (NSCLC), and targeted therapies have been approved for patients with select EGFR mutations. However, there are additional EGFR mutations for which effective therapies have not yet been identified, and the frequency and impact of atypical EGFR mutations on drug sensitivity is unknown.

[0005] Epidermal growth factor receptor (EGFR) mutations occur in 10-15% of patients with non-small cell lung cancer (NSCLC) and can be divided into classical and atypical mutations. 1-4 Classical EGFR mutations include L858R and exon 19 deletions (Ex19del), and patients with these mutations show marked improvement in clinical endpoints when treated with first-, second-, and third-generation TKIs. 5-7 The current standard of care for patients with classic EGFR-mutated NSCLC is treatment with the third-generation TKI osimertinib. 8A Phase III trial of osimertinib demonstrated an objective response rate (ORR) of 80% and a median progression-free survival (mPFS) of 18.9 months in treatment-naïve patients. 7 and median overall survival (mOS) of 38.6 months. 9 , i.e., resulting in significantly improved clinical outcomes compared with previous generation EGFR TKIs.

[0006] Other EGFR mutations in the kinase domain (exons 18–21) have also been validated as oncogenic drivers of NSCLC, but treatment options for these NSCLCs are limited. Patients with atypical EGFR mutations experience heterogeneous and reduced responses to EGFR inhibitors. 1、3、4、10-15 In a phase II study (KCSG-LU15-09) of treatment-naive patients harboring atypical EGFR mutations, osimertinib treatment resulted in a 50% ORR and mPFS of 8.2 months. 16 , Studies of acquired osimertinib resistance have identified exon 18 17-22 and 20 23-28 Currently, the only atypical EGFR mutations for which there is an FDA-approved treatment are EGFR S768I, L861Q, and G719X, for which afatinib is thought to be effective based on retrospective studies. 29-32 Atypical EGFR mutations, for which there are no FDA-approved TKIs, are often considered a single entity, and there are no clear established guidelines for EGFR TKI treatment for patients with these mutations, resulting in patients often receiving cytotoxic chemotherapy. Clinical trial design and treatment of patients with atypical EGFR mutations often rely on the exon location of the mutation to predict treatment, but heterogeneity in drug sensitivity even at a single exon location has been clearly observed. 1、11、12、33-36 .

[0007] There is a recognized need for systems for identifying and classifying EGFR mutations that predict response to cancer therapy for patient treatment and clinical trial design, as well as methods for using such systems to predict the efficacy of cancer therapy in order to more effectively treat cancer. Summary of the Invention

[0008] overview Aspects of the disclosure address a particular need in the art by providing a method for treating a subject with cancer (e.g., lung cancer) and a method for predicting patient response to cancer therapy. Thus, in some aspects, provided herein is a method for treating a subject with cancer, e.g., lung cancer, comprising administering an effective amount of one or more kinase inhibitors from one or more kinase classes to a subject determined to have one or more EGFR mutations from analysis of tumor DNA from the subject. Also disclosed is a method for treating a subject with cancer, e.g., lung cancer, comprising (a) detecting one or more EGFR mutations in tumor DNA from the subject; and (b) administering an effective amount of one or more kinase inhibitors from one or more kinase classes depending on the EGFR mutations detected. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the EGFR mutation is a classic-like mutation, an exon 20 loop near-loop insertion mutation (Ex20ins-NL), an exon 20 loop far-loop insertion mutation (Ex20ins-FL), a T790M-like susceptibility (T790M-like 3S) mutation, a T790M-like resistance (T790M-like 3R) mutation, or a P-loop αC helix compression (PACC) mutation.

[0009] The embodiments of the present disclosure include a method for treating a subject with cancer, a method for improving the efficacy of a kinase inhibitor used to treat a subject with cancer, a method for identifying a subject with cancer as a candidate for treatment with a specific kinase inhibitor, a method for identifying EGFR mutations, a method for classifying one or more EGFR mutations, and a method and composition for treating a subject with lung cancer. The methods of the present disclosure may include one, two, three, four, five, six, or more of the following steps: determining that a subject has cancer, providing one or more kinase inhibitors to the subject, providing an EGFR inhibitor to the subject, providing an alternative therapy to the subject, providing two or more types of cancer therapy to the subject, identifying one or more kinase inhibitors as needing improved efficacy, detecting one or more EGFR mutations in tumor DNA from the subject, identifying a subject as a candidate for treatment with one or more specific kinase inhibitors, identifying a subject as sensitive to one or more specific kinase inhibitors, identifying a subject as resistant to one or more specific kinase inhibitors. Certain embodiments of the present disclosure may exclude one or more of the above elements and / or steps.

[0010] Provided herein in some aspects is a method for classifying a cancer sample, comprising: (a) detecting EGFR mutations in the cancer sample; and (b) classifying the cancer sample by: (i) classifying the cancer sample as having EGFR mutations A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R (ii) classic-like EGFR-mutated cancers with EGFR mutations of Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del, or G773R T790M; (iii) T790M-like 3S EGFR mutant cancers, where the EGFR mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q. EGFR-mutated cancer; (iv) EGFR mutations are A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD(v) exon 20ins-NL EGFR mutation cancers with EGFR mutations H773_V774 insNPH, H773_V774 insSVDNR, or H773_V774 insSVDNR; (v) exon 20ins-NL EGFR mutation cancers with EGFR mutations H773_V774 insNPH, H773_V774 insSVDNR, or H773_V774 insSVDNR; or (vi) exon 20ins-FL EGFR mutant cancer with an EGFR mutation A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768IDisclosed are methods comprising classifying the cancer sample as a P-loop αC-helix compacted EGFR mutation cancer, the cancer being V774M, T751_I759 delinsN, V769L, V769M, or V774. Any one or more of the EGFR mutations may be excluded from the embodiments of the present disclosure. In some embodiments, the method further comprises classifying the cancer sample as sensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the method further comprises classifying the cancer sample as insensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20.

[0011] In some embodiments, the method further comprises classifying the cancer sample as sensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for a mutation associated with EGFR exon 20 if the cancer sample is classified as a classical-like EGFR mutant cancer sample. In some embodiments, the method further comprises classifying the cancer sample as (i) sensitive to a third generation EGFR inhibitor; and (ii) insensitive to a first generation EGFR inhibitor or a second generation EGFR inhibitor if the cancer sample is classified as a T790M-like 3S EGFR mutant cancer. In some embodiments, the method further comprises classifying the cancer sample as insensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, or a third generation EGFR inhibitor if the cancer sample is classified as a T790M-like 3R EGFR mutant cancer. In some embodiments, the method further comprises classifying the cancer sample as (i) sensitive to a second generation EGFR inhibitor, or an EGFR inhibitor specific for a mutation associated with EGFR exon 20, if the cancer sample is classified as an exon 20ins-NL EGFR mutant cancer; and (ii) insensitive to a third generation EGFR inhibitor. In some embodiments, the method further comprises classifying the cancer sample as insensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, and a third generation EGFR inhibitor, if the cancer sample is classified as an exon 20ins-FL EGFR mutant cancer. In some embodiments, the method further comprises classifying the cancer sample as (i) sensitive to a second generation EGFR inhibitor; and (ii) insensitive to a third generation EGFR inhibitor, if the cancer sample is classified as a P-loop αC-helix compression EGFR mutant cancer.

[0012] In some aspects, the present disclosure provides a method for classifying cancer samples, comprising: (a) detecting the EGFR mutation in cancer samples, which is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M or T790M; and (b) classifying the cancer samples as T790M-like 3S-mutated cancer.In some embodiments, the method further comprises classifying the cancer samples as sensitive to EGFR inhibitors. In some embodiments, the EGFR inhibitor is a third generation EGFR inhibitor. In some embodiments, the method further comprises classifying the cancer sample as insensitive to the EGFR inhibitor. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor or a second generation EGFR inhibitor.

[0013] In some aspects, disclosed herein is a method for classifying cancer samples, comprising: (a) detecting EGFR mutation in cancer samples, which is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q; and (b) classifying the cancer samples as T790M-like 3R EGFR mutation cancer. In some embodiments, the method further comprises classifying the cancer samples as insensitive to EGFR inhibitors. In some embodiments, the EGFR inhibitor is a first-generation EGFR inhibitor, a second-generation EGFR inhibitor, or a third-generation EGFR inhibitor.

[0014] Provided herein in some aspects is a method for classifying a cancer sample, comprising: (a) detecting a nucleotide sequence encoding one or more of the following nucleotide sequences in a cancer sample: A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY Disclosed is a method comprising: (a) detecting an EGFR mutation that is H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP; and (b) classifying the cancer sample as an exon 20 loop proximal insertion EGFR mutation cancer. In some embodiments, the method further comprises classifying the cancer sample as sensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a second generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the method further comprises classifying the cancer sample as insensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor or a third generation EGFR inhibitor.

[0015] In some aspects, disclosed herein is a method for classifying cancer samples, comprising: (a) detecting EGFR mutations in cancer samples, which are H773_V774 insNPH, H773_V774 insAH, H773dupH, V774_C775 insHV, V774_C775 insPR; and (b) classifying the cancer sample as exon 20 loop distal insertion EGFR mutation cancer. In some embodiments, the method further comprises classifying the cancer sample as insensitive to EGFR inhibitors. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, or a third generation EGFR inhibitor.

[0016] Provided herein in some aspects is a method for classifying a cancer sample, comprising: (a) detecting a genomic DNA sequence of a cancer sample that is ... S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R Disclosed is a method comprising: (a) detecting an EGFR mutation that is T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M; and (b) classifying the cancer sample as a P-loop αC helix compression EGFR mutant cancer. In some embodiments, the method further comprises classifying the cancer sample as sensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a second generation EGFR inhibitor. In some embodiments, the method further comprises classifying the cancer sample as insensitive to an EGFR inhibitor. In some embodiments, the EGFR inhibitor is a third generation EGFR inhibitor.

[0017] In some embodiments, the method further comprises, prior to (a), obtaining a cancer sample from the subject. In some embodiments, the method further comprises extracting tumor DNA from the cancer sample. In some embodiments, (a) comprises sequencing the tumor DNA from the cancer sample. In some embodiments, the cancer sample is a lung cancer sample. In some embodiments, the lung cancer sample is a non-small cell lung cancer sample.

[0018] Disclosed herein in some aspects is a method for treating a subject for cancer, comprising administering an effective amount of an EGFR inhibitor to a subject determined to have a classical-like EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the EGFR inhibitor is Erlotinib, Gefitinib, AZD3759, Sapatinib, Lapatinib, Tucatinib, Afatinib, Dacomitinib, Neratinib, Tarlox-TKI, Tarloxotinib, Osimertinib, Nazartinib, Olmutinib, Rocelitinib, Nacotinib, Lazertinib, TAS 6417, AZ5104 or TAK-788 (Mobocertinib). In some embodiments, the classical-like EGFR mutations are A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, L858R R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M.

[0019] In some aspects, disclosed herein is a method for treating a subject for cancer, comprising administering an effective amount of an EGFR inhibitor to a subject determined to have a T790M-like 3S EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the EGFR inhibitor is a third-generation EGFR inhibitor. In some embodiments, the EGFR inhibitor is osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, or lazertinib. In some embodiments, the EGFR inhibitor is not a first-generation or second-generation EGFR inhibitor. In some embodiments, the EGFR inhibitor is not erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, or tarloxotinib. In some embodiments, the T790M-like 3S EGFR mutation is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M.

[0020] In some aspects, disclosed herein is a method for treating a subject for cancer, comprising administering an effective amount of a tyrosine kinase inhibitor to a subject determined to have a T790M-like 3R EGFR mutation from analysis of tumor DNA from the subject, wherein the tyrosine kinase inhibitor is not an EGFR inhibitor. In some embodiments, the tyrosine kinase inhibitor is a PKC inhibitor. In some embodiments, the PKC inhibitor is ruboxistaurin, midostaurin, sotrastaurin, chelerythrine, myabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostatin 1, or tamoxifen. In some embodiments, the tyrosine kinase inhibitor is an ALK inhibitor. In some embodiments, the ALK inhibitor is AZD3463, brigatinib, crizotinib, ceritinib, alectinib, lorlatinib, ensartinib, entrectinib, repotrectinib, verizatinib, arcotinib, foritinib, CEP-37440, TQ-B3139, PLB1003, TPX-0131, or ASP-3026. In some embodiments, the T790M-like 3R EGFR mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q.

[0021] Disclosed herein in some aspects is a method for treating a subject for cancer, comprising administering an effective amount of an EGFR inhibitor to a subject determined to have an exon 20 loop proximal insertion EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the EGFR inhibitor is a second generation EGFR inhibitor or an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the EGFR inhibitor is afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS 6417, AZ5104 or TAK-788 (mobocertinib). In some embodiments, the EGFR inhibitor is not a first generation or third generation EGFR inhibitor. In some embodiments, the EGFR inhibitor is not erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, or lazertinib. In some embodiments, the exon 20 loop proximal insertion EGFR mutation is A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP.

[0022] Disclosed herein in some aspects is a method for treating a subject for cancer, comprising administering an effective amount of an EGFR inhibitor to a subject determined to have an exon 20 loop distal insertion EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the EGFR inhibitor is an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the EGFR inhibitor is TAS 6417, AZ5104 or TAK-788 (mobocertinib). In some embodiments, the exon 20 loop distal insertion EGFR mutation is H773_V774 insNPH, H773_V774 insAH, H773dupH, V774_C775 insHV, V774_C775 insPR.

[0023] Disclosed herein in some aspects is a method for treating a subject for cancer, comprising administering an effective amount of an EGFR inhibitor to a subject determined to have a P-loop αC-helix compaction EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20. In some embodiments, the EGFR inhibitor is erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS 6417, AZ5104, or TAK-788 (mobocertinib). In some embodiments, the EGFR inhibitor is a second generation EGFR inhibitor. In some embodiments, the EGFR inhibitor is afatinib, dacomitinib, neratinib, tarlox-TKI or tarloxotinib. In some embodiments, the EGFR inhibitor is not a second generation EGFR inhibitor. In some embodiments, the EGFR inhibitor is not afatinib, dacomitinib, neratinib, tarlox-TKI or tarloxotinib.In some embodiments, the P-loop αC-helix compression EGFR mutations are A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M.

[0024] In some embodiments, the subject has lung cancer. In some embodiments, the subject has non-small cell lung cancer. In some embodiments, the subject does not have lung cancer.

[0025] Provided herein in some aspects is a method for treating a subject for lung cancer comprising administering an effective amount of osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, or a combination thereof to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, wherein the EGFR mutation is selected from the group consisting of Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M.

[0026] Also provided herein, in some aspects, is a method for treating a subject for lung cancer, comprising: (a) detecting, in tumor DNA from the subject, Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I Disclosed are methods that include (a) detecting an EGFR mutation, or an EGFR mutation that is T790M; and (b) administering to the subject an effective amount of osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, or a combination thereof.

[0027] In some embodiments, the method includes administering osimertinib to the subject. In some embodiments, the method includes administering nazartinib to the subject. In some embodiments, the method includes administering olmutinib to the subject. In some embodiments, the method includes administering loceritinib to the subject. In some embodiments, the method includes administering nacotinib to the subject. In some embodiments, the method includes administering lazertinib to the subject.

[0028] In some embodiments, the EGFR mutation is Ex19del T790M. In some embodiments, the EGFR mutation is Ex19del T790M L718V. In some embodiments, the EGFR mutation is Ex19del T790M G724S. In some embodiments, the EGFR mutation is G719A T790M. In some embodiments, the EGFR mutation is G719S T790M. In some embodiments, the EGFR mutation is H773R T790M. In some embodiments, the EGFR mutation is I744_E749del insMKK. In some embodiments, the EGFR mutation is L747_K754 delinsATSPE. In some embodiments, the EGFR mutation is L858R T790M L792H. In some embodiments, the EGFR mutation is L858R T790M V843I. In some embodiments, the EGFR mutation is L858R T790M. In some embodiments, the EGFR mutation is S768I T790M. In some embodiments, the EGFR mutation is T790M.

[0029] In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0030] Provided herein in some aspects is a method for treating a subject for lung cancer, comprising administering to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject an effective amount of afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib), or a combination thereof, wherein the EGFR mutation is selected from the group consisting of A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H ... V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP.

[0031] Also provided herein, in some aspects, is a method for treating a subject for lung cancer, comprising: (a) detecting any of the following in tumor DNA from the subject: A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY Disclosed are methods that include (a) detecting an EGFR mutation that is H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP; and (b) administering to the subject an effective amount of afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib) or a combination thereof.

[0032] In some embodiments, the method includes administering afatinib to the subject. In some embodiments, the method includes administering dacomitinib to the subject. In some embodiments, the method includes administering neratinib to the subject. In some embodiments, the method includes administering a Tarlox-TKI to the subject. In some embodiments, the method includes administering tarloxotinib to the subject. In some embodiments, the method includes administering TAS6417 (CLN-081) to the subject. In some embodiments, the method includes administering AZ5104 to the subject. In some embodiments, the method includes administering TAK-788 (mobocertinib) to the subject.

[0033] In some embodiments, the EGFR mutation is A767_V769dupASV. In some embodiments, the EGFR mutation is A767_S768insTLA. In some embodiments, the EGFR mutation is S768_D770dupSVD. In some embodiments, the EGFR mutation is S768_D770dupSVD L858Q. In some embodiments, the EGFR mutation is S768_D770dupSVD R958H. In some embodiments, the EGFR mutation is S768_D770dupSVD V769M. In some embodiments, the EGFR mutation is V769_D770insASV. In some embodiments, the EGFR mutation is V769_D770insGSV. In some embodiments, the EGFR mutation is V769_D770insGVV. In some embodiments, the EGFR mutation is V769_D770insMASVD. In some embodiments, the EGFR mutation is D770_N771insNPG. In some embodiments, the EGFR mutation is D770_N771insSVD. In some embodiments, the EGFR mutation is D770del insGY. In some embodiments, the EGFR mutation is D770_N771 insG. In some embodiments, the EGFR mutation is D770_N771 insY H773Y. In some embodiments, the EGFR mutation is N771dupN. In some embodiments, the EGFR mutation is N771dupN G724S. In some embodiments, the EGFR mutation is N771_P772insHH. In some embodiments, the EGFR mutation is N771_P772insSVDNR. In some embodiments, the EGFR mutation is P772_H773insDNP.

[0034] In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0035] Provided herein in some aspects is a method for treating a subject for lung cancer, comprising administering an effective amount of afatinib, dacomitinib, neratinib, tarlox-TKI, tarloxotinib, or a combination thereof to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, wherein the EGFR mutation is selected from the group consisting of A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M.

[0036] Also provided herein, in some aspects, is a method for treating a subject for lung cancer, comprising: (a) detecting any of the following in tumor DNA from the subject: A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R and (b) administering to the subject an effective amount of afatinib, dacomitinib, neratinib, tarlox-TKI, tarloxotinib or a combination thereof.

[0037] In some embodiments, the method includes administering afatinib to the subject. In some embodiments, the method includes administering dacomitinib to the subject. In some embodiments, the method includes administering neratinib to the subject. In some embodiments, the method includes administering a Tarlox-TKI to the subject. In some embodiments, the method includes administering tarloxotinib to the subject.

[0038] In some embodiments, the EGFR mutation is A750_I759del insPN. In some embodiments, the EGFR mutation is E709_T710del insD. In some embodiments, the EGFR mutation is E709A. In some embodiments, the EGFR mutation is E709A G719A. In some embodiments, the EGFR mutation is E709A G719S. In some embodiments, the EGFR mutation is E709K. In some embodiments, the EGFR mutation is E709K G719S. In some embodiments, the EGFR mutation is E736K. In some embodiments, the EGFR mutation is E746_A750del A647T. In some embodiments, the EGFR mutation is E746_A750del R675W. In some embodiments, the EGFR mutation is E746_T751del insV S768C. In some embodiments, the EGFR mutation is Ex19del C797S. In some embodiments, the EGFR mutation is Ex19del G796S. In some embodiments, the EGFR mutation is Ex19del L792H. In some embodiments, the EGFR mutation is Ex19del T854I. In some embodiments, the EGFR mutation is G719A. In some embodiments, the EGFR mutation is G719A D761Y. In some embodiments, the EGFR mutation is G719A L861Q. In some embodiments, the EGFR mutation is G719A R776C. In some embodiments, the EGFR mutation is G719A S768I. In some embodiments, the EGFR mutation is G719C S768I. In some embodiments, the EGFR mutation is G719S. In some embodiments, the EGFR mutation is G719S L861Q. In some embodiments, the EGFR mutation is G719S S768I. In some embodiments, the EGFR mutation is G724S. In some embodiments, the EGFR mutation is G724S Ex19del. In some embodiments, the EGFR mutation is G724S L858R. In some embodiments, the EGFR mutation is G779F. In some embodiments, the EGFR mutation is I740dupIPVAK. In some embodiments, the EGFR mutation is K757M L858R. In some embodiments, the EGFR mutation is K757R. In some embodiments, the EGFR mutation is L718Q.In some embodiments, the EGFR mutation is Ex19del. In some embodiments, the EGFR mutation is L718Q L858R. In some embodiments, the EGFR mutation is L718V. In some embodiments, the EGFR mutation is L718V L858R. In some embodiments, the EGFR mutation is L747_S752del A755D. In some embodiments, the EGFR mutation is L747P. In some embodiments, the EGFR mutation is L747S. In some embodiments, the EGFR mutation is L747S L858R. In some embodiments, the EGFR mutation is L747S V774M. In some embodiments, the EGFR mutation is L858R C797S. In some embodiments, the EGFR mutation is L858R L792H. In some embodiments, the EGFR mutation is L858R T854S. In some embodiments, the EGFR mutation is N771G. In some embodiments, the EGFR mutation is R776C. In some embodiments, the EGFR mutation is R776H. In some embodiments, the EGFR mutation is E709_T710del insD S22R. In some embodiments, the EGFR mutation is S752_I759del V769M. In some embodiments, the EGFR mutation is S768I. In some embodiments, the EGFR mutation is S768I L858R. In some embodiments, the EGFR mutation is S768I L861Q. In some embodiments, the EGFR mutation is S768I V769L. In some embodiments, the EGFR mutation is S768I V774M. In some embodiments, the EGFR mutation is T751_I759 delinsN. In some embodiments, the EGFR mutation is V769L. In some embodiments, the EGFR mutation is V769M. In some embodiments, the EGFR mutation is V774M. In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0039] In some embodiments of the methods disclosed herein, the method further comprises administering an additional cancer therapy to the subject. In some embodiments, the additional cancer therapy comprises chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the additional cancer therapy comprises an anaplastic lymphoma kinase (ALK) inhibitor. In some embodiments, the ALK inhibitor is brigatinib or AZD3463. In some embodiments, the additional cancer therapy comprises a protein kinase C (PKC) inhibitor. In some embodiments, the PKC inhibitor is ruboxistaurin, midostaurin, or sotrastaurin.

[0040] Also disclosed herein, in some aspects, is a method for treating a subject for non-small cell lung cancer, the method comprising administering to a subject, who has been previously determined to have a classical-like EGFR mutation from analysis of tumor DNA from the subject, an effective amount of a composition comprising: (a) a third generation EGFR inhibitor, (b) a second generation EGFR inhibitor, (c) a first generation EGFR inhibitor, or (d) an EGFR inhibitor specific for a mutation associated with EGFR exon 20.

[0041] Further, in some aspects, a method for treating a subject for non-small cell lung cancer is disclosed, comprising administering an effective amount of a composition comprising (a) a third generation EGFR inhibitor, (b) a PKC inhibitor, or (c) an ALK inhibitor to a subject previously determined to have a T790M-like 3S EGFR mutation from analysis of tumor DNA from the subject. In some embodiments, the method does not include administering a second generation EGFR inhibitor to the subject. In some embodiments, the method does not include administering a first generation EGFR inhibitor to the subject.

[0042] Disclosed herein in some aspects is a method for treating a subject for non-small cell lung cancer, comprising administering to a subject previously determined to have a T790M-like 3R EGFR mutation from analysis of tumor DNA from the subject an effective amount of a composition comprising (a) a PKC inhibitor, or (b) an ALK inhibitor. In some aspects, the method further comprises administering to the subject a third generation EGFR inhibitor. In some aspects, the method does not comprise administering to the subject a third generation EGFR inhibitor. In some embodiments, the method does not comprise administering to the subject a second generation EGFR inhibitor. In some embodiments, the method does not comprise administering to the subject a first generation EGFR inhibitor.

[0043] Also disclosed herein in some aspects is a method for treating a subject for non-small cell lung cancer, comprising administering to a subject, who has been previously determined to have an exon 20 loop proximal insertion EGFR mutation from analysis of tumor DNA from the subject, an effective amount of a composition comprising (a) a second generation EGFR inhibitor, or (b) an EGFR inhibitor specific for a mutation associated with EGFR exon 20. In some aspects, the method does not include administering to the subject a first generation EGFR inhibitor. In some embodiments, the method does not include administering to the subject a third generation EGFR inhibitor.

[0044] Further, in some embodiments, a method for treating a subject for non-small cell lung cancer is disclosed, the method comprising: (a) administering to a subject, the subject having a pre-determined exon 20 loop distal insertion EGFR mutation from analysis of tumor DNA from the subject, an effective amount of a composition comprising an EGFR inhibitor specific for a mutation associated with EGFR exon 20. In some embodiments, the method does not include administering to the subject a first generation EGFR inhibitor. In some embodiments, the method does not include administering to the subject a second generation EGFR inhibitor. In some embodiments, the method does not include administering to the subject a third generation EGFR inhibitor.

[0045] Disclosed herein, in some aspects, is a method for treating a subject for non-small cell lung cancer, the method comprising administering to a subject, who has been previously determined to have a P-loop αC-helix compression EGFR mutation from analysis of tumor DNA from the subject, an effective amount of a composition comprising (a) a second generation EGFR inhibitor, (b) a first generation EGFR inhibitor, or (c) an EGFR inhibitor specific for a mutation associated with EGFR exon 20. In some embodiments, the composition comprises a second generation EGFR inhibitor. In some embodiments, the method does not include administering to the subject a third generation EGFR inhibitor.

[0046] "Individual," "subject," and "patient" are used interchangeably and can refer to a human or non-human.

[0047] As used herein, "treatment" refers to both therapeutic and prophylactic measures, the purpose of which is to prevent or slow (reduce) the growth, development, or spread of undesirable physiological changes or diseases, such as cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, a delay or slowing of disease progression, improvement or alleviation of disease state, and remission (partial or complete), whether detectable or not. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Subjects in need of treatment include those already suffering from the condition or disease, as well as those susceptible to suffering from the condition or disease, or those in whom the condition or disease is to be prevented. The outcome of treatment can be determined by methods known in the art, such as determining relief of pain as measured by a reduced need for administration of opiates or other analgesics, determining reduction in tumor burden, determining functional recovery, or other methods known in the art.

[0048] Throughout this specification, the word "about" is used to indicate that a value includes the inherent variation of error in the measurement or quantification method.

[0049] The use of the words "a" or "an" used in conjunction with the word "comprising" can mean "one," but it is also not inconsistent with the meaning of "one or more," "at least one," and "one or more than one."

[0050] The term "and / or" means "and" or "or." For example, A, B and / or C includes A only, B only, C only, A and B in combination, A and C in combination, B and C in combination, or A, B and C in combination. In other words, "and / or" functions as an inclusive disjunction.

[0051] "Comprising" (and any form of includes, e.g., "including"), "having" (and any form of has, e.g., "having"), "including" (and any form of includes, e.g., "including") or "containing" (and any form of contains, e.g., "containing") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0052] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout the specification. Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the disclosure.

[0053] Any method related to a therapeutic, diagnostic or physiological purpose or effect may also be described in "use" claim language, such as the "use" of any compound, composition or agent described herein to achieve or realize the described therapeutic, diagnostic or physiological purpose or effect.

[0054] It is contemplated that any embodiment described herein can be implemented with respect to any method or composition of the disclosure, and any method or composition of the disclosure can be implemented with respect to any embodiment described herein. Additionally, the compositions of the present disclosure can also be used to achieve the methods of the disclosure.

[0055] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0056] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0057] [Figure 1A] EGFR mutations can be divided into four distinct subgroups based on drug sensitivity and structural changes. Figure 1A. Heatmap from unsupervised hierarchical clustering of log(mutant / WT) ratios from Ba / F3 cells expressing the indicated mutations 72 hours after treatment with the indicated drugs. To determine mutant / WT ratios, IC50 values ​​for each drug and cell line were calculated and then compared to the average IC50 value of Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF to maintain viability). □ represents the average of n=3 replicates. For co-occurring mutations, the order of exons 1, 2 and 3 was assigned arbitrarily. Groups were assigned based on structural predictions. [Figure 1B]EGFR mutations can be divided into four different subgroups based on drug sensitivity and structural changes. Figure 1B-1E. In silico mutation mapping of (Figure 1B) classical-like, (Figure 1C) T790M-like, (Figure 1D) exon 20 loop insertion (red / blue) and WT (gray / green) as well as (Figure 1E) PACC mutant. [Figure 1C] See legend to Figure 1B. [Figure 1D] See legend to Figure 1B. [Figure 1E] See legend to Figure 1B. [Figure 1F] EGFR mutations can be divided into four different subgroups based on drug sensitivity and structural alterations. Figure 1F. Dot plot of ρ values ​​of Spearman correlation of mutations to exon-based group averages or structure-function-based averages by drug. Dots represent each mutation, bars represent mean ρ value ± standard deviation (SD), p values ​​were determined using paired Student's t-test. [Diagram 2] Structure-function-based grouping predicts drug and mutation susceptibility better than exon-based grouping. Bar graph of Spearman rho values ​​for the indicated mutations compared to exon-based groups (yellow) or structure-function-based groups (green). The delta of the two rho values ​​is shown as an overlapping grey bar. As the delta bar shifts to the right, the Spearman rho value is higher for the structure-function-based group and as the grey bar shifts to the left, the Spearman rho value is higher for the exon-based group. [Figure 3A]Heatmaps generated through supervised clustering by structure-function-based groups cluster drug sensitivity more correctly than exon-based groups. Figures 3A-3B. Heatmap supervised clustering by (Figure 3A) exon-based or (Figure 3B) structure-function-based groups of log (mutant / WT) ratios from Ba / F3 cells expressing the indicated mutations 72 hours after treatment with the indicated drugs. To determine mutant / WT ratios, IC50 values ​​were calculated for each drug and cell line and then compared to the average IC50 value of Ba / F3 cells expressing WT EGFR (+10 ng / ml EGF to maintain viability). □ represents the average of n=3 replicates. For co-occurring mutations, the order of exons 1, 2, and 3 was assigned arbitrarily. Groups were assigned based on structural predictions. [Figure 3B] See legend to Figure 3A. [Figure 4A] Classical-like EGFR mutations are not predicted to alter the drug binding pocket and are the most sensitive to third-generation EGFR TKIs. Figures 4A-4B. In silico model of WT EGFR (PDB 2ITX) visualized as both (Figure 4A) a ribbon model and (Figure 4B) a space-filling model. Residues important for receptor signaling and drug binding are highlighted. [Figure 4B] See legend to Figure 4A. [Figure 4C] Classical-like EGFR mutations are not predicted to alter the drug binding pocket and are the most sensitive to third-generation EGFR TKIs (Figures 4C-4D). (Figure 4C) An in silico model of L861Q overlapping with WT (gray) and L861R (blue) and (Figure 4D) a space-filling model show that the R861 substitution is distal to the drug binding pocket and has minimal impact on the overall structure of EGFR compared to WT. [Figure 4D] See legend to Figure 4C. [Figure 4E]Classical-like EGFR mutations are not predicted to alter the drug binding pocket and are the most sensitive to third-generation EGFR TKIs. Figure 4E. Dot plots of mutant / WT IC50 values ​​for Ba / F3 cells expressing classical-like EGFR mutations and treated with the indicated classes of EGFR TKIs. Dots represent the mean of n=3 replicate mutant / WT IC50 values ​​for individual cell lines expressing classical-like mutations with individual drugs. Bars represent the mean mutant / WT IC50 values ​​± SEM for each class of EGFR TKI and all classical-like cell lines. p values ​​were determined by analysis of variance with unequal SD determined by Brown-Forsythe test to determine differences in SD. Differences between groups were determined using the Holm-Sidak multiple comparison test. [Figure 4F] Classic-like EGFR mutations are not predicted to alter the drug binding pocket and are the most sensitive to third generation EGFR TKIs. Figure 4F. Tumor growth curves for PDXs harboring EGFR L858R E709K compound mutations treated with the indicated inhibitors. Tumors were measured three times a week and symbols are mean tumor volume ± SEM. Mice were randomized into six groups. Drugs were administered to mice 5 days a week and on day 28 mice were euthanized and tumors harvested. [Figure 4G] Classic-like EGFR mutations are not predicted to alter the drug binding pocket and are the most sensitive to third-generation EGFR TKIs. Figure 4G. Dot plot of percent change in tumor volume at day 28 for tumors described in Figure 4F. Dots represent each tumor and bars represent mean ± SEM per group. Statistical differences were determined by ordinary one-way ANOVA with post-hoc Tukey multiple comparison test to determine differences between groups. [Diagram 5]Exon 20 loop insertions are a distinct class of EGFR mutation. Figure 5A. Dot plots of mutant / WT IC50 values ​​for Ba / F3 cells expressing exon 20 loop insertion mutations treated with the indicated classes of EGFR TKIs. Dots represent the mean of n=3 replicate mutant / WT IC50 values ​​for individual cell lines expressing exon 20 loop insertion mutations with individual drugs. Bars represent the mean mutant / WT IC50 values ​​± SEM for each class of EGFR TKI and all exon 20 loop insertion cell lines. p values ​​were determined by analysis of variance with unequal SDs determined by Brown-Forsythe test to determine differences in SD. Differences between groups were determined using the Holm-Sidak multiple comparison test. Figure 5B. Tumor growth curves for PDXs harboring EGFR S768dupSVD exon 20 loop insertion mutations treated with the indicated inhibitors. Tumors were measured three times a week and symbols are the mean tumor volume ± SEM. Mice were randomized into four groups. Mice were administered drugs 5 days per week and euthanized on day 21 to harvest tumors. Figure 5C. Dot plot of percent change in tumor volume on day 21 for tumors described in Figure 5B. Dots represent individual tumors and bars represent mean ± SEM per group. Statistical differences were determined by ordinary one-way ANOVA with post-hoc Tukey's multiple comparison test to determine differences between groups. [Figure 6A] Drug repurposing can overcome T790M-like resistance mutations. Figure 6A. Heatmap from unsupervised hierarchical clustering of log(mutant / WT) ratios from Ba / F3 cells expressing the indicated mutations 72 hours after treatment with the indicated drugs. Squares represent the mean of n=3 replicates. For co-occurring mutations, the order of exons 1, 2, and 3 was assigned arbitrarily. Groups were assigned based on hierarchical clustering and known resistance mutations. [Figure 6B]Drug repurposing can overcome T790M-like resistance mutations. Figures 6B-6C. Dot plots of mutant / WT IC50 values ​​for Ba / F3 cells expressing (Figure 6B) T790M-like 3S (third-generation EGFR TKI sensitive) and (Figure 6C) T790M-like 3R (third-generation EGFR TKI resistant) EGFR mutations treated with the indicated classes of EGFR TKIs. Dots represent the mean of n=3 replicate mutant / WT IC50 values ​​for individual cell lines expressing classic-like mutations with individual drugs. Bars represent the mean mutant / WT IC50 values ​​± SEM for each class of EGFR TKI and all cell lines. p values ​​were determined by analysis of variance with unequal SD determined by Brown-Forsythe test to determine differences in SD. Differences between groups were determined using the Holm-Sidak multiple comparison test. [Figure 6C] See legend to Figure 6B. [Figure 7A] PACC mutations are robustly sensitive to second-generation TKIs. Figure 7A. In silico modeling of EGFR G179S (PDB 2ITN, purple) with osimertinib in the reactive conformation (green) and G719S in the predicted conformation (orange) shows destabilizing TKI-protein interactions at the indole ring. [Figure 7B] PACC mutations are robustly sensitive to second-generation TKIs. Figure 7B. Dot plots of mutant / WT IC50 values ​​for Ba / F3 cells expressing PACC mutations treated with the indicated classes of EGFR TKIs. Dots represent the mean of n=3 replicate mutant / WT IC50 values ​​for individual cell lines expressing PACC mutations with individual drugs. Bars represent the mean mutant / WT IC50 values ​​± SEM for each class of EGFR TKI and all PACC cell lines. p values ​​were determined by analysis of variance with unequal SD determined by Brown-Forsythe test to determine differences in SD. Differences between groups were determined using the Holm-Sidak multiple comparison test. [Figure 7C]PACC mutations are robustly sensitive to second generation TKIs. Figure 7C. Tumor growth curves for PDXs harboring EGFR S768dupSVD exon 20 loop insertion mutations treated with the indicated inhibitors. Tumors were measured three times a week and symbols are mean tumor volume ± SEM. Mice were randomized into six groups. Drugs were administered to mice 5 days a week and on day 28 mice were euthanized and tumors harvested. [Figure 7D] PACC mutations are robustly sensitive to second-generation TKIs. Figure 7D. Computed tomography (CT) scan of a patient with NSCLC harboring a G719S E709K compound mutation before and 4 weeks after afatinib treatment. Arrows indicate resolution of pleural effusion in the right lobe and reduction in pleural effusion and tumor size in the left lobe. [Figure 7E] PACC mutations are robustly sensitive to second-generation TKIs. Figure 7E. Heatmap from unsupervised hierarchical clustering of log(mutant / WT) ratios from Ba / F3 cells expressing the indicated mutations 72 hours after treatment with the indicated drugs. Squares represent the mean of n=3 replicates. For co-occurring mutations, the order of exons 1, 2, and 3 was assigned arbitrarily. Groups were assigned based on predicted mutation impact. [Figure 7F] PACC mutations are robustly sensitive to second-generation TKIs. Figure 7F. Dot plots of mutant / WT IC50 values ​​for Ba / F3 cells expressing classic EGFR mutations (white bars) or classic EGFR mutations and acquired PACC mutations (colored bars) treated with the indicated classes of EGFR TKIs. Dots represent the mean of n=3 replicate mutant / WT IC50 values ​​for individual cell lines expressing the indicated mutations with individual drugs. Bars represent the mean mutant / WT IC50 values ​​± SEM for each class of EGFR TKI and the indicated cell line. p values ​​were determined by analysis of variance with unequal SD determined by Brown-Forsythe test to determine differences in SD. Differences between groups were determined using the Holm-Sidak multiple comparison test. [Figure 7G]PACC mutations are robustly sensitive to second-generation TKIs. Figure 7G. In silico modeling of EGFR Ex19del G796S (purple) with osimertinib in a reactive conformation (blue) and G719S in a predicted conformation (orange) shows destabilizing TKI-protein interactions in the hinge region (yellow), which displaces the reactive group of osimertinib (arrow). [Figure 8A] PACC mutations alter the orientation of the P-loop and / or the αC-helix and are sensitive to second-generation TKIs. Figure 8A. Overlap of the G719S (PDB 2ITN, green) and WT EGFR (PDB 2ITX, grey) crystal structures shows a significant shift of F723 in the P-loop (red arrow), which orients the benzyl ring in a downward position and condenses the P-loop into the drug-binding pocket. Additionally, G719S has an inward shift of the αC-helix compared to the WT crystal structure. [Figure 8B] PACC mutations alter the orientation of the P-loop and / or αC-helix and are sensitive to second-generation TKIs. Figure 8B. Space-filling model of G719S (PDB 2ITN) at the P-loop (red), αC-helix (blue), hinge region (orange), C797 (yellow) and DFG motif (green) highlighted to show the steric hindrance of the drug-binding pocket caused by the shifted P-loop. [Figure 8C] PACC mutations alter the orientation of the P-loop and / or αC-helix and are sensitive to second-generation TKIs. Figure 8C. In silico homology model of EGFR L718Q (pink) with the predicted osimertinib structure shows that Q718 interferes with the interaction of osimertinib (green) with M793, shifting the Michael acceptor (reactive group, green arrow) out of alignment with C797 (yellow arrow). [Figure 8D] PACC mutations alter the orientation of the P loop and / or αC helix and are sensitive to second-generation TKIs. Figure 8D. In silico modeling of EGFR G719S. [Figure 8E]PACC mutations alter the orientation of the P-loop and / or αC-helix and are sensitive to second-generation TKIs. Figure 8E. Dot plot of percent change in tumor volume at day 28 for tumors described in Figure 3C. Dots represent each tumor and bars represent mean ± SEM per group. Statistical differences were determined by ordinary one-way ANOVA with post-hoc Tukey's multiple comparison test to determine differences between groups. [Figure 8F] PACC mutations alter the orientation of the P loop and / or αC helix and are sensitive to second generation TKIs. Figure 8F. In silico modeling of EGFR Ex19del G796S (purple). [Figure 9A] Structure-function groups predict patient outcome better than exon-based groups. Figure 9A. Kaplan-Meier plots of afatinib treatment duration for patients with NSCLC tumors harboring atypical EGFR mutations (N=358 patients) stratified by structure-based groups. Figures 9A-9B. Classic-like N=58, T790M-like N=68, Ex20ins N=76, PACC N=156. If the mutation was not explicitly described, those patients were excluded from the structure-function-based analysis. [Figure 9B] Structure-function groups predict patient outcomes better than exon-based groups. Figure 9B. Forest plot of hazard ratios calculated from the Kaplan-Meier plot in Figure 9A. Hazard ratios and p-values ​​were calculated using the Mantel-Cox log-rank test. Data represent hazard ratios ± 95% CI. Figures 9A-9B. Classical-like N=58, T790M-like N=68, Ex20ins N=76, PACC N=156. If a mutation was not explicitly described, those patients were excluded from the structure-function-based analysis. [Figure 9C] Structure-function groups predict patient outcome better than exon-based groups. Figure 9C. Kaplan-Meier plot of PFS for N=44 patients (N=13 treated with first-generation EGFR TKI, N=21 treated with second-generation EGFR TKI, or N=10 treated with third-generation EGFR TKI) with NSCLC tumors harboring PACC mutations. [Figure 9D] Structure-function groups predict patient outcome better than exon-based groups. Figure 9D. Forest plot of hazard ratios calculated from the Kaplan-Meier plot in panel C and Extended Data Figure E. Hazard ratios and p-values ​​were calculated using the Mantel-Cox log-rank test. PACC N=44: 1st N=13, 2nd N=21 and 3rd N=10; non-PACC N=40: 1st N=20, 2nd N=6 and 3rd N=14. [Figure 10A] Structure-function groups identify patients with greater benefit to second-generation TKIs than exon-based groups. Figures 10A-10B. Overall response rates to afatinib stratified by (Figure 10A) structure-function-based group (N=507: classical-like N=91, T790M-like N=103, Ex20ins N=120, and PACC N=193) or (Figure 10B) exon-based group (N=528: exon 18 N=133, exon 19 N=22, exon 20 N=294, exon 21 N=79). If no mutation was explicitly described (N=21), those patients were excluded from the structure-function-based analysis. [Figure 10B] See legend to Figure 10A. [Figure 10C] Structure-function groups identify patients with greater benefit to second-generation TKIs than exon-based groups. Figure 10C. Kaplan-Meier plots of afatinib treatment duration for patients (N=364) with NSCLC tumors harboring atypical EGFR mutations stratified by exon-based groups. Figures 10C-10D. Exon 18 N=87, Exon 19 N=19, Exon 20 N=195, and Exon 21 N=63). [Figure 10D]The structure-function group identifies patients with greater benefit to second-generation TKIs than the exon-based group. Figure 10D. Forest plot of hazard ratios calculated from the Kaplan-Meier plot in Figure 10C. Hazard ratios and p-values ​​were calculated using the Mantel-Cox log-rank test. Data represent hazard ratios ± 95% CI. Figures 10C-10D. Exon 18 N=87, exon 19 N=19, exon 20 N=195 and exon 21 N=63). [Figure 10E] Structure-function groups identify patients with greater benefit to second-generation TKIs than exon-based groups. Figure 10E. Kaplan-Meier plot of PFS for patients (N=40) with NSCLC tumors harboring non-PACC atypical EGFR mutations treated with first-generation (N=20), second-generation (N=6), or third-generation (N=14) EGFR TKIs. [Figure 11] Representative space-filling models of the disclosed EGFR mutation subgroups showing changes in the overall shape of the drug binding pocket are shown. The most common mutations for each group are shown, and drug sensitivity or selectivity is listed from most selective or sensitive to resistant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description The present disclosure is based, at least in part, on the surprising discovery that four distinct structure-function-based groups of EGFR mutations predict patient outcomes following cancer therapy, e.g., treatment with kinase inhibitors, better than classical grouping of mutations by exon in the EGFR gene in which the mutations occur. In particular, each of the four groups of mutations corresponds to a different class of drug that is particularly effective in treating cancers expressing EGFR mutations from each group, which can be repurposed for patient treatment. Moreover, the structure-function-based groups also outperform exon-based groupings in predicting mutations with similar sensitivity to drug classes.

[0059] Thus, in some embodiments, a method for treating a subject for cancer, e.g., lung cancer, is disclosed that includes administering an effective amount of one or more kinase inhibitors from one or more kinase classes to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, where the EGFR mutation is a classical-like mutation, an exon 20 loop insertion specific mutation, a T790M-like susceptibility (T790M-like 3S) mutation, a T790M-like resistance (T790M-like 3R) mutation, or a P-loop αC helix compression mutation. Also disclosed is a method for treating a subject for cancer, e.g., lung cancer, comprising: (a) detecting an EGFR mutation in tumor DNA from the subject that is a classical-like mutation, an exon 20 loop insertion specific mutation (e.g., exon 20ins-NL or exon 20ins-FL), a T790M-like susceptibility (T790M-like 3S) mutation, a T790M-like resistance (T790M-like 3R) mutation, or a P-loop αC helix compression mutation; and (b) administering an effective amount of one or more kinase inhibitors from one or more kinase classes depending on the EGFR mutation detected.

[0060] I. Treatment Aspects of the present disclosure relate to compositions comprising a therapeutically effective amount of one or more cancer therapies and administration of such compositions to a subject or patient in need thereof. In some embodiments, the one or more cancer therapies comprise one or more kinase inhibitors.

[0061] The composition of the present disclosure can be used for in vivo, in vitro or ex vivo administration.The administration route of the composition can be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, inhalation or a combination of two or more administration routes.Cancer therapy can be administered via the same or different administration routes.

[0062] As used herein, the term "cancer" can be used to refer to solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer can occur in the blood, bladder, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0063] The cancer may specifically be of the following histological types, but is not limited to: neoplasm (malignant); carcinoma; carcinoma (undifferentiated); giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma (malignant); cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; adenocarcinoma (familial polyposis coli); solid tumor; carcinoid tumor (malignant); bronchioloalveolar carcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma ;Granular cell carcinoma;Follicular adenocarcinoma;Papillary follicular adenocarcinoma;Non-encapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Skin adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Earwax gland carcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease (breast);Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma (malignant);Ovarian stromal tumor (malignant);Theca cell tumor (malignant);Granulosa cell tumor (malignant);Androblastoma (malignant);Sertoli cell carcinoma;Leydig cell tumor (malignant);Lydig cell tumor (malignant);Lipid Cell tumors (malignant);paraganglioma (malignant);extramammary paraganglioma (malignant);pheochromocytoma;glomerular sarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus (malignant);sarcoma;fibrosarcoma;fibrous histiocytoma (malignant);myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumors (malignant);mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma (malignant);Brenner tumor (malignant);phyllodes tumor (malignant);synovial sarcoma;mesothelioma (malignant);dysgerminoma;fetal carcinoma; teratoma (malignant); ovarian goiter (malignant); choriocarcinoma; mesonephroma (malignant); angiosarcoma; hemangioendothelioma (malignant); Kaposi's sarcoma; hemangiopericytoma (malignant); lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma (malignant); mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor (malignant); ameloblastic odontosarcoma; ameloblastoma (malignant); ameloblastic fibrosarcoma; pinealoma (malignant); chordoma; glioma (malignant); ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; anaplastic neuroectodermal tumor;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neuroblastoma;Meningioma (malignant);Neurofibrosarcoma;Neurilemoma (malignant);Granular cell tumor (malignant);Malignant lymphoma;Hodgkin's disease;Hodgkin's;Side granuloma;Malignant lymphoma (small lymphocytic);Malignant lymphoma (diffuse large cell);Malignant lymphoma (follicular);Mycosis fungoides;Other specified non-Hodgkin's lymphoma;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lymphoblastic leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myeloid sarcoma and hairy cell leukemia. ;

[0064] In some embodiments, methods for treating lung cancer are disclosed. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma. In some embodiments, the non-small cell lung cancer is large cell carcinoma. In some embodiments, the non-small cell lung cancer is adenosquamous carcinoma. In some embodiments, the non-small cell lung cancer is sarcomatoid carcinoma. In some embodiments, the lung cancer is small cell lung cancer. In some aspects, methods for treating cancers that are not lung cancer are disclosed.

[0065] In some embodiments, the cancer therapy includes a local cancer therapy. In some embodiments, the cancer therapy includes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a local cancer therapy.

[0066] A. Kinase Inhibitors In some embodiments, the one or more cancer therapies include one or more kinase inhibitors. In some embodiments, the disclosed methods include administering one or more kinase inhibitors to a subject or patient in need thereof. As used herein, kinase inhibitor refers to a pharmaceutical compound that inhibits kinases. Examples of kinases that can be inhibited by the kinase inhibitors of the present disclosure include epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) and protein kinase C (PKC). Kinases are part of many cellular functions, including cell signaling, growth and division. Specifically, tyrosine kinases are responsible for the activation of many proteins through signaling cascades that result from the phosphorylation of proteins by tyrosine kinases. Kinase inhibitors inhibit the phosphorylation and subsequent activation of proteins by tyrosine kinases.

[0067] Kinase inhibitors act by competing with adenosine triphosphate, i.e. the phosphorylated entity, the substrate or both, or by acting allosterically, i.e. binding to a site outside the active site and influencing its activity by a conformational change. Recently, it has been shown that TKIs deprive tyrosine kinases of access to the Cdc37-Hsp90 molecular chaperone system on which they depend for cellular stability, leading to ubiquitination and degradation of the tyrosine kinase.

[0068] The amount of one or more kinase inhibitors delivered to a patient can be variable. In one suitable embodiment, the kinase inhibitor can be administered in an amount effective to cause the arrest or regression of cancer in the host. In other embodiments, the kinase inhibitor can be administered in an amount that is 2-10,000 times less than the chemotherapeutic effective amount of the kinase inhibitor. For example, the kinase inhibitor can be administered in an amount that is about 20 times, about 500 times, or about 5000 times less than the chemotherapeutic effective amount of the kinase inhibitor.

[0069] Kinase inhibitors of the present disclosure can be tested in vivo for the desired therapeutic activity when used alone or in combination with another cancer therapy, and to determine effective doses.For example, such compounds can be tested in suitable animal model systems, including rats, mice, chickens, cows, monkeys, rabbits, etc., before testing in humans.In vitro tests can also be used to determine suitable compounds and dosages, as described in the Examples.

[0070] The composition of one or more kinase inhibitors may or may not be customized to address any kinase inhibitor sensitivity or resistance of the cancer determined based on the analysis of the subject's genome for one or more mutations in the EGFR gene of the subject with cancer.The composition may be administered to the subject without performing a pre-analysis of the genome.The kinase inhibitor composition may include any one or more kinase inhibitors associated with an effective therapy for treating cancer.

[0071] A subject may be administered one or more kinase inhibitor compositions, including compositions comprising one or more kinase inhibitors that overcome any sensitivity or resistance of the cancer as determined based on an analysis of the subject's genome for one or more mutations in the EGFR gene of the subject with cancer. The kinase inhibitors may be administered to treat the cancer and / or to enhance a therapy for treating the cancer.

[0072] The kinase inhibitor composition may be administered alone or in combination with one or more additional therapeutic agents disclosed herein. "In combination" administration with one or more additional therapeutic agents includes both simultaneous (concurrent) administration and sequential administration in any order. The kinase inhibitor composition and one or more additional therapeutic agents may be administered as one composition or as two separate compositions administered simultaneously or sequentially. Administration may be chronic or intermittent, as deemed appropriate by the supervising physician, including taking into account changes in undesirable side effects.

[0073] In some embodiments, the kinase inhibitor of the present disclosure is a tyrosine kinase inhibitor (TKI).Embodiments of the present disclosure include administering at least one, two, three, four, five or six classes of TKI to a subject with cancer.In some embodiments, the one, two, three, four, five or six TKI classes include a first generation EGFR TKI, a second generation EGFR TKI, a third generation EGFR TKI, an EGFR TKI specific for mutations associated with EGFR exon 20, an anaplastic lymphoma kinase (ALK) inhibitor or a protein kinase C (PKC) inhibitor.

[0074] In some embodiments, the TKI is a first generation EGFR TKI, including erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib and icotinib. In some aspects, the first generation EGFR inhibitor is erlotinib. In some aspects, the first generation EGFR inhibitor is gefitinib. In some aspects, the first generation EGFR inhibitor is AZD3759. In some aspects, the first generation EGFR inhibitor is sapatinib. In some aspects, the first generation EGFR inhibitor is lapatinib. In some aspects, the first generation EGFR inhibitor is tucatinib. In some aspects, the first generation EGFR inhibitor is icotinib. Any one or more of these EGFR inhibitors may be excluded from certain embodiments. As used herein, "first-generation EGFR TKI" (also used interchangeably herein as "first-generation EGFR TKI", "first-generation EGFR inhibitor" and "first generation EGFR inhibitor") refers to an EGFR inhibitor that can bind non-covalently to EGFR protein and cannot bind covalently to EGFR. In some embodiments, the TKI is a second-generation EGFR TKI, including afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189 and sutetinib. In some aspects, the second-generation EGFR inhibitor is afatinib. In some aspects, the second-generation EGFR inhibitor is dacomitinib. In some aspects, the second-generation EGFR inhibitor is neratinib. In some aspects, the second-generation EGFR inhibitor is a Tarlox-TKI. In some aspects, the second-generation EGFR inhibitor is tarloxotinib. In some aspects, the second generation EGFR inhibitor is BDTX189. In some aspects, the second generation EGFR inhibitor is stetinib. Any one or more of these EGFR inhibitors may be excluded from certain embodiments.As used herein, "second-generation EGFR TKI" (also used interchangeably herein as "second-generation EGFR TKI", "second-generation EGFR inhibitor" or "second-generation EGFR inhibitor") refers to an EGFR inhibitor that can covalently bind to wild-type EGFR protein and certain mutant EGFR proteins, but cannot bind to (or has low affinity for) T790M mutant EGFR. In some embodiments, the TKI is a third-generation EGFR TKI, including osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, WZ4002, almonertinib, fulmonertinib, abivertinib, alfultinib, mavereltinib, abivertinib, olafertinib and resibertinib. In some aspects, the third-generation EGFR inhibitor is osimertinib. In some aspects, the third generation EGFR inhibitor is nazartinib. In some aspects, the third generation EGFR inhibitor is olmutinib. In some aspects, the third generation EGFR inhibitor is loceritinib. In some aspects, the third generation EGFR inhibitor is nacotinib. In some aspects, the third generation EGFR inhibitor is lazertinib. In some aspects, the third generation EGFR inhibitor is WZ4002. In some aspects, the third generation EGFR inhibitor is almonertinib. In some aspects, the third generation EGFR inhibitor is fulmonertinib. In some aspects, the third generation EGFR inhibitor is abivertinib. In some aspects, the third generation EGFR inhibitor is alfultinib. In some aspects, the third generation EGFR inhibitor is mavereltinib. In some aspects, the third generation EGFR inhibitor is abivertinib. In some aspects, the third generation EGFR inhibitor is orafeltinib. In some aspects, the third generation EGFR inhibitor is resibertinib. Any one or more of these EGFR inhibitors may be excluded from certain embodiments.As used herein, "third-generation EGFR TKI" (also used interchangeably herein as "third-generation EGFR TKI", "third-generation EGFR inhibitor" and "third-generation EGFR inhibitor") refers to an EGFR inhibitor that can covalently bind to T790M mutant EGFR protein in addition to wild-type EGFR and other mutant EGFR proteins. In some embodiments, the EGFR TKI is an EGFR TKI specific for mutations associated with EGFR exon 20, including TAS 6417, AZ5104, TAK-788 (mobocertinib) and DZD9008. In some aspects, the EGFR inhibitor specific for mutations associated with EGFR exon 20 is TAS 6417. In some aspects, the EGFR inhibitor specific for mutations associated with EGFR exon 20 is AZ5104. In some aspects, the EGFR inhibitor specific for mutations associated with EGFR exon 20 is TAK-788 (mobocertinib). In some aspects, the EGFR inhibitor specific for mutations associated with EGFR exon 20 is DZD9008. Any one or more of these EGFR inhibitors may be excluded from certain embodiments. EGFR TKI specific for mutations associated with EGFR exon 20 (or "EGFR inhibitor") refers to an EGFR inhibitor that can covalently bind to exon 20 insertion mutant EGFR. In some aspects, the TKI of the present disclosure is not an EGFR inhibitor. In some embodiments, the TKI is an ALK inhibitor, including AZD3463, brigatinib, crizotinib, ceritinib, alectinib, lorlatinib, ensartinib, entrectinib, repotrectinib, verizatinib, arcotinib, folitinib, CEP-37440, TQ-B3139, PLB1003, TPX-0131 and ASP-3026. In some aspects, the ALK inhibitor is AZD3463. In some aspects, the ALK inhibitor is brigatinib. In some aspects, the ALK inhibitor is crizotinib. In some aspects, the ALK inhibitor is ceritinib. In some aspects, the ALK inhibitor is alectinib. In some aspects, the ALK inhibitor is lorlatinib.In some aspects, the ALK inhibitor is ensartinib. In some aspects, the ALK inhibitor is entrectinib. In some aspects, the ALK inhibitor is repotrectinib. In some aspects, the ALK inhibitor is verizatinib. In some aspects, the ALK inhibitor is arcotinib. In some aspects, the ALK inhibitor is folitinib. In some aspects, the ALK inhibitor is CEP-37440. In some aspects, the ALK inhibitor is TQ-B3139. In some aspects, the ALK inhibitor is PLB1003. In some aspects, the ALK inhibitor is TPX-0131. In some aspects, the ALK inhibitor is ASP-3026. Any one or more of these ALK inhibitors may be excluded from certain embodiments. In some embodiments, the TKI is a PKC inhibitor, including ruboxistaurin, midostaurin, sotrastaurin, chelerythrine, myabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostatin 1, and tamoxifen. In some aspects, the PKC inhibitor is ruboxistaurin, midostaurin, sotrastaurin, chelerythrine, myabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostatin 1, tamoxifen. Any one or more of these PKC inhibitors may be excluded from certain embodiments.

[0075] In some embodiments, the TKI is erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, TAS 6417, AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, AZD3463, brigatinib, ruboxistaurin, midostaurin, sotrastaurin, or combinations thereof.Embodiments of the present disclosure include administration of at least one, two, three, four, five, or more TKIs to a subject with cancer. In some embodiments, the one or more TKIs include two or more of erlotinib, gefitinib, AZD3759, sapatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, TAS 6417, AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, AZD3463, brigatinib, ruboxistaurin, midostaurin, and sotrastaurin.

[0076] In some embodiments, the EGFR inhibitor is gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, TAS 6417, AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, or lazertinib.Embodiments of the present disclosure include administration of at least one, two, three, four, five, or more EGFR inhibitors to a subject with cancer.

[0077] In some embodiments, the methods disclosed herein further comprise administering to the subject an additional cancer therapy. In some embodiments, the additional cancer therapy comprises chemotherapy, radiation therapy, or immunotherapy. In some embodiments, the additional cancer therapy comprises an ALK inhibitor. In some embodiments, the ALK inhibitor is brigatinib or AZD3463. In some embodiments, the additional cancer therapy comprises a PKC inhibitor. In some embodiments, the PKC inhibitor is ruboxistaurin, midostaurin, or sotrastaurin.

[0078] In some embodiments, the kinase inhibitors of the present disclosure are EGFR inhibitors designed to be active in one or more types of EGFR mutant cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in classical-like EGFR mutant lung cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in T790M-like 3S EGFR mutant lung cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in T790M-like 3R EGFR mutant lung cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in exon 20ins-NL EGFR mutant lung cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in exon 20ins-FL EGFR mutant lung cancer. In some embodiments, the kinase inhibitors are EGFR inhibitors designed to be active in P-loop αC-helix compression EGFR mutant lung cancer.

[0079] In some embodiments, the kinase inhibitors of the present disclosure are EGFR inhibitors that are preferentially effective in one or more types of EGFR mutant cancer. An EGFR inhibitor that is "preferentially effective" in a type of EGFR mutant cancer refers to an EGFR inhibitor that has increased efficacy in killing cancer cells with a given type of EGFR mutation compared to cancer cells with a different type of EGFR mutation. For example, an EGFR inhibitor that is preferentially effective in classical-like EGFR mutant lung cancer refers to an EGFR inhibitor that has increased efficacy in killing cancer cells with classical-like EGFR mutations (e.g., A702T) compared to cancer cells with P-loop αC helix compacting EGFR mutations (e.g., V769L). In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in classical-like EGFR mutant lung cancer. In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in T790M-like 3S EGFR mutant lung cancer. In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in T790M-like 3R EGFR mutant lung cancer. In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in exon 20ins-NL EGFR mutant lung cancer. In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in exon 20ins-FL EGFR mutant lung cancer. In some embodiments, the kinase inhibitor is an EGFR inhibitor that is preferentially effective in P-loop αC-helix compression EGFR mutant lung cancer.

[0080] B. Radiation Therapy In some embodiments, radiation therapy such as ionizing radiation is administered to the subject as a therapeutic agent.As used herein, "ionizing radiation" refers to radiation that contains particles or photons that have sufficient energy to cause ionization (gain or loss of electrons) or can generate sufficient energy to cause ionization (gain or loss of electrons) through nuclear interaction.A preferred non-limiting example of ionizing radiation is X-rays.The means for delivering X-rays to target tissue or cells are well known in the art.

[0081] In some embodiments, the radiation therapy can include external radiation therapy, internal radiation therapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some embodiments, the external radiation therapy includes three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some embodiments, the internal radiation therapy includes interstitial brachytherapy, intracavitary brachytherapy, or intracavitary radiation therapy. In some embodiments, the radiation therapy is administered to the primary tumor.

[0082] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 110, 111, 112, 1 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein), at most 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein) or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 Gy (or any derivable range therein). In some embodiments, ionizing radiation is administered in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein). When more than one dose is administered, the doses may be separated by about 1, 4, 8, 12, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks (or any derivable range therein).

[0083] In some embodiments, the amount of radiation therapy administered to a subject can be presented as a total dose of radiation therapy, which is then administered in fractionated doses.For example, in some embodiments, the total dose is 50 Gy, which is administered in 10 fractionated doses of 5 Gy each.In some embodiments, the total dose is 50-90 Gy, which is administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of radiation is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 100, 101, 102, 103, 104, 105, 106, 107, 1 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140 or 150 Gy (or any derivable range therein), at most 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 1 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135,140 or 150 Gy (or any derivable range therein) or about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140 or 150 Gy (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 (or any derivable range therein), at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 (or any derivable range therein), at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 ( or any derivable range therein) or just 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 ... 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (or any derivable range therein) fractionated doses are administered. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day.29 or 30 (or any derivable range therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 (or any derivable range therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 (or any derivable range therein) fractionated doses are administered per week.

[0084] C. Cancer Immunotherapy In some embodiments, the method includes the administration of cancer immunotherapy as a therapeutic agent. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be classified as active, passive or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAA). Such molecules are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting TAA. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes and cytokines. Various immunotherapies are known in the art, examples of which are provided below.

[0085] 1. Checkpoint Inhibitors and Combination Therapies Embodiments of the present disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below. "Checkpoint inhibitor therapy" (also referred to as "immune checkpoint blockade therapy", "immune checkpoint therapy", "ICT", "checkpoint blockade immunotherapy" or "CBI") disclosed herein refers to cancer therapy that includes providing one or more immune checkpoint inhibitors to a subject having or suspected of having cancer.

[0086] PD-1 can act within the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during immune responses. The inhibitors of the present disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0087] Aliases for "PD-1" include CD279 and SLEB2. Aliases for "PDL1" include B7-H1, B7-4, CD274 and B7-H. Aliases for "PDL2" include B7-DC, Btdc and CD273. In some embodiments, the PD-1, PDL1 and PDL2 are human PD-1, PDL1 and PDL2.

[0088] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partner. In certain aspects, the PDL2 binding partner is PD-1. The inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, such as those described in U.S. Patent Application Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0089] In some embodiments, the PD-1 inhibitor 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 pidilizumab. In some embodiments, the PD-1 inhibitor 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 PDL1 inhibitor comprises AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA® and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, 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. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0090] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, such as rHIgM12B7.

[0091] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab or pidilizumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2 and CDR3 domains of the VH region of nivolumab, pembrolizumab or pidilizumab and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab or pidilizumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on PD-1, PDL1 or PDL2 as the above-mentioned antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97 or 99% (or any derivable range therein) of variable region amino acid sequence identity with the above-mentioned antibody.

[0092] 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 it binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. T cell activation via T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors of the present disclosure may block one or more functions of CTLA-4, B7-1 and / or B7-2 activity. In some embodiments, the inhibitors block the interaction between CTLA-4 and B7-1. In some embodiments, the inhibitors block the interaction between CTLA-4 and B7-2.

[0093] 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.

[0094] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in US8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), US6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Applications WO2001 / 014424, WO2000 / 037504 and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0095] An additional anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0096] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2 and CDR3 domains of the VH region of tremelimumab or ipilimumab and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on PD-1, B7-1 or B7-2 as the above-mentioned antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97 or 99% (or any derivable range therein) of variable region amino acid sequence identity with the above-mentioned antibody.

[0097] Another immune checkpoint that can be targeted in the methods provided herein is lymphocyte activation gene 3 (LAG3), also known as CD223 and lymphocyte activation 3. The complete mRNA sequence of human LAG3 has Genbank accession number NM_002286. LAG3 is a member of the immunoglobulin superfamily found on the surface of activated T cells, natural killer cells, B cells and plasmacytoid dendritic cells. The main ligand of LAG3 is MHC class II, which has been reported to negatively regulate T cell proliferation, activation and homeostasis in a manner similar to CTLA-4 and PD-1, and also play a role in Treg suppressive function. LAG3 also helps to maintain CD8+ T cells in a tolerogenic state and cooperates with PD-1 to maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in dendritic cell maturation and activation. The inhibitors of the present disclosure can block one or more functions of LAG3 activity.

[0098] In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 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.

[0099] Anti-human LAG3 antibody (or VH and / or VL domain derived therefrom) suitable for use in the present method can be generated using methods well known in the art.Alternatively, anti-LAG3 antibody recognized in the art can be used.For example, anti-LAG3 antibody can include GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017 or GSK2831781. The following anti-LAG3 antibodies can be used in the methods disclosed herein: US9,505,839 (BMS-986016, also known as leratolimab); US10,711,060 (IMP-701, also known as LAG525); US9,244,059 (IMP731, also known as H5L7BW); US10,344,089 (25F7, also known as LAG3.1); WO2016 / 028672 (MK-4280, also known as 28G-10); WO2017 / 019894(BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195(REGN3767); Yu, X., et al., mAbs, 2019; 11:6(LBL-007).These and other anti-LAG-3 antibodies useful in the claimed disclosure are described, for example, in WO2016 / 028672, WO2017 / 106129, WO2017062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220562, WO2017 / 220571, WO2017 / 220572, WO2017 / 220573, WO2017 / 220574, WO2017 / 220575, WO2017 / 220576, WO2017 / 220577, WO2017 / 220578, WO2017 / 220579 ... WO2017 / 220569, WO2018 / 071500, WO2017 / 015560; WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227 and WO2014 / 140180. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 can also be used.

[0100] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2 and CDR3 domains of the VH region of an anti-LAG3 antibody and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97 or 99% (or any derivable range therein) of variable region amino acid sequence identity with the above-mentioned antibodies.

[0101] Another immune checkpoint that can be targeted in the methods provided herein is Hepatitis A Virus Cell Receptor 2 (HAVCR2) and T-cell immunoglobulin mucin domain containing 3 (TIM-3), also known as CD366. The complete mRNA sequence of human TIM-3 has Genbank accession number NM_032782. TIM-3 is found on CD4+ Th1 and CD8+ Tc1 cells that produce surface IFNγ. The extracellular region of TIM-3 consists of a single variable immunoglobulin domain (IgV) that is distal to the membrane and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint that, together with other inhibitory receptors including PD-1 and LAG3, mediates T-cell exhaustion. TIM-3 has also been shown to be a CD4+ Th1-specific cell surface protein that controls macrophage activation. The inhibitors of the present disclosure may block one or more functions of TIM-3 activity.

[0102] In some embodiments, the immune checkpoint inhibitor is an anti-TIM-3 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.

[0103] Anti-human TIM-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including MBG453, TSR-022 (also known as covolimab) and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed disclosure can be found, for example, in US9,605,070, US8,841,418, US2015 / 0218274 and US2016 / 0200815. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 can also be used.

[0104] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2 and CDR3 domains of the VH region of an anti-TIM-3 antibody and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97 or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.

[0105] 2. Activation of costimulatory molecules In some embodiments, the immunotherapy comprises an agonist of a costimulatory molecule. In some embodiments, the agonist comprises an activator of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Agonists include agonist antibodies, polypeptides, compounds, and nucleic acids.

[0106] 3. Dendritic cell therapy Dendritic cell therapy involves dendritic cells presenting tumor antigens to lymphocytes, which then activate and prime the lymphocytes to elicit an antitumor response and kill other cells that present the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, they aid in cancer antigen targeting. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0107] One way to induce dendritic cells to present tumor antigens is by inoculation with autologous tumor lysates or short peptides (small pieces of proteins that correspond to protein antigens on cancer cells). These peptides are often administered in combination with adjuvants (highly immunogenic substances) to enhance immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0108] Dendritic cells can also be activated in vivo by inducing tumor cells to express GM-CSF, which can be accomplished by genetically engineering the tumor cells to produce GM-CSF or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0109] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of disassembled tumor cells). These cells are injected (with optional adjuvants) to induce an immune response.

[0110] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. An antigen is added to the antibody to induce dendritic cells to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0111] 4. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine new specificities with immune cells to target cancer cells. Typically, these receptors transfer the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to the treatment of using such transformed cells for cancer therapy.

[0112] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to artificially generate T cells targeted to markers found on cancer cells. Scientists can extract T cells from humans, genetically modify them, and return them to the patient to attack cancer cells. Once engineered into CAR-T cells, T cells act as "living drugs." CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule, which then activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells and not normal cells are targeted. The specificity of CAR-T cells is determined by the choice of the molecule that is targeted.

[0113] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtageneciloreucel (Yescarta).

[0114] 5. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. Cytokines can modulate the immune response. Tumors often use cytokines to grow themselves and dampen the immune response. This immunomodulatory effect allows cytokines to be used as drugs to induce an immune response. Two commonly used cytokines are interferons and interleukins.

[0115] Interferons are produced by the immune system. They are usually involved in antiviral responses but are also used in cancer. Interferons are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ).

[0116] Interleukins have a range of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0117] 6. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization by the infusion of T cells (adoptive cell transfer). T cells are found in blood and tissues and are usually activated when they find a foreign pathogen. Specifically, T cells are activated when their surface receptors encounter cells that display a portion of a foreign protein on their surface antigen. They can be either infected cells or antigen-presenting cells (APCs). They are found in normal and tumor tissues, where they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs, such as dendritic cells, that present tumor antigens. These cells can attack tumors, but the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0118] Several methods have been developed to generate and obtain tumor-targeting T cells. T cells specific for tumor antigens can be extracted from tumor samples (TILs) or filtered from the blood. Subsequent activation and culture is performed ex vivo, and the product is reinfused. Activation can occur through gene therapy or by exposing the T cells to tumor antigens.

[0119] It is contemplated that the cancer treatment may exclude any of the cancer treatments described herein.Furthermore, aspects of the present disclosure include patients who have been previously treated with the therapies described herein, patients who are currently being treated with the therapies described herein, or patients who have never been treated with the therapies described herein.In some aspects, the patient is a patient who has been determined to be resistant to the therapies described herein.In some aspects, the patient is a patient who has been determined to be sensitive to the therapies described herein.

[0120] D. Oncolytic Viruses In some embodiments, the additional therapy includes an oncolytic virus as a therapeutic agent. An oncolytic virus is a virus that preferentially infects and kills cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are not only believed to cause direct destruction of tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy.

[0121] E. Polysaccharides In some embodiments, the additional therapy includes polysaccharides as therapeutic agents.Certain compounds found in mushrooms, mainly polysaccharides, can upregulate the immune system and may have anti-cancer properties.For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophages, NK cells, T cells and immune system cytokines, and are being investigated in clinical trials as immune adjuvants.

[0122] F. Neoantigens In some embodiments, the additional therapy includes neoantigen administration as a therapeutic agent. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, identified using RNA sequencing data, is higher in tumors with high mutational burden. The levels of transcripts associated with natural killer cell and T cell cytolytic activity positively correlate with mutational burden in many human tumors.

[0123] G. Chemotherapy In some embodiments, the additional therapy includes chemotherapy as a therapeutic agent. Suitable classes of chemotherapeutic agents include (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dacarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related substances (e.g., 6-mercaptopurine). , 6-thioguanine, pentostatin), (c) natural products such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., interferon-α), and (d) miscellaneous agents such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and corticosteroid synthesis inhibitors (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.

[0124] Cisplatin has been widely used to treat cancer, such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents and, in certain embodiments, is administered at a dose of about 15 mg / m 2 ~about 20mg / m 2In clinical use, an effective dose comprising is used for 5 days every 3 weeks for a total of 3 courses. In some embodiments, the amount of cisplatin delivered to a cell and / or subject with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount delivered when cisplatin is used alone.

[0125] Other suitable chemotherapeutic agents include microtubule inhibitors, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector and doxorubicin has been determined to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combination therapy of the construct with doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0126] Doxorubicin is poorly absorbed and is preferably administered intravenously. In certain embodiments, suitable intravenous dosages for adults include about 60 mg / m2 to about 75 mg / m2 at intervals of about 21 days, or about 25 mg / m2 to about 30 mg / m2 on each of 2 or 3 consecutive days (repeated at intervals of about 3 to about 4 weeks), or about 20 mg / m2 once a week. In elderly patients, the lowest dose should be used if there is previous bone marrow suppression caused by previous chemotherapy or neoplastic bone marrow infiltration, or if the drug is used in combination with other myelopoiesis suppressing drugs.

[0127] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN® available from Mead Johnson and NEOSTAR® available from Adria) is another suitable chemotherapeutic agent. Suitable oral dosages for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous dosages include, for example, about 40 mg / kg to about 50 mg / kg initially divided over a period of about 2 to about 5 days, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration or into body cavities.

[0128] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU), and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject at a dosage of about 7.5 to about 1000 mg / m2. Furthermore, 5-FU administration schedules may be of various durations, such as up to 6 weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0129] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and is therefore useful in the present disclosure for these cancers.

[0130] The amount of chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause cessation or regression of cancer in the host when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is 2-10,000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 times, about 500 times, or about 5000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure may be tested in vivo for the desired therapeutic activity when combined with the construct and for the determination of effective dosages. For example, such compounds may be tested in suitable animal model systems, including rats, mice, chickens, cows, monkeys, rabbits, and the like, prior to testing in humans. In vitro testing may also be used to determine suitable combinations and dosages, as described in the Examples.

[0131] H. Surgery In some embodiments, the additional therapy includes surgery as a therapeutic agent. Approximately 60% of cancer patients undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, which physically removes, excises and / or destroys all or part of the cancerous tissue, and may be combined with other therapies, such as the treatment of this embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapy. Tumor resection refers to the physical removal of at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microsurgery (Mohs surgery).

[0132] When part or all of the cancerous cells, tissues or tumors are removed, a cavity may form in the body. Treatment may be achieved by perfusion, direct injection or local application of additional anti-cancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also be at various dosages.

[0133] I. Anti-EGFR Antibodies and Antibody-Drug Conjugates In certain aspects, one or more anti-EGFR antibodies or antibody-like molecules (including, for example, antibody drug conjugates) are contemplated for combination with one or more EGFR inhibitors of the present disclosure. For example, the therapeutic method of the present disclosure may include treatment of a patient with one or more EGFR inhibitors (which may be selected based on the patient's EGFR mutation classification) in combination with one or more anti-EGFR antibodies (or antibody drug conjugates thereof). Anti-EGFR antibodies are known in the art, for example, cetuximab and amivantamab. In certain aspects, a method of treating a subject with an exon 20 loop insertion EGFR mutant cancer (e.g., a cancer with a mutation in Tables 2.1-2.5) is disclosed, comprising providing both a second generation EGFR inhibitor and amivantamab (or antibody drug conjugates thereof).

[0134] J. Angiogenesis Inhibitors In certain aspects, one or more angiogenesis inhibitors are considered to be combined with one or more EGFR inhibitors of the present disclosure.For example, the therapeutic method of the present disclosure can include the treatment of patients with one or more EGFR inhibitors (which can be selected based on the EGFR mutation classification of patients) combined with one or more angiogenesis inhibitors.Angiogenesis inhibitors are known in the art, for example, ramucirumab and bevacizumab.

[0135] K. Other Agents It is contemplated that other therapeutic agents may be combined with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be combined with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Additionally, it is contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may also be combined with certain aspects of the present embodiment to improve the efficacy of the treatment.

[0136] II. Cancer Treatment Aspects of the disclosure relate to methods including the treatment of a subject having or suspected of having cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma. In some embodiments, the non-small cell lung cancer is squamous cell carcinoma. In some embodiments, the non-small cell lung cancer is large cell carcinoma. In some embodiments, the non-small cell lung cancer is adenosquamous carcinoma. In some embodiments, the non-small cell lung cancer is sarcomatoid carcinoma. In some embodiments, the lung cancer is small cell lung cancer. In some embodiments, the cancer is not lung cancer.

[0137] In certain embodiments, the tumor DNA of a subject with cancer is analyzed or measured or evaluated for one or more mutations in the EGFR gene, regardless of which mutations are actually present and / or absent. One or more mutations in the EGFR gene can be analyzed or measured in any suitable manner. For example, a mutation in the EGFR gene ("EGFR mutation") can be identified by sequencing DNA and / or RNA (e.g., mRNA) from a sample. In certain cases, a cancer with one or more mutations in the EGFR gene has increased sensitivity (or decreased resistance) to one or more kinase inhibitors from one or more kinase inhibitor classes. In certain cases, a cancer with one or more mutations in the EGFR gene has decreased sensitivity (or increased resistance) to one or more kinase inhibitors from one or more kinase inhibitor classes.

[0138] In certain embodiments, the disclosed methods include treating a subject having cancer (e.g., lung cancer, such as non-small cell lung cancer) by administering a therapeutically effective amount of a composition comprising one or more kinase inhibitors from one or more kinase inhibitor classes. In some embodiments, the one or more kinase inhibitors are of the same class. In some embodiments, the one or more kinase inhibitors are of different classes. As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and / or "effective dose," and refers to an amount of an agent sufficient to produce a desired result or have a desired effect in the particular condition being treated. In some embodiments, a therapeutically effective amount is an amount sufficient to ameliorate at least one symptom, behavior, or event associated with a pathological, abnormal, or otherwise undesirable condition, or an amount sufficient to prevent or reduce the likelihood of such a condition occurring or recurring, or an amount sufficient to slow the progression of such a condition. For example, in some embodiments, an effective amount refers to an amount of a composition comprising one or more kinase inhibitors that can treat or prevent cancer in a subject. An effective amount can vary depending on the organism or individual being treated. The appropriate effective amount to be administered for a particular application of the disclosed method can be determined by those skilled in the art using the guidance provided herein. The term "treatment", "treat" or "treating" as used herein refers to an intervention that seeks to change the natural course of the subject undergoing treatment, and may be performed for prevention or during the pathological course of disease or condition. Treatment may work to achieve one or more of various desired results, such as preventing the occurrence or recurrence of disease, alleviating or reducing the severity of symptoms, reducing any direct or indirect pathological effects of disease, preventing the spread of disease, slowing down the rate of disease progression, improving or alleviating disease state, and remission or improving prognosis.

[0139] The disclosed methods include compositions and methods for treating cancer (e.g., lung cancer, such as non-small cell lung cancer) with one or more kinase inhibitors based on the sensitivity (or resistance) of the cancer to one or more kinase inhibitors from one or more kinase inhibitor classes. In some embodiments, the cancer is more sensitive (or less resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes than to a kinase inhibitor from a different kinase inhibitor class. In some cases, the methods are used in subjects where it is uncertain whether the cancer is more sensitive (or less resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes, and in other cases, the methods are used in subjects where the cancer is known to be more sensitive (or less resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes. In other cases, the disclosed methods are used routinely or in the general therapeutic interest of the subject, even if the cancer has been determined to be more sensitive (or less resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes for the subject. In some cases, the methods are used in subjects where it is uncertain whether the cancer is more insensitive (or more resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes, and in other cases, the methods are used in subjects where the cancer is known to be more insensitive (or more resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes. In other cases, the methods of the disclosure are used routinely or in the general therapeutic interest of the subject, even if the cancer has been determined to be more insensitive (or more resistant) to one or more kinase inhibitors from one or more kinase inhibitor classes for the subject.

[0140] The selection of one or more kinase inhibitors from one or more kinase inhibitor classes to be used to treat cancer (e.g., lung cancer) can be the result of analysis of tumor DNA from a subject with cancer for one or more mutations in the EGFR gene. In some cases, the selection of one or more kinase inhibitors from one or more kinase inhibitor classes is the result of analysis of tumor DNA from a subject with cancer for one or more mutations in the subject's EGFR gene, and the results of the analysis determine the one or more kinase inhibitors from one or more kinase inhibitor classes to be used to treat the cancer. In some embodiments, the one or more mutations are classical-like EGFR mutations. In some embodiments, the one or more mutations are exon 20 loop insertion (ex20ins) mutations. In some embodiments, the one or more mutations are T790M-like 3S mutations. In some embodiments, the one or more mutations are T790M-like 3R mutations. In some embodiments, the one or more mutations are P-loop αC helix compression (PACC) mutations.

[0141] A. Classic-like EGFR mutations In some cases, a subject having cancer (e.g., lung cancer, such as non-small cell lung cancer) is determined to have one or more classical-like EGFR mutations, and the one or more kinase inhibitors selected to treat the cancer may include a first generation EGFR TKI, a second generation EGFR TKI, a third generation EGFR TKI, or an EGFR TKI specific for mutations associated with EGFR exon 20. Classical-like EGFR mutations include, but are not limited to, the mutations provided in Tables 1.1-1.5 below. Classical-like EGFR mutations of the present disclosure may include any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-one, thirty-two, thirty-three, or thirty-four of the mutations in Tables 1.1-1.5. Any one or more of the mutations in Tables 1.1-1.5 may be excluded from the aspects of the present disclosure. "Classical-like" EGFR mutations refer to EGFR mutations that are distant from the ATP-binding pocket of the EGFR protein. Also provided are examples of the response of cells containing classical-like EGFR mutations to the TKIs disclosed herein. The data shows the IC of a given TKI for cells with classical-like EGFR mutations. 50 IC values ​​for cells with wild-type EGFR 50 It expresses the ratio to the value.

[0142] (Table 1.1) Response of cells containing classical-like EGFR mutations to first-generation TKIs TIFF2024506839000001.tif99159

[0143] (Table 1.2) Response of cells containing classical-like EGFR mutations to second-generation TKIs TIFF2024506839000002.tif104155

[0144] Table 1.3 Response of cells containing classical-like EGFR mutations to third-generation TKIs TIFF2024506839000003.tif150164

[0145] Table 1.4 Response of cells containing classical-like EGFR mutations to Ex20ins-specific TKIs TIFF2024506839000004.tif109133

[0146] Table 1.5. List of exemplary classical-like EGFR mutations TIFF2024506839000005.tif19288

[0147] Thus, in some embodiments, methods are disclosed for treating a subject for lung cancer (e.g., NSCLC) comprising administering an effective amount of one or more EGFR inhibitors (e.g., erlotinib, gefitinib, AZD3759, sapatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, or combinations thereof) to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, wherein the EGFR mutation is a classical-like mutation. In some embodiments, the classical-like EGFR mutations are A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, L858R R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M. Also disclosed is a method for treating a subject for cancer, the method comprising: (a) detecting an EGFR mutation in tumor DNA from the subject, the EGFR mutation being a classical-like mutation; and (b) administering to the subject an effective amount of one or more EGFR inhibitors (e.g., erlotinib, gefitinib, AZD3759, sapatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, or a combination thereof).In some aspects, classic-like EGFR mutations include A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, L858R R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M. Any one or more of the above EGFR mutations may be excluded from aspects of the disclosure.

[0148] In some embodiments, the method includes administering erlotinib to the subject. In some embodiments, the method includes administering gefitinib to the subject. In some embodiments, the method includes administering AZD3759 to the subject. In some embodiments, the method includes administering sapatinib to the subject. In some embodiments, the method includes administering afatinib to the subject. In some embodiments, the method includes administering dacomitinib to the subject. In some embodiments, the method includes administering neratinib to the subject. In some embodiments, the method includes administering a Tarlox-TKI to the subject. In some embodiments, the method includes administering tarloxotinib to the subject. In some embodiments, the method includes administering TAS6417 (CLN-081) to the subject. In some embodiments, the method includes administering AZ5104 to the subject. In some embodiments, the method includes administering TAK-788 (mobocertinib) to the subject. In some embodiments, the method includes administering osimertinib to the subject. In some embodiments, the method includes administering nazartinib to the subject. In some embodiments, the method includes administering olmutinib to the subject. In some embodiments, the method includes administering loceritinib to the subject. In some embodiments, the method includes administering nacotinib to the subject. In some embodiments, the method includes administering lazertinib to the subject.

[0149] In some embodiments, the EGFR mutation is A702T. In some embodiments, the EGFR mutation is A763insFQEA. In some embodiments, the EGFR mutation is A763insLQEA. In some embodiments, the EGFR mutation is D761N. In some embodiments, the EGFR mutation is E709A L858R. In some embodiments, the EGFR mutation is E709K L858R. In some embodiments, the EGFR mutation is E746_A750del A647T. In some embodiments, the EGFR mutation is E746_A750del L41W. In some embodiments, the EGFR mutation is E746_A750del R451H. In some embodiments, the EGFR mutation is Ex19del E746_A750del. In some embodiments, the EGFR mutation is K754E. In some embodiments, the EGFR mutation is L747_E749del A750P. In some embodiments, the EGFR mutation is L747_T751del L861Q. In some embodiments, the EGFR mutation is L833F. In some embodiments, the EGFR mutation is L833V. In some embodiments, the EGFR mutation is L858R. In some embodiments, the EGFR mutation is L858R A289V. In some embodiments, the EGFR mutation is L858R E709V. In some embodiments, the EGFR mutation is L858R L833F. In some embodiments, the EGFR mutation is L858R P100T. In some embodiments, the EGFR mutation is L858R P848L. In some embodiments, the EGFR mutation is L858R R108K. In some embodiments, the EGFR mutation is L858R R324H. In some embodiments, the EGFR mutation is L858R R324L. In some embodiments, the EGFR mutation is L858R S784F. In some embodiments, the EGFR mutation is L858R S784Y. In some embodiments, the EGFR mutation is L858R T725M. In some embodiments, the EGFR mutation is L858R V834L. In some embodiments, the EGFR mutation is L861Q. In some embodiments, the EGFR mutation is L861R. In some embodiments, the EGFR mutation is S720P. In some embodiments, the EGFR mutation is S784F. In some embodiments, the EGFR mutation is S811F.In some embodiments, the EGFR mutation is T725M.

[0150] In some embodiments, the subject has been previously treated with a cancer therapy. In some embodiments, the cancer therapy included chemotherapy. In some embodiments, the subject has been determined to be resistant to the cancer therapy.

[0151] B. Exon 20 loop insertion mutation In some cases, a subject having cancer (e.g., lung cancer, such as non-small cell lung cancer) is determined to have one or more exon 20 loop insertion (Ex20ins) EGFR mutations, and the kinase inhibitor selected may include a second generation TKI or a TKI specific for a mutation associated with EGFR exon 20. In some aspects, the Ex20ins EGFR mutation is an Ex20ins loop proximal (NL) mutation. Ex20ins EGFR mutations include, but are not limited to, the mutations provided in Tables 2.1-2.5 below. An "Ex20ins" EGFR mutation refers to an EGFR mutation that is an insertion mutation in exon 20 of the EGFR gene, including a mutation at the C-terminus of the αC helix of the EGFR protein. Also provided are examples of the response of cells containing an Ex20ins EGFR mutation to the TKIs disclosed herein. The data shows the IC of a given TKI for cells with an Ex20ins EGFR mutation. 50 IC values ​​for cells with wild-type EGFR 50 It expresses the ratio to the value.

[0152] (Table 2.1) Response of cells harboring Ex20ins EGFR mutations to first-generation TKIs TIFF2024506839000006.tif48159

[0153] (Table 2.2) Response of cells containing Ex20ins EGFR mutations to second-generation TKIs TIFF2024506839000007.tif58155

[0154] (Table 2.3) Response of cells containing Ex20ins EGFR mutations to third-generation TKIs TIFF2024506839000008.tif67160

[0155] (Table 2.4) Response of cells containing Ex20ins EGFR mutations to Ex20ins-specific TKIs TIFF2024506839000009.tif63142

[0156] Table 2.5. List of exemplary exon 20 loop insertion EGFR mutations TIFF2024506839000010.tif254158

[0157] Thus, in some embodiments, methods are disclosed for treating a subject for lung cancer comprising administering an effective amount of one or more second generation or exon 20 loop insertion specific EGFR inhibitors (e.g., afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib) or combinations thereof) to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, wherein the EGFR mutation is an exon 20 loop proximal insertion (ex20ins-NL) mutation. In some aspects, the ex20ins-NL mutation is A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP. Also disclosed is a method for treating a subject for lung cancer, comprising: (a) detecting an EGFR mutation in tumor DNA from the subject, the EGFR mutation being an exon 20ins-NL mutation; and (b) administering to the subject an effective amount of one or more second generation or exon 20 loop insertion specific EGFR inhibitors (e.g., afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, TAS6417 (CLN-081), AZ5104, TAK-788 (mobocertinib) or combinations thereof).In some aspects, the ex20ins-NL mutation is A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP. Further disclosed are methods that include administering an EGFR inhibitor to a subject determined to have an exon 20 loop proximal insertion EGFR mutation from analysis of tumor DNA from the subject.

[0158] In some embodiments, the method includes administering afatinib to the subject. In some embodiments, the method includes administering dacomitinib to the subject. In some embodiments, the method includes administering neratinib to the subject. In some embodiments, the method includes administering a Tarlox-TKI to the subject. In some embodiments, the method includes administering tarloxotinib to the subject. In some embodiments, the method includes administering TAS6417 (CLN-081) to the subject. In some embodiments, the method includes administering AZ5104 to the subject. In some embodiments, the method includes administering TAK-788 (mobocertinib) to the subject.

[0159] In some embodiments, the EGFR mutation is A767_V769dupASV. In some embodiments, the EGFR mutation is A767_S768insTLA. In some embodiments, the EGFR mutation is S768_D770dupSVD. In some embodiments, the EGFR mutation is S768_D770dupSVD L858Q. In some embodiments, the EGFR mutation is S768_D770dupSVD R958H. In some embodiments, the EGFR mutation is S768_D770dupSVD V769M. In some embodiments, the EGFR mutation is V769_D770insASV. In some embodiments, the EGFR mutation is V769_D770insGSV. In some embodiments, the EGFR mutation is V769_D770insGVV. In some embodiments, the EGFR mutation is V769_D770insMASVD. In some embodiments, the EGFR mutation is D770_N771insNPG. In some embodiments, the EGFR mutation is D770_N771insSVD. In some embodiments, the EGFR mutation is D770del insGY. In some embodiments, the EGFR mutation is D770_N771 insG. In some embodiments, the EGFR mutation is D770_N771 insY H773Y. In some embodiments, the EGFR mutation is N771dupN. In some embodiments, the EGFR mutation is N771dupN G724S. In some embodiments, the EGFR mutation is N771_P772insHH. In some embodiments, the EGFR mutation is N771_P772insSVDNR. In some embodiments, the EGFR mutation is P772_H773insDNP.

[0160] In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0161] C. T790M-like mutation In some cases, a subject with cancer (e.g., lung cancer) is determined to have one or more T790M-like EGFR mutations. A "T790M-like" EGFR mutant contains at least one mutation in the hydrophobic cleft; the addition of one or more known resistance mutations can reduce sensitivity to classical EGFR TKIs. In some cases, a subject with cancer is determined to have one or more T790M-like EGFR mutations, but does not contain a resistance mutation referred to herein as a "T790M-like 3S" mutant (i.e., C797S, which confers resistance to classical EGFR TKIs). 37,38 , L718X 18,24 or L792H 23,24 ) is not detected, and the kinase inhibitors selected may include third generation TKIs, TKIs specific for mutations associated with EGFR exon 20, ALK inhibitors and / or PKC inhibitors. T790M-like 3S EGFR mutations include, but are not limited to, the mutations provided in Tables 3.1-3.5 below. T790M-like 3S EGFR mutations may refer to EGFR mutations present in the hydrophobic core of the EGFR protein. Also provided are examples of the response of cells containing T790M-like 3S EGFR mutations to the TKIs disclosed herein. The data shows the IC of a given TKI for cells with T790M-like 3S EGFR mutations. 50 IC values ​​for cells with wild-type EGFR 50 It expresses the ratio to the value.

[0162] Table 3.1 Response of cells containing T790M-like 3S EGFR mutations to first-generation TKIs TIFF2024506839000011.tif102160

[0163] Table 3.2 Response of cells containing T790M-like 3S EGFR mutations to second-generation TKIs TIFF2024506839000012.tif97148

[0164] Table 3.3 Response of cells containing T790M-like 3S EGFR mutations to third-generation TKIs TIFF2024506839000013.tif100160

[0165] Table 3.4 Response of cells containing T790M-like 3S EGFR mutations to Ex20ins-specific TKIs TIFF2024506839000014.tif93142

[0166] Table 3.5. List of exemplary T790M-like 3S EGFR mutations TIFF2024506839000015.tif82158

[0167] Thus, in some embodiments, methods are disclosed for treating a subject for lung cancer comprising administering to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject an effective amount of one or more third generation or EGFR inhibitors, PKC inhibitors and / or ALK inhibitors (e.g., osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, brigatinib, AZD3463, ruboxistaurin, midostaurin, sotrastaurin, or combinations thereof), wherein the EGFR mutation is a T790M-like 3S mutation. In some aspects, the T790M-like 3S mutation is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M. Also disclosed is a method for treating a subject for lung cancer, comprising: (a) detecting an EGFR mutation in tumor DNA from the subject, the EGFR mutation being a T790M-like 3S mutation; and (b) administering to the subject an effective amount of one or more third generation EGFR inhibitors, PKC inhibitors and / or ALK inhibitors (e.g., osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, brigatinib, AZD3463, ruboxistaurin, midostaurin, sotrastaurin, or combinations thereof).Furthermore, disclosed is a method comprising administering an EGFR inhibitor to a subject determined to have a T790M-like 3S EGFR mutation from analysis of tumor DNA from the subject.

[0168] In some embodiments, the method includes administering osimertinib to the subject. In some embodiments, the method includes administering nazartinib to the subject. In some embodiments, the method includes administering olmutinib to the subject. In some embodiments, the method includes administering loceritinib to the subject. In some embodiments, the method includes administering nacotinib to the subject. In some embodiments, the method includes administering lazertinib to the subject. In some embodiments, the method includes administering brigatinib to the subject. In some embodiments, the method includes administering AZD3463 to the subject. In some embodiments, the method includes administering ruboxistaurin to the subject. In some embodiments, the method includes administering midostaurin to the subject. In some embodiments, the method includes administering sotrastaurin to the subject.

[0169] In some embodiments, the EGFR mutation is Ex19del T790M. In some embodiments, the EGFR mutation is Ex19del T790M L718V. In some embodiments, the EGFR mutation is Ex19del T790M G724S. In some embodiments, the EGFR mutation is G719A T790M. In some embodiments, the EGFR mutation is G719S T790M. In some embodiments, the EGFR mutation is H773R T790M. In some embodiments, the EGFR mutation is I744_E749del insMKK. In some embodiments, the EGFR mutation is L747_K754 delinsATSPE. In some embodiments, the EGFR mutation is L858R T790M L792H. In some embodiments, the EGFR mutation is L858R T790M V843I. In some embodiments, the EGFR mutation is L858R T790M. In some embodiments, the EGFR mutation is S768I T790M. In some embodiments, the EGFR mutation is T790M.

[0170] In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0171] In some cases, a subject with cancer (e.g., lung cancer) has one or more T790M-like EGFR mutations and one or more resistance mutations referred to herein as "T790M-like 3R" mutations (i.e., C797S, C798S, C79 ... 37,38 , L718X 18,24 or L792H 23,24 ), and the kinase inhibitor selected may include an ALK inhibitor or a PKC inhibitor. T790M-like 3R EGFR mutations include, but are not limited to, the mutations provided in Tables 4.1-4.5 below. T790M-like 3R EGFR mutations may refer to EGFR mutations that include a mutation in the hydrophobic core of the EGFR protein (e.g., T790M) and a mutation outside the hydrophobic core of the EGFR protein (e.g., C797S, L718X, or L792H). Also provided are examples of the response of cells containing T790M-like 3R EGFR mutations to the TKIs disclosed herein. The data shows the IC of a given TKI for cells with a T790M-like 3R EGFR mutation. 50 IC values ​​for cells with wild-type EGFR 50 It expresses the ratio to the value.

[0172] Table 4.1 Response of cells containing T790M-like 3R EGFR mutations to first-generation TKIs TIFF2024506839000016.tif67160

[0173] Table 4.2 Response of cells containing T790M-like 3R EGFR mutations to second-generation TKIs TIFF2024506839000017.tif64161

[0174] Table 4.3 Response of cells containing T790M-like 3R EGFR mutations to third-generation TKIs TIFF2024506839000018.tif67160

[0175] Table 4.4 Response of cells containing T790M-like 3R EGFR mutations to Ex20ins-specific TKIs TIFF2024506839000019.tif85142

[0176] Table 4.5. List of exemplary T790M-like 3R EGFR mutations TIFF2024506839000020.tif39158

[0177] Thus, in some embodiments, a method for treating a subject for lung cancer is disclosed, comprising administering an effective amount of one or more PKC inhibitors and / or ALK inhibitors (e.g., brigatinib, AZD3463, ruboxistaurin, midostaurin, sotrastaurin, or a combination thereof) to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject, wherein the EGFR mutation is a T790M-like 3R mutation. In some aspects, the T790M-like 3R mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, L858R T790M L718Q, or L858R T790M L718V. Further disclosed is a method comprising administering a tyrosine kinase inhibitor to a subject determined to have a T790M-like 3R EGFR mutation from analysis of tumor DNA from the subject, wherein the tyrosine kinase inhibitor is not an EGFR inhibitor.

[0178] Also disclosed is a method for treating a subject for lung cancer, comprising: (a) detecting an EGFR mutation in tumor DNA from the subject, the EGFR mutation being a T790M-like 3R mutation; and (b) administering to the subject an effective amount of one or more PKC inhibitors and / or ALK inhibitors (e.g., brigatinib, AZD3463, ruboxistaurin, midostaurin, sotrastaurin, or a combination thereof). In some aspects, the T790M-like 3R mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q.

[0179] In some embodiments, the method includes administering brigatinib to the subject. In some embodiments, the method includes administering AZD3463 to the subject. In some embodiments, the method includes administering ruboxistaurin to the subject. In some embodiments, the method includes administering midostaurin to the subject. In some embodiments, the method includes administering sotrastaurin to the subject.

[0180] In some embodiments, the EGFR mutation is Ex19del T790M C797S. In some embodiments, the EGFR mutation is Ex19del T790M L792H. In some embodiments, the EGFR mutation is G724S T790M. In some embodiments, the EGFR mutation is L718Q T790M. In some embodiments, the EGFR mutation is L858R T790M C797S. In some embodiments, the EGFR mutation is L858R T790M L718Q.

[0181] In some embodiments, the subject has been previously treated with a cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759, or sapatinib. In some embodiments, the cancer therapy includes afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib. In some embodiments, the cancer therapy includes TAS6417 (CLN-081), AZ5104, or TAK-788 (mobocertinib). In some embodiments, the cancer therapy includes osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to the cancer therapy.

[0182] D. PACC mutation In some embodiments, a subject with cancer (e.g., lung cancer) is determined to have one or more P-loop αC-helix compression (PACC) mutations, including mutations spanning EGFR exons 18-21, such as, for example, G719X, L747X, S768I, L792X, and T854I, and the kinase inhibitor selected may include a second generation TKI. PACC EGFR mutations include, but are not limited to, the mutations provided in Tables 5.1-5.5 below. A "PACC" EGFR mutation refers to an EGFR mutation that resides within the ATP binding pocket and / or at the C-terminus of the αC-helix. Also provided are examples of the response of cells containing PACC EGFR mutations to the TKIs disclosed herein. The data shows the IC of a given TKI for cells with a PACC EGFR mutation. 50 IC values ​​for cells with wild-type EGFR 50 It expresses the ratio to the value.

[0183] Table 5.1 Response of cells containing PACC EGFR mutations to first-generation TKIs TIFF2024506839000021.tif198160

[0184] Table 5.2 Response of cells containing PACC EGFR mutations to second-generation TKIs TIFF2024506839000022.tif198161

[0185] Table 5.3 Response of cells containing PACC EGFR mutations to third-generation TKIs TIFF2024506839000023.tif117160TIFF2024506839000024.tif183160

[0186] Table 5.4 Response of cells containing PACC EGFR mutations to Ex20ins-specific TKIs TIFF2024506839000025.tif202155

[0187] Table 5.5. List of exemplary PACC EGFR mutations TIFF2024506839000026.tif67158TIFF2024506839000027.tif243158TIFF2024506839000028.tif17158

[0188] Thus, in some embodiments, a method is disclosed for treating a subject for lung cancer comprising administering to a subject determined to have an EGFR mutation from analysis of tumor DNA from the subject an effective amount of one or more second generation EGFR inhibitors (e.g., afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, or a combination thereof), wherein the EGFR mutation is a PACC mutation. In some aspects, PACC mutations include A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M.Also disclosed is a method for treating a subject for lung cancer, the method comprising: (a) detecting an EGFR mutation in tumor DNA from the subject, the EGFR mutation being a PACC mutation; and (b) administering to the subject an effective amount of one or more second-generation EGFR inhibitors (e.g., afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, or a combination thereof). In some aspects, PACC mutations include A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M. Further disclosed is a method comprising administering a tyrosine kinase inhibitor to a subject determined to have a P-loop αC helix compression EGFR mutation from analysis of tumor DNA from the subject, wherein the tyrosine kinase inhibitor is not an EGFR inhibitor.

[0189] In some embodiments, the method includes administering afatinib to the subject. In some embodiments, the method includes administering dacomitinib to the subject. In some embodiments, the method includes administering neratinib to the subject. In some embodiments, the method includes administering a Tarlox-TKI to the subject. In some embodiments, the method includes administering tarloxotinib to the subject.

[0190] In some embodiments, the EGFR mutation is A750_I759del insPN. In some embodiments, the EGFR mutation is E709_T710del insD. In some embodiments, the EGFR mutation is E709A. In some embodiments, the EGFR mutation is E709A G719A. In some embodiments, the EGFR mutation is E709A G719S. In some embodiments, the EGFR mutation is E709K. In some embodiments, the EGFR mutation is E709K G719S. In some embodiments, the EGFR mutation is E736K. In some embodiments, the EGFR mutation is E746_A750del A647T. In some embodiments, the EGFR mutation is E746_A750del R675W. In some embodiments, the EGFR mutation is E746_T751del insV S768C. In some embodiments, the EGFR mutation is Ex19del C797S. In some embodiments, the EGFR mutation is Ex19del G796S. In some embodiments, the EGFR mutation is Ex19del L792H. In some embodiments, the EGFR mutation is Ex19del T854I. In some embodiments, the EGFR mutation is G719A. In some embodiments, the EGFR mutation is G719A D761Y. In some embodiments, the EGFR mutation is G719A L861Q. In some embodiments, the EGFR mutation is G719A R776C. In some embodiments, the EGFR mutation is G719A S768I. In some embodiments, the EGFR mutation is G719C S768I. In some embodiments, the EGFR mutation is G719S. In some embodiments, the EGFR mutation is G719S L861Q. In some embodiments, the EGFR mutation is G719S S768I. In some embodiments, the EGFR mutation is G724S. In some embodiments, the EGFR mutation is G724S Ex19del. In some embodiments, the EGFR mutation is G724S L858R. In some embodiments, the EGFR mutation is G779F. In some embodiments, the EGFR mutation is I740dupIPVAK. In some embodiments, the EGFR mutation is K757M L858R. In some embodiments, the EGFR mutation is K757R. In some embodiments, the EGFR mutation is L718Q.In some embodiments, the EGFR mutation is Ex19del. In some embodiments, the EGFR mutation is L718Q L858R. In some embodiments, the EGFR mutation is L718V. In some embodiments, the EGFR mutation is L718V L858R. In some embodiments, the EGFR mutation is L747_S752del A755D. In some embodiments, the EGFR mutation is L747P. In some embodiments, the EGFR mutation is L747S. In some embodiments, the EGFR mutation is L747S L858R. In some embodiments, the EGFR mutation is L747S V774M. In some embodiments, the EGFR mutation is L858R C797S. In some embodiments, the EGFR mutation is L858R L792H. In some embodiments, the EGFR mutation is L858R T854S. In some embodiments, the EGFR mutation is N771G. In some embodiments, the EGFR mutation is R776C. In some embodiments, the EGFR mutation is R776H. In some embodiments, the EGFR mutation is E709_T710del insD S22R. In some embodiments, the EGFR mutation is S752_I759del V769M. In some embodiments, the EGFR mutation is S768I. In some embodiments, the EGFR mutation is S768I L858R. In some embodiments, the EGFR mutation is S768I L861Q. In some embodiments, the EGFR mutation is S768I V769L. In some embodiments, the EGFR mutation is S768I V774M. In some embodiments, the EGFR mutation is T751_I759 delinsN. In some embodiments, the EGFR mutation is V769L. In some embodiments, the EGFR mutation is V769M. In some embodiments, the EGFR mutation is V774M.

[0191] In some embodiments, the subject has been previously treated with cancer therapy. In some embodiments, the cancer therapy includes erlotinib, gefitinib, AZD3759 or sapatinib. In some embodiments, the cancer therapy includes osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib. In some embodiments, the cancer therapy includes chemotherapy. In some embodiments, the subject has been determined to be resistant to cancer therapy.

[0192] In some embodiments, the disclosed methods include identifying one or more subjects as candidates for treatment with one or more kinase inhibitors from one or more kinase inhibitor classes based on the presence or absence of one or more mutations in the EGFR gene of the subject's tumor. For example, in some embodiments, a method is disclosed that includes identifying a subject having cancer (e.g., lung cancer) as a candidate for treatment with one or more kinase inhibitors from one or more kinase inhibitor classes by determining that the efficacy of one or more kinase inhibitors from one or more kinase inhibitor classes is or will be optimal. In some cases, the one or more kinase inhibitors from one or more kinase inhibitor classes are or will be optimal when the subject is determined to have one or more mutations in the EGFR gene in the subject's tumor that confer increased sensitivity (or decreased resistance) to the one or more kinase inhibitors. In some cases, the one or more kinase inhibitors from one or more kinase inhibitor classes are or will be suboptimal when the subject is determined to have one or more mutations in the EGFR gene in the subject's tumor that confer decreased sensitivity (or increased resistance) to the one or more kinase inhibitors. In some embodiments, the disclosed methods include determining an optimal cancer treatment for a subject for whom current or previous cancer treatments are or have been suboptimal, hi some embodiments, the subject is given multiple types of cancer therapy, e.g., multiple kinase inhibitor therapies.

[0193] In certain aspects, the disclosure relates to methods of predicting sensitivity or resistance in a subject having cancer to one or more kinase inhibitors from one or more kinase inhibitor classes based on analysis of one or more of the following biomarkers in the subject's tumor: (1) classical-like EGFR mutations; (2) exon 20 loop proximal insertion (Ex20ins-NL) EGFR mutations; (3) exon 20 loop distal insertion (Ex20ins-FL) EGFR mutations; (4) T790M-like 3S EGFR mutations; (5) T790M-like 3R EGFR mutations; or (6) PACC EGFR mutations.

[0194] In some aspects, the disclosure relates to methods of predicting therapy outcome, including the likelihood of sensitivity or resistance to one or more kinase inhibitors from one or more kinase inhibitor classes, for a subject having cancer (e.g., lung cancer) and in need of treatment with one or more kinase inhibitors from one or more kinase inhibitor classes. Such analysis of (1), (2), (3), (4), (5) or (6) above results in a decision of whether or how to best treat the cancer, or which one or more kinase inhibitors from one or more kinase inhibitor classes should be administered to treat the cancer.

[0195] Thus, in some embodiments, the likelihood of sensitivity or resistance to a given kinase inhibitor is determined based on the analysis of the tumor DNA of a subject with cancer (e.g., lung cancer) for one or more mutations in the subject's EGFR gene.In such cases, as a result of the tumor DNA analysis, the subject is administered a targeted therapeutic strategy for treating the cancer.For example, the subject can be administered a therapeutically effective amount of one or more kinase inhibitors from one or more kinase inhibitor classes.

[0196] If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject has one or more classical-like EGFR mutations, the subject may have an increased likelihood of sensitivity (or a decreased likelihood of resistance) to one or more first-generation EGFR TKIs, second-generation EGFR TKIs, third-generation EGFR TKIs, or EGFR TKIs specific for mutations associated with EGFR exon 20, and the subject may optionally be provided with a therapeutically effective amount of one or more first-generation EGFR TKIs, second-generation EGFR TKIs, third-generation EGFR TKIs, or EGFR TKIs specific for mutations associated with EGFR exon 20. If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject does not have one or more classical-like EGFR mutations, the subject may have a decreased likelihood of sensitivity (or an increased likelihood of resistance) to one or more first-generation EGFR TKIs, second-generation EGFR TKIs, third-generation EGFR TKIs, or EGFR TKIs specific for mutations associated with EGFR exon 20, the subject may optionally be provided with a therapeutically effective amount of one or more alternative kinase inhibitors.

[0197] If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject has one or more ex20ins-NL EGFR mutations, the subject may have an increased likelihood of sensitivity (or a decreased likelihood of resistance) to one or more second-generation EGFR TKIs or EGFR TKIs specific for mutations associated with EGFR exon 20, and the subject may optionally be provided with a therapeutically effective amount of one or more second-generation EGFR TKIs or EGFR TKIs specific for mutations associated with EGFR exon 20. If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject does not have one or more ex20ins-NL EGFR mutations, the subject may have an increased likelihood of sensitivity (or a decreased likelihood of resistance) to one or more second-generation EGFR TKIs or EGFR TKIs specific for mutations associated with EGFR exon 20, and the subject may optionally be provided with a therapeutically effective amount of one or more alternative kinase inhibitors.

[0198] If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject has one or more T790M-like 3S EGFR mutations, the subject may have an increased likelihood of sensitivity (or decreased likelihood of resistance) to one or more third generation EGFR TKIs, EGFR TKIs specific for mutations associated with EGFR exon 20, ALK inhibitors or PKC inhibitors, and the subject may optionally be provided with a therapeutically effective amount of one or more third generation EGFR TKIs, EGFR TKIs specific for mutations associated with EGFR exon 20, ALK inhibitors or PKC inhibitors. If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject does not have one or more T790M-like 3S EGFR mutations, the subject may have a decreased likelihood of sensitivity (or an increased likelihood of resistance) to one or more third generation EGFR TKIs, EGFR TKIs specific for mutations associated with EGFR exon 20, ALK inhibitors or PKC inhibitors, and the subject may, in some cases, be provided with a therapeutically effective amount of one or more alternative kinase inhibitors.

[0199] If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject has one or more T790M-like 3R EGFR mutations, the subject may have an increased likelihood of sensitivity (or a decreased likelihood of resistance) to one or more ALK inhibitors or PKC inhibitors, and the subject may optionally be provided with a therapeutically effective amount of one or more ALK inhibitors or PKC inhibitors. If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject does not have one or more T790M-like 3R EGFR mutations, the subject may have an increased likelihood of sensitivity (or an increased likelihood of resistance) to one or more ALK inhibitors or PKC inhibitors, and the subject may optionally be provided with a therapeutically effective amount of one or more alternative kinase inhibitors.

[0200] If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject has one or more PACC EGFR mutations, the subject may have an increased likelihood of sensitivity (or a decreased likelihood of resistance) to one or more second-generation EGFR TKIs, and the subject may optionally be provided with a therapeutically effective amount of one or more second-generation EGFR TKIs. If analysis of tumor DNA of a subject having cancer (e.g., lung cancer) for one or more mutations in the EGFR gene indicates that the subject does not have one or more PACC EGFR mutations, the subject may have an increased likelihood of sensitivity (or an increased likelihood of resistance) to one or more second-generation EGFR TKIs, and the subject may optionally be provided with a therapeutically effective amount of one or more alternative kinase inhibitors.

[0201] III. Sample preparation In certain aspects, the method includes collecting a sample (also referred to as a "biological sample") from a subject. The collection methods provided herein may include methods of biopsy, such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, incisional biopsy, excision biopsy, punch biopsy, shaved biopsy, or skin biopsy. In certain embodiments, the sample is collected from a biopsy from lung tissue by any of the aforementioned biopsy methods. In other embodiments, the sample may be collected from any of the tissues provided herein, including non-cancerous or cancerous tissues and non-cancerous or cancerous tissues from serum, gallbladder, mucosa, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. In some aspects, the sample is a cancerous or non-cancerous lung tissue sample. Alternatively, the sample may be collected from any other source, including blood, sweat, hair follicle, oral tissue, tears, menstrual secretions, feces, or saliva. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, may collect the biological sample for testing. Additionally, the biological sample may be collected without the assistance of a medical professional.

[0202] Samples include, but are not limited to, subject tissues, cells or biological materials derived from cells. Biological samples may be heterogeneous or homogeneous populations of cells or tissues. Biological samples may be collected using any method known in the art that can provide suitable samples for the analytical methods described herein. Samples may be collected by non-invasive methods, including skin or cervical scraping, oral mucosa swabbing, saliva collection, urine collection, fecal collection, menstrual secretions, tears or semen collection.

[0203] The sample may be taken by methods known in the art. In certain embodiments, the sample is taken by biopsy. In other embodiments, the sample is taken by swabbing, endoscopy, scraping, phlebotomy, or any other method known in the art. In some cases, the sample may be taken, stored, or transported using the components of the kit of the method. In some cases, multiple samples, such as multiple lung tissue samples, may be taken for diagnosis by the method described herein. In other cases, multiple samples, for example, one or more samples from one tissue type (e.g., lung) and one or more samples from another specimen (e.g., serum or blood), may be taken for diagnosis by the method. In some cases, multiple samples, for example, one or more samples from one tissue type (e.g., lung) and one or more samples from another specimen (e.g., serum or blood), may be taken at the same time or at different times. Samples may be taken at different times and stored and / or analyzed by different methods. For example, samples may be taken and analyzed by routine staining or any other cytological analysis method, by sequencing (e.g., DNA or RNA sequencing), by microarray, or by any other genetic analysis method.

[0204] In some embodiments, the biological sample may be collected by a doctor, nurse, or other medical professional, such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may prescribe the appropriate test or assay to be performed on the sample. In certain aspects, the molecular profiling provider may provide an opinion as to which assay or test is most appropriately prescribed. In further aspects of the method, the patient or subject may collect the biological sample for testing, such as collecting a whole blood sample, a urine sample, a fecal sample, an oral sample, or a saliva sample, without the assistance of a medical professional.

[0205] In other cases, samples are taken by invasive procedures including biopsy, needle aspiration, endoscopy or phlebotomy. Methods of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy or large core biopsy. In some embodiments, multiple samples may be taken by the methods herein to ensure a sufficient amount of biological material.

[0206] In some cases, the biological sample is a cell-free sample (e.g., a serum sample). In such cases, the biological sample may contain cell-free nucleic acids, such as DNA (e.g., cell-free tumor DNA, cell-free fetal DNA) or RNA (e.g., cell-free tumor RNA, cell-free fetal RNA). In some embodiments, the cell-free biological sample contains or is suspected of containing DNA or RNA from lung cancer.

[0207] Also, the general method for taking biological samples is known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is incorporated herein by reference in its entirety, describe the general method of biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of lung or suspected lung tumor or neoplasm. In some cases, fine needle aspiration sampling procedure can be guided by using ultrasound, X-ray or other imaging device.

[0208] In some aspects of the method, the molecular profiling provider may obtain the biological sample directly from the subject, from a medical professional, from a third party, or from a kit provided by the molecular profiling provider or a third party. In some cases, the subject, medical professional, or third party may obtain the biological sample and send it to the molecular profiling provider, which then collects the biological sample. In some cases, the molecular profiling provider may provide appropriate containers and additives for storing and transporting the biological sample to the molecular profiling provider.

[0209] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. Alternatively, an individual may collect a sample using an over-the-counter (OTC) kit. An OTC kit may include a means for collecting the sample as described herein, a means for storing the sample in preparation for testing, and instructions for correct use of the kit. In some cases, molecular profiling services are included in the purchase price of the kit. In other cases, molecular profiling services are charged separately. A sample suitable for use by a molecular profiling contractor may be any material including tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of the individual to be tested. Methods are provided for determining the suitability and / or appropriateness of a sample.

[0210] In some embodiments, the subject may be referred to a specialist, such as an oncologist, surgeon, or endocrinologist. The specialist may also collect a biological sample for testing, or refer the individual to a testing center or research facility for submitting a biological sample. In some cases, a medical professional may refer the subject to a testing center or research facility for submitting a biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling company may collect the sample.

[0211] IV. Assay Methods A. Sequencing In some embodiments, the methods of the disclosure include a sequencing method. Exemplary sequencing methods include those described below.

[0212] 1. Massively Parallel Signature Sequencing (MPSS) The first of the next-generation sequencing technologies, Massively Parallel Signature Sequencing (or MPSS), was developed at Lynx Therapeutics in the 1990s. MPSS was a bead-based method that read sequences in 4-nucleotide increments using a complex method of adapter ligation followed by adapter decoding. The intrinsic nature of the MPSS output was typical of later "next-generation" data types, containing hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used to sequence cDNA for the measurement of gene expression levels.

[0213] 2. Polony Sequencing Polony sequencing, developed in the laboratory of George M. Church at Harvard University, was one of the first next-generation sequencing systems and was used to sequence an entire genome in 2005. In vitro paired-tag libraries were combined with emulsion PCR, automated microscopy and ligation-based sequencing chemistry to sequence the E. coli genome with >99.9999% accuracy and approximately one-ninth the cost of Sanger sequencing.

[0214] 3. 454 Pyrosequencing A parallel version of pyrosequencing amplifies DNA in droplets in an oil solution (emulsion PCR), where each droplet contains a single DNA template attached to a single primer-coated bead that subsequently forms a clonal colony. The sequencer contains many picoliter-volume wells, each containing a single bead and sequencing enzyme. Pyrosequencing uses luciferase to generate light to detect individual nucleotides added to the nascent DNA, and uses the combined data to generate sequence readouts. The technology offers intermediate read lengths and price per base compared to Sanger sequencing on the one hand, and Solexa and SOLiD on the other.

[0215] 4. Illumina (Solexa) Sequencing In this method, DNA molecules and primers are first attached to a slide and amplified with polymerase to form localized clonal DNA colonies (later coined "DNA clusters"). To determine the sequence, four reversible terminator bases (RT bases) are added and unincorporated nucleotides are washed away. After the camera captures an image of the fluorescently labeled nucleotides, the dye, along with the terminal 3' blocker, is chemically removed from the DNA to allow the next cycle to begin. Unlike pyrosequencing, the DNA strand is extended one nucleotide at a time and image acquisition can be performed with a split-second delay, allowing very large arrays of DNA colonies to be captured by sequential images taken from a single camera.

[0216] Decoupling the enzymatic reaction from image capture allows for optimal throughput and theoretically unlimited sequencing capacity. Therefore, in an optimal configuration, the final achievable instrument throughput is determined only by the camera analog-to-digital conversion rate multiplied by the number of cameras divided by the number of pixels per DNA colony required for optimal visualization (~10 pixels / colony).

[0217] 5. SOLiD Sequencing The SOLiD technology uses sequencing by ligation, where a pool of all possible oligonucleotides of fixed length is labeled according to the sequencing position. The oligonucleotides are annealed and ligated; preferential ligation by DNA ligase for matching sequences generates a signal that provides information about the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing a single copy of the same DNA molecule, are placed on a glass slide. The result is sequences of quantity and length that are comparable to Illumina sequencing.

[0218] 6. Ion Torrent Semiconductor Sequencing Ion Torrent Systems Inc. has developed a system based on standard sequencing chemistry, but with a novel semiconductor-based detection system. This sequencing method is based on the detection of hydrogen ions released during DNA polymerization, in contrast to the optical methods used in other sequencing systems. A single type of nucleotide is filled into the microwell containing the template DNA strand to be sequenced. If the introduced nucleotide is complementary to the primary template nucleotide, it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion, which triggers a highly sensitive ion sensor, which indicates that a reaction has occurred. If homopolymeric repeats are present in the template sequence, multiple nucleotides are incorporated in a single cycle. This causes the release of a corresponding number of hydrogens and a proportionally higher electronic signal.

[0219] 7. DNA nanoball sequencing DNA nanoball sequencing is a type of high-throughput sequencing technique used to determine the whole genome sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. The nucleotide sequence is then determined using unlinked sequencing by ligation. This DNA sequencing method allows for sequencing a large number of DNA nanoballs per run at a low reagent cost compared to other next-generation sequencing platforms. However, only a short DNA sequence is determined from each DNA nanoball, making it difficult to map short reads to a reference genome. The technique has been used in several genome sequencing projects.

[0220] 8. Heliscope Single Molecule Sequencing Heliscope sequencing is a single molecule sequencing method developed by Helicos Biosciences. It uses DNA fragments with polyA tail adapters attached to the flow cell surface. The next step involves extension-based sequencing by periodic washing of the flow cell with fluorescently labeled nucleotides (similar to the Sanger method, one nucleotide at a time type). The reads are performed by the Heliscope sequencer.

[0221] 9. Single Molecule Real-Time (SMRT) Sequencing The SMRT sequencing method is based on the sequencing by synthesis technique. DNA is synthesized in a zero mode waveguide (ZMW) - a small well-like container with a capture tool located at the bottom of the well. Sequencing is performed with an unmodified polymerase (attached to the bottom of the ZMW) and fluorescently labeled nucleotides that flow freely in solution. The wells are constructed in such a way that only the fluorescence generated by the bottom of the well is detected. The fluorescent label is cleaved from the nucleotide when incorporated into the DNA strand, leaving an unmodified DNA strand. This technique allows reads of more than 20,000 nucleotides, with an average read length of 5 kilobases.

[0222] B. Additional Assays In some embodiments, the methods include amplifying and / or sequencing one or more target genomic regions using at least one pair of primers specific for the target genomic regions. In other embodiments, an enzyme, such as a primase or a combined primase / polymerase enzyme, is added to the amplification step to synthesize the primers.

[0223] In some embodiments, arrays can also be used to detect nucleic acids of the present disclosure. Arrays include solid supports to which nucleic acid probes are attached. Arrays usually include a plurality of different nucleic acid probes attached to the surface of a substrate at different known positions. These arrays, also referred to as "microarrays" or colloquially as "chips", have been reviewed in the art, for example, U.S. Patent Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186, and Fodor et al., 1991, each of which is incorporated herein by reference in its entirety for all purposes. Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261, which is incorporated herein by reference in its entirety for all purposes. Although in certain aspects planar array surfaces are used, arrays can be made on surfaces of virtually any shape or on multiple surfaces. Arrays can be nucleic acids on beads, gels, polymer surfaces, fibers such as fiber optics, glass or any other suitable substrate. See U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are incorporated by reference in their entirety for all purposes.

[0224] A nucleic acid array can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes that can hybridize to different and / or same biomarkers. Multiple probes for the same gene can be used on a single nucleic acid array. Probes for other disease genes can also be included in the nucleic acid array. The density of the probes on the array can be in any range. In some embodiments, the density can be 50, 100, 200, 300, 400, 500 or more per cm2 (or any derivable range therein) or at least 50, 100, 200, 300, 400, 500 or more per cm2 (or any derivable range therein).

[0225] Particularly contemplated are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these technologies include quantitative methods for rapid and accurate analysis of a large number of genes. By tagging genes with oligonucleotides or using fixed probe arrays, chip technology can be used to separate target molecules as high-density arrays and screen these molecules based on hybridization (see also Pease et al., 1994 and Fodor et al, 1991). It is contemplated that this technology can be used in conjunction with the evaluation of the expression level of one or more cancer biomarkers for diagnostic, prognostic and treatment methods.

[0226] Certain embodiments may involve the use of arrays or data generated from arrays. The data may be readily available. Moreover, arrays may be prepared to generate data that can then be used in correlation studies.

[0227] In addition to the use of arrays and microarrays, it is contemplated that a number of different assays can be used to analyze nucleic acids. Such assays include, but are not limited to, nuclear amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, digital PCR, ddPCR (droplet digital PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged-Amplicon deep sequencing), PAP (Pyrophosphorolysis-activation polymerization), next-generation RNA sequencing, Northern hybridization, Hybridization Protection Assay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and / or Bridge Litigation Assay (Genaco).

[0228] Amplification primers or hybridization probes can be prepared to be complementary to the genomic regions, biomarkers, probes or oligos described herein. The term "primer" or "probe" as used herein is meant to encompass any nucleic acid capable of priming the synthesis of a nascent nucleic acid in a template-dependent process and / or pairing with a single strand or a portion of an oligo of the present disclosure. Typically, primers are oligonucleotides of 10-20 and / or 30 nucleic acids in length, although longer sequences can be used. Primers can be provided in double-stranded or single-stranded form.

[0229] The use of probes or primers of 13-100 nucleotides in length, particularly 17-100 nucleotides or in some aspects up to 1-2 kilobases or more in length, allows for the formation of double-stranded molecules that are stable and selective. Molecules having complementary sequences over a contiguous stretch of more than 20 bases in length may be used to increase the stability and / or selectivity of the resulting hybrid molecules. Nucleic acid molecules for hybridization may be designed with one or more complementary sequences of 20-30 nucleotides in length, or longer if desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragments by chemical means or by introducing selected sequences into a recombinant vector for recombinant production.

[0230] In one embodiment, each probe / primer comprises at least 15 nucleotides. For example, each probe can comprise at least 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any derivable range therein) or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any derivable range therein). Nucleotides can have these lengths and have identical or complementary sequences to genes described herein. In particular, each probe / primer has a relatively high sequence complexity and does not have ambiguous residues (undefined "n" residues). The probe / primer can hybridize to the target gene (including its RNA transcript) under stringent or highly stringent conditions. It is contemplated that the probe or primer may have inosines or other design features that accommodate the recognition of more than one human sequence for a particular biomarker.

[0231] In one embodiment, quantitative RT-PCR (TaqMan, ABI, etc.) is used to detect and compare the level or abundance of nucleic acids in samples. The concentration of target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before PCR is initiated. By measuring the concentration of the PCR product of the target DNA in PCR reactions that have completed the same number of cycles and are in the linear range, it is possible to measure the relative concentration of a particular target sequence in the original DNA mixture. This direct proportional relationship between the concentration of the PCR product and the relative abundance in the starting material is true in the linear range portion of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mixture and is independent of the original concentration of the target DNA. Therefore, sampling and quantification of the amplified PCR product may be performed when the PCR reaction is in the linear portion of its curve. In addition, the relative concentration of amplifiable DNA may be normalized to some independent standard / control, which may be based on either internally present or externally introduced DNA species. The abundance of a particular DNA species may be determined relative to the average abundance of all DNA species in the sample.

[0232] In one embodiment, PCR amplification utilizes one or more internal PCR standards.Internal standards can be housekeeping genes that are abundant in cells, and can be specifically GAPDH, GUSB and β-2 microglobulin.These standards can be used to normalize expression levels, so that the expression levels of different gene products can be directly compared.Those skilled in the art will know how to normalize expression levels using internal standards.

[0233] V. Administration of Therapeutic Compositions Therapies provided herein include the administration of a combination of therapeutic agents comprising at least one or more kinase inhibitors from one or more kinase inhibitor classes. In some embodiments, at least one, two, three, four, five or six TKI classes are administered. In some embodiments, the one, two, three, four, five or six TKI classes include a first generation EGFR TKI, a second generation EGFR TKI, a third generation EGFR TKI, an EGFR TKI specific for mutations associated with EGFR exon 20, an ALK inhibitor or a PLC inhibitor. In some embodiments, the TKI is erlotinib, gefitinib, AZD3759, sapatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, TAS 6417, AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, AZD3463, brigatinib, ruboxistaurin, midostaurin or sotrastaurin. In some embodiments, at least one, two, three, four, five or more TKIs are administered to a subject with cancer. In some embodiments, the one or more TKIs include two or more of erlotinib, gefitinib, AZD3759, sapatinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, TAS 6417, AZ5104, TAK-788 (mobocertinib), osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, AZD3463, brigatinib, ruboxistaurin, midostaurin, or sotrastaurin. Any one or more TKIs may be excluded from certain embodiments of the present disclosure.

[0234] The embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. In some embodiments, different therapies are administered sequentially (at different times) or in parallel (at the same time). Different therapies may be administered as one composition or as more than one composition, for example, two compositions, three compositions, or four compositions. In some embodiments, the therapies are administered as separate compositions. In some embodiments, the therapies are in the same composition. In some embodiments of the methods disclosed herein, a single dose of cancer therapy is administered. In some embodiments of the methods disclosed herein, multiple doses of cancer therapy are administered. Various combinations of agents may be used. For example, a first generation TKI is designated as "A" and a second generation TKI is designated as "B". TIFF2024506839000029.tif18128

[0235] The compositions of the present disclosure can be prepared according to standard techniques and may include water, buffered water, saline, glycine, glucose, isotonic sucrose solution, etc., glycoproteins such as albumin, lipoproteins, globulins, etc., to enhance stability. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparations are combined with a sterile aqueous solution before administration. The compositions may contain pharma- ceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, isotonicity agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Preparation of compositions containing cancer therapy will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. Moreover, for animal (eg, human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0236] The composition is a pharma- ceutically acceptable or pharmacologically acceptable composition. The term "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0237] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of undesired microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0238] The cancer therapy or therapeutic substance of the present disclosure may be administered by the same or different administration routes.In some embodiments, the cancer therapy is administered intraarterially, intravenously, intraperitoneally, subcutaneously, intramuscularly, intratumorally, locally, orally, transdermally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly or intranasally.The appropriate dosage can be determined based on the type of disease to be treated, the severity and course of the disease, the clinical condition of the subject, the medical history of the subject and response to treatment, and the discretion of the attending physician.

[0239] Treatment may include various "unit doses". A unit dose is defined as comprising a predetermined amount of therapeutic composition calculated to produce the desired response as described above in connection with its administration, i.e., appropriate route and regimen. The amount administered and the specific route and formulation are within the ability of those skilled in the clinical art to determine and depend on the desired outcome and / or protection. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0240] Generally, the composition is administered in a manner compatible with the dosage form and in an amount that is therapeutically or prophylactically effective for the subject being treated.The exact amount of therapeutic composition is also dependent on the judgment of the practitioner and is specific to each individual.The suitable regimen for initial administration and booster administration also varies, but an initial administration is typically followed by subsequent administrations.Factors that affect dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (whether symptom relief or cure), and the efficacy, stability and toxicity of the particular therapeutic agent or other therapy that the subject may be undergoing.

[0241] The amount administered, both according to the number of treatments and the unit dose, depends on the desired treatment effect. Effective dose is understood to refer to the amount required to achieve a specific effect. In the implementation of certain embodiments, it is believed that a dose ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, doses are contemplated to include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, mg / day, or any range derivable therein. Furthermore, such doses may be administered multiple times per day or over multiple days, weeks, or months.

[0242] In certain embodiments, an effective dose of the pharmaceutical composition is a dose capable of providing a blood level of about 1 μM to 150 μM. In other embodiments, an effective dose provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any derivable range therein). In other aspects, the dose can provide the following blood levels of the agent resulting from the therapeutic agent being administered to a subject: about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μM, or any derivable range therein, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100 μM or any derivable range therein or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 μM or any derivable range therein or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any derivable range therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of the therapeutic agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels described herein may refer to the unmetabolized therapeutic agent.

[0243] It will be understood and appreciated by those skilled in the art that dosage units of μg or mg per kg body weight can be converted and expressed in comparable concentration units of μg / ml or mM (blood levels), such as 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions made regarding uptake and concentration measurements are well known and will allow one skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0244] VI. Kits Certain aspects of the present invention also relate to kits comprising the disclosed compositions or compositions for implementing the disclosed methods. In some embodiments, the kits can be used to evaluate one or more biomarkers. In certain embodiments, the kits can be used to evaluate one or more biomarkers. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 133, 144, 150 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors (or any derivable values ​​or ranges and combinations therein) or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors (or any derivable values ​​or ranges and combinations therein). In some embodiments, there are kits for assessing biomarker activity in a cell.

[0245] The kit may contain components that may be individually packaged or disposed in containers, such as tubes, bottles, vials, syringes, or other suitable container means.

[0246] Individual components may also be provided in concentrated amounts in the kit. In some embodiments, components are provided individually at the same concentration as they are in solution with other components. The concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or higher.

[0247] Kits for using the probes, synthetic nucleic acids, non-synthetic nucleic acids and / or inhibitors of the present disclosure for prognostic or diagnostic applications are included as part of the present disclosure.Specifically, any such molecules corresponding to any of the biomarkers identified herein are contemplated, including nucleic acid primers / primer sets and probes that are identical to or complementary to all or a portion of the biomarker, which may include non-coding sequences of the biomarker and coding sequences of the biomarker.

[0248] In certain aspects, negative and / or positive control nucleic acids, probes and inhibitors are included in some kit embodiments. In addition, the kits may include samples that are negative or positive controls for one or more biomarkers.

[0249] Additionally, any embodiment of the disclosure that includes a particular biomarker by name is considered to include embodiments that include a biomarker having a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the designated nucleic acid.

[0250] An embodiment of the present disclosure includes a kit for analyzing a pathological sample by evaluating a biomarker profile of the sample, the kit comprising two or more biomarker probes in suitable container means, the biomarker probes detecting one or more of the biomarkers identified herein. The kit may further comprise a reagent for labeling nucleic acid in the sample. The kit may also comprise a labeling reagent comprising at least one of amine-modified nucleotides, poly(A) polymerase and poly(A) polymerase buffer. The labeling reagent may comprise an amine-reactive dye.

[0251] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein, and that the various aspects may be combined. The claims as originally filed are contemplated to include claims that are multiple dependent on any filed claim or combination of filed claims. EXAMPLES

[0252] The following examples are included to demonstrate aspects of the present disclosure. It will be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present disclosure. However, those skilled in the art will understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed, and these changes can also produce the same or similar results without departing from the spirit and scope of the present disclosure.

[0253] 1. Structure-function-based groups predict EGFR TKI sensitivity more accurately than exon-based groups To determine the impact of EGFR mutations on TKI sensitivity, we generated a panel of 76 cell lines expressing EGFR mutations spanning exons 18-21 and screened them against 18 known EGFR inhibitors representing first (non-covalent), second (covalent) and third (covalent, T790M targeting) generation TKIs as well as compounds under investigation for exon 20ins. Through hierarchical clustering of in vitro selectivity against WT EGFR and mutational mapping of EGFR mutations, we observed four distinct subgroups of EGFR mutations: classical-like mutations distant from the ATP-binding pocket (Figure 1A, B), T790M-like mutations in the hydrophobic core (Figure 1A, C), Ex20ins located at the C-terminus of the αC-helix (Figure 1A, D), and a fourth group predicted to be P-loop αC-helix compression (PACC) mutations, located on the inner surface of the ATP-binding pocket and at the C-terminus of the αC-helix (Figure 1A, E). For various mutations, the structure-function-based group predicted drug sensitivity better than the exon-based group as determined by Spearman correlation (p<0.0001, Fig. 1F, Fig. 2). Supervised clustering by structure-function-based group maintained separate groupings in the heatmap. However, supervised clustering by exon location seemed to disrupt the drug sensitivity patterns on the heatmap (Fig. 3). Classical-like atypical EGFR mutations were predicted to have little effect on the overall structure of EGFR compared to WT EGFR (Fig. 4A-4D) and were sensitive and selective to all classes of EGFR TKIs, especially third-generation TKIs, in vitro (Fig. 4E) and in vivo (Fig. 4F, Fig. 4G). Ex20ins mutations were resistant to first- and third-generation TKIs and sensitive only to selected second-generation TKIs (e.g., Tarlox-TKI) and ex20ins-specific TKIs in vitro (Fig. 5A) and in vivo (Fig. 5C, Fig. 5D). These findings demonstrate that structure-function-based groups can predict drug class sensitivity for a given mutation and can predict which mutation groups are most sensitive to a given inhibitor more effectively than traditional exon-based groupings.

[0254] 2. EGFR TKI-resistant T790M-like mutations can be inhibited by ALK and PKC inhibitors Although all T790M-like mutants had at least one mutation in the hydrophobic core, there were two distinct subgroups of T790M-like mutants, namely, third-generation TKI-sensitive (T790M-like 3S) and third-generation TKI-resistant (T790M-like 3R) (Figure 6A). T790M-like 3S mutants showed high selectivity for third-generation TKIs and some exon 20-specific inhibitors, and moderate selectivity for ALK and PKC inhibitors (Figure 6B). T790M-like 3R mutants were characterized by T790M and known drug resistance mutations (i.e., C797S 37,38 , L718X 18,24 or L792H 23,24 ), which was resistant to classical EGFR TKIs but retained selectivity for selected ALK and PKC inhibitors (Figure 6C). Collectively, these data demonstrate that T790M-like mutants contained at least one mutation in the hydrophobic cleft known to convey resistance to first- and second-generation EGFR TKIs, but that the addition of known resistance mutations reduced sensitivity to classical EGFR TKIs, which could be overcome by drug repurposing with ALK or PKC inhibitors.

[0255] 3. PACC mutations are most sensitive to second-generation EGFR TKIs The PACC mutations consisted of mutations spanning exons 18-21, including mutations such as G719X, L747X, S768I, L792X, and T854I. The PACC mutations were predicted to affect the total volume of the ATP and drug binding pockets through changes in the orientation of the P-loop or αC-helix (Figure 5A, Figure 5B). In silico analysis of the interaction of osimertinib with the PACC mutations G719S and L718Q predicted that the change in the orientation of the P-loop would shift the position of TKI stabilizing points such as V726 and F723, tilting the indole ring of osimertinib away from the P-loop compared to the reactive conformation of osimertinib, destabilizing drug binding (Figure 7A, Figure 5C). In contrast, second-generation EGFR TKIs do not interact with the P-loop of EGFR, maintaining key interaction points in the hydrophobic cleft of the PACC mutants (Figure 5C, Figure 5D). Comparing the selectivity of PACC mutations with first-, second- and third-generation and ex20ins-specific EGFR TKIs, we found that second-generation EGFR TKIs were significantly more selective for PACC mutations than any other class of TKIs (Figure 7B). We also found that in vivo, mice carrying PDXs with G719A mutations were resistant to the third-generation TKI osimertinib, but most sensitive to second-generation EGFR TKIs (Figure 7C, Figure 5E). Finally, patients with combined PACC mutations E709K G719S showed clinical benefit and tumor shrinkage with afatinib treatment after progression on osimertinib (Figure 7D). Taken together, these data demonstrated that PACC mutations are a distinct subgroup of EGFR mutations; resistant to third-generation EGFR TKIs; and sensitive to second-generation EGFR TKIs.

[0256] Similarly, acquired PACC mutations co-occurring with classical primary EGFR mutations retained sensitivity to second-generation EGFR TKIs while acquiring resistance to third-generation EGFR TKIs (Figure 7E, Figure 7F). As mentioned above, acquired PACC mutations showed allele specificity in acquired drug resistance (Figure 7E). In silico analysis of acquired mutations such as G796S co-occurring with Ex19del predicted that they confer resistance to third-generation EGFR TKIs such as osimertinib by shifting the hinge region of the receptor to prevent osimertinib stabilization at M793 and displacing the acrylamide group of osimertinib away from C797, preventing binding (Figure 7G). However, second-generation inhibitors were predicted to be relatively unaffected by the shift in the hinge region of the receptor, maintaining the orientation of the acrylamide group closer to C797 (Figure 5F). Within the MD Anderson GEMINI database, one patient with lung adenocarcinoma harboring an EGFR L858R mutation was identified who received first-line osimertinib treatment and subsequently developed an EGFR-dependent resistance mechanism. A biopsy at the time of progression identified a PACC mutation (Figure 6A, Figure 6B). The patient acquired an L718V mutation, was treated with a second-generation EGFR TKI, and experienced clinical benefit with stable disease and tumor shrinkage (Figure 6A, Figure 6B). Collectively, these data demonstrate that both primary and acquired PACC mutations are sensitive to second-generation EGFR TKIs in preclinical models and patients, and that structure-function-based grouping can identify novel groupings of mutations for which older generation EGFR TKIs showed the greatest selectivity.

[0257] 4. Structure-function-based subgroups predict patient outcomes to TKIs better than exon-based subgroups To determine whether structure-function-based groups could better identify patients most likely to benefit from a given drug compared with exon-based groups, we used a publicly available database of clinical outcomes in patients with NSCLC harboring atypical EGFR mutations treated with afatinib. 39,40A retrospective analysis of ORR and duration of treatment (DoT) of 847 patients was performed. Although structure-function-based grouping showed clear differences between sensitive (classical-like and PACC) and resistant (T790M-like and Ex20ins) subgroups (ORR 63% vs. 20%), the exon-based group showed less variation between groups (Figure 10A, 10B). Further structure-function-based grouping identified more patient subgroups that required significantly longer DoT for afatinib treatment than the exon-based group (Figure 9A, 9B and Figure 10C, 10D). The exon-based grouping identified patients with exon 18 mutations that required longer DoT than patients with mutations in exons 20 or 21 (Figure 10C, 10D). On the other hand, structure-function-based grouping identified that patients with PACC mutations experienced significantly longer DoT than any other subgroup, and that patients with classic-like mutations required significantly longer DoT than patients with exon 20 loop insertions or T790M-like mutations (Figure 9A,B). These data demonstrate that structure-based grouping better identifies which patient groups will derive the greatest benefit from a given drug than exon-based grouping.

[0258] To determine whether structure-based grouping can identify which class of inhibitor provides the greatest benefit to patients with atypical EGFR mutations compared to traditional grouping, a retrospective analysis of mPFS of patients with atypical EGFR mutations treated with first-, second- or third-generation EGFR TKIs in the MD Anderson GEMINI database was performed. To determine whether structure-based grouping can identify which class of inhibitor provides the greatest benefit to patients with atypical EGFR mutations, a retrospective analysis of mPFS of patients with atypical EGFR mutations treated with first-, second- or third-generation EGFR TKIs in the MD Anderson GEMINI database was performed. Patients with PACC mutations treated with second-generation EGFR TKIs showed significantly longer PFS than patients treated with first- or third-generation EGFR TKIs (19.3 months vs. 8.5 months and 4.1 months, respectively, Figure 9C, Figure 9D). In contrast, in patients with non-PACC atypical mutations, progression-free survival did not differ significantly between EGFR TKI classes (Figure S1E), confirming that PACC mutations are highly sensitive to second-generation EGFR TKIs, as predicted by preclinical modeling. These data demonstrate that structure-based grouping can better identify which class of EGFR TKI provides the greatest benefit to patients with specific groups of mutations.

[0259] 5. Conclusion The diversity of atypical EGFR mutations and their higher prevalence than previously recognized highlight the need for comprehensive next-generation sequencing (NGS) for NSCLC patients. As described herein, EGFR mutations, including atypical mutations, can be divided into four distinct subgroups based on structure and function, with the structure / function-based group being more predictive of drug sensitivity and patient outcomes than the exon-based group. These four subgroups are: "classical-like," "T790M-like," "exon 20 loop insertion," and "P-loop αC helix compression" (or "PACC"). The four subgroups, including descriptions and exemplary mutations, are provided in FIG. 11. "Exon 20 loop insertion" mutations are further divided into exon 20 loop proximal insertion (Es20ins-NL) mutations and exon 20 loop distal insertion (Ex20ins-FL) mutations. "T790M-like" mutations are further divided into T790M-like 3S mutations and T790M-like 3R mutations.

[0260] Previous studies have shown activity of second-generation EGFR TKIs in patients with selected exon 18 mutations. 33,34 , Structure / function-based grouping identified a larger subgroup of EGFR mutations, namely PACC mutations, for which second-generation EGFR TKIs were more selective than third-generation EGFR TKIs. Clinically, second-generation EGFR TKIs were associated with WT EGFR inhibition and related adverse events, but 15,35,36 , Most second-generation EGFR TKIs are administered at maximum tolerated doses to produce plasma concentrations 10-100-fold higher than those required to inhibit PACC mutations. Unlike osimertinib, second-generation EGFR TKIs have limited CNS activity, demonstrating the need for novel EGFR TKIs with reduced WT EGFR inhibition and CNS activity capable of inhibiting PACC mutations.

[0261] These studies demonstrated that structure / function-based groups can identify classes of drugs that may be effective across the entire group of mutations, reflecting the observation that mutations in different regions of a gene can induce similar changes in protein structure. For example, L718Q, S768I, and T854I are in exons 18, 20, and 21, respectively, but are all PACC mutations that have similar structural effects on drug binding. Conversely, mutations within the same exon may induce completely different changes. L747_K754del-insATSPE, L747P, and E746-A750del mutations are in exon 19, but are T790M-like, PACC, and classical mutations, respectively, with obvious differences in drug sensitivity and structural effects. A clinical challenge for physicians treating patients with EGFR-mutated cancers is to properly identify their mutations and match them with the best EGFR TKI. The classification presented here provides a framework by which clinicians can more effectively individualize EGFR TKI therapy, informed by internet-based tools or companies providing NGS reports.Finally, these findings support the notion that in cancers harboring polymorphic oncogenes, the adoption of structure / function-based approaches may improve clinical trial design and drug development.

[0262] 6. Exemplary Methods Ba / F3 cell generation, drug screening and IC 50Approximation. Ba / F3 cells were obtained as a gift from Dr. Gordon Mills (MD Anderson Cancer Center) and maintained in RPMI (Sigma) containing 10% FBS, 1% penicillin / streptomycin and 10 ng / ml recombinant mIL-3 (R&D Biosystems). To establish stable Ba / F3 cell lines, Ba / F3 cells were transduced with retroviruses containing mutant EGFR plasmids for 12-24 hours. Retroviruses were generated using Lipofectamine 2000 (Invitrogen) transfection of Phoenix 293T-ampho cells (Orbigen) with pBabe-Puro-based vectors listed in Table 6 below. Vectors were generated by GeneScript or Bioinnovatise using the Addgene parent vectors listed in Table 6 below.

[0263] Table 6. EGFR mutant vectors used to generate cell lines TIFF2024506839000030.tif124160

[0264] After 48-72 hours of transduction, 2 μg / ml puromycin (Invitrogen) was added to Ba / F3 cell lines in complete RPMI. To select EGFR-positive cell lines, cells were stained with PE-EGFR (Biolegend) and sorted by FACS. After sorting, EGFR-positive cells were maintained in RPMI containing 10% FBS, 1% penicillin / streptomycin and 1 ng / ml EGF to support cell viability. Drug screening was performed as previously described. 41,42Immediately, cells were seeded in 384-well plates (Greiner Bio-One) at 2000–3000 cells per well in technical triplicates. Seven different concentrations of TKI or DMSO vehicle were added to reach a final volume of 40 μL per well. After 72 h, 11 μL of Cell Titer Glo (Promega) was added to each well. Plates were incubated for a minimum of 10 min and bioluminescence was measured using a FLUOstar OPTIMA plate reader (BMG LABTECH). Raw bioluminescence values ​​were normalized to DMSO control treated cells and values ​​were plotted in GraphPad Prism. Normalized data were fitted to a variable slope using nonlinear regression and IC was calculated by GraphPad Prism by interpolation of the concentration at 50% inhibition. 50 Drug screens were performed with technical triplicates and duplicate or triplicate biological replicates on each plate. For each drug, IC values ​​for the mutant cell lines were determined. 50 Values ​​are the mean IC of Ba / F3 cells expressing WT EGFR supplemented with 10 ng / ml EGF. 50 The mutant to WT ratio (Mut / WT) for each drug was calculated by dividing by the value. Statistical differences between groups were determined by analysis of variance as described in the figure legends. Table 7 shows an overview of all drugs tested.

[0265] Table 7. Summary of compounds tested TIFF2024506839000031.tif90160TIFF2024506839000032.tif242160TIFF2024506839000033.tif242160TIFF2024506839000034.tif54160

[0266] In silico mutation mapping and docking experiments. Wild-type EGFR (2ITX) in complex with AMP-PNP and EGFR in complex with AMP-PNP retrieved from the PDB. L858RThe X-ray structures of the mutant (2ITV) were used for MD simulations. All crystallographic ligands, ions and water molecules were removed from the X-ray structures. Missing side chain atoms and loops in these structures were replaced with the Schrodinger Prime homology module 43 Constructed using EGFR L858R The activation loop region (862–876) missing in the mutant structure was constructed using the activation loop from another EGFR structure (5XGN). The exon 19 deletion mutant (ΔELREA) was modeled on wild-type EGFR using the Prime program, followed by MM / GBSA-based loop refinement of the β3-αC loop region. Prime side chain predictions were used with Schrodinger and backbone sampling to refine all double mutants (EGFR L858R / L718Q , EGFR Ex19del / L718Q , EGFR L858R / L792H , EGFR Ex19del / L792H ) side chain prediction was performed, and the mutated residues were then minimized. The structure was finally synthesized using the “QuickPrep” module in MOE. 44 Pymol software was used for visualization of mutation positions on WT (2ITX) EGFR and alignment with EGFR D770insNPG (4LRM) or EGFR G719S (2ITN).

[0267] Heatmap generation and group Spearman correlation. R and the ComplexHeatmap package (R Foundation for Statistical Computing, Vienna, Austria, Complex Heatmap) 45Heatmaps and hierarchical clustering were generated by plotting the median log(Mut / WT) values ​​for each cell line and each drug using . Hierarchical clustering was determined by the Euclidean distance between Mut / WT ratios. In the case of co-occurring mutations, exon order was assigned arbitrarily, and in the case of acquired mutations, exons were assigned in the order in which the mutations were observed clinically. Structure-function groups were assigned based on the predicted effect of the mutation on the receptor conformation. Mutation correlations were determined by correlating the median log(Mut / WT) value of each mutation and drug against the mean of the median log(Mut / WT) values ​​of the structure-function-based or exon-based group to which the mutation belonged, using Spearman's rho. For each correlation, the tested mutation was removed from the mean structure-function-based and exon-based groups. Mean rho values ​​were compared by two-tailed Student's t-test.

[0268] PDX generation and in vivo experiments. Patient-derived xenografts were generated as part of the MD Anderson Cancer Center Lung Cancer Moon Shots program and were performed in accordance with Good Animal Practices and with the approval of the MD Anderson Cancer Center Institutional Animal Care and Use Committee (Houston, TX) under protocol number PA140276. 46 Surgical samples were washed with serum-free RPMI supplemented with 1% penicillin-streptomycin and then implanted into the right flank of 5-week-old NSG mice within 2 hours of resection. Tumors were cultured as described for EGFR mutations. 46 , and verified by DNA fingerprinting and qPCR. To grow tumors, 5-6 week old female NSG mice (NOD.Cg-Prkdcscid IL2rgtmWjl / Szj) were purchased from Jax Labs (#005557). Fragments of NSCLC tumors expressing EGFR G719S or L858R / E709K were implanted into 6-8 week old female NSG mice. When tumors reached 2000 mm 3Once tumors reached a mass of 100–1500 mm, they were harvested and reimplanted into the right flank of 6- to 8-week-old female NSG mice. Tumors were measured three times weekly and tumor volumes ranged from 275–325 mm for the EGFR G719S model to 150–1500 mm for the EGFR G719S model. 3 , 150~175mm for L858R / E709K models 3 Upon reaching a median age of 10 days, mice were randomized into treatment groups. Treatment groups included vehicle control (0.5% methylcellulose, 0.05% Tween-80 in dH2O), 100 mg / kg erlotinib, 20 mg / kg afatinib, 5 mg / kg osimertinib, and 20 mg / kg osimertinib. During treatment, body weights and tumor volumes were measured three times a week, and mice were treated five days a week (Monday-Friday). Mice were given a drug holiday if they lost more than 10% of their body weight or if their total body weight was less than 20 grams.

[0269] Case study of a patient treated with a second-generation TKI. Patients were enrolled under the GEMINI protocol (PA13-0589) approved by the MD Anderson Institutional Review Board.

[0270] Retrospective analysis of ORR and duration of treatment with afatinib. Uncommon EGFR Database 39Responses to afatinib and duration of afatinib treatment were tabulated from 803 patients in the study. Objective response rates were reported for 529 patients. Patients were stratified by either structure-function-based or exon-based groups, and ORR was determined by counting the number of patients reported to have a complete or partial response. Statistical differences between subgroups (structure-based or exon-based) were determined using Fisher's exact test. Duration of treatment was provided to the Uncommon EGFR database for 746 patients. Patients were stratified by structure-function-based and exon-based groups, and median DoT was calculated using the Kaplan-Meier method. Statistical differences, hazard ratios, and p-values ​​for Kaplan-Meier plots were generated using GraphPad Prism software and the Mantel-Cox log-rank test. If a mutation was not explicitly stated (i.e., exon 19 mutation), those patients were excluded from the structure-function-based analysis but included in the exon-based analysis.

[0271] Retrospective analysis of PFS in patients with atypical mutations. There were 333 patients with NSCLC identified in the MD Anderson GEMINI database whose tumors expressed atypical mutations. Of these patients, 81 had received at least one EGFR tyrosine kinase inhibitor treatment and did not harbor exon 20 loop insertion mutations. Clinical parameters were extracted from the respective databases. PFS was calculated with respect to the first EGFR TKI received, including patients who had received prior chemotherapy. PFS was defined as the time from initiation of the first EGFR TKI to radiological progression or death. Median PFS was calculated using the Kaplan-Meier method, and hazard ratios and p-values ​​were calculated using the Mantel-Cox log-rank test.

[0272] Tables 8.1-8.4 show the EGFR mutations analyzed in the described studies and the subgroups assigned. The EGFR mutations of the present disclosure can be, but are not limited to, those listed in Table 8.1, Table 8.2, Table 8.3 or Table 8.4.

[0273] Table 8.1. List of classical-like EGFR mutations TIFF2024506839000035.tif172128

[0274] Table 8.2 List of PACC EGFR mutations TIFF2024506839000036.tif174128TIFF2024506839000037.tif111128

[0275] Table 8.3. List of exon 20 loop insertion EGFR mutations TIFF2024506839000038.tif119128

[0276] Table 8.4. List of T790M-like EGFR mutations TIFF2024506839000039.tif83128

[0277] Table 9. Patient characteristics TIFF2024506839000040.tif124160TIFF2024506839000041.tif236160TIFF2024506839000042.tif236160 TIFF2024506839000043.tif236160TIFF2024506839000044.tif236160TIFF2024506839000045.tif193160

[0278] All methods disclosed and claimed herein can be accomplished and carried out without undue experimentation in light of this disclosure. Although the compositions and methods of the present disclosure have been described in terms of specific embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that the same or similar results can be achieved by substituting certain chemically and physiologically related agents for the agents described herein. All such similar substitutes and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the claims.

[0279] 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. TIFF2024506839000046.tif194160TIFF2024506839000047.tif238160TIFF20245068390 00048.tif231160TIFF2024506839000049.tif231160TIFF2024506839000050.tif128160

Claims

1. (a) detecting an EGFR mutation in a cancer sample; and (b) subjecting the cancer sample to one of the following: (i) a P-loop αC-helix compression EGFR mutant cancer, wherein the EGFR mutation is G719A, G719A S768I, G719C S768I, G719S, G719S S768I, or S768I; (ii) The EGFR mutation is A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A D761Y, G719A L861Q, G719A R776C, G719S L861Q, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del insD S22R, S752_I759del V769M, S768I L858R, S768I L861Q, S768I P-loop αC-helix compression EGFR mutant cancers that are V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774; (iii) The EGFR mutation is A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, L858R Classic-like EGFR mutant cancers with R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M; (iv) T790M-like 3S EGFR mutant cancer, wherein the EGFR mutation is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M; (v) T790M-like 3R EGFR mutant cancer, wherein the EGFR mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q; (vi) The EGFR mutation is A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, N771dupN Exon 20ins-NL EGFR mutant cancer, where the mutation is G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP; or (vii) exon 20ins-FL EGFR mutant cancer, wherein the EGFR mutation is H773_V774 insNPH, H773_V774 insAH, H773dupH, V774_C775 insHV, or V774_C775 insPR. Classification stage 16. A method for classifying a cancer sample, comprising:

2. The method of claim 1, further comprising the steps of: (a) if the cancer sample is classified as a P-loop αC-helix compression EGFR mutant cancer, classifying the cancer sample as (i) sensitive to a second generation EGFR inhibitor, and (ii) insensitive to a third generation EGFR inhibitor; (b) classifying the cancer sample as sensitive to an EGFR inhibitor; (c) classifying the cancer sample as insensitive to an EGFR inhibitor; (d) if the cancer sample is classified as a classic-like EGFR mutant cancer sample, classifying the cancer sample as sensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for a mutation associated with EGFR exon 20; (e) if the cancer sample is classified as a T790M-like 3S EGFR mutant cancer, classifying the cancer sample as (i) sensitive to a third generation EGFR inhibitor; and (ii) insensitive to a first generation EGFR inhibitor or a second generation EGFR inhibitor; (f) if the cancer sample is classified as a T790M-like 3R EGFR mutant cancer, classifying the cancer sample as insensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, or a third generation EGFR inhibitor; (g) if the cancer sample is classified as an exon 20ins-NL EGFR mutant cancer, classifying the cancer sample as (i) sensitive to a second generation EGFR inhibitor, or an EGFR inhibitor specific for a mutation associated with EGFR exon 20; and (ii) insensitive to a third generation EGFR inhibitor; or (h) if the cancer sample is classified as an exon 20ins-FL EGFR mutant cancer, classifying the cancer sample as insensitive to a first generation EGFR inhibitor, a second generation EGFR inhibitor, and a third generation EGFR inhibitor.

3. 3. The method of claim 2, wherein the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20.

4. (a) A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, L858R detecting an EGFR mutation that is R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M; and (b) classifying the cancer sample as a classic-like EGFR mutant cancer.

16. A method for classifying a cancer sample, comprising:

5. The method of claim 4, further comprising classifying the cancer sample as sensitive to an EGFR inhibitor.

6. 6. The method of claim 5, wherein the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, a third generation EGFR inhibitor, or an EGFR inhibitor specific for mutations associated with EGFR exon 20.

7. (a) detecting an EGFR mutation in a cancer sample that is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M; and (b) classifying the cancer sample as a T790M-like 3S mutant cancer.

16. A method for classifying a cancer sample, comprising:

8. 8. The method of claim 7, further comprising classifying the cancer sample as (i) sensitive to an EGFR inhibitor, or (ii) insensitive to an EGFR inhibitor.

9. The method described in claim 8, wherein the EGFR inhibitor (i) is a third generation EGFR inhibitor, and / or the EGFR inhibitor (ii) is a first generation EGFR inhibitor or a second generation EGFR inhibitor.

10. (a) detecting an EGFR mutation in a cancer sample that is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q; and (b) classifying the cancer sample as a T790M-like 3R EGFR mutant cancer.

16. A method for classifying a cancer sample, comprising:

11. The method of claim 10, further comprising classifying the cancer sample as insensitive to an EGFR inhibitor.

12. 12. The method of claim 11, wherein the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, or a third generation EGFR inhibitor.

13. (a) A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, D770_N771 insY H773Y, N771dupN, and N771dupN in cancer samples. detecting an EGFR mutation that is G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP; and (b) classifying the cancer sample as an exon 20 loop proximal insertion EGFR mutant cancer.

16. A method for classifying a cancer sample, comprising:

14. 14. The method of claim 13, further comprising classifying the cancer sample as (i) sensitive to an EGFR inhibitor, or (ii) insensitive to an EGFR inhibitor.

15. The method described in claim 14, wherein the EGFR inhibitor (i) is a second-generation EGFR inhibitor or an EGFR inhibitor specific for a mutation associated with EGFR exon 20, and / or the EGFR inhibitor (ii) is a first-generation EGFR inhibitor or a third-generation EGFR inhibitor.

16. (a) detecting the following EGFR mutations in a cancer sample: H773_V774 insNPH, H773_V774 insAH, H773dupH, V774_C775 insHV, V774_C775 insPR; and (b) classifying the cancer sample as an exon 20 loop far-loop insertion EGFR mutant cancer.

16. A method for classifying a cancer sample, comprising:

17. 17. The method of claim 16, further comprising classifying the cancer sample as insensitive to an EGFR inhibitor.

18. 18. The method of claim 17, wherein the EGFR inhibitor is a first generation EGFR inhibitor, a second generation EGFR inhibitor, or a third generation EGFR inhibitor.

19. (a) A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, G719C in cancer samples. S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del detecting an EGFR mutation that is insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M; and (b) classifying the cancer sample as a P-loop αC-helix compression EGFR mutant cancer.

16. A method for classifying a cancer sample, comprising:

20. 20. The method of claim 19, further comprising classifying the cancer sample as (i) sensitive to an EGFR inhibitor, or (ii) insensitive to an EGFR inhibitor.

21. The method described in claim 20, wherein the EGFR inhibitor (i) is a second-generation EGFR inhibitor and / or the EGFR inhibitor (ii) is a third-generation EGFR inhibitor.

22. A method described in any one of claims 1 to 21, wherein the cancer sample is obtained from a subject.

23. 23. The method of claim 22, further comprising extracting tumor DNA from the cancer sample.

24. 24. The method of any one of claims 1 to 23, wherein (a) comprises sequencing tumor DNA from a cancer sample.

25. The method of any one of claims 1 to 24, wherein the cancer sample is a lung cancer sample.

26. 26. The method of claim 25, wherein the lung cancer sample is a non-small cell lung cancer sample.

27. ​​A pharmaceutical composition comprising an EGFR inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the EGFR inhibitor to a subject determined to have a classical-like EGFR mutation from analysis of tumor DNA from the subject.

28. EGFR inhibitors, (i) a first-generation EGFR inhibitor, a second-generation EGFR inhibitor, a third-generation EGFR inhibitor, or an EGFR exon 20-specific mutation-specific EGFR inhibitor; or (ii) erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, icotinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189, sutetinib, osimertinib, nazartinib, olmutinib, rocelitinib, nacotinib, lazertinib, WZ4002, almonertinib, fulmonertinib, abivertinib, alfultinib, mavereltinib, abivertinib, olafertinib, resibertinib, TAS 6417, AZ5104, TAK-788 (mobocertinib), or DZD9008; 28. The pharmaceutical composition of claim 27.

29. Classic-like EGFR mutations include A702T, A763insFQEA, A763insLQEA, D761N, E709A L858R, E709K L858R, E746_A750del A647T, E746_A750del L41W, E746_A750del R451H, Ex19del E746_A750del, K754E, L747_E749del A750P, L747_T751del L861Q, L833F, L833V, L858R, L858R A289V, L858R E709V, L858R L833F, L858R P100T, L858R P848L, and L858R 29. The pharmaceutical composition of any one of claims 27-28, wherein the amino acid sequence is R108K, L858R R324H, L858R R324L, L858R S784F, L858R S784Y, L858R T725M, L858R V834L, L861Q, L861R, S720P, S784F, S811F, or T725M.

30. A pharmaceutical composition comprising an EGFR inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the EGFR inhibitor to a subject determined to have a T790M-like 3S EGFR mutation from analysis of tumor DNA from the subject.

31. EGFR inhibitors, (i) It is a third-generation EGFR inhibitor; (ii) osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, WZ4002, almonertinib, fulmonertinib, abivertinib, alflutinib, mavereltinib, abivertinib, orafeltinib, or resibertinib; (iii) is neither a first-generation nor a second-generation EGFR inhibitor; (iv) Not erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, icotinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189, or stetinib; 31. The pharmaceutical composition of claim 30.

32. 32. The pharmaceutical composition of any one of claims 30 to 31, wherein the T790M-like 3S EGFR mutation is Ex19del T790M, Ex19del T790M L718V, Ex19del T790M G724S, G719A T790M, G719S T790M, H773R T790M, I744_E749del insMKK, L747_K754 delinsATSPE, L858R T790M L792H, L858R T790M V843I, L858R T790M, S768I T790M, or T790M.

33. A pharmaceutical composition comprising a tyrosine kinase inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the tyrosine kinase inhibitor to a subject determined to have a T790M-like 3R EGFR mutation from analysis of tumor DNA from the subject, wherein the tyrosine kinase inhibitor is not an EGFR inhibitor.

34. 34. The pharmaceutical composition of claim 33, wherein the tyrosine kinase inhibitor is a PKC inhibitor or an ALK inhibitor.

35. the PKC inhibitor is ruboxistaurin, midostaurin, sotrastaurin, chelerythrine, myabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostatin 1, or tamoxifen, and / or the ALK inhibitor is AZD3463, brigatinib, crizotinib, ceritinib, alectinib, lorlatinib, ensartinib, entrectinib, repotrectinib, verizatinib, arcotinib, folitinib, CEP-37440, TQ-B3139, PLB1003, TPX-0131, or ASP-3026; 35. The pharmaceutical composition of claim 34.

36. The pharmaceutical composition of any one of claims 33 to 35, wherein the T790M-like 3R EGFR mutation is Ex19del T790M C797S, Ex19del T790M L792H, G724S T790M, L718Q T790M, L858R T790M C797S, or L858R T790M L718Q.

37. A pharmaceutical composition comprising an EGFR inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the EGFR inhibitor to a subject determined to have an exon 20 loop proximal insertion EGFR mutation from analysis of tumor DNA from the subject.

38. EGFR inhibitors, (i) A second-generation EGFR inhibitor or an EGFR inhibitor specific for EGFR exon 20-associated mutations; (ii) afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189, stetinib, TAS 6417, AZ5104, TAK-788 (mobocertinib), or DZD9008; (iii) is not a first-generation or third-generation EGFR inhibitor; or (iv) is not erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, icotinib, osimertinib, nazartinib, olmutinib, loceritinib, nacotinib, lazertinib, WZ4002, almonertinib, fulmonertinib, abivertinib, alflutinib, mavereltinib, abivertinib, orafeltinib, or resibertinib; 38. The pharmaceutical composition of claim 37.

39. Exon 20 loop proximal insertion EGFR mutations were A767_V769dupASV, A767_S768insTLA, S768_D770dupSVD, S768_D770dupSVD L858Q, S768_D770dupSVD R958H, S768_D770dupSVD V769M, V769_D770insASV, V769_D770insGSV, V769_D770insGVV, V769_D770insMASVD, D770_N771insNPG, D770_N771insSVD, D770del insGY, D770_N771 insG, and D770_N771 insY 38. The pharmaceutical composition of any one of claims 36 to 37, wherein the amino acid sequence is H773Y, N771dupN, N771dupN G724S, N771_P772insHH, N771_P772insSVDNR, or P772_H773insDNP.

40. A pharmaceutical composition comprising an EGFR inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the EGFR inhibitor to a subject determined to have an exon 20 loop distal insertion EGFR mutation from analysis of tumor DNA from the subject.

41. EGFR inhibitors, (i) An EGFR inhibitor specific for EGFR exon 20-associated mutations; (ii) TAS 6417, AZ5104, TAK-788 (mobocertinib), or DZD9008; 41. The pharmaceutical composition of claim 40.

42. The pharmaceutical composition of any one of claims 40 to 41, wherein the exon 20 loop distal insertion EGFR mutation is H773_V774 insNPH, H773_V774 insAH, H773dupH, V774_C775 insHV, or V774_C775 insPR.

43. A pharmaceutical composition comprising an EGFR inhibitor for use in a method for treating a subject for cancer, the method comprising the step of administering an effective amount of the EGFR inhibitor to a subject determined to have a P loop αC helix compression EGFR mutation from analysis of tumor DNA from the subject.

44. EGFR inhibitors, (i) a first-generation EGFR inhibitor, a second-generation EGFR inhibitor, or an EGFR exon 20-specific EGFR inhibitor; (ii) erlotinib, gefitinib, AZD3759, sapatinib, lapatinib, tucatinib, icotinib, afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189, stetinib, TAS 6417, AZ5104, TAK-788 (mobocertinib), or DZD9008; (iii) is a second-generation EGFR inhibitor; (iv) afatinib, dacomitinib, neratinib, Tarlox-TKI, tarloxotinib, BDTX189; or stetinib; (v) is not a second-generation EGFR inhibitor; or (vi) Not afatinib, dacomitinib, neratinib, tarlox-TKI, tarloxotinib, BDTX189, or stetinib; 44. The pharmaceutical composition of claim 43.

45. P-loop αC-helix compacting EGFR mutations include A750_I759del insPN, E709_T710del insD, E709A, E709A G719A, E709A G719S, E709K, E709K G719S, E736K, E746_A750del A647T, E746_A750del R675W, E746_T751del insV S768C, Ex19del C797S, Ex19del G796S, Ex19del L792H, Ex19del T854I, G719A, G719A D761Y, G719A L861Q, G719A R776C, G719A S768I, and G719C S768I, G719S, G719S L861Q, G719S S768I, G724S, G724S Ex19del, G724S L858R, G779F, I740dupIPVAK, K757M L858R, K757R, L718Q, Ex19del, L718Q L858R, L718V, L718V L858R, L747_S752del A755D, L747P, L747S, L747S L858R, L747S V774M, L858R C797S, L858R L792H, L858R T854S, N771G, R776C, R776H, E709_T710del 45. The pharmaceutical composition of any one of claims 43 to 44, wherein the amino acid sequence is insD S22R, S752_I759del V769M, S768I, S768I L858R, S768I L861Q, S768I V769L, S768I V774M, T751_I759 delinsN, V769L, V769M, or V774M.

46. The pharmaceutical composition of any one of claims 27 to 45, wherein the subject has lung cancer.

47. 47. The pharmaceutical composition of claim 46, wherein the subject has non-small cell lung cancer.

Images