阅读说明：本技术 对携带酪氨酸激酶抑制剂抗性egfr突变的癌细胞具有抗肿瘤活性的化合物 (Compounds with antitumor activity against cancer cells carrying tyrosine kinase inhibitor resistant EGFR mutations ) 是由 J·罗比丘 M·尼尔森 J·V·海马赫 于 2020-04-16 设计创作，主要内容包括：本公开提供了通过施用第二代喹唑啉胺衍生物类酪氨酸激酶抑制剂来治疗确定具有奥希替尼抗性EGFR突变的患者的癌症的方法。(The present disclosure provides methods of treating cancer in patients determined to have an axitinib-resistant EGFR mutation by administering a second generation quinazolinamine derivative tyrosine kinase inhibitor.)

1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a quinazolinamine derivative Tyrosine Kinase Inhibitor (TKI), wherein the subject has been determined to have one or more Epidermal Growth Factor Receptor (EGFR) TKI resistant mutations.

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

3. The method of any one of claims 1-2, wherein the quinazolinamine derivative TKI is a covalently bound quinazolinamine TKI.

4. The method of claim 3, wherein the covalently bound quinazolinamine TKI is afatinib, tasotinib-TKI, dactinib, pelitinib, or alitinib.

5. The method of any one of claims 1-2, wherein the quinazolinamine derivative TKI is a non-covalently bound quinazolinamine TKI.

6. The method of claim 5, wherein the non-covalently bound quinazolinamine TKI is sapatinib, AZD3759, tilinib, TAK-285, or gefitinib.

7. The method of any one of claims 1-6, wherein the quinazolinamine derivative TKI is formulated as a tablet.

8. The method of any one of claims 1-7, wherein the one or more EGFR TKI-resistant mutations comprise a point mutation, insertion, and/or deletion of 1-18 nucleotides at exon 18, 19, 20, or 21.

9. The method of any one of claims 1-8, wherein the one or more EGFR TKI resistance mutation(s) comprise one or more point mutations, insertions and/or deletions of 3-18 nucleotides between amino acids 688-728 of exon 18.

10. The method of claim 9, wherein the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724.

11. The method of claim 9 or 10, wherein the one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and/or G724S.

12. The method of any one of claims 8-11, wherein the one or more EGFR exon 18 mutations comprise E709A, E790K, L718Q, L718V, G719A, G719S, S720, and/or G724S.

13. The method of any one of claims 1-12, wherein the one or more EGFR TKI resistance mutations comprises one or more point mutations, insertions and/or deletions of 3-18 nucleotides between amino acids 729-761 of exon 19.

14. The method of claim 13, wherein the one or more EGFR exon 19 mutations are located at one or more residues selected from I744, L747, a755, K757, and/or D761.

15. The method of claim 13 or 14, wherein the one or more EGFR exon 19 mutations comprise I744V, I744T L747S, L747FS, a755T, K757R, and/or D761N.

16. The method of any one of claims 1 to 15, wherein the one or more EGFR TKI resistance mutations comprises one or more point mutations, insertions and/or deletions of 3 to 18 nucleotides between amino acids 763 and 823 of exon 20.

17. The method of claim 16, wherein the one or more EGFR exon 20 mutations are located at one or more residues selected from a763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776.

18. The method of claim 16 or 17, wherein the one or more EGFR exon 20 mutations comprises D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, a763insLQEA, L792H, G796D, S784F, C775Y, and/or S811F.

19. The method of any one of claims 16-18, wherein the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, a763insLQEA, L792H, G796D, G796S, S784F, C775Y, and/or S811F.

20. The method of any one of claims 1 to 18, wherein the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions and/or deletions of 3 to 18 nucleotides between amino acids 824 and 875 of exon 21.

21. The method of claim 20, wherein the one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, L844, and L858.

22. The method of any one of claims 20 or 21, wherein the one or more EGFR exon 21 mutations may comprise L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and/or T854I.

23. The method of any one of claims 1-22, wherein the subject has been determined to have 2,3 or 4 EGFR TKI resistance mutations.

24. The method of any one of claims 1-23, wherein the subject has been previously administered a TKI.

25. The method of claim 24, wherein the subject is resistant to a previously administered TKI.

26. The method of claim 24 or 25, wherein the TKI is lapatinib, erlotinib, gefitinib, norcetinib, temotinib, nagutinib, axitinib, ibrutinib or azatinib.

27. The method of any one of claims 24-26, wherein the TKI is gefitinib, erlotinib, or axitinib.

28. The method of any one of claims 24-27, wherein the TKI is axitinib.

29. The method of any one of claims 1-28, wherein the one or more EGFR TKI resistance mutations is located at residues E709, L718, G719, G724, C797, V843, T854, L861, and/or L792.

30. The method of any one of claims 1-29, wherein the subject has been determined to be free of EGFR mutations at residues C797 or T790.

31. The method of any one of claims 1-30, wherein the subject is determined to be free of EGFR mutation at residue T790.

32. The method of any one of claims 1-29, wherein the subject has a T790 mutation.

33. The method of claim 32, wherein the subject has a T790 mutation in combination with at least one additional mutation.

34. The method of claim 31, wherein the subject is determined to have a mutation at residue C797.

35. The method of any one of claims 1-34, wherein the one or more EGFR TKI resistance mutations is selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid.

36. The method of any one of claims 1-35, wherein the one or more EGFR TKI resistance mutations are selected from the group consisting of L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

37. The method of any one of claims 1-36, wherein the subject is determined to have an EGFR TKI resistance mutation by analyzing a genomic sample from the patient.

38. The method of claim 37, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

39. The method of any one of claims 1-38, wherein the presence of the EGFR TKI resistance mutation is determined by nucleic acid sequencing or PCR analysis.

40. The method of any one of claims 1-39, wherein a quinazolinamine derivative TKI is administered orally.

41. The method of any one of claims 1-40, wherein the quinazolinamine derivative TKI is administered daily.

42. The method of claim 41, wherein the quinazolinamine derivative TKI is administered continuously.

43. The method of claim 42, wherein the quinazolinamine derivative TKI is administered in a 28 day cycle.

44. The method of any one of claims 1-43, further comprising administering an additional anti-cancer therapy.

45. The method of claim 44, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy.

46. The method of claim 44, wherein the quinazolinamine derivative TKI and/or the anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in a slow release agent, in a controlled release agent, in a delayed release agent, as a suppository or sublingually.

47. The method of claim 44, wherein administering the quinazolinamine derivative TKI and/or anti-cancer therapy comprises local, regional or systemic administration.

48. The method of claim 44, wherein the quinazolinamine derivative TKI and/or anti-cancer therapy is administered two or more times.

49. The method of any one of claims 1-48, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic cancer, glioma, sarcoma, carcinoma of the malignant epithelium, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, Lifamenil tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal gland tumor, osteosarcoma tumor, multiple neuroendocrine tumors type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, cervical cancer, or cervical cancer, Colon cancer, rectal cancer, or skin cancer.

50. The method of any one of claims 1-49, wherein the cancer is non-small cell lung cancer.

51. The method of any one of claims 1-50, wherein the patient is a human.

52. A pharmaceutical composition for a subject who has been determined to have one or more EGFR TKI resistance mutations, comprising a quinazolinamine derivative class TKI.

53. The composition of claim 52, wherein the composition is further defined as an oral composition.

54. The composition of claim 52 or 53, wherein the composition comprises 5-25mg of the quinazolinamine derivative TKI.

55. The composition of any one of claims 52-54, wherein the composition is formulated as a tablet.

56. The composition of any one of claims 52-55, wherein the one or more EGFR TKI-resistant mutations comprise a point mutation, insertion, and/or deletion of 1-18 nucleotides at exon 18, 19, 20, or 21.

57. The composition of any one of claims 52-56, wherein the subject has been determined to have 2,3, or 4 EGFR TKI resistance mutations.

58. The composition of any one of claims 52-57, wherein the one or more EGFR TKI resistance mutation is located at residue E709, L718, G719, G724, C797, V843, T854, L861, and/or L792.

59. The composition of any one of claims 52-58, wherein the subject has been determined to be free of EGFR mutations at residues C797 or T790.

60. The composition of any one of claims 52-59, wherein the one or more EGFR TKI resistance mutation is selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid.

61. The composition of any one of claims 52-59, wherein the one or more EGFR TKI resistance mutation is selected from the group consisting of L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

62. The composition of any one of claims 52-61, wherein the subject is being treated with an anti-cancer therapy.

63. A method of predicting the response of a subject having cancer to a quinazolinamine derivative type TKI alone or a combination of a quinazolinamine derivative type TKI and a second anticancer therapy, comprising detecting an EGFR TKI resistance mutation in a genomic sample obtained from the patient, wherein if the sample is positive for the presence of the EGFR TKI resistance mutation, the patient is predicted to have a good response to the quinazolinamine derivative type TKI alone or the combination of the quinazolinamine derivative type TKI and an anticancer therapy.

64. The method of claim 63, wherein the EGFR TKI resistance mutation is located at residue E709, L718, G719, G724, C797, V843, T854, L861, and/or L792.

65. The method of claim 63 or 64, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

66. The method of any one of claims 63-65, wherein the presence of a HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis.

67. The method of any one of claims 63-66, wherein the EGFR TKI resistance mutation is selected from the group consisting of G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid.

68. The method of any one of claims 63-67, wherein the EGFR TKI resistance mutation is selected from the group consisting of L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

69. The method of any one of claims 63-68, wherein a good response to the quinazolinamine derivative TKI alone or in combination with an anti-cancer therapy comprises reducing tumor size or burden, blocking tumor growth, reducing tumor-associated pain, reducing cancer-associated pathology, reducing cancer-associated symptoms, cancer non-progression, increasing disease-free interval, extending time to progression, inducing remission, reducing metastasis, or increasing patient survival.

70. The method of any one of claims 63-69, further comprising administering the quinazolinamine derivative class TKI alone or in combination with a second anticancer therapy to the patient predicted to respond well.

71. The method of any one of claims 63-70, wherein the quinazolinamine derivative TKI is administered orally.

72. The method of any one of claims 63-71, wherein the quinazolinamine derivative TKI is administered at a dose of 5-25 mg.

73. The method of any one of claims 63-72, wherein the quinazolinamine derivative TKI is formulated as a tablet.

1. Field of the invention

The present invention relates generally to the fields of molecular biology and medicine. In particular, it relates to methods of treating patients having tyrosine kinase inhibitor resistant EGFR mutations.

2. Description of the related Art

Approximately 10% of non-small cell lung cancers (NSCLC) have Epidermal Growth Factor Receptor (EGFR) mutations that result in increased sensitivity to Tyrosine Kinase Inhibitors (TKIs) such as gefitinib, erlotinib and ositinib. Recently, oxitinib was approved for first-line therapy of EGFR mutant NSCLC4, but primary resistance (de novo resistance) and acquired resistance remain therapeutic obstacles in many patients. A series of atypical and acquired EGFR mutations could potentially lead to ocitinib resistance. Studies have shown that these atypical and acquired resistance mutations alter the confirmation of the drug binding pocket, leading to altered binding affinity of the drug to the receptor, the pocket being in front of the oxitinib near the solvent. Thus, there is an unmet need for new therapies for treating resistant EGFR mutant cancers.

Background

SUMMARY

Embodiments of the present disclosure provide methods and compositions for treating cancer in patients with resistant EGFR mutations. In a first embodiment, there is provided a method of treating cancer in a subject comprising administering to the subject an effective amount of a quinazolinamine derivative Tyrosine Kinase Inhibitor (TKI), wherein the subject has been determined to have one or more Epidermal Growth Factor Receptor (EGFR) TKI resistant mutations. In some aspects, the patient is a human.

In certain aspects, the quinazolinamine derivative TKIs a covalently bound quinazolinamine TKI. In some aspects, the covalently bound quinazolinamine TKI is afatinib, tasoxotinib (tarloxotinib) -TKI, dacatinib, pelitinib, or alitinib (allitinib). In some aspects, the quinazolinamine derivative TKIs a non-covalently bound quinazolinamine TKI. In some aspects, the non-covalently bound quinazolinamine TKI is sapatinib (sapatinib), AZD3759, tilinib (Varlitinib), TAK-285, or gefitinib. In certain aspects, the quinazolinamine derivative TKI is formulated as a tablet.

In certain aspects, the one or more EGFR TKI resistance mutations comprise a point mutation, insertion, and/or deletion of 1-18 nucleotides at exon 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 688-728 of exon 18. In particular aspects, the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. In particular aspects, the one or more EGFR exon 18 mutations comprise E709A, E790K, L718Q, L718V, G719A, G719S, S720P, and/or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 729-761 of exon 19. In certain aspects, the one or more EGFR exon 19 mutations are located at one or more residues selected from I744, L747, a755, K757, and/or D761. In particular aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, a755T, K757R, and/or D761N. In some aspects, the one or more EGFR TKI resistance mutation comprises one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 763-823 of exon 20. In certain aspects, the one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of a763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. In some aspects, the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, a763insLQEA, L792H, G796D, G796S, S784F, C775Y, and/or S811F. In certain aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 824-875 of exon 21. In particular aspects, the one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, L844, and L858. In some aspects, the one or more EGFR exon 21 mutations may comprise L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and/or T854I. In some aspects, the subject has been determined to have 2,3, or 4 EGFR TKI resistance mutations. In certain aspects, the one or more EGFR TKI resistance mutations are located at residues E709, L718, G719, G724, C797, V843, T854, L861, and/or L792. In some aspects, the subject has been determined to be free of EGFR mutations at residues C797 or T790. In particular aspects, the subject is determined to be free of EGFR mutation at residue T790. In other aspects, the subject is determined to have a T790 mutation alone or a combination of a T790 mutation and another mutation. In certain aspects, the subject is determined to have a mutation at residue C797. In some aspects, the one or more EGFR TKI resistance mutations are selected from G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid. In particular aspects, the one or more EGFR TKI resistance mutations are selected from L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

In some aspects, the subject has been previously administered a TKI. In certain aspects, the subject is resistant to a previously administered TKI. In some aspects, the TKI is lapatinib, afatinib, dacatinib, axitinib, ibrutinib, azatinib (nazatinib), omotinib, rocitinib (rociletinib), nanocitinib (naquotinib), or lenatinib. In particular aspects, the TKI is oxitinib, ibrutinib, azatinib, omotinib, norcetinib, or nautitinib. In a particular aspect, the TKI is ocitinib.

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

In some aspects, the quinazolinamine derivative TKI is administered orally. In certain aspects, the quinazolinamine derivative TKI is administered daily. In particular aspects, the quinazolinamine derivative TKI is administered continuously. In some aspects, the quinazolinamine derivative TKI is administered in a 28 day cycle.

In further aspects, the method further comprises administering an additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In particular aspects, the bosutinib (poziotiib) and/or the anticancer therapy are administered intravenously, subcutaneously, intraosseously, orally, transdermally, in a sustained release formulation, in a controlled release formulation, in a delayed release formulation, as a suppository or sublingually. In some aspects, administering bosutinib and/or the anti-cancer therapy comprises local (local), regional (regional) or systemic administration. In particular aspects, the bosutinib and/or the anti-cancer therapy is administered two or more times.

In some aspects, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma of the epithelium (carcinoma), lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, islet cell cancer, li fanmanic tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma tumor, multiple neuroendocrine tumors type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In a particular aspect, the cancer is non-small cell lung cancer.

Also provided herein are pharmaceutical compositions comprising a quinazolinamine derivative class TKI for subjects who have been determined to have one or more EGFR TKI resistance mutations. In some aspects, the composition is further defined as an oral composition. In certain aspects, the composition comprises 5-25mg of the quinazolinamine derivative TKI. In some aspects, the composition is formulated as a tablet. In some aspects, the subject is being treated with an anti-cancer therapy.

In certain aspects, the one or more EGFR TKI resistance mutations comprise a point mutation, insertion, and/or deletion of 1-18 nucleotides at exon 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutation(s) comprise one or more point mutations, insertions and/or deletions of 3-18 nucleotides between amino acids 688-728 of exon 18. In particular aspects, the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. In particular aspects, the one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and/or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 729-761 of exon 19. In certain aspects, the one or more EGFR exon 19 mutations are located at one or more residues selected from I744, L747, a755, K757, and/or D761. In particular aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, a755T, K757R, and/or D761N. In some aspects, the one or more EGFR TKI resistance mutation comprises one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 763-823 of exon 20. In certain aspects, the one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of a763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. In some aspects, the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, a763insLQEA, L792H, G796D, S784F, C775Y, and/or S811F. In certain aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 824-875 of exon 21. In particular aspects, the one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, L844, and L858. In some aspects, the one or more EGFR exon 21 mutations may comprise L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and/or T854I. In some aspects, the subject has been determined to have 2,3, or 4 EGFR TKI resistance mutations. In certain aspects, the one or more EGFR TKI resistance mutations are located at residues E709, L718, G719, G724, C797, V843, T854, L861, and/or L792. In some aspects, the subject has been determined to be free of EGFR mutations at residues C797 or T790. In particular aspects, the subject is determined to be free of EGFR mutation at residue T790. In other aspects, the subject is determined to have a T790 mutation alone or a combination of a T790 mutation and another mutation. In certain aspects, the subject is determined to have a mutation at residue C797. In some aspects, the one or more EGFR TKI resistance mutations are selected from G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid. In particular aspects, the one or more EGFR TKI resistance mutations are selected from L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

Another embodiment provides a method of predicting the response of a subject with cancer to a quinazolinamine derivative TKI alone or in combination with a second anticancer therapy, comprising detecting an EGFR TKI resistance mutation in a genomic sample obtained from the patient, wherein if the sample is positive for the presence of the EGFR TKI resistance mutation, the patient is predicted to respond well to the quinazolinamine derivative TKI alone or in combination with the anticancer therapy. In some aspects, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain aspects, the presence of a HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis.

In certain aspects, the one or more EGFR TKI resistance mutations comprise a point mutation, insertion, and/or deletion of 1-18 nucleotides at exon 18, 19, 20, or 21. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 688-728 of exon 18. In particular aspects, the one or more EGFR exon 18 mutations are located at one or more residues selected from the group consisting of E709, L718, G719, S720, and G724. In particular aspects, the one or more EGFR exon 18 mutations comprise E709A, L718Q, L718V, G719A, G719S, S720P, and/or G724S. In some aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 729-761 of exon 19. In certain aspects, the one or more EGFR exon 19 mutations are located at one or more residues selected from I744, L747, a755, K757, and/or D761. In particular aspects, the one or more EGFR exon 19 mutations comprise I744V, I744T, L747S, L747FS, a755T, K757R, and/or D761N. In some aspects, the one or more EGFR TKI resistance mutation comprises one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 763-823 of exon 20. In certain aspects, the one or more EGFR exon 20 mutations are located at one or more residues selected from the group consisting of a763, S768, V769, H773, D770, V774, C775, S784, L792, G796, C797, S811, and R776. In some aspects, the one or more EGFR exon 20 mutations comprise D770insNPG, S784F, R776C, S768I, V774M, S768I, H773insAH, H773insNPH, V774A, V769L, V769M, S768dupSVD, a763insLQEA, L792H, G796D, S784F, C775Y, and/or S811F. In certain aspects, the one or more EGFR TKI resistance mutations comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 824-875 of exon 21. In particular aspects, the one or more EGFR exon 21 mutations are located at one or more residues selected from the group consisting of L833, V834, G836, V843, T854, L861, L862, L844, and L858. In some aspects, the one or more EGFR exon 21 mutations may comprise L833F, V834L, L858R, L861Q, V843I, L861R, L862V, L844V, L861Q, G836S, and/or T854I. In some aspects, the subject has been determined to have 2,3, or 4 EGFR TKI resistance mutations. In certain aspects, the one or more EGFR TKI resistance mutations are located at residues E709, L718, G719, G724, C797, V843, T854, L861, and/or L792. In some aspects, the subject has been determined to be free of EGFR mutations at residues C797 or T790. In particular aspects, the subject is determined to be free of EGFR mutation at residue T790. In other aspects, the subject is determined to have the T790 mutation alone or a combination of the T790 mutation and another mutation. In certain aspects, the subject is determined to have a mutation at residue C797. In some aspects, the one or more EGFR TKI resistance mutations are selected from G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid. In particular aspects, the one or more EGFR TKI resistance mutations are selected from L861Q, G719S, L858R/L792H, L858R/C797S, and Ex19 del/C797S.

Another embodiment provides a favorable response to a quinazolinamine derivative TKI, alone or in combination with an anti-cancer therapy, including reducing tumor size or burden, blocking tumor growth, reducing tumor-associated pain, reducing cancer-associated pathology, reducing cancer-associated symptoms, cancer progression-free, increasing disease-free interval, extending time to progression, inducing remission, reducing metastasis, or increasing patient survival.

In further aspects, the methods further comprise administering to the patient predicted to have a good response a quinazolinamine derivative-based TKI, alone or in combination with a second anticancer therapy. In some aspects, the quinazolinamine derivative TKI is administered orally. In certain aspects, the quinazolinamine derivative TKI is administered at a dose of 5-25 mg. In some aspects, the quinazolinamine derivative TKI is formulated as a tablet.

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

Brief description of the drawings

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

FIGS. 1A-1D: among the atypical EGFR mutations in vitro, second generation quinazolinyl TKIs are more potent and selective than third generation TKIs such as oxitinib. (FIG. 1A) Log IC of Ba/F3 cells expressing primary (primary) atypical mutations spanning exons 18-21 were treated with the indicated inhibitors for 72 hours₅₀Heat map of values. Mutations are sequenced from top to bottom from maximum resistance to most sensitivity, and drugs from lowest IC₅₀Value to highest IC₅₀The values are ordered from left to right. (FIG. 1B) IC of Ba/F3 cells expressing a primary atypical mutation spanning exons 18-21₅₀Value divided by IC of WT EGFR (+10ng/ml EGF) expressing Ba/F3 cells treated with the indicated inhibitors for 72 hours₅₀Heat map of ratio of values. The mutation order remained the same as panel a, but the drugs were reordered from left to right from maximum to minimum selectivity. (FIG. 1C) IC of atypical mutant Ba/F3-expressing cells₅₀Mean of values ± bar graph of SEM, separated by drug class. N-40 cell lines and the symbol represents each cell line. Statistical significance was determined by ANOVA. (FIG. 1D) mean + -SEM bar graph of mutant/WT EGFR ratio for atypical mutant Ba/F3-expressing cells, separated by drug class. N-40 cell lines, symbols represent each cell line. Statistical significance was determined by ANOVA.

FIGS. 2A-2D: the P-loop exon 18 mutation results in primary resistance to ocitinib in vivo, but does not result in resistance to other EGFR TKIs. (FIG. 2A) PDX model of NSCLC harboring an EGFR exon 18P-loop mutation (G719A) tumor growth curves treated with indicated inhibitors for 28 days. (FIG. 2B) mean volume percent change in G719A tumors at the end of the 28 day experiment with indicated inhibitor. + -. bar graph of SEM. symbols represent individual mice.A significant difference was determined by ANOVA and Tukey assays for multiple comparisons (FIG. 2C) tumor growth curves for 28 days with indicated inhibitor for NSCLC PDX models with non-P-loop exon 18EGFR mutation (E709K L858R) (FIG. 2D) mean volume percent change in E709K/L858R tumors at the end of the 28 day experiment with indicated inhibitor. + -. bar graph of SEM. symbols represent individual mice.

FIGS. 3A-3D: acquired atypical mutations drive resistance to oxitinib, but are sensitive to quinazoline TKI, and drug sensitivity/resistance to concurrent mutations may be driven by primary mutations. (FIG. 3A) Log IC of Ba/F3 cells expressing acquired atypical mutations spanning exons 18-21 for 72 hours treated with the indicated inhibitors₅₀Heat map of values. Mutations are sequenced from top to bottom from maximum resistance to most sensitivity, and drugs from lowest IC₅₀Value to highest IC₅₀The values are ordered from left to right. (FIG. 3B) IC of Ba/F3 cells expressing acquired atypical mutations spanning exons 18-21₅₀Value divided by IC of WT EGFR (+10ng/ml EGF) expressing Ba/F3 cells treated with the indicated inhibitors for 72 hours₅₀Heat map of ratio of values. The mutation sequence remained the same as panel a, with the drugs re-ordered from left to right from maximum to minimum selectivity. (FIGS. 3C-D) Bar graphs of mutant/WT ratios for different drug classes with primary mutations Ex19del (FIG. 3C) and L858R (FIG. 3D) and without primary mutations. For individual primary mutations (open bars), the symbols represent the mutant/WT average ratio for each drug, while for primary mutations + atypical mutations (solid bars), the symbols represent the mutant/WT average ratio for all drugs in the indicated drug class for each mutation. Due to the sample size asymmetry, statistical differences were determined using a non-parametric student's t-test.

FIGS. 4A-4D: whether primary (primary) or tertiary (tertiary) mutation, the T790M mutation drives resistance to first and second generation drugs, but unique third generation TKI and drug re-targeting can overcome tertiary T790M positive resistance. (FIG. 4A) Log IC of Ba/F3 cells expressing EGFR mutations coincident with T790M mutations and treated with the indicated typical EGFR inhibitors for 72 hours₅₀Heat map of values. Mutations are sequenced from top to bottom from maximum resistance to most sensitivity, and drugs from lowest IC₅₀Value to highest IC₅₀The values are ordered from left to right.(FIG. 4B) IC of Ba/F3 cells expressing EGFR mutation Simultaneous with T790M mutation₅₀Value divided by IC of WT EGFR (+10ng/ml EGF) expressing Ba/F3 cells treated with the indicated typical EGFR inhibitor for 72 hours₅₀Heat map of ratio of values. The order of mutations remained the same as panel a, but for typical EGFR inhibitors, the drugs were reordered from left to right from maximum selectivity to minimum selectivity. (FIG. 4C) Log IC of Ba/F3 cells expressing EGFR mutation coincident with T790M mutation and treated with indicated reutilizing inhibitor for 72 hours₅₀Heat map of values. The mutation sequence remained the same as Panel A and the drug was taken from the lowest IC₅₀Value to highest IC₅₀The values are arranged from left to right. (FIG. 4D) IC of Ba/F3 minicells expressing EGFR mutations co-occurring with the T790M mutation₅₀The values were divided by the IC of Ba/F3 cells expressing WTFEGFR (+10ng/ml EGF) treated with the indicated reuptake inhibitors for 72 hours₅₀Heat map of ratio of values. The mutation order remained the same as panel a, but the drugs were reordered from left to right from maximum to minimum selectivity.

Description of the exemplary embodiments

The present study identified ositinib-resistant EGFR mutations throughout various malignancies (e.g., NSCLC). Drug susceptibility of resistant mutations to TKI was evaluated systematically. Resistant EGFR mutations were found to be sensitive to the second generation quinazolinamine derivative class TKI.

Accordingly, certain embodiments of the present disclosure provide methods for treating cancer patients having an axitinib-resistant EGFR mutation. In particular, the methods of the invention comprise administering a second generation quinazolinamine derivative-based TKI to a patient identified as having one or more oxitinib-resistant EGFR mutations, e.g., exon 18, 19, 20, or 21 mutations, such as acquired atypical and/or typical mutations. The size and flexibility (flexibility) of TKI of the second generation quinazoline amine derivative overcomes steric hindrance, so that EGFR mutants are inhibited at low nanomolar concentrations. Therefore, the second generation quinazolinamine derivative TKI is a potent EGFR inhibitor useful for targeting the esitinib resistant EGFR mutation.

I. Definition of

As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, the words "a" or "an" when used in conjunction with the word "comprising" may mean one or more.

The use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive, but the present disclosure supports the definition of alternatives and "and/or" only. As used herein, "another" may mean at least a second/second or more/more.

As used herein, "substantially free" with respect to specified components is used herein to mean that none of the specified components are intentionally formulated into the composition and/or are present only as contaminants or in trace amounts. The total amount of the specified components resulting from any unwanted contamination of the composition is typically below 1%, more preferably below 0.1%, and still more preferably below 0.01% of the composition. Most preferred are compositions wherein no measurable amount of a given component is detectable by standard analytical methods.

The term "predominantly" should be understood to mean that a method or composition includes only the specified steps or materials, as well as those materials or steps that do not materially affect the basic and novel characteristics of such methods and compositions.

The term "substantially free" is used to indicate that 98% of the listed components and less than 2% of the composition or particle are substantially free of components.

The terms "substantially" or "approximately" as used herein may be used to modify any quantitative comparison, value, measurement, or other representation that may be permissive for change without resulting in a change in the basic function to which it is related.

The term "about" generally means within a standard deviation of the stated value as determined using standard analytical techniques for measuring the stated value. The term may also be used with ± 5% of the stated value.

"treating" or "treating" includes (1) inhibiting the disease (e.g., arresting further development of pathology and/or symptomatology) in a subject or patient experiencing or exhibiting disease pathology or symptomatology, (2) ameliorating the disease (e.g., reversing pathology and/or symptomatology) in a subject or patient experiencing or exhibiting disease pathology or symptomatology, and/or (3) achieving any measurable reduction in the disease in a subject or patient experiencing or exhibiting disease pathology or symptomatology. For example, the treatment may comprise administering an effective amount of a TKI of the second generation quinazolinamine derivative class.

"prophylactic treatment" includes: (1) reducing or alleviating the risk of developing a disease in a subject or patient who may be at risk for the disease and/or who is predisposed to the disease but does not yet experience or exhibit any or all of the pathologies or symptomatologies of the disease, (2) slowing the onset of a disease pathology or symptomatology in a subject or patient who may be at risk for the disease and/or who is predisposed to the disease but does not yet experience or exhibit any or all of the pathologies or symptomatologies of the disease.

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

The term "effective" as used in the specification and/or claims means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" when used in the context of treating a patient or subject with a compound refers to an amount of the compound sufficient to effect such treatment or prevention of a disease when the compound is administered to the subject or patient to treat or prevent the disease.

As used herein, the term "IC₅₀"refers to 50% of the inhibitory dose to obtain maximal response. Such quantitative measurements indicate how much of a particular drug or other substance (inhibitor) is needed to carry out a given biological, biochemical or chemical process (or processA component of process, i.e., an enzyme, cell receptor, or microorganism) inhibits half.

An "anti-cancer" agent can adversely affect a cancer cell/tumor in a subject, for example, by promoting cancer cell killing, inducing cancer cell apoptosis, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cells, promoting an immune response against a cancer cell or tumor, arresting or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

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

"hybridization" or "hybridization" refers to the binding between nucleic acids. Hybridization conditions may vary depending on the sequence homology of the nucleic acids to be bound. Therefore, if the sequence homology between the subject nucleic acids is high, stringent conditions are used. If the sequence homology is low, mild conditions are used. When hybridization conditions are stringent, hybridization specificity increases, and this increase in hybridization specificity results in a decrease in the yield of non-specific hybridization products. However, under mild hybridization conditions, the hybridization specificity decreases, and this decrease in hybridization specificity results in an increase in the yield of non-specific hybridization products.

"Probe" or "probes" refers to a polynucleotide that is at least eight (8) nucleotides in length and forms a hybrid structure with a target sequence due to the complementarity of at least one sequence in the probe with a sequence in the target region. The polynucleotide may comprise DNA and/or RNA. In certain embodiments, the probe is detectably labeled. The size of the probes can vary widely. Typically, probes are, for example, at least 8-15 nucleotides in length. Other probes are, for example, at least 20, 30 or 40 nucleotides in length. Still other probes are somewhat longer, e.g., at least 50, 60, 70, 80, or 90 nucleotides in length. The probe may also have any specific length falling within the aforementioned range. Preferably, the probe does not contain a sequence complementary to a sequence used to prime the target sequence during the polymerase chain reaction.

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

"modified ribonucleotide" or deoxyribonucleotide refers to molecules that can be used to replace a naturally occurring base in a nucleic acid, including, but not limited to, modified purines and pyrimidines, rare bases, convertible nucleosides, structural analogs of purines and pyrimidines, labeled, derivatized and modified nucleosides and nucleotides, conjugated nucleosides and nucleotides, sequence modifications, end modifications, spacer modifications, and nucleotides having backbone modifications including, but not limited to, ribomodified nucleotides, phosphoramidates, phosphorothioates, phosphoramidites, methylphosphonates, methylphosphonites, phosphorodites, phosphorodithioates, phosphorodites, phosphoroamidites, phosphorodites, phosphoroamidites, phosphoroamidates, phosphoroamidites, phosphoroamidates, phosphoroamidites, phosphoroamidates, and other molecules, and the like, Peptide nucleic acids, achiral and neutral internucleotide linkages (intemucleotide).

"variant" refers to a polynucleotide or polypeptide that differs by the exchange, deletion, or insertion of one or more nucleotides or amino acids, relative to the wild type or the most prevalent form in a population of subjects, respectively. The number of nucleotides or amino acids exchanged, deleted or inserted may be 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, for example 25, 30, 35, 40, 45 or 50.

"primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be a DNA oligonucleotide, an RNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic or modified nucleotides. The upper and lower limits of primer length are empirically determined. The lower limit of the primer length is the minimum length required to form a stable duplex upon hybridization to a target nucleic acid under nucleic acid amplification reaction conditions. Under such hybridization conditions, very short primers (typically less than 3-4 nucleotides in length) will not form thermodynamically stable duplexes with the target nucleic acid. The upper limit is generally determined by the likelihood of duplex formation in regions of the target nucleic acid other than the predetermined nucleic acid sequence. Generally, suitable primer lengths range from about 10 to about 40 nucleotides in length. In certain embodiments, for example, the length of the primer may be 10-40, 15-30, or 10-20 nucleotides in length. When placed under appropriate conditions, the primer is capable of acting as a point of initiation of synthesis on the polynucleotide sequence.

"detect", "detectable" and grammatical equivalents thereof refer to a means of determining the presence and/or quantity and/or identity of a target nucleic acid sequence. In some embodiments, the detection occurs upon amplification of the target nucleic acid sequence. In other embodiments, sequencing of a target nucleic acid can be characterized as "detecting" the target nucleic acid. Labels attached to the probes may include any of a variety of different labels known in the art that are detectable, for example, chemically or physically. Labels that can be attached to the probes can include, for example, fluorescent materials and luminescent materials.

"amplifying", "amplifying" and grammatical equivalents thereof refer to any method of replicating at least a portion of a target nucleic acid sequence in a template-dependent manner, including, but not limited to, a number of techniques for amplifying nucleic acid sequences in a linear or exponential manner. Exemplary means for performing the amplification step include Ligase Chain Reaction (LCR), Ligase Detection Reaction (LDR), post-ligation Q-replicase amplification, PCR, primer extension, Strand Displacement Amplification (SDA), hyperbranched (hyperbranched) strand displacement amplification, Multiple Displacement Amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplification (two-step multiplexed amplification), Rolling Circle Amplification (RCA), recombinase-polymerase amplification (RPA) (twist dx, Cambridg, UK), and self-sustained sequence replication (3SR), including multiplexed forms or combinations thereof, such as, but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/LDR, LCR/PCR, PCR/CCR (also known as combined chain reaction-CCR), and the like. A description of such techniques can be found, inter alia, in Sambrook et al, molecular Cloning,3^rdEdition (molecular cloning, third Edition).

As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

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

Resistant EGFR mutations

Certain embodiments of the present disclosure relate to determining whether a subject has one or more axitinib-resistant EGFR mutations, e.g., exon 18, 19, 20, or 21 mutations. The subject may have 2,3, 4, or more EGFR exon 20 mutations. The one or more EGFR mutations can be at one or more residues selected from the group consisting of E709, L718, G719, G724, C797, V843, T854, L861, and L792. The one or more EGFR mutations may be G719X, E709X, G724S, L718X, L861Q, T8541, V8431, C797S, and/or L792X, wherein X is any amino acid. Methods of mutation detection are known in the art and include PCR analysis and nucleic acid sequencing as well as FISH and CGH. In particular aspects, EGFR mutations are detected by DNA sequencing, e.g., from tumor DNA or circulating free DNA from plasma.

EGFR exon 18 mutations may comprise one or more point mutations, insertions and/or deletions of 3-18 nucleotides between amino acids 688-728 of exon 18, which deletions are amino acid in-frame deletions. The one or more EGFR exon 18 mutations may be located at one or more residues selected from the group consisting of E709, L718, G719, S720 and G724. The one or more EGFR exon 18 mutations may comprise E709A, L718Q, L718V, G719A, G719S, S720P, and G724S.

EGFR exon 19 mutations may be 3-18 nucleotides at one or more points of mutation, insertion and/or deletion between amino acids 729-761 of exon 19, which is an amino acid in-frame deletion. The one or more EGFR exon 19 mutations may be located at one or more residues selected from I744, L747, a755, K757, and/or D761. The one or more EGFR exon 19 mutations may comprise I744V, I744T L747S, L747FS, a755T, K757R, and/or D761N.

EGFR exon 20 mutations may comprise one or more point mutations, insertions, and/or deletions of 3-18 nucleotides between amino acids 763-823. The one or more EGFR exon 20 mutations may be located at one or more residues selected from G724, a763, S768, V769, H773, V774, C775, S784, G796, C797, S811, and R776. The one or more EGFR exon 20 mutations may comprise S784F, R776C, S768I, V774M, S768I, H773insAH, V774A, V769M, S768dupSVD, a763insLQEA, G796D, S784F, C775Y, and/or S811F.

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

In some aspects, the subject may have or develop a mutation at EGFR residue C797 that may result in resistance to TKIs, e.g., TKIs of the second generation quinazolinamine derivative class. Thus, in certain aspects, the subject is determined to have no mutation at EGFR C797 and/or T790, e.g., C797S and/or T790M. In some aspects, oxitinib may be administered to a subject having a T790 mutation, e.g., T790M, and chemotherapy and/or radiation therapy may be administered to a subject having a C797 mutation, e.g., C797S. For example, if C797S was obtained in the context of typical EGFR mutations (e.g., L858R or exon 19 deletions) while T790M was absent, these mutations may be sensitive to quinazolinamine TKIs. However, if the C797S mutation is obtained along with the T790M mutation or exon 20 insertion mutation, these mutations may be resistant to quinazolinamine TKIs. Furthermore, if the T790M mutation is obtained with a canonical mutation (e.g., L858R or exon 19 deletion), these mutations may be resistant to quinazolinamine TKIs, but not to oxitinib. Furthermore, in vitro, when the T790M mutation was obtained together with the exon 18 point mutation (G719X/T790M), these mutations appeared to be sensitive to quinazolinamine TKI. In some aspects, the L858R/C797S, Ex19del/C797S, or G719X/T790M mutants are sensitive to quinazolinamine TKI. However, in certain aspects, the L858R/T790M/C797S, exon 19 deletion/T790M/C797 s, and exon 20 insertion + C797S or T790M mutants are resistant to EGFR TKI.

The patient sample can be any body tissue or fluid that includes nucleic acid from a subject's lung cancer. In certain embodiments, the sample is a blood sample comprising circulating tumor cells or cell-free DNA. In other embodiments, the sample may be a tissue, such as lung tissue. The lung tissue may be from tumor tissue and may be freshly frozen or Formalin Fixed Paraffin Embedded (FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

Samples suitable for use in the methods described herein contain genetic material, such as genomic dna (gdna). Genomic DNA is typically extracted from biological samples such as blood or mucosal scrapings (scrapings) of the inner walls of the mouth, but may also be extracted from other biological samples including urine, tumors or cough (expecterants). The sample itself typically comprises nucleated cells (e.g., blood cells or buccal cells) or tissue removed from the subject, including normal tissue or tumor tissue. Methods and reagents for obtaining, processing and analyzing samples are known in the art. In some embodiments, the sample is obtained with the aid of a healthcare provider, e.g., drawing blood. In some embodiments, the sample is obtained without the assistance of a healthcare provider, e.g., in the case of a non-invasive sample, such as a sample containing buccal cells obtained using a buccal swab or brush, or a mouthwash sample.

In some cases, biological samples may be treated for DNA isolation. For example, DNA in a cell sample or tissue sample can be separated from other components of the sample. Cells can be harvested from a biological sample using standard techniques known in the art. For example, cells can be harvested by centrifuging a cell sample and resuspending the pelleted cells. The cells may be resuspended in a buffer solution, such as Phosphate Buffered Saline (PBS). After centrifugation of the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, such as gDNA. See, e.g., Ausubel et al (2003). The sample may be concentrated and/or purified to isolate the DNA. All samples obtained from the subject, including those that have undergone any type of further processingAre considered to have been obtained from the subject. Genomic DNA can be extracted from a biological sample using conventional methods, including, for example, phenol extraction. Or may use a technique such asTissue kit (Qiagen, Chatsworth, Calif.) andgenomic DNA is extracted using a genomic DNA purification kit (Promega). Non-limiting examples of sample sources include urine, blood, and tissue.

Methods known in the art can be used to determine whether a resistant EGFR mutation described herein is present. For example, gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and/or arrays may be used to detect the presence or absence of an insertion mutation. Amplification of the nucleic acid can be accomplished using methods known in the art, such as PCR, if desired. In one example, a sample (e.g., a sample comprising genomic DNA) is obtained from a subject. The DNA in the sample is then examined to determine the identity of the insertional mutation described herein. Insertional mutations can be detected by any of the methods described herein, for example by sequencing, or by hybridizing genes in genomic DNA, RNA or cDNA to nucleic acid probes, for example DNA probes (which include cDNA and oligonucleotide probes) or RNA probes. Nucleic acid probes can be designed to specifically or preferentially hybridize to a particular variant.

A set of probes generally refers to a set of primers, typically primer pairs, for detecting a target genetic variation (e.g., EGFR mutation) used in the operable treatment recommendations of the present disclosure, and/or detectably labeled probes. Primer pairs are used in amplification reactions to define amplicons of the target genetic variation across each of the above genes. The set of amplicons is detected from a set of matched probes. In exemplary embodiments, the methods of the invention can use taqman (Roche Molecular Systems, Pleasanton, Calif.) assays for detecting a set of target genetic variations, such as EGFR mutations. In one embodiment, the set of probes is a set of primers for generating an amplicon that is detected by a nucleic acid sequencing reaction, such as a next generation sequencing reaction. In these embodiments, for example, AmpliSEQTM (Life Technologies/Ion Torrent, Carlsbad, Calif.) or TruSEQTM (Illumina, San Diego, Calif.) technology may be employed.

Analysis of nucleic acid markers can be performed using techniques known in the art, including but not limited to sequence analysis and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, 1992), solid phase sequencing (Zimmerman et al, 1992), mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, 1998), and sequencing by hybridization (Chee et al, 1996; Drmanac et al, 1993; Drmanac et al, 1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. In addition, next generation sequencing methods can be performed using commercially available kits and instruments from companies such as Life Technologies/Ion Torrent PGM or Proton, Illumina HiSEQ or MiSEQ, and Roche/454 next generation sequencing systems.

Other methods of nucleic acid analysis may include direct manual sequencing (Church and Gilbert, 1988; Sanger et al, 1977; U.S. Pat. No. 5,288,644); automatic fluorescence sequencing; single strand conformation polymorphism assay (SSCP) (Schafer et al, 1995); clamp Denaturing Gel Electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformation Sensitive Gel Electrophoresis (CSGE); denaturing Gradient Gel Electrophoresis (DGGE) (Sheffield et al, 1989); denaturing high performance liquid chromatography (DHPLC, Underhill et al, 1997); infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318); mobility change analysis (Orita et al, 1989); restriction enzyme analysis (Flavell et al, 1978; Geever et al, 1981); real-time quantitative PCR (Raca et al, 2004); heteroduplex analysis; chemical Mismatch Cleavage (CMC) (Cotton et al, 1985); rnase protection assays (Myers et al, 1985); using a polypeptide recognizing a nucleotide mismatch, such as the mutS protein of e.coli (e.coli); allele-specific PCR, and combinations of these methods. See, for example, U.S. patent publication No. 2004/0014095, which is incorporated herein by reference in its entirety.

In one example, a method of identifying an EGFR mutation in a sample comprises contacting nucleic acids from the sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding a mutant EGFR protein or a fragment thereof comprising a mutation, and detecting the hybridization. In particular embodiments, the probe is administered with, for example, a radioisotope(s) (ii)³H、³²P or³³P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent. In particular embodiments, the probe is an antisense oligomer, such as PNA, morpholino-phosphoramidate, LNA, or 2' -alkoxyalkoxy. Probes can be from about 8 nucleotides to about 100 nucleotides, or from about 10 to about 75, or from about 15 to about 50, or from about 20 to about 30 nucleotides. In another aspect, the probes of the present disclosure are provided in a kit for identifying EGFR mutations in a sample, the kit comprising an oligonucleotide that specifically hybridizes to or adjacent to a site of a mutation in the EGFR gene. The kit may also include instructions for using the second generation quinazolinamine derivative class TKIs to treat patients with tumors containing EGFR mutations based on the results of hybridization assays using the kit.

In another aspect, a method of detecting EGFR mutation in a sample comprises amplifying from the sample a nucleic acid corresponding to the EGFR gene, or fragment thereof, suspected of containing a mutation, and comparing the electrophoretic mobility of the amplified nucleic acid to the electrophoretic mobility of the corresponding wild-type EGFR gene, or fragment thereof. Differences in mobility indicate the presence of mutations in the amplified nucleic acid sequence. Electrophoretic mobility can be measured on polyacrylamide gels.

Alternatively, the nucleic acid can be analyzed using Enzymatic Mutation Detection (EMD) (Del Tito et al, 1998) to detect mutations. EMD Using phage resolvase T₄Endonuclease VII, which scans along double-stranded DNA until it detects and cleaves structural distortions caused by base pair mismatches resulting from point mutations, insertions and deletions. The presence of the mutation is indicated by detecting the two short fragments formed by cleavage by the resolvase, for example by gel electrophoresis. The advantage of the EMD method is that a single protocol is used to identify reactions directly from PCRThe point mutations, deletions and insertions determined eliminate the need for sample purification, thereby reducing hybridization time and increasing signal-to-noise ratio. Mixed samples containing up to 20-fold excess of normal DNA and fragments up to 4kb in size can be analyzed. However, EMD scanning does not identify the specific base change that occurred in the mutation-positive sample, and additional sequencing procedures are required to identify the mutation, if necessary. Similar resolvase T may be used as demonstrated in U.S. Pat. No. 5,869,245₄CEL I enzyme of endonuclease VII.

Methods of treatment

Also provided herein are methods of treating or delaying progression of cancer in an individual comprising administering to a subject determined to have a resistant EGFR mutation an effective amount of a second generation quinazolinamine derivative TKI or a structurally similar inhibitor. The subject may have more than one EGFR mutation.

Examples of cancers that are expected to be treated include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-lung cancer lesions, colon cancer, melanoma, and bladder cancer. In a particular aspect, the cancer is non-small cell lung cancer.

In some embodiments, the subject is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disorder described herein). In one embodiment, the subject is in need of an enhanced immune response. In certain embodiments, the subject is, or is at risk of, impaired immune function. For example, the subject is receiving or has received chemotherapy treatment and/or radiation therapy. Alternatively, or in combination, the subject is suffering from or at risk of suffering from impaired immune function due to infection.

Certain embodiments relate to administering a second generation quinazolinamine derivative class TKI to a subject determined to have an axitinib resistant EGFR mutation. The second generation quinazolinamine derivative TKIs may be administered orally, for example in tablet form. The second generation quinazolinamine derivative TKI may be administered in a dose of 4-25mg, for example in a dose of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 mg. Administration may be daily, every other day, every 3 days, or weekly. Administration may be continuous, for example, on a 28 day cycle.

The oxitinib, chemotherapy and/or radiation therapy can be administered alone or in combination with a second generation quinazolinamine derivative class TKI. Oxitinib may be administered at a dose of 25-100mg, e.g. about 40mg or 80 mg. Administration may be daily, every other day, every 2 days, every 3 days, or once per week. Oxitinib may be administered orally, e.g. in the form of a tablet.

A. Pharmaceutical composition

Also provided herein are pharmaceutical compositions and formulations for determining subjects with resistant EGFR mutations, comprising a second generation quinazolinamine derivative class TKI and a pharmaceutically acceptable carrier.

Can be prepared by mixing the active ingredient (e.g., antibody or polypeptide) with the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22)^ndedition (remington pharmacology, 22 th edition), 2012) to prepare the pharmaceutical compositions and formulations described herein in lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbonic quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, e.g. sugar caneSugar, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., soluble human PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r) ((r))Baxter International, Inc.). Certain exemplary shasegps (including rHuPH20) and methods of use are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

B. Combination therapy

In certain embodiments, the compositions and methods of the present embodiments relate to second generation quinazolinamine derivative TKIs in combination with at least one additional therapy. The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing therapies. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy.

In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side-effect limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of a therapeutic side-effect, such as an antiemetic agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma radiation. In some embodiments, the additional therapy is a therapy that targets the PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventive agent. The additional therapy may be one or more chemotherapeutic agents known in the art.

The second generation quinazolinamine derivative TKIs may be administered before, during, after, or in various combinations relative to additional cancer therapies, e.g., immune checkpoint therapies. The administration interval can range from simultaneous to several minutes to several days to several weeks. In embodiments where the second generation quinazolinamine derivative TKI is provided to the patient separately from the additional therapeutic agent, it is generally ensured that there is not a significant period of time between each delivery, such that the two compounds are still able to produce a beneficial combined effect in the patient. In this case, it is contemplated that the antibody therapy and the anti-cancer therapy can be provided to the patient within about 12-24 or 72 hours of each other, more specifically, within about 6-12 hours of each other. In some cases, significant prolongation of treatment time may be required if the interval between each administration is from several days (2, 3, 4,5, 6, or 7) to several weeks (1, 2,3, 4,5, 6, 7, or 8).

Various combinations may be employed. For the following examples, the second generation quinazolinamine derivative TKI is "a" and the anti-cancer therapy is "B":

administration of any compound or therapy of the present embodiments to a patient should follow the general protocol for administering such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

1. Chemotherapy

A wide variety of chemotherapeutic agents may be used according to embodiments of the present invention. The term "chemotherapy" refers to the treatment of cancer with drugs. "chemotherapeutic agent" is used to refer to a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified according to their activity pattern within the cell, e.g., whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly cross-link DNA, intercalate DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodidopa (benzodipa), carboquone, metoclopramide (meteedopa), and radopa (uredopa); ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphamide), and trimethylolmelamine (trimethlomelamine); polyacetylenyl (acetogenin) (especially bullatacin and bullatacin); camptothecin (including the synthetic analog topotecan); bryostatins; cariostatin (callystatin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including the synthetic analogs KW-2189 and CB1-TM 1); eleutherobin (eleutherobin); (ii) coprinus atramentarius alkali; sarcodictyin (sarcodictyin); spongistatin (spongistatin); nitrogen mustards such as chlorambucil, chlorophosphamide (cholphosphamide), estramustine, ifosfamide, dichloromethyldiethylamine (mechlorothamine), mechlorethamine hydrochloride, melphalan, neonebixin, benzene mustarol, prednimustine, trofosfamide and uramustine; nitrosoureas such as carmustine, chlorouramicin, fotemustine, lomustine, nimustine and ranimustine (ranirnustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin (calicheamicin), particularly calicheamicin γ l and calicheamicin ω I1); daptomycin (dynemicin), including daptomycin a; bisphosphonates, such as clodronate; esperamicin (esperamicin); and the neocarcinostatin chromophore and related tryptophane diyne antibiotic chromophores, aclacinomycin (aclacinomycin), actinomycin, amtricin, azaserine, bleomycin, actinomycin C (cactinomycin), karabicin (carabicin), carminomycin, oncomycin, tryptomycin, actinomycin D (dactinomycin), daunomycin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolo-doxorubicin and deoxydoxorubicin), epirubicin, isosbixacin, idarubicin, milbemycin, mitomycins such as mitomycin C, mycophenolic acid, norramycin, olivomycin (VOIMYCIN), pelomycin (peplomycin), Pofilomycin (potfiomycin), purines, and doxorubicin (laqueque) Rodobicin, streptavidin, streptozotocin, tubercidin, ubenimex, netastatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as carpoterone, drotandrosterone propionate, epithioandrostanol, meiandrane, and testolactone; anti-adrenalines, such as mitotane and trostane; folic acid supplements, such as furinic acid (frillinic acid); acetic acid glucurolactone; (ii) an aldophosphamide glycoside; (ii) aminolevulinic acid; eniluracil; amsacrine, atomoxetine (bestraucil); a bisantrene group; edatrexate (edatraxate); desphosphamide (defofamine); colchicine; a sulphinoquinone; eflomixine (elformithine); ammonium etiolate; an epothilone; ethydine; gallium nitrate; a hydroxyurea; lentinan; lonidamine (lonidainine); maytansinoids, such as maytansinoids and ansamitocins; mitoguazone; mitoxantrone; molindol (mopidanmol); diamine nitrene (nitrerine); pentostatin; methionine; pirarubicin; losoxanthraquinone; podophyllinic acid; 2-ethyl hydrazide; (ii) procarbazine; PSK polysaccharide complex; lezoxan; rhizomycin; a texaphyrin; a germanium spiroamine; alternarionic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; trichothecene toxins (especially T-2 toxin, echinocandin a (veracurin a), myrmecin a, and trichostatin (anguidine)); a urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; a gamma cytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination compounds such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novatron (novantrone); (ii) teniposide; edatrexae; daunomycin; aminopterin; (ii) Hirodad; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, navelbine (navelbine), farnesyl-protein transferase inhibitors, antiplatin (transplatinum), and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

2. Radiotherapy

Other factors that cause DNA damage and have been widely used include the generally known targeted delivery of gamma rays, X-rays, and/or radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and ultraviolet irradiation. It is likely that all of these factors will cause extensive damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. The dose of X-rays ranges from a prolonged (3-4 weeks) dose of 50-200 roentgens per day to a single dose of 2000-6000 roentgens. The dosage range of the radioisotope varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted and the uptake by the tumor cells.

3. Immunotherapy

It will be appreciated by those skilled in the art that additional immunotherapies may be combined or used in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules that target and destroy cancer cells. RituximabIs that it is trueFor example. The immune effector can be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may act as an effector of the therapy, or it may recruit other cells to actually affect cell killing. The antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin a chain, cholera toxin, pertussis toxin, etc.) and used as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

Antibody-drug conjugates have become a breakthrough approach for the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody Drug Conjugates (ADCs) comprise a monoclonal antibody (MAb) covalently linked to a cell killing drug. This approach combines the high specificity of mabs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" mabs that deliver a payload (drug) to tumor cells with enriched levels of antigen. Targeted delivery of drugs also minimizes their exposure to normal tissues, thereby reducing toxicity and increasing the therapeutic index. Two ADC drugs approved by FDA, namely 2011 approved(Brentuximab vedotin) and approved in 2013(trastuzumab-maytansine conjugate (trastuzumab emtansine) or T-DM1) validated the method. Currently more than 30 ADC drug candidates are in various stages of clinical trials for cancer treatment (Leal et al, 2014). As antibody engineering and linker-payload (linker-payload) optimization matures more and more, the discovery and development of new ADCs is more and more dependent on the identification and validation of new targets and the generation of targeted mabs suitable for this approach. Two criteria for ADC targets are upregulation/high levels of expression in tumor cells and robust internalization.

In one aspect of immunotherapy, tumor cells must carry a marker that is suitable for targeting, i.e., not present on most other cells. There are many tumor markers, and in the context of this embodiment, any of these markers may be suitable for being targeted. Common tumor markers include CD20, carcinoembryonic Antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen (Sialyl Lewis Antigen), MucA, MucB, PLAP, laminin receptor, erb B and p 155. Another aspect of immunotherapy is the combination of anti-cancer effects with immunostimulating effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ -IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

Examples of immunotherapy include immunoadjuvants such as Mycobacterium bovis (Mycobacterium bovis), Plasmodium falciparum (Plasmodium falciparum), dinitrochlorobenzene and aromatics (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoules et al, 1998), cytokine therapies such as interferons alpha, beta and gamma, IL-1, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, such as TNF, IL-1, IL-2 and p53(Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD 20, anti-ganglioside GM2, and anti-p 185(Hollander, 2012; Hanibuchi et al, 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either increase signal (e.g., co-stimulatory molecules) or decrease signal. Immune checkpoint blockade inhibitory immune checkpoints that can be targeted include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T Lymphocyte Attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4(CTLA-4, also known as CD152), indoleamine 2, 3-dioxygenase (IDO), Killer Immunoglobulin (KIR), lymphocyte activation gene 3(LAG3), programmed death factor 1(PD-1), T cell immunoglobulin and mucin domain 3(TIM-3), and T cell activation inhibitor Ig V domain (VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and/or CTLA-4.

The immune checkpoint inhibitor may be a drug, such as a recombinant form of a small molecule, ligand or receptor, or in particular an antibody, such as a human antibody (e.g., international patent publication WO 2015016718; pardol, Nat Rev Cancer,12(4): 252-. Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular antibodies in chimeric, humanized or human form may be used. As the skilled artisan will appreciate, certain antibodies referred to in this disclosure may use alternative names and/or equivalent names. Such alternative and/or equivalent names are interchangeable within the context of the present invention. For example, it is well known that palivizumab (lambrolizumab) is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In particular aspects, the PD-1 ligand binding partner is PDL1 and/or PDL 2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In particular aspects, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist may 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 axis antagonists for use in the methods provided herein are known in the art, for example, as described in U.S. patent publication nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected fromAuto-nivolumab, pembrolizumab and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising PDL1 or PDL2 fused to the extracellular portion of a constant region (e.g., the Fc region of an immunoglobulin sequence) or a PD-1 binding moiety). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 andis an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, Pabollizumab,And SCH-900475, are anti-PD-1 antibodies described in WO 2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342.

Another immune checkpoint that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4(CTLA-4), also known as CD 152. The Genbank accession number of the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is present on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also referred to as B7-1 and B7-2, respectively) on antigen presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and may be important to their function. Activation of T cells by T cell receptors and CD28 results in increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

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.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the methods of the invention can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies may be used. For example, the methods disclosed herein may use anti-CTLA-4 antibodies disclosed below: U.S. patent No. 8,119,129; international patent publication Nos. WO01/14424, WO98/42752, and WO 00/37504(CP675,206, also known as tremelimumab (tremelimumab); formerly known as tiximumab); U.S. patent No. 6,207,156; hurwitz et al, 1998; camacho et al, 2004; and Mokyr et al, 1998. The teachings of each of the foregoing 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 patent application nos. WO2001014424 and WO2000037504 and U.S. patent No. 8,017,114; which is incorporated herein by reference in its entirety.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also referred to as 10D1, MDX-010, MDX-101, and) Or antigen-binding fragments and variants thereof (see, e.g., WO 01/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of ipilimumab VH region and the CDR1, CDR2, and CDR3 domains of ipilimumab VL region. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the above antibody. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity to ipilimumab).

Other molecules that are useful for modulating CTLA-4 include: CTLA-4 ligands and receptors (e.g., as described in U.S. patent nos. 5,844,905, 5,885,796 and international patent application nos. WO1995001994 and WO1998042752, which are incorporated herein by reference in their entirety) and immunoadhesins (e.g., as described in U.S. patent No. 8,329,867, which is incorporated herein by reference).

4. Surgery

Approximately 60% of cancer patients will undergo certain types of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the therapies, chemotherapies, radiation therapies, hormonal therapies, gene therapies, immunotherapies, and/or replacement therapies of embodiments of the present invention. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and surgery controlled by a microscope (Mohs' surgery).

After removal of some or all of the cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local area application of other anti-cancer therapies. 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 have different dosages.

5. Other agents

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to enhance the efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions (GAP junctions), cytostatic and differentiating agents, cytostatic agents, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of intercellular junctions increases the anti-hyperproliferative effect on the adjacent hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to increase the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to enhance the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of embodiments of the invention to improve therapeutic efficacy.

IV. reagent kit

Kits for detecting an axitinib-resistant EGFR mutation (e.g., those disclosed herein) are also within the scope of the present disclosure. An example of such a kit may include a set of esitinib-resistant EGFR mutation-specific primers. The kit may further comprise instructions for using the primers to detect the presence or absence of a specific axitinib-resistant EGFR mutation described herein. The kit may further comprise instructions for diagnostic purposes indicating that a positive identification of an austenitib resistant EGFR mutation described herein in a sample from a cancer patient indicates sensitivity to a second generation quinazolinamine derivative class TKI or a structurally similar inhibitor. The kit may further comprise instructions indicating that a positive identification of an austenitib resistant EGFR mutation described herein in a sample from a cancer patient indicates that the patient should be treated with a second generation quinazolinamine derivative class TKI or a structurally similar inhibitor.

V. examples

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1-identification of drugs for cancer cells with an oxicetinib-resistant EGFR mutation

A panel of Ba/F3 cell lines expressing either the axitinib or erlotinib resistance mutations, including atypical EGFR mutations and typical EGFR mutations spanning exons 18-21, was generated. The transformability of the mutations was then assessed by continued cell viability following IL-3 deprivation. Activated EGFR mutant Ba/F3 cells were then screened for their ability to antagonize EGFR TKI. Cell viability was determined by the Cell Titer Glo assay.

The second generation quinazolinamine derivative TKI inhibits the proliferation of Ba/F3 cell line expressing atypical mutation such as L861Q, G719S, L858R/L792H, L858R/C797S and Ex19del/C797S, IC₅₀Value of<3nM。

In another study, a PDX model of NSCLC harboring the EGFR exon 18P-loop mutation (G719A) was treated with the indicated inhibitors for 28 days. It was further found that the P-loop exon 18 mutation resulted in primary resistance to oxitinib in vivo, but not to other EGFR TKIs (fig. 2).

Ba/F3 cells expressing acquired atypical mutations spanning exons 18-21 were treated with inhibitors for 72 hours. It was observed that acquired atypical mutations drive resistance to oxitinib, but are sensitive to quinazoline TKI, and that drug sensitivity/resistance of co-occurring mutations may be driven by primary mutations (figure 3). It has also been shown that the T790M mutation drives resistance to first and second generation drugs, regardless of primary or tertiary mutation, but unique third generation TKI and drug re-targeting can overcome tertiary T790M positive drug resistance.

TABLE 5 EGFR mutation vectors

Thus, the second generation quinazolinamine derivative TKI is a potent inhibitor of primary and acquired atypical ocitinib resistant EGFR mutant NSCLC, including L861Q, G719S, L858R/L792H, L858R/C797S and Ex19 del/C797S. Current studies indicate that second generation TKIs overcome the axitinib resistance of atypical EGFR mutant NSCLC.

Example 2 materials and methods

Ba/F3 cell line production and IL-3 deprivation: the Ba/F3 cell line was established as described previously (Robichaux et al, 2018). Briefly, stable Ba/F3 cell lines were generated by retroviral transduction of the Ba/F3 cell line for 12 hours. Retroviruses were generated by transfecting the pBabe-Puro-based vectors (Addge and BioInnovatise) summarized in Table 1 into Phoenix 293T-ampho cells (Orbigen) using Lipofectamine 2000 (Invitrogen). Three days after transduction, 2. mu.g/ml puromycin (Invitrogen) was added to the RPMI medium. The Cell lines were then grown in the absence of IL-3 for two weeks and Cell viability was assessed every three days using the Cell Titer Glo assay (Progema). The resulting stable cell line was maintained in RPMI-1640 medium containing 10% FBS but no IL-3.

Cell viability assay and IC₅₀Evaluation: cell viability was determined using the Cell Titer Glo assay (Promega) as described previously (Robichaux et al, 2018). Briefly, 2000-3000 cells per well were seeded in 384-well plates (Greiner Bio-One) in triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitor or vehicle alone to a final volume of 40 μ L per well. After 3 days, 11 μ L Cell Titer Glo was added to each well. The plates were shaken for 15 minutes and bioluminescence was measured using a FLUOstar OPTIMA multimodal microplate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO-treated cells and the normalized values were plotted in GraphPad Prism using non-linear regressionNormalized data with variable slope was fitted. IC for 50% inhibition calculated by GraphPad Prism₅₀The value is obtained.

All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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