阅读说明：本技术 对携带her2外显子21插入的癌细胞具有抗肿瘤活性的化合物 (Compounds having antitumor activity against cancer cells carrying an insertion of exon 21 of HER2 ) 是由 J·罗比丘 J·V·海马赫 于 2020-03-27 设计创作，主要内容包括：本公开提供了通过施用第三代酪氨酸激酶抑制剂(例如,波齐替尼)治疗确定具有HER2外显子21突变的患者的癌症的方法。(The present disclosure provides methods of treating cancer in patients identified as having a HER2 exon 21 mutation by administering a third generation tyrosine kinase inhibitor (e.g., bosutinib).)

1. A method of treating cancer in a subject comprising administering to the subject an effective amount of bosutinib, wherein the subject has been determined to have one or more HER2 exon 21 mutations.

2. The method of claim 1, wherein the bosutinib is further identified as bosutinib hydrochloride.

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

4. The method of any one of claims 1-3, wherein the one or more HER2 exon 21 mutations comprise point mutations, insertions and/or deletions of 1-18 nucleotides between amino acids 832 and 883.

5. The method of any one of claims 1-4, wherein the subject has been determined to have 2,3, or 4 HER exon 21 mutations.

6. The method of any one of claims 1-5, wherein the subject has been previously administered a tyrosine kinase inhibitor.

7. The method of claim 6, wherein the subject is resistant to a previously administered tyrosine kinase inhibitor.

8. The method of claim 7, wherein the tyrosine kinase inhibitor is lapatinib, afatinib, dacomitinib, oxitinib, ibrutinib, azatinib, or lenatinib.

9. The method of claim 4, wherein the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869.

10. The method of claim 4, wherein the one or more HER2 exon 21 mutations are located at V842 and/or R868 residues.

11. The method of any one of claims 1-10, wherein the subject has been determined to have no EGFR mutation at C797 residue.

12. The method of any one of claims 1-11, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R.

13. The method of any one of claims 1-12, wherein the one or more HER2 exon 21 mutations is V842I and/or R868W.

14. The method of any one of claims 1-13, wherein the subject is determined to have a HER2 exon 21 mutation by analyzing a genomic sample from a patient.

15. The method of claim 15, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

16. The method of any one of claims 1-15, wherein the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing or PCR analysis.

17. The method of any one of claims 1-16, wherein the bosutinib is administered orally.

18. The method of any one of claims 1-17, wherein the bosutinib is administered at a dose of 5-25 mg.

19. The method of any one of claims 1-18, wherein the bosutinib is administered at a dose of 8mg, 12mg, or 16 mg.

20. The method of any one of claims 1-19, wherein the bosutinib is administered daily.

21. The method according to any one of claims 1-20, wherein the bosutinib is administered continuously.

22. The method of any one of claims 1-21, wherein the bosutinib is administered in a 28 day cycle.

23. The method of any one of claims 1-22, further comprising administering an additional anti-cancer therapy.

24. The method of claim 23, wherein the additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

25. The method of claim 23 or 24, wherein the bosutinib and/or anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, in a sustained release agent, in a controlled release agent, in a delayed release agent, as a suppository, or sublingually.

26. The method of any one of claims 23-25, wherein administering bosutinib and/or the anti-cancer therapy comprises local, regional or systemic administration.

27. The method according to any one of claims 23-26, wherein two or more times bosutinib and/or anti-cancer therapy are administered.

28. The method of any one of claims 1-27, wherein the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary tract cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic cancer, glioma, sarcoma, epithelial cell tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, bile duct cancer, pheochromocytoma, islet cell cancer, li famenil tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, 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, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, cervical cancer, or a combination thereof, Colon cancer, rectal cancer, or skin cancer.

29. The method of any one of claims 1-28, wherein the cancer is non-small cell lung cancer.

30. The method of any one of claims 1-29, wherein the patient is a human.

31. A pharmaceutical composition for identifying a subject having one or more HER exon 21 mutations comprising bosutinib.

32. The composition of claim 31, wherein the composition is further defined as an oral composition.

33. The composition according to claim 31 or 32, wherein the composition comprises 5-25 mg of bosutinib.

34. The composition of any one of claims 31-33, wherein the composition comprises 8mg, 12mg, or 16mg of bosutinib.

35. The composition of any one of claims 31-34, wherein the bosutinib is further determined as bosutinib hydrochloride.

36. The composition of any one of claims 31-35, wherein the composition is formulated as a tablet.

37. The composition of any one of claims 31-36 wherein the one or more HER2 exon 21 mutations comprise point mutations, insertions and/or deletions of 1-18 nucleotides between amino acids 832 and 883.

38. The composition of any one of claims 31-37, wherein the subject has been determined to have 2,3, or 4 HER exon 21 mutations.

39. The composition of claim 37, wherein the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869.

40. The method of claim 37, wherein the one or more HER2 exon 21 mutations are located at V842 and/or R868 residues.

41. The composition of any one of claims 31-40, wherein the subject has been determined to have no EGFR mutation at C797 residues.

42. The composition of any one of claims 31-41, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R.

43. The method of any one of claims 31-42, wherein the one or more exon 21 mutations is V842I and/or R868W.

44. The composition of any one of claims 31-43, wherein the subject is being treated with an anti-cancer therapy.

45. A method of predicting the response of a subject having cancer to bociclib alone or a combination of bociclib and a second anti-cancer therapy comprising detecting a HER2 exon 21 mutation in a genomic sample obtained from said patient, wherein the patient is predicted to have a good response to the combination of bociclib alone or bociclib and the second anti-cancer therapy if the sample is positive for the presence of the HER2 exon 21 mutation.

46. The method of claim 45, wherein the HER exon 21 mutation is further determined as an exon 20 insertion mutation.

47. The method of claim 45 or 46, wherein the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue.

48. The method of any one of claims 45-47, wherein the presence of a HER exon 21 mutation is determined by nucleic acid sequencing or PCR analysis.

49. The method according to claim 48 wherein the HER2 exon 21 mutation comprises a point mutation, insertion and/or deletion of 1-18 nucleotides between amino acids 832 and 883.

50. The method of claim 49, wherein the HER2 exon 21 mutation is located at one or more residues selected from the group consisting of V842, R868, and L869.

51. The method of claim 49, wherein the one or more HER2 exon 21 mutations are located at V842 and/or R868 residues.

52. The method of any one of claims 45-51, wherein the one or more HER2 exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R.

53. The method of any one of claims 45-52, wherein a good response to either Boletinib alone or a combination of Boletinib and a second anticancer therapy comprises reducing tumor size or burden, blocking tumor growth, reducing tumor-associated pain, reducing cancer-associated pathology, reducing cancer-associated symptoms, no progression of cancer, increased disease-free interval, increased time to progression, induction of remission, reduction of metastasis, or increased patient survival.

54. The method of any one of claims 45-53, further comprising administering to the patient predicted to have a good response, either poezinib alone or a combination of poezinib and a second anti-cancer therapy.

55. The method of claim 54, wherein the bosutinib is administered orally.

56. The method of claim 54 or 55, wherein the bosutinib is administered at a dose of 5-25 mg.

57. The method of any one of claims 54-56, wherein the bozitinib is administered at a dose of 8mg, 12mg, or 16 mg.

58. The method of any one of claims 54-57, wherein said bosutinib is further determined as bosutinib hydrochloride.

59. The method of any one of claims 54-58, wherein the pozzertib hydrochloride is formulated as a tablet.

1. Field of the invention

The present invention relates generally to the fields of molecular biology and medicine. More specifically, the invention relates to methods of treating patients having a HER2 exon 21 mutation.

2. Description of the related Art

Erb-b2 receptor tyrosine kinase 2(ERBB2) (also known as human epidermal growth factor receptor 2(HER2)) amplification occurs in many cancer types, and targeting agents such as trastuzumab, pertuzumab, trastuzumab mettanatin conjugate (T-DM1), lapatinib and neratinib have been shown to improve clinical outcome compared to chemotherapy alone (Vogel et al, 2002). Activating mutations in ERBB2(HER2) have been reported to be present in many cancer types (Kris et al, 2015). Although there is FDA approved targeted therapy against cancers carrying HER2 amplification, there is no approved targeted therapy specifically directed against HER2 mutations. However, the non-small cell lung cancer (NSCLC) guidelines of the national integrated cancer network in the united states suggest the use of extensive molecular profiling to test newly diagnosed patients for HER2 mutations (Ettinger et al, 2018).

Recent clinical studies of targeting agents against HER2 mutant cancers have focused on covalent second generation Tyrosine Kinase Inhibitors (TKIs), such as afatinib, lenatinib, and dacatinib. SuMMIT pan-cancer studies report that patients receiving treatment with neratinib had an Objective Response Rate (ORR) of less than 15% for all HER2 mutations (Hyman et al, 2018). However, in several studies, breast cancer patients have an ORR of 12.5% -32% of the single drug neratinib when patients are stratified by cancer type (Hyman et al, 2018; Ma et al, 2017); while lung cancer patients had a response rate of 0% -4% to lenatinib as a single drug (Hyman et al, 2018; Mazieres et al, 2015), demonstrating the cancer-specific difference in HER2 inhibitory efficacy. Interestingly, HER2 targeting agents appear to trigger variation specificity differences within a single cancer type. In the sumit trial, patients with point mutations in the HER2 kinase domain had 21.4% ORR for neratinib, while patients with exon 20 insertion had 7.1% ORR for neratinib (Hyman et al, 2018). Furthermore, whereas dactinib has an ORR of 11.5% for HER2 mutant NSCLC, no response occurred in patients with HER2 exon 20 insertion mutation p.y772dupyvma (Kris et al, 2015), NSCLC patients positive for exon 20 insertion had rates of 18.2% and 18.8% for afatinib in two separate afatinib studies.

Studies with HER2 monoclonal antibody and drug-antibody conjugates showed similar results. Pan cancer study MyPathway examined the efficacy of the combination of the anti-HER 2 monoclonal antibodies trastuzumab and pertuzumab in 35 different tumor types and reported 11% ORR for all HER2 mutations and cancer types. In this study, only 21% of NSCLC patients responded to 1 cholangiocarcinoma patient out of the 35 tumor types enrolled. Furthermore, in a pan-HER 2 mutant NSCLC study in which the efficacy of T-DM1 was tested, patients carrying the exon 20 insertion mutation had 54.5% ORR, but patients with the exon 19 mutation did not respond in part. These cancer-and variation-specific patient prognostic differences indicate that there is an unmet need for a detailed, systematic understanding of HER2 mutation profiles for different cancer types and for finding effective therapies for the various HER2 mutations identified.

Preclinical studies of HER2 activating mutations also reported different sensitivities to various TKIs. Studies of mutations in the extracellular domain of HER2 indicate that these mutations are associated with resistance to non-covalent inhibitors (such as lapatinib), but exhibit strong sensitivity to covalent TKIs (including neratinib, afatinib, and axitinib), while mutations within exon 19 indicate different sensitivity to lapatinib and covalent inhibitors. In addition, studies have shown that the HER2 exon 20 mutation is broadly resistant to non-covalent and covalent TKIs (such as oxitinib, azatinib, norcetib and temotinib). Furthermore, covalent quinazolinamine-based TKIs (lenatinib, afatinib, and dacatinib) induce different responses to individual HER2 exon 20 mutations. However, only the unusual HER2 mutation showed sensitivity to clinically relevant concentrations of these TKIs. Recently, it has been reported that pozzatinib effectively inhibits the HER2 exon 20 insertion mutation at concentrations attainable in patients, and that pozzatinib treatment induced a radiological response in one patient carrying the HER2 exon 20 mutation. However, a single HER2 TKI targeting the most common variant of HER2 mutant cancers has not been found.

Background

The invention was made with government support under fund number CA190628 awarded by the national institutes of health. The government has certain rights in this invention.

SUMMARY

Embodiments of the present disclosure provide methods and compositions for treating cancer in a patient having a HER2 exon 21 mutation. In one embodiment, there is provided a method of treating cancer in a subject comprising administering to the subject an effective amount of bosutinib, wherein the subject has been determined to have one or more HER exon 21 mutations. In a particular aspect, the subject is a human.

In some aspects, bosutinib is further identified as bosutinib hydrochloride. In certain aspects, the bosutinib hydrochloride is formulated as a tablet.

In certain aspects, the one or more HER2 exon 21 mutations include one or more point mutations, insertions, and/or deletions between amino acids 832 and 883 of 1-18 nucleotides. In some aspects, the subject has been determined to have 2,3, or 4 HER2 exon 21 mutations. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842 and R868. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

In some aspects, the subject is resistant to or has demonstrated resistance to a previously administered tyrosine kinase inhibitor. In certain aspects, the tyrosine kinase inhibitor is lapatinib, afatinib, dactinib, axitinib, ibrutinib, azatinib, or lenatinib.

In certain aspects, the bosutinib may be administered orally. In some aspects, the bosutinib is administered at a dose of 5mg-25mg, e.g., 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, or 25 mg. In certain aspects, the bosutinib is administered at a dose of 8mg, 12mg, or 16 mg. In some aspects, the bosutinib is administered once daily. In certain aspects, the administration of bosutinib is continuous. In some aspects, the bosutinib is administered on a 28 day cycle.

In certain aspects, the subject is determined to have a HER2 exon 21 mutation by analyzing a genomic sample from the subject. In some aspects, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In particular aspects, the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing (e.g., DNA sequencing of circulating free DNA in tumor tissue or plasma) or PCR analysis.

In certain aspects, the method further comprises administering an additional anti-cancer therapy. In some aspects, the anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In certain aspects, the bosutinib and/or the anti-cancer 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. 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 (e.g., daily, every other day, or weekly).

In some aspects, the cancer is an oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary tract cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, bile duct cancer, pheochromocytoma, islet cell cancer, limemann's tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, 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.

In another embodiment, a pharmaceutical composition comprising poecitinib for use in identifying a patient having one or more HER2 exon 21 mutations is provided. In certain aspects, the one or more HER2 exon 21 mutations include point mutations, insertions, and/or deletions of 1-18 nucleotides between amino acids 832-883. In certain aspects, the subject has been determined to have 2,3, or 4 HER2 exon 21 mutations.

In some aspects, bosutinib is further identified as bosutinib hydrochloride. In certain aspects, the bosutinib hydrochloride is formulated as a tablet.

In some aspects, the bosutinib is administered orally. In some aspects, the bosutinib is administered at a dose of 5mg-25mg (e.g., 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, or 25 mg). In some aspects, the bosutinib is administered at a dose of 8mg, 12mg, or 16 mg. In certain aspects, the bosutinib is administered once daily. In some aspects, the administration of bosutinib is continuous. In some aspects, the bosutinib is administered on a 28 day cycle.

In some aspects, the subject is resistant to or has demonstrated resistance to a previously administered tyrosine kinase inhibitor. In certain aspects, the tyrosine kinase inhibitor is lapatinib, afatinib, dactinib, oxitinib, ibrutinib, azatinib, or lenatinib.

In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842 and R868. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I and R868W. In some aspects, the patient is receiving an anti-cancer therapy.

In yet another embodiment, there is provided a method of predicting the response of a subject with cancer to bozitinib alone or a combination of bozitinib and an anti-cancer therapy comprising detecting the HER2 exon 21 mutation in a genomic sample obtained from said patient, wherein a patient is predicted to have a good response to bozitinib alone or a combination of bozitinib and an anti-cancer therapy if the sample is positive for the presence of the HER2 exon 21 mutation. In some aspects, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In certain aspects, the presence of the HER2 exon 21 mutation is determined by nucleic acid sequencing or PCR analysis. In certain aspects, the HER2 exon 21 mutation comprises one or more point mutations, insertions and/or deletions between amino acids 832 and 883 of 1-18 nucleotides. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842 and R868. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

In certain aspects, a good response to a combination of bozitinib or bozitinib alone and 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, no progression of cancer, an increase in disease-free interval, an increase in time to progression, inducing remission, reducing metastasis, or increasing patient survival. In yet another aspect, the patient predicted to have a good response is administered either pozzinib alone or a combination of pozzinib and an anti-cancer therapy.

In some aspects, bosutinib is further identified as bosutinib hydrochloride. In certain aspects, the bosutinib hydrochloride is formulated as a tablet.

In some aspects, the bosutinib is administered orally. In some aspects, the bosutinib is administered at a dose of 5mg-25mg (e.g., 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, or 25 mg). In some aspects, the bosutinib is administered at a dose of 8mg, 12mg, or 16 mg. In certain aspects, the bosutinib is administered once daily. In some aspects, the administration of bosutinib is continuous. In some aspects, the bosutinib is administered on a 28 day cycle.

In some aspects, the subject is resistant to or has demonstrated resistance to a previously administered tyrosine kinase inhibitor. In certain aspects, the tyrosine kinase inhibitor is lapatinib, afatinib, dactinib, axitinib, ibrutinib, azatinib, or lenatinib.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from 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-1J: the HER2 mutation occurs in a variety of cancer types, with mutational hot spots appearing across the entire receptor. Bar graph of weighted average of HER2 mutation (a) and HER2 exon 20 mutation (B) frequencies listed by cancer. Bars represent weighted mean ± SEM. The size of the dots represents the number of patients in each database. The frequency of HER2 mutations detected by cfDNA reported by guard Health was normalized against the clinical sensitivity reported by Odegaard et al 2018.

FIGS. 2A-2H: the HER2 mutation hotspot varies by cancer type. Pie charts of the position frequency of the HER2 mutation in (a) all cancers (N-2338), (B) lung cancer (N-177), (C) breast cancer (N-143), and (D) colorectal cancer (N-219) reported in the cbioport and MD Anderson databases. (E) Lollipop profile of the 10 most common HER2 mutations in all cancers reported by cBioPortal and MD Anderson (N2338 HER2 mutations). The length of the bars is related to the frequency of the mutations. (E-H) lollipop plots of the 10 most common HER2 mutations across NSCLC (F, N177), breast (G, N143) and colorectal (H, N219) in the cbioport and MD Anderson databases; the length of the bars correlates with the reported mutation frequency.

FIGS. 3A-3C: the most common variant of HER2 in the tyrosine kinase domain is the activating mutation. Stable Ba/F3 cell lines expressing HER2 exon 19(a), HER2 exon 20(B), and HER2 exon 21(C) mutations were grown for 14 days without IL-3 for cell viability. Cell viability was determined every 3 days by the Cell Titer Glo assay. Mean ± SEM are plotted for each cell line (n-3 biologically independent experiments).

FIGS. 4A-4F: bosutinib was the most potent inhibitor of HER2 mutations detected in Ba/F3 cells. (A) Calculated logIC of Ba/F3 cells stably expressing the indicated mutation and 72 hours after drug treatment in GraphPad₅₀Heat map of values. Cell viability was determined by the Cell Titer Glo assay (N.gtoreq.3). All Ba/F3 cell lines (B) expressing HER2 mutations, HER2 exon 19 mutant cell line (C), HER2 exon 20 mutant cell line (D) or HER2 exon 21 mutant cell line (E) average IC 72 hours after drug treatment with afatinib, lenatinib, tasotetinib-TKI or bosutinib₅₀The value is obtained. Bars represent mean. + -. SEM (N.gtoreq.3). (C-E) statistical significance between groups was determined using ANOVA and Dunn multiple comparison tests. Mean IC of Ba/F3 cells expressing L755S or L755P treated with indicated inhibitors₅₀The value (F). Dots represent mean. + -. SEM (N.gtoreq.3). Statistical significance was determined by paired t-test.

FIGS. 5A-5D: molecular dynamics simulation of HER2 mutants revealed a possible mechanism for drug sensitivity reduction of the Y772dupYVMA and L755P mutations. (A)150ns accelerated the alpha-C-helix position of HER 2V 777L and the Y772dupYVMA exon 20 mutant during molecular dynamics simulation. (B) HER2 exon 20 mutants are a population percentage of molecular dynamics snapshots of the α -C-helix "in" conformation versus the α -C-helix "out" conformation. (C) Molecular dynamics snapshots of V777L and Y772dupYVMA mutants. There was a slight difference in the conformation of the P-loop and kinase hinge, but there was a significant shift in the α -C-helix position (V777L for the "out" position and Y772dupYVMA for the "in" position). (D) Molecular dynamics snapshots of L755P and L755S HER2 mutants. The L755P mutant lacks backbone hydrogen bonds to V790, resulting in kinase hinge instability and contraction of the P-loop toward the binding site.

FIGS. 6A-6F: human cell lines expressing the HER2 mutation were also most sensitive to bosutinib. MCF10A cells expressing exon 20 insertion mutations, HER 2G 776delinsVC (a), HER 2Y 772dupYVMA (B), HER 2G 778dupGSP (C), were treated with indicated inhibitors for 72 hours of dose response curves. (D) Histogram of MCF10A HER2 selectivity index. For each indicated drug, the IC of the cell line was mutated₅₀Value divided by the average IC of cell lines expressing HER2 WT₅₀The value is obtained. Dots represent mean. + -. SEM for each cell line, bars represent mean. + -. min/max for all three cell lines (N.gtoreq.3 for each cell line). (E) Dose response curves of CW-2 large intestine cells carrying HER2 exon 19 mutation L755S treated with indicated inhibitors for 72 hours. The (a-C, E) curves represent mean ± SEM, N ═ 3. (F) Histogram of CW-2 tumor volume at day 21. Mice were treated with vehicle control (N ═ 5), 30mg/kg of lenatinib (N ═ 5), 20mg/kg of afatinib (N ═ 5) or 5mg/kg of pozzatinib (N ═ 5), 5 days/week, and were randomized to 350mm indicated by the dashed line³A tumor. Dots represent individual tumors, bars represent mean ± SEM. Statistical significance was determined using one-way ANOVA.

FIGS. 7A-7D: NSCLC patients with HER2 mutation had a positive response rate of 42% to pozitinib. (A) Cascade of responses of the first 12 HER2 exon 20 patients in NCT 03066206. Objective partial responses (from left: lines 7, 8,10, 11 and 12) are shown, undetermined responses (line 9) are shown, disease stabilization (lines 3-6) and disease progression (lines 1-2) are shown. (B) Kaplan-meier plots of progression free survival for the first 12 HER2 exon 20 patients show that up to 12 months of 2018, mPFS was 5.6 months. (C) CT scans of patients with HER 2Y 772dupYVMA mutation 1 day before and 8 weeks after treatment with bosutinib. (D) PET scans of patients with HER 2L 755P mutant NSCLC 1 day before and 4 weeks after treatment with bosutinib. Patients were previously treated and progressed on platinum-based chemotherapy in combination with trastuzumab, nivolumab and anti-TDM 1, but target lesions were reduced by 12% after treatment with bosutinib.

FIGS. 8A-8G: treatment with bosutinib induces cell surface accumulation of HER2, and combination treatment with bosutinib and T-DM1 enhances anti-tumor activity. (A) FACS analysis of HER2 receptor expression on MC10A cell lines expressing HER 2Y 772dupYVMA, HER 2G 778dupGSP and HER 2G 776delinsVC 24 hours after 10nM treatment with polazinib. Bars represent mean ± SEM, significant differences between DMSO and pozzertib treated groups were determined by student's t-test. (B) IC of MCF10A cell lines expressing HER 2Y 772dupYVMA, HER 2G 778dupGSP and HER 2G 776delinsVC after treatment with bosutinib, T-DM1 or bosutinib and the indicated dose of T-DM1₅₀Histogram of values. Bars represent mean ± SEM (n ═ 3 independent experiments), and significant differences were determined by one-way ANOVA and post hoc dunne multiple comparisons. (C) Tumor growth curves of HER 2Y 772dupYVMA NSCLC PDX treated with indicated inhibitors. Bozitinib treatment was administered 5 days per week, T-DM1 was administered 1 time at the beginning of treatment. (D) Kaplan-Meier curve of Progression Free Survival (PFS), where PFS is defined as tumor doubling from the optimal response. Significant differences between groups were determined using the Mantel-Cox log rank test. At the time of euthanasia, mice were examined. (E) Dot plots of percent change in tumor volume at day 15 for mice treated with indicated inhibitors. (F) Graph of the number of tumor-bearing mice in each group at day 15 and day 45. (G) Tumor volume spider plots of HER 2Y 772dupYVMA mice treated with indicated inhibitors. Dotted line indicates the point of random fetching (300 mm)³)。

FIGS. 9A-9D: in the Guardant, cBioPortal and MD Anderson databases, exon 20 insertion mutation diversity varies depending on the type of cancer. Pie charts of HER2 exon 20 insertion mutation frequency in N ═ 517(a) for all cancer types. Further by cancer type: (B) lung cancer, N-362, (C) breast cancer, N-30, (D) other cancers, N-125 analyzed the frequency of exon 20 insertion mutations.

FIGS. 10A-10B: the common HER2 mutation is constitutively phosphorylated and p-HER2 expression is independent of drug sensitivity. (A) Relative p-HER2 expression was determined by the ratio of p-HER2 to total HER2 as determined by ELISA. Bars represent mean ± SEM, and n is 3. And ND is lower than the detection limit. (B) Relative HER2 and bosutinib IC were plotted against the Ba/F3 HER2 mutant cell line₅₀Correlation of values. Pearson correlations and p-values were determined by GraphPad Prism (n-3).

FIGS. 11A-11B: molecular modeling showed that the binding pocket size of HER2 mutants was different. (A) The HER2 kinase domain exon 19, 20 and 21 protein backbones are blue, pink and orange, respectively. The ligand in the template X-ray structure (PDB 3PP0) is in the form of a green rod and provides a marker of mutated residues/insertion positions. (B) Curves of binding pocket volume from HER2 mutants accelerated molecular dynamics simulation.

FIG. 12: bosutinib inhibits p-HER2 in HER2 mutant cell lines. Western blot of G776 delinsVC-expressing MCF10A cells 2 hours after treatment with the indicated drugs and doses.

FIG. 13: bosutinib inhibits tumor growth of exon 19 mutated colorectal cancer xenografts. CW-2 cells carrying the HER 2L 755S mutation were injected into the flank of 6-week-old female nu/nu nude mice. When the tumor reaches 350mm³At time, mice were randomly assigned to 4 groups: 20mg/kg afatinib, 5mg/kg bosutinib, 30mg/kg lenatinib or vehicle control. Tumor volume was measured three times a week, and mice received drug from monday to friday (5 days per week). Symbols represent mean ± SEM at each time point. Statistical significance was determined using two-way ANOVA and Tukey multiple comparison tests. Asterisks indicate significant differences between vehicle and either poecitinib or neratinib. Starting on day 10 where a significant difference was first detected, the p-value for each comparison is listed below.

Description of the exemplary embodiments

This study identified the frequency of the most common genomic variants of HER2 mutation in various malignancies. The activation potential of the 16 most common HER2 mutations was systematically demonstrated and their drug susceptibility to the 11 commonly used EGFR and HER2 TKI was evaluated. Insertional mutation of exon 20 and p.l755p (but not p.l755s) mutation of exon 19 were found to be refractory to many tested TKIs. Molecular dynamics modeling of drug-resistant HER2 variants, L755P and exon 20 insertions showed that these mutations affect the conformational state of the receptor, reducing the overall size of the drug binding pocket. Furthermore, bosutinib was identified as a potent inhibitor of all HER2 mutations evaluated. Furthermore, this study showed that bosutinib was clinically active in NSCLC patients carrying the most resistant HER2 variant, exon 20 insertion, exon 21 mutation, and L755P. Finally, these studies indicate that bosutinib-mediated cell surface receptor aggregation enhances T-DM1 activity, and that T-DM1 activity may be beneficial in enhancing anti-tumor activity in vivo, leading to complete regression of tumors in the PDX model of HER 2-mutated NSCLC.

Accordingly, certain embodiments of the present disclosure provide methods of treating cancer patients having a HER2 exon 21 mutation. In particular, the method comprises administering to a patient identified as having a HER exon 21 mutation, either bosutinib (also known as HM781-36B) or afatinib. The size and flexibility of poecitinib overcome steric hindrance, inhibiting HER2 exon 21 mutant at nanomolar low concentrations. Therefore, poecitinib or afatinib, as well as structurally similar inhibitors, are potent HER2 inhibitors useful for targeting HER2 exon 21 insertion that is resistant to irreversible TKI generation 2 and 3.

I. Definition of

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

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.

The term "about" means the stated value. + -. 5%.

"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 pathology or symptomatology of the disease, (2) ameliorating the disease (e.g., reversing pathology and/or symptomatology) in a subject or patient experiencing or exhibiting pathology or symptomatology of the disease, and/or (3) effecting any measurable reduction of the disease in a subject or patient experiencing or exhibiting pathology or symptomatology of the disease. For example, treatment may comprise administering an effective amount of boertinib or afatinib.

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

As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or 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 this specification and/or claims means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" refers to the amount of the compound that, when administered to a subject or patient to treat or prevent a disease, is sufficient to effect such treatment or prevention of the disease.

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

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

The term "insertion" or "insertional mutation" refers to the addition of one or more nucleotide base pairs to a DNA sequence. For example, the HER2 exon 21 insertion mutation includes one or more insertions of 1-18 nucleotides between amino acids 832-883.

"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 test 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.

"detection", "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, rolling circle amplificationAmplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, 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/LCR (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).

HER2 exon 21 mutations

Certain embodiments of the present disclosure relate to determining whether a subject has one or more HER2 exon 21 mutations, in particular one or more insertion mutations shown in figure 2. The subject may have 2,3, 4, or more HER2 exon 21 mutations. 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, the exon 21 mutation is detected by DNA sequencing, e.g., from a tumor or circulating free DNA from plasma.

The HER2 exon 21 mutation may include one or more point mutations, insertions and/or deletions of 1-18 nucleotides between amino acids 832-883. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842, R868, and L869. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I, R868W, and L869R. In some aspects, the one or more HER2 exon 21 mutations are located at one or more residues selected from the group consisting of V842 and R868. In some aspects, the one or more exon 21 mutations are selected from the group consisting of V842I and R868W.

In certain aspects, the subject may have or develop a mutation at EGFR residue C797, which may result in resistance to TKIs (e.g., bosutinib). Thus, in certain aspects, a 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).

The patient sample can be any body tissue or body fluid that includes nucleic acid from a lung cancer of a subject.

In certain embodiments, the sample will be 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 purifiedTo isolate the DNA. All samples obtained from the subject, including those that have undergone any type of further processing, are considered to be 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 the HER2 exon 21 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, and/or detectably labeled probes for detecting a target genetic variation (e.g., HER2 exon 21 mutation) used in the operable treatment recommendations of the present disclosure. 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 for detecting a set of target genetic variations, such as HER2 exon 21 mutations^TM(Roche Molecular Systems, Pleasanton, Calif.). 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, AmpliSEQ can be employed^TM(Life Technologies/Ion Torrent, Carlsbad, Calif.) or TruSEQ^TM(Illumina, San Diego, Calif.).

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 a HER2 mutation in a sample comprises contacting nucleic acid from the sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding a mutant HER2 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 a HER2 mutation in a sample, the kit comprising an oligonucleotide that specifically hybridizes to or adjacent to a site of a mutation in the HER2 gene. The kit may further comprise instructions for treating a patient having a tumor comprising a HER2 insertion mutation with either poetinib or afatinib based on the results of a hybridization assay using the kit.

In another aspect, a method of detecting an exon 21 mutation in a sample comprises amplifying from said sample a nucleic acid corresponding to exon 21 of the HER2 gene or a 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 HER2 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 a mismatch of base pairs caused by point mutations, insertions and deletionsThe structure of (2) is distorted. 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 the use of a single protocol to identify point mutations, deletions and insertions determined directly from the PCR reaction eliminates the need for sample purification, thereby reducing hybridization time and improving 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 for treating or delaying progression of cancer in an individual comprising administering to a subject determined to have a HER2 exon 21 mutation (e.g., exon 21 insertion) an effective amount of bosutinib, afatinib, or a structurally similar inhibitor. The subject may have more than one HER exon 21 mutation.

Examples of cancers contemplated 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, lung pre-neoplastic 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 determining that administration of polazinib (also known as HM781-36B, HM781-36 and 1- [4- [4- [ (3, 4-dichloro-2-fluoroanilino) -7-methoxyquinazolin-6-yl ] -oxypiperidin-1-yl ] prop-2-en-1-one) to a subject having a mutation in exon 21 of HER2 polazinib is a quinazoline-based pan-HER inhibitor that irreversibly blocks signaling through tyrosine kinase receptors of the HER family, including HER1, HER2, and HER4, polazinib, or a structurally similar compound (e.g., U.S. patent No. 8,188,102 and U.S. patent publication No. 20130071452; incorporated herein by reference) can be used in the present methods.

The bosutinib, such as bosutinib hydrochloride, may be administered orally, e.g., in the form of a tablet. The bovatinib may be administered in a dose of 4mg-25mg, for example in a dose of 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg or 24 mg. Administration may be daily, every other day, every 3 days, or weekly. The administration may be on a continuous schedule, such as a 28 day cycle.

In some aspects, oxitinib may be administered to a subject having a T790 mutation (e.g., T790M), and chemotherapy and/or radiation therapy as described herein may be administered to a subject having a C797 mutation (e.g., C797S). Oxitinib, chemotherapy and/or radiotherapy may be administered alone or in combination with bosutinib. Oxitinib may be administered at a dose of 25mg to 100mg, e.g. about 40mg or 80 mg. Administration may be daily, every other day, every 2 days, every 3 days, or once a week. Oxitinib may be administered orally, e.g. in the form of a tablet.

Afatinib may be administered at a dose of 10mg-50mg, for example 10mg, 20mg, 30mg, 40mg or 50 mg. Afatinib may be administered daily, every other day, every 2 days, every 3 days, or once weekly. Afatinib may be administered orally, for example in the form of a tablet.

A. Pharmaceutical composition

Also provided herein are pharmaceutical compositions and formulations for determining subjects with a HER2 exon 21 mutation (e.g., exon 21 insertion) comprising bortinib or afatinib and a pharmaceutically acceptable carrier.

Can be prepared by mixing the extract with the desired purityThe active ingredient (e.g., antibody or polypeptide) is combined 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, such as sucrose, 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 embodiments of the invention relate to poecitinib or afatinib 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 bocetinib or afatinib 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 boertinib or afatinib is provided to the patient separately from the additional therapeutic agent, it is generally ensured that there is not a long period of time between each delivery so that the two compounds can still produce a beneficial combined effect on 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 examples below, the bocetinib or afatinib is "a" and the anti-cancer therapy is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A。

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. RituximabThis is an 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 delivery of the payload (drug) to the enrichment vehicle(ii) a "armed" MAb of tumor cells at the level 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 from 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 describedPDL2-Fc fusion soluble receptors 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 WO00/37504 (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 HER2 exon 21 mutations (such as those disclosed herein) are also within the scope of the present disclosure. An example of such a kit may include a set of exon 21 mutation specific primers. The kit may further comprise instructions for using the primers to detect the presence or absence of a particular HER2 exon 21 mutation described herein. The kit may further comprise instructions for diagnostic purposes indicating that a positive identification of the HER2 exon 21 mutation described herein in a sample from a cancer patient indicates sensitivity to the tyrosine kinase inhibitors bovatinib or afatinib or a structurally similar inhibitor. The kit may further comprise instructions that a positive identification of a HER2 exon 21 mutation described herein in a sample from a cancer patient indicates that the patient should be treated with boratinib or afatinib 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 the exon 21 mutation of HER2

The HER2 mutation is most common in bladder, gastric, and biliary tract cancers: to understand the diversity of HER2 mutations in different cancer types, several databases were queried, including cohorts (cowrt) from the cnbioportal, MD Anderson cancer center and basic Medicine company (Foundation Medicine) and cfDNA cohorts from Guardant Health. In all databases, all non-synonymous HER2 mutations were analyzed for 25 different cancer types (table 2). A weighted average frequency of HER2 mutations was calculated. Similar to what was observed in the AACR GENIE database (Meric-Bernstam et al, 2018), HER2 mutations were most common in bladder cancer (8.3%), cholangiocarcinoma (5.3%) and gastric cancer (4.5%) (fig. 1A); and HER2 exon 20 mutations were most common in small bowel cancer (1.8%), lung cancer (1.5%) and breast cancer (0.9%) (fig. 1B).

The HER2 mutation is most common in the tyrosine kinase domain of HER2, and the mutation hot spot varies with malignancy: next, the frequency of mutations in different regions of HER2 receptor reported by cbioport and MD Anderson was analyzed. In all cancer types, HER2 mutations were most common in the tyrosine kinase domain (46%), including mutations in exon 20 (20%), exon 19 (11%) and exon 21 (9%) (fig. 2A). Furthermore, the extracellular domain mutation accounted for 37% of the HER2 mutations. Among all the cancers queried, the most common HER2 mutations were p.s310f/Y (11.0%), p.y772_ a775dupYVMA (5.7%), p.l755p/S (4.6%), p.v842i (4.4%), and p.v777l/M (4.0%) (fig. 2E). In lung cancer, most of the HER2 mutations occurred within exon 20 (48%), with Y772_ a775dupYVMA accounting for 34% of all HER2 mutations (fig. 2B, 2F). In breast cancer, most of the HER2 mutations occurred within exon 19 (37%), with the L755 mutation being the most common, accounting for 22% of the HER2 mutations (fig. 2C). However, unlike lung cancer where one variant predominates, the mutation diversity of the exon 19 mutation is greater in breast cancer (fig. 2G). In colorectal cancer, HER2 mutations were most common in exon 21 (23%) and the extracellular domain (23%), with the V842I variant in exon 21 being most common (19%) (fig. 2D, 2H).

Y772dupYVMA is the most common HR2 exon 20 insertion mutation in all cancer types: the HER2 exon 20 mutation is the most common mutation within the HER2 tyrosine kinase domain (accounting for 16% of all HER2 mutations and 43% of tyrosine kinase domain mutations), and the HER2 exon 20 insertion mutation remains a clinical challenge. To understand the diversity and prevalence of exon 20 insertions, the incidence of HER2 exon 20 insertion sequences was analyzed by cancer type in the cbiportal, MD Anderson, and Guardant Health databases. Y772dupYVMA insertions were the most common HER2 exon 20 insertions, accounting for 70% of all HER2 exon 20 insertions, and p.g778dupgsp (14%) and p.g776del inspvc (9%) insertions were the second and third most common (fig. 9A). Exon 20 insertion mutations (N ═ 362) in NSCLC showed the greatest diversity of exon 20 insertion mutations (fig. 9B), and exon 20 insertion mutations (N ═ 30) in breast cancer showed the least diversity of the inserted sequences, with only three different variants reported (fig. 9C). Other rare insertion mutations were observed in other cancer types, but replication at Y772 and G778 occurred most frequently in each cancer type analyzed (fig. 9D).

A frequently detected change in HER2 is an activating mutation: to evaluate the functional impact of the common HER2 mutation, Ba/F3 cells were made to stably express the 16 most commonly detected HER2 mutations of exons 19, 20 and 21. It was found that 16 tested mutations in HER2 all induced IL-3 independent survival of Ba/F3 cells (FIGS. 3A-3C). Furthermore, expression of these 16 HER2 mutations resulted in expression of phosphorylated HER2 (fig. 10A), indicating that these mutations result in receptor activation.

Bosutinib is the most potent TKI tested and inhibits the most common HER2 mutation in vitro: although recent reports have highlighted the effectiveness of covalent quinazolinamine-based TKIs (i.e. afatinib, dactinib, bosertinib, lenatinib) in preclinical models of HER2 mutant disease, clinical studies with afatinib, dactinib, and lenatinib have lower ORR and cancer-specific and variant-specific differences in patient outcome. To systematically assess drug sensitivity of the most commonly detected HER2 variants, a panel of HER2 mutant Ba/F3 cells was screened against 11 covalent and non-covalent EGFR and HER2 TKI. The HER2 mutant showed robust resistance to the non-covalent inhibitors lapatinib and sapatinib (fig. 4A). Covalent TKI, ocitinib, ibrutinib and azatinib are ineffective in inhibiting the cellular viability of cells expressing the exon 20 mutation; however, these TKIs did show activity on cells expressing the D769 variant (fig. 4A). In contrast, the covalent quinazolinamine-based TKI, afatinib, lenatinib, dacatinib, tasotinib-TKI, and pozzatinib had inhibitory activity against HER2 mutants of all three exons (fig. 4A). Among all HER2 mutant variants and TKIs tested, boresinib had the lowest average IC₅₀And is significantly more effective than afatinib, lenatinib, or tasotinib-TKI in reducing cell viability (fig. 4B). Furthermore, although the efficacy of bosutinib for HER2 exon 19 and 20 mutations was significantly higher than afatinib, lenatinib, or tasotinib-TKI, the average IC for exon 21 mutants was higher₅₀There were no significant differences (fig. 4C-4E), indicating that the mutation position affects drug binding. Furthermore, within exon 19, the L755S and L755P variants had significant differences in drug susceptibility for all TKIs tested (fig. 4F), suggesting that specific amino acid changes at this site affect drug binding affinity.

HER2 mutation position and amino acid changes affect drug binding affinity: to further understand how mutation positions and amino acid changes can affect drug binding affinity and inhibitory potency, molecular dynamics simulations were used to examine how these mutations affect the structure and dynamics of the HER2 kinase domain. Molecular models of L755S, L755P, Y772dupYVMA, and V777L HER2 mutants (fig. 11A) were constructed using publicly available X-ray constructs (PDB 3PP0) as templates and subjected to accelerated molecular dynamics to increase protein conformation sampling. In these HER2 mutants, the range of protein conformations sampled, particularly with respect to the P-loop and α -C-helix positions, varied. Even between exon 20 mutations there is a clear difference, especially in the α -C-helical region, where the α -C-helical conformation changes in duration between "in" (active conformation with smaller binding pocket) and "out" (inactive conformation with larger binding pocket). The V777L mutant sampled largely the "outer" conformation, while the Y772dupYVMA mutant sampled both the "inner" and "outer" conformations (fig. 5A). Overall, these differences in conformational state resulted in the Y772dupYVMA mutant existing 10 times more frequently in the "in" conformation than the V777L mutant (fig. 5B), and, on average, the binding pocket size of Y772dupYVMA was smaller compared to V777L (fig. 5C and 11B). Furthermore, the smaller binding pocket of Y772drupYVMA compared to V777L may be responsible for the weaker potency of neratinib against Y772dupYVMA because neratinib contains pyridine rings oriented towards an α -C-helix.

Further analysis of HER2 mutant binding pocket volumes (fig. 10B) indicated that mutations of the same residues may have distinct effects on protein conformation. In particular, the mutated proline residue of L755P lacks a hydrogen bond donor that disrupts backbone hydrogen bonds between the β 3 and β 5 chains between L755 and V790, respectively. The lack of stability between these two β -strands results in β -sheet instability and structural rearrangement of the kinase hinge region (fig. 5D). In particular, the L800 residue of L755P protrudes into the active site, greatly reducing the pocket size. The conformational changes in the β 3 chain also cause inward collapse of the P-loop, further reducing pocket volume and making the mutant less susceptible to most TKIs. Furthermore, changes in hinge mobility may also play a role in kinase activation. These significant changes in the validation of the L755P mutant were in contrast to the performance of the L755S mutant, the conformation and pocket volume profile of the L755S mutant was more similar to wild-type HER2 (fig. 11B).

HER2 mutant human cancer cell line showed enhanced sensitivity to poezetinib: clinical studies detecting HER2 inhibitors showed cancer type-specific differences in drug sensitivity (Hyman et al, 2018). To determine whether the covalent, quinazolinamine-based TKIs were active in the HER2 mutant disease model, the group of EGFR/HER2 TKIs were tested in human cancer cell lines. Pre-neoplastic MCF10A mammary epithelial cells were transfected with the HER2 exon 20 mutation and evaluated for in vitro sensitivity to 12 EGFR/HER2 TKIs. MCF10A cells expressing the G776del insVC, Y772dupYVMA or G778dupGSP HER2 mutations were the most sensitive to bosutinib, IC₅₀Values were 12nM, 8.3nM and 4.5nM, respectively (FIGS. 6A-6C). In contrast, tasotinib-TKI and lenatinib produced average IC's of 21nM and 150nM, respectively₅₀Values (FIGS. 6A-6C) indicating 2.6-fold and 19-fold (p) potency of Boletinib as tasotinib-TKI and lenatinib, respectively<0.001). Furthermore, western blotting of MCF10AHER 2G 776delinsVC cells with bosutinib and lenatinib showed that 10nM of bosutinib (but not lenatinib) completely inhibited p-HER2 (fig. 12A). Since wild-type (WT) HER2 did not transform Ba/F3 cells to grow independently of IL-3, MCF10A cells were used to determine the selectivity of TKI for mutant HER2 compared to WT HER 2. To this end, the selectivity index (SI, mutant IC) for each inhibitor was calculated₅₀value/WT IC₅₀Values), and found that pozitinib was the most mutation selective TKI tested in MCF10A cell line (SI ═ 0.028), followed by pyrroltinib (SI ═ 0.063) and tasotinib-TKI (SI ═ 0.111) (fig. 6D). Consistent with the data obtained using Ba/F3 cells (fig. 3C), the differences in sensitivity between bosutinib, tasotinib-TKI and lenatinib were less pronounced but significant (p ═ 0.02 and p ═ 0.0004) in the HER2 exon 19 mutant colorectal cancer (CW-2) model, with average IC (p ═ 0.02 and p ═ 0.0004), with the data obtained using Ba/F3 cells (fig. 3C)₅₀Values were 3.19nM, 4.24nM and 68.8nM, respectively (FIG. 6E). Furthermore, on day 21, on a xenografted mouse model of CW-2 colorectal cells, the animals treated with bosutinib (5mg/kg) showed a 58% reduction in tumor volume compared to the vehicle treated group (p ═ 0.011). In contrast, animals treated with neratinib (30mg/kg) showed an increase in tumor volume (28%; compared to vehicle control (p ═ 0.023)) Furthermore, afatinib (20mg/kg) treatment did not significantly affect tumor growth compared to vehicle control (fig. 6F, 13).

Bosutinib has anti-tumor activity in HER2 mutant NSCLC patients: based on these preclinical data and previously published work on exon 20 mutations (Robichaux et al, 2018), a phase II clinical trial initiated by one investigator on EGFR and HER2 exon 20 mutant NSCLC (NCT03066206) has been initiated. Patients were treated with 16mg of bosutinib orally once a day until progression, death or withdrawal. Objective responses were assessed every eight weeks according to RECIST v 1.1. Of the first 12 evaluable patients carrying the exon 20 insertion mutation of HER2, 6/12 (50%) had the best response of Partial Response (PR). After 2 months, repeated scans of 5/12 confirmed this response (confirmed objective response rate, 42%) (fig. 7A). Of these 12 patients, 2 patients had disease Progression (PD) at first response assessment, resulting in 83% Disease Control Rate (DCR). By 12 months 2018, 10 of 12 patients progressed, with median PFS of the first 12 patients being 5.6 months (fig. 7B). To date, all patients enrolled in the study carried one of the two most common HER2 exon 20 insertions (Y772dupYVMA and G778dupGSP) (fig. 7A). Representative images of one NSCLC patient with the Y772dupYVMA mutation before and after treatment (8 weeks) showed robust tumor shrinkage of the right lung (fig. 7C). Patient characteristics including previous treatment line numbers can be seen in table 3. In addition, one NSCLC patient carrying the HER2 exon 19 point mutation (L755P) who received a number of pre-treatments received an isosexual regimen (C-IND 18-0014). The patient received 16mg of bosutinib daily and the tumor shrunk by four weeks (fig. 7D, white frame). Patient Stable Disease (SD) (-12% reduction in target lesions) according to RECIST v 1.1. The patient continued to receive pazitinib for more than 7 months under disease control until imaging indicated disease progression and discontinued pazitinib. The patient was in good clinical condition at the end of the treatment with bosutinib and continued to receive further systemic treatment.

The combination treatment of the bosutinib and the T-DM1 enhances the anti-tumor activity: HER2 TKI lapatinib studies previously performed in a HER2 positive breast cancer model and in the EGFR processEGFR inhibitor studies in a degenerative NSCLC model have shown that TKI treatment results in increased cell surface receptor accumulation and increased cell surface HER2/EGFR increases sensitivity to antibody-dependent cellular cytotoxicity (ADCC). To determine whether wave-zitinib treatment increased total HER2 receptor expression on the cell surface, cell surface HER2 expression was analyzed by FACS 24 hours after low dose wave-zitinib treatment. It was found that on average, bosutinib treatment increased cell surface HER2 expression 2-fold (fig. 8A, p)<0.0001). Next, it was examined whether the combination of bosutinib and T-DM1 reduced cell viability in vitro and found that, although T-DM1 alone did not inhibit cell viability of MCF10A HER2 mutant cell lines, the combination of T-DM1 and bosutinib resulted in IC₅₀The values were significantly lower than each agent alone, dose-dependent (fig. 8B). To validate these results in vivo, low dose of bosutinib in combination with a single dose of T-DM1 was tested in the HER2 mutant NSCLC PDX model (HER 2Y 772dupYVMA) (fig. 8C). To assess response to treatment, Progression Free Survival (PFS), defined as the time from optimal response to tumor doubling, was determined. Median pfs (mPFS) was 3 days for mice receiving vehicle control, while mPFS was 15 days and 27 days for mice receiving low dose of politinib or T-DM1, respectively. However, mice (14/20) treated with a single dose of T-DM1 in combination with low dose of bosutinib remained tumor-free at day 45 (fig. 8D). Furthermore, at the optimal response (day 15), the combination of low dose of pozzatinib (2.5mg/kg) and single dose of T-DM1(10mg/kg) resulted in complete tumor regression in 20/20 mice (100%) compared to 2/9 mice receiving T-DM1 alone or 0/12 mice receiving low dose of pozzotinib (fig. 8C-8F). By day 30, tumor growth resumed in all mice receiving T-DM1 alone; however, there were no signs of tumor recurrence in 14/20 mice receiving combination therapy (fig. 8F, 8G).

Here, HER2 mutations that occur in various tumor types were reported, but the specific mutation hot spots varied from malignancy to malignancy. Furthermore, the sensitivity to HER2 TKIs heterogeneous at the mutation site, with HER2 exon 20 insertions and the L755P mutation being resistant to most HER2 TKIs, probably due to a reduced drug binding pocket volume. Furthermore, bosutinib was identified as a potent pan-HER 2 mutation selective inhibitor with clinical efficacy in NSCLC patients carrying a HER2 exon 20 insertion and a L755P mutation. Finally, it was recognized that bosutinib treatment induced the accumulation of HER2 on the cell surface, and that the combination of bosutinib and T-DM1 enhanced antitumor activity in vitro and in vivo.

Pan-cancer analysis showed that the HER2 mutation hotspot varied with cancer type and had different sensitivity to HER2 TKI in vitro, which may affect clinical efficacy. In the sumit test, lenatinib was most effective in breast cancer patients, and most of the responders were positive for mutations L755S, V777L, or L869R. These mutations and low IC in vitro Ba/F3 drug screening₅₀The values are correlated. In contrast, colorectal cancer patients do not respond to lenatinib. Consistent with this clinical observation, the V842I mutation was found to be the most common HER2 mutation in colorectal cancer cases, and this specific mutation was insensitive to neratinib in drug screening assays. These data indicate that the different TKI sensitivities between malignancies can be explained in part by the cancer-specific mutational hot spots, which directly affect drug sensitivity. However, there are still key issues as to why the distribution of HER2 mutations varies from tumor type to tumor type and whether a given mutation produces a similar drug response in different tumor types. Data from the sumit assay show that although specific exon 20 insertions are associated with lenatinib sensitivity in breast cancer patients, these same mutations are associated with drug resistance in all other cancer types, demonstrating that there may be potential mechanisms for these tumor type-specific sensitivity differences that could be worth further investigation.

The exon 20 insertion mutation and the exon 19L755P mutation were resistant to most HER2 TKIs. In vitro drug screening showed that exon 20 insertion mutation and L755P mutation had the highest IC for each TKI detected₅₀The value is obtained. Molecular dynamics simulations show that these mutation-induced conformational changes affect the overall size and mobility of the drug binding pocket. In conclusion, these in vitro and in silico results are consistent with clinical observations that patients with the exon 20 insertion mutation of HER2 have historically responded poorly to TKI. Frequent insertions in exon 20Of lung cancer in patients carrying a HER2 exon 20 insertion mutation have 0%, 11.5% and 18.2% -18.8% response rates to lenatinib, dacatinib and afatinib, respectively. Furthermore, although the L755S mutation has been shown to be responsive to neratinib, the L755P mutation is very resistant to both TKI and antibody-drug conjugates.

Example 2 materials and methods

Prevalence and variation frequency analysis of HER2 mutation: to determine the frequency of each HER2 mutation reported in the MD Anderson cancer center, cbioport, basic medical company, or Guardant Health databases, each database was queried individually, then the frequencies were weighted according to the total number of patients in each database and reported as a weighted average. To determine the frequency of HER2 mutations for different cancer types in cbioport, all non-overlapping studies were selected and derived. For the overlap study, only the largest dataset was used. To determine the HER2 mutation frequency in the MD Anderson Cancer center, the databases of the individualized Cancer treatment Institute (Institute for Personalized Cancer Therapy) were queried for all HER2 mutations independent of Cancer type. To determine the frequency of HER2 exon 20 mutations from basic medicine companies, de-identification data of patients with HER2 deletions, frame shifts, insertions and point mutations were tabulated and cancer types with fewer than 5 mutations were excluded. Finally, to determine the frequency of HER2 exon 20 mutations in Guardant Health, samples with ERBB2 exon 20 mutations tested during the 5 months of 2015 10 to 2018 (a panel of 70 and 73 genes) in the Guardant360 clinical database were queried.Is a comprehensive cfDNA NGS test certified by CLIA and CAP/NYSDOH, which reports SNV, insertion deletion (indel) and fusion, and the SNV has 73 genes. The frequencies reported by Guardant Health were then normalized to the clinical sensitivity correction reported by Odegaard et al, 2018. Specifically, frequency was divided by the percent clinical sensitivity (85.9%).

Ba/F3 cell line production and IL-3 deprivation: the Ba/F3 cell line was established as described previously (Robichaux et al, 2018). Briefly, the Ba/F3 cell line was transduced by reverse transcription for 12 hours to generate a stable Ba/F3 cell line. Retroviruses were generated by transfecting the pBabe-Puro-based vector summarized in Table 1(Addge and BioInnovase) into Phoenix293T-ampho cells (Orbigen) using Lipofectamine 2000 (Invitrogen). 3 days after transduction, 2. mu.g/ml puromycin (Invitrogen) was added to the RPMI medium. After 5 days of selection, cells were stained with FACS-sorted FITC-HER2 (Biolegend). 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 lines were maintained in IL-3 free RPMI-1640 medium with 10% FBS.

Cell viability assay and IC₅₀And (3) estimating: cell viability was determined as described previously using the Cell Titer Glo assay (Promega) (robichoux et al, 2018). Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in triplicate. Cells were treated with 7 different concentrations of tyrosine kinase inhibitor or vehicle alone to a final volume of 40 μ L/well. After 3 days, 11. mu.L of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes and bioluminescence was determined using a FLUOstar OPTIMA multimodal microplate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO-treated cells and normalized values were plotted in GraphPad Prism using non-linear regression fitted to normalized data with variable slopes. IC at 50% inhibition calculated by GraphPad Prism₅₀The value is obtained.

ELISA for phosphorylation and total HER2 and conjugation to IC₅₀Correlation of values: the proteins were harvested from the parental Ba/F3 cell line and each Ba/F3 cell line expressing the HER2 mutation as described above. Mu.g/ml protein was added to each ELISA plate and the ELISA was performed as described in the manufacturer's instructions for phosphorylated HER2(Cell Signaling, #7968) and total HER2(Cell Signaling, # 7310). The relative p-HER2 expression was determined by the ratio of p-HER2 to total HER2 as determined by ELISA. The relative p-HER2 ratio was compared to the calculated Bo-zitinib IC as described above₅₀Values are plotted. Pearson correlations and p-values were determined by GraphPad Prism.

Tyrosine kinase inhibitors and T-DM 1: all inhibitors were purchased from seleck Chemical, except EGF816 and pyrroltinib, which were purchased from MedChem Express. All inhibitors were dissolved in DMSO at a concentration of 10mM and stored at-80 ℃. Inhibitors were limited to two freeze-thaw cycles before being discarded. T-DM1 was purchased from the MD Anderson cancer center facility pharmacy and reconstituted.

Molecular dynamics simulation: the protein structure model of the HER2 mutant was constructed by introducing in silico mutations in the PDB 3PP 0X-ray structure using the MOE computer program (Chemical Computing Group). The NAMD simulation software package was used for classical and accelerated molecular dynamics simulations. The supplemental information portion provides more detailed information.

Human cell lines: MCF10A cells were purchased from ATCC and cultured in DMEM/F12 medium supplemented with 1% penicillin/streptomycin, 5% horse serum (sigma), 20ng/ml EGF, 0.5mg/ml hydrocortisone and 10. mu.g/ml insulin. A stable cell line was established by reverse transcription transduction and the pBabe-Puro-based vector summarized in Table 1(Addge and BioInnovartise) was transfected into Phoenix293T-ampho cells (Orbigen) using Lipofectamine 2000(Invitrogen) to generate retroviruses. 2 days after transduction, puromycin (Invitrogen) at 0.5. mu.g/ml was added to the RPMI medium. After 14 days of selection, cells were detected in the cell viability assay described above. CW-2 cells were supplied by the Riken cell line database of MTA and stored in RPMI containing 10% FBS and 1% penicillin/streptomycin.

In vivo xenograft study: by mixing 1X10 in 50% matrigel⁶Cell injection into 6-week-old female nu/nu nude mice establishment of CW-2 cell line xenografts. When the tumor reaches 350mm³At time, mice were randomly assigned to 4 groups: 20mg/kg Afatinib, 5mg/kg bositinib, 30mg/kg lenatinib or vehicle control (0.5% methylcellulose, 2% Tween 80 dH)₂O solution). Tumor volumes were measured three times per week. Mice received drug on monday through friday (5 days per week), but dosing was started on wednesday, with 2 days of drug withdrawal allowed after the first 3 days of dosing.

Y772dupYVMA PDX mice were purchased from Jax Labs (model # TM 01446). Will be from expressing HER 2Y 772dupYVMAIs inoculated into 5 to 6 week old female NSG mice (Jax Labs # 005557). Mice were measured 3 times per week when tumor volume reached 200mm³-300mm³At time, mice were randomly assigned to 4 treatment groups: vehicle control (0.5% methylcellulose, 0.05% tween 80 dH₂O solution), 2.5mg/kg of bosutinib, 10mg/kg of T-DM1, or a combination of 2.5mg/kg of bosutinib and 10mg/kg of T-DM 1. Tumor volume and body weight were measured three times per week. 2.5mg/kg of Bozitinib-treated mice received oral administration one to five weeks (5 days per week). On the randomized days, 10mg/kg T-DM1 treated mice received an Intravenous (IV) dose of T-DM 1. Mice treated with the combination of bosutinib and T-DM1 received an IV dose of T-DM1 and 2.5mg/kg bosutinib at 5 days per week starting 3 days after T-DM1 administration. Mice were allowed to receive drug holidays if they lost more than 10% or less than 20g of body weight. Progression-free survival was defined as tumor doubling from the optimal response measured twice in succession. Complete regression is defined as a reduction in tumor burden of greater than 95%, and for mice with complete tumor regression, tumor doubling is defined as a doubling of greater than 75mm measured two or more consecutive times³. The experiments were completed in accordance with Good Animal Practices (Good Animal Practices) and were approved by the institutional Animal care and use committee (Houston, TX) for MD Anderson cancer.

Table 1: vectors for generating stable cell lines

Table 2: the total number of patients classified by cancer type in each database.

Table 3: patient characteristics and number of lines of prior treatment.

FACS: MCF10A cells overexpressing the HER2 mutation were plated overnight in 6-well plates and then treated with 10nM of polazitinib. After 24 hours, the cells were washed twice with PBS and trypsinized. The cells were then resuspended in PBS containing 0.5% FBS and then stained with Biolegend anti-HER 2-FITC antibody (#324404) on ice for 45 minutes. Cells were washed twice with PBS containing 0.5% FBS and analyzed by flow cytometry. IgG and unstained controls were used for gating.

Western blotting: for western blotting, cells were washed in PBS and then lysed in RIPPA lysis buffer (ThermoFisher) and mixed tablet with protease inhibitor (Roche). Proteins (30. mu.g-40. mu.g) were loaded onto gels purchased from BioRad. Detection was performed using a BioRad semi-dry transfer tank, followed by antibodies against pHER2, HER2, pPI3K, PI3K, p-AKT, p-ERK1/2 and ERK1/2(1: 1000; Cell Signaling). Blots were detected with anti-vinculin or β -actin antibody (Sigma-Aldrich) as loading control and exposed using ECL western blot substrate (Promega).

HER2 expression level and Ba/F3 mutant IC₅₀The correlation of (a): proteins were harvested from Ba/F Cell lines and ELISA was performed on total HER2(Cell Signaling, #7310) according to the manufacturing instructions. The relative expression determined by ELISA was directed to the IC calculated as described above₅₀Values are plotted. Pearson correlations and p-values were determined by GraphPad Prism.

Clinical trial and CIND identifier: patients provided written informed consent to treatment with bosutinib in a syngeneic regimen (MD Anderson cancer center CIND-18-0014) or clinical trial NCT 03066206. The protocol was approved by the MD Anderson cancer central agency review board and the U.S. food and drug administration.

***

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.

Reference to the literature

The following references are specifically incorporated by reference herein to the extent that they provide exemplary procedures or other details that supplement those set forth herein.

Arcila et al.，Clin Cancer Res 18：4910-8，2012.

Arcila et al.，Mol Cancer Ther 12(2)：220-229，2013.

Austin-Ward and Villaseca，Revista Meddica de Chile，126(7)：838-845，1998.

Ausubel et al.，Current Protocols in Molecular Biology，John Wiley&amp；Sons，New York，NY，2003.

Bukowski et al.，Clinical Cancer Res.，4(10)：2337-2347，1998.

Camacho et al.J Clin Oncology 22(145)：Abstract No.2505(antibody CP-675206)，2004.

Cha et al.lnt J Cancer 130：2445-54，2012.

Chee et al.，Science，274：610-614，1996.

Cho et al.，Cancer Res 73：6770-9，2013.

Christodoulides et al.，Microbiology，144(Pt 11)：3027-3037，1998.

Church and Gilbert，Proc.Natl.Acad.Sei.USA 81：1991-1995(1988).

Cotton et al.，Proc.Natl.Acad.Sci.USA 85：4397-4401(1985).

Davidson et al.，J.Immunother.，21(5)：389-398，1998.

Davies et al.，Plos One8，2013.

Del Tito et al，Clinical Chemistry 44：731-739，1998.

Drmanac et al.，Nat.Biotechnol.，16：54-58，1998.

Drmanac et al.，Science，260：1649-1652，1993.

Ettinger et al.，J Natl Compr Canc Netw 16：807-21，2018.

Flavell et al.，Cell 15：25(1978).

Fu et al.，Nat.Biotechnol.，16：381-384，1998/

Geever et al.，Proc.Natl.Acad.Sci.USA 78：5081(1981).

Hanibuchi et al.，Int.J.Cancer，78(4)：480-485，1998.

Hellstrand et al.，Acta Oncologica，37(4)：347-353，1998.

Hollander，Front.Immun.，3：3，2012.

Hong et al.，J Biol Chem 282：19781-7，2007.

Hui and Hashimoto，Infection Immun.，66(11)：5329-5336，1998.

Hurwitz et al.Proc Natl Acad Sci USA 95(17)：10067-10071，1998.

Hyman et al.，Nature 554：189-94，2018.

International patent publication No. WO99/57318

International patent publication No. WO1995001994

International patent publication No. WO1998042752

International patent publication No. WO2000037504

International patent publication No. WO2001014424

International patent publication No. WO2009/101611

International patent publication No. WO2009/114335

International patent publication No. WO2010/027827

International patent publication No. WO2011/066342

International patent publication No. WO2015016718

International patent publication No. WO00/37504

International patent publication No. WO01/14424

International patent publication No. WO98/42752

Kosaka et al.，Cancer Res 2017.

Kris et al.，Ann Oncol 26：1421-7，2015.

Kris et al.，Ann Oncol 26：1421-7，2015.

Leal，M.，Ann N Y Acad Sci 1321，41-54，2014.

Lynch et al.，N Engl J Med.350(21)：2129-2139，2004.

Ma et al.，J Clin Oncol 33，2015.

Maemondo et al.，N Engl J Med 362：2380-8，2010.

Meric-Bernstam et al.，Clin Cancer Res，2018.

Mitsudomi and Yatabe，Cancer Sci.98(12)：1817-1824，2007.

Mokyr et al.Cancer Res 58：5301-5304，1998.

Oxnard et al.，J Thorac Oncol.8(2)：179-184，2013.

Paez et al.，Science 304(5676)：1497-1500，2004.

Pao et al.，Proc Natl Acad Sci USA 101(36)：13306-13311，2004.

Pardoll，Nat Rev Cancer，12(4)：252-64，2012.

Perera et al.，Proc Natl Acad Sci U S A 106：474-9，2009.

Qin et al.，Proc.Natl.Acad.Sci.USA，95(24)：14411-14416，1998.

Raca et al.，Genet Test 8(4)：387-94(2004).

Robichaux et al.，Nat Med 24：638-46，2018.

Sanger et al.，Proc.Natl.Acad.Sci.USA 74：5463-5467(1977).

Sears et al.，Biotechniques，13：626-633，1992.

Sheffield et al.，Proc.Natl.Acad.Sci.USA 86：232-236(1989).

Shen et al.，J Recept Signal Transduct Res 36：89-97.2016.

Thress et al.，Nat Med 21：560-2，2015.

U.S. Pat. No. 4,870,287

U.S. Pat. No. 5,288,644

U.S. Pat. No. 5,739,169

U.S. Pat. No. 5,760,395

U.S. Pat. No. 5,801,005

U.S. Pat. No. 5,824,311

U.S. Pat. No. 5,830,880

U.S. Pat. No. 5,844,905

U.S. Pat. No. 5,846,945

U.S. Pat. No. 5,869,245

U.S. Pat. No. 5,885,796

U.S. Pat. No. 6,207,156

U.S. Pat. No. 8,008,449

U.S. Pat. No. 8,017,114

U.S. Pat. No. 8,119,129

U.S. Pat. No. 8,188,102

U.S. Pat. No. 8,329,867

U.S. Pat. No. 8,354,509

U.S. Pat. No. 8,735,553

U.S. patent publication No. 2004/0014095

U.S. patent publication No. 2005/0260186

U.S. patent publication No. 2006/0104968

U.S. patent publication No. 20110008369

U.S. patent publication No. 20130071452

U.S. patent publication No. 2014022021

U.S. patent publication No. 20140294898

Underhill et al.，Genome Res.7：996-1005(1997).

Vogel et al.，J Clin Oncol 20：719-26，2002.

Yang et al.，Int J Cancer 2016.

Yasuda et al.，Sci Transl Med 5(216)：216ra177，2013.

Zimmerman et al.，Methods Mol.Cell.Biol.，3：39-42，1992.

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