Methods for treating gefitinib-resistant cancer

Abstract

The present invention relates to a method for treating gefitinib and / or erlotinib resistant cancer. Cancer patients are monitored for cancer development after treatment with gefitinib and / or erlotinib. The development of cancer indicates that the cancer is resistant to gefitinib and / or erlotinib. Once it is shown that the cancer is developing, a pharmaceutical composition containing an irreversible epidermal growth factor receptor (EGFR) inhibitor is administered to the patient. In a preferred embodiment, the irreversible EGFR inhibitor is EKB-569, HKI-272 and HKI-357.

Description

[0001] The present application is a divisional application of an invention patent application with an application date of February 2, 2006, application number 201810778846.6, and name “Method for treating gefitinib-resistant cancer”. Background of the Invention

[0002] Epithelial cell cancers, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, spleen cancer, testicular cancer, thymic cancer, etc., are diseases characterized by the abnormal accelerated growth of epithelial cells. This accelerated growth initially causes tumor formation. Finally, transfer to different organ positions may also occur. Although progress has been made in the diagnosis and treatment of multiple cancers, these diseases still result in significant mortality.

[0003] Lung cancer is the leading cause of cancer death in industrial countries. Cancer that begins in the lungs is divided into two main types, non-small cell lung cancer and small cell lung cancer, according to the performance of cells under a microscope. Non-small cell lung cancer (squamous cell carcinoma, adenocarcinoma and large cell cancer) spreads to other organs generally slower than small cell lung cancer. About 75% of lung cancer cases are classified as non-small cell lung cancer (e.g., adenocarcinoma), while the other 25% of lung cancer cases are small cell lung cancer. Non-small cell lung cancer (NSCLC) is the main cause of cancer death in the United States, Japan and Western Europe. For patients with advanced disease, chemotherapy provides moderate help in terms of survival rate, but pays the price of significant toxicity, and strongly requires the therapeutic agent (Schiller JH etc., N Engl J Med, 346:92-98,2002) that specifically targets the key gene damage involved in tumor growth.

[0004] Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) membrane-bound protein expressed on the surface of epithelial cells. EGFR is a family of growth factor receptors for many protein tyrosine kinases (a group of cell cycle regulatory molecules). (WJ Gullick et al., 1986, Cancer Res., 46: 285-292). When the ligand of EGFR (or EGF or TGF-α) binds to the extracellular domain, EGFR is activated, resulting in the autophosphorylation of the intracellular tyrosine kinase domain of the receptor (S. Cohen et al., 1980, J. Biol. Chem., 255: 4834-4842; ABS Schreiber et al., 1983, J. Biol. Chem., 258: 846-853).

[0005] EGFR is a growth-promoting oncogene, the protein product of erbB or ErbBl, which is a member of the ERBB family of protooncogenes, and is believed to play a key role in the occurrence and development of many human cancers. In particular, enhanced expression of EGFR is observed in breast cancer, bladder cancer, lung cancer, head cancer, neck cancer, gastric cancer, and glioblastoma. The ERBB family of oncogenes encodes four structurally related transmembrane receptors, namely EGFR, HER-2 / neu (erbB2), HER-3 (erbB3), and HER-4 (erbB4). Clinically, it has been reported that ERBB oncogene amplification and / or receptor overexpression in tumors are associated with disease recurrence and poor patient prognosis, as well as with the response to therapy. (L. Harris et al., 1999, Int. J. Biol. Markers, 14: 8-15; and J. Mendelsohn and J. Baselga, 2000, Oncogene, 19: 6550-6565).

[0006] EGFR is composed of three basic domains, namely the extracellular domain (ECD, which is glycosylated and contains a ligand-binding pocket with two cysteine-rich regions; a short transmembrane domain and an intracellular domain with intrinsic tyrosine kinase activity. The transmembrane region connects the ligand-binding domain to the intracellular domain. Amino acid and DNA sequence analysis and studies on the non-glycosylated form of EGFR have shown that the main chain mass of the EGFR protein is 132 kDa and has 1186 amino acid residues (ALUllrich et al., 1984, Nature, 307:418-425; J. Downward et al., 1984, Nature, 307:521-527; C. R. Carlin et al., 1986, MoI. Cell. Biol., 6:257-264; and FLV Mayes and MD Waterfield, 1984, The EMBO J., 3:531-537).

[0007] The binding of EGF or TGF-α to EGFR activates the signal transduction pathway and leads to cell proliferation. The dimerization, conformational changes and internalization of EGFR molecules play a role in conducting intracellular signals, causing cell growth regulation (G. Carpenter and S. Cohen, 1979, Ann. Rev. Biochem., 48: 193-216). Genetic changes that affect the regulation of growth factor receptor function or lead to overexpression of receptors and / or ligands cause cell proliferation. In addition, it has been determined that EGFR plays a role in cell differentiation, enhancement of cell motility, protein secretion, new blood vessel formation, invasion, metastasis and drug resistance of cancer cells to chemotherapeutic agents and radiation. (M.-J. Oh et al., 2000, Clin. Cancer Res., 6: 4760-4763).

[0008] A variety of EGFR inhibitors have been identified, including some that are already in clinical trials for the treatment of various cancers. For a recent overview, see de Bono, JS and Rowinsky, EK (2002), "The ErbB Receptor Family: A Therapeutic Target For Cancer", Trends in Molecular Medicine, 8, S 19-26.

[0009] A group of promising targets for therapeutic intervention in cancer treatment include members of the HER-kinase axis. They are often upregulated in solid epithelial tumors such as prostate, lung and breast tumors, and also upregulated in glioblastoma. The epidermal growth factor receptor (EGFR) is a member of the HER-kinase axis and has been a target of choice for the development of a variety of different cancer therapies. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are among these therapies because reversible phosphorylation of tyrosine residues is required for EGFR pathway activation. In other words, EGFR-TKIs block cell surface receptors that are responsible for triggering and / or maintaining cell signaling pathways that cause tumor cell growth and differentiation. In particular, it is believed that these inhibitors interfere with the EGFR kinase domain, called HER-I. Among the more promising EGFR-TKIs, there are three series of compounds: quinazolines, pyridopyrimidines, and pyrrolopyrimidines.

[0010] Two more advanced compounds in clinical development include Gefitinib (compound ZD 1839 developed by AstraZeneca UK Ltd.; available under the trade name IRESSA; hereinafter "IRESSA") and Erlotinib (compound OSI-774 developed by Genentech, Inc. and OSI Pharmaceuticals, Inc., available under the trade name TARCEVA; hereinafter "TARCEVA"); both have produced encouraging clinical results. Conventional cancer treatment with both IRESSA and TARCEVA involves oral administration of no more than 500 mg of each compound per day. In May 2003, IRESSA was the first of these products to enter the U.S. market, when it was approved for the treatment of patients with advanced non-small cell lung cancer.

[0011] IRESSA is an orally active quinazoline that works by directly inhibiting tyrosine kinase phosphorylation on the EGFR molecule. It competes for adenosine triphosphate (ATP) binding sites, resulting in inhibition of the HER-kinase axis. The exact mechanism of the IRESSA reaction is not fully understood, however, studies suggest that the presence of EGFR is a necessary prerequisite for its action.

[0012] A significant limitation in the use of these compounds is that their recipients may develop resistance to their therapeutic effects after an initial response to therapy, or they may not respond to EGFR-TKIs to any detectable degree. Response rates to EGFR-TKIs vary among different ethnic groups. At the low end of EGFR-TKI responders, in certain populations, only 10-15% of patients with advanced non-small cell lung cancer respond to EGFR kinase inhibitors. Therefore, a better understanding of the molecular mechanisms underlying the sensitivity of IRESSA and TARCEVA will greatly benefit targeted therapies for those patients most likely to benefit from such therapies.

[0013] There is a clear need in the art for satisfactory treatment of cancer, and particularly epithelial cell cancers, such as lung cancer, ovarian cancer, breast cancer, brain cancer, colon cancer and prostate cancer, which incorporates the benefits of TKI therapy and overcomes the non-response exhibited by patients. Such treatment can have a profound impact on the health of patients, and particularly elderly patients, in whom cancer is particularly common. Brief description of the invention

[0014] The inventors of the present invention have surprisingly found that irreversible EGFR inhibitors are effective in treating cancer in patients who no longer respond to gefitinib and / or erlotinib therapy. Therefore, in one embodiment, the present invention provides a method for treating gefitinib and / or erlotinib-resistant cancer. In this embodiment, the development of patient cancer is monitored at a time point after the initial administration of gefitinib and / or erlotinib treatment. The development of cancer is an indication of a cancer that is resistant to gefitinib and / or erlotinib treatment, and a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor is administered to the patient.

[0015] In a preferred embodiment, the irreversible EGFR inhibitor is EKB-569, HKI-272 or HKI-357. Alternatively, the irreversible EGFR inhibitor may be any compound that binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0016] The development of cancer can be monitored using methods well known to those skilled in the art. For example, the development of cancer can be monitored by visual inspection of the cancer, such as X-rays, CT scans, or MRI. Alternatively, the development of cancer can be monitored by detecting tumor biomarkers.

[0017] In one embodiment, the patient is monitored at multiple time points throughout cancer treatment. For example, by analyzing the progression of the cancer at a second time point and comparing this analysis with the analysis at a first time point, the progression of the cancer can be monitored. The first time point can be before or after initial treatment with gefitinib and / or erlotinib, and the second time point is after the first point. Increased growth of the cancer indicates progression of the cancer.

[0018] In one embodiment, the development of cancer is monitored by analyzing the size of the cancer. In one embodiment, the size of the cancer is analyzed by visual inspection of the cancer using X-rays, CT scans, or MRI. In one embodiment, the size of the cancer is monitored by detecting tumor biomarkers.

[0019] In one embodiment, the cancer is an epithelial cell cancer. In one embodiment, the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

[0020] In one embodiment, the size of the cancer is monitored at additional time points, and the additional time points are subsequent to the second time point.

[0021] In one embodiment, the later time point is at least 2 months after the previous time point. In one embodiment, the later time point is at least 6 months after the previous time point. In one embodiment, the later time point is at least 10 months after the previous time point. In one embodiment, the later time point is at least one year after the previous time point.

[0022] In another embodiment, the present invention provides a method for treating cancer, the method comprising administering a pharmaceutical composition comprising an irreversible EGFR inhibitor to a patient having a mutation in EGFR, i.e., a methionine substitution of the threonine at position 790 (T790M) of SEQ ID. NO. 1. The T790M mutation confers resistance to treatment with gefitinib and / or erlotinib.

[0023] A brief description of the graph

[0024] Figure 1A-1B Shown are EGFR sequence analysis in recurrent metastatic lesions from two NSCLC patients with acquired gefitinib resistance. Figure 1A Sequence analysis of Case 1 is shown. The T790M mutation in EGFR is present in recurrent liver lesions after the development of clinical gefitinib resistance. (Left) No mutation was detected in the primary lung lesion at the time of diagnosis. (Right) Both the primary lung tumor and the recurrent liver lesion harbor the L858R gefitinib-sensitive mutation. It is noteworthy that the L858R mutation is present in both the primary and recurrent lesions at the expected heterozygous mutation ratio, however T790M is detectable at low levels compared to the wild-type allele. The polymorphism (G / A) shown in the same tracing demonstrates the equivalent reproduction of the two alleles in the non-clonal PCR product (SEQ ID NOS 3&4 are disclosed in order of appearance, respectively). Figure 1B Sequence analysis of case 2 is shown. The T790M mutation was present in a minority of gefitinib-resistant cells. (Left) The T790M mutation was not detected by sequencing non-clonal PCR products either in the lung primary tumor or in eight recurrent liver lesions from this case. Heterozygosity at adjacent polymorphisms (G / A) confirmed amplification of both EGFR alleles from these samples. The heterozygous gefitinib-sensitive mutation, L861Q, was detected in the primary lung tumor as well as in each of the eight recurrent liver lesions in the expected proportion (SEQ ID NOS 3 & 5 are disclosed in order of appearance, respectively).

[0025] Figure 2A-2C We show that bronchoalveolar carcinoma cell lines acquire resistance to gefitinib and persistent sensitivity to irreversible ERBB family inhibitors. Figure 2AShown is the inhibition of proliferation of bronchoalveolar cancer cell lines with wild-type EGFR (NCI-Hl 666) with an activating delE746-A750 mutation in EGFR (NCI-Hl 650) or two representative gefitinib-resistant subclones of NCI-Hl 650 (G7 and Cl 1) by tyrosine kinase inhibitors. The effect of the reversible inhibitor gefitinib was compared with the irreversible inhibitor HKI-357. Comparable results were observed with other irreversible inhibitors. Cell numbers were determined by crystal violet staining after 72 h of exposure to the indicated drug concentrations and incubation in 5% FCS with 100 ng / ml EGFR. Each data point represents the average of four samples. Figure 2B Shown are the chemical structures of the EGFR reversible inhibitor gefitinib, the EGFR irreversible inhibitor EKB-569, and two EGFR and ERBB2 irreversible dual inhibitors HKI-272 and HKI-357. Figure 2C Shown are drug-resistant NCI-HI 650 cells developed after treatment with different concentrations of gefitinib or the irreversible ERBB inhibitor EKB-569. Colonies were stained after 12 days of culture in the presence of inhibitors.

[0026] Figures 3A-3D Shows sustained dependence on EGFR and ERBB2 signaling and altered receptor trafficking in gefitinib-resistant cells. Figure 3A Cell viability after siRNA-mediated knockdown of EGFR and ERBB2 in a bronchoalveolar cell line with wild-type EGFR (NCI-H1 666) is shown, compared with the viability of cells with an activating delE746-A750 mutation in EGFR (NCI-H1 650) and two gefitinib-resistant derivatives (G7 and C1 1). Viable cells were counted after 72 h of double-stranded RNA treatment, and the fraction relative to cells treated with nonspecific siRNA is shown, with standard deviations calculated from triplicate samples. Figure 3B Shown in the cell that uses increasing concentration of gefitinib or irreversible inhibitor HKI-357 to process, then carries out 2-h pulse with EGF, suppresses EGFR autophosphorylation (Y1068) and phosphorylation of downstream effector AKT and MAPK (ERK).Compare parental cell line NCI-H1650 with representative gefitinib-resistant system G7.Total AKT and MAPK are shown as control; For the total EGFR level of the lower limit detected in these cells, use tubulin as loading control. Figure 3CShow and sensitive NCI-H1 650 parental cell line comparison, the EGFR internalization phenomenon of the change in gefitinib-resistant NCI-H 1650 (G7) cell.After adding ligand 5 and 20min, use the EGF mark EGFR of rhodamine mark.The internalization phenomenon of the enhancement of EGFR in NCI-H1 650 (G7) cell is most obvious (Zeiss microscope, x 63 magnification) when 20min. Figure 3D Immunoblot showing EGFR internalized from NCI-H1 650 parental cells and the resistant derivative G7 after biotinylation and chase for more than 20 min pulse labeling of cell surface proteins. The enhanced intracellular EGFR in NCI-H1 650 (G7) cells was compared with the unchanged ferroportin receptor (TR) internalization phenomenon.

[0027] Figure 4A-4B Demonstrating the efficacy of an irreversible ERBB inhibitor in inhibiting the T790M EGFR mutant. Figure 4A Show gefitinib and two irreversible inhibitors (HKI-357 and HKI-272) inhibit EGFR autophosphorylation (Y1068) and NCI-H1975 bronchial alveolar cell line (containing sensitizing mutation (L858R) and mutation associated with resistance (T790M) both) in the downstream effector AKT and MAPK (ERK) phosphorylation ability comparison.Total EGFR, AKT and MAPK are shown as loading controls. Figure 4B Shown is the inhibition of proliferation of NCI-H1975 cells harboring L858R and T790M mutations with three irreversible ERBB family inhibitors compared to gefitinib.

[0028] Figure 5 The nucleotide sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 1) of EGFR are shown.

[0029] Figure 6 It was shown that like gefitinib, HKI 357 and EKB 569 (labeled "Wyeth") demonstrated enhanced cell killing ability against NSCLC cells harboring EGFR mutations, but unlike gefitinib, were less prone to generate clones resistant to these drugs in vitro and they retained their potency against gefitinib-resistant clones. DETAILED DESCRIPTION OF THE INVENTION

[0030] Cancers resistant to gefitinib and erlotinib

[0031] Gefitinib (compound ZD 1839, developed by AstraZeneca UK Ltd.; available under the trade name IRESSA) and erlotinib (compound OSI-774, developed by Genentech, Inc. and OSI Pharmaceuticals, Inc.; available under the trade name TARCEVA) have produced surprising clinical responses in non-small cell lung cancers (NSCLCs) that contain activating mutations in the EGF receptor (EGFR) (1-3), the target of these competitive inhibitors of ATP binding (4, 5). The potency of these tyrosine kinase inhibitors may result from both alterations in ATP cleavage (cleft) associated with these mutations, which result in enhanced inhibition of the mutant kinase by these drugs, and from the biological dependence of these cancer cells on the enhanced survival signals transduced by the mutant receptors, a phenomenon described as "oncogene addiction" (6, 7).

[0032] Although responses to both gefitinib and erlotinib can last up to 2–3 years, the median duration of response in most NSCLC cases is only 6–8 months (8–10). The mechanisms underlying acquired drug resistance are not well understood. By analogy, secondary kinase domain mutations effectively inhibit drug binding in patients with imatinib (GLEEVEC), which inhibits the BCR-ABL kinase involved in chronic myeloid leukemias (CMLs), the C-KIT kinase involved in gastrointestinal stromal tumors (GISTs), and the FIP1L1-PDGFR-α kinase in idiopathic hypereosinophilic syndrome (HES) (11–16). However, recurrent NSCLC is not easily biopsied; therefore, only limited clinical specimens are available for analysis. Recently, a single secondary mutation in the EGFR kinase domain, T790M, was reported in three of six cases of recurrent disease after gefitinib or erlotinib therapy (17,18). Codon 315 of BCR-ABL, similar to EGFR codon 790, is frequently mutated in imatinib-resistant CML (11,12), and mutations of corresponding residues in C-XJr (codon 670) and FIP1L1-PDGFR-a (codon 674) are associated with imatinib-resistant GIST and HES, respectively (15,16). Early in vitro models of resistance to EGFR inhibitors suggested that codon 790 mutations in the wild-type receptor would similarly suppress inhibition by EGFR tyrosine kinase inhibitors (19). More recently, transfected EGFR proteins containing activating mutations were shown to reduce inhibition by gefitinib and erlotinib together with the T790M substitution (17,18). Although the T790M mutation appears to contribute to acquired resistance in some cases of NSCLC, its potential mechanism of treatment failure in the absence of secondary EGFR mutations remains unclear.

[0033] In contrast to the cytoplasmic kinase BCR-ABL, signal transduction by membrane-bound EGFR involves a complex ligand binding pathway, homodimerization and heterodimerization of the receptor with ERBB2 and other family members, followed by internalization and recycling of the ligand-bound receptor or ubiquitin-mediated receptor degradation (20). Important EGF-dependent signaling is thought to occur during the internalization process, which is also associated with the dissociation of the EGFR complex at low pH in intracellular vesicles. Thus, multiple factors regulate the intensity and quality of signal transduction by the receptor, and changes in EGFR trafficking are closely linked to the regulation of EGF-dependent cellular responses (20).

[0034] The present invention is based on the discovery that gefitinib-resistant cancers may include those in which the T790M EGFR mutation is present only in a subset of resistant tumor cells and those in which no T790M mutation is observed, but enhanced EGFR internalization is observed. The present invention is also based on the discovery that irreversible EGFR inhibitors covalently cross-linked to the receptor are effective in inhibiting cancers with T790M mutations, as well as in cancers with altered EGFR transport that renders such cancers resistant to treatment with gefitinib and / or erlotinib. Therefore, the present invention provides methods of treating gefitinib- and / or erlotinib-resistant cancers comprising administering an irreversible EGFR inhibitor.

[0035] Methods of treating patients

[0036] In one embodiment, the present invention provides a method for treating gefitinib / erlotinib-resistant cancer. The method comprises administering to a patient in need of such treatment an effective amount of certain irreversible EGFR inhibitors, including EKB-569 (4-anilinoquinoline-3-carbonitrile; Greenberger et al., 11 th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Amsterdam, November 7-10, 2000, Abstract 388; Wyeth), HKI-357 (a derivative of 4-anilinoquinoline-3-carbonitrile; Tsou et al., J. Med. Chem. 2005, 48: 1107-1131; Wyeth) and / or HKI-272 (a derivative of 4-anilinoquinoline-3-carbonitrile; Rabindran et al., Cancer Res. 2004, 64, 3958-3965; Wyeth). In a preferred embodiment, the present invention provides a method comprising administering an effective amount of EKB-569 to a patient in need of such treatment. In a preferred embodiment, the present invention provides a method comprising administering an effective amount of HKI-357 to a patient in need of such treatment.

[0037] The treatment may also include a combination of therapies, including, but not limited to, a tyrosine kinase inhibitor in combination with other tyrosine kinase inhibitors, chemotherapy, radiation therapy, and the like.

[0038] Cancers can be initially diagnosed as gefitinib / erlotinib sensitive or predicted to be gefitinib / erlotinib sensitive using the methods described in Lynch et al., 2004; 350: 2129-2139. Gefitinib / erlotinib sensitivity can be predicted by EGFR mutations present in the tumor, including, for example, deletion of residues 747 (lysine) to 749 (glutamic acid) in combination with mutations in 750 (alanine), deletion of residues 747 (lysine) to 750 (alanine), substitution of arginine for leucine at residue 858, substitution of glutamine for leucine at residue 861.

[0039] Cancer can be diagnosed as resistant to gefitinib and / or erlotinib after initiation of treatment with gefitinib and / or erlotinib. Alternatively, cancer can be diagnosed as resistant to gefitinib and / or erlotinib before initial treatment with these compounds. Gefitinib and / or erlotinib resistance in a tumor can occur after treatment with gefitinib and / or erlotinib, e.g., 6 months or more. Alternatively, gefitinib and / or erlotinib resistance in a tumor can be diagnosed less than 6 months after initiation of treatment with gefitinib and / or erlotinib. Diagnosis of gefitinib and / or erlotinib resistance can be accomplished by monitoring the progression of the tumor during treatment with gefitinib and / or erlotinib. The progression of a tumor can be determined by comparing the status of the tumor between various time points after initiation of treatment, or by comparing the status of the tumor between a time point after initiation of treatment and a time point before initial treatment with gefitinib and / or erlotinib. During treatment with gefitinib and / or erlotinib, the development of tumors can be monitored by visual inspection, for example, using radiography, for example, X-rays, CT scans or other monitoring methods known to technicians, including palpitations of cancer or methods of monitoring tumor biomarker levels. The development of cancer during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. An increase in tumor biomarker levels indicates tumor development. Thus, an increase in tumor biomarker levels during treatment with gefitinib and / or erlotinib indicates resistance to gefitinib and / or erlotinib. Detection of new tumors or detection of metastasis indicates tumor development. Tumor shrinkage stops, indicating tumor development. The growth of cancer is represented by, for example, an increase in tumor size, metastasis or detection of new cancer and / or an increase in tumor biomarker levels.

[0040] The development of gefitinib and / or erlotinib resistance can be monitored by testing circulating tumor cells from the patient's circulation or other body fluids for the presence of mutations associated with gefitinib and / or erlotinib resistance. The presence of mutations associated with gefitinib and / or erlotinib resistance in tumor cells from the patient is indicative of a gefitinib and / or erlotinib resistant tumor.

[0041] In one embodiment, the patient's tumor comprises a mutation indicating sensitivity to gefitinib and / or erlotinib, but it is resistant to treatment with gefitinib and / or erlotinib. In one embodiment, the patient's tumor comprises a mutation indicating sensitivity to gefitinib and / or erlotinib, and comprises a mutation indicating resistance to gefitinib and / or erlotinib, such as a T790M mutation, i.e., in EGFR, a methionine residue replaces a natural threonine residue, such as an enhanced EGFR internalization phenomenon. In one embodiment, the patient's tumor does not comprise a mutation indicating sensitivity to gefitinib and / or erlotinib and does not comprise a mutation indicating resistance to gefitinib and / or erlotinib, such as a T790M mutation in EGFR, such as an enhanced EGFR internalization phenomenon.

[0042] When used in connection with the administration of a drug, an "effective amount" refers to an amount that results in a beneficial effect in at least a statistically significant portion of patients, such as improvement of symptoms, cure, reduction in disease burden, reduction in tumor or cell number, prolongation of life, improvement in quality of life, or other effect typically recognized as positive by a physician familiar with treating a particular type of disease or condition.

[0043] The effective dose of the active ingredient used may vary depending on the specific compound used, the mode of administration, and the severity of the condition being treated. The technician will note that the effective dose for each patient may vary depending on the severity of the disease, individual genetic variation, or metabolic rate. However, generally, satisfactory results are obtained when the compound of the present invention is administered at a daily dose of about 0.5 to about 1000 mg / kg body weight, optionally in divided doses 2-4 times a day, or in a sustained-release form. The total daily dose is planned to be about 1-1000 mg, preferably about 2-500 mg. The dosage form suitable for oral administration includes about 0.5-1000 mg of the active compound closely mixed with a pharmaceutically acceptable solid or liquid carrier. The dosage regimen can be adjusted to provide the best therapeutic effect. For example, multiple divided doses may be given daily or the dosage may be reduced proportionally according to the indication of the emergency situation of the treatment.

[0044] The route of administration may be intravenous (IV), intramuscular (IM), subcutaneous (SC), intradermal (ID), intraperitoneal (IP), intrathecal (IT), intrapleural, intrauterine, rectal, vaginal, topical, intratumoral, etc. The compounds of the invention may be administered parenterally by injection or gradual infusion over time and may be transported by peristaltic means.

[0045] Administration can be through mucosal or transdermal means. For transmucosal or transdermal administration, an appropriate permeation barrier penetrant is used in the formulation. Such penetrants are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to promote penetration. Transmucosal administration can be by nasal sprays, for example, or using suppositories. For oral administration, the compounds of the present invention are formulated into conventional oral administration forms such as capsules, tablets and tonics.

[0046] For topical administration, the pharmaceutical composition (kinase activity inhibitor) is formulated into ointments, salves, gels, or creams generally known in the art.

[0047] The therapeutic compositions of the invention, such as irreversible EGFR inhibitors, are, for example, administered intravenously by conventional unit dose injection. The term "unit dose" when used to refer to the therapeutic compositions of the invention refers to physically discrete units suitable as single doses for administration to a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

[0048] The composition is administered in a manner consistent with the dosage form and in an effective therapeutic amount. The amount and frequency of administration will depend on the patient being treated, the capacity of the patient's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required to be administered depends on the judgment of the practitioner and will vary from individual to individual.

[0049] Therapeutic compositions for implementing the methods of the invention are described herein, such as, irreversible EGFR inhibitors. Any formulation or drug delivery system generally known to those skilled in the art may be used, containing an active ingredient suitable for the intended use. Suitable pharmaceutically acceptable carriers for oral, rectal, topical or parenteral (including inhalation, subcutaneous, peritoneal, intramuscular and intravenous) administration are known to those skilled in the art. The carrier must be pharmaceutically acceptable, meaning compatible with the other ingredients in the formulation and harmless to the recipient.

[0050] As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, as they refer to compositions, carriers, diluents, and reagents, are used interchangeably and represent that the material is capable of being administered, or does not produce undesirable physiological effects in mammals.

[0051] Formulations suitable for parenteral administration conveniently include sterile aqueous preparations of the active compound, which are preferably isotonic with the blood of the recipient. Thus, such preparations may conveniently contain distilled water, 5% dextrose in distilled water or saline. Useful formulations also include concentrated solutions or solids containing the compound, which are diluted with an appropriate solvent to provide the above-mentioned solutions suitable for parenteral administration.

[0052] For enteral administration, the compound may be mixed with an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; such as powders or granules; or aqueous or non-aqueous liquid suspensions or solutions, such as syrups, elixirs, emulsions or draughts. Suitable carriers may be starch or powdered sugar and include lubricants, flavoring agents, binders and other materials of the same nature.

[0053] Tablets may be prepared by compression or molding, optionally with one or more adjuvants. Compressed tablets may be prepared by compressing the active compound in a free-flowing form such as a powder or granules, optionally mixed with an adjuvant such as a binder, lubricant, inert diluent, surfactant or dispersant, in a suitable machine. Molded tablets may be prepared by molding a mixture of the active compound in powder form with any suitable carrier in a suitable machine.

[0054] Syrups or suspensions may be prepared by adding the active compound to a concentrated sugar, such as sucrose in water, with any adjuvants added. Such adjuvants may include flavoring agents, agents to prevent sugar crystallization or agents to increase the solubility of any other ingredients, such as polyols, for example, glycerol or sorbitol.

[0055] Formulations for rectal administration may be presented as a suppository with a conventional carrier as a suppository base, such as cocoa butter or Witepsol S55 (Trademark of Dynamite Nobel Chemical, Germany).

[0056] Formulations for oral administration may contain enhancers. Orally acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoyl carnitine, polyethylene glycol monolauryl-9, phosphatidylcholine, cyclodextrins and their derivatives; bile salts such as sodium deoxycholate, sodium taurocholate, glycocholate and sodium fusidate; chelating agents include EDTA, citric acid and salicylates; and fatty acids (e.g., oleic acid, dodecanoic acid, acylcarnitines, mono- and diglycerides). Other oral absorption enhancers include benzalkonium chloride, benzethonium chloride, CHAPS (3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate), Big-CHAPS (N,N-bis(3-D-glucosamidopropyl)-choleamine), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols and alkyl alcohols. A particularly preferred oral absorption enhancer for use in the present invention is sodium lauryl sulfate.

[0057] Alternatively, the present compound can be administered in the form of liposomes or microspheres (or microparticles). Methods for preparing liposomes or microspheres for administration to patients are well known to those skilled in the art. U.S. Patent No. 4,789,734 describes a method for encapsulating biological raw materials in liposomes, the contents of which are incorporated herein by reference. Importantly, the raw material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, surfactants are added if necessary, and the raw material is dialyzed or ultrasonically treated when necessary. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

[0058] Microspheres formed from polymers or proteins are familiar to those skilled in the art and can be tailored to be suitable for direct entry into the bloodstream through the gastrointestinal tract. Alternatively, the compound can be incorporated and the microspheres or microsphere composites implanted for slow release over a period of days to months. See, e.g., U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214 and Jein, TIPS 19:155-157 (1998), the contents of which are incorporated herein by reference.

[0059] In one embodiment, the tyrosine kinase inhibitor of the present invention can be formulated into liposomes or microparticles of suitable size to gather in the capillary bed after intravenous administration. When the liposomes or microparticles are placed in the capillary bed around the ischemic tissue, the drug can be administered locally to the most effective site. Suitable liposomes targeting ischemic tissue are generally less than about 200 nanometers and are also typically unilamellar vesicle liposomes (vesicles), as disclosed in, for example, U.S. Patent No. 5,593,688 entitled "Liposomal targeting of ischemic tissue" by Baldescliweiler, the contents of which are incorporated herein by reference.

[0060] Preferred microparticles are those prepared from biodegradable polymers such as polyglycolide, polylactic acid and copolymers thereof. One skilled in the art can readily determine an appropriate carrier system based on a variety of factors including the desired drug release rate and the desired dosage.

[0061] In one embodiment, the preparation is administered directly to the interior of the blood vessel via a catheter. For example, administration can be performed through the hole of the catheter. In those embodiments where the active compound has a relatively long half-life (approximately 1 day to 1 week or longer), the preparation can be incorporated into a biodegradable polymeric hydrogel, such as those disclosed in U.S. Patent No. 5,410,016 to Hubbell et al. These polymeric hydrogels can be delivered to the tissue lumen and the active compound is released over time when the polymer degrades. If desired, the polymeric hydrogel can have microparticles or liposomes containing the active compound dispersed therein, unless there is another mechanism to provide controlled release of the active compound.

[0062] The preparations may conveniently be in unit dosage form and may be prepared by any method familiar to the pharmaceutical art. All methods include the step of mixing the active compound with a carrier which may comprise one or more adjuvants. Typically, the preparations are prepared by uniformly and intimately mixing the active compound with a liquid carrier or a finely ground solid carrier and then, if desired, shaping the product into the desired unit dosage form.

[0063] The preparation may also contain one or more optional auxiliary agents used in the field of pharmaceutical preparations, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, suspending agents, preservatives (including antioxidants), etc.

[0064] The compound (i.e., irreversible EGFR inhibitor) in the present method can be administered to the respiratory tract in the form of a nasal inhalant or an aerosol or a solution for a nebulizer or a fine powder for insufflation, alone or mixed with an inert carrier such as lactose. In such a case, the particles of the active compound are preferably less than 50 microns, preferably less than 10 microns, and more preferably 2-5 microns in diameter.

[0065] Generally for nasal administration, a weak acid pH will be preferred. Preferably, the pH of the composition of the present invention is about 3-5, more preferably about 3.5 to about 3.9 and most preferably 3.7. Adjustment of pH is achieved by adding a suitable acid such as hydrochloric acid.

[0066] The preparation of pharmaceutical compositions containing active ingredients dissolved or dispersed therein is well known in the art and need not be limited to the preparation. Typically such compositions are prepared as injectable or liquid solutions or suspensions, however, solid forms suitable for solutions or suspensions can also be prepared, which are dissolved in liquids prior to use. The preparations can also be emulsified.

[0067] The active ingredient can be mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient in an amount suitable for use in the treatment methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. Furthermore, if desired, the composition may contain a small amount of auxiliary substances such as wetting agents or emulsifiers, pH buffers, and the like that enhance the effectiveness of the active ingredient.

[0068] The irreversible kinase inhibitors of the present invention may include pharmaceutically acceptable salts of their components. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0069] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no substances other than the active ingredient and water, or containing a buffer such as sodium phosphate, saline or both at physiological pH, such as phosphate-buffered saline. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol and other solutes.

[0070] Liquid compositions may also contain liquid phases other than water and which exclude water. Examples of such other liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions.

[0071] definition:

[0072] The terms "ErbB 1," "epidermal growth factor receptor," and "EGFR" are used interchangeably herein and refer to native sequence EGFR as disclosed, for example, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987), including variants thereof (e.g., deletion mutant EGFR in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR protein product. As used herein, the EGFR protein has been disclosed as GenBank Accession No. NP_005219 (SEQ ID NO.: 1) encoded by the erbB1 gene (GenBank Accession No. NM_005228 (SEQ ID NO: 2)). erbB1

[0073] The nucleotide and amino acid sequences of EGFR can be found in Figure 5 Found in.

[0074] As used herein, the term "nucleic acid variation with increased kinase activity" refers to a variation (ie, mutation) in the nucleotide sequence of a gene that results in increased kinase activity. The increased kinase activity is a direct result of the variation in the nucleic acid and is associated with the protein encoded by the gene.

[0075] As used herein, the term "drug" or "compound" refers to a chemical entity or biological product, or a combination of chemical entities or biological products, that is administered to a patient to treat, prevent or control a disease or condition. The chemical entity or biological product is preferably, but not necessarily, a low molecular weight compound, but may also be a larger compound, for example, a nucleic acid, an oligomer of amino acids, or a carbohydrate, including but not limited to proteins, oligonucleotides, ribozymes, DNA enzymes, glycoproteins, siRNAs, lipoproteins, aptamers, modifications thereof, and combinations thereof.

[0076] As used herein, the terms "effective" and "efficacy" include both pharmacological effects and physiological safety. Pharmacological effect refers to the ability of a treatment to produce a desired biological effect in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (usually side effects) at the cellular, organ and / or biological level caused by the administration of the treatment. "Poor effect" means that the treatment results in a therapeutically significantly lower level of pharmacological effect and / or a therapeutically higher level of adverse physiological effect.

[0077] Nucleic acid molecules can be isolated from a particular biological sample using any of a variety of methods familiar in the art, with the specific isolation process selected being appropriate for the particular biological sample. For example, nucleic acid molecules can be obtained from solid materials using freeze-thaw and alkaline lysis; nucleic acid molecules can be obtained from urine using heating and alkaline lysis; and nucleic acids can be obtained from blood using proteinase K extraction (Rolff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994).

[0078] As used herein, "cancer" in a subject or patient refers to the presence of cells that have characteristic features typical of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In certain instances, cancer cells will be present in the form of a tumor, or such cells may be present locally in an animal or circulate in the bloodstream as independent cells. Example

[0079] Compounds. Compounds used herein include EKB-569, HKI-357, and HKI-272 as described in the following documents: U.S. Pat. No. 6,002,008; Greenberger et al., Proc. th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, Clinical.Cancer Res.Vol.6Supplement, Nov.2000, ISSN1078-0432; Rabindran et al., Cancer Res. 64:3958-3965 (2004); Holbro and Hynes, Ann. Rev. Pharm. Tox. 44: 195-217 (2004); and Tejpar et al., J. Clin. Oncol. ASCO Annual Meeting Proc. Vol. 22, No. 14S: 3579 (2004).

[0080] Relapsed NSCLC Analysis and Generation of Gefitinib-Resistant NCI-H1650 Cells. Relapsed NSCLC clinical specimens were obtained by autopsy after obtaining appropriate consent. The complete kinase domain of EGFR was sequenced after analysis of non-clonal PCR products. Multiple clones of exon 20 were sequenced to detect codon 790. Mutation analysis of EGFR (exons 1-28), ERBB2 (exons 1-24), PTEN (exons (Qxons) 1-9), Kras (codons 12, 13 and 61) and p53 (exons 5-8) in gefitinib-resistant clones and the parental NCI-H1650 cell line was performed by automated sequencing of individual exons using dye terminator chemistry (BIGDYE version 1.1, Applied Biosystems) and flanking intronic sequences with bidirectional sequencing (PCR conditions available on request). Sequencing reactions were performed on an ABI3100 sequencer (Applied Biosystems) and electropherograms were analyzed by using SEQUENCE NAVIGATOR and FACTURA software (Applied Biosystems).

[0081] To generate drug-resistant subclones of NCI-H1650 cells, these cells were treated with ethyl methanesulfonate (EMS; 600 μg / ml), allowed to recover for 72 h, and then seeded in 20 μM gefitinib at a density of 10 cm per 10-cm 2 Plate 6x10 4 Compared with the irreversible inhibitor, these cells were relatively resistant to gefitinib, which was confirmed by incubating 5x10 4 Cells were seeded in six-well plates with 5% FCS and 100 ng / ml EGF (Sigma), and then fixed with 4% formaldehyde for 72 h, stained with 0.1% crystal violet, and the cell population was quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). For micro-intervention RNA (siRNA) knockdown experiments, cells were transfected with double-stranded RNA oligonucleotides targeting EGFR, ERBB2 (two SMART pools from Dharmacon, Lafayette, CO), or non-specific controls (LRT1B) were performed using X-treme gene transfection reagents (Roche Applied Science). After 72 h, cells were stained with crystal violet and analyzed with an Odyssey infrared scanner.

[0082] Immunoblotting and signaling studies. Inhibition of EGFR signaling by increasing concentrations of gefitinib or irreversible inhibitors was determined by the following assay: 9x10 4 Cells were seeded in 24-well plates, drugs were added to medium containing 5% FCS for 15 min, then pulsed with 100 ng / ml EGF for 2-h, and lysates were harvested. Lysates were prepared in 2x gel loading buffer, sonicated, boiled, and then separated by 10% SDS / PAGE, followed by electrotransfer to polyvinylidene fluoride (PVDF) membranes and immunoblotted. Antibodies used were phospho-EGFR Yl 068 and phosphorylated mitogen-activated protein kinase (MAPK) (Cell Signaling Technology, Beverly, MA), phospho-AKT (BioSource International, Camarillo, CA), and total EGFR, MAPK, AKT, and tubulin (Santa Cruz Biotechnology).

[0083] EGFR internalization analysis. To demonstrate EGFR internalization by fluorescence microscopy, cells were grown on coverslips and incubated with 1 ng / ml recombinant human (rh) EGF (Molecular Probes, Eugene, OR) for various time intervals before being fixed in 4% paraformaldehyde for 10 min. Coverslips were washed with PBS and mounted with ProLong Gold antifade reagent (Molecular Probes). To quantify EGFR internalization by cell surface biotinylation, cells were grown to confluence, pretreated with cyclohexamide, incubated with 1.5 mg / ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce) for 1 h on ice, washed with blocking buffer (50 nM NH4CL / 1 mM MgCl2A) 1 mM CaCl2 in PBS) to quench free sulfo-NHS-SS-biotin, and then washed several times with PBS. The cells were then incubated at 37°C in culture medium for various time intervals to allow internalization of the biotinylated molecules, washed twice on ice with glutathione solution (50 mM glutathione / 75 mM NaCl / 75 mM NaOH / 1% BSA) for 20 min to strip all biotin groups from the cell surface, and then the cells were scraped and lysed in 500 μM radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.4, with 150 mM NaCL / 0.1% SDS / 1% Triton X-100) supplemented with NaF, sodium orthovanadate, and protease inhibitors. The cell extracts were centrifuged, and the supernatants were incubated with streptavidin beads (Sigma) to collect the biotinylated proteins, which were then analyzed by SDS / PAGE and immunoblotting with anti-EGFR antibody (SC-03, Santa Cruz Biotechnology) or anti-transferrin receptor antibody (Santa Cruz Biotechnology).

[0084] Results and discussion

[0085] Analysis of recurrent lung cancer with acquired resistance to gefitinib. Recurrent gefitinib-resistant NSCLC occurred in two patients whose tumors already contained activating mutations of the EGFR kinase at diagnosis and who had shown a dramatic initial clinical response to the drug (1). In both cases, metastatic disease developed in the liver 1-2 years after initial treatment, leading to the death of the patients. In case 1, analysis of the large liver metastases obtained at autopsy showed persistence of the sensitizing EGFR mutation (L858R) and the presence of a newly acquired T790M mutation ( Figure 1A Interestingly, analysis of nonclonal PCR products showed that the initial L858R mutation was present at an abundance consistent with a heterozygous mutation present in all tumor cells, whereas the secondary T790M mutation was seen at approximately one-fifth the abundance of the corresponding wild-type allele. Thus, this resistance-associated mutation appears to be present in only a subset of cells in recurrent tumors.

[0086] Case 2 relates to eight obvious recurrence metastases in liver after gefitinib therapy failure.In all these independent injuries, the L861Q EGFR mutation of sensitization exists with the ratio of expected heterozygous mutation.By the analysis of the non-cloned PCR product from any of these transfers, no secondary EGFR mutation is detected.However, after PCR product subclone, it is found that in two of four metastatic tumor analyses (T790M, 2 in 50 clones of self-injury 1 sequencing and 1 in 56 clones of self-injury 2 sequencing), T790M mutation exists with very low frequency, but from two other recurrence metastases (0 in 55 clones of self-injury 3 sequencing and 0 in 59 clones of self-injury 4 sequencing) or primary tumor (0 in 75 clones), it is not found ( Figure 1B and Table 1). Taken together, these results are consistent with previous reports that T790M mutations are present in some but not all cases of acquired gefitinib resistance (3 of 7 tumors; see Refs. 17, 18, and 21). Furthermore, as noted previously (18), even in some cases with this resistance-associated mutation, it appears to be present in only a small fraction of tumor cells in recurrent lesions. These observations suggest that additional mechanisms of resistance are involved in cases without secondary EGFR mutations and that such mechanisms coexist with T790M mutations in other cases.

[0087] Generation of gefitinib-resistant cell lines sensitive to irreversible inhibitors. Given the strong correlation between clinical responses of EGFR-mutant NSCLC and the enhanced gefitinib sensitivity of NSCLC cell lines with these mutations (2, 6, 22, 23), as well as the limited availability of clinical samples from relapsed patients, the applicants established an in vitro gefitinib resistance model. The applicants cultured the bronchoalveolar carcinoma cell line NCI-H1650 with an in-frame deletion of the EGFR kinase (delE746-A750) in 20 μM gefitinib, either pre-exposed or not pre-exposed to the mutagen ethyl methanesulfonate. This cell line exhibited a 100-fold enhanced sensitivity to gefitinib compared to certain NSCLC lines expressing wild-type EGFR (6). While the vast majority of these cells were effectively killed by 20 μM gefitinib, at ~10 ～5Resistant clones were readily observed with a frequency of 1.47 fold, regardless of mutagen treatment. Forty-nine independent resistant clones were isolated, showing an average 50-fold reduction in sensitivity to gefitinib ( Figure 2A All showed persistence of sensitizing mutations without changes in EGFR expression, and none had acquired secondary EGFR mutations or new mutations in ERBB2, p53, Kras, or PTEN. Gefitinib-resistant clones demonstrated considerable resistance to related anilinoquinazoline inhibitors. However, strikingly, they showed resistance to ERBB family ( Figure 2A ) of the three inhibitors: EGFR and ERBB2 (for EGFR, IC 50 values ​​of 92 and 34 nM, respectively, and for ERBB2, 59 and 33 nM, respectively) for the dual inhibitors HKI-272 (24) and HKI-357 (compound 7f in reference 25), and EGFR (for EGFR, IC 50 The value is 39 nM and for ERBB2, IC 50 Value 1.3 μM) selective inhibitor EKB-569 (26) (Wyeth) ( Figure 2B All three drugs are irreversible inhibitors, most likely via covalent bonding to the cys773 residue in the catalytic domain of EGFR or cys805 of ERBB2. Like gefitinib, these compounds demonstrated enhanced killing of NSCLC cells harboring EGFR mutations compared to cells expressing wild-type receptors ( Figure 2A ). However, in contrast to gefitinib, for which resistant clones are readily generated even at high drug concentrations, the applicants were unable to establish cell clones resistant to the irreversible inhibitor at concentrations of about 10 μM, even after mutagenesis with ethyl methanesulfonate ( Figure 2C ).

[0088] Gefitinib-resistant cells rely on EGFR and ERBB2 expression. In order to gain insight into the mechanism of gefitinib resistance and sustained sensitivity to irreversible inhibitors, the applicant first determines whether the resistant cell line still relies on EGFR for their viability. The applicant has previously shown that siRNA-mediated knockout of EGFR triggers apoptosis in cells containing mutant EGFRs, but does not trigger apoptosis in cells with wild-type alleles (6). Significantly, after transfection with siRNA-targeted EGFR, parental NCI-H1 650 cells and their gefitinib-resistant derivatives show comparable reductions in cell viability ( Figure 3A). Thus, the acquisition of gefitinib-resistance does not involve EGFR-independent activation of downstream effectors. Because HKI-272 and HKI-357 target both EGFR and ERBB2, the applicants also tested inhibition of this related receptor. Knockout of ERBB2 in NCI-H1650 and its gefitinib-resistant derivatives also caused loss of viability ( Figure 3A ), suggesting a role for EGFR-ERBB2 heterodimers in transducing essential survival signals in tumor cells harboring EGFR mutations. Inhibition of EGFR by irreversible inhibitors alone appears to be sufficient to induce apoptosis in gefitinib-resistant cells, as demonstrated by the efficacy of EKB-569, which primarily targets EGFR (26). However, given the potential complementary effects of targeting EGFR and ERBB2 by using siRNA and the availability of irreversible inhibitors targeting these family members, the potential benefit of dual inhibition has been considered.

[0089] The applicants compared the ability of gefitinib and irreversible ERBB family inhibitors to inhibit signal transduction via the downstream effectors of EGFR that mediate its proliferation and survival pathways. In inhibiting EGFR autophosphorylation (detected at residue Y1068), and in AKT and MAPK phosphorylation in parental NCI-H1650 cells containing delE746-A750 EGFR mutations, HKI-357 was 10 times more potent than gefitinib ( Figure 3B In the gefitinib-resistant derivative, NCI-H1650 (G7), gefitinib showed a significantly reduced inhibitory effect on AKT phosphorylation, a key EGFR signaling effector associated with gefitinib response (6), whereas HKI-357 was shown to have sustained activity ( Figure 3B ).

[0090] Altered EGFR internalization in gefitinib-resistant clones. Given the lack of secondary mutations in EGFR and the continued susceptibility of gefitinib-resistant cells to siRNA-mediated inhibition of EGFR, the applicants examined whether the mechanism underlying the differential inhibition of EGFR signaling by reversible and irreversible inhibitors in gefitinib-resistant cells might be related to alterations in receptor trafficking, which has been well documented as a regulator of EGFR-dependent signaling (20). Indeed, as measured by fluorescein-labeled EGF internalization ( Figure 3C Analysis of EGFR trafficking in NCI-H1650-derived resistant cells demonstrated a consistent enhancement of EGFR internalization, as measured by both quantification of cytoplasmic biotinylated EGFR ( Figure 3DSuch an effect was not observed in the transferrin receptor, suggesting that this is not the result of a general change in all receptor processing. Although further work is needed to elucidate the precise mechanism of this change in EGFR trafficking, many regulatory proteins have been implicated in this complex process, and these results suggest that the ability of gefitinib to inhibit EGFR activation compromises these cells, while the effects of irreversible inhibitors are not detectably affected.

[0091] The inhibition of T790M EGFR signal transduction caused by irreversible inhibitors and the enhanced cell killing power. The inhibition of EGFR signal transduction enhancement by irreversible ERBB inhibitors produces such a possibility that these drugs can also show sustained activity in the case of cells containing T790M secondary mutations in EGFR. Therefore, the applicant tested the effect of these inhibitors on the NCI-H1 975 bronchial alveolar carcinoma cell line containing L858R and T790M mutations in EGFR (18). It is significant that the cell line is derived from patients who are not treated with EGFR inhibitors, indicating that the mutation is not uniquely associated with acquired drug resistance. In the inhibition of EGFR autophosphorylation and downstream signal transduction caused by ligand-, when measured by AKT and MAPK phosphorylation, HKI-357 and HKI-272 are significantly more effective than gefitinib ( Figure 4A ). Similarly, all three irreversible inhibitors inhibited proliferation in this cell line under conditions of resistance to gefitinib ( Figure 4B ). Thus, irreversible ERBB inhibitors appear to be effective in cells containing T790 MEGFR as well as in cells with altered transmission of the wild-type receptor.

[0092] Our results confirm reports of the T790M mutation in EGFR as a secondary mutation previously reported in sensitive NSCLCs harboring activating mutations and associated with the emergence of acquired resistance (17,18). However, this mutation is present in only a subset of cases, even though tumors harboring the T790M mutation may contain only a small fraction of cells with this mutation. These observations imply that multiple resistance mechanisms may coexist in recurrent tumors after an initial response to gefitinib or similar reversible EGFR inhibitors. Furthermore, these findings suggest that resistance mechanisms independent of T790M may be equally, if not more potent than the T790M substitution itself in conferring drug resistance and may explain why recurrent tumors rarely exhibit clonality to T790M (17,18). In vitro mechanisms of acquired gefitinib resistance do not remarkably frequently involve secondary EGFR mutations but are instead associated with altered receptor trafficking. However, it should be noted that the applicant did not test the EGFR transmission in all the resistant clones established in vitro by the applicant, and there may be other mechanisms that may work on gefitinib resistance in some clones. Nevertheless, in fact, all gefitinib-resistant clones showed comparable sensitivity to irreversible ERBB inhibitors.

[0093] Applicants' results suggest that there is a marked difference between competitive EGFR inhibitors such as gefitinib, whose efficacy is limited by the rapid development of resistance in vitro, and irreversible inhibitors, to which acquired resistance appears to be rare ( Figure 2C ). The applicant speculates that the enhanced internalization of ligand-bound EGFR in drug-resistant cells may be associated with the dissociation of gefitinib-EGFR complexes at low pH in intracellular vesicles. In contrast, irreversible cross-linking of receptors will not be affected by such changes in receptor transport. After cells are passaged up to 20 generations in the absence of drugs, the acquired resistance to gefitinib is stable and persistent, suggesting that genetic or epigenetic changes in genes regulating EGFR turnover can explain this phenomenon. Because receptor transport is not easy to study using available clinical samples, it may be necessary to identify such genetic changes before possible clinical relevance. Nevertheless, such a mechanism may work on acquired gefitinib-resistance in patients with recurrent disease who do not have secondary mutations in EGFR.

[0094] Irreversible ERBB inhibitors also appear to be effective in overcoming gefitinib resistance mediated by the T790M mutation, which is presumed to maintain the effects mediated by inhibitor binding regardless of the alteration of this important residue. While this work was underway, another EGFR irreversible inhibitor [CL-387,785, Calbiochem (27)] was shown to inhibit the kinase activity of the T790M EGFR mutant (17). The potency of CL-387,785 in the presence of T790M is thought to be a result of the lack of a chloride at position 3 of the aniline group, which is present in gefitinib and presumably sterically hinders binding to the mutant methionine at codon 790. However, EKB-569, HKI-272, and HKI-357 all have a chloride moiety at this position on the aniline ring, suggesting that their shared ability to irreversibly bind to EGFR may explain their potency, rather than a lack of specific steric interaction with T790M (24-26). Thus, these irreversible inhibitors could prove to be broadly effective across multiple resistance mechanisms, in addition to the T790M mutation.

[0095] Table 1. EGFR T790M mutation was present at a very low frequency in the recurrent tumor of case 2

[0096]

[0097] Sequencing of a large number of cloned PCR products revealed that the minority alleles in two of the four cases of liver damage contained the T790M mutation.

[0098] References cited throughout this specification are hereby incorporated by reference in their entireties. Specific implementation method:

[0100] 1. A method for treating gefitinib and / or erlotinib-resistant cancer, the method comprising the following steps:

[0101] a. monitoring the patient for the development of cancer at a time point after the patient is initially treated with gefitinib and / or erlotinib, wherein the development of the cancer is an indication of a cancer that is resistant to treatment with gefitinib and / or erlotinib; and

[0102] b. administering a pharmaceutical composition comprising an irreversible epidermal growth factor receptor (EGFR) inhibitor to a cancer patient resistant to treatment with gefitinib and / or erlotinib.

[0103] 2. The method of embodiment 1, wherein the irreversible EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0104] 3. The method of embodiment 1, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0105] 4. The method of embodiment 1, wherein the progression of the cancer is monitored by visual inspection of the cancer.

[0106] 5. The method of embodiment 4, wherein the visual detection of cancer is performed by X-ray, CT scan or MRI.

[0107] 6. The method of embodiment 1, wherein the progression of the cancer is monitored by detecting tumor biomarkers.

[0108] 7. The method of embodiment 1, wherein monitoring the progression of the cancer comprises comparing the cancer at a second time point to the cancer at a first time point, wherein the second time point is after the first time point, wherein the first time point is before or after initial treatment with gefitinib and / or erlotinib, and wherein increased growth of the cancer indicates progression of the cancer.

[0109] 8. The method of embodiment 7, wherein the cancer at another time point is compared to the cancer at a previous time point.

[0110] 9. The method of embodiment 1, wherein the cancer is epithelial cell cancer.

[0111] 10. The method of embodiment 1, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer, and bladder cancer.

[0112] 11. A method for treating cancer, comprising the following steps:

[0113] a. Giving gefitinib and / or erlotinib to cancer patients;

[0114] b. monitor the patient's cancer progression; and

[0115] c. Once cancer development is demonstrated, the patient is given an irreversible EGFR inhibitor.

[0116] 12. The method of embodiment 11, wherein the irreversible EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0117] 13. The method of embodiment 11, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 (SEQ ID NO: 1) of EGFR.

[0118] 14. The method of embodiment 11, wherein the progression of the cancer is monitored by visual inspection of the cancer.

[0119] 15. The method of embodiment 14, wherein visual detection of cancer is performed using X-ray, CT scan or MRI.

[0120] 16. The method of embodiment 11, wherein the progression of the cancer is monitored by tumor biomarker detection.

[0121] 17. The method of embodiment 11, wherein monitoring the progression of the cancer comprises comparing the cancer at a second time point to the cancer at a first time point, wherein the second time point is after the first time point, wherein the first time point is before or after initial treatment with gefitinib and / or erlotinib, wherein increased growth of the cancer indicates progression of the cancer.

[0122] 18. The method of embodiment 17, wherein the cancer at the additional time point is compared to the cancer at the previous time point.

[0123] 19. The method of embodiment 11, wherein the cancer is epithelial cell cancer.

[0124] 20. The method of embodiment 11, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer and bladder cancer.

[0125] 21. The method of embodiment 11, wherein the irreversible EGFR inhibitor is administered simultaneously with the reversible EGFR inhibitor.

[0126] 22. The method of embodiment 21, wherein the reversible EGFR inhibitor is gefitinib or erlotinib.

[0127] 23. A method for treating cancer, the method comprising administering a pharmaceutical composition containing an irreversible EGFR inhibitor to a cancer patient, the patient having a mutation in EGFR (SEQ ID NO: 1), wherein the mutation is a substitution of methionine for threonine at position 790.

[0128] 24. The method of embodiment 23, wherein the EGFR inhibitor is selected from EKB-569, HKI-272 and HKI-357.

[0129] 25. The method of embodiment 23, wherein the irreversible EGFR inhibitor binds to cysteine ​​773 of EGFR (SEQ ID NO: 1).

[0130] 26. The method of embodiment 23, wherein the cancer is epithelial cell cancer.

[0131] 27. The method of embodiment 23, wherein the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, head and neck cancer, esophageal cancer, lung cancer, non-small cell lung cancer, nervous system cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, pancreatic cancer, genitourinary cancer and bladder cancer.

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Sequence Listing <110> THE GENERAL HOSPITAL CORPORATION Wyeth Company (WYETH) <120> Methods for treating gefitinib-resistant cancer <130> 030258-057642-PCT <140> PCT / US2006 / 003717 <141> 2006-02-02 <150> 60 / 671,989 <151> 2005-04-15 <150> 60 / 649,483 <151> 2005-02-03 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 1210 <212> PRT <213> Homo sapiens <400> 1 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45 Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60 Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys 65 70 75 80 Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95 Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110 Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125 Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140 His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu 145 150 155 160 Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175 Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190 Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205 Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220 Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys 225 230 235 240 Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255 Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270 Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285 Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300 Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu 305 310 315 320 Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 385 390 395 400 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 465 470 475 480 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 545 550 555 560 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 625 630 635 640 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670 Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685 Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700 Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser 705 710 715 720 Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735 Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750 Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765 Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780 Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp 785 790 795 800 Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815 Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830 Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845 Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860 Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp 865 870 875 880 Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895 Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910 Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925 Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940 Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys 945 950 955 960 Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975 Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990 Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005 Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe 1010 1015 1020 Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala 1025 1030 1035 1040 Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln 1045 1050 1055 Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp 1060 1065 1070 Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro 1075 1080 1085 Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser 1090 1095 1100 Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser 1105 1110 1115 1120 Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro 1125 1130 1135 Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp 1140 1145 1150 Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp 1155 1160 1165 Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn 1170 1175 1180 Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val 1185 1190 1195 1200 Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 1205 1210 <210> 2 <211> 3878 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (246)..(3875) <400> 2 cccggcgcag cgcggccgca gcagcctccg ccccccgcac ggtgtgagcg cccgacgcgg 60 ccgaggcggc cggagtcccg agctagcccc ggcggccgcc gccgcccaga ccggacgaca 120 ggccacctcg tcggcgtccg cccgagtccc cgcctcgccg ccaacgccac aaccaccgcg 180 cacggccccc tgactccgtc cagtattgat cgggagagcc ggagcgagct cttcggggag 240 cagcg atg cga ccc tcc ggg acg gcc ggg gca gcg ctc ctg gcg ctg ctg 290 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu 1 5 10 15 gct gcg ctc tgc ccg gcg agt cgg gct ctg gag gaa aag aaa gtt tgc 338 Ala Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys 20 25 30 caa ggc acg agt aac aag ctc acg cag ttg ggc act ttt gaa gat cat 386 Gln Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His 35 40 45 ttt ctc agc ctc cag agg atg ttc aat aac tgt gag gtg gtc ctt ggg 434 Phe Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly 50 55 60 aat ttg gaa att acc tat gtg cag agg aat tat gat ctt tcc ttc tta 482 Asn Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu 65 70 75 aag acc atc cag gag gtg gct ggt tat gtc ctc to gcc ctc aac aca 530 Lys Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr 80 85 90 95 gtg gag cga att cct ttg gaa aac ctg cag atc atc aga gga aat atg 578 Val Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met 100 105 110 tac tac gaa aat tcc tat gcc tta gca gtc tta tct aac tat gat gca 626 Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala 115 120 125 aat aaa acc gga ctg aag gag ctg ccc atg aga aat tta cag gaa atc ​​674 Asn Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile 130 135 140 ctg cat ggc gcc gtg cgg ttc agc aac aac cct gcc ctg tgc aac gtg 722 Leu His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val 145 150 155 gag agc atc cag tgg cgg gac ata gtc agc agt gac ttt ctc agc aac 770 Glu Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn 160 165 170 175 atg tcg atg gac ttc cag aac cac ctg ggc agc tgc caa aag tgt gat 818 Met Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp 180 185 190 cca agc tgt ccc aat ggg agc tgc tgg ggt gca gga gag gag aac tgc 866 Pro Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys 195 200 205 cag aaa ctg acc aaa atc atc tgt gcc cag cag tgc tcc ggg cgc tgc 914 Gln Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys 210 215 220 cgt ggc aag tcc ccc agt gac tgc tgc cac aac cag tgt gct gca ggc 962 Arg Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly 225 230 235 tgc aca ggc ccc cgg gag agc gac tgc ctg gtc tgc cgc aaa ttc cga 1010 Cys Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg 240 245 250 255 gac gaa gcc acg tgc aag gac acc tgc ccc cca ctc atg ctc tac aac 1058 Asp Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn 260 265 270 ccc acc acg tac cag atg gat gtg aac ccc gag ggc aaa tac agc ttt 1106 Pro Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe 275 280 285 ggt gcc acc tgc gtg aag aag tgt ccc cgt aat tat gtg gtg aca gat 1154 Gly Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp 290 295 300 cac ggc tcg tgc gtc cga gcc tgt ggg gcc gac agc tat gag atg gag 1202 His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu 305 310 315 Gaa Gac Gcc Gtc Cgc Aag Tgt Aag Aag Tgc Gaa Ggg Cct Tgc Cgc Aaa 1250 Glu Asp Gly Val Arg Lys Cys Lys Glu Gly Pro Cys Arg Lys 320 325 330 335 gtg tgt aac gga ata ggt att ggt gaa ttt aaa gac tca ctc tcc ata 1298 Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile 340 345 350 aat gct acg aat at aaa cac ttc aaa aac tgc acc tcc atc agt ggc 1346 Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly 355 360 365 gat ctc cac atc ctg ccg gtg gca ttt agg ggt gac tcc ttc aca cat 1394 Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His 370 375 380 act cct cct ctg gat cca cag gaa ctg gat att ctg aaa acc gta aag 1442 Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys 385,390,395 gaa atc ​​aca ggg ttt ttg ctg att cag gct tgg cct gaa aac agg acg 1490 Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr 400 405 410 415 gac ctc cat gcc ttt gag aac cta gaa atc ​​ata cgc ggc agg acc aag 1538 Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys 420 425 430 caa cat ggt cag ttt tct ctt gca gtc gtc agc ctg aac ata aca tcc 1586 Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser 435 440 445 ttg gga tta cgc tcc ctc aag gag ata agt gat gga gat gtg ata att 1634 Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile 450 455 460 tca gga aac aaa aat ttg tgc tat gca aat aca ata aac tgg aaa aaa 1682 Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys 465 470 475 ctg ttt ggg acc tcc ggt cag aaa acc aaa att ata agc aac aga ggt 1730 Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly 480 485 490 495 gaa aac agc tgc aag gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc 1778 Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser 500 505 510 ccc gag ggc tgc tgg ggc ccg gag ccc agg gac tgc gtc tct tgc cgg 1826 Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg 515 520 525 aat gtc agc cga ggc agg gaa tgc gtg gac aag tgc aac ctt ctg gag 1874 Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu 530 535 540 ggt gag cca agg gag ttt gtg gag aac tct gag tgc ata cag tgc cac 1922 Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His 545 550 555 cca gag tgc ctg cct cag gcc atg aac atc acc tgc aca gga cgg gga 1970 Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly 560 565 570 575 cca gac aac tgt atc cag tgt gcc cac tac att gac ggc ccc cac tgc 2018 Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys 580 585 590 gtc aag acc tgc ccg gca gga gtc atg gga gaa aac aac acc ctg gtc 2066 Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val 595 600 605 tgg aag tac gca gac gcc ggc cat gtg tgc cac ctg tgc cat cca aac 2114 Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn 610 615 620 tgc acc tac gga tgc act ggg cca ggt ctt gaa ggc tgt cca acg aat 2162 Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn 625 630 635 ggg cct aag atc ccg tcc atc gcc act ggg atg gtg ggg gcc ctc ctc 2210 Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu 640 645 650 655 ttg ctg ctg gtg gtg gcc ctg ggg atc ggc ctc ttc atg cga agg cgc 2258 Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg 660,665,670 cac atc gtt cgg aag cgc acg ctg cgg agg ctg ctg cag gag agg gag 2306 His Ile Val Arg Lys Arg Thr Leu Arg Leu Leu Gln Glu Arg Glu 675,680,685 ctt gtg gag cct ctt aca ccc agt gga gaa gct ccc aac caa gct ctc 2354 Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu 690,695,700 ttg agg atc ttg aag gaa act gaa ttc aaa aag atc aaa gtg ctg ggc 2402 Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Ile Lys Val Leu Gly 705 710 715 tcc ggt gcg ttc ggc acg gtg tat aag gga ctc tgg atc cca gaa ggt 2450 Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly 720 725 730 735 gag aaa gtt aaa at ccc gtc gct atc aag gaa tta aga gaa gca aca 2498 Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Lion Arg Glu Ala Thr 740,745,750 tct ccg aaa gcc aac aag gaa atc ctc gat gaa gcc tac gtg atg gcc 2546 Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala 755 760 765 agc gtg gac aac ccc cac gtg tgc cgc ctg ctg ggc atc tgc ctc acc 2594 Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr 770 775 780 tcc acc gtg cag ctc atc acg cag ctc atg ccc ttc ggc tgc ctc ctg 2642 Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu 785 790 795 gac tat gtc cgg gaa cac aaa gac aat att ggc tcc cag tac ctg ctc 2690 Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu 800 805 810 815 aac tgg tgt gtg cag atc gca aag ggc atg aac tac ttg gag gac cgt 2738 Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg 820 825 830 cgc ttg gtg cac cgc gac ctg gca gcc agg aac gta ctg gtg aaa aca 2786 Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr 835 840 845 ccg cag cat gtc aag atc aca gat ttt ggg ctg gcc aaa ctg ctg ggt 2834 Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly 850 855 860 gcg gaa gag aaa gaa tac cat gca gaa gga ggc aaa gtg cct atc aag 2882 Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys 865 870 875 tgg atg gca ttg gaa tca att tta cac aga atc tat acc cac cag agt 2930 Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser 880 885 890 895 gat gtc tgg agc tac ggg gtg acc gtt tgg gag ttg atg acc ttt gga 2978 Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly 900 905 910 tcc aag cca tat gac gga atc cct gcc agc gag atc tcc tcc atc ctg 3026 Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu 915 920 925 gag aaa gga gaa cgc ctc cct cag cca ccc ata tgt acc atc gat gtc 3074 Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val 930,935,940 tac atg atc atg gtc aag tgc tgg atg ata gac gca gat agt cgc cca 3122 Tyr Met Ile Met Val Lys Trp Met Ile Asp Ala Asp Ser Arg Pro 945,950,955 aag ttc cgt gag ttg atc atc gaa ttc tcc aaa atg gcc cga gac ccc 3170 Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro 960 965 970 975 cag cgc tac ctt gtc att cag ggg gat gaa aga atg cat ttg cca agt 3218 Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser 980,985,990 cct aca gac tcc aac ttc tac cgt gcc ctg atg gat gaa gaa gac atg 3266 Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met 995 1000 1005 gac gac gtg gtg gat gcc gac gag tac ctc atc cca cag cag ggc ttc 3314 Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020 ttc agc agc ccc tcc acg tca cgg act ccc ctc ctg agc tct ctg agt 3362 Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser 1025 1030 1035 gca acc agc aac aat tcc acc gtg gct tgc att gat aga aat ggg ctg 3410 Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu 1040 1045 1050 1055 caa agc tgt ccc atc aag gaa gac agc ttc ttg cag cga tac agc tca 3458 Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser 1060 1065 1070 gac ccc aca ggc gcc ttg act gag gac agc ata gac gac acc ttc ctc 3506 Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu 1075 1080 1085 cca gtg cct gaa tac ata aac cag tcc gtt ccc aaa agg ccc gct ggc 3554 Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly 1090 1095 1100 tct gtg cag aat cct gtc tat cac aat cag cct ctg aac ccc gcg ccc 3602 Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro 1105 1110 1115 agc aga gac cca cac tac cag gac ccc cac agc act gca gtg ggc aac 3650 Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn 1120 1125 1130 1135 ccc gag tat ctc aac act gtc cag ccc acc tgt gtc aac agc aca ttc 3698 Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe 1140 1145 1150 gac agc cct gcc cac tgg gcc cag aaa ggc agc cac caa att agc ctg 3746 Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu 1155 1160 1165 gac aac cct gac tac cag cag gac ttc ttt ccc aag gaa gcc aag cca 3794 Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro 1170 1175 1180 aat ggc atc ttt aag ggc tcc aca gct gaa aat gca gaa tac cta agg 3842 Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg 1185 1190 1195 gtc gcg cca caa agc agt gaa ttt att gga gca tga 3878 Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 1200 1205 1210 <210> 3 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 3 tgcarctcat cacgca 16 <210> 4 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 4 tgcarctcat caygca 16 <210> 5 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 5 tgcaactcat catgca 16

Claims

1. Use of an irreversible EGFR inhibitor in the preparation of a medicament for treating a patient with non-small cell lung cancer, wherein the patient with non-small cell lung cancer has an EGFR (SEQ ID NO: 1) mutant, wherein the irreversible EGFR inhibitor covalently binds to the cysteine ​​at position 773 in the ligand-binding pocket of EGFR and inhibits the EGFR mutant, wherein the EGFR mutant comprises a threonine at position 790 replaced by methionine, wherein the irreversible EGFR inhibitor is selected from EKB-569 and HKI-357.

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