Treatment of cancer resistant to EGFR tki using c-met inhibitor

Abstract

The present disclosure provides methods of treating a cancer in a subject. In one embodiment, the method comprises administering to the subject a therapeutic effective amount of a c-Met inhibitor, wherein the subject has been determined to have a c-MET gene amplification and an activating EGFR mutation.

Description

TREATMENT OF CANCER RESISTANT TO EGFR TKI USING C-MET INHIBITORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US provisional application 63 / 387,503, filed December 15, 2022, the disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention generally relates to cancer treatment. In particular, the present invention relates to methods for treating a cancer patient using c-Met inhibitor when the patient is resistant to the treatment of EGFR tyrosine kinase inhibitor.BACKGROUND

[0003] The epidermal growth factor receptor (EGFR) gene, also called ERBB1, encodes a 170-kDa transmembrane tyrosine kinase receptor. The EGFR is activated by binding to its ligands such as epidermal growth factor or transforming growth factor-alpha, resulting in homodimerization or heterodimerization with another member of the EGFR family. This receptor activation is followed by phosphorylation of specific tyrosine residues within the cytoplasmic tail, stimulating the downstream signaling pathway that regulates cell proliferation, migration, adhesion, differentiation and survival. Gene amplification and / or protein overexpression of EGFR have been observed in a variety of solid tumors, including lung, colorectal, urinary bladder, breast, head, neck, esophageal and gastric carcinomas. In some tumors such as non-small cell lung carcinoma and colorectal carcinoma, increased EGFR expression is associated with advanced stage and an unfavorable prognosis.

[0004] The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), such as Gefitinib and Erlotinib, Osimertinib are competitive inhibitors of the tyrosine kinase domain of EGFR that bind to its adenosine-5' triphosphate-binding site. Certain somatic activating mutations of the EGFR gene, increased gene copy number and clinical and pathological features have been associated with dramatic tumor responses and favorable clinical outcomes with these agents in cancer patients. However, some cancer patients harboring activating EGFR mutations still demonstrate resistance to EGFR TKI treatment. For example, although Osimertinib has offered substantial benefit for the classical plus T790M EGFR-mutant NSCLC patients, drug resistance develops and poses a critical challenge for long-term survival. The resistance is rooted to either on-target secondary EGFR mutations or compensatory oncogenic pathways bypassing EGFR TKIs intervention.

[0005] Therefore, there is an urgent need to develop new methods for treating cancer patients carrying EGFR activating mutations including the patients who will develop resistance to EGFR TKI after initial responses.SUMMARY

[0006] The present disclosure in one aspect provides a method of treating a subject having a cancer. In one embodiment, the method comprises administering to the subject a therapeutic effective amount of a c-Met inhibitor, wherein the subject has been determined to have a c-MET gene amplification and an activating EGFR mutation.

[0007] In some embodiments, the c-Met inhibitor is selected from the group consisting of Vebreltinib (APL-101), Savolitinib (volitinib), Crizotinib, Cabozantinib, PLB1001, bozitinib, SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF- 04217903, MK2461, GSK1363089 (XL880, foretinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, Tepotinib, HQP-8361, merestinib, ARGX-111, onartuzumab, rilotumumab, emibetuzumab, and XL184. In some embodiments, the c-Met inhibitor is Vebriltinib or Savolitinib. In some embodiments, the c-Met inhibitor is Vebriltinib.

[0008] In some embodiments, the activating EGFR mutation referred to in the present disclosure is selected from the group consisting of deletions in exon 19 (exl9del), L858R, T790M, exon 20 insertion (ex20ins), V765A, T783A, S768I, L861Q, and / or G719X (e.g., G719S, G719A and G719C). In some embodiments, the activating EGFR mutation is L858R, or exl9del, or T790M.

[0009] In some embodiments, the method provided herein further comprises administering to the subject a therapeutic effective amount of an EGFR tyrosine kinase inhibitor. In some embodiments, the EGFR tyrosine kinase inhibitor is selected from the group consisting of Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rociletinib, Omutinib, Neratinib, Lapatinib, Nazartinib, Naquotinib, Mavelertinib, Mobocertinib, Vandetanib and Avitinib. In some embodiments, the EGFR tyrosine kinase inhibitor is Osimertinib.

[0010] In some embodiments, the subject being treated by the method provided herein has been determined to be resistant to treatment of an EGFR tyrosine kinase inhibitor. In some embodiments, the EGFR tyrosine kinase inhibitor is selected from the group consisting of Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rociletinib, Omutinib,Neratinib, Lapatinib, Nazartinib, Naquotinib, Mavelertinib, Mobocertinib, Vandetanib and Avitinib. In some embodiments, the EGFR tyrosine kinase inhibitor is Erlotinib.

[0011] In some embodiments, the cancer being treated by the method provided herein is selected from the groups consisting of a lung cancer, a melanoma, a renal cancer, a liver cancer, a myeloma, a prostate cancer, a breast cancer, a colorectal cancer, a pancreatic cancer, a thyroid cancer, a hematological cancer, a leukemia and a non-Hodgkin’s lymphoma. In some embodiments, the cancer is a non-small cell lung cancer (NSCLC).BRIEF DESCRIPTION OF DRAWING

[0012] FIG. 1 shows that Vebriltinib (APL-101) monotherapy is effective for treatment of tumors harboring activating EGFR mutations with co-occurring c-Met amplification.

[0013] FIG. 2 shows that Vebriltinib (APL-101) induced c-Met protein degradation in tumors harboring both activating EGFR mutation and c-Met amplification.

[0014] FIG. 3 shows that Vebriltinib (APL-101) monotherapy is effective for treatment of tumors harboring activating EGFR mutations with acquired resistance to EGFR TKI due to c-Met amplification.

[0015] FIG. 4 shows that Vebriltinib (APL-101) and EGFR TKI (Osimertinib) combination (combo) treatment may be more effective than c-MET inhibitor monotherapy in tumors harboring EGFR activating mutation with acquired resistance to EGFR TKI.

[0016] FIG. 5 shows that Vebriltinib (APL-101) induced c-Met protein degradation in tumors harboring both activating EGFR mutation and c-Met amplification.

[0017] FIG. 6 shows that Vebriltinib (APL-101) and EGFR TKI combo treatment may lead to more durable response than EGFR TKI alone in tumors harboring EGFR activating mutations even when the tumors are not resistant to EGFR TKI.DETAILED DESCRIPTION OF THE INVENTION

[0018] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0020] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0021] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0022] Definitions

[0023] The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

[0024] As used herein, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0025] As used herein, an “antibody” encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies (e.g., humanized murine antibodies), and heteroconjugate antibodies (e.g., bispecific antibodies). Fragments of antibodies include those that bind antigen, (e.g., Fab', F(ab')2, Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Hl.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent orbispecific molecules, diabodies, triabodies, and tetrabodies. The term “antibody” further includes both polyclonal and monoclonal antibodies.

[0026] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

[0027] As used herein, the term “cancer” refers to any diseases involving an abnormal cell growth and includes all stages and all forms of the disease that affects any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. In general, cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells. As used herein, cancer types include, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, breast cancer, Burkitt's lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing's sarcoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukaemia, liver cancer, lung cancer, medulloblastoma, melanoma, neuroblastoma, nonHodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, , skin cancer, stomach cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thyroid cancer, vaginal cancer, visual pathway and hypothalamic glioma.

[0028] The term “cancer sample” includes a biological sample or a sample from a biological source that contains one or more cancer cells. Biological samples include samples from body fluids, e.g., blood, plasma, serum, or urine, or samples derived, e.g., by biopsy, from cells, tissues or organs, preferably tumor tissue suspected to include or essentially consist of cancer cells.

[0029] It is noted that in this disclosure, terms such as “comprises”, “comprised”, “comprising”, “contains”, “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Terms such as “consisting essentially of’ and “consists essentiallyof’ have the meaning attributed in United States Patent law; they allow for the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms “consists of’ and “consisting of’ have the meaning ascribed to them in United States Patent law; namely that these terms are close ended.

[0030] The term “c-Met” refers to a proto -oncogene that encodes a protein known as hepatocyte growth factor receptor (HGFR). c-Met protein is composed of the a chain and P chain generated by cleaving a precursor of c-Met (pro c-Met) and forms a dimer by a disulfide linkage. c-Met is a receptor penetrating a cell membrane and the entire a chain and a part of the P chain are present extracellularly (see, e.g., Mark, et al., The Journal of Biological Chemistry (1992) 267:26166-71; Ayumi I, Journal of Clinical and Experimental Medicine (2008) 224:51-55). See also GenBank Accession No: NP 000236.2 for human c-Met and its a chain and P chain. It has been shown that abnormal c-Met activation in cancer correlates with poor prognosis, where aberrantly active c-Met triggers tumor growth, formation of new blood vessels that supply the tumor with nutrients, and cancer spread or other organs.

[0031] A “c-Met inhibitor,” as used herein, refers an agent that can suppress the expression or activity of c-Met protein. Examples of c-Met inhibitor include, without limitation Crizotinib, Cabozantinib, Tepotinib, AMG337, Vebriltinib (also referred to as APL-101, PLB1001, bozitinib), SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ- 38877605, PF-04217903, MK2461, GSK1363089 (XL880, foretinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, Capmatinib), E7050, BMS-777607, Savolitinib (Volitinib), HQP-8361, merestinib, ARGX-111, onartuzumab, rilotumumab, emibetuzumab XL184 and compounds disclosed in US20150218171.

[0032] The terms “determining,” “assessing,” “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi -quantitative determinations. Where either a quantitative and semi -quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.

[0033] As used herein, the term “effective amount” or “therapeutically effective amount” means the amount of agent that is sufficient to prevent, treat, reduce and / or ameliorate the symptoms and / or underlying causes of any disorder or disease, or the amount of an agent sufficient to produce a desired effect on a cell. In one embodiment, a “therapeutically effective amount” is an amount sufficient to reduce or eliminate a symptom of a disease. In another embodiment, a therapeutically effective amount is an amount sufficient to overcome the disease itself.

[0034] The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0035] The term “sample” as used herein refers to a biological sample that is obtained from a subject and contains RNA transcripts. Examples of sample include, without limitation, cells, such as cancer cells, tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) and paraffin embedded tissues, and bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc. In certain embodiments, the sample can be a biological sample comprising cancer cells. In some embodiments, the sample is a fresh or archived sample obtained from a tumor, e.g., by a tumor biopsy or fine needle aspirate. The sample also can be any biological fluid containing cancer cells. The collection of a sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy.

[0036] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0037] As used herein, the term “toxin” means an antigenic poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof.

[0038] The term “treatment,” “treat,” or “treating” refers to a method of reducing the effects of a cancer (e.g., breast cancer, lung cancer, ovarian cancer or the like) or symptom ofcancer. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a cancer or symptom of the cancer. For example, a method of treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between 10 and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0039] EGFR TKIs and Activating EGFR mutations

[0040] The epidermal growth factor receptor (EGFR) family, also referred to as the HER or ErbB family, consists of four receptor tyrosine kinases (TKs) — EGFR (HERl / ErbBl), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) — that regulate many developmental, metabolic and physiological processes. The binding of EGFR with various cognate ligands, including EGF, transforming growth factor-a, amphiregulin and others, leading to homodimerization of two EGFRs or the heterodimerization of EGFR with other family members, most commonly HER2, thus increasing the intracellular EGFR TK activity. The activation of EGFR TK leads to the autophosphorylation of the intracellular domain of EGFR, and the phosphor-tyrosine residues thus formed serve as a docketing site for various adapter molecule, resulting in the activation of the Ras / mitogen-activated protein kinase pathway, the PI3K / Akt pathway and signal transducers and activators of transcription signaling pathways.

[0041] In tumor cells, the TK activity of EGFR may be dysregulated by various oncogenic mechanisms, including EGFR gene mutation, increased gene copy number and EGFR protein overexpression. Improper activation of EGFR TK results in increased malignant cell survival, proliferation, invasion and metastasis. EGFR overexpression is observed in tumors from more than 60% of patients with metastatic non-small-cell lung cancer (NSCLC) and is correlated with poor prognosis. EGFR activation mutations are drivers for NSCLC with overall prevalence ranges from 10% to 50% worldwide (Molecular Diagnostic & Therapy (2022) 26:7-18). These findings have provided a rationale for the development of anticancer agents that inhibit the activity of EGFR tyrosine kinase.

[0042] EGFR TKIs

[0043] First-generation EGFR TKIs, including Gefitinib, Erlotinib and Icotinib, reversibly bind to EGFR and inhibit the binding of ATP to the TK domain. This blockhampers cell proliferation, ultimately leading to cell death. The structures of Gefitinib, Erlotinib and Icotinib are shown below.

[0046] Icotinib

[0047] The second-generation EGFR TK inhibitors, including Afatinib andDacomitinib, are irreversible inhibitors, which covalently bind to EGFR. The structures of the second-generation EGFR TKIs are shown below.

[0051] Despite the initial high response rates, many patients on the first and second- generation EGFR TKIs will become resistant to treatment. Various mechanisms of acquired resistance have been identified and these can be divided into secondary mutations in EGFR, the activation of alternative signaling pathways, and phenotypic or histologic transformation. The commonest mechanism of acquired resistance is EGFR T790M mutation, which accounts for 50-60% of secondary resistance to primary EGFR TKI therapy. Given the limited efficacy of the first and second-generation EGFR TKIs, the third-generation EGFR TKIs were developed. These include Osimertinib, Nazartinib, Olmutinib, Mavelertinib, YH5448, Avitinib and Rociletinib. The structures of the third-generation EGFR TKIs are shown below.

[0056]

[0057] Rociletinib

[0058] Activating EGFR Mutations

[0059] Treatment with the EGFR TK inhibitors (TKIs), such as Gefitinib and Erlotinib, results in dramatic antitumor activity in a subset of patients with NSCLC. Sequencing of the EGFR gene revealed that a majority of tumors responding to EGFR TKIs harbored mutations in the TK domain of EGFR gene. The mutations in the TK domain of EGFR gene are referred to as “activating EGFR mutations” because they lead to a ligandindependent activation of TK activity.

[0060] The activating EGFR mutations are commonly found in the first four exons (18 through 21) of the TK domain of EGFR gene. These mutations generally fall into three major classes, with the majority of EGFR TKI-sensitizing mutations falling into class I and II. Class I mutations are in-frame deletions in exon 19 (exl9del); these deletions almost always include amino-acid residues leucine-747 to glutamic acid-749 (ALRE). Class II mutations are single-nucleotide substitutions that cause an amino-acid alteration. The predominant singlepoint mutation is in exon 21, which substitutes an arginine for a leucine at codon 858 (L858R). L858R has the highest prevalence of any single-point activating mutation in EGFR TK. Other class II activating mutations include those result in a glycine-719 (G719) change to serine, alanine or cysteine (G719X). Class III mutations are in-frame duplications and / or insertions in exon 20 (ex20ins). A variety of other activating mutations have been detected with low frequency, including V765A, S768I and T783A in exon 20 and L861Q in exon 21.

[0061] In some embodiments, the activating EGFR mutation referred to in the present disclosure is selected from the group consisting of ex!9del, L858R, ex20ins, and T790M.

[0062] C -Met Gene Amplification

[0063] Many cancer patients treated with the EGFR TKIs acquire resistance to treatment, resulting the major obstacle to long-term disease remission in the clinic. C-Met gene amplification has been known as a resistance mechanism to first- or second-generation EGFR TKIs in addition to the appearance of T790M mutation. The present disclosure in one aspect provides a method of using a c-Met inhibitor to treat cancer patients having both activating EGFR mutation and c-Met gene amplification.

[0064] The proto-oncogene c-MET encodes for the receptor tyrosine kinase (RTK) c- Met. Cells of epithelial-endothelial origin widely express c-MET, where it is essential for embryonic development and tissue repair. Hepatocyte growth factor (HGF) is the only known ligand for the c-Met receptor and is expressed mainly in cells of mesenchymal origin. Under normal conditions, c-Met dimerizes and auto-phosphorylates upon ligand binding, which in turn creates active docking sites for proteins that mediate downstream signaling leading to the activation of the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3 -kinase (PI3K)-AKT, v-src sarcoma viral oncogene homolog (SRC), signal transducer and activator of transcription (STAT) signaling pathways. Such activation evokes a variety of pleiotropic biological responses leading to increased cell growth, scattering and motility, invasion, protection from apoptosis, branching morphogenesis, and angiogenesis. However, under pathological conditions improper activation of c-Met may confer proliferative, survival and invasive / metastatic abilities of cancer cells.

[0065] Deregulation and the consequent aberrant signaling of c-Met may occur by different mechanisms including gene amplification and activating mutations. It has been reported that c-Met is overexpressed in a variety of carcinomas including lung, breast, ovary, kidney, colon, thyroid, live rand gastric carcinomas. Such overexpression could be the result of transcription activation, hypoxia-induced overexpression, or as a result of c-Met gene amplification. Gene amplification is a frequent genetic alteration of c-Met and has been reported as associated with a poor prognosis in NSCLC, colorectal and gastric cancer.

[0066] Detection of Activating EGFR Mutation and c-Met Gene Amplification

[0067] The activating EGFR mutations and / or the c-Met gene amplification in a cancer patient can be detected by proper methods known in the art including without limitation, a amplification assay, a hybridization-based assay, a sequencing-based assay and an immunoassay.

[0068] Amplification assay

[0069] A nucleic acid amplification assay involves copying a target nucleic acid (e.g., DNA or RNA), thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification may be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using the polymerase chain reaction ("PCR", see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (Innis et al., eds, 1990)), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR); quantitative PCR, such as TaqMan®, nested PCR, ligase chain reaction (See Abravaya, K., et al., Nucleic Acids Research, 23:675- 682, (1995), branched DNA signal amplification (see, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S14, (1993), amplifiable RNA reporters, Q-beta replication (see Lizardi et al., Biotechnology (1988) 6: 1197), transcription-based amplification (see, Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86: 1173-1177), boomerang DNA amplification, strand displacement activation, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87: 1874-1878), rolling circle replication (U.S. Patent No. 5,854,033), isothermal nucleic acid sequence based amplification (NASBA), and serial analysis of gene expression (SAGE).

[0070] In certain embodiments, the nucleic acid amplification assay is a PCR-based method. PCR is initiated with a pair of primers that hybridize to the target nucleic acid sequence to be amplified, followed by elongation of the primer by polymerase which synthesizes the new strand using the target nucleic acid sequence as a template and dNTPs as building blocks. Then the new strand and the target strand are denatured to allow primers to bind for the next cycle of extension and synthesis. After multiple amplification cycles, the total number of copies of the target nucleic acid sequence can increase exponentially.

[0071] In certain embodiments, intercalating agents that produce a signal when intercalated in double stranded DNA may be used. Exemplary agents include SYBR GREEN™ and SYBR GOLD™. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.

[0072] In certain embodiments, a detectably labeled primer or a detectably labeled probe can be used, to allow detection of the activating EGFR mutation corresponding to that primer or probe. In certain embodiments, multiple labeled primers or labeled probes with different detectable labels can be used to allow simultaneous detection of multiple activating EGFR mutation.

[0073] Hybridization assay

[0074] Nucleic acid hybridization assays use probes to hybridize to the target nucleic acid, thereby allowing detection of the target nucleic acid. Non-limiting examples of hybridization assay include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplexed hybridization-based assays.

[0075] In certain embodiments, the probes for hybridization assay are detectably labeled. In certain embodiments, the nucleic acid-based probes for hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray, and can hybridize to the target nucleic acid molecules which are detectably labeled.

[0076] In certain embodiments, hybridization assays can be performed by isolating the nucleic acids (e.g., RNA or DNA), separating the nucleic acids (e.g. by gel electrophoresis) followed by transfer of the separated nucleic acid on suitable membrane filters (e.g. nitrocellulose filters), where the probes hybridize to the target nucleic acids and allows detection. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7. The hybridization of the probe and the target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing hybridized filters to photographic film.

[0077] In some embodiments, hybridization assays can be performed on microarrays. Microarrays provide a method for the simultaneous measurement of the levels of large numbers of target nucleic acid molecules. The target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having multiple immobilized nucleic acid probes arrayed at a density of up to several million probes per square centimeter of the substrate surface. The RNA or DNA in the sample is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative levels of the RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316.

[0078] Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261. Although a planar array surface is often employed the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. PatentNos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. Useful microarrays are also commercially available, for example, microarrays from Affymetrix, from Nano String Technologies, QuantiGene 2.0 Multiplex Assay from Panomics.

[0079] In certain embodiments, hybridization assays can be in situ hybridization assay. In situ hybridization assay is useful to detect the presence of c-Met gene amplification. Probes useful for in situ hybridization assay can be mutation or gene fusion specific probes, which hybridize to a specific activating EGFR mutation to detect the presence or absence of the specific mutation of interest. Methods for use of unique sequence probes for in situ hybridization are described in U.S. Pat. No. 5,447,841, incorporated herein by reference. Probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. See, e.g., U.S. Pat. No. 5,776,688 to Bittner, et al., which is incorporated herein by reference. Any suitable microscopic imaging method can be used to visualize the hybridized probes, including automated digital imaging systems. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.

[0080] Sequencing methods

[0081] Sequencing methods useful in the measurement of the activating EGFR mutation of interest and / or the c-Met gene amplification involves sequencing of the target nucleic acid. Any sequencing known in the art can be used to detect the EGFR mutation and / or the c-Met gene amplification. In general, sequencing methods can be categorized to traditional or classical methods and high throughput sequencing (next generation sequencing). Traditional sequencing methods include Maxam-Gilbert sequencing (also known as chemical sequencing) and Sanger sequencing (also known as chain-termination methods).

[0082] High throughput sequencing, or next generation sequencing, by using methods distinguished from traditional methods, such as Sanger sequencing, is highly scalable and able to sequence the entire genome or transcriptome at once. High throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437 (7057): 376-80 (2005)). Sequence-by-synthesis involves synthesizing a complementary strand of the target nucleic acid by incorporating labeled nucleotide or nucleotide analog in a polymerase amplification. Immediately after or upon successful incorporation of a label nucleotide, a signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide isremoved before the incorporation, detection and identification steps are repeated. Examples of sequence-by-synthesis methods are known in the art, and are described for example in U.S. Pat. No. 7,056,676, U.S. Pat. No. 8,802,368 and U.S. Pat. No. 7,169,560, the contents of which are incorporated herein by reference. Sequencing-by-synthesis may be performed on a solid surface (or a microarray or a chip) using fold-back PCR and anchored primers. Target nucleic acid fragments can be attached to the solid surface by hybridizing to the anchored primers, and bridge amplified. This technology is used, for example, in the Illumina® sequencing platform.

[0083] Pyrosequencing involves hybridizing the target nucleic acid regions to a primer and extending the new strand by sequentially incorporating deoxynucleotide triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. Each base incorporation is accompanied by release of pyrophosphate, converted to ATP by sulfurylase, which drives synthesis of oxyluciferin and the release of visible light. Since pyrophosphate release is equimolar with the number of incorporated bases, the light given off is proportional to the number of nucleotides adding in any one step. The process is repeated until the entire sequence is determined.

[0084] In certain embodiments, the EGFR mutation and the c-Met gene amplification described herein is detected by transcriptome shotgun sequencing (RNA sequencing) and exome sequencing (DNA sequencing).

[0085] Immunoassay

[0086] Immunoassays used herein typically involves using antibodies that specifically bind to c-Met protein. Such antibodies can be obtained using methods known in the art (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al, Nature (1989) 341 :544-546), or can be obtained from commercial sources. Examples of immunoassays include, without limitation, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunoprecipitations, sandwich assays, competitive assays, immunofluorescent staining and imaging, immunohistochemistry (H4C), and fluorescent activating cell sorting (FACS). For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds., 7thed. 1991). Moreover, the immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7thed. 1991).

[0087] In certain embodiments, the c-Met expression level is measured as the level of a subset of c-Met protein, such as the level of modified c-Met protein, e.g. phosphorylated c- Met protein. In such cases, the c-Met expression level can be detected using antibodies that specifically bind to the modified c-Met protein.

[0088] Any of the assays and methods provided herein for the measurement of the c-Met expression level can be adapted or optimized for use in automated and semi -automated systems, or point of care assay systems.

[0089] The c-Met expression level described herein can be normalized using a proper method known in the art. For example, the c-Met expression level can be normalized to a standard level of a standard marker, which can be predetermined, determined concurrently, or determined after a sample is obtained from the subject. The standard marker can be run in the same assay or can be a known standard marker from a previous assay. For another example, the c-Met expression level can be normalized to an internal control which can be an internal marker, or an average level or a total level of a plurality of internal markers.

[0090] Treatment with c-Met Inhibitor

[0091] A “c-Met inhibitor,” as used herein, refers to an agent that can suppress the expression or activity of c-Met protein. In certain embodiments, c-Met inhibitor is selected from the group consisting of Crizotinib, Cabozantinib, Tepotinib, AMG337, Vebriltinib (APL-101, PLB1001, bozitinib), SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ- 38877605, PF-04217903, MK2461, GSK1363089 (XL880, foretinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, savolitinib (volitinib), HQP-8361, merestinib, ARGX-111, onartuzumab, rilotumumab, emibetuzumab, and XL184.

[0092] In certain embodiments, c-Met inhibitor is Vebriltinib (APL-101, previously named CBT-101, see US20150218171, which is incorporated in its entirety by reference), which has the following formula:

[0093] In certain embodiments, c-Met inhibitor can be formulated with a pharmaceutically acceptable carrier. The carrier, when present, can be blended with c-Met inhibitor in any suitable amounts, such as an amount of from 5% to 95% by weight of carrier, based on the total volume or weight of c-Met inhibitor and the carrier. In some embodiments,the amount of carrier can be in a range having a lower limit of any of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70% or 75%, and an upper limit, higher than the lower limit, of any of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95%. The amount of carrier in a specific embodiment may be determined based on considerations of the specific dose form, relative amounts of c-Met inhibitor, the total weight of the composition including the carrier, the physical and chemical properties of the carrier, and other factors, as known to those of ordinary skill in the formulation art.

[0094] The c-Met inhibitor may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the c-Met inhibitor may be administered in conjunction with other treatments. The c-Met inhibitor may be encapsulated or otherwise protected against gastric or other secretions, if desired.

[0095] A suitable, non-limiting example of a dosage of the c-Met inhibitor disclosed herein is from about 1 mg / kg to about 2400 mg / kg per day, such as from about 1 mg / kg to about 1200 mg / kg per day, 75 mg / kg per day to about 300 mg / kg per day, including from about 1 mg / kg to about 100 mg / kg per day. Other representative dosages of such agents include about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1300 mg / kg, 1400 mg / kg, 1500 mg / kg, 1600 mg / kg, 1700 mg / kg, 1800 mg / kg, 1900 mg / kg, 2000 mg / kg, 2100 mg / kg, 2200 mg / kg, and 2300 mg / kg per day. In some embodiments, the dosage of the c-Met inhibitor in human is about 400 mg / day given every 12 hours. In some embodiments, the dosage of the c-Met inhibitor in human ranges 300-500 mg / day, 100-600 mg / day or 25-1000 mg / day. The effective dose of c-Met inhibitor disclosed herein may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0096] In one embodiment, the method further comprises administering at least one additional therapeutic agent selected from the group consisting of a modulator of immune checkpoint, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radio-sensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.

[0097] As used herein, the term “immune checkpoint” or “cancer immune checkpoint” refers to a molecule in the immune system that either turns up a signal (i.e., co-stimulatory molecules) or turns down a signal (i.e., inhibitory molecule) of an immune response. In certain embodiments, the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, LAG-3, TIM-1, CTLA-4, VISTA, B7-H2, B7-H3, B7-H4, B7-H6, 284, ICOS, HVEM, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-4, BTLA, SIRP alpha (CD47), CD48, 284 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT and A2aR.

[0098] In certain embodiments, the modulator of immune checkpoint is a monoclonal antibody against the immune checkpoint. In certain embodiments, the immune checkpoint is PD-1 orPD-Ll. In certain embodiments, the anti -PD-1 antibody is selected from those disclosed in PCT application publication No. WO2016 / 014688, which is incorporated in its entirety by reference. In certain embodiments, the anti-PD-1 antibody is APL-501 (previously named as CBT-501, see WO2016 / 014688), GB226 or genolimzumab. In certain embodiments, the anti-PD-Ll antibody is selected from those disclosed in PCT application publication No. W02016 / 022630, which is incorporated in its entirety by reference. In certain embodiments, the anti-PD-Ll antibody is APL-502 (previously named as CBT-502, see WO2016 / 022630) or TQB2450.

[0099] Anti-cancer Agents Other Than c-Met Inhibitor

[0100] The method of present disclosure also involves administering to the subject a combination of a c-Met inhibitor and a second anti-cancer agent other than the c-Met inhibitor. In some embodiments, the second anti-cancer agent is an EGFR TKI, which has been disclosed above.

[0101] Other anti -cancer agent that can be used in combination with the c-Met inhibitor in the method disclosed herein include, without limitation: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g. cytoxan®), chlorambucil (CHL; e.g. leukeran®), cisplatin (CisP; e.g. platinol®) busulfan (e.g. myleran®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites, such as methotrexate (MTX), etoposide (VP16; e.g. vepesid®), 6 -mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5- fluorouracil (5-FU), capecitabine (e.g.Xeloda®), dacarbazine (DTIC), and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g. adriamycin®), daunorubicin (daunomycin), bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g. taxol®) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such asdexamethasone (DEX; e.g. decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin, folinic acid, raltitrexed, and other folic acid derivatives, and similar, diverse antitumor agents. The following agents may also be used as additional agents: arnifostine (e.g. ethyol®), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lornustine (CCNU), doxorubicin lipo (e.g. doxil®), gemcitabine (e.g. gemzar®), daunorubicin lipo (e.g. daunoxome®), procarbazine, mitomycin, docetaxel (e.g. taxotere®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl -camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, and chlorambucil.

[0102] In some embodiment, the drug used in the method disclosed herein include, without limitation: Alymsys® (Bevacizumab), Avastin® (Bevacizumab), Camptosar® (Irinotecan Hydrochloride), Capecitabine, Cetuximab, Cyramza® (Ramucirumab), Eloxatin® (Oxaliplatin), Erbitux® (Cetuximab), 5-FU (Fluorouracil Injection), Fluorouracil Injection, Ipilimumab, Irinotecan Hydrochloride, Keytruda® (Pembrolizumab), Leucovorin Calcium, Lonsurf® (Trifluridine and Tipiracil Hydrochloride), Mvasi® (Bevacizumab), Opdivo® (Nivolumab), Oxaliplatin, Panitumumab, Pembrolizumab, Ramucirumab, Regorafenib, Stivarga® (Regorafenib), Trifluridine and Tipiracil Hydrochloride, Vectibix® (Panitumumab), Xeloda® (Capecitabine), Yervoy® (Ipilimumab), Zaltrap® (Ziv- Aflibercept), Zirabev® (Bevacizumab), Ziv-Aflibercept.

[0103] The drug described herein may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the drug may be administered in conjunction with other treatments.

[0104] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended tolimit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.Example 1

[0105] This example is an in vivo anti -tumor efficacy study of Vebriltinib in LU0858 NSCLC patient-derived xenograft (PDX), a tumor model harboring EGFR L858R mutation with MET amplification. This study illustrates that Vebriltinib monotherapy is effective for treatment of tumors carrying activating EGFR mutations, resistant to EGFR TKI, and with co-occurring c-Met amplification.

[0106] Materials and Methods

[0107] BALB / c nude mice, female, age 6-8 weeks were provided by Shanghai Slack Experimental Animal Co., Ltd. (Shanghai, China). c-MET TKI Vebriltinib was provided by Crown Biotechnology (Taicang) Co., Ltd. (Taicang, China). EGFR TKI Erlotinib was provided by Nanjing Anji Biotechnology Co., Ltd. (Nanjing China). LU0858 (EGFR L858R, MET amplified) PDX tumor was provided by Crown Biotechnology (Taicang) Co., Ltd. (Taicang, China).

[0108] Mice (BALB / c nude) bearing LU0858 tumors were treated with indicated doses of Vebriltinib (0-21 mg / kg, orally once a day) or with EGFR inhibitor, Erlotinib (50 mg / kg orally once a day) for 21 days. Tumor volumes and body weight of mice were measured after treatment initiation. Tumor samples were harvested at the end of the study to assess pharmacodynamic inhibition of Vebriltinib on phospho-MET and total MET proteins by western blotting and IHC.

[0109] Results

[0110] Tumor growth was inhibited by Vebriltinib (at 7 and 21 mg / kg) to the end of treatment with ED50 of 3.0 mg / kg (FIG. 1). No significant tumor shrinkage was observed with Erlotinib alone, suggesting this EGFR mutant PDX is resistance to EGFR TKI. Data values are presented as mean ± SEM of n=8; * P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnetf s post-tests). In addition, all mice tolerated well to the treatments in this study as assessed by body weight changes. Western blot analysis of tumor lysates (upper panel) and immunohistochemistry of tumor tissue IHC (lower panel)demonstrated inhibition of both phospho-MET and total MET proteins by western blotting and IHC from tumors treated with 21 mg / kg APL-101 (FIG. 2).Example 2

[0111] This example is an in vivo anti -tumor efficacy study of Vebriltinib in LD1- 0025-200662 NSCLC patient-derived xenograft (PDX), a tumor model harboring EGFR exl9del mutation with MET amplification. This study illustrates that Vebriltinib monotherapy is effective for treatment of tumors carrying activating EGFR mutations, resistant to EGFR TKI, and with co-occurring c-Met amplification.

[0112] Materials and Methods

[0113] NU / NU nude mice, female, age 5-6 weeks were provided by Beijing Vital River Laboratory Animal Technology co. LTD. (Beijing, China). c-MET TKI Vebriltinib was provided by Laurus Lab Ltd. (Visakhapatnam, India). c-MET TKI Savolitinib was provided by Selleckchem (Texas, USA). EGFR TKI Osimertinib was provided by Selleckchem (Texas, USA). EGR TKI APL-122 (NT-113) was provided by Inogent Laboratories Private Limited (Hyderabad, India). LD 1-0025-200662 (EGFR exl9del, MET amplified) PDX tumor was provided by Xi’an LideBiotech CO., LTD (Xi’an, China).

[0114] NU / NU nude mice bearing LD 1-0025-200662 tumors were treated with Vebriltinib, Osimertinib, Savolitinib, or APL-122 as single-agent, or in combo treatment. All compounds were administrated at the same dose of 10 mg / kg orally once a day for 21 days. Tumor volumes and body weight of mice were measured after treatment initiation (Day 0). Mice were continuously raised, and tumor volumes and body weight of mice measured to observer tumor re-growth until day 70.

[0115] Results

[0116] Tumor growth was completely inhibited by Vebriltinib or Savolinib as singleagent or in combo with Osimertinib or APL-122 (FIG. 3). No significant tumor shrinkage was observed with Osimertinib or APL-122 as single-agent, suggesting this EGFR mutant PDX is resistance to EGFR TKI. Tumor re-growth was observed in tumors treated with Savolinib alone or in combo with Osimertinib after dosing stopped. In contrast, no tumor regrowth was observed in tumors treated with Vebriltinib alone or in combo with Osimertinib or APL-122 after dosing stopped. Data values are presented as mean ± SEM of n=8; * P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnett's post-tests). In addition, all mice tolerated well to the treatments in this study as assessed by body weight changes.Example 3

[0117] This example is an in vivo anti -tumor efficacy study of Vebriltinib in LD1- 0025-361336 NSCLC PDX, a tumor model harboring EGFR exl9del mutation with MET amplification. This study illustrates that Vebriltinib and EGFR TKI (Osimertinib) combo treatment is more effective than c-MET inhibitor monotherapy in some EGFR TKI resistant tumor.

[0118] Materials and Methods

[0119] NCG immunodeficient mice, female, age 5-6 weeks were provided by GemPharmatech Co., LTD (Nanjing, China). c-MET TKI APL-101 was provided by Laurus Lab Ltd. (Visakhapatnam, India). c-MET TKI Savolitinib was provided by Selleckchem (Texas, USA). EGFR TKI Osimertinib was provided by Selleckchem (Texas, USA). EGR TKI APL-122 (NT-113) was provided by Inogent Laboratories Private Limited (Hyderabad, India). LD1-0025-361336 (EGFR exl9del, MET amplified) PDX tumor was provided by Xi’an LideBiotech CO., LTD (Xi’an, China).

[0120] NCG immunodeficient mice bearing LD1-0025-361336 tumors were treated with APL-101, Osimertinib, Savolitinib, or APL-122 as single-agent, or in combo treatment. All compounds were administrated at the same dose of 10 mg / kg orally once a day for 28 days. Tumor volumes and body weight of mice were measured after treatment initiation for 28 days. Tumor samples were harvested at the end of the study to assess pharmacodynamic inhibition of Vebriltinib on phospho-MET and total MET proteins by western blotting.

[0121] Results

[0122] Tumor growth was partially inhibited by Osimertinib or APL-122 as singleagent, suggesting that this EGFR mutant PDX is partially resistant to EGFR TKI. APL-101 or Savolitinib single-agent only partially inhibited the tumor growth, in contrast to its complete anti -tumor effect on LU0858 and LD 1-0025-200662 (FIG. 4). Combo treatment of c-MET TKI (APL-101 or Savolitinib) with EGFR TKI (Osimertinib or APL-122) showed complete tumor inhibition, suggesting that c-MET TKI and EGFR TKI combo treatment is more effective than c-MET inhibitor monotherapy in EGFR mutant tumors with partial resistance to EGFR TKI (FIG. 4). As shown in FIG. 5, The stronger anti-tumor effects of c-MET and EGFR TKI Combo treatment is associated with c-Met protein degradation. Data values are presented as mean ± SEM of n=8; * P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnett's post-tests). In addition, all mice tolerated well to the treatments in this study as assessed by body weight changes.Example 4

[0123] This example is an in vivo anti -tumor efficacy study of Vebriltinib in LD1- LU1868 NSCLC PDX, a tumor model harboring EGFR T790M mutation with low c-MET expression level and no evidence of MET amplification. This study illustrates that Vebriltinib and EGFR TKI (Osimertinib) combo treatment may lead to more durable response than EGFR TKI alone in EGFR mutant NSCLC responding to EGFR TKI.

[0124] Materials and Methods

[0125] BALB / c Nude mice, female, age 6-7 weeks were provided by GemPharmatech Co., LTD. (Nanjing, China). c-MET TKI Vebriltinib was provided by Laurus Lab Ltd. (Visakhapatnam, India). EGFR TKI Osimertinib was provided by Selleckchem (Texas, USA). LUI 868 (EGFR T790M) PDX tumor was provided by Crown Biotechnology (Taicang) Co., Ltd. (Taicang, China).

[0126] BALB / c Nude mice bearing LUI 868 tumors were treated with Vebriltinib or Osimertinib as single-agent, or in combo treatment. All compounds were administrated at the same dose of 10 mg / kg orally once a day for 40 days. Mice were continuously raised after dosing stopped, and tumor volumes and body weight of mice measured to observer tumor regrowth for 74 days.

[0127] Results

[0128] Tumor growth was completely inhibited by Osimertinib as single-agent or in combo with Vebriltinib, suggesting that this EGFR mutant PDX is sensitive to EGFR TKI. Vebriltinib treatment as single agent did not show any anti-tumor effects. However, addition of Vebriltinib to Osimertinib treatment showed longer duration of tumor suppression than Osimertinib alone after dosing stopped as shown in FIG. 6. Data values are presented as mean ± SEM of n=5; * P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnett's post-tests). In addition, all mice tolerated well to the treatments in this study as assessed by body weight changes.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having a cancer, the method comprising: administering to the subject a therapeutic effective amount of a c-Met inhibitor, wherein the subject has been determined to have a c-MET gene amplification and an activating EGFR mutation.

2. The method of claim 1, wherein the c-Met inhibitor is selected from the group consisting of Vebriltinib (APL-101), Savolitinib (volitinib), Crizotinib, Cabozantinib, PLB1001, bozitinib, SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF- 04217903, MK2461, GSK1363089 (XL880, foretinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, Tepotinib, HQP-8361, merestinib, ARGX-111, onartuzumab, rilotumumab, emibetuzumab, and XL184.

3. The method of claim 1, wherein the c-Met inhibitor is Vebriltinib or Savolitinib.

4. The method of claim 1, wherein the c-Met inhibitor is Vebriltinib.

5. The method of claim 1, wherein the activating EGFR mutation is a deletion of EGFR exon 19, L858R, or T790M.

6. The method of claim 1, wherein the activating EGFR mutation is L858R.

7. The method of claim 1, further comprising administering to the subject a therapeutic effective amount of an EGFR tyrosine kinase inhibitor.

8. The method of claim 7, wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rociletinib, Omutinib, Neratinib, Lapatinib, Nazartinib, Naquotinib, Mavelertinib, Mobocertinib, Vandetanib and Avitinib.

9. The method of claim 7, wherein the EGFR inhibitor is Osimertinib.

10. The method of claim 1, wherein the subject has been determined to be resistant to treatment of an EGFR tyrosine kinase inhibitor.

11. The method of claim 10, wherein the EGFR inhibitor is selected from the group consisting of Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib,Rociletinib, Omutinib, Neratinib, Lapatinib, Nazartinib, Naquotinib, Mavelertinib, Mobocertinib, Vandetanib and Avitinib. The method of claim 10, wherein the EGFR inhibitor is Erlotinib. The method of claim 1, wherein the cancer is selected from the groups consisting of a lung cancer, a melanoma, a renal cancer, a liver cancer, a myeloma, a prostate cancer, a breast cancer, a colorectal cancer, a pancreatic cancer, a thyroid cancer, a hematological cancer, a leukemia and a non -Hodgkin’s lymphoma. The method of claim 1, wherein the cancer is a non-small cell lung cancer (NSCLC). A method of treating a subject having a cancer, the method comprising: administering to the subject a therapeutic effective amount of a c-Met inhibitor and a therapeutic effective amount of an EGFR tyrosine kinase inhibitor, wherein the subject has been determined to have an activating EGFR mutation responding to treatment with EGFR inhibitors. The method of claim 15, wherein the subject is not detected as having MET amplification. The method of claim 15, wherein the c-Met inhibitor is selected from the group consisting of Vebriltinib (APL-101), Savolitinib (volitinib), Crizotinib, Cabozantinib, PLB1001, bozitinib, SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF-04217903, MK2461, GSK1363089 (XL880, foretinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, Tepotinib, HQP- 8361, merestinib, ARGX-111, onartuzumab, rilotumumab, emibetuzumab, and XL184. The method of claim 15, wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rociletinib, Omutinib, Neratinib, Lapatinib, Nazartinib, Naquotinib, Mavelertinib, Mobocertinib, Vandetanib and Avitinib.

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