Treatment of cancer resistant to EGFR TKI using c-MET inhibitors

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application 63 / 387,503, filed on December 15, 2022, the disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention generally relates to cancer treatment. Specifically, the present invention relates to methods for treating cancer patients with c-Met inhibitors when the patients are resistant to treatment with EGFR tyrosine kinase inhibitors. Background Art

[0004] The epidermal growth factor receptor (EGFR) gene (also known as ERBB1) encodes a 170-kDa transmembrane tyrosine kinase receptor. EGFR is activated by binding to its ligands, such as epidermal growth factor or transforming growth factor α, such that it homodimerizes or heterodimerizes with another member of the EGFR family. This receptor activation is followed by phosphorylation of specific tyrosine residues within the cytoplasmic tail, stimulating downstream signaling pathways that regulate 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 cancer, colorectal cancer, bladder cancer, breast cancer, head and neck cancer, esophageal cancer, and gastric cancer. In some tumors, such as non-small cell lung cancer and colorectal cancer, increased EGFR expression is associated with advanced stage and poor prognosis.

[0005] Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), such as Gefitinib, Erlotinib, and Osimertinib, are competitive inhibitors of the tyrosine kinase domain of EGFR, binding to its adenosine-5'-triphosphate binding site. In cancer patients, certain somatic activating mutations of the EGFR gene, gene copy number increases, and clinical and pathological features are associated with significant tumor responses and favorable clinical outcomes with the use of these agents. However, some cancer patients carrying activating EGFR mutations still exhibit resistance to EGFR TKI treatment. For example, although Osimertinib provides substantial benefits to patients with classical plus T790M EGFR mutant NSCLC, the development of resistance poses a critical challenge to long-term survival. Resistance results from on-target secondary EGFR mutations or compensatory oncogenic pathways that bypass EGFR TKI intervention.

[0006] Therefore, there is an urgent need to develop new methods for treating cancer patients carrying activating EGFR mutations, including those who develop resistance to EGFR TKIs after an initial response. Summary of the Invention

[0007] One aspect of the present disclosure provides a method for treating a subject suffering from cancer. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a c-Met inhibitor, wherein the subject has been determined to have c-MET gene amplification and an activating EGFR mutation.

[0008] 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 Vebreltinib or Savolitinib. In some embodiments, the c-Met inhibitor is Vebreltinib.

[0009] In some embodiments, the activating EGFR mutations mentioned in the present disclosure are selected from the group consisting of: exon 19 deletion (ex19del), 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 ex19del or T790M.

[0010] In some embodiments, the methods provided herein further comprise administering to the subject a therapeutically 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, olmutinib, neratinib, lapatinib, nazartinib, naquotinib, mavelertinib, mobocertinib, vandetanib, and avitinib. In some embodiments, the EGFR tyrosine kinase inhibitor is osimertinib.

[0011] In some embodiments, it has been determined that a subject treated by the methods provided herein is resistant to treatment with 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, olmutinib, neratinib, lapatinib, nazartinib, naquotinib, mavelertinib, mobocertinib, vandetanib, and avitinib. In some embodiments, the EGFR tyrosine kinase inhibitor is erlotinib.

[0012] In some embodiments, the cancers treated by the methods provided herein are selected from the group consisting of: lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancers, leukemia, and non-Hodgkin's lymphoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shows that APL-101 monotherapy is effective in treating tumors carrying activating EGFR mutations with co-occurring c-Met amplification.

[0014] Figure 2 Shows that APL-101 induces c-Met protein degradation in tumors co-carrying activating EGFR mutations and c-Met amplification.

[0015] Figure 3It is shown that the single-agent therapy of Vanpire (APL-101) is effective in treating tumors carrying activating EGFR mutations with acquired resistance to EGFR TKI due to c-Met amplification.

[0016] Figure 4 It is shown that in tumors carrying EGFR activating mutations with acquired resistance to EGFR TKI, the combination therapy of Vanpire (APL-101) and EGFR TKI (Osimertinib) may be more effective than the single-agent therapy of c-MET inhibitor.

[0017] Figure 5 It is shown that Vanpire (APL-101) induces c-Met protein degradation in tumors simultaneously carrying activating EGFR mutations and c-Met amplification.

[0018] Figure 6 It is shown that in tumors carrying EGFR activating mutations, even when the tumors are not resistant to EGFR TKI, the combination therapy of Vanpire (APL-101) and EGFR TKI may produce a more durable response than EGFR TKI alone. Detailed Description

[0019] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus can of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive, as the scope of the present disclosure will be defined only by the appended claims.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure 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.

[0021] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and incorporated herein by reference to disclose and describe the methods and / or materials. 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. Additionally, the provided publication dates may differ from the actual publication dates, which may need to be independently confirmed.

[0022] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and shown herein has discrete components and features that can be readily separated from or combined with the features of any of several other embodiments without departing from the scope or spirit of the disclosure. Any of the recited methods can be performed in the recited order of events or in any other order that is logically possible.

[0023] Definitions

[0024] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms used herein are intended to have the meanings commonly understood by those skilled in the chemical and pharmaceutical arts. In some instances, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from the definitions commonly understood in the art.

[0025] As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents.

[0026] As used herein, "antibody" encompasses both naturally occurring immunoglobulins and non-naturally occurring immunoglobulins, including, for example, single-chain antibodies, chimeric antibodies (e.g., humanized murine antibodies), and heteroconjugate antibodies (e.g., bispecific antibodies). Antibody fragments include fragments that bind to an antigen (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). See also, e.g., Pierce Catalog and Handbook, 1994 - 1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term "antibody" also includes bivalent or bispecific molecules, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies. The term "antibody" further includes both polyclonal and monoclonal antibodies.

[0027] As used herein, the term "administer" means to provide an agent or composition to a subject and includes, but is not limited to, administration by a medical professional and self-administration.

[0028] As used herein, the term "cancer" refers to any disease involving abnormal cell growth and includes all stages and all forms of diseases affecting 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 all stages and grades of cancer, including pre-metastatic and post-metastatic cancer. Generally, cancers can be classified according to the tissue or organ in which the cancer is located or originated and the morphology of the cancerous tissue and cells. As used herein, cancer types include acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, 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 tumors, Ewing's sarcoma, gastric (stomach) cancer, glioma, head and neck cancer, gastric cardia cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, liver cancer, lung cancer, medulloblastoma, melanoma, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, skin cancer, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thyroid cancer, vaginal cancer, visual pathway and hypothalamic glioma.

[0029] The term "cancer sample" includes a biological sample or a sample from a biological source containing one or more cancer cells. Biological samples include samples from body fluids, such as blood, plasma, serum, or urine, or samples from cells, tissues, or organs, such as by biopsy, preferably tumor tissue suspected of containing cancer cells or consisting essentially of cancer cells.

[0030] Note that, in the present disclosure, terms such as "comprises", "comprised", "comprising", "contains" or "containing" have the meaning given by United States patent law; the terms are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Terms such as "consisting essentially of" and "consists essentially of" have the meaning given by United States patent law; the terms permit 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 given in United States patent law; that is, the terms are closed-ended.

[0031] The term "c-Met" refers to a proto-oncogene that encodes a protein called hepatocyte growth factor receptor (HGFR). The c-Met protein is composed of an alpha chain and a beta chain produced by cleavage of the precursor of c-Met (pro c-Met), and forms a dimer through a disulfide bond. c-Met is a receptor that penetrates the cell membrane, and the entire alpha chain and part of the beta chain are extracellular (see, for example, Mark et al., The Journal of Biological Chemistry (1992) 267:26166-71; Ayumi I, Journal of Clinical and Experimental Medicine (2008) 224:51-55). For human c-Met and its alpha and beta chains, also see GenBank accession number: NP _ 000236.2. It has been shown that abnormal c-Met activation in cancer is associated with poor prognosis, where abnormally active c-Met triggers tumor growth, the formation of new blood vessels that nourish the tumor, and the spread of cancer to other organs.

[0032] As used herein, the term "c-Met inhibitor" refers to an agent that can inhibit the expression or activity of c-Met protein. Examples of c-Met inhibitors include, but are not limited to, crizotinib, cabozantinib, tepotinib, AMG337, Wanbirui (also known as APL-101, PLB1001, Breytinib), SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF-04217903, MK2461, GSK1363089 (XL880, Furitinib), AMG458, tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, savolitinib (volitinib), HQP-8361, merestinib, ARGX-111, onartuzumab, rituximab, emibetuzumab, XL184, and the compounds disclosed in US20150218171.

[0033] The terms "determine", "evaluate", "measure", and "detect" are used interchangeably and are intended to refer to quantitative and semi-quantitative determinations. In cases where quantitative and semi-quantitative determinations are intended, the phrases "determine the level of a polynucleotide or polypeptide of interest" or "detect a polynucleotide or polypeptide of interest" may be used.

[0034] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an agent sufficient to prevent, treat, alleviate, and / or ameliorate the symptoms and / or underlying cause 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" refers to the amount sufficient to reduce or eliminate the symptoms of a disease. In another embodiment, a therapeutically effective amount is the amount sufficient to overcome the disease itself.

[0035] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0036] As used herein, the term "sample" refers to a biological sample obtained from a subject and containing RNA transcripts. Examples of samples include, but are not limited to, cells such as cancer cells, tissues such as biopsy tissues (e.g., biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, kidney tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue), and paraffin-embedded tissues, as well as body fluids such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal lavage fluid, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, etc. In certain embodiments, the sample can be a biological sample containing cancer cells. In some embodiments, the sample is a fresh or archived sample obtained from a tumor, e.g., by tumor biopsy or fine needle aspiration. The sample can also be any biological fluid containing cancer cells. Collection of a sample from a subject is performed according to standard protocols generally followed by a hospital or clinic, such as during a biopsy procedure.

[0037] As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include pre-birth and post-birth forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a human presenting to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient". The subject can have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0038] As used herein, the term "toxin" means an antigenic toxin or venom of plant or animal origin. An example is diphtheria toxin or a portion thereof.

[0039] The term "treatment" (treatment, treat, or treating) refers to a method of alleviating the effects of cancer (e.g., breast cancer, lung cancer, ovarian cancer, etc.) or cancer symptoms. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of cancer or cancer symptoms. For example, if one or more symptoms of a subject's disease are reduced by 10% compared to a control, the method of treating the disease is considered a treatment. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to a natural or control level. It should be understood that treatment does not necessarily mean a cure or complete elimination of the disease, condition, or symptoms of the disease or condition.

[0040] EGFR TKI and activating EGFR mutations

[0041] The epidermal growth factor receptor (EGFR) family (also known as the HER or ErbB family) consists of four receptor tyrosine kinases (TKs) - EGFR (HER1 / ErbB1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4) - which regulate many developmental, metabolic, and physiological processes. Binding of EGFR to various cognate ligands, including EGF, transforming growth factor-α, amphiregulin, etc., results in either homodimerization of two EGFRs or heterodimerization of EGFR with other family members (most commonly HER2), thereby increasing intracellular EGFR TK activity. Activation of EGFR TK leads to autophosphorylation of the intracellular domain of EGFR, and the resulting phospho-tyrosine residues serve as docking sites for various adaptor molecules, triggering activation of signal transducers and activators of the Ras / mitogen-activated protein kinase pathway, the PI3K / Akt pathway, and the transcriptional signaling pathway.

[0042] In tumor cells, the TK activity of EGFR may be dysregulated by various oncogenic mechanisms, including EGFR gene mutations, increased gene copy number, and overexpression of the EGFR protein. Aberrant activation of EGFR TK causes increased survival, proliferation, invasion, and metastasis of malignant cells. EGFR overexpression has been observed in tumors from more than 60% of patients with metastatic non-small cell lung cancer (NSCLC) and is associated with poor prognosis. EGFR-activating mutations are drivers of NSCLC, with a global overall prevalence ranging from 10% to 50% (Molecular Diagnostic & Therapy (2022) 26:7–18). These findings provide the rationale for developing anticancer agents that inhibit the activity of EGFR tyrosine kinase.

[0043] EGFR TKI

[0044] First-generation EGFR TKIs (including gefitinib, erlotinib, and icotinib) reversibly bind to EGFR and inhibit the binding of ATP to the TK domain. This blockade impedes cell proliferation and ultimately leads to cell death. The structures of gefitinib, erlotinib, and icotinib are shown below.

[0045]

[0046] Second-generation EGFR TK inhibitors (including afatinib and dacomitinib) are irreversible inhibitors that bind covalently to EGFR. The structures of second-generation EGFR TKIs are shown below.

[0047]

[0048]

[0049] Despite an initially high response rate, many patients using first-generation and second-generation EGFR TKIs will develop resistance to the treatment. Various mechanisms of acquired resistance have been identified, and these mechanisms can be classified into secondary mutations in EGFR, activation of alternative signaling pathways, and phenotypic or histological transformation. The most common mechanism of acquired resistance is the EGFR T790M mutation, which accounts for 50–60% of secondary resistance to primary EGFR TKI therapy. Given the limited efficacy of first-generation and second-generation EGFR TKIs, third-generation EGFR TKIs have been developed. These include osimertinib, nazartinib, olmutinib, marvolitinib, YH5448, ivosertinib, and rocitinib. The structures of third-generation EGFR TKIs are shown below.

[0050]

[0051]

[0052] Treatment with EGFR TK inhibitors (TKIs) such as gefitinib and erlotinib produces significant antitumor activity in subsets of patients with NSCLC. Sequencing of the EGFR gene has revealed that most tumors responsive to EGFR TKIs carry mutations in the TK domain of the EGFR gene. Mutations in the TK domain of the EGFR gene are referred to as "activating EGFR mutations" because they result in ligand-independent activation of TK activity.

[0053] Activating EGFR mutations are typically found in the first four exons (18 to 21) of the TK domain of the EGFR gene. These mutations generally fall into three major classes, with most EGFR TKI-sensitizing mutations belonging to classes I and II. Class I mutations are in-frame deletions in exon 19 (ex19del); these deletions almost always include the amino acid residues leucine-747 to glutamate-749 (ΔLRE). Class II mutations are single nucleotide substitutions that cause amino acid changes. The major single-point mutation is in exon 21, which substitutes arginine for leucine at codon 858 (L858R). L858R has the highest prevalence among any single-point activating mutations in EGFR TK. Other class II activating mutations include mutations that cause glycine-719 (G719) to 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 low-frequency activating mutations have been detected, including V765A, S768I, and T783A in exon 20 and L861Q in exon 21.

[0054] In some embodiments, the activating EGFR mutations referred to in the present disclosure are selected from the group consisting of: ex19del, L858R, ex20ins, and T790M.

[0055] C - Met gene amplification

[0056] Many cancer patients treated with EGFR TKIs develop resistance to the treatment, which poses a major obstacle to long-term disease remission clinically. In addition to the emergence of the T790M mutation, C-Met gene amplification has also been recognized as a resistance mechanism to first- or second-generation EGFR TKIs. On the one hand, the present disclosure provides a method of treating cancer patients with both activating EGFR mutations and C-Met gene amplification using a c-Met inhibitor.

[0057] The proto-oncogene c-MET encodes the receptor tyrosine kinase (RTK) c-Met. c-MET is widely expressed in epithelial-endothelial-derived cells, which are essential for embryonic development and tissue repair. Hepatocyte growth factor (HGF) is the only known ligand for the c-Met receptor and is mainly expressed in mesenchymal-derived cells. Under normal conditions, c-Met dimerizes and autophosphorylates upon ligand binding, thereby generating active docking sites for proteins that mediate downstream signal transduction, 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 causes a variety of pleiotropic biological responses, resulting in increased cell growth, dispersion and motility, invasion, prevention of apoptosis, branching morphogenesis, and angiogenesis. However, under pathological conditions, inappropriate activation of c-Met may confer on cancer cells the ability to proliferate, survive, and invade / metastasize.

[0058] Dysregulation of c-Met and subsequent abnormal signal transduction may occur through different mechanisms, including gene amplification and activating mutations. It has been reported that c-Met is overexpressed in a variety of cancers, including lung cancer, breast cancer, ovarian cancer, kidney cancer, colon cancer, thyroid cancer, liver cancer, and gastric cancer. Such overexpression may be the result of transcriptional activation, hypoxia-induced overexpression, or c-Met gene amplification. Gene amplification is a common genetic alteration of c-Met and has been reported to be associated with poor prognosis in NSCLC, colorectal cancer, and gastric cancer.

[0059] Detection of activating EGFR mutations and C - Met gene amplification

[0060] Activating EGFR mutations and / or c-Met gene amplifications in cancer patients can be detected by suitable methods known in the art, including but not limited to amplification assays, hybridization-based assays, sequencing-based assays, and immunoassays.

[0061] Amplification assay

[0062] Nucleic acid amplification assays involve replicating a target nucleic acid (e.g., DNA or RNA), thereby increasing the copy number of the amplified nucleic acid sequence. The amplification can be exponential or linear. Exemplary nucleic acid amplification methods include but are not limited to amplification using polymerase chain reaction (“PCR,” see U.S. Pat. Nos. 4,683,195 and 4,683,202; “PCR Protocols: A Guide To Methods And Applications” (edited by Innis et al., 1990)), reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR); quantitative PCR, such as 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 reporter genes, Q-β 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. Pat. No. 5,854,033), nucleic acid sequence-based amplification (NASBA) based on isothermal conditions, and serial analysis of gene expression (SAGE).

[0063] In certain embodiments, the nucleic acid amplification assay is a PCR-based method. PCR is initiated by a pair of primers that hybridize to the target nucleic acid sequence to be amplified, and then the primers are extended by a polymerase that uses the target nucleic acid sequence as a template and dNTPs as building blocks to synthesize new strands. Then, the new strands and the target strands are denatured to allow primer binding, and the next cycle of extension and synthesis is carried out. After multiple amplification cycles, the total copy number of the target nucleic acid sequence can increase exponentially.

[0064] In certain embodiments, intercalating agents that produce a signal when intercalated into double-stranded DNA can be used. Exemplary agents include SYBR GREEN TM and SYBR GOLD TM . Since these reagents are not template-specific, it is assumed that the signal is produced based on template-specific amplification. This can be confirmed by monitoring the signal as a function of temperature, since the melting point of the template sequence is typically much higher than, for example, primer dimers and the like.

[0065] In certain embodiments, detectably labeled primers or detectably labeled probes can be used to allow detection of activating EGFR mutations corresponding to the primer or probe. In certain embodiments, multiple labeled primers or labeled probes having different detectable labels can be used to allow simultaneous detection of multiple activating EGFR mutations.

[0066] Hybridization assays

[0067] Nucleic acid hybridization assays use probes to hybridize to target nucleic acids, thereby allowing detection of the target nucleic acid. Non-limiting examples of hybridization assays include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplex hybridization-based assays.

[0068] In certain embodiments, the probes used in hybridization assays are detectably labeled. In certain embodiments, nucleic acid-based probes used in hybridization assays are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray and can hybridize to target nucleic acid molecules that are detectably labeled.

[0069] In certain embodiments, hybridization assays can be performed by isolating nucleic acids (e.g., RNA or DNA), separating the nucleic acids (e.g., by gel electrophoresis), and subsequently transferring the separated nucleic acids to a suitable membrane filter (e.g., a nitrocellulose filter), where the probe hybridizes to the target nucleic acid and allows detection. See, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, 1989, Chapter 7. Hybridization of the probe to 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 the hybridized filter to photographic film.

[0070] In some embodiments, the hybridization assays can be performed on a microarray. The microarray provides a method for simultaneously measuring the levels of a large number of target nucleic acid molecules. The target nucleic acid can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acid can be hybridized to the microarray, which comprises a substrate having a plurality of immobilized nucleic acid probes arranged at a density of up to several million probes per square centimeter of the substrate surface. The RNA or DNA in the sample hybridizes to the complementary probes on the array and is then detected by laser scanning. The hybridization intensity of each probe on the array is determined and converted into a quantitative value representing the relative level of the RNA or DNA. See U.S. Patent Nos. 6,040,138, 5,800,992, 6,020,135, 6,033,860, and 6,344,316.

[0071] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261. Although planar array surfaces are commonly employed, the arrays can be constructed on surfaces of virtually any shape or even on multiple surfaces. The arrays can be peptides or nucleic acids on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992. The arrays can be encapsulated in such a way as to allow diagnosis or other manipulation of the fully enclosed device. Useful microarrays are also commercially available, for example, from Affymetrix, microarrays from Nano String Technologies, and the QuantiGene 2.0 multiplex assay from Panomics.

[0072] In certain embodiments, the hybridization assay can be an in situ hybridization assay. The in situ hybridization assay can be used to detect the presence of c-Met gene amplification. The probe that can be used for the in situ hybridization assay can be a mutant or gene fusion specific probe that hybridizes with a specific activating EGFR mutation to detect the presence or absence of the specific mutation of interest. The use of unique sequence probes for in situ hybridization is described in U.S. Patent No. 5,447,841, which is incorporated herein by reference. The probe can be observed with a fluorescence microscope and appropriate filters for each fluorophore, or multiple fluorophores can be observed by using a dual bandpass or triple bandpass filter set. See, for example, U.S. Patent 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 probe, including automated digital imaging systems. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the probe.

[0073] Sequencing methods

[0074] Sequencing methods that can be used to measure the activating EGFR mutation and / or c-Met gene amplification of interest involve sequencing of the target nucleic acid. Any sequencing known in the art can be used to detect the EGFR mutation and / or c-Met gene amplification. Generally, sequencing methods can be classified into 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 method).

[0075] By using methods different from traditional methods such as Sanger sequencing, high-throughput sequencing or next-generation sequencing is highly scalable and capable of sequencing an entire genome or transcriptome at once. High-throughput sequencing involves sequencing by synthesis, sequencing by ligation, and ultra-deep sequencing (as described in Marguiles et al., Nature 437(7057):376-80(2005)). Sequencing by synthesis involves synthesizing a complementary strand of a target nucleic acid by incorporating labeled nucleotides or nucleotide analogs in polymerase amplification. Immediately after or simultaneously with the successful incorporation of a labeled nucleotide, the signal of the label is measured and the identity of the nucleotide is recorded. Before repeating the incorporation, detection, and identification steps, the detectable label on the incorporated nucleotide is removed. Examples of sequencing by synthesis methods are known in the art and are described in, for example, U.S. Patent No. 7,056,676; U.S. Patent No. 8,802,368 and U.S. Patent No. 7,169,560, the contents of which are incorporated herein by reference. Sequencing by synthesis can be performed on a solid surface (or microarray or chip) using loop-back PCR and anchored primers. Target nucleic acid fragments can be ligated to the solid surface by hybridizing with the anchored primers and subjected to bridge amplification. For example, this technique is used for sequencing platforms.

[0076] Pyrosequencing involves hybridizing a target nucleic acid region with a primer and extending a new strand by sequentially incorporating deoxynucleotide triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. The incorporation of each base is accompanied by the release of pyrophosphate, which is converted to ATP by sulfurylase, which drives the synthesis of oxidized luciferin and the release of visible light. Since the pyrophosphate release is equimolar to the number of incorporated bases, the emitted light is proportional to the number of nucleotides added in any one step. The process is repeated until the entire sequence is determined.

[0077] In certain embodiments, the EGFR mutations and c-Met gene amplifications described herein are detected by transcriptome shotgun sequencing (RNA sequencing) and exome sequencing (DNA sequencing).

[0078] Immunoassay

[0079] The immunoassays used herein generally involve the use of antibodies that specifically bind to the 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, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), immunoprecipitation, sandwich assays, competitive assays, immunofluorescent staining and imaging, immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS). For a review of immunology and immunoassay procedures, see Basic and Clinical Immunology (edited by Stites and Terr, 7th ed. 1991). In addition, immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (edited by Maggio 1980); and Harlow and Lane, supra. For a review of immunoassays in general, see Methods in Cell Biology: Antibodies in Cell Biology, Volume 37 (edited by Asai 1993); Basic and Clinical Immunology (edited by Stites and Terr, 7th ed. 1991).

[0080] In certain embodiments, the c-Met expression level is measured as the level of a subset of the c-Met protein, such as the level of a modified c-Met protein (e.g., phosphorylated c-Met protein). In such cases, an antibody that specifically binds to the modified c-Met protein can be used to detect the c-Met expression level.

[0081] Any assay and method provided herein for measuring the c-Met expression level can be adapted or optimized for automated and semi-automated systems or point-of-care assay systems.

[0082] The c-Met expression levels described herein can be normalized using suitable methods known in the art. For example, the c-Met expression levels can be normalized relative to the standard levels of a standard marker, which can be pre-determined, contemporaneously determined, or determined after obtaining a sample from a subject. The standard marker can be run in the same assay or can be a known standard marker in a previous assay. As another example, the c-Met expression levels can be normalized relative to an internal control, which can be an internal marker or the average or total level of a plurality of internal markers.

[0083] Treatment with c - Met inhibitors

[0084] As used herein, a "c-Met inhibitor" refers to an agent that can inhibit the expression or activity of the c-Met protein. In certain embodiments, the c-Met inhibitor is selected from the group consisting of crizotinib, cabozantinib, tepotinib, AMG337, vanpirecetin (APL-101, PLB1001, buritinib), SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF-04217903, MK2461, GSK1363089 (XL880, fruquintinib), AMG458, tivantinib (ARQ197), INCB28060 (INC280, capmatinib), E7050, BMS-777607, savolitinib (volitinib), HQP-8361, merestinib, ARGX-111, onartuzumab, rituximab, emactuzumab, and XL184.

[0085] In certain embodiments, the c-Met inhibitor is vanpirecetin (APL-101, previously named CBT-101, see US20150218171, which is incorporated herein by reference in its entirety), which has the following formula:

[0086]

[0087] In certain embodiments, the c-Met inhibitor can be formulated with a pharmaceutically acceptable carrier. When present, the carrier can be blended with the c-Met inhibitor in any suitable amount, such as an amount of 5% to 95% by weight of the carrier, based on the total volume or weight of the c-Met inhibitor and the carrier. In some embodiments, the amount of the carrier can be within a range having a lower limit of any one of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, or 75% and an upper limit of any one of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95% above the lower limit. As is known to those of ordinary skill in the art of formulation, the amount of the carrier in a particular embodiment can be determined based on considerations of the particular dosage form, the relative amount of the c-Met inhibitor, the total weight of the composition containing the carrier, the physical and chemical properties of the carrier, and other factors.

[0088] The c-Met inhibitor can be administered in any desired and effective manner: orally, or topically to the eye in the form of an ointment or drops, or parenterally or otherwise administered in any suitable manner, such as intraperitoneally, subcutaneously, topically, intradermally, by inhalation, intratracheally, rectally, vaginally, sublingually, intramuscularly, intravenously, intraarterially, intrathecally, or intralymphatically. Additionally, the c-Met inhibitor can be administered in combination with other therapies. If desired, the c-Met inhibitor can be encapsulated or otherwise protected from the effects of gastric or other secretions.

[0089] Suitable non-limiting examples of the dosage of the c-Met inhibitor disclosed herein are 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, from 75 mg / kg 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 a human body is about 400 mg / day, administered once every 12 hours. In some embodiments, the dosage of the c-Met inhibitor in a human body is in the range of 300 - 500 mg / day, 100 - 600 mg / day, or 25 - 1000 mg / day. The effective dosage of the c-Met inhibitor disclosed herein can be administered as two, three, four, five, six, or more sub-dosages at appropriate intervals during a day.

[0090] In one embodiment, the method further comprises administering at least one additional therapeutic agent selected from the group consisting of immune checkpoint modulators, cytotoxic agents, toxins, radionuclides, immunomodulators, photoactive therapeutic agents, radiosensitizers, hormones, antiangiogenic agents, and combinations thereof.

[0091] As used herein, the term "immune checkpoint" or "cancer immune checkpoint" refers to a molecule in the immune system that upregulates an immune response (i.e., a costimulatory molecule) or downregulates an immune response (i.e., an inhibitory molecule). 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, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-4, BTLA, SIRPα (CD47), CD48, 284 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR.

[0092] In certain embodiments, a modulator of an immune checkpoint is a monoclonal antibody directed against the immune checkpoint. In certain embodiments, the immune checkpoint is PD-1 or PD-L1. In certain embodiments, the anti-PD-1 antibody is selected from those disclosed in PCT Application Publication No. WO2016 / 014688, which is hereby incorporated by reference in its entirety. In certain embodiments, the anti-PD-1 antibody is APL-501 (previously named CBT-501, see WO2016 / 014688), GB226, or genolimzumab. In certain embodiments, the anti-PD-L1 antibody is selected from those disclosed in PCT Application Publication No. WO2016 / 022630, which is hereby incorporated by reference in its entirety. In certain embodiments, the anti-PD-L1 antibody is APL-502 (previously named CBT-502, see WO2016 / 022630) or TQB2450.

[0093] Anticancer agents other than c - Met inhibitors

[0094] The methods of the present disclosure also relate to administering to a 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.

[0095] Other anti-cancer agents that can be used in combination with a c-Met inhibitor in the methods disclosed herein include, but are not limited to: alkylating agents or agents having alkylating effects, such as cyclophosphamide (CTX; e.g., ) chlorambucil (CHL; e.g., ) cisplatin (CisP; e.g., ) busulfan (e.g., ) Melphalan, Carmustine (BCNU), Streptozotocin, Triethylenemelamine (TEM), Mitomycin C, etc.; Antimetabolites such as Methotrexate (MTX), Etoposide (VP16; e.g., ) 6-Mercaptopurine (6MP), 6-Thioguanine (6TG), Cytarabine (Ara-C), 5-Fluorouracil (5-FU), Capecitabine (e.g., ) Dacarbazine (DTIC), etc.; Antibiotics such as Actinomycin D, Doxorubicin (DXR; e.g., ) Daunorubicin (Daunomycin), Bleomycin, Mithramycin, etc.; Alkaloids such as Vincristine (VCR), Vinblastine, etc.; And other antitumor agents such as Paclitaxel (e.g., ) and Paclitaxel derivatives, cytostatic agents, glucocorticoids (such as Dexamethasone (DEX; e.g., )) and corticosteroids (such as Prednisone), nucleoside enzyme inhibitors (such as Hydroxyurea), amino acid depletion enzymes (such as Asparaginase), leucovorin, folinic acid, Raltitrexed and other folic acid derivatives, and various similar antitumor agents. The following agents can also be used as additional agents: Amifostine (e.g., ) Dactinomycin, Chlorambucil, Streptozocin, Cyclophosphamide, Lomustine (CCNU), Liposomal Doxorubicin (e.g., ) Gemcitabine (e.g., ) Liposomal Daunorubicin (e.g., ) Procarbazine, Mitomycin, Docetaxel (e.g., ) aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon α, interferon β, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, teniposide, testosterone, thioguanine, thiotepa, uracil mustard, vinorelbine, and chlorambucil.

[0096] In some embodiments, the drugs used in the methods disclosed herein include, but are not limited to: (Bevacizumab) (Bevacizumab) (Irinotecan Hydrochloride), capecitabine, Cetuximab, (Ramucirumab) (Oxaliplatin) (Cetuximab), 5-FU (Fluorouracil Injection), Fluorouracil Injection, Ipilimumab, Irinotecan Hydrochloride, (Pembrolizumab), calcium folinate (Trifluridine and Tipiracil Hydrochloride) (Bevacizumab) (Nivolumab), Oxaliplatin, Panitumumab, Pembrolizumab, Ramucirumab, Regorafenib, (Regorafenib), Trifluridine and Tipiracil Hydrochloride (Panitumumab), (Capecitabine), (Ipilimumab), (Ziv-Aflibercept), (Bevacizumab), Ziv-Aflibercept.

[0097] The drugs described herein can be administered in any desired and effective manner: oral ingestion, or topical administration to the eye in the form of an ointment or drops, or parenteral or other administration in any suitable manner, such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intralung, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal or intralymphatic administration. Additionally, the drugs can be administered in combination with other treatments.

[0098] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall, in whole or in part, within the scope of the invention. These specific compositions, materials, and methods are not intended to limit the invention but are only used to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive faculty and without departing from the scope of the invention. It should be understood that many variations can be made in the procedures described herein while still remaining within the bounds of the invention. The intention of the inventors is that such variations are included within the scope of the invention.

[0099] Example 1

[0100] This example is an in vivo anti-tumor efficacy study of Vimurui in LU0858 NSCLC patient-derived xenografts (PDX), which is a tumor model carrying an EGFR L858R mutation with MET amplification. This study shows that Vimurui monotherapy is effective against tumors carrying activating EGFR mutations, resistant to EGFR TKIs, and having concurrent c-Met amplification.

[0101] Materials and methods

[0102] BALB / c nude mice (female, 6 - 8 weeks old) were provided by Shanghai Slack Experimental Animal Co., Ltd. (Shanghai, China). The c-MET TKI, Wanbirui, was provided by Crown Biotechnology (Taicang) Co., Ltd. (Taicang, China). The EGFR TKI, erlotinib, was provided by Nanjing Anji Biotechnology Co., Ltd. (Nanjing, China). The LU0858 (EGFR L858R, MET amplified) PDX tumor was provided by Crown Biotechnology (Taicang) Co., Ltd. (Taicang, China).

[0103] Mice (BALB / c nude mice) bearing LU0858 tumors were treated with indicated doses of Wanbirui (0 - 21 mg / kg, orally once daily) or with the EGFR inhibitor erlotinib (50 mg / kg, orally once daily) for 21 days. Tumor volume and body weight of the mice were measured after the start of treatment. Tumor samples were collected at the end of the study, and the pharmacodynamic inhibition of Wanbirui on phospho-MET and total MET proteins was evaluated by Western blot and IHC.

[0104] Results

[0105] At the end of treatment with the ED50 of 3.0 mg / kg, Wanbirui (at 7 mg / kg and 21 mg / kg) inhibited tumor growth ( Figure 1 ). No significant tumor shrinkage was observed with erlotinib alone, indicating that this EGFR mutant PDX is resistant to EGFR TKI. Data values are presented as mean ± SEM for n = 8; *P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnett's post-test). Additionally, as evaluated by body weight changes, all mice tolerated the treatments in this study well. Western blot analysis of tumor lysates (upper inset) and immunohistochemistry of tumor tissues by IHC (lower inset) showed that both phospho-MET and total MET proteins from tumors treated with 21 mg / kg APL-101 were inhibited ( Figure 2 ).

[0106] Example 2

[0107] This example is the in vivo anti-tumor efficacy study of Vanpire in LD1-0025-20066 non-small cell lung cancer (NSCLC) patient-derived xenografts (PDX), and the PDX is a tumor model carrying an EGFR ex19del mutation with MET amplification. This study shows that Vanpire monotherapy is effective in treating tumors with activating EGFR mutations, resistant to EGFR TKIs, and with concurrent c-Met amplification.

[0108] Materials and methods

[0109] NU / NU nude mice (female, 5-6 weeks old) were provided by Beijing Vital River Laboratory Animal Technology co., LTD. (Beijing, China). The c-MET TKI Vanpire was provided by Laurus Lab Ltd. (Visakhapatnam, India). The c-MET TKI Savolitinib was provided by Selleckchem (Texas, USA). The EGFR TKI Osimertinib was provided by Selleckchem (Texas, USA). The EGFR TKI APL-122 (NT-113) was provided by Inogent Laboratories Private Limited (Hyderabad, India). The LD1-0025-20066 (EGFR ex19del, MET amplified) PDX tumor was provided by Xi'an Lide Biotech CO., LTD (Xi'an, China).

[0110] NU / NU nude mice carrying LD1-0025-20066 tumors were treated with Vanpire, Osimertinib, Savolitinib, or APL-122 as single agents or in combination therapies. All compounds were administered at the same dose of 10 mg / kg orally once a day for 21 days. The tumor volume and body weight of the mice were measured after the start of treatment (day 0). The mice were continuously housed, and the tumor volume and body weight of the mice were measured to observe tumor regrowth until day 70.

[0111] Results

[0112] Vanpire or savolitinib, either as a single agent or in combination with osimertinib or APL-122, can completely inhibit tumor growth( Figure 3 ). No significant tumor shrinkage was observed with osimertinib or APL-122 as single agents, indicating that this EGFR mutant PDX is resistant to EGFR TKIs. After discontinuation of dosing, tumor regrowth was observed in tumors treated with either savolitinib alone or in combination with osimertinib. In contrast, no tumor regrowth was observed after discontinuation of dosing in tumors treated with either vanpire alone or in combination with osimertinib or APL-122. Data values are presented as mean ± SEM for n = 8; *P < 0.05 relative to vehicle control (one-way ANOVA followed by Dunnett's post hoc test). Additionally, as evaluated by body weight changes, all mice tolerated the treatments in this study well.

[0113] Example 3

[0114] This example is a study on the in vivo antitumor efficacy of vanpire in LD1-0025-361336 NSCLC PDX, which is a tumor model carrying an EGFR ex19del mutation with MET amplification. This study shows that in some EGFR TKI-resistant tumors, combination therapy with vanpire and an EGFR TKI (osimertinib) is more effective than c-MET inhibitor monotherapy.

[0115] Materials and methods

[0116] NCG immunodeficient mice (female, 5 - 6 weeks old) were provided by Jiangsu GEM Pharmatech Co., Ltd. (GemPharmatech Co., LTD) (Nanjing, China). The c-MET TKI APL-101 was provided by Laurus Labs Limited (Visakhapatnam, India). The c-MET TKI savolitinib was provided by Selleckchem (Texas, USA). The EGFR TKI osimertinib was provided by Selleckchem (Texas, USA). The EGFR TKI APL-122 (NT-113) was provided by Inogent Labs Pvt Ltd (Hyderabad, India). The LD1-0025-361336 (EGFR ex19del, MET-amplified) PDX tumor was provided by Xi'an Lead Biotechnology Co., Ltd. (Xi'an, China).

[0117] NCG immunodeficient mice bearing the LD1-0025-361336 tumor were treated with APL-101, osimertinib, savolitinib, or APL-122 as single agents or in combination. All compounds were administered at the same dose of 10 mg / kg orally once daily for 28 days. Tumor volume and body weight of the mice were measured starting from the beginning of treatment for 28 days. Tumor samples were collected at the end of the study, and the pharmacodynamic inhibition of vanpire by phospho-MET and total MET proteins was evaluated by Western blot.

[0118] Results

[0119] Osimertinib or APL-122 as single agents partially inhibited tumor growth, indicating that this EGFR mutant PDX has partial resistance to EGFR TKIs. APL-101 or savolitinib as single agents only partially inhibited tumor growth. In contrast, they had a complete anti-tumor effect on LU0858 and LD1-0025-200662 ( Figure 4 ). The combination treatment of c-MET TKI (APL-101 or savolitinib) and EGFR TKI (osimertinib or APL-122) showed complete tumor inhibition, indicating that in EGFR mutant tumors with partial resistance to EGFR TKIs, the combination treatment of c-MET TKI and EGFR TKI is more effective than c-MET inhibitor monotherapy ( Figure 4 ). As Figure 5 shown, the stronger anti-tumor effect of the c-MET and EGFR TKI combination treatment is associated with c-MET protein degradation. Data values are presented as mean ± SEM for n = 8; *P < 0.05 versus vehicle control (one-way ANOVA followed by Dunnett's post hoc test). Additionally, as evaluated by body weight change, all mice tolerated the treatments in this study well.

[0120] Example 4

[0121] This example is an in vivo anti-tumor efficacy study of vanpire in LD1-LU1868 NSCLC PDX, which is a tumor model carrying an EGFR T790M mutation with low c-MET expression levels and no evidence of MET amplification. This study showed that in EGFR mutant NSCLC responsive to EGFR TKIs, the combination treatment of vanpire and EGFR TKI (osimertinib) may produce a more durable response than EGFR TKI alone.

[0122] Materials and methods

[0123] BALB / c nude mice (female, 6 - 7 weeks old) were provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Nanjing, China). The c-MET TKI, Vanpire, was provided by Laurus Labs Limited (Visakhapatnam, India). The EGFR TKI, Osimertinib, was provided by Selleckchem (Texas, USA). The LU1868 (EGFR T790M) PDX tumors were provided by Crown Bioscience (Taicang) Co., Ltd. (Taicang, China).

[0124] BALB / c nude mice bearing LU1868 tumors were treated with Vanpire or Osimertinib as single agents or in combination therapy. All compounds were administered at the same dose of 10 mg / kg orally once a day for 40 days. After the dosing was stopped, the mice were continuously housed, and the tumor volume and body weight of the mice were measured to observe tumor regrowth for 74 days.

[0125] Results

[0126] Osimertinib as a single agent or in combination with Vanpire completely inhibited tumor growth, indicating that this EGFR mutant PDX is sensitive to EGFR TKI. Treatment with Vanpire as a single agent did not show any anti-tumor effect. However, after the dosing was stopped, adding Vanpire to the Osimertinib treatment showed a longer duration of tumor inhibition than Osimertinib alone, as Figure 6 shown. Data values are presented as mean ± SEM for n = 5; *P < 0.05 vs. vehicle control (one-way ANOVA followed by Dunnett's post hoc test). Additionally, as evaluated by body weight changes, all mice tolerated the treatments in this study well.

Claims

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

2. The method according to claim 1, wherein the c-Met inhibitor is selected from the group consisting of: Vanpire (APL-101), Savolitinib (Volitinib), Crizotinib, Cabozantinib, PLB1001, Burutinib, SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF-04217903, MK2461, GSK1363089 (XL880, Furitinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, Capmatinib), E7050, BMS-777607, Tepotinib, HQP-8361, Maritinib, ARGX-111, Onartuzumab, Rituximab, Emactuzumab, and XL184.

3. The method according to claim 1, wherein the c-Met inhibitor is Vanpire or Savolitinib.

4. The method according to claim 1, wherein the c-Met inhibitor is Vanpire.

5. The method according to claim 1, wherein the activating EGFR mutation is an EGFR exon 19 deletion, L858R, or T790M.

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

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

8. The method according to claim 7, wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of: Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rositinib, Omotinib, Neratinib, Lapatinib, Nazartinib, Nacotinib, Mavotinib, Mobocertinib, Vandetanib, and Ivotinib.

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

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

11. The method according to claim 10, wherein the EGFR inhibitor is selected from the group consisting of: Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rositinib, Omotinib, Neratinib, Lapatinib, Nazartinib, Nacotinib, Mavotinib, Mobocertinib, Vandetanib, and Ivotinib.

12. The method according to claim 10, wherein the EGFR inhibitor is Erlotinib.

13. The method according to claim 1, wherein the cancer is selected from the group consisting of: lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia, and non-Hodgkin lymphoma.

14. The method according to claim 1, wherein the cancer is non-small cell lung cancer (NSCLC).

15. A method for treating a subject suffering from cancer, the method comprising: administering to the subject a therapeutically effective amount of a c-Met inhibitor and a therapeutically effective amount of an EGFR tyrosine kinase inhibitor, wherein the subject has been determined to have an activating EGFR mutation responsive to treatment with an EGFR inhibitor.

16. The method according to claim 15, wherein the subject is not detected to have MET amplification.

17. The method according to claim 15, wherein the c-Met inhibitor is selected from the group consisting of: Vanpire (APL-101), Savolitinib (Volitinib), Crizotinib, Cabozantinib, PLB1001, Brivanib, SU11274, PHA665752, K252a, PF-2341066, AM7, JNJ-38877605, PF-04217903, MK2461, GSK1363089 (XL880, Furitinib), AMG458, Tivantinib (ARQ197), INCB28060 (INC280, Capmatinib), E7050, BMS-777607, Tepotinib, HQP-8361, Meritinib, ARGX-111, Onartuzumab, Rituximab, Emactuzumab, and XL184.

18. The method according to claim 15, wherein the EGFR tyrosine kinase inhibitor is selected from the group consisting of: Erlotinib, Gefitinib, Icotinib, Afatinib, Dacomitinib, Osimertinib, Rositinib, Omotitinib, Neratinib, Lapatinib, Nazartinib, Nacotitinib, Mavotinib, Mobocertinib, Vandetanib, and Ivacaftor.

Images