Patent Grant
10393748
This application claims the benefit of Korean Patent Application No. 10-2014-0001799 filed on Jan. 7, 2014, and Korean Patent Application No. 10-2015-0000956 filed on Jan. 6, 2015, the entire disclosures of which are hereby incorporated by reference.
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 146,702 bytes ASCII (Text) file named “719114 ST25-Revised” created Feb. 1, 2016.
Provided are methods for predicting and/or monitoring the effect of a c-Met inhibitor by analyzing one or more biomarkers; a composition for predicting and/or monitoring the effect of a c-Met inhibitor, comprising a material detecting the biomarker; a composition for selecting a subject suitable for the application of a c-Met inhibitor, comprising a material detecting the biomarker; a method for predicting and/or monitoring the effect of a c-Met inhibitor using one or more biomarkers; a method for selecting a subject suitable for the application of a c-Met inhibitor using one or more biomarkers; and a method for preventing and/or treating cancer comprising administering a c-Met inhibitor to the selected subject.
A biomarker generally refers to a measured characteristic (e.g., a naturally occurring protein) which may be used as an indicator of some change caused in an organism by an external factor (e.g., injury). Active studies have recently been made to apply biomarkers to the diagnosis of various diseases, such as cancer, stroke, dementia, etc., and the prediction or monitoring of therapeutic effects of some agents. Among biomarkers relevant to drug development are pharmacodynamic markers (PD markers) for indicating whether drugs are functionally effective in vivo, and predictive markers for indicating the most likely response to particular drugs before administration. The use of such markers is helpful in establishing the clinical strategy of drugs. For example, a predictive marker, designed to indicate sensitivity or resistance to drug action, may be applied to the selection of patients to allow for more effective drug therapy while the activity of a drug in individual patients can be monitored with a pharmacodynamic marker, which together can lead to the establishment of effective therapeutic strategies. Further, even in the absence of a predictive marker, a pharmacodynamic marker permits the early monitoring of responses to a drug, thus allowing one to identify drug-effective groups from drug-ineffective groups at an early stage. Consequentially, more effective and successful drug therapies can be developed. In addition, when applied to the monitoring of responses to a drug as a function of concentrations, a pharmacodynamic marker can be an index for calculating suitable doses of the drug.
To date, cancer is one of the leading causes of death. Although the development of medical techniques has brought about a remarkable progress in cancer therapy, the 5-year survival rate of all cancers has only improved by 10% over the past two decades. This is because cancer characteristics, such as rapid growth, metastasis, etc., make it difficult to diagnose and treat within a suitable time. The identification of suitable biomarkers that provide information regarding the efficacy of a cancer therapy can greatly impact the ability to provide the most suitable cancer therapies at the most optimal times. For example, patients with lung cancer may differ from each other in cancer classification, genotype, and protein secretion, and thus must be treated with different, proper therapeutics. For chemotherapy using a specific drug, a corresponding biomarker, if present, would reduce the number of erroneous trials and increase possibility of success. In this regard, it is very important to explore biomarkers for predicting or monitoring the effect of anti-cancer therapeutics. A proper biomarker, if successfully exploited, can make a great contribution to the utility and value of anti-cancer drugs and the success rate of treatment with them.
c-Met is a hepatocyte growth factor (HGF) receptor. Hepatocyte growth factor (HGF) acts as a multi-functional cytokine which binds to the extracellular domain of the c-Met receptor to regulate cell division, cell motility, and morphogenesis in various normal and tumor cells. The c-Met receptor is a membrane receptor that possesses tyrosine kinase activity. c-Met is a proto-oncogene, that encodes the representative receptor tyrosine kinase. Occasionally, it takes part in a variety of mechanisms responsible for the development of cancer such as oncogenesis, metastasis, migration, angiogenesis, and invasion of cancer cells, etc., irrespectively of the ligand HGF, and thus has attracted intensive attention as a target for anti-cancer therapy. Thus targeted therapies, such as antibodies against c-Met, have been developed.
A therapy with a developed c-Met targeting drug might be more effective treating cancer, with an elevated probability of success if there is a biomarker that is capable of predicting and monitoring the therapeutic effect of the drug to select patients suitable for the drug therapy and to monitor patient responses to the drug. Thus, the use of biomarkers could be applied to the establishment of effective therapeutic strategies.
Provided is a method for predicting and/or monitoring the effect of a c-Met inhibitor, including measuring the existence (presence/absence), the level, and/or the mutation of at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins, in a biological sample.
Also provided is a method for selecting a subject suitable for the application of a c-Met inhibitor, including measuring the existence, the level, and/or the mutation of at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins, in a biological sample.
Another embodiment provides a method for inhibiting c-Met, and a method of preventing and/or treating a cancer, which methods including administering a c-Met inhibitor to the selected subject selected by the disclosed methods as being suitable for application of the c-Met inhibitor.
Provided is the use of at least one of selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins in predicting the effect of a c-Met inhibitor and/or monitoring the effect of administered c-Met inhibitors.
One embodiment provides a method for predicting and/or monitoring the effect of a c-Met inhibitor, comprising the analysis of at least one biomarker selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins.
Another embodiment provides a method for selecting a subject to which a c-Met inhibitor is applicable (i.e., will exhibit efficacy), comprising the analysis of at least one biomarker selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins.
Another embodiment provides a composition and a kit for predicting and/or monitoring the effect of a c-Met inhibitor, comprising a material interacting with at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins.
Another embodiment provides a composition and a kit for selecting a subject to which a c-Met inhibitor is applicable, comprising a material interacting with at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins.
Another embodiment provides a method for predicting and/or monitoring the efficacy of c-Met inhibitor or for providing information on the prediction and/or monitoring of the efficacy of a c-Met inhibitor, comprising measuring existence, level, and/or mutation of at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins, in a biological sample.
Another embodiment provides a method for selecting a subject suitable for the application of a c-Met inhibitor, comprising measuring for existence, level and/or mutation of at least one selected from the group consisting of IL-8, b-IG-H3, MIF, KRAS/BRAF, and nucleic acids encoding the proteins, in a biological sample obtained from a subject.
Another embodiment provides a method for preventing and/or treating cancer, comprising administering a c-Met inhibitor to a subject, wherein the subject has a high level of IL-8 or a nucleic acid encoding IL-8 compared to a reference sample in which a c-Met inhibitor is not effective, as defined below; shows a decreased level of IL-8, bIG-H3, MIF, and/or nucleic acids encoding them after administering a c-Met inhibitor (compared to that of before the administration); and/or has a mutation of KRAS, BRAF, and/or nucleic acids encoding them. For example, the subject may be selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor. This preventing or treating method may further comprise selecting a subject suitable for the application of the c-Met inhibitor, prior to the administration.
Another embodiment provides a method for inhibiting c-Met, comprising administering a c-Met inhibitor to a subject, wherein the subject has a high level of IL-8 or a nucleic acid encoding IL-8 compared to a reference sample in which a c-Met inhibitor is not effective, as defined below; shows a decreased level of IL-8, bIG-H3, MIF, and/or nucleic acids encoding them after administering a c-Met inhibitor (compared to that of before the administration); and/or has a mutation of KRAS, BRAF, and/or nucleic acids encoding them. For example, the subject may be selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor. This inhibiting method may further comprise selecting a subject suitable for the application of the c-Met inhibitor, prior to the administration.
As used herein, the term “efficacy” of a c-Met inhibitor refers to a c-Met inhibitory effect and/or the pharmaceutical activity of a c-Met inhibitor resulting in the prevention, amelioration, reduction or treatment of a c-Met-related disease, for instance, a cancer. As regards cancer the efficacy refers to an anticancer effect, for example, an ability to induce the reduction or death of cancer cells or tissues, and the suppression of cancer cell migration and/or invasion responsible for cancer metastasis. In other word, the efficacy may refer to responsiveness of a subject, who is administered with a c-Met inhibitor, to the c-Met inhibitor.
As used herein, the term “predicting an efficacy of a c-Met inhibitor” may refer to determining whether or not or how much the c-Met inhibitor exhibits its desired efficacy (e.g., an antitumor or anticancer efficacy) on a subject who is treated therewith.
As used herein, the wording “a subject suitable for the application of the c-Met inhibitor” may refer to “a subject suitable for administration, treatment with a c-Met inhibitor”, “a subject with a disease (e.g., cancer) that is likely to respond to a c-Met inhibitor”, or an individual that is likely to respond to a c-Met inhibitor.
The expression level of a certain protein may be determined by quantitatively analyzing the protein or the gene (nucleic acid) encoding it, such as DNA, cDNA or mRNA. As used herein, the term “gene” refers to any nucleic acid encoding the protein, whether genomic DNA, cDNA, or RNA (mRNA). The determination of KRAS and/or BRAF mutations may be achieved at a gene level or a protein level.
IL-8 (Interleukin 8) is a chemokine functioning as an important mediator of immune responses. IL-8, also known as neutrophil chemotactic factor, has two primary functions: it induces chemotaxis in target cells, primarily neutrophils but also other granulocytes, causing them to migrate toward the site of infection; and also phagocytosis once they have arrived. IL-8 is also known to be a potent promoter of angiogenesis contributing to the proliferation and metastasis of tumor cells. In one exemplary embodiment of the present invention, a response to a c-Met inhibitor varies depending on the expression level of IL-8, and a difference in the expression level of IL-8 between biological samples before and after treatment with an c-Met inhibitor is dependent on the response to the c-Met inhibitor (see Example 1). Briefly, a higher level of IL-8 or its gene in a biological sample indicates that a c-Met inhibitor, e.g., an anti-c-Met antibody, more efficiently exerts its function on the biological sample or a patient from which the biological sample is sourced. In addition, a biological sample in which a c-Met inhibitor, for example, an anti-c-Met antibody, functions well (drug response group), a decrease in the level of IL-8 or its gene after the application of the c-Met inhibitor, compared to before the application, is typically observed.
That is, an expression level of IL-8 can be used to predict the efficacy of an c-Met inhibitor and thus to select a subject suitable for treatment with the c-Met inhibitor, or a change in the expression level of IL-8 according to, or before and after treatment with a c-Met inhibitor allows for the evaluation (identification) or monitoring of the efficacy of the c-Met inhibitor, thereby being useful in determining whether the c-Met inhibitor should continue to be applied, or in establishing the therapeutic strategy of the c-Met inhibitor with regard to dose, dosing interval, and dosing number. Based on this finding, the use of IL-8 as a marker for indicating and/or monitoring the efficacy of a c-Met inhibitor is suggested.
The IL-8 may originate from vertebrates including mammals, such as rodents, e.g., mice, rat, etc., and primates, e.g., humans, monkeys, etc., birds, reptiles, amphibians, and fishes. For example, the IL-8 may be selected from the group consisting of NP_000575.1, AAH13615.1, AAA59158.1, NP_001028137.1, AAA80141.2, and AAA86711.1. An IL-8-encoding gene (cDNA or mRNA) may be selected from among NM_000584.3, BC013615.1 (from 41 to 340 positions), NC_000004, NG_029889, NC_018915.2, AC_000136, M28130.1, NM_001032965.1, S78555.1, and U19849.1.
One embodiment is directed to the analysis of a biomarker for the prediction and/or monitoring of the efficacy of a c-Met inhibitor comprising the analysis of (e.g., measuring the level of) at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
Another embodiment is directed to the analysis of a biomarker for the selection of a subject suitable to the application of a c-Met inhibitor, comprising the analysis of (e.g., measuring the level of) at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
A further is directed to a composition and a kit for the prediction and/or monitoring of the efficacy of a c-Met inhibitor, comprising a material capable of interacting with at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
A further embodiment addresses a composition and a kit for the selection of a subject suitable to the application of a c-Met inhibitor, comprising a material interacting with at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
Another embodiment is a method for predicting and/or monitoring the efficacy of a c-Met inhibitor or for offering information on the prediction and/or monitoring of the efficacy of a c-Met inhibitor, comprising measuring a biological sample for a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
Another embodiment provides a method for selecting a subject suitable to the application of a c-Met inhibitor or for offering information on the selection of a subject suitable to the application of a c-Met inhibitor, comprising measuring a biological sample for a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8.
Provided according to one embodiment is a method for predicting the efficacy of a c-Met inhibitor, for offering information on the prediction of the efficacy of a c-Met inhibitor, for selecting a subject suitable to the application of a c-Met inhibitor, or for offering information of the selection of a subject suitable to the application of a c-Met inhibitor, comprising measuring a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample. In the method for predicting the efficacy of a c-Met inhibitor or for offering information on the efficacy of a c-Met inhibitor, when a biological sample is measured to have a high level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8, the biological sample or a patient from which the biological sample is originated can be predicted to allow the c-Met inhibitor to effectively exert its activity or may be considered to be suitable for the application of the c-Met inhibitor. Hence, the method for predicting the efficacy of a c-Met inhibitor or for offering information on the efficacy of a c-Met inhibitor may further comprise determining that the c-Met inhibitor exhibits its efficacy in the biological sample or the patient when the biological sample is observed to have a high level of at least one selected from the group IL-8 or a nucleic acid (e.g., gene) encoding IL-8 after the measurement. In addition, the method for selecting a subject suitable to the application of a c-Met inhibitor or for offering information on the selection may further comprise determining the biological sample or the patient as a subject suitable for the application of the c-Met inhibitor when the biological sample is observed to have a high level of at least one selected from the group IL-8 or a nucleic acid (e.g., gene) encoding IL-8 after the measurement.
“A high level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8” may refer to a large amount of IL-8 protein and/or a nucleic acid (e.g., gene) encoding IL-8 (DNA, cDNA or mRNA), compared to a biological sample (reference sample) from a patient in which a c-Met inhibitor to be applied does not exert therapeutic activity. For example, when the level of at least one selected from IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a test biological sample from a test patient is at least about 1.5-, about 2-, about 3-, about 4-, or about 5-fold, for example, about 1.5- to about 100-fold, about 2- to about 100-fold, about 3- to about 100-fold, about 4- to about 100-fold, or about 5- to about 100-fold greater in weight than that in a reference biological sample on which the c-Met inhibitor to be applied does not exert its efficacy (e.g., a c-Met inhibitory efficacy or an anticancer efficacy), the c-Met inhibitor is predicted to effectively exert its efficacy (e.g., a c-Met inhibitory efficacy or an anticancer efficacy) in the test biological sample or the test patient, or the test biological sample or the test patient can be considered as a subject suitable to the application of the c-Met inhibitor. The c-Met inhibitor may be, for example, an anti-c-Met antibody. The reference biological sample can be any sample useful for comparing the expression of the c-Met biomarker and/or c-Met inhibitor efficacy, such as a biological sample comprising cells, such as a mammalian cancer cell, in which the c-Met inhibitor does not have its efficacy (e.g., a c-Met inhibitory and/or anticancer efficacy). Examples of the reference biological sample on which the c-Met inhibitor does not exert its efficacy include, but are not limited to, the Lovo cell line (CCL-229, ATCC), and the HT-29 cell line (HTB-38, ATCC). Therefore, the method for predicting the efficacy of a c-Met inhibitor (or for offering information on the prediction of the efficacy of a c-Met inhibitor) or for selecting a subject suitable to the application of a c-Met inhibitor (or for offering information of the selection of a subject suitable to the application of a c-Met inhibitor) may further comprise a step of measuring a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a reference biological sample. The measurement of the level of IL-8 and/or a nucleic acid (e.g., gene) encoding IL-8 (e.g., quantitative analysis) may be performed by the same method in both the test biological sample and the reference biological sample.
Measuring the biological sample for a level of at least one selected from among IL-8 and a nucleic acid (e.g., gene) encoding IL-8 may comprise i) treating (reacting or contacting) the biological sample with a material interacting with at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8; and ii) quantitatively analyzing the reaction mixture to determine the level of at least one selected from IL-8 and a nucleic acid (e.g., gene) encoding IL-8. In an embodiment, prior to step i), a step of preparing a biological sample may be further performed, wherein the preparation step may comprise obtaining (isolating) a biological sample from a patient or obtaining a biological sample which has been isolated from a patient. In step i), the interacting material may be at least one selected from the group consisting of compounds (a small molecular chemical; including any general label such as a fluorescent, a dye, etc.), proteins (antibodies, aptamers, etc.), nucleic acids (DNA, RNA, etc.), and the like, which bind to IL-8 or IL-8 gene, and for example, the interacting material may be a compound (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), an antibody, or an aptamer, all binding (e.g., specifically binding) to IL-8, a polynucleotide (e.g., a primer, a probe, an aptamer) binding to a part or entirety of a nucleic acid (e.g., gene) encoding IL-8, or any combination thereof; and optionally, may be conjugated with a general label, such as a fluorescent, a secondary antibody, a bead (e.g., a magnetic bead or polystyrene bead), a dye, or any combination thereof. The step i) may be configured to form a complex by adding the interacting material to the biological sample. In step ii), the reaction mixture may be the complex resulting from interaction (binding) between at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-band the interacting material, which can be obtained in step i). Quantitatively analyzing may comprise quantifying the complex, the label conjugated to the complex, or the IL-8 or nucleic acid (e.g., gene) encoding IL-8 segregated from the complex after the isolation of the complex from the biological sample.
Provided in accordance with another embodiment is a method for monitoring (evaluating or identifying) the efficacy of a c-Met inhibitor or for offering information on the monitoring (evaluation or identification) of the efficacy of a c-Met inhibitor, comprising measuring a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample. In the method for monitoring the efficacy of a c-Met inhibitor or for offering information on the monitoring of the efficacy of a c-Met inhibitor, levels of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in biological samples from a patient before and after treatment with the c-Met inhibitor (or in a c-Met inhibitor-untreated biological sample and a c-Met inhibitor-treated biological sample) may be measured and compared to each other. When the post-treatment level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 is lower (decreased) compared to the pre-treatment level, the c-Met inhibitor is determined to exert its efficacy in the patient from which the biological sample is sourced (obtained), or when the post-treatment level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 is the same with or higher (increased) compared to the pre-treatment level, it is determined that the c-Met inhibitor does not exert its efficacy or a resistance to the c-Met inhibitor occurs in the patient from which the biological sample is sourced (obtained). Therefore, the method for monitoring the efficacy of a c-Met inhibitor or for offering information on the monitoring of the efficacy of a c-Met inhibitor may further comprise 1) comparing the levels of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample from the c-Met inhibitor-treated group with that in a biological sample from the untreated group, and/or 2) determining that the c-Met inhibitor exerts its efficacy in the biological sample-derived patient when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the biological sample of the c-Met inhibitor-treated group is reduced, compared to that of the untreated group (in this case, it may be determined to maintain (continue) the administration of a c-Met inhibitor to the subject), and/or 3) determining that the c-Met inhibitor does not exert its efficacy or a resistance to the c-Met inhibitor occurs in the biological sample-derived patient when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the biological sample of the c-Met inhibitor-treated group is maintained or increased (in this case, it may be determined to stop the administration of a c-Met inhibitor to the subject). The c-Met inhibitor may be an anti-c-Met antibody. The c-Met inhibitor-treated group and the untreated group mean the same biological sample after and before treatment with a c-Met inhibitor, respectively, or aliquots of the same biological sample which are treated with and are not treated with a c-Met inhibitor (for example, treated only with a vehicle), respectively. Unless stated otherwise, the terms “c-Met inhibitor-untreated group (or biological sample)” and “biological sample before treatment with a c-Met inhibitor” or “pre-treatment biological sample” are identical to each other while the terms “c-Met inhibitor-treated group (or biological sample)” and “biological sample after treatment with a c-Met inhibitor” or “post-treatment biological sample” share the same meaning.
The level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 may be measured by quantifying an IL-8 protein and/or a nucleic acid (e.g., gene) encoding IL-8 (DNA, cDNA or mRNA). For example, when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or in the biological sample after treatment with a c-Met inhibitor is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt % compared to that (considering 100 wt %) of the c-Met inhibitor-untreated group or the pre-treatment biological sample, the c-Met inhibitor-treated group, or the post-treatment sample biological, is determined to decrease in the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8, compared to the c-Met inhibitor-untreated or the pre-treatment biological sample. When the level of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or the post-treatment biological sample is about 0 wt % of that in the c-Met inhibitor-untreated group or the pre-treatment biological sample there is no detection (absence) of at least one selected from the IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or the post-treatment biological sample. Levels of IL-8 and/or a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-untreated group (or pre-treatment) and the c-Met inhibitor-treated group (or post-treatment) may be measured by conducting the same procedure on sample samples from the same patient.
Monitoring the efficacy of an c-Met inhibitor, IL-8 and/or a nucleic acid (e.g., gene) encoding IL-8 may be quantitatively analyzed within a relatively short period of time, for example, a week, 5 days, 3 days, 2 days or 1 day after administration of the c-Met inhibitor, and comparison between levels of the marker before and after administration thereof can evaluate the efficacy of c-Met inhibitor. Henceforth, the advantage of establishing a more effective therapeutic strategy can be achieved, since it is possible to evaluate the efficacy of the c-Met inhibitor in an early stage of therapy.
In one embodiment, the measuring of a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample may comprise 1) determining a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample from a patient prior to treatment with a c-Met inhibitor (or a c-Met inhibitor untreated biological sample which is a part of biological sample from a patient) and 2) determining a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in a biological sample from the patient after treatment with a c-Met inhibitor (or a c-Met inhibitor treated biological sample which is the other part of biological sample from a patient). Each of the steps 1) and 2) may comprise i) applying (adding) a material interacting with at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 to the biological sample, and ii) quantitatively analyzing the resulting reaction mixture to determine a level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8. In an embodiment, prior to the step i), a step of preparing a biological sample may be further performed, wherein the preparation step may comprise obtaining (isolating) a biological sample from the patient or obtaining a biological sample which has been isolated from a patient. In step i), as will be further elucidated below, the interacting material may be at least one selected from the group consisting of compounds (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), proteins (antibodies, aptamers, etc.) nucleic acid (DNA, RNA, etc.), and the like, binding to IL-8 or IL-8 gene, and for example, the interacting material may be at least one selected from the group consisting of a compound, an antibody (e.g., antibodies for Intracellular staining (Human CXCL8/IL-8 Phycoerythrin MAb; #IC208P; R&D systems), etc.), an aptamer, all specifically binding to IL-8, and a polynucleotide (e.g., a primer (e.g., a primer set used in RT-PCR for IL-8 gene (ACCESSION NO: BC013615.1; Forward primer: 5′-ATG ACT TCC AAG CTG GCC GTG GCT-3′ (SEQ ID NO: 113) and Reverse primer: 5′-TCT CAG CCC TCT TCA AAA ACT TCT-3′ (SEQ ID NO: 114), etc.), a probe, an aptamer) binding to a part or entirety of a gene encoding IL-8, and optionally, may be conjugated with a label, such as a fluorescent or a dye. The step i) may be configured to form a complex by applying (adding) the interacting material to the biological sample. In step ii), the reaction mixture may be a complex resulting from interaction (binding) between at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 and the interacting material, which can be obtained in step i). The quantitatively analyzing step may comprise quantifying the complex, the marker conjugated to the complex, or the IL-8 or the gene segregated from the complex after the isolation of the complex from the biological sample. The quantitative analysis of IL-8 may be performed by any general quantifying means of proteins, such as immunochromatography, immunohistochemistry, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA; e.g., Platinum ELISA Human IL-8/NAP-1 (#BMS204/3, eBioscience), etc.), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microarray, surface plasmon resonance (SPR), flow cytometry assay (e.g., Cytometric Bead Array (CBA) assay (e.g., IL-8 flex set (#558277), human inflammatory cytokine kit (#551811); BD biosciences), Intracellular staining (e.g., using antibodies such as Human CXCL8/IL-8 Phycoerythrin MAb (#IC208P; R&D systems)), etc.), Luminex assay, and the like, and the quantitative analysis of IL-8 gene may be performed by any general quantifying means of genes (DNA or RNA), such as PCR (e.g., qPCR, RT-PCR, etc.), mRNA microarray, and the like, but not limited thereto.
The monitoring method may be useful in determining whether the c-Met inhibitor continues to be used, and/or suitable dosing conditions of the c-Met inhibitor (dose, dosing interval, number of doses, etc.). For example, when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or in the post-treatment biological sample is reduced, compared to the c-Met inhibitor-untreated group or the pre-treatment biological sample, for example, when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or in the post-treatment biological sample is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt % of that of the c-Met inhibitor-untreated group or the pre-treatment biological sample, the use of c-Met inhibitor may be determined to be continued. In addition, when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or in the post-treatment biological sample is reduced, compared to the c-Met inhibitor-untreated group or the pre-treatment biological sample, for example, when the level of at least one selected from the group consisting of IL-8 and a nucleic acid (e.g., gene) encoding IL-8 in the c-Met inhibitor-treated group or in the post-treatment biological sample is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt %, of that of the c-Met inhibitor-untreated group or the pre-treatment biological sample, the dosing condition of the c-Met inhibitor may be determined to be suitable. The dosing condition may be at least one selected from the group consisting of a dose, a dosing interval, and a number of doses.
Another embodiment provides a method for inhibiting c-Met, comprising administering a c-Met inhibitor to a subject who has a high level of IL-8 and/or a nucleic acid encoding IL-8 (wherein the high level is defined above) and/or shows a decreased level of IL-8 and/or a nucleic acid encoding IL-8 after administration of a c-Met inhibitor (compared to before administration of the c-Met inhibitor). The subject may be 1) selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor or 2) determined by the above method of monitoring efficacy of a c-Met inhibitor as a subject wherein a c-Met inhibitor exerts its efficacy thereon.
Another embodiment provides a method for preventing and/or treating cancer, comprising administering a c-Met inhibitor to a subject who has a high level of IL-8 or a nucleic acid encoding IL-8 (wherein the high level is defined above) and/or shows a decreased level of IL-8 and/or a nucleic acid encoding IL-8 after administration of a c-Met inhibitor (compared to before administration of the c-Met inhibitor). The subject may be 1) selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor or 2) determined by the above method of monitoring efficacy of a c-Met inhibitor as a subject wherein a c-Met inhibitor exert its efficacy thereon.
The method for inhibiting c-Met or the method for preventing and/treating cancer may further comprise selecting a subject to which the c-Met inhibitor is applicable, prior to the administering step. Details of the selection are as described above. The c-Met inhibitor may be an anti-c-Met antibody.
In an embodiment, the method for inhibiting c-Met or for preventing and/or treating cancer may comprise:
identifying a subject to which a c-Met inhibitor is applicable; and
administering a pharmaceutically effective amount of the c-Met inhibitor to the subject.
In another embodiment, the method for inhibiting c-Met or for preventing and/or treating cancer may comprise:
measuring a level of IL-8 and/or a nucleic acid (e.g., gene) encoding IL-8 in a biological sample to select a c-Met inhibitor-applicable subject; and
administering a pharmaceutically effective amount of the c-Met inhibitor to the subject.
With regard to dosing conditions of the c-Met inhibitor, such as doses, dosing intervals and/or number of doses, they may be determined in the method for monitoring a c-Met inhibitor for efficacy.
Some anti-cancer agents such as sunitinib, cis-platin, paclitaxel, etc. often cannot exert their anticancer effects in individuals with a high level of IL-8 because they provoke resistance. In contrast, c-Met inhibitors, for example, anti-c-Met antibodies, particularly, those described below, can exhibit high anticancer activity in individuals with a high level of IL-8, thereby allowing for the suggestion of more effective therapeutic strategies.
bIG-H3, also known as TGFBI (transforming growth factor beta-induced), is RGD-containing protein that binds to type I, II and IV collagens. The RGD motif is found in ECM (extracellular matrix). Serves as a ligand binding to a3b1 integrin, bIG-H3 is involved in cell adhesion, and migration. bIG-H3 is a soluble protein and plays a role in cancer metastasis and angiogenesis in the tumor microenvironment. No reports have been made on a relationship between c-Met and bIG-H3, so far.
MIF (Macrophage migration inhibitory factor) is an important regulator of innate immunity and is classified as an inflammatory cytokine.
In the Example section of the specification, a difference in the expression level of bIG-H3 and/or MIF between c-Met inhibitor-treated groups and untreated groups or between a post-treatment group and a pre-treatment group was observed to vary depending in response to the c-Met inhibitor (refer to Examples 2 and 3). In detail, when a c-Met inhibitor, e.g., an anti-c-Met antibody functions well (drug responsive group), the level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins is lower after application of the c-Met inhibitor than before the application.
In other words, expression levels of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins before or after the application of a c-Met inhibitor are measured to assay (identify) or monitor the effect of the c-Met inhibitor, so that the measurements can be effectively used to determine whether the c-Met inhibitor should continue to be applied or not, or to establish a therapeutic strategy with regard to, for example, doses, dosing intervals, dose numbers of the c-Met inhibitor. Based on this finding, bIG-H3 and/or MIF as appear to be viable pharmacodynamic markers for c-Met inhibitors.
The bIG-H3 may originate from vertebrates including mammals, such as rodents, e.g., mice, rat, etc., and primates, e.g., humans, monkeys, etc., birds, reptiles, amphibians, and fishes. For example, the bIG-H3 may be selected from the group consisting of, but not limited to, human bIG-H3 (NP_000349.1, AAC24944.1, AAC08449.1, AAH00097.1, etc.), mouse bIG-H3 (NP_033395.1, AAI29901.1, AAI29902.1, etc.), rat bIG-H3 (NP_446254.1, etc.), and zebrafish bIG-H3 (NP_878282.1, etc.). A bIG-H3 encoding gene (cDNA or mRNA) may be selected from the group consisting of but not limited to, NM_000358.2, NM_009369.4, NM_053802.1, and NM_182862.1.
MIF may originate from vertebrates including mammals, birds, reptiles, amphibians, and fishes. For example, the MIF may be selected from the group consisting of, but not limited to, human MIF (NP_002406.1, etc.), monkey MIF (e.g., NP_001028087.1, etc.), sheep MIF (NP_001072123.1, etc.), rodent MIF (e.g., mouse MIF (NP_034928.1, etc.), rat MIF (NP_112313.1, etc.), NP_001266756.1, etc.), cow MIF (NP_001028780.1, etc.), zebrafish MIF (NP_001036786.1, etc.), pig MIF (NP_001070681.1, etc.), frog MIF (NP_001083650.1, NP_001107147.1, etc.), and fish MIF (e.g., NP_001118053.1, NP_001135019.1, NP_001027889.1). An MIF-encoding gene (cDNA or mRNA) may be selected from among, but not limited to, NM_002415.1, NM_001032915.1, NM_001078655.1, NM_010798.2, NM_031051.1, NM_001279827.1, NM_001033608.1, NM_001043321.1, NM_001077213.2, NM_001090181.1, NM_001113675.1, NM_001124581.1, NM_001141547.1, and NM_001032717.1.
An embodiment provides a biomarker for evaluating the effect of a c-Met inhibitor, comprising at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins.
Another embodiment provides a composition and a kit for evaluating the effect of a c-Met inhibitor, comprising at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins.
Another embodiment provides a method for evaluating (or identifying or monitoring) the efficacy of a c-Met inhibitor or for offering information on the evaluation (or identifying or monitoring) of the efficacy of a c-Met inhibitor, comprising measuring a level of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins in a biological sample.
In the method for monitoring the efficacy of a c-Met inhibitor or for offering information on the monitoring of the efficacy of a c-Met inhibitor, levels of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in biological samples from a patient before and after treatment with the c-Met inhibitor (or in a c-Met inhibitor treated group biological sample and a c-Met inhibitor untreated biological sample) are measured and compared. When the post-treatment level of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins is lower (decreased) compared to the pre-treatment level, the c-Met inhibitor is determined to exert its activity in the patient from which the biological sample is sourced (obtained), whereas when the post-treatment level of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins is the same with or higher (increase) compared to the pre-treatment level, it is determined that the c-Met inhibitor does not exert its efficacy or a resistance to the c-Met inhibitor occurs in the patient from which the biological sample is sourced (obtained). Therefore, the method for monitoring the efficacy of a c-Met inhibitor or for offering information on the monitoring of the efficacy of a c-Met inhibitor may further comprise 1) comparing the level of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins in a biological sample from the c-Met inhibitor-treated group with that in a biological sample from the untreated group, and/or 2) determining that the c-Met inhibitor exerts its efficacy in the biological sample-derived patient when the level of at least one selected from the group consisting of bIG-H3 or MIF and a nucleic acid (e.g., gene) encoding bIG-H3 or MIF in the biological sample of the c-Met inhibitor-treated group is reduced, compared to that of the untreated group (in this case, it may be determined to maintain (continue) the administration of a c-Met inhibitor to the subject), and/or 3) determining that the c-Met inhibitor does not exert its efficacy or a resistance to the c-Met inhibitor occurs in the biological sample-derived patient when the level of at least one selected from the group consisting of bIG-H3, MIF, and nucleic acids encoding the proteins in the biological sample of the c-Met inhibitor-treated group is maintained or increased (in this case, it may be determined to stop the administration of a c-Met inhibitor to the subject). The c-Met inhibitor may be an anti-c-Met antibody.
The level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins may be measured by quantifying an bIG-H3 protein, an MIF protein and nucleic acids (DNA, cDNA or mRNA) encoding the proteins using the quantification techniques described, above. For example, when the level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in the c-Met inhibitor-treated group or in the biological sample after treatment with a c-Met inhibitor is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt % of that of the c-Met inhibitor-untreated group or the pre-treatment biological sample,” the c-Met inhibitor-treated group, or the post-treatment sample biological is determined to decrease in the level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins, compared to the c-Met inhibitor-untreated or the pre-treatment biological sample. When the level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in the c-Met inhibitor-treated group or the post-treatment biological sample is about 0 wt % of that in the c-Met inhibitor-untreated group or the pre-treatment biological sample, there is no detection (absence) of at least one selected from the bIG-H3, MIF and nucleic acids encoding the proteins in the c-Met inhibitor-treated group or the post-treatment biological sample. Levels of bIG-H3, MIF and nucleic acids encoding the proteins in the c-Met inhibitor-untreated group (or pre-treatment) and the c-Met inhibitor-treated group (or post-treatment) may be measured by conducting the same procedure on sample samples from the same patient.
bIG-H3, MIF and nucleic acids encoding the proteins may be quantitatively analyzed within a relatively short period of time, for example, one month, two weeks, a week, 5 days, 3 days, 2 days or 1 day after administration of the c-Met inhibitor, and comparison between levels of the marker after and before administration thereof can evaluate the efficacy of c-Met inhibitor. Henceforth, the advantage of establishing a more effective therapeutic strategy can be achieved, since it makes it possible to evaluate the efficacy of the c-Met inhibitor in an early stage of therapy.
In one embodiment, the measuring of a level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in a biological sample may comprise 1) determining a level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in a biological sample from a patient prior to treatment with a c-Met inhibitor (or in a c-Met inhibitor untreated biological sample which is a part of biological sample from a patient) and 2) determining a level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins in a biological sample from the patient after treatment with a c-Met inhibitor (or in a c-Met inhibitor treated biological sample which is a part of biological sample from a patient). Each of the steps 1) and 2) may comprise i) applying (adding) a material interacting with at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins to the biological sample; and ii) quantitatively analyzing the resulting reaction mixture to determine a level of at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins. In an embodiment, prior to the step i), a step of preparing a biological sample may be further performed, wherein the preparation step may comprise obtaining (isolating) a biological sample from the patient or obtained a biological sample which has been isolated from a patient. In step i), as will be further elucidated below, the interacting material may be at least one selected from the group consisting of compounds (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), proteins (antibodies, aptamers, etc.) nucleic acid (DNA, RNA, etc.), and the like, binding to bIG-H3, MIF, bIG-H3 gene or MIF gene, and for example, the interacting material may be at least one selected from the group consisting of a compound, an antibody, an aptamer, all specifically binding to bIG-H3 or MIF, and a polynucleotide (e.g., a primer, a probe, an aptamer) binding to a part or entirety of a gene encoding bIG-H3 or MIF, and optionally, may be conjugated with a label, such as a fluorescent, a secondary antibody, a bead (e.g., a magnetic or polystyrene bead), or a dye. The step i) may be configured to form a complex by applying (adding) the interacting material to the biological sample. In step ii), the reaction mixture may be a complex resulting from interaction (binding) between at least one selected from the group consisting of bIG-H3, MIF and nucleic acids encoding the proteins and the interacting material, which can be obtained in step i). The quantitative analysis step may comprise quantifying the complex, the label conjugated to the complex, or the bIG-H3 or the MIF, or the nucleic acid encoding same segregated from the complex after the isolation of the complex from the biological sample. The quantitative analysis of bIG-H3 or MIF may be performed by any general quantifying means of proteins, such as immunochromatography, immunohistochemistry, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microchip, surface plasmon resonance (SPR), flow cytometry assay (e.g., Cytometric Bead Array (CBA) assay, human inflammatory cytokine kit; BD biosciences), Intracellular staining, Luminex assay, and the like, and the quantitative analysis of IL-8 gene may be performed by any general quantifying means of genes (DNA or RNA), such as PCR, (e.g., qPCR, RT-PCR, etc.), mRNA microarray, and the like, but not limited thereto.
The evaluating method may be useful in determining whether the c-Met inhibitor continues to be used, and/or suitable dosing conditions of the c-Met inhibitor (dose, dosing interval, and number of dosing). For example, when the level of at least one selected from the group consisting of bIG-H3, MIF and genes in the c-Met inhibitor-treated group or in the post-treatment biological sample is reduced, compared to the c-Met inhibitor-untreated group or the pre-treatment biological sample, e.g., when the level of at least one selected from the group consisting of bIG-H3, MIF and genes in the c-Met inhibitor-treated group or in the post-treatment biological sample is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt % of that of the c-Met inhibitor-untreated group or the pre-treatment biological sample, the use of c-Met inhibitor may be determined to be continued. In addition, when the level of at least one selected from the group consisting of bIG-H3, MIF and genes in the c-Met inhibitor-treated group or in the post-treatment biological sample is reduced, compared to the c-Met inhibitor-untreated group or the pre-treatment biological sample, e.g., when the level of at least one selected from the group consisting of bIG-H3, MIF and genes in the c-Met inhibitor-treated group or in the post-treatment biological sample is about 0 to about 80 wt %, about 0 to about 70 wt %, about 0 to about 60 wt %, about 0 to about 50 wt %, about 0 to about 40 wt %, about 0 to about 30 wt %, about 0 to about 20 wt %, or about 0 to about 10 wt % of that of the c-Met inhibitor-untreated group or the pre-treatment biological sample, the dosing condition of the c-Met inhibitor may be determined to be suitable. The dosing condition may be at least one selected from the group consisting of a dose, a dosing interval, and a number of doses. The dosing condition may be determined using the techniques described, above.
Another embodiment provides a method for inhibiting c-Met, comprising administering a c-Met inhibitor to a subject in need thereof. Another embodiment provides a method for preventing and/or treating cancer, comprising administering a c-Met inhibitor to a subject in need thereof. In the method for inhibiting c-Met or for preventing and/or treating cancer, the subject may be one showing a decreased level of bIG-H3, MIF, and/or nucleic acids encoding them after administering the c-Met inhibitor (compared to before the administration). For example, the subject may be 1) selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor or 2) determined by the above method of monitoring efficacy of a c-Met inhibitor as a subject wherein a c-Met inhibitor exert its efficacy thereon.
In the method for inhibiting c-Met or for preventing and/or treating cancer, the administration of the c-Met inhibitor may be conducted according to the suitable dosing conditions determined by the evaluating method, such as a dose, a dosing interval, and/or a number of doses.
Levels of IL-8, bIG-H3, MIF, and nucleic acids (e.g., genes) encoding them can be measured by any general protein or gene assay method using a material interacting with the proteins or the genes. The material interacting with any of the proteins or the genes may be a compound, an antibody, or an aptamer, all specifically binding to IL-8, bIG-H3, or MIF 8 proteins, or a polynucleotide (e.g., a primer, a probe, an aptamer) binding to a part or entirety of a gene encoding IL-8, bIG-H3, or MIF proteins. For example, the levels of IL-8, bIG-H3, or MIF protein may be measured by detecting enzymatic reactions, fluorescence, luminescence and/or radioactivity using a compound (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), an antibody and/or an aptamer, which binds (e.g., specifically binds) to IL-8, bIG-H3, or MIF proteins. In detail, the level of the proteins may be determined using an analysis technique selected from among, but not limited to, immunochromatography, immunohistochemistry (IHC), immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (ETA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microarray, surface plasmon resonance (SPR), flow cytometry assay (e.g., Cytometric Bead Array (CBA) assay (BD biosciences), Intracellular staining, etc.), Luminex assay, and the like. In addition, the level of a gene encoding IL-8, bIG-H3 or MIF may be measured by a typical gene assay method using a primer, probe or aptamer hybridizable with the gene, for example, by polymerase chain reaction (PCR; e.g., qPCR, RT-PCR), FISH (fluorescent in situ hybridization), or microarray assay. In one embodiment, the primer is designed to detect a fragment of successive base pairs out of the gene encoding IL-8, bIG-H3 or MIF5 (full-length DNA, cDNA or mRNA), for example, a fragment of about 5 bp to about 2000 bp or 5 to 1000 bp, e.g., about 10 bp to about 1500 bp, about 10 bp to about 1000 bp, about 10 to about 500 bp, about 20 to about 200 bp, or about 50 to about 200 bp, and may be a pair of primers having nucleotide sequences which are respectively hybridizable with (e.g., complementary to) 3′- and 5′-terminal regions ranging in size from about 5 to about 100 bp, e.g., about 5 to about 50 bp, about 5 to about 30 bp, or about 10 to about 25 bp. The probe or the aptamer hybridizable with the gene may have a nucleotide sequence with a size of from about 5 bp to about 2000 bp, from about 5 bp to about 1500 bp, from about 5 bp to about 1000 bp, from about 5 bp to about 500 bp, from about 5 to about 100 bp, from about 5 to about 50 bp, from about 5 to about 30 bp, or from about 5 to about 25 bp, which is hybridizable with (or complementary to) a fragment of the IL-8, bIG-H3 or MIF-encoding gene (full-length DNA, cDNA or mRNA). As used herein, the term “hybridizable” means pertaining to complementarily binding to a specific region of the gene, with a sequence complementarity of 80% or higher, e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% between the primer, probe or aptamer and the gene region.
The RAS-RAF pathway is a representative cell proliferation signaling pathway of which cancer cells take advantage in their growth. For this reason, the RAS-RAF pathway is a main target of anticancer drugs. Hence, a mutation of the RAS-RAF pathway may be an important factor that can affect the efficacy of a drug targeting the pathway. No reports have been made on a relationship between c-Met and RAS-RAF mutation, so far.
In one exemplary embodiment of the present invention, a response to a c-Met inhibitor varies depending on the mutation of RAS and/or RAF (see Example 4). In greater detail, when a biological sample is observed to have a RAS and/or RAF mutation, a c-Met inhibitor, e.g., an anti-c-Met antibody does not work in the biological sample or the patient from which the biological sample originates.
In other words, detection of the mutation of RAS and/or RAF may be effectively used to predict the efficacy of a c-Met inhibitor or to select a subject to which a c-Met inhibitor is applicable. Based on this finding, RAS-RAF is first suggested as a predictive maker for c-Met inhibitors.
RAS, a member of the GTPase superfamily, may be selected from the group consisting of KRAS, NRAS, and HRAS, all of which originate from vertebrates including mammals, such as rodents, e.g., mice, rat, etc., and primates, e.g., humans, monkeys, etc., birds, reptiles, amphibians, and fishes. For example, KRAS may be selected from the group consisting of, but not limited to, human KRAS (NP_004976.2, NP_203524.1, etc.), mouse KRAS (NP_067259.4, etc.), zebrafish KRAS (NP_001003744.1, etc.), frog KRAS (NP_001095209.1, etc.), cow KRAS (NP_001103471.1, etc.), chicken KRAS (NP_001243091.1, etc.), monkey KRAS (NP_001248441.1, etc.), NP_001028153.1, NP_113703.1, and NP_001008034.1. A KRAS-encoding gene (cDNA or mRNA) may be selected from among, but not limited to, NM_004985.4, NM_033360.3, NM_021284.6, NM_001003744.1, NM_001101739.1, NM_001110001.1, NM_001256162.1, NM_001261512.2, NM_001032981.2, NM_031515.3, and NM_001008033.1.
The mutation of KRAS connected with the prediction of the effect of c-Met inhibitors may be a substitution on the codon 12 GGT (corresponding to Gly at position 12 of the amino acid sequence NP_004976) of wild-type KRAS (accession number: NM_004985) and/or on the codon 13 GGC (corresponding to Gly at position 13 of the amino acid sequence NP_004976), as shown in Table 1, below.
In addition, the mutation may be a substitution for at least one amino acid residue selected from the group consisting of Gly at position 13, Ile at position 24, Ala at position 59, Gln at position 61, and Ala at position 146 on the amino acid sequence of NP_004976, and/or a mutation in a KRAS coding nucleic acid inducing the amino acid mutation described above. For example, the mutation of KRAS may comprise at least one selected from the group consisting of G12A, G12D, G12R, G12C, G12S, G12V, A146T, A59T, L23R, G13N, G13D, I24F, Q61L, Q61H, G13C, and Q61K on the amino acid sequence of NP_004976, mutations in a KRAS coding nucleic acid inducing the amino acid mutation described above, or a combination thereof.
RAF may be selected from the group consisting of BRAF, c-RAF A-RAF, V-RAF, KSR1, and KSR2, all of which originate from vertebrates including mammals, such as rodents, e.g., mice, rats, etc., and primates, e.g., humans, monkeys, etc., birds, reptiles, amphibians, and fishes. For example, BRAF may be selected from the group consisting of, but not limited to, human BRAF (NP_004324.2, etc.), mouse BRAF (NP_647455.3, etc.), frog BRAF (NP_001083526.1, etc.), zebrafish BRAF (NP_991307.2, etc.), rat BRAF (NP_579817.1, etc.), and chicken KRAS (NP_990633.1, etc.). The BRAF-encoding gene (cDNA or mRNA) may be selected from the group consisting of but not limited to, NM_004333.4, NM_139294.5, NM_001090057.1, NM_205744.3, NM_133283.1, and NM_205302.1.
The mutation of BRAF connected with the prediction of the effect of c-Met inhibitors may be a substitution of at least one selected from the group consisting of the amino acids at positions 596 (G), 600 (V), 601 (K), 469 (G), 466 (G), 581 (N), 597 (L), 464 (G), and the like, mutations in a BRAF coding nucleic acid inducing the amino acid mutation described above, or a combination thereof. For example, the mutation of BRAF connected with the prediction of the effect of c-Met inhibitors may comprise at least one following mutation on the basis of wild-type BRAF (accession number NP_004324): G596R (base G at position 596 substituted by R), V600E, V600K, V600R, K601E, K601E, G469A, V600M, G469A, V600L, G466V, V600D, G469V, D594G, D594N, N581S, L597V, L597S, L597Q, K601N, G466V, G466E, G464V, G469E, etc.; mutations in a BRAF coding nucleic acid inducing the amino acid mutation described above; or a combination thereof.
An embodiment provides a composition and a kit for the prediction of the efficacy of a c-Met inhibitor, comprising a material detecting a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins.
Another embodiment provides a composition and a kit for the selection of a subject suitable to the application of a c-Met inhibitor, comprising a material detecting a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins.
Also, provided according to another embodiment is a method for predicting the efficacy of a c-Met inhibitor, for offering information on the prediction of the efficacy of a c-Met inhibitor, for selecting a subject suitable to the application of a c-Met inhibitor, or for offering information of the selection of a subject suitable to the application of a c-Met inhibitor, comprising detecting a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins in a biological sample. In the method for predicting the efficacy of a c-Met inhibitor or for offering information on the efficacy of a c-Met inhibitor, when a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins is not detected in a biological sample, the biological sample or a patient from which the biological sample is originated can be predicted to allow the c-Met inhibitor to effectively exert its activity or may be considered to be suitable for the application of the c-Met inhibitor. Hence, the method for predicting the efficacy of a c-Met inhibitor or for offering information on the efficacy of a c-Met inhibitor may further comprise estimating that when a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins in a biological sample, the c-Met inhibitor would exert its efficacy in the biological sample or the patient. In addition, the method for selecting a subject suitable to the application of a c-Met inhibitor or for offering information on the selection may further comprise determining the biological sample or the patient as a subject suitable for the application of the c-Met inhibitor when a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins is not detected biological sample.
Detecting a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins in the biological sample may comprise i) treating the biological sample with a material detecting a mutation of at least one selected from the group consisting of KRAS protein, BRAF protein and nucleic acids encoding the proteins; and ii) analyzing the reaction mixture to determine the presence or absence of the mutation. In an embodiment, prior to the preparing step i), a step of preparing a biological sample may be further performed, wherein the preparation step may comprise obtaining (isolating) a biological sample from a patient or obtaining a biological sample which has been isolated from a patient. In step i), the detective material may be at least one selected from the group consisting of 1) a polynucleotide (e.g., a probe or aptamer) comprising a nucleotide sequence hybridizable with (e.g., complementary to) a mutation-containing fragment of a KRAS gene and/or BRAF gene (full-length DNA, cDNA or mRNA) ranging in size from about 5 bp to about 2000 bp, from about 5 bp to about 1500 bp, from about 5 bp to about 1000 bp, from about 5 bp to about 500 bp, from about 5 bp to about 100 bp, from about 5 bp to about 50 bp, from about 5 bp to about 30 bp, or from about 5 bp to about 25 bp, and a pair of primers having nucleotide sequences which are respectively hybridizable with (e.g., complementary to) 3′- and 5′-terminal regions ranging in size from about 5 bp to about 100 bp, from about 5 bp to about 50 bp, from about 5 bp to about 30 bp, or from about 5 bp to about 25 bp, out of the gene fragment with a size of about 5 bp to about 2000 bp or about 5 bp to about 1000 bp, e.g., from about 10 bp to about 1500 bp, from about 10 bp to about 1000 bp, from about 10 bp to about 500 bp, from about 20 to about 200 bp, or from about 50 to about 200 bp; 2) a compound (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), an antibody, or an aptamer, all binding (e.g., specifically binding) to the KRAS or BRAF protein having the amino acid mutation. Optionally, the detective material may be conjugated with a label, such as a fluorescent, a secondary antibody, a bead (e.g., a magnetic bead or a polystyrene bead), a dye or a combination thereof. As used herein, the term “hybridizable” means pertaining to complementarily binding to a specific region of the gene, with a sequence homology of 80% or higher, e.g., 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% between the primer, probe or aptamer and the gene region. The step i) may be configured to form a complex by adding (applying) the detective material to the biological sample.
In step ii), the reaction mixture may be the complex resulting from hybridization between a gene selected from a KRAS gene and a BRAF gene (which is mutated) and the detective material or resulting from an interaction between a compound (a small molecular chemical; e.g., general label such as a fluorescent, a dye, etc.), an antibody, or an aptamer, and a KRAS or BRAF protein having the amino acid mutation (e.g., substitution), which can be obtained in step i). The analyzing may comprise detecting the presence of the complex or the label conjugated to the complex, or base or amino acid sequencing the KRAS, BRAF, KRAS gene and/or BRAF gene fragment segregated from the complex after the isolation of the complex from the biological sample.
Another embodiment provides a method for inhibiting c-Met, comprising administering a c-Met inhibitor to a subject who has a mutation of at least one of KRAS, BRAF, and nucleic acids encoding them. For example, the subject may be selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor.
Another embodiment provides a method for preventing and/or treating cancer, comprising administering a c-Met inhibitor to a subject who has a mutation of at least one of KRAS, BRAF, and nucleic acids encoding them. For example, the subject may be selected by the above method for selecting a subject suitable for the application of a c-Met inhibitor.
The method for inhibiting c-Met or the method for preventing and/treating cancer may further comprise selecting a subject to which the c-Met inhibitor is applicable, prior to the administering step. Details of the selection are as described above. The c-Met inhibitor may be an anti-c-Met antibody.
In an embodiment, the method for inhibiting c-Met or for preventing and/or treating cancer may comprise:
identifying a subject to which a c-Met inhibitor is applicable; and
administering a pharmaceutically effective amount of the c-Met inhibitor to the subject.
In another embodiment, the method for inhibiting c-Met or for preventing and/or treating cancer may comprise:
detecting a mutation on KRAS, BRAF, or nucleic acid encoding them in a biological sample to select a c-Met inhibitor-applicable subject; and
administering a pharmaceutically effective amount of the c-Met inhibitor to the selected c-Met inhibitor-applicable subject. As used herein, the mutation of KRAS or BRAF protein may refer to the above described amino acid mutation (e.g., substitution), and the mutation of a nucleic acid encoding KRAS or BRAF protein may refer to a mutation (e.g., substitution) in the nucleic acid which induce the mutation of KRAS or BRAF protein as described above.
From the point of view of using a c-Met inhibitor such as an anti-c-Met antibody, a certain expression level of c-Met in cancer cells is prerequisite for the c-Met inhibitor therapy.
Accordingly, the composition and/or kit for predicting an efficacy of a c-Met inhibitor, selecting a subject for applying a c-Met inhibitor, or monitoring an efficacy of a c-Met inhibitor may further comprise a substance interacting with at least one selected from the group consisting of c-Met and c-Met coding gene, in addition to a substance interacting with at least one selected from the group consisting of IL-8, bIG-H3, MIF, KRAS/BRAF, and coding genes thereof. The interacting substance with c-Met and/or c-Met coding gene may be at least one selected from the group consisting of chemicals (small molecules; e.g., general label such as a fluorescent, a dye, etc.), proteins, peptides, nucleic acids (polynucleotides, oligonucleotides, etc.), and the like, which are specifically interact with (or bind to) c-Met and/or c-Met coding gene. For example, the substance interacting with c-Met and/or c-Met coding gene may be at least one selected from the group consisting of chemicals, antibodies, and aptamers, which specifically bind to c-Met, and nucleic acids (e.g., primers, probes, aptamers, etc.) which bind to an whole or a part (e.g., about 5 to about 100 bp, about 5 to about 50 bp, about 5 to about 30 bp, or about 5 to about 25 bp) of c-Met coding gene, which may be labeled with a general labeling material, such as a florescence, a secondary antibody, beads (e.g., a magnetic bead or polystyrene bead), a coloring material, and the like.
In addition, the method for predicting the efficacy of a c-Met inhibitor (or for offering information on the prediction of the efficacy of a c-Met inhibitor) or the method for selecting a subject suitable to the application of a c-Met inhibitor (or for offering information of the selection of a subject suitable to the application of a c-Met inhibitor) may further comprise measuring a level of c-Met protein or a gene thereof (e.g., full-length DNA, cDNA, mRNA) in the biological sample from the patient. The steps of measuring the level of c-Met and/or c-Met coding gene and measuring the level of at least one selected from the group consisting of IL-8, bIG-H3, MIF, KRAS/BRAF, and coding genes thereof may be performed simultaneously or sequentially in any order. Details of the measuring step are as described above. For example, a western blotting technique may be employed. In this regard, when a predetermined amount (e.g., about 10 μg) of proteins obtained from a biological sample (e.g., cancer cells or tissues) is loaded on SDS PAGE gel, transferred onto the membrane, and reacted with an anti-c-Met antibody, and then, exposed on ECL reaction for a certain time (e.g., about 30 sec), the detection of a band may indicate that a prerequisite for the c-Met inhibitor therapy is established. In another embodiment, if the c-Met level, which is determined by immunohistochemistry (IHC) using a tissue section (e.g., (Formalin-fixed paraffin-embedded tissues (FFPE), etc.) of a tumor tissue, is +2 or more, the tumor tissue can be determined that a c-Met inhibitor can exhibit its therapeutic efficacy thereon. In another embodiment, when a biological sample is found to have a c-Met mRNA level of about 12,000 or higher, about 13,000 or higher, or about 14,000 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”), a prerequisite for the c-Met inhibitor therapy may be established. Cancer cells characterized by a high expression level of c-Met include cells from lung cancer, breast cancer, brain cancer, stomach cancer, liver cancer, and kidney cancer. However, any cancer cell, although derived from different kinds, may be a target of the c-Met inhibitor therapy if it expresses a high level of c-Met according to personal characteristics of patients.
In another embodiment, a biological sample used in the method for predicting the efficacy of a c-Met inhibitor (or for offering information on the prediction of the efficacy of a c-Met inhibitor) or the method for selecting a subject suitable to the application of a c-Met inhibitor (or for offering information of the selection of a subject suitable to the application of a c-Met inhibitor) may be a tissue, a cell or body fluid (blood, serum, urine, saliva, etc.) which shows a high expression level of c-Met, for example, a c-Met level of about 12,000 or higher, about 13,000 or higher, or about 14,000 or higher, as measured by Affymetrix array (Affymetrix GeneChip Human Genome U133 Plus 2.0 array; using the primer set “203510_at”).
The method of predicting, monitoring, or selecting may further comprising administering a c-Met inhibitor to a subject, who is selected by the selecting method, or who is predicted or monitored that a c-Met inhibitor exerts its efficacy therein.
The method of predicting, monitoring, or selecting may further comprise optimizing the application of a c-Met inhibitor or a therapy with a c-Met inhibitor by 1) determining whether or not a c-Met inhibitor is administered to a subject according to the determination of the determining step of the predicting or selecting method (e.g., determining to administer a c-Met inhibitor to the subject when the level of IL-8 and/or a nucleic acid encoding IL-8 in a biological sample obtained (separated) from the subject is higher than that of a reference sample or when a mutation of at least one of KRAS, BRAF, and nucleic acids encoding the proteins is not present in the biological sample, or determining not to administer a c-Met inhibitor to the subject when the level of IL-8 and/or a nucleic acid encoding IL-8 in a biological sample obtained (separated) from the subject is equal to or lower than that of a reference sample or when a mutation of at least one of KRAS, BRAF, and nucleic acids encoding the proteins is present in the biological sample, or 2) determining whether the administration of a c-Met inhibitor to the subject is maintained (continued) or stopped according to the determination of the determining step of the monitoring method (e.g., determining to maintain the administration of c-Met inhibitor to the subject, when the level of IL-8, bIG-H3, MIF, and/or nucleic acids encoding the proteins in the biological sample after the administration of a c-Met inhibitor is decreased compared to before the administration, or determining to stop the administration of a c-Met inhibitor, when the level of IL-8, bIG-H3, MIF, and/or nucleic acids encoding the proteins in the biological sample after the administration of a c-Met inhibitor is equal to or increased compared to before the administration.
As used herein, the term “c-Met inhibitor-applicable subject” or “subject suitable for the application of the c-Met inhibitor” means a patient to which a c-Met inhibitor therapy is applicable suitably, and the term “subject” may be selected from the group consisting of mammals, such as rodents, e.g., mice, rats, etc., and primates, e.g., humans, monkeys, etc., e.g., a cancer patient. The biological sample may be a patient itself (mammals, such as primates, e.g., humans, monkeys, etc., or rodents, e.g., mice, rats, etc.) or a cell, a tissue or body fluid (e.g., blood, serum, urine, saliva, etc.) isolated from the patient or artificially an artificial culture thereof. The biological sample may be blood or a serum. The reference sample may be selected from the group consisting of mammal, mammalian cells or tissues (including tumor (e.g., cancer) cells or tissues), and the like, on which a c-Met inhibitor has no efficacy (e.g., a c-Met inhibitory efficacy or an anti-cancer efficacy).
As used herein, the term “c-Met inhibitor” may refer to any agent capable of inhibiting activity and/or expression of c-Met, or blocking c-Met signaling by inhibiting a ligand of c-Met. In an particular embodiment, the c-Met inhibitor may be at least one selected from the group consisting of an anti-c-Met antibody or an antigen-binding fragment thereof, crizotinib (PF-02341066; 3-((R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine), cabozantinib (XL-184; N-(4-(6,7-dimethoxyquinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), foretinib (N-(3-fluoro-4-(6-methoxy-7-(3-morpholinopropoxyl)quinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), PHA-665752((R,Z)-5-(2,6-dichlorobenzylsulfonyl)-3-((3,5-dimethyl-4-(2-(pyrrolidin-1-ylmethyl)pyrrolidine-1-carbonyl)-1H-pyrrol-2-yl)methylene)indolin-2-one), SU11274((Z)—N-(3-chlorophenyl)-3-((3,5-dimethyl-4-(1-methylpiperazine-4-carbonyl)-1H-pyrrol-2-yl)methylene)-N-methyl-2-oxoindoline-5-sulfonamide), SGX-523(6-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-ylthio)quinoline), PF-04217903(24443-(quinolin-6-ylmethyl)-3H-[1,2,3]triazolo[4,5-b]pyrazin-5-yl)-1H-pyrazol-1-yl)ethanol), EMD 1214063(Benzonitrile, 3-[1,6-Dihydro-1-[[3-[5-[(1-Methyl-4-Piperidinyl)Methoxy]-2-PyriMidinyl]Phenyl]Methyl]-6-Oxo-3-Pyridazinyl]), golvatinib (N-(2-fluoro-4-((2-(4-(4-methylpiperazin-1-yl)piperidine-1-carboxamido)pyridin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide), INCB28060(2-fluoro-N-methyl-4-(7-(quinolin-6-ylmethyl)imidazo[1,2-b][1,2,4]triazin-2-yl)benzamide), MK-2461(N-((2R)-1,4-Dioxan-2-ylmethyl)-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide), tivantinib (ARQ 197; (3R,4R)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione), NVP-BVU972(6-[[6-(1-Methyl-1H-pyrazol-4-yl)imidazo[1,2-b]pyridazin-3-yl]methyl]quinoline), AMG458({1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide}), BMS 794833(N-(4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)-5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide), BMS 777607(N-[4-[(2-Amino-3-chloropyridin-4-yl)oxy]-3-fluorophenyl]-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide), MGCD-265(N-(3-Fluoro-4-(2-(1-methyl-1H-imidazol-4-yl)thieno[3,2-b]pyridin-7-yloxy)phenylcarbamothioyl)-2-phenylacetamide), AMG-208(7-Methoxy-4-[(6-phenyl-1,2,4-triazolo[4,3-b]pyridazin-3-yl)methoxy]quinoline), BMS-754807((2S)-1-[4-[(5-Cyclopropyl-1H-pyrazol-3-yl)amino]pyrrolo[2,1-f][1,2,4]triazin-2-yl]-N-(6-fluoro-3-pyridinyl)-2-methyl-2-pyrrolidinecarboxamide), JNJ-38877605(6-[Difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]quinoline), and pharmaceutically acceptable salts thereof; or any combination thereof.
The anti-c-Met antibody or an antigen-binding fragment thereof may be any antibody which specifically recognizes c-Met as an antigen and/or specifically binds to c-Met, or an antigen-binding fragment thereof. For example, the anti-c-Met antibody may be any antibody that acts on c-Met to induce intracellular internalization and degradation of c-Met, or antigen-binding fragment thereof. The anti-c-Met antibody may recognize any specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope.
Herein, unless stated otherwise, the term “anti-c-Met antibody” may be intended to cover not only an anti-c-Met antibody in a complete form (e.g., an IgG form) but also its antigen-binding fragment. The antigen-binding fragment thereof may be at least one selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab′, and F(ab′)2 of an anti-c-Met antibody.
“c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF). c-Met may be derived (obtained) from any species, particularly a mammal, for instance, primates such as human c-Met (e.g., GenBank Accession No. NP_000236.2), monkey c-Met (e.g., Macaca mulatta, GenBank Accession No. NP_001162100), or rodents such as mouse c-Met (e.g., GenBank Accession No. NP_032617.2), rat c-Met (e.g., GenBank Accession No. NP_113705.1), and the like. The c-Met protein may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession No. NM_000245.2, a polypeptide having the amino acid sequence identified as GenBank Accession No. NP_000236.2 or extracellular domains thereof. The receptor tyrosine kinase c-Met participates in various mechanisms, such as cancer incidence, metastasis, migration of cancer cells, invasion of cancer cells, angiogenesis, and the like.
c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and includes a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin identity/homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region having the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79), is a loop region between the second and the third beta propellers within the epitopes of the SEMA domain. This region acts as an epitope for the anti-c-Met antibody.
The term “epitope,” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including about 5 or more contiguous (consecutive on primary, secondary (two-dimensional), or tertiary (three-dimensional) structure) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, about 5 to about 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide having about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide includes at least the amino sequence of SEQ ID NO: 73 (EEPSQ) which serves as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.
The epitope having the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third beta propellers within the SEMA domain of a c-Met protein. The epitope having the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.
Thus, the anti-c-Met antibody may specifically bind to an epitope which includes about 5 to about 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.
In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may comprise or consist essentially of:
(i) at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence comprising 8-19 consecutive amino acids within the amino acid sequence of SEQ ID NO: 2 comprising amino acid residues from the 3rd to 10th positions of the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence comprising 6-13 consecutive amino acids within the amino acid sequence of SEQ ID NO: 85 comprising amino acid residues from the 1st to 6th positions of the amino acid sequence of SEQ ID NO: 85, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
(ii) at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 15, the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence comprising 9-17 consecutive amino acids within the amino acid sequence of SEQ ID NO: 89 comprising amino acid residues from the 1st to 9th positions of the amino acid sequence of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;
(iii) a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or
Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:
Formula I: Xaa1-Xaa2-Tyr-Tyr-Met-Ser (SEQ ID NO: 4), wherein Xaa1 is absent or Pro or Ser, and Xaa2 is Glu or Asp,
Formula II: Arg-Asn-Xaa3-Xaa4-Asn-Gly-Xaa5-Thr (SEQ ID NO: 5), wherein Xaa3 is Asn or Lys, Xaa4 is Ala or Val, and Xaa5 is Asn or Thr,
Formula III: Asp-Asn-Trp-Leu-Xaa6-Tyr (SEQ ID NO: 6), wherein Xaa6 is Ser or Thr,
Formula IV: Lys-Ser-Ser-Xaa7-Ser-Leu-Leu-Ala-Xaa8-Gly-Asn-Xaa9-Xaa10-Asn-Tyr-Leu-Ala (SEQ ID NO: 7), wherein Xaa7 is His, Arg, Gln, or Lys, Xaa8 is Ser or Trp, Xaa9 is His or Gln, and Xaa10 is Lys or Asn,
Formula V: Trp-Xaa11-Ser-Xaa12-Arg-Val-Xaa13 (SEQ ID NO: 8), wherein Xaa11 is Ala or Gly, Xaa12 is Thr or Lys, and Xaa13 is Ser or Pro, and
Formula VI: Xaa14-Gln-Ser-Tyr-Ser-Xaa15-Pro-Xaa16-Thr (SEQ ID NO: 9), wherein Xaa14 is Gly, Ala, or Gln, Xaa15 is Arg, His, Ser, Ala, Gly, or Lys, and Xaa16 is Leu, Tyr, Phe, or Met.
In one embodiment, the CDR-H1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. The CDR-H2 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 25, and SEQ ID NO: 26. The CDR-H3 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 85.
The CDR-L1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 106. The CDR-L2 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. The CDR-L3 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89.
In another embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may comprise or consisting essentially of:
a heavy variable region comprising or consisting essentially of a polypeptide (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, a polypeptide (CDR-H2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 25, and SEQ ID NO: 26, and a polypeptide (CDR-H3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 85; and
a light variable region comprising or consisting essentially of a polypeptide (CDR-L1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 106, a polypeptide (CDR-L2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, and a polypeptide (CDR-L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89.
In an embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may comprise or consist essentially of a heavy variable region comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 74, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94, and a light variable region comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 75, SEQ ID NO: 88, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 107.
In another embodiment, the anti-c-Met antibody or an antigen-binding fragment may be Onartuzumab (MetMab), LY2875358 (Heavy chain: SEQ ID NO: 111; Light chain: SEQ ID NO: 112), Rilotumumab (AMG102), or an antigen-binding fragment thereof, and the like.
Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected into humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.
The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.
The anti c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic. The antibody may be monoclonal.
An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ϵ) type, which may be further categorized as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.
As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region VH that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, CH1, CH2, and CH3, and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region VL that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.
The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDRs may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.
The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. In one embodiment, the antigen-binding fragment may be selected from the group consisting of scFv, (scFv)2, Fab, Fab′, and F(ab′)2, but not be limited thereto.
Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region CH1, includes one antigen-binding site.
The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of CH1.
The F(ab′)2 antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.
Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be a polypeptide comprising about 1 to about 100 or about 2 to about 50 amino acids, wherein the amino acids may be selected from any amino acids without limitation.
The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)2 fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.
The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.
When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin may be replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.
In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid residue of the amino acid sequence of the hinge region so that it exhibits enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100 (U7-HC6), 101 (U6-HC7), 102 (U3-HC9), 103 (U6-HC8), or 104 (U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). Preferably, the hinge region includes the amino acid sequence of SEQ ID NO: 100 or 101.
In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, under Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the entire disclosure of which is incorporated herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.
In the anti-c-Met antibody, the rest portion of the light chain and the heavy chain portion other than the CDRs, the light chain variable region, and the heavy chain variable region, e.g., a light chain constant region and/or a heavy chain constant region, may be from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).
By way of further example, the anti-c-Met antibody may comprise or consist essentially of:
(a) a heavy chain comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1st to 17th positions is a signal peptide), the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1st to 17th positions is a signal peptide), the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1st to 17th positions is a signal peptide), and the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66; and
(b) a light chain comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1st to 20th positions is a signal peptide), the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1st to 20th positions is a signal peptide), the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.
For example, the anti-c-Met antibody may be selected from the group consisting of:
(i) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;
(ii) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and (b) a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;
(iii) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;
(iv) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and (b) a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70;
(v) an antibody comprising a heavy chain comprising (a) the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70;
(v) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70;
(vi) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 108;
(vii) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 108; and
(viii) an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 108.
The polypeptide comprising the amino acid sequence of SEQ ID NO: 70 is a light chain including human kappa (κ) constant region, and the polypeptide comprising the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide comprising the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide comprising the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) of SEQ ID NO: 108 with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibit increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.
In another embodiment, the anti c-Met antibody may comprising a light chain complementarity determining region comprising the amino acid sequence of SEQ ID NO: 106, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107, or a light chain comprising the amino acid sequence of SEQ ID NO: 108.
The c-Met inhibitor may be applied (administered) together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be those commonly used for the formulation of the inhibitor (e.g., antibodies), which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The c-Met inhibitor may further comprise one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and preservative.
The c-Met inhibitor may be administered orally or parenterally. The parenteral administration may selected from the group consisting of intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, the c-Met inhibitor for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the c-Met inhibitor may be administered using an optional device that enables an active substance to be delivered to target cells.
The term “the pharmaceutically effective amount” as used herein may refer to an amount of which the active ingredient can exert pharmaceutically significant effects. The pharmaceutically effective amount of the c-Met inhibitor for a single dose may be prescribed in a variety of ways, depending on factors such as formulation methods, administration manners, age of patients, body weight of patients, gender of patients, pathologic conditions of patients, diets, administration time, administration interval, administration mute, excretion speed, and reaction sensitivity. For example, the pharmaceutically effective amount of the c-Met inhibitor for a single dose may be in ranges of 0.001 to 100 mg/kg, or 0.02 to 10 mg/kg, but not limited thereto. The pharmaceutically effective amount for the single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container.
The c-Met inhibitor may be used in the prevention and/or treatment of a cancer. The cancer may be associated with over-expression and/or abnormal activation of c-Met. The cancer may be a solid cancer or a blood cancer. The cancer may be, but not limited to, one or more selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head or neck cancer, brain cancer, osteosarcoma, and the like. The cancer may be a primary cancer or a metastatic cancer. The cancer may be a c-Met inhibitor (e.g., an anti-c-Met antibody) resistant cancer.
The prevention and/or treatment of cancer may be achieved by at least one effect of the c-Met inhibitor selected from cancer cell death, inhibition of cancer cell proliferation, inhibition of migration, invasion, or metastasis of cancer, improvement of symptoms associated with cancer, etc.
Biomarkers capable of predicting and/or monitoring an efficacy of a c-Met inhibitor may lead to improvement of the therapeutic effect of the c-Met inhibitor, and making it possible to provide a patient-tailored treatment which is optimized for individual patient, thereby increasing the therapeutic efficacy.
Hereafter, the present invention will be described in detail by examples.
The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.
1.1. Production of “AbF46”, a Mouse Antibody to c-Met
1.1.1. Immunization of Mice
To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.
1.1.2. Cell Fusion and Production of a Hybridoma
Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×108 cells) were mixed with myeloma cells (Sp2/0) (1×108 cells), followed by spinning to yield a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×105 cells/mL in a selection medium (HAT medium). 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO2 incubator to establish a hybridoma cell population.
1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein
From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.
Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.
The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.
Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).
1.1.4. Production and Purification of a Monoclonal Antibody
The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.
First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS (fetal bovine serum) were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO2 incubator.
After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with a filter (Amicon). The antibody was stored in PBS before use in the following examples.
1.2. Construction of chAbF46, a Chimeric Antibody to c-Met
A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Reference Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.
In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.
Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/mL. After 24 hours, when the cell number reached to 1×106 cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.
Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.
After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).
1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46
1.3.1. Heavy Chain Humanization
To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST search (www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has an identity/identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).
For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).
1.3.2. Light Chain Humanization
To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has an identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.
To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a BLAST search. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.
For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A BLAST search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.
Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.
Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/ml, and after 24 hours, when the cell number reached to 1×106 cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (invtrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.
After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples comprised a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).
1.4. Construction of scFV Library of huAbF46 Antibody
For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker comprising the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.
After expression, the product was found to exhibit specificity to c-Met.
1.5. Construction of Library Genes for Affinity Maturation
1.5.1. Selection of Target CDRs and Synthesis of Primers
The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 2 below.
For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).
1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met
The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.
The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.
1.6. Selection of Antibody with Improved Affinity from Libraries
After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 3 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.
1.7. Conversion of Selected Antibodies into IgG
Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment comprising L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment comprising L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.
Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/mL. After 24 hours, when the cell number reached to 1×106 cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). I In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.
After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).
1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1
Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.
The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain comprising the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain comprising a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain comprising the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.
For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.
Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/mL. After 24 hours, when the cell number reached to 1×106 cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.
After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (U6-HC7) and huAbF46-H4-A1 (IgG2 Fc) were representatively selected for the following examples, and referred as anti-c-Met antibody L3-1Y and L3-1Y/IgG2, respectively.
1.1. Assay for Predicting and Evaluating Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using IL-8
1.1.1. Construction of Cancer Cell-Implanted Mouse Model
For use in assays for predicting and evaluating the efficacy of an anti-c-Met antibody, xenograft mouse models were prepared by implanting various conventional cancer cell lines (Lovo, HT-29, EBC-1, MKN45, and Hs746T) to mice.
Briefly, the human colorectal cancer cell line HT29 (ATCC HTB-38) or Lovo (ATCC CCL-229) were injected s.c. at a dose of 5×106 cells into BALB/C nude mice (4-5 week old, male; Shanghai SLAC Laboratory Animal Co. Ltd.) to construct xenograft mouse models. For modeling, 15 mice were allocated to each group. Also, the human stomach cancer cell lines Hs746T (ATCC HTB-135) and MKN45 (ATCC RCB1001), and the human non-small cell lung cancer cell line EBC-1 (ATCC JCRB0820) were used in the same manner to construct xenograft mouse models.
That is, mouse models implanted, respectively, with Lovo, HT-29, EBC-1, Hs746T, and MKN45 were constructed, and used in the following experiments.
1.1.2. Prediction of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using IL-8
After the anti-c-Met antibody L3-1Y/IgG2 prepared in Reference Example 1 was administered to Lovo-, HT-29-, EBC-1-, MKN45-, or Hs746T-implanted mouse models constructed in Example 1.1.1, tumor sizes were measured to identify groups which were responsive to L3-1Y/IgG2 (responder; effective anticancer activity) and were not responsive (non-responder; no anticancer activity).
Briefly, the anti-c-Met antibody was injected to each group of the mouse models constructed in Example 1.1.1 from 7-10 days after implantation. The injection was conducted at regular intervals of 7 days for 4 weeks via an intravenous route. Tumor volumes were measured and recorded twice a week. For a control, PBS was injected. The anti-c-Met antibody was injected at a dose of 0.2, 1, 5, or 10 mg/kg to HT29- or Lovo-implanted groups, and at a dose of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 mg/kg to EBC-1-, MKN45-, or Hs746T-implanted groups. Results are depicted in
An examination was made of a difference in serum IL-8 levels between the responder and the non-responder groups. Serum IL-8 levels of the Lovo-, the HT-29-, the EBC-1- and the Hs746T-implanted mouse models were compared to those of the control mice (injected with PBS only; at a dose of 100 μL every 7 days for 4 weeks), and relationship between serum IL-8 level and responsiveness to L3-1Y/IgG2 was investigated.
Briefly, sera from the control of each model were analyzed using a BD Cytometric Bead Array kit. Whole blood was collected from the mouse models in a BD vacutainer SST™ tube. After collection of the whole blood, the tube was mixed by gently inverting for 5 times, and then, allowed to clot for 30 minutes at room temperature. After the incubation, the clot was removed by centrifuge the tube at 1500×g for 10 minutes in a refrigerated centrifuge. The serum sample is obtained by transfer the supernatant into a new tube. Experiments were performed with a human inflammatory cytokine kit. According to the manufacture's guideline, mouse serum was diluted 1/4 (v/v) in the assay diluent buffer and incubated for 1.5 hrs with biotinylated anti-human IL-8 antibody-coated beads (BD Cytometric Bead Array (CBA) Human inflammatory cytokines kit, #551811, BD biosciences). Thereafter, the sample was mixed with 1 mL of the wash buffer containing in the BD Cytometric Bead Array (CBA) Human inflammatory cytokines kit, and centrifuged. After removal of the supernatant, the residue was incubated for 1.5 hrs with the streptavidin-PE of the kit, mixed with 1 mL of the wash buffer. After centrifugation (200 g, 5 min), the supernatant containing unreacted materials was discarded. The precipitate was suspended in 200 μL of wash buffer before FACS analysis (BD FACS CantoII flow cytometer, BD Bioscience). From the data thus obtained, serum IL-8 concentrations were calculated using the FCAP array program (BD Biosciences), and quantified by multiplication by the dilution factor.
The results are shown in
1.1.3. Evaluation of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using IL-8
To examine the relationship between the change of IL-8 level and the responsiveness to an anti-c-Met antibody, the changes of serum IL-8 levels after treatment of the anti-c-Met antibody in cancer cell-implanted mouse models were monitored.
Briefly, among the serum samples obtained from the mouse xenograft models of Example 1.1.2, the vehicle-treated sample (PBS-treated, control) and the anti-c-Met antibody treated samples with high and low doses were prepared. To this end, serum samples were taken from HT29 and Lovo models (non-responder groups) injected at doses of 0.2 mg/kg and 5 mg/kg and from EBC-1 and Hs746T models (responder groups) injected at doses of 1 mg/kg and 3 mg/kg, and examined for the relationship between the anticancer efficacy of the anti-c-Met antibody and the change of IL-8 level after anti-c-Met antibody treatment. Serum IL-8 levels were analyzed with reference to Example 1.1.2.
Results are depicted in
1.2. Assay for Predicting and Evaluating Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues by Measuring the Serum Level of IL-8.
1.2.1. Construction of Mouse Model Implanted with Patient-Derived Tumor Tissues.
For use in examining whether IL-8 can be used as a reliable biomarker in predicting or evaluating the efficacy of an anti-c-Met antibody when it is applied to patients, mouse models implanted with patient-derived tumor tissue suffering from cancer (lung cancer) were constructed.
This experiment was performed in the special company Oncotest (Oncotest GmbH, Freibrug Germany). Briefly, patient-derived lung cancer cell lines (non-small cell lung cancer (NSCLC)) LXFA297, 526, 623, 983, 1041 and 1647 were cultured and injected s.c. into NMRI nude mice (4-6 week old, female; Harlan) to construct xenograft mouse models. The construction was carried out according to the Oncotest SOP “Subcutaneous implantation” guideline. 10 mice were allocated to each group. After implantation, the mouse models were randomized to reduce measurement deviations of average tumor volumes between groups, and then injected with the drug. Experiments using patient-derived gastric cancer cell lines, GXF214 and GXF251, and patient-derived renal cancer cell lines, RXF488, RXF1114 and RXF1220 were performed referring to the above method.
The mouse models implanted with patient-derived tumor tissues were tested for responsiveness to an anti-c-Met antibody while L3-1Y/IgG2 was injected thereto.
Briefly, to monitor the anticancer efficacy of the antibody, the tumor volumes were regularly measured. Anticancer efficacy was calculated by dividing tumor volumes of each antibody-treated group (5 mg/kg, iv) by those of the control group (PBS injected) after completion of the injection (% inhibition=100−(tumor volume of test group/tumor volume of control*100)). The results are summarized in Tables 4 and 5.
A reduction of tumor volume by 30% or greater upon treatment with L3-1Y/IgG2 was considered statistically significant, which led to determining the antibody as being efficacious, and the group as a responder.
As is understood from data of Tables 4 and 5, the lung cancer cell-implanted mouse models LXFA526, LXFA623, and LXFA1647, and the kidney cancer cell-implanted mouse model RXF1114 were found to respond to L3-1Y/IgG2 (responder) while the other mouse models implanted with patient-derived tumor tissue were not responsive to the antibody (non-responder)
1.2.2. Prediction of the Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues Using Measurement of the Serum Level of IL-8
Differences in the basal level of serum IL-8 between the responders and the non-responders were examined. In this regard, serum samples were taken from the vehicle groups of mouse models constructed in Example 1.2.1 and the IL-8 levels in the serum samples were used to examine relationship with responsiveness to L3-1Y/IgG2.
Briefly, serum samples taken from the mouse models implanted with patient-derived tumor tissues were classified according to test group. IL-8 levels were measured in vehicle treated groups from the 6 samples of lung cancer models, 2 samples of stomach cancer models, 3 samples of kidney cancer models. Measurement of the IL-8 levels was carried out in the same manner as in Example 1.1.2.
The results are shown in
1.2.3. Evaluation of the Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues Using Measurement of the Serum Level of IL-8
To examine the relationship between IL-8 level and responsiveness to an anti-c-Met antibody, changes of serum IL-8 levels after treatment of an anti-c-Met antibody in mouse models implanted with patient-derived tumor tissues were monitored.
Briefly, serum samples taken from the mouse models implanted with patient-derived tumor tissues were classified according to test group. IL-8 levels were measured in 6 samples of lung cancer models, 2 samples of stomach cancer models, 3 samples of kidney cancer models after injection of the anti-c-Met antibody (5 mg/kg, iv) and in samples of the control (PBS injected), and examined for relationship between the anticancer efficacy of the anti-c-Met antibody and the change of IL-8 level after the anti-c-Met antibody treatment. Measurement of the IL-8 levels was carried out in the same manner as in Example 1.1.2.
Results are depicted in
1.3. Change in IL-8 Level with Time after Anti-c-Met Antibody Injection
1.3.1. Construction of Cancer Cell-Implanted Mouse Model and Assay for Anticancer Activity of Anti-c-Met Antibody in the Mouse Model
The role of IL-8 as a pharmcodynamic marker of anti-c-Met antibodies was confirmed in xengraft mouse models which were prepared by implanting cancer cell lines known to be responsive to the antibody.
As opposed to Example 1.1, this experiment was configured to examine the responsiveness in a condition under which tumor volumes were little changed. After implantation, tumors were increased to a size of 1000 mm3 and, then, the antibody was injected in various doses to the mice models. According time after injection, serum samples and tumor tissues were taken from three mice per group. In addition, tumor volumes were measured at regular intervals of time.
Briefly, xenograft mouse models implanted with the human stomach cell line Hs746T (Accession No. HTB-135) or human non-small cell lung cancer line EBC-1 (Accession No. JCRB0820) were employed. Each cancer cell line was injected s.c. at a dose of 5×106 cells to 150 Balb/c nude mice (Shanghai SLAC Laboratory Animal Co. Ltd.). All of the experimental animals were male and 5-6 weeks old upon the implantation, and bred in an SPF condition. In order to increase the tumor to a predetermined size, the mice implanted with the cancer cells were treated with the drug (antibody) only 16-20 days after the implantation. When the tumor grew to a size of about 1000 mm3, the mice were randomly divided into groups of 30: PBS-treated group (Group 1) and anti-c-Met antibody-treated groups (Groups 2, 3, and 4). The treated groups were classified according to the dose of the antibody: 1 mg/kg (Group 2), 3 mg/kg (Group 3) and 10 mg/kg (Group 4). The antibody and the vehicle were administered twice at regular intervals of 7 days. Each sample was withdrawn 1 hr, 1 day (24 hrs), 2 days (48 hrs), 3 days (72 hrs), and 7 days (168 hrs) after the first injection. The same sampling time schedule was repeated after the second injection: 1 hr (169 hrs), 1 day (192 hrs), 2 days (216 hrs), 3 days (240 hrs), and 7 days (336 hrs). With regard to sampling, serum and tissues were taken from three mice which were sacrificed at each time point. Tumor volumes were also measured upon sampling to monitor the anticancer activity of the antibody (PharmaLegacy Laboratories Vivarium).
Anticancer activity data obtained above are depicted in
As is apparent from the data of
1.3.2 Evaluation of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using Measurement of the Serum Level of IL-8
The following experiments were conducted with samples taken from the EBC-1- or Hs746T-implanted mouse models prepared in Example 1.3.1. To examine whether the anti-c-Met antibody-induced responsiveness of IL-8 is simply reflect the shrunken tumor size as a result from the efficacy of anti-cMet Ab, time course serum samples were analyzed from the EBC-1- or Hs746T-implanted mouse models to measure the short-term response to L3-1Y/IgG2.
Briefly, serum samples were taken in an amount of 100 μL from each of 3 mice per group according to time after antibody dosing (see
The results are summarized in
As can be seen in
1.4. Test of Efficacies of c-Met Inhibitors Other than Anti-c-Met Antibodies
It was verified whether or not the change in IL-8 level can be applicable to a monitoring of responsiveness to a c-Met targeting drug other than anti-c-Met antibodies, as a marker.
In particular, EBC-1 (human lung cancer cell line; Accession No. JCRB0820) and Hs746T (human stomach cancer cell line; Accession No. HTB-135), on which anti-c-Met antibody L3-1Y/IgG2 displays anticancer efficacies (anti-c-Met antibody responder groups), and Lovo (human colon cancer cell line; Accession No. CCL-229) and HT29 (human colon cancer cell line; Accession No. HTB-38), on which anti-c-Met antibody L3-1Y/IgG2 displays no anticancer efficacies (anti-c-Met antibody non-responder groups), were selected and cultured. The 4 cell lines were seeded on a 96 well plate at the same amount (1×104 cells/well) and cultured for 24 hours (media: RPMI (Gibco, Invitrogen), culture conditions: 37° C., 5% CO2). The cultured cells were treated with representative c-Met inhibitor, crizotinib (Selleck chemical) and PHA665752((R,Z)-5-(2,6-dichlorobenzylsulfonyl)-3-((3,5-dimethyl-4-(2-(pyrrolidin-1-ylmethyl)pyrrolidine-1-carbonyl)-1H-pyrrol-2-yl)methylene)indolin-2-one), respectively. For comparison, human control IgG (chrompure human IgG (009-000-003, JACKSON IMMUNORESEARCH LABS INC.)) as a control and anti-c-Met antibody L3-1Y/IgG2 (Reference Example) were used respectively. Each of the inhibitors (or antibody) was treated at 10-fold serial diluted concentrations from 1000 nM to 0.01 nM. 72 hours after the treatment, the level of IL-8 in cell culture of each treatment group was assayed by Cytometric Bead Array (CBA) method using BD Cytometric Bead Array kit (see Example 1.1.2). In addition, in order to verify the anticancer efficacy of the c-Met inhibitors, the cell proliferation (%) was measured at 72 hours after c-Met inhibitor treatment through Cell-Titer Glo (CTG) assay.
The obtained results of cell proliferation (%) (i.e., results of CTG assay) at 72 hours were shown in
In addition, at the concentration of 10 nM, from which the responsiveness to the inhibitors can be observed, the level of IL-8 in a cell culture of each treatment group was measured by Cytometric Bead Array (CBA) method using BD Cytometric Bead Array kit (see, Example 1.1.2), and the obtained results are shown in
These results indicate that the decrease in IL-8 level can also be useful as a maker for evaluation of the efficacy of not only anti-c-Met antibodies but also c-Met inhibitors other than anti-c-Met antibodies.
2.1. Selection of bIG-H3 as Pharmacodynamic Marker for Anti-c-Met Antibody
Proteins which showed a significant difference in the expression level between an anti-c-Met antibody-treated group and an untreated group responsive to the anti-c-Met antibody were selected as possible biomarkers for evaluating the efficacy of the anti-c-Met antibody, using an antibody array membrane capable of detecting 119 soluble receptors (a human soluble receptor array kit non-hematopoietic panel, cat# ARY012; R&D; alternatively, a human cytokine array kit (e.g., #ARY005; R&D) can be used).
In mouse models which were constructed with U87MG (Accession No. HTB-14) in the same manner as in Example 1.1.1, changes in tumor size with treatment with the anti-c-Met antibody L3-1Y/IgG2 were monitored. The results are shown in
Of the U87MG cells which were identified to respond to the anti-c-Met antibody, differences in protein expression level between anti-c-Met antibody-treated cells groups and untreated cells groups were investigated.
In this regard, serum samples taken from mouse models which were constructed with U87MG (Accession No. HTB-14) in the same manner as in Example 1.1.1 were incubated with the antibody array membrane (R&D, cat# ARY012) to examine responsiveness to the anti-c-Met antibody. In the membrane, 200 μl of the mouse serum was incubated for overnight at 4° C. with the buffer and the capture antibody, followed by developing the membrane in chemiluminescence immunoassay. The antibody L3-1Y/IgG2 was intravenously injected at a dose of 10 mg/ml every 7 days for 4 weeks from 7-10 days after implantation.
The results are depicted in
2.2. Evaluation of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using bIG-H3
2.2.1. Construction of Cancer Cell-Implanted Mouse Model
For use in assays for evaluating the efficacy of an anti-c-Met antibody, xenograft mouse models were prepared by implanting various cancer cell lines (anti-c-Met antibody non-responsive cell lines: Lovo (Accession No. CCL-229), HT-29 (Accession No. HTB-38), PC3 (Accession No. CRL-1435), BxPC3 (Accession No. CRL-1687), MDAMB231 (Accession No. HTB-26); anti-c-Met antibody responsive cell lines: Hs746T (Accession No. HTB-135), MKN45 (Accession No. RCB1001), EBC-1 (Accession No. JCRB0820), U87MG (Accession No. HTB-14), MHCC97H (Accession No. NB100-122)) to mice in the same manner as in Example 1.1.1.
Of the mouse models, Hs746T-, MKN45- and EBC-1-implanted mouse models were found to have responsiveness to the anti-c-Met antibody, as demonstrated in Example 1.1.2.
2.2.2. Evaluation of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using bIG-H3
To examine relationship between bIG-H3 level and responsiveness to anti-c-Met antibody, changes of serum bIG-H3 levels with treatment with an anti-c-Met antibody in cancer cell-implanted mouse models were monitored.
Briefly, serum samples taken from mouse models in a group were pooled, and quantitatively analyzed for bIG-H3 level using a bIG-H3 ELISA kit (R&D) in comparison with the control (injected with vehicle (PBS) only). More specifically, Whole blood was collected from the mouse models in a BD vacutainer SST™ tube. After collection of the whole blood, the tube was mixed by gently inverting for 5 times, and then, allowed to clot for 30 minutes at room temperature before centrifuging for 15 minutes at 1000×g to obtain serum. For individual ELISA assay, MIF (R&D, DMF00B) and IG-H3(R&D, DY2935) ELISA kit were used following manufacture's protocol. Briefly, 100 μL Assay Diluent RD1-53 was added to each well. The serum sample was added in coated plate at the amount of 50 μL per well, and then, incubated 2 hours at room temperature. After washing step, 200 ul of antibody conjugate was added to each well and incubated 2 hours and further incubated for 30 minutes after adding substrate solution. Wavelength at 450 nm was read using microplate reader.
Results are depicted in
2.3. Evaluation of Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues Using bIG-H3
2.3.1. Construction of Mouse Model Implanted with Patient-Derived Tumor Tissue
With reference to Example 1.2.1, patient-derived lung cancer cell-implanted mouse models LXFA297, LXFA526, LXFA623, LXFA983, LXFA1041, and LXFA1647 were constructed. Of them, LXFA526, LXFA623, and LXFA1647 were observed to be responsive to the anti-c-Met antibody as shown in Table 4.
2.3.2. Evaluation of Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues Using bIG-H3
To examine the relationship between bIG-H3 level and responsiveness to an anti-c-Met antibody, changes of serum bIG-H3 levels with treatment with an anti-c-Met antibody in patient-derived tumor tissues-implanted mouse models were monitored.
Briefly, serum samples taken from mouse models in a group were pooled, and quantitatively analyzed for bIG-H3 level using a bIG-H3 ELISA kit (R&D) in comparison with the control (injected with vehicle (PBS) only), in the same manner as in Example 2.2.2 (refer to Example 1.1.2).
Results are depicted in
Cancer tissues isolated from L3-1Y/IgG2-treated or untreated groups of the animal models were homogenized and lysed in 500 μg of a lysis buffer (Roche). After centrifugation, the cell soup (cell lysate) was harvested and quantified. The cell soup was loaded in an amount corresponding to 1 ug of the total cell protein to plates of the bIG-H3 ELISA kit (R&D). The plates were previously coated with a capture antibody for bIG-H3. The HRP-conjugated detection antibody was applied to the plates before reading O.D 450.
The results are depicted in
3.1. Selection of MIF as Pharmacodynamic Marker for Anti-c-Met Antibody
A protein which showed a significant difference in the expression level between an anti-c-Met antibody-treated group and an untreated group of the LXFA623 mouse model responsive to the anti-c-Met antibody (refer to Example 1.2.1 and Table 4) was selected as a marker for evaluating the efficacy of the anti-c-Met antibody, using an antibody array membrane capable of detecting 46 cytokines (R&D ARY005).
Briefly, 200 μl of each of serum samples taken from the antibody-treated group (L3-1Y/IgG2, 5 mg/kg) and the untreated group (PBS injected, control) of the LXFA623 mouse model were incubated overnight 4° C. with the buffer provided in the kit, followed by developing dots in chemiluminescence immunoassay. This experiment was performed with antibody array membrane (R&D ARY005).
The results are depicted in
3.2. Evaluation of the Efficacy of Anti-c-Met Antibody in Cancer Cell-Implanted Mouse Model Using MIF
In the same manner as in Example 2.2.1, xenograft mouse models were constructed by implanting various cancer cell lines (non-responder: Lovo (Accession No. CCL-229), HT-29 (Accession No. HTB-38), PC3 (Accession No. CRL-1435), BxPC3 (Accession No. CRL-1687), MDAMB231 (Accession No. HTB-26); responder: Hs746T (Accession No. HTB-135), MKN45 (Accession No. RCB1001), EBC-1 (Accession No. JCRB0820), U87MG (Accession No. HTB-14), MHCC97H (Accession No. NB100-122)).
To examine relationship between MIF level and responsiveness to anti-c-Met antibody, changes of MIF levels with treatment with an anti-c-Met antibody in cancer cell-implanted mouse models were monitored.
Briefly, serum samples taken from mouse models in each group, e.g., L3-1Y/IgG2-treated group or untreated group (PBS injected, control) of the xenograft animal models, were pooled, and quantitatively analyzed for MIF level using an MIF ELISA kit (R&D). The experiment was conducted according to the manufacturer's manual.
Results are depicted in
3.3. Evaluation of Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissues Using MIF
With reference to Example 1.2.1, patient-derived lung cancer cell-implanted mouse models LXFA297, LXFA526, LXFA623, LXFA983, LXFA1041, and LXFA1647 were constructed. Of them, LXFA526, LXFA623, and LXFA1647 were observed to be responsive to the anti-c-Met antibody as shown in Table 4.
To examine the relationship between MIF level and responsiveness to an anti-c-Met antibody, changes of serum MIF levels with treatment with an anti-c-Met antibody in patient-derived tumor tissues-implanted mouse models were monitored.
Briefly, MIF levels were using an MIF ELISA kit in the same manner as in Example 3.2
Results are depicted in
4.1. Assay for Predicting Efficacy of Anti-c-Met Antibody in Mouse Model Implanted with Patient-Derived Tumor Tissue Using RAS-RAF Mutation
4.1.1. Construction of Mouse Model Implanted with Patient-Derived Tumor Tissue
For use in predicting the efficacy of anti-c-Met antibodies, xenograft mouse models implanted with patient-derived tumor tissues were constructed.
Briefly female NMRI nude mice (Charles River or Harlan, 4-6 weeks old) were implanted s.c. with cancer tissues extracted from patients (LXFA: lung cancer, GXF: stomach cancer, RXF: kidney cancer, GXA: gastric cancer, LXFL: lung large cell carcinoma, OVXF: ovarian cancer, PAXF: pancreatic cancer, CXF: colon cancer). When the tumor grew to a volume of 80 to 200 mm3, the mice were randomly divided into groups of 10. L3-1Y/IgG2 was injected i.v. at a dose of 5 mg/kg while PBS was used for a control group. On Day 0, 7, 14, 21, 28, and 35 after randomization, the antibody was administered. When the tumor volume reached 2000 mm3, the mice were sacrificed and used in the following experiments.
4.1.2. Measurement of c-Met Expression Level in Patient-Derived Tumor Tissues-Implanted Mouse Model
c-Met expression levels in the patient-derived cancer cell-implanted mouse models were measured using a Western blotting method.
Briefly, tumor tissues isolated from the animal models were homogenized and lysed in 500 μl of a lysis buffer (Roche). After centrifugation, the cell soup (cell lysate) was harvested and quantified. The cell soup was loaded in an amount corresponding to 10 μg of the total cell protein (SDS-PAGE), followed by exposure to the membrane (Cell Signal; including the antibody)
The results are depicted in
4.1.3. Determination of RAS-RAF Mutation and Responsiveness to Anti-c-Met Antibody in Patient-Derived Tumor Tissues-Implanted Mouse Model
The patient-derived tumor tissues-implanted mouse models were examined for the presence of mutation of K-RAS and B-RAF, and for responsiveness to the anti-c-Met antibody.
DNA isolated from cancer cells was amplified by PCR using a Sequenom™ system including the primers to discriminate the wild-type from mutants. Mutants were detected mostly at G12V, G12C, Q61H, and G12C for K-RAS, and at G596R and D594A for B-RAF.
This experiment was carried out with reference to Example 1.2.1.
Results are summarized in
As can be seen in Table 6, when one or more mutations are present in K-RAS or B-RAF, the anti-c-Met antibody is not allowed to sufficiently its activity in spite of a high expression level of c-Met (e.g., LXFA 2201). This indicates that the presence or absence of mutations in K-RAS or B-RAF is a critical factor to predicting the efficacy of the anti-c-Met antibody.
4.2. Assay for Predicting Efficacy of Anti-c-Met Antibody in Cancer Cell Line Using RAS-RAF Mutation (In Vitro)
4.2.1. Preparation of Cancer Cell Line
NCI-H441 (HTB-114), NCI-H1993 (CRL-5909), NCI-H1373 (CRL-5866), A549 (CCL-185), HCC827 (CRL-2868), and EBC-1 (Accession No. JCRB0820) cells were seeded at a density of 1×104 cells/well to plates and incubated with the antibody L3-1Y/IgG2 (1 μg/ml) for 72 hrs. Cell growth was compared between antibody-treated and untreated groups.
4.2.2. Examination of Cancer Cell-Implanted Mouse Model for Presence of RAS-RAF Mutation and Responsiveness to Anti-c-Met Antibody
The cancer cell lines were examined for the presence of mutations in K-RAS and B-RAF and responsiveness to anti-c-Met antibody.
Briefly, DNA isolated from the cancer cells was amplified using a Sequenom™ system including the primers to discriminate the wild-type from mutants. Mutations were detected mostly at G12V, G12C, and G12S for K-RAS, and at G596R and D594A for B-RAF.
Results are summarized in Table 7.
As can be seen in Table 7, the anti-c-Met antibody can exert its anticancer activity in cancer models only when both K-RAS and B-RAF are wild-types.
5.1. Test of In Vivo Efficacy of Antibody
Fourteen (14) mouse xenograft models, which were subjected to subcutaneously grafting of patient derived tumor tissues (Non-small cell lung cancer: NSCLC), were examined by Oncotest GmbH (Germany).
To each of the 14 mouse xenograft models, L3-1Y/IgG2 and empty vehicle (control) were administered, respectively, and the size of the tumor tissues was measured to determine the responsiveness to the antibody.
When the tumor size in the mouse xenograft models reaches 100 mm3 or more, the mouse xenograft models were administered (iv) with L3-1Y/IgG2 (PBS buffer in case of control) at the amount of 5 mg/kg once a week for 6 weeks in total, and the tumor size reaches 2000 mm3 or more, the experiment was stopped.
The tumor size (mm3) was calculated by the following formula:
Tumor size (mm3)=(major axis*minor axis*minor axis)*½
The determination of responder (efficacy) group was conducted by ANOVA analysis where the hypothesis corresponding to p-value of 0.05 or less was excluded. That is, the hypothesis that the tumor size distribution is not changed in a drug (antibody)-administered group compared to non-administered group was subjected to ANOVA testing, and the group where p-value is more than 0.05 was determined as a non-responder (non-efficacy) group, and the group where p-value is 0.05 or less was determined as a responder (efficacy) group.
The obtained results for the responsiveness of the xenograft models to L3-1Y/IgG2 were summarized in Table 8:
5.2. Test of Prediction Accuracy to the Efficacy of the Antibody
To the 14 mouse xenograft models were subjected to following 7 tests (see Examples 5.2.1 to 5.2.7). In each test, the prediction accuracy, prediction sensitivity, and prediction specificity were calculated as follows:
Prediction Accuracy (%)=[(TP+TN)/14]*100
Prediction Sensitivity (%)=(TP/the number of antibody efficacy positive (responder) models)*100
Prediction Specificity (%)=(TN/the number of antibody efficacy negative (non-responder) models)*100
[TP (true positive): the number of models that are determined as being efficacy positive in both antibody efficacy test (Example 5.2) and each test (one of Examples 5.2.1 to 5.2.7); and
TN (true negative): the number of models that are determined as being efficacy negative in both antibody efficacy test (Example 5.2) and each test (one of Examples 5.2.1 to 5.2.7)]
The results of 7 tests are shown in Table 9 as below, wherein:
TP (true positive) refers to the case that is determined as being efficacy positive in both antibody efficacy test (Example 5.2) and each test (one of Examples 5.2.1 to 5.2.7);
TN (true negative) refers to the case that is determined as being efficacy negative in both antibody efficacy test (Example 5.2) and each test (one of Examples 5.2.1 to 5.2.7);
FP (false positive) refers to the case that is determined as being efficacy negative in antibody efficacy test (Example 5.2) but efficacy positive in each test (one of Examples 5.2.1 to 5.2.7); and
FN (false negative) refers to the case that is determined as being efficacy positive in antibody efficacy test (Example 5.2) but efficacy negative in each test (one of Examples 5.2.1 to 5.2.7).
The mouse tumor tissues that have been subject to the experiment of Example 5.1 were extracted, and a part of them was used in preparing a formalin fixed paraffin embedded (FFPE) block. The prepared FFPE block was used for measuring the level of surface expression c-MET by Ventana MET IHC (immunohistochemistry) (using sp44 (Ventana, Catalog Number 790-4430), which is an assay used for a clinical trial (phase III) of metmab (Genentech); see US2013089541 A1) according to a standard protocol of the manufacturer.
The case, where the IHC score according to the Ventana MET IHC assay is 2˜3, was determined as a responder (efficacy or positive) group.
A modified Ventana MET IHC assay was performed, wherein the assay was identical to the Ventana MET IHC described Example 5.2.1, except that the Ventana antibody sp44 was used by 5/6 dilution. The case, where the IHC score according the modified Ventana MET IHC assay is 2˜3, was determined as a responder (efficacy or positive) group.
The level of IL-8 in serum of each mouse xenograft model was quantified referring to Examples 1.1.2. The case, where the level of serum IL-8 is 500 pg/ml or more, was determined as a responder (efficacy or positive) group, and the case, where the level of serum IL-8 is less than 500 pg/ml, was determined as a non-responder (non-efficacy or negative) group.
The results from Example 5.2.2 (efficacy prediction by the modified Ventana MET IHC) and Example 5.2.3 (efficacy prediction using IL-8) were combined to predict the efficacy of anti-c-Met antibody L3-1Y/IgG2. In particular, the case, where the IHC score according to the modified Ventana MET IHC assay of Example 5.2.2 is 2˜3 and the serum IL-8 level according to Example 5.2.3 is 500 pg/ml or more, was determined as a responder (efficacy or positive) group, and the other case was determined as a non-responder (non-efficacy or negative) group.
The results from Examples 5.2.1, 5.2.2, 5.2.3, and 5.2.4 are summarized in Table 9, as below:
The 14 mouse xenograft models were subject to the experiment according to Example 4.1.3, to examine the presence or absence of K-RAS and/or B-RAF mutation. The case, where both of K-RAS and B-RAF are wild-type, was determined as a responder (efficacy or positive) group, and the case, where at least one of K-RAS and B-RAF is mutated, was determined as a non-responder (non-efficacy or negative) group.
The results from Example 5.2.3 (efficacy prediction using IL-8) and Example 5.2.5 (efficacy prediction according to K-RAS and B-RAF mutation) were combined to predict the efficacy of anti-c-Met antibody L3-1Y/IgG2. In particular, the case, where the serum IL-8 level according to Example 5.2.3 is 500 pg/ml or more and both of K-RAS and B-RAF are wild-type, was determined as a responder (efficacy or positive) group, and the other case was determined as a non-responder (non-efficacy or negative) group.
The results from Example 5.2.2 (efficacy prediction by the modified Ventana MET IHC), Example 5.2.3 (efficacy prediction using IL-8), and Example 5.2.5 (efficacy prediction according to K-RAS and B-RAF mutation) were combined to predict the efficacy of anti-c-Met antibody L3-1Y/IgG2. In particular, the case, where when the IHC score according to the modified Ventana MET IHC assay of Example 5.2.2 is 2˜3, the serum IL-8 level according to Example 5.2.3 is 500 pg/ml or more, and both of K-RAS and B-RAF are wild-type, the mouse xenograft model was determined as a responder (efficacy or positive) group, and the other case was determined as a non-responder (non-efficacy or negative) group.
The results from Examples 5.2.5, 5.2.6, and 5.2.7 are summarized in Table 10, as below:
As shown in Tables 9 and 10, when the modified c-MET IHC assay, IL-8 marker, and KRAS-BRAF mutation marker are respectively used, the prediction accuracy is 64.29%, 71.43%, and 71.43%, respectively, which are all excellent compared to that of the control (Example 5.2.1; 28.57%). A combination of at least two of the modified c-MET IHC assay, IL-8 marker, and KRAS-BRAF mutation marker shows better prediction accuracy. In particular, the combination of all of the modified c-MET IHC assay, IL-8 marker, and KRAS-BRAF mutation marker shows excellent prediction accuracy (100%).
While one or more embodiments of the present invention have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
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