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    • 专利摘要:
    Method for predicting the therapeutic response to serine-threonine kinase inhibitor drugs BRAF. The present invention relates to an in vitro method that makes it possible to predict the therapeutic response to treatment with BRAF inhibitor drugs, such as vemurafenib, in cancer patients associated with oncogenic mutations in said kinase, such as BRAF positive melanoma. This method is based on the measurement of cytoplasmic ERK1/2 levels, through the detection and quantification of ERK1/2 phosphorylated in serine 301/284 respectively, in a biological sample isolated from the patient. The invention also provides an antibody specific to serine phosphorylated ERK1/2 301/284 and a kit comprising it, which are useful in the described method. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2793801A1
    申请号:ES201930419
    申请日:2019-05-13
    公开日:2020-11-16
    发明作者:Baraja Pedro Crespo；Martínez Berta Casar
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universidad de Cantabria；
    IPC主号:
    专利说明:

    [0004] The present invention falls within the field of oncology, specifically within the methods of predicting the response to BRAF serine-threonine kinase inhibitor drugs in cancer patients carrying oncogenic mutations in BRAF (positive BRAF). These methods make it possible to identify early which patients will benefit from said treatment prior to its administration. In particular, the invention provides a biomarker, as well as a specific antibody against it, useful for predicting which patients will show sensitivity and, therefore, an adequate clinical response, to subsequent treatment with BRAF inhibitors.
    [0006] BACKGROUND OF THE INVENTION
    [0008] 60% of melanomas carry oncogenic mutations in the serine / threonine kinase BRAF. Currently, the only effective treatment for these cases is through the administration of specific kinase inhibitors (for example, vemurafenib and dabrafenib). Although the majority of cases respond to treatment, for reasons still unknown, approximately 30% of patients are refractory to such therapy, so these patients are subjected to useless treatment, not without adverse side effects. Furthermore, considering that such treatment costs around € 30,000 per patient, these cases represent an unnecessary and completely sterile waste of financial resources for health systems.
    [0010] The existence of patients refractory to treatment with BRAF inhibitor drugs makes it necessary to develop predictive methods that can be applied in clinical practice to identify these patients at an early stage. This approach would help to design effective treatment strategies tailored to each patient.
    [0012] The characterization of molecular markers that make it possible to discriminate the cases that are capable of responding to treatment with BRAF inhibitors and those that do not, among all patients with BRAF-positive melanoma, is of crucial importance. Be able to determine early if the patient will present a response favorable to treatment before administering it would be very useful in clinical practice to be able to select the appropriate treatment for each individual case. This in turn would allow to improve the prognosis of the subject.
    [0014] However, no biomarker or methodology is currently available to reliably and simply predict the clinical response to treatment with BRAF inhibitor drugs in these patients before proceeding with their administration. In other words, there are no methods that can be applied in clinical practice that allow the identification of non-responding patients, which makes it difficult to predict the efficacy of therapy.
    [0016] A few years ago it was reported that the response to vemurafenib is positively correlated with the cytoplasmic levels of MAP kinase ERK1 / 2 (Bollag G et al., 2010, Nature, 467: 596-599). In this regard, it should also be noted that cytoplasmic ERK1 / 2 levels are correlated with a good prognosis in invasive breast tumors and in small cell lung tumors (Blackhall FH. Et al., 2007, Clin Cancer Res, 9: 2241- 2247; Nakopoulou L. et al., 2007, APMIS, 113: 693-701). Unfortunately, to date this concept has not been able to be brought into practice, as there is no easy way to discriminate between cytoplasmic ERK1 / 2 and nuclear ERK1 / 2, except for cumbersome microscopic techniques (Bollag G et al., 2010, Nature, 467: 596-599), impractical for routine diagnosis.
    [0018] Therefore, it is necessary to have adequate tools that can be used in clinical practice that make it possible to selectively identify cytoplasmic ERK1 / 2 versus nuclear ERK1 / 2, since these tools would allow predicting the response of cancer patients, preferably melanoma , BRAF positive to the therapeutic administration of BRAF inhibitor drugs, such as vemurafenib.
    [0020] DESCRIPTION OF THE INVENTION
    [0022] The present invention solves the problem raised above by means of a method that makes it possible to discriminate cytoplasmic ERK1 / 2 from nuclear ERK1 / 2. This method uses, in particular, the detection and quantification, in isolated samples of BRAF positive cancer patients, of ERK1 / 2 phosphorylated in serine 301/284 respectively, which have been shown in the present invention to be present only in the cytoplasmic fraction of ERK. Quantification of the levels of phosphorylated ERK1 / 2 in serine 301/284 thus makes it possible to predict the clinical response to subsequent treatment with BRAF inhibitors.
    [0024] The inventors of the present invention have identified a new phosphorylation site in ERK2, specifically in serine 284 (serine 301 in ERK1) and, after generating an antibody that specifically recognizes ERK1 and ERK2 phosphorylated in serine 301/284 respectively, they have verified that said phosphorylation occurs, solely and exclusively, in the cytoplasmic fraction of ERK1 / 2. Thus, using protein electrophoresis techniques, it is demonstrated in the present invention that the levels of ERK1 / 2 phosphorylated in serine 301/284 are much higher in melanoma cell lines that respond adequately to treatment with the BRAF inhibitor drug vemurafenib, in comparison with the levels found in lines resistant to said drug. Therefore, the quantification of the levels of ERK1 / 2 phosphorylated in serine 301/284 in biological samples isolated from cancer patients carrying oncogenic mutations in BRAF (BRAF positive) allows predicting the therapeutic response to the administration of BRAF inhibitors.
    [0026] Likewise, the serine phosphorylated anti-ERK1 / 2 antibody 301/284 generated in the present invention can be used in the method of predicting the therapeutic response to BRAF inhibitors proposed here for the easy and rapid stratification of cases of cancer, preferably melanoma, BRAF positive based on its response potential to BRAF inhibitor therapy.
    [0028] Therefore, one aspect of the invention refers to the use of levels of phosphorylated ERK1 in serine 301 and levels of phosphorylated ERK2 in serine 284 as a biomarker to predict in vitro the therapeutic response to treatment with an inhibitor drug of serine-threonine kinase BRAF in a patient.
    [0030] "Predicting the therapeutic response" refers to determining, before administering the treatment, whether the BRAF inhibitor drug will induce a favorable, positive or adequate response in the subject or patient once treated with it, that is, after administering the A "positive or adequate response" to a BRAF inhibitor drug occurs when an improvement or reduction in symptoms of the tumor, preferably cancer, more preferably melanoma, in the patient. Said positive or adequate response to treatment with a BRAF inhibitor occurs, in particular, when the levels of total ERK1 / 2 activity, evaluated by the levels of phosphorylation of its TEY phosphorylation motif, are decreased in an isolated biological sample. of the patient after drug administration compared to said activity levels measured in a biological sample isolated from the same patient prior to drug administration. Said positive or adequate response to treatment with a BRAF inhibitor also preferably occurs when there is disappearance, or decrease, of macroscopic metastatic lesions.
    [0032] The term "in vitro" means that the methods and uses described in the invention are performed entirely outside the human or animal body.
    [0034] The term "subject", "individual" or "patient", as used in the present invention, preferably refers to a human or non-human mammal, such as primates, equines, rodents, ruminants, cats or dogs. Preferably, the patient to whom the invention relates is a human being.
    [0036] "ERK1 / 2" preferably refers to human ERK1 (MAPK1) and ERK2 (MAPK3) (MAPK1 (UniprotKB: P28482) and MAPK3 (UniprotKB: P27361) according to the HUGO nomenclature). They are MAP kinases involved in various cellular functions, such as regulation of meiosis, mitosis, and postmitosis in differentiated cells. A variety of stimuli, including growth factors, cytokines, viral infection, carcinogenic agents, etc., activate the RAS-ERK signaling pathway. ERK1 / 2 are rapidly phosphorylated upon activation of cell surface receptor tyrosine kinases, such as the epidermal growth factor receptor. This phosphorylation leads to the activation of its kinase activity.
    [0038] The "ERK1 / 2 phosphorylated at serine 301 or at serine 284, respectively" are the ERK1 and ERK2 enzymes that have incorporated a phosphate group at said specific positions in their amino acid sequence.
    [0040] The enzyme "serine-threonine kinase BRAF" is the enzyme, preferably human, (UniprotKB: P15056), more preferably encoded by the 7q34 gene. The expression "BRAF positive" refers, in the present invention, to the presence of a substitution (oncogenic) mutation at residue V600 of the BRAF amino acid sequence, preferably to the presence of the V600E substitution.
    [0042] In another preferred embodiment, the serine-threonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib, more preferably vemurafenib.
    [0044] The drug “vemurafenib” (PLX4032 or RG7204) is a low molecular weight chemical compound that is administered orally, an inhibitor of the activity of the enzyme serine-threonine kinase BRAF. The therapeutic indication for this BRAF inhibitor is in unresectable or metastatic melanoma with a positive BRAF V600 mutation. It is marketed under the name Zelboraf®.
    [0046] The drug “dabrafenib” is also a BRAF inhibitor and its therapeutic indication is in unresectable or metastatic melanoma with a positive BRAF V600 mutation, as treatment alone or in combination with trametinib, and in advanced non-small cell lung cancer with mutation of BRAF V600 positive, in combination with trametinib. It is marketed under the name Taflinar®.
    [0048] Oncogenic mutations in the gene encoding the BRAF enzyme, which lead to the substitution of the amino acid Valine (V) at position 600, preferably by the aspartic amino acid (E), lead to the constitutive activation of this protein, which promotes cell proliferation in the absence of growth factors that are normally required for such proliferation. Oncogenic mutations in BRAF have been very frequently identified in specific cancers, being present, for example, in 60% of melanomas. The most frequently observed oncogenic mutation in BRAF is V600E, which represents approximately 90% of the mutations in BRAF observed in melanoma.
    [0050] Therefore, in another preferred embodiment, the patient referred to in the present invention suffers from cancer, in particular BRAF positive cancer, that is to say, it is a cancer patient that has an oncogenic mutation in BRAF where said mutation is preferably in V600, more preferably the mutation is V600E.
    [0051] More preferably, the cancer is melanoma, even more preferably BRAF V600 mutation positive unresectable or metastatic melanoma, or non-small cell lung cancer, even more preferably BRAF V600 mutation positive advanced non-small cell lung cancer. In a particular embodiment, the cancer is melanoma.
    [0053] In the most preferred embodiment of the present invention, the BRAF inhibitor is vemurafenib and the patient is suffering from melanoma.
    [0055] As the levels of cytoplasmic ERK1 / 2 correlate, not only with the therapeutic response to treatment with a BRAF inhibitor drug, but also with a good prognosis in breast tumors, preferably invasive, and in lung tumors, preferably small cells (Blackhall FH. Et al., 2007, Clin Cancer Res, 9: 2241-2247; Nakopoulou L. et al., 2007, APMIS, 113: 693-701), another aspect of the invention refers to the use of ERK1 levels serine 301 phosphorylated and serine 284 phosphorylated ERK2 levels as a biomarker for the in vitro prognosis of breast, preferably invasive, and / or lung, preferably small cell tumors in a patient.
    [0057] The term "prognosis" refers to the method by which a prediction is made of the events that will occur in the development or course of a disease, preferably cancer, including relapse or metastatic dissemination capacity.
    [0059] Another aspect of the invention relates to an in vitro method , hereinafter "method of the invention", for predicting the therapeutic response to treatment with a serine-threonine kinase inhibitor drug BRAF in a patient, wherein said method comprises the following stages:
    [0060] to. Quantify levels of serine 301 phosphorylated ERK1 and serine 284 phosphorylated ERK2 in a biological sample isolated from the patient (collected prior to administering the BRAF inhibitor treatment), b. Compare the levels quantified in step (a) with a reference value, where said reference value comes from the quantification of levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from a patient that no responds to treatment with a serine-threonine kinase inhibitor BRAF, and
    [0061] c. Assign the patient of stage (a) to the group of individuals who will respond adequately to treatment when the quantification value obtained in stage (a) is significantly higher than the reference value.
    [0063] The term "quantify" refers to the measurement of the amount or concentration of ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284 in the sample.
    [0065] The term "amount" refers to, but is not limited to, the absolute or relative amount of serine 301 phosphorylated ERK1 and serine 284 phosphorylated ERK2 in the sample, as well as any other value or parameter related to or that can be derived from it. These other values or parameters comprise, for example, intensity values of a signal obtained from any physical or chemical property of ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284 obtained by indirect or direct measurements, for example, by mass spectroscopy or by nuclear magnetic resonance.
    [0067] Thus, the quantification of the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284, as described in the present invention, can be carried out as a direct or indirect measurement. Direct measurement refers to the measurement of the intensity of a signal directly obtained from the presence of ERK1 phosphorylated in serine 301 and ERK2 phosphorylated in serine 284. This signal is directly correlated with the number of product molecules present in the sample. This signal, also called "intensity signal", can be obtained, for example, by measuring an intensity value derived from a physical or chemical property of the product. Indirect measurement refers to the measurement obtained from a secondary component (i.e., a component that is different from ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284) or a measurement derived from, for example, responses cells, ligands, substrates, tags or enzymatic reaction products associated with ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284 or their activities.
    [0068] Methods for detecting and quantifying levels of phosphorylated ERK1 in serine 301 and levels of phosphorylated ERK2 in serine 284 are known to those of skill in the art, for example, by massive phosphoproteomics studies employing the technique of mass spectrometry.
    [0070] This quantification of levels of phosphorylated ERK1 in serine 301 and levels of phosphorylated ERK2 in serine 284 can be performed, but not limited to, by incubation or in situ hybridization with specific antibodies against (which recognize) ERK1 phosphorylated in serine 301 and / or ERK2 phosphorylated at serine 284, or an immunologically active fragment thereof, in assays such as Western blot, electrophoresis gels, membrane blotting and hybridization with specific probes, immunoprecipitation assays, protein arrays, preferably microarrays based on antibodies, flow cytometry, immunofluorescence, immunohistochemistry, chemiluminescence, immunoassays such as ELISA, radioimmunoassay (RIA), or any other enzymatic method, by incubation with a specific ligand or substrate, lateral flow devices or Luminex®, NMR or any other technique diagnostic imaging using paramagnetic nanoparticles or another type of nanoparticle as functionalized, by chromatographic techniques preferably combined with mass spectrometry, colorimetry or isoelectric focusing. Measurement of the levels of phosphorylated ERK1 in serine 301 and of phosphorylated ERK2 in serine 284 can be carried out by the specific recognition of any fragment of the enzymes thus phosphorylated by means of probes and / or antibodies.
    [0072] Preferably, the quantification of the levels of ERK1 phosphorylated in serine 301 and of ERK2 phosphorylated in serine 284 is carried out in the present invention by immunoassay based on specific antibodies against said forms of ERK, more preferably by Western blot. For the immunoassay, the antibodies employed may be labeled or unlabeled, for example, they may be labeled with a secondary antibody, an enzyme, an enzyme substrate, radioisotopes, magnetic labels, fluorescence, or the like, and / or they may be immobilized on a support such as a microtiter plate or bio-chip.
    [0074] An "immunoassay" or "immunohistochemical assay" is a biochemical assay that detects and / or quantifies the amount or concentration of one or more peptides of interest (in the context of the present invention, ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284), in a sample. To do this, the reaction uses one or more antibodies specific to the antigens to be detected. Quantification of the protein can be carried out by any method known in the art, for which the antigen and / or the antibody will preferably be labeled. The immunoassay to which the present invention relates can be competitive or non-competitive. In a competitive immunoassay the signal detected will be inversely proportional to the concentration of antigen in the sample. In a non-competitive immunoassay (such as a sandwich ELISA) the detected signal is directly proportional to the concentration of antigen in the sample.
    [0076] Examples of useful immunoassays to be applied in the method of the present invention are, but are not limited to, immunobloting (Western blot), immunoprecipitation, ELISA, multiplex assay, dipstick assay, on-line immunoassay (LIA), radioimmunoassay (RIA), immunoradiometric assay. (IRMA), immunofluorescence, immunohistochemistry, chemiluminescence, passive hemagglutination, immunolabeling with gold particles (transmission electron microscopy), lipopolysaccharide (LPS) chips or protein or x-map, immunoquantitative real-time PCR (iqPCR), electrochemiluminescent labels, Label-free immunoassays (e.g. surface plasmon resonance), photoacoustic immunoassay, cloned donor enzyme immunoassay (CEDIA), lateral flow immunochromatographic assays, magnetic immunoassay (MIA), envelope-based fiber optic immunoassay (SOFIA), immunoassay based on CD / DVD or agglutination-PCR (ADAP).
    [0078] The ELISA assay is based on the immobilization of an antigen or antibody on a solid support, so that this system is subsequently put in contact with a fluid phase that contains the complementary reagent, which can be linked to a label or marker compound. The ELISA technique referred to in the present invention can be direct, indirect or sandwich.
    [0080] The antibody used in the methods described herein is preferably conjugated to a detection system or is linked to a secondary antibody which is coupled to a detection system. The signal produced as a consequence of the labeling reaction can be measured, for example, but not limited to, by spectrophotometry, chemiluminescence, spectrofluorometry, bioluminescence, deference calorimetry !, analytical ultracentrifugation, interferometry, etc. Preferably, the technique employed in the present invention is chemiluminescence.
    [0082] The term "collected prior to administering the BRAF inhibitor treatment" refers to a biological sample from the patient when the patient has not yet received prior treatment with BRAF inhibitors, preferably vemurafenib.
    [0084] The term "isolated biological sample" refers to, but is not limited to, any biological tissue and / or fluid obtained or extracted from a subject or patient that comprises tumor cells, that is, cells derived from a tumor. The biological sample can be, for example, a tissue from a tumor biopsy or a sample from a fine needle aspirate, or it can be a biological fluid, for example, blood, plasma, serum, lymph, ascites fluid, urine, saliva or glandular exudate. The biological sample can be fresh, frozen, fixed or fixed and embedded in paraffin.
    [0086] The term "reference value", as used in the method of the invention described above, is any value or range of values derived from the quantification of the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in an isolated biological sample from a patient known not to respond adequately to treatment with a BRAF inhibitor, preferably vemurafenib, or in a mixture of isolated biological samples from such patients.
    [0088] The "comparison" referred to in step (b) of the method of the invention can be carried out manually or in an automated manner.
    [0090] An amount "significantly greater" than a reference value can be determined by various statistical tools, such as, for example, but not limited to, determination of confidence intervals, determination of the p-value, Student's t test, Fisher's discriminant functions, Kruskal-Wallis, ANOVA, Bonferroni, Mann-Whitney, etc.
    [0092] Steps (a) and (b) of the method of the invention can be totally or partially computerized. Furthermore, the method of the invention may comprise other additional, optional steps, for example related to the pre-treatment of biological samples before their analysis.
    [0094] In another preferred embodiment of the method of the invention, the serineathreonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib, more preferably vemurafenib.
    [0096] In another preferred embodiment, the patient to whom the method of the invention is to be applied suffers from cancer, in particular BRAF positive cancer, that is, it is a cancer patient that has an oncogenic mutation in BRAF where said mutation is preferably in the V600, most preferably the mutation is V600E.
    [0098] More preferably, the cancer is melanoma, even more preferably BRAF V600 mutation positive unresectable or metastatic melanoma, or non-small cell lung cancer, even more preferably BRAF V600 mutation positive advanced non-small cell lung cancer. In a particular embodiment, the cancer is melanoma.
    [0100] In a particular embodiment of the method of the invention, the serineathreonine kinase inhibitor BRAF is vemurafenib and the patient suffers from melanoma.
    [0102] In another preferred embodiment, the patient to whom the method of the invention is to be applied is human.
    [0104] In another preferred embodiment, the quantification of the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 is carried out, in the methods described herein, by using an antibody that recognizes ERK1 phosphorylated in serine 301 and ERK2 phosphorylated in serine 284 specific against the peptide consisting of SEQ ID NO: 1 (NRLFPNADSKALDLLDKML), that is, by means of the antibody of the invention described below.
    [0106] Another aspect of the invention relates to an in vitro method for the prognosis of breast, preferably invasive, and / or lung tumors, preferably small cells, in a patient, where said method comprises the following steps: to. Quantify the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from the patient, b. Compare the levels quantified in step (a) with a reference value, where said reference value comes from the quantification of levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from a patient diagnosed with the same type of cancer as the patient in step (a) and who has a poor prognosis, and
    [0107] c. Assign the patient from stage (a) to the group of individuals who will have a good prognosis when the quantification value obtained in stage (a) is significantly higher than the reference value from stage (b).
    [0109] By "poor prognosis" is understood the relapse, metastasis and / or absence of improvement in the symptoms of the tumor, preferably cancer.
    [0111] The antibody described in the present invention, which recognizes ERK1 phosphorylated at serine 301 and ERK2 phosphorylated at serine 284 and which is specific against the peptide of SEQ ID NO: 1, has been designed by the inventors of the present invention to develop the methods described here. Therefore, another aspect of the invention refers to an antibody, which recognizes ERK1 phosphorylated in serine 301 and ERK2 phosphorylated in serine 284, specific against the peptide consisting of SEQ ID NO: 1 (NRLFPNADSKALDLLDKML). Hereinafter this antibody will be referred to as "antibody of the invention".
    [0113] This antibody of the invention is polyclonal, that is, it has been generated by immunizing an animal, preferably a non-human mammal, more preferably a rabbit, with the peptide (antigen) designed by the inventors consisting of SEQ ID NO: 1. Methods for the production and purification of polyclonal antibodies by immunizing an animal with the corresponding antigen are widely known to those skilled in the art, and any of them could be used to obtain the antibody of the invention.
    [0115] In the peptide consisting of SEQ ID NO: 1, the serine at position 9 is phosphorylated, preferably said serine has been chemically phosphorylated. Said peptide is 100% homologous or identical between the ERK1 and ERK2 paralogs, so the The antibody of the invention raised against it recognizes (binds, hybridizes with) both serine phosphorylated proteins 301 and 284 respectively.
    [0117] Another aspect of the invention refers to a peptide consisting of SEQ ID NO: 1 (NRLFPNADSKALDLLDKML), where the serine at position 9 is phosphorylated. Another aspect of the invention refers to the use of said peptide for the generation of the antibody of the invention.
    [0119] Preferably, the antibody of the invention is labeled. The term "labeled", as used in the present description, refers to the fact that the antibody is conjugated to a tag or detection system. A large number of tags are known in the state of the art that can be conjugated to an antibody. Examples of such labels are, but are not limited to, radioisotopes [eg 32P, 35S or 3H], fluorescent or luminescent markers [eg fluorescein (FITC), rhodamine, texas red, GFP, phycoerythrin (PE), allophycocyanin, 6-carboxyfluorescein (6-FAM), 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2 ', 4', 7 ', 4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N, N, N', N'-tetramethyl-6-carboxyrodamine (TAMRA)]; secondary antibodies or fragments of antibodies [eg, F (ab) 2) fragments], affinity tags [eg, biotin, avidin, agarose, bone morphogenetic protein (BMP), haptens], enzymes or enzyme substrates [for e for example, alkaline phosphatase (AP) and horseradish peroxidase (HRP)].
    [0121] The advantage of labeling antibodies is that they can be detected and their signal quantified.
    [0123] Preferably, the antibody of the invention is immobilized on a support. The term "immobilized", as used in the present description, refers to the fact that the antibody is bound to a support without losing its immunoreaction activity with its antigen (peptide of SEQ ID NO: 1). Preferably, the support is the surface of a matrix (for example, a nylon or latex matrix), a membrane, a microtiter plate (for example, 96-well) or support made of plastic, silicone, glass or the like, or beads (for example spheres, preferably agarose spheres or small superparamagnetic microspheres composed of biodegradable matrices), gel, cellulosic support, adsorption resin or the like. In general, any solid surface that allows direct or indirect binding, by covalent or non-covalent bonding, of the antibody of the invention can be employed. Furthermore, the support can be in the form of a rod, test strip, paper, latex bead, microsphere, array, multi-well plate or the like.
    [0125] More preferably, the antibody of the invention is labeled and immobilized. Even more preferably, said labeling is through a secondary antibody conjugated with horseradish peroxidase.
    [0127] The antibody described in the present invention can be human, humanized, recombinant, monoclonal, chimeric, conjugated, etc. Immunologically active fragments of the antibody of the invention are also included within the scope of the present invention. Examples of such fragments are, but are not limited to, a Fab, F (ab ') 2, Fab', F sc or Fv fragment.
    [0129] Another aspect of the invention refers to a kit, useful to be used in the methods described herein, hereinafter "kit of the invention", which comprises the antibody of the invention, where said antibody may or may not be labeled and / or or immobilized as described above.
    [0131] This kit preferably comprises all those elements necessary to predict the therapeutic response to treatment with a BRAF inhibitor drug in a patient or to predict (the evolution of) breast tumors, preferably invasive, and / or lung, preferably small cells, in a patient, according to the methods described above. Thus, the kit of the invention can comprise elements such as, for example but not limited to, preservation solutions, buffers, diluents, filters, vehicles, enzymes, such as polymerases, cofactors necessary to obtain an optimal activity of said enzymes, etc. The kit can comprise all those supports and containers necessary for the implementation of the methods described herein. The kit can further comprise other molecules, genes, proteins or probes, suitable as positive or negative controls. Preferably, the kit of the invention further comprises instructions on how to carry out the methods described herein, labels to identify the different elements included in the kit and their application and / or lists of the components included in the kit.
    [0132] The kit of the invention can comprise, in different combinations, primers or oligonucleotides and control probes, secondary antibodies that serve for labeling through their conjugation with a detection system and / or control antibodies that serve for the normalization of the levels of expression, reagents, detection signals, instruments necessary for the processing and start-up of arrays, fluorochromes, labeling probes, substrates necessary for the activation or initiation of the labeling reaction by the selected conjugation system, blocking solutions, de stop and / or wash, or the like. The control probes, primers and / or antibodies can be specific for genes, proteins or peptides constitutively expressed by the cells in the biological sample analyzed, so that their application makes it possible to ensure that the values of the expression levels of ERK1 phosphorylated in serine 301 and ERK2 phosphorylated at serine 284 measured in the sample are reliable and correct.
    [0134] The kit may also comprise suitable means to house and preserve the biological sample to be analyzed in the methods described herein. Such means may be, for example, a container suitable for containing a sample from a tumor biopsy or aspirate. Thus, said kit may comprise a container such as bottles, vials, syringes or test tubes. Said container can be made of, for example, but not limited to, glass or plastic or any other suitable material for the preservation of the biological sample under optimal conditions.
    [0136] Another aspect of the invention relates to the use of the antibody of the invention or the kit of the invention to predict in vitro the therapeutic response to treatment with a serine-threonine kinase inhibitor drug BRAF in a patient, or to predict in vitro ( the evolution of) breast tumors, preferably invasive, and / or lung, preferably small cell, in a patient, more preferably by the methods described above.
    [0138] In a preferred embodiment of such use, the serine threonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib, preferably vemurafenib.
    [0140] In another preferred embodiment, the patient suffers from cancer, in particular BRAF positive cancer, that is, it is a cancer patient presenting an oncogenic mutation in BRAF, where said mutation is preferably V600, more preferably V600 mutation is V600E.
    [0142] More preferably, the cancer is melanoma, even more preferably BRAF V600 mutation positive unresectable or metastatic melanoma, or non-small cell lung cancer, even more preferably BRAF V600 mutation positive advanced non-small cell lung cancer. In a particular embodiment, the cancer is melanoma.
    [0144] In the most preferred embodiment, the serine-threonine kinase inhibitor BRAF is vemurafenib and the patient is suffering from melanoma.
    [0146] In another preferred embodiment, the patient is human.
    [0148] Another aspect of the invention relates to an in vitro method for obtaining information or data useful for predicting the therapeutic response to treatment with a serine-threonine kinase inhibitor drug BRAF in a patient, where said method comprises the following steps:
    [0149] to. Quantify levels of serine 301 phosphorylated ERK1 and serine 284 phosphorylated ERK2 in a biological sample isolated from the patient (collected prior to administering BRAF inhibitor treatment), and
    [0150] b. Compare the levels quantified in step (a) with a reference value, where said reference value comes from the quantification of levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from a patient unresponsive to treatment with a serine-threonine kinase inhibitor BRAF.
    [0152] These steps (a) and (b) can be carried out as explained above for the method of the invention.
    [0154] Another aspect of the present invention relates to a method for treating a patient suffering from BRAF positive cancer, preferably BRAF positive melanoma, comprising: (a) identifying the patient as a responder or non-responder to treatment with BRAF inhibitors, preferably by treatment with vemurafenib, according to the method of the invention; and (b) administering said treatment to the patient when in step (a) it has been determined that he is a responding patient thereto.
    [0155] Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to limit the scope of protection of the present invention.
    [0157] DESCRIPTION OF THE FIGURES
    [0159] Fig. 1. Specificity of the antibody of the invention (anti-phospho ser284 / 301 antibody). HEK294 cells were transfected with human (Hs) or zebrafish (Dr) ERK2, where the homologous residue of serine 284 is a proline, therefore, not phosphorylatable. It is observed that the antibody only recognizes human ERK2, in cells under stimulation, in this case with EGF. An anti-FLAG antibody was used as a control.
    [0161] Fig. 2. ERK1 / 2 phosphorylated in ser 301/284 are located exclusively in the cytoplasm. HeLa cells were stimulated with EGF for 5 min, and the subcellular localization of phosphorylated endogenous ERK1 / 2 in the aforementioned residues was analyzed by immunofluorescence and confocal microscopy. It is observed that ERK1 / 2 phosphorylated in these positions are completely excluded from the nucleus, marked with DAPI.
    [0163] Fig. 3. Correlation between levels of ERK1 / 2 phosphorylated in ser 301/284 and sensitivity to vemurafenib. From the BRAF positive melanoma cell line M249, a subline resistant to vemurafenib (vemR) was obtained and the levels of phospho-ser 284/301 were analyzed in this subline in comparison with the parental line. It is observed that these levels are much higher in the parental line, sensitive to vemurafenib.
    [0165] Fig. 4. Correlation between levels of phosphorylated ERK1 / 2 in ser 301/284 and sensitivity to vemurafenib. The sensitivity to vemurafenib was evaluated in different melanoma lines, analyzing the reduction in the total activity of ERK1 / 2, measured by the decrease in the levels of canonical phosphorylation (p-ERK). It is observed that the most sensitive lines are 8505C and A375P. Phospho-ser 284/301 levels are also analyzed in these lines. It is observed that these levels, before treatment with vemurafenib, are much higher than those observed in the resistant lines MELJUSO and SKMEL2.
    [0167] Fig. 5. Vemurafenib sensitivity of the melanoma lines used in Fig.
    [0168] 4, evaluated by the concentration necessary to stop proliferation (GI50). The most sensitive lines are seen to be 8505C and A375P, which show the highest levels of phospho-ser 284/301 in FIG. Four.
    [0170] EXAMPLES
    [0172] Next, the invention will be illustrated by means of tests carried out by the inventors, which show the effectiveness of the method of the invention and the antibody generated to be used in said method.
    [0174] Example 1. Material and methods.
    [0176] The cell lines used in the tests were:
    [0178] HEK293T: Human kidney embryonic cells, T antigen positive.
    [0179] HeLa: epithelial cells of cervical cancer.
    [0180] A375p: melanoma epithelial cells, show a BRAF mutation.
    [0181] SKMEL2: melanoma epithelial cells, show BRAF mutation.
    [0182] Parental M249 and Vemurafenib-resistant M249: melanoma epithelial cells, mutation in BRAF.
    [0183] Mel JUSO: melanoma epithelial cells.
    [0185] The cell lines were grown in DMEM culture medium supplemented with 10% Fetal Bovine Serum and antibiotics (Penicillin and Streptomycin).
    [0187] Western blot:
    [0189] To obtain the total protein extracts, the cells were lysed with the adequate volume of lysis buffer (20 mM HEPES pH 7.5, 10 Mm EGTA, 40 mM P-glycerophosphate, 1% non-ionic detergent NP40, 2.5 mM MgCl2 , 1mM orthovanadate, 1mM DTT and extemporaneously 10qg / ml aprotinin and 10qg / ml leupeptin). The cells they were collected and centrifuged at 12,000 rpm, for 10 minutes and at 4 ° C. The protein extracts were separated from the rest of the components of the cells and the protein concentration of each lysate was quantified. For the determination of the amount of protein, the Bradford method was used. Approximately 50 pg of protein were taken, to which 5X Laemli buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 20 mM DTT and 0.005% bromophenol blue) was added. After boiling the samples for five minutes, they were electrophoresed on a vertical polyacrylamide gel -SDS (sodium dodecyl sulfate) -PAGE of 12% acrylamide. The proteins were transferred to a nitrocellulose membrane setting the amperage at 400 mA for 1 hour. Once the transfer was finished, the membranes were incubated for one hour, at room temperature and with shaking, in a TBS-T solution with 4% BSA, to block non-specific sites. After that, the filters were incubated with the phospho-specific primary antibody of the invention (0.2-0.4 pg / ml diluted in 4% BSA in TBS-T) for one hour at room temperature or overnight at 4 ° C under stirring. Two washes with TBS-T were carried out for a total of fifteen minutes, after which the filters were incubated with the corresponding secondary antibody conjugated to peroxidase, diluted 1: 5,000 or 1: 10,000 in 0.4% BSA in TBS-T for one hour at room temperature. Two TBS-T washes were carried out again and the protein was detected by chemiluminescence using the ECL kit. An autoradiography of the filters with Kónica films was carried out, which allowed the detection of a band where the primary antibody had specifically recognized the phosphorylated protein of interest. As internal loading control, a specific antibody was used that recognizes the total protein of interest.
    [0191] Immunofluorescence
    [0193] Cells were grown in DMEM 10% Fetal Bovine Serum until subconfluence on sterile 10 mm diameter glass coverslips. At the time of immunofluorescence, cells were washed with 1X PBS and fixed with a 4% solution of paraformaldehyde in 1X PBS, for 10 minutes at room temperature. Cells were permeabilized by incubating for 5 minutes with a 0.5% dilution of Triton X-100 in PBS, followed by three 1X PBS washes of five minutes each. The cells were then incubated with the phosphospecific primary antibody of the invention at a concentration 1/100 for one hour in a humid chamber. After Three washes with PBS, five minutes each, the secondary antibody conjugated to the FITC fluorophore was added and incubated for 45-50 minutes in a humid chamber and in the dark. After that time, two new washes were carried out, each lasting five minutes, with 1X PBS. Finally, a drop of Prolong mounting medium with DAPI was added to a slide and the cover was then placed on top with the cells facing down. Cells were examined by confocal microscopy (Leica TCS SP8). The images were digitized and processed using the Fiji Image J program.
    [0195] Immunohistochemistry
    [0197] Tissue samples or cell pellets were fixed with 4% parapholmaldehyde and embedded in paraffin. The sections were mounted on positively charged slides (Genex-brand®), recommended for immunohistochemistry. Dewaxing was achieved by passing the sections through xylene (10 min), and decreasing graduations of ethyl alcohol (100 ° 10 min, 96 ° 5 min, and 70 ° 5 min). A blocking of the endogenous peroxidase activity was carried out by incubating the sections in 3% hydrogen peroxide solution in methanol for 15 min, and incubation in distilled water for 10 min. Subsequently, the sections were incubated with a 1% BSA bovine serum albumin solution in TBST buffer for 30 min with the intention of blocking non-specific binding sites. Subsequently, the sections were incubated with the phosphospecific antibody of the invention at a 1: 100 dilution in PBS, overnight at 4 ° C, in a humid chamber. The development of the reaction was carried out by the DAKO chromogen DAB technique. Contrast staining was performed by immersing the sections in Mayer's hematoxylin for 1 min; they were then placed under a stream of running water for development. Mounting was done with aqueous mounting medium (VectaMount AQ, Vector Lab Ind). Observation of the preparations was made on a Nikon microscope. The photographs were taken with an Olympus C4000 digital camera.
    [0199] Example 2. Results.
    [0201] To obtain an antibody that specifically recognized the phosphorylated ser 301/284 of ERK1 / 2, respectively, rabbits were immunized with the peptide consisting of SEQ ID NO: 1 mentioned above, following the protocols to use routinely used for this purpose. The immunoreactivity of the resulting serum was analyzed by western blot (Fig. 1) in HEK293 cells transfected with plasmids encoding human (Hs) or zebrafish (Dr) ERK2. In the latter, the residue corresponding to serine 284 is a proline (proline 293), so it is not phosphorylatable, and serves as a negative control. It was observed that after stimulation with EGF for 5 min, which induces the phosphorylation of canonical TEY residues in both human ERK2 and zebrafish, the phosphorylation of ser 284 is detected only in human ERK2.
    [0203] Once the specificity of the antibody was demonstrated, the cell sublocalization of ERK1 / 2 phosphorylated in ser 301/284 was analyzed. For this, immunofluorescence was performed in HeLa cells, fasting (starved) or stimulated with EGF for 5 min (Fig. 2). It was observed, by confocal microscopy, that endogenous ERK1 / 2 phosphorylated in being 301/284 are located exclusively in the cytoplasm, being completely excluded from the cell nucleus, stained by DAPI.
    [0205] Subsequently, the correlation of levels of phosphorylated ERK1 / 2 in being 301/284 with sensitivity towards BRAF inhibitors in melanoma cells carrying BRAF mutations was analyzed. For this purpose, the M249 cell line was used, from which a subline resistant to vemurafenib (vemR) was obtained. The levels of endogenous ERK1 / 2 phosphorylated in ser 301/284 were analyzed by western blot (FIG. 3) comparing the resistant subline with the parental line, sensitive to vemurafenib. It was observed that the levels of phospho-ser 301/284 are much higher in the parental line, sensitive to vemurafenib, demonstrating a positive correlation between the levels of phospho-ser 301/284 and the response to the antitumor compound.
    [0207] To verify the previous point, the correlation between the phosphorylation levels of being 284/301 in ERK1 / 2 and the sensitivity to vemurafenib in different melanoma lines was evaluated. It was observed that the reduction in the total activity of ERK1 / 2, measured by the decrease in the levels of canonical phosphorylation (p-ERK) by western blot, after the treatment of the different cell lines with vemurafenib, was correlated with the higher levels of phospho-ser 284/301. It is observed that said levels before treatment with vemurafenib are much higher in the most sensitive lines, 8505C and A375P, than those observed in the most resistant lines, MELJUSO and SKMEL2 (FIG. 4). This correlation is also observed when evaluating the concentration of vemurafenib necessary to stop the proliferation (GI50) of the different melanoma cell lines (FIG. 5). It is observed that the lines most sensitive to treatment with vemurafenib are 8505C and A375P, which show the highest levels of phospho-ser 284/301 in FIG 4.
    权利要求:
    Claims (21)
    [1]
    1. Use of levels of phosphorylated ERK1 in serine 301 and levels of phosphorylated ERK2 in serine 284 as a biomarker to predict in vitro response to treatment with a serine-threonine kinase inhibitor BRAF in a patient.
    [2]
    2. Use according to claim 1, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib.
    [3]
    3. Use according to any of claims 1 or 2, wherein the patient suffers from cancer.
    [4]
    4. Use according to claim 3, wherein the cancer is melanoma or non-small cell lung cancer.
    [5]
    Use according to any one of claims 1 to 4, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and the patient suffers from melanoma.
    [6]
    6. Use according to any one of claims 1 to 5, wherein the patient is human.
    [7]
    7. In vitro method to predict the response to treatment with a serine-threonine kinase inhibitor BRAF in a patient, where said method comprises the following steps:
    to. Quantify the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from the patient, b. Compare the levels quantified in step (a) with a reference value, where said reference value comes from the quantification of levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 in a biological sample isolated from a unresponsive patient to treatment with a serine-threonine kinase inhibitor BRAF, and
    c. Assign the patient of stage (a) to the group of individuals who will respond adequately to treatment when the quantification value obtained in stage (a) is significantly higher than the reference value.
    [8]
    8. Method according to claim 7, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib.
    [9]
    9. Method according to any of claims 7 or 8, wherein the patient suffers from cancer.
    [10]
    10. Method according to claim 9, wherein the cancer is melanoma or non-small cell lung cancer.
    [11]
    Method according to any one of claims 7 to 10, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and the patient suffers from melanoma.
    [12]
    12. Method according to any of claims 7 to 11, wherein the patient is human.
    [13]
    Method according to any of claims 7 to 12, wherein the quantification of the levels of phosphorylated ERK1 in serine 301 and of ERK2 phosphorylated in serine 284 is carried out by using a specific antibody against the peptide consisting of SEQ ID NO: 1.
    [14]
    14. A specific antibody against the peptide consisting of SEQ ID NO: 1.
    [15]
    15. Kit comprising the antibody according to claim 14.
    [16]
    16. Use of the antibody according to claim 14 or the kit according to claim 15 to predict in vitro the response to treatment with a serine-threonine kinase inhibitor BRAF in a patient.
    [17]
    17. Use according to claim 16, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and / or dabrafenib.
    [18]
    18. Use according to any of claims 16 or 17, wherein the patient suffers from cancer.
    [19]
    19. Use according to claim 18, wherein the cancer is melanoma or non-small cell lung cancer.
    [20]
    20. Use according to any one of claims 16 to 19, wherein the serine-threonine kinase inhibitor BRAF is vemurafenib and the patient suffers from melanoma.
    [21]
    21. Use according to any one of claims 16 to 20, wherein the patient is human.
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    WO2020229717A1|2020-11-19|
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