Methods for predicting response to inhibitors of EGFR kinase activity

Abstract

The present invention relates to a method for selecting patients who may respond to treatment with a tyrosine kinase inhibitor (TKI).The present invention further relates to a diagnostic kit for predicting the response of EGFR kinase activity to an inhibitor.Furthermore, the present invention relates to the use of phosphorylated T59 on SHP2 as a biomarker for predicting EGFR kinase activity.

Description

[Technical Field]

[0001] The present invention relates to a method for selecting patients who may respond to treatment with a tyrosine kinase inhibitor (TKI). The present invention further relates to a diagnostic kit for predicting the response of EGFR kinase activity to an inhibitor. Furthermore, the present invention relates to the use of phosphorylated T59 on SHP2 as a biomarker for predicting EGFR kinase activity. BACKGROUND OF THE INVENTION

[0002] Phosphorylation of specific tyrosine residues in proteins is often a hallmark of intracellular signal transduction. Enzymes capable of catalyzing such reactions are called tyrosine kinases. Tyrosine kinase inhibitors (TKIs) are drugs that inhibit protein activation via tyrosine phosphorylation. Growth factor receptor tyrosine kinases play a role in the pathogenesis and progression of various disorders and diseases, including human malignancies. These receptors are anchored by transmembrane domains in the membrane of cells that express them. The extracellular domains bind to growth factors. When growth factors bind to the extracellular domains, a signal is transmitted from the extracellular to the intracellular kinase domains. This signal transduction contributes to various pleiotropic responses, including induction of DNA synthesis, changes in gene expression, and cell growth, proliferation, and differentiation.

[0003] The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and a member of the HER / ErbB family, which consists of four members: EGFR (HER1 / ErbB1), HER2 / neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). This receptor consists of three parts: a glycosylated extracellular ligand-binding domain, a single-chain transmembrane domain, and an intracellular protein-tyrosine kinase domain. Ligand binding to the extracellular domain leads to receptor dimerization and activation of the protein-tyrosine kinase. Several growth factors, such as EGF and transforming growth factor α (TGF-α), bind to the epidermal growth factor receptor. EGFR is widely distributed on the cell surface of mammalian epithelial cells, fibroblasts, glial cells, and keratinocytes. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation, and differentiation. Malfunction of protein-tyrosine kinases such as EGFR, or abnormalities in the activity or cellular localization of key components of associated signaling pathways, can both lead to the development of tumors, diabetes, immune deficiencies, and cardiovascular disease.

[0004] Some malignant tumors are associated with mutations or increased expression of members of the ErbB family. Overexpression of EGFR and ErbB2 is closely associated with cell proliferation in certain types of tumors of epithelial origin, such as glioblastoma (including glioblastoma multiforme), lung cancer (adenocarcinoma including bronchoalveolar cell carcinoma (BAC) and non-small cell lung cancer (NSCLC)), as well as breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, bladder cancer, colon cancer, colorectal cancer, kidney cancer, and head and neck cancer. Amplification and / or overexpression of EGF receptor on the membrane of tumor cells is associated with poor prognosis in these patients.

[0005] These observations have spurred research aimed at inhibiting the function of human EGFR or HER2 as a therapeutic approach to treat cancer. For example, anti-EGFR and anti-HER2 antibodies have shown promising results in human cancer treatment. EGFR-related drugs currently available on the market include gefitinib (Iressa), erlotinib (Tarceva), and afatinib (Gilotrif) (see M.L. Uribe et al., Cancer 2021, 13, 2748). These are orally administered quinazoline derivatives that act as selective EGFR tyrosine kinase inhibitors (EGFR-TKIs) and are used to treat EGFR mutation-responsive (ERBB1 mutation-responsive) lung cancer. Lapatinib (Tykerb) is an orally active quinazoline derivative that acts as a dual EGFR / HER2 inhibitor and is used to treat ErbB2-overexpressing breast cancer. The aforementioned TKIs competitively bind to the phosphorylation site of tyrosine kinases in the intracellular segment, blocking the interaction between the phosphorylation site and ATP, inhibiting tyrosine phosphorylation and a series of downstream signaling events, thereby inhibiting tumor cell growth. The reversible EFGR inhibitors gefitinib and erlotinib have shown favorable therapeutic effects in patients with non-small cell lung cancer harboring EGFR mutations, significantly extending their progression-free survival (PFS) and overall survival (OS). Trastuzumab, pertuzumab, amivantam (Librevant), and trastuzumab-emtansine are intravenously administered monoclonal antibodies that target the extracellular domain and are used to treat ErbB2-positive breast cancer. Trastuzumab-emtansine is an antibody-drug conjugate that delivers cytotoxic drugs to cells overexpressing ErbB2. Cetuximab and panitumumab are monoclonal antibodies that target ErbB1 and are used to treat colorectal cancer.

[0006] However, not all subjects respond to EGFR therapy, potentially exposing patients to the harmful side effects associated with EGFR-targeted therapy without benefiting from it.

[0007] WO2007 / 106432 describes a method for predicting the response of a subject with an EGFR-mediated disease or condition (e.g., a cancer of epithelial origin, such as NSCLC) to an EGFR kinase inhibitor. According to this method, the amount of phosphorylation of residues Y1068 and T1148 in EGFR is measured. High phosphorylation levels of these two residues are used as an indicator that a subject is likely to respond to a drug that inhibits EGFR kinase activity. However, this method has not been established in practice. Currently, there is no companion diagnostic test that uses this phosphorylation site on EGFR to select responders for anti-tumor treatment.

[0008] Another group of enzymes that plays an important role in signal transduction cascades alongside tyrosine kinases are protein tyrosine phosphatases (PTPs), which can remove phosphate groups from phosphorylated tyrosine residues on proteins. One member of the PTP family is SHP2 (Src homology region 2 domain-containing phosphatase-2), also known as protein tyrosine phosphatase non-receptor type 11 (PTPN11). SHP2 contains two tandem Src homology region 2 domains, which function as phosphotyrosine-binding domains and mediate the interaction of SHP2 with its substrates. As a member of the SH2 family, SHP2 can be activated by various upstream activities and mediate phosphotyrosine signaling in various cellular pathways (see Z. Song et al., Acta Pharmaceutica Sinica B, 11(1), 2021, 13-29).

[0009] J. Biol. Chem., Vol. 270, No. 36, 1995, pp. 21277-21284, describes the association of SH2 domain protein tyrosine phosphatases with EGFR in human tumor cells.

[0010] Generally, Western blot reagents or Western blot assays are known from the prior art.

[0011] Zhang et al., Cancer Research, vol. 81, no. 11, 2021, pp. 3051-3066, studied the proteome and phosphoproteome of a series of isogenic EGFR-mutated lung adenocarcinoma cell lines sensitive or resistant to these drugs. They determined whether changes in SHP2 phosphorylation were associated with PI3K / AKT and MAPK signaling in TKI-resistant cells. Relative quantification of phosphorylated proteins normalized to total protein expression was performed on Western blots.

[0012] Li et al., Oncology Reports, vol. 33, no. 2, 2014, pp. 951-957, discusses biomarkers for predicting response to tyrosine kinase inhibitors. Western blot analysis was used to measure phosphorylation levels.

[0013] WO2007 / 027916 discloses phosphorylation sites identified in signal transduction proteins and pathways in human tumors. This document further provides phosphorylation site-specific antibodies and heavy isotope-labeled peptides (AQUA peptides) for selectively detecting and quantifying these phosphorylation sites / proteins. These antibodies were tested by ELISA and Western blot assays.

[0014] Grimes et al., Science Signaling, vol. 11, no. 531, 2018, presents a study integrating protein phosphorylation, acetylation, and methylation data sets to outline signaling networks in lung cancer. Immunoprecipitation with a tyrosine-specific antibody was used. However, the level of T59 in SHP2 cannot be experimentally measured using the antibody and method referenced in this paper. In addition to T59, SHP2 has at least seven other known threonine residues that can be phosphorylated. Using the described antibody, it is not possible to measure the signal present only at T59; only the total level of threonine phosphorylation across all proteins can be measured.

[0015] This result was confirmed by the inventors of the present invention. Specifically, Figures 4 and 5 of the present application show that neither the total phosphothreonine signal nor the total phosphorylation signal on SHP2 serves as a good classifier of EGFR activity. Therefore, although the reference antibody can be used to measure all phosphothreonine residues (PTN11) on SHP2, it will perform poorly as a classifier.

[0016] In summary, these prior art documents describe potential biomarkers designed to identify cancers resistant to therapy (negative selection criteria), rather than biomarkers that identify patients with an active EGFR signaling pathway (positive selection criteria), which are the subject of the present invention. In contrast, the present invention defines a novel method for defining patients who will benefit from EGFR therapy, without further subdividing this group into responders and non-responders (see Figure 6 of the present invention).

[0017] Furthermore, whereas the prior art is not designed to define EGFR activity (and thus a positive selection criterion for anti-EGFR therapy), the present invention tests whether the phosphorylation of the aforementioned biomarkers HER3 and ERK1 / 2 can predict EGFR activity defined by genomic mutation status. HER3 and ERK1 / 2 are known signaling hubs, and their phosphorylation depends on multiple input signals and phosphorylates many output proteins. Therefore, while they may be phosphorylated in EGFR-active cells, they are also phosphorylated under many other conditions and in many other molecular states. Furthermore, they are not expected to form the basis of an accurate predictor of EGFR activity. This is confirmed by the present invention (see Figures 4 and 5 of the present invention). Notably, the data show that HER3 and ERK1 / 2 perform significantly worse than pT59 on PTN11.

[0018] Because patient responses to anticancer therapies, especially biologic therapies, vary, there is a need to find ways to predict which treatment regimen will be most appropriate for a particular patient. In particular, it would be desirable to have a method for stratifying patients, specifically distinguishing between responders and non-responders, to identify classes of subjects who may benefit from EGFR-targeted therapy.

[0019] It is therefore an object of the present invention to provide a method for predicting the response of a subject suffering from a disease or disorder to an inhibitor of EGFR kinase activity.

[0020] In particular, an object of the present invention is to provide a method for predicting whether a patient suffering from a cancer that is a candidate for treatment with an inhibitor of EGFR kinase activity will respond (be sensitive or amenable to treatment) with an inhibitor of EGFR kinase activity, which method is particularly applicable to patients suffering from cancers of epithelial cell origin, in particular NSCLC (non-small cell lung cancer).

[0021] This method should overcome the shortcomings of the current state of the art. In particular, the objective of the present invention is to provide a method for predicting response to inhibitors of EGFR kinase activity. Current state-of-the-art methods are based on mutation analysis, which has biological limitations. EGFR overactivity can be caused not only by mutations but also by other mechanisms. IHC (immunohistochemistry)-based methods would be a superior alternative, allowing for testing and identifying more patients who may benefit from TKI treatment.

[0022] It is a further object of the present invention to provide methods for identifying markers that can predict the response of subjects with overactive EGFR to inhibitors of EGFR kinase activity.

[0023] There are tumor-activating mutations that can be used to identify patients who will respond to TKI therapy. For example, there are EGFR mutations present in a subgroup of NSCLC adenocarcinoma patients (approximately 15%), which can be used as a marker of EGFR activity to identify patients who will respond to TKI treatment.

[0024] The phosphorylation sites used in accordance with the present invention were identified from an NSCLC tumor dataset by comparing NSCLC cases annotated with wild-type EGFR and mutant, activated EGFR. In this analysis, the phosphorylation signals of the phosphorylation sites were measured by mass spectrometry. Surprisingly, phosphorylation of the T59 amino acid on SHP2 showed the most statistically significant effect between the two groups, providing a signal strong enough on its own to clearly distinguish samples containing activated EGFR from the majority of the cohort.

[0025] The method according to the invention has the following advantages: The method according to the invention makes it possible to determine the drug sensitivity of an individual patient before treatment and, optionally in combination with genomic analysis, serves as the basis for individualized targeted therapy. - Identifying NSCLC patients with activated EGFR based on measuring p-site signaling rather than mutation testing. The phosphorylation site (p-site) at T59 on SHP2 can serve as a biomarker. - This method can identify patients with activating EGFR that would be missed by conventional mutation testing. - Companion diagnostics based on immunohistochemistry may be a useful alternative in routine laboratory or clinical settings. - More patients who could benefit from TKI treatment could be tested and identified. The stability of the phosphorylation sites used according to the invention is not affected by the ischemic time. Although the stability of the p-sites may limit their practical use, in this case the ischemic time is not a limiting factor for the method of the invention. Summary of the Invention

[0026] The present invention relates to a method for predicting the response of a subject suffering from a disease or disorder to an inhibitor of EGFR kinase activity, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising residue T59 with a control level; wherein a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to respond to an agent that inhibits the EGFR kinase activity.

[0027] In particular, the present invention relates to a method for predicting the response of a subject suffering from non-small cell lung cancer (NSCLC) to an inhibitor of EGFR kinase activity, the method comprising the steps of: a) obtaining a sample from a tumor of a subject with non-small cell lung cancer (NSCLC); b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising said residue T59 with a control level; wherein a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to control levels indicates that the subject is likely to respond to an agent that inhibits EGFR kinase activity.

[0028] The present invention further relates to a method for selecting a subject suffering from a disease or disorder that may respond to an inhibitor of EGFR kinase activity, the method comprising the steps of: a) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising said residue T59 with a control level; wherein a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to control levels indicates that the subject is likely to respond to an agent that inhibits EGFR kinase activity.

[0029] In particular, the present invention further relates to a method for selecting a subject suffering from non-small cell lung cancer (NSCLC) that may respond to an inhibitor of EGFR kinase activity, the method comprising the steps of: a) obtaining a sample from a tumor of a subject with non-small cell lung cancer (NSCLC); b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising said residue T59 with a control level; wherein a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to control levels indicates that the subject is likely to respond to an agent that inhibits EGFR kinase activity.

[0030] The present invention further relates to a method for identifying overactive EGFR in a subject, the method comprising the steps of: i) obtaining a sample from a tumor of a subject having a cancer of epithelial origin; ii) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; iii) comparing the phosphorylation level of at least one phosphorylation site comprising residue T59 with a control level; Here, a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to have a hyperactive EGFR.

[0031] In particular, the present invention further relates to a method for identifying overactive EGFR in a subject, the method comprising the steps of: i) obtaining a sample from a tumor of a subject with non-small cell lung cancer (NSCLC); ii) measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, said phosphorylation site including residue T59; iii) comparing the phosphorylation level of at least one phosphorylation site comprising residue T59 with a control level; Here, a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to have a hyperactive EGFR.

[0032] The present invention also relates to a kit for predicting the response of a subject suffering from a disease or disorder to an EGFR kinase inhibitor, optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, said phosphorylation site including residue T59.

[0033] In particular, the present invention also relates to a kit for predicting the response of a subject suffering from non-small cell lung cancer (NSCLC) to an EGFR kinase inhibitor, optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, the phosphorylation site including residue T59.

[0034] The present invention also relates to a kit for predicting hyperactive EGFR to an EGFR kinase inhibitor in a subject suffering from a disease or disorder, the kit optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, the phosphorylation site including residue T59.

[0035] In particular, the present invention also relates to a kit for predicting hyperactive EGFR to an EGFR kinase inhibitor in a subject suffering from non-small cell lung cancer (NSCLC), the kit optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, the phosphorylation site comprising residue T59.

[0036] The present invention also relates to the use of phosphorylated T59 on SHP2 as a marker that can predict response to inhibitors of EGFR kinase activity in subjects with overactive EGFR, which use involves measuring amino acid residues in one or more proteins from the EGFR signaling pathway that are over- or under-phosphorylated compared to control levels.

[0037] In particular, the present invention also relates to the use of phosphorylated T59 on SHP2 as a marker that can predict response to inhibitors of EGFR kinase activity in subjects with overactive EGFR, which use involves measuring the phosphorylation of T59 on SHP2 in one or more proteins from the EGFR signaling pathway that are over- or under-phosphorylated compared to control levels.

[0038] Another embodiment of the present invention relates to a method for predicting whether a patient suffering from non-small cell lung cancer (NSCLC) is suitable for anti-EGFR therapy, the method comprising the steps of: a) obtaining a sample from a tumor of a subject with non-small cell lung cancer (NSCLC); b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising said residue T59 with a control level; wherein a significantly elevated phosphorylation level of at least one phosphorylation site comprising residue T59 compared to a control level indicates that the subject is suitable for anti-EGFR therapy.

[0039] In the following description of suitable and preferred embodiments of the present invention, the term "predicting the response of a subject suffering from a disease, in particular non-small cell lung cancer (NSCLC)" can be replaced with the term "predicting whether a patient is suitable for anti-EGFR therapy" unless otherwise defined.

[0040] In the sense of the present invention, SHP2 and PTPN11 are used synonymously.

[0041] "Protein" is used interchangeably with polypeptide and includes fragments and domains of proteins as well as whole proteins.

[0042] By "phosphorylatable amino acid" or phosphorylation is meant any amino acid that can be modified by the addition of a phosphate group, and includes both forms of such amino acids.

[0043] By "phosphorylatable peptide sequence" is meant a peptide sequence that includes at least one phosphorylatable amino acid.

[0044] In the sense of the present invention, the term "subject" refers to any human or animal. Animals (non-human) include all vertebrates, e.g., mammals and non-mammals, including cows, sheep, pigs, goats, horses, poultry, dogs, cats, non-human primates, rodents, etc. In one embodiment, the subject is a human subject.

[0045] Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation. The EGF receptor (EGFR) binds to EGF, forming a protein-ligand interaction.

[0046] The terms "p-site," "phosphorylation site," and "site of phosphorylation" are used synonymously.

[0047] A first embodiment of the present invention is a method for predicting the response of a subject suffering from a disease or disorder, particularly non-small cell lung cancer, to an inhibitor of EGFR kinase activity.

[0048] A particular embodiment of the present invention is a method for predicting whether a (human) patient suffering from cancer who is a candidate for treatment with an inhibitor of EGFR kinase activity will respond to treatment with an inhibitor of EGFR kinase activity, comprising steps a) to c) as defined above and below, wherein phosphorylation of at least one phosphorylation site in the SHP2 protein, including residue T59, indicates that the patient is likely to respond to treatment with an agent that inhibits EGFR kinase activity.

[0049] Step a) In step a), a sample is taken from a tumor of a subject with a cancer of epithelial origin.

[0050] Suitable samples include tissues or cells from tumors of subjects with cancers of epithelial cell origin. Preferably, the samples include biopsy samples such as tumor biopsies, primary tissues, and metastatic tissues. In particular, the specimens can be obtained by needle biopsy, image-guided biopsy, surgical (excision) biopsy, shave / punch biopsy, endoscopic biopsy, laparoscopic biopsy, and combinations thereof.

[0051] The term "taking a sample from a tumor" as used herein refers to a sample taken from a patient for diagnostic purposes. This tumor sample can be obtained from the patient by conventional means known to those skilled in the art, i.e., by biopsy (taken by aspiration or puncture, excision, or other surgical methods leading to biopsy or excised cellular material). In this way, it is possible to obtain tissue for the method of the present invention. However, the tumor will not be (completely) removed during sample collection. In the sense of the present invention, "taking a sample from a tumor" is not intended to refer to a therapeutic treatment method.

[0052] In a preferred embodiment, the subject has an overactive EGFR.

[0053] EGFR hyperactivity is not synonymous with EGFR overexpression. Hyperactivity of the epidermal growth factor receptor (EGFR) is observed in many cancers, sometimes accompanied by gene amplification. In other words, EGFR hyperactivity refers to increased EGFR-driven phosphorylation activity, not increased EGFR expression itself.

[0054] A sample is classified as EGFR hyperactive based on its somatic mutation profile. In particular, a sample is determined to be EGFR hyperactive if it has at least one activating mutation as defined below.

[0055] Hyperactive EGFR is caused by mutations in or protein interactions with EGFR that result in conformational changes of the receptor leading to an active signaling state that contributes to pathology.

[0056] In a first preferred embodiment, hyperactive EGFR is caused by a mutation in the EGFR gene. The EGFR gene is located on chromosome 7p11.2 and has 28 exons encoding a 464-amino acid transmembrane receptor protein. Within the EGFR gene, exons 5-7 and 13-16 encode the ligand-binding domain, exons 18-24 encode the tyrosine kinase domain, and autophosphorylation occurs in the region encoded by exons 25-28. In particular, the mutation affects at least one of the aforementioned domains. In lung cancer patients who may respond positively to TKI therapy, EGFR mutations are particularly observed in exons 18-21.

[0057] Said EGFR mutations are in particular selected from mutations altering L858, T790, G719, L861, S768; deletions in exon 19.

[0058] In particular, the mutations include: - point mutant L858R, - point mutation T790M, - point mutation G719A / C, - point mutant L861Q, - point mutation S768I, and equivalent mutations, e.g. - Exon 19 deletion.

[0059] Other equivalent mutations include mutations in the functional portion where L858 is located, such as mutations in the tyrosine kinase portion of EGFR or mutations in or near the ATP binding pocket of EGFR.

[0060] In a second preferred embodiment, hyperactive EGFR is caused by protein interactions with EGFR, resulting in a conformational change of the receptor into a state of active signaling that contributes to the pathology.

[0061] Preferably, the disease or disorder from which the subject is suffering is cancer, particularly cancer of epithelial origin, including primary cancer and secondary (metastatic) disease.

[0062] In particular, said cancer is selected from glioblastoma, melanoma, lung cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, pancreatic cancer, bladder cancer, head cancer, neck cancer, colon cancer, colorectal cancer, and kidney cancer.

[0063] In particular, said disease or disorder is non-small cell lung cancer (NSCLC).

[0064] Step b) In step b) of the method of the present invention, the phosphorylation level of the phosphorylation site residue T59 in the SHP2 protein is measured.

[0065] The phosphorylation level can be measured by targeted mass spectrometry on tumor tissue.

[0066] Said determination can also be carried out by the development of antibodies and / or IHC assays that specifically target the p site.

[0067] Antibodies for use in the present invention may be generated by any suitable method known in the art, including, but not limited to, polyclonal, monoclonal, humanized, phage display-derived, or chimeric antibodies.

[0068] Step c) In step c) of the method of the invention, the phosphorylation level of at least one phosphorylation site comprising residue T59 in the SHP2 protein is compared with a control level.

[0069] Thus, a significant increase in the phosphorylation level of at least one phosphorylation site including residue T59 compared to control levels indicates that the subject is likely to respond to an agent that inhibits EGFR kinase activity.

[0070] Thereby, a significantly elevated phosphorylation level of at least one phosphorylation site comprising residue T59 compared to a control level indicates that said subject has a hyperactive EGFR.

[0071] The control level refers to the level of phosphorylation at a specific amino acid residue, particularly an amino acid side chain, of a protein in a non-cancerous tissue. In other words, the control level refers to the level of phosphorylation at a specific amino acid residue of a protein in a normal, non-tumorous tissue.

[0072] A "significantly elevated" level of phosphorylation (relative to a control level) is one whose difference from the control level is statistically significant using suitable and well-known statistical methods in the art, e.g., phosphorylation of at least one phosphorylation site including residue T59 of SHP2 in a subject with hyperactive EGFR.

[0073] Methods for measuring the phosphorylation level of an amino acid residue are conventional and routine. Typically, this measurement relies on the existence of a set of antibodies specific for either the unphosphorylated or phosphorylated form of a particular amino acid residue in the context of a protein of interest (e.g., EGFR). Such antibodies are commercially available or can be routinely generated using conventional procedures. In one embodiment, a synthetic peptide containing the target amino acid (either the unphosphorylated or phosphorylated form) from the protein of interest is used as an antigen to prepare a suitable antibody. The antibody can be polyclonal or monoclonal. The antibody is selected and validated to detect only the phosphorylated form of the protein and not the unphosphorylated form of the native or denatured protein, or vice versa.

[0074] Such antibodies can be used in a variety of ways. For example, whole-cell lysates can be prepared from patient samples and spotted in an array format onto a suitable substrate, such as a nitrocellulose strip or glass slide. Preferably, proteins in the sample are denatured before spotting. Typically, the cells are spotted in serial dilutions, such as two-fold serial dilutions, to provide a wide dynamic range. Appropriate controls, such as positive and control-level controls, can be included. Each array is then probed with an appropriate detectable antibody, as described above, to measure and / or quantify which amino acid residues in the various proteins of interest are phosphorylated. Methods of immunoquantification are conventional. Other suitable assays using such antibodies to assess the level and / or degree of phosphorylation of residues of interest include, for example, Western blots, ELISA assays, immunohistochemistry, mass spectrometry, and other conventional assays. Suitable methods include those capable of detecting phosphorylated proteins in very small samples (e.g., approximately 200 cells). Alternatively, methods suitable for larger sample sizes (e.g., FFPE tissue sections) can be used.

[0075] Assays for measuring the presence and / or level of phosphorylated residues can be readily adapted to a high-throughput format, if desired, using, for example, robotics. The methods of the invention can further comprise measuring the activation state of EGFR.

[0076] That is, the presence or absence of activating mutations can be determined in patient samples. For example, subjects who have both an activating mutation and hyperphosphorylation of SHP2 residue T59 can be determined to be responders according to the present invention and therefore can be selected for EGFR therapy.

[0077] Other conditions mediated by EGFR can also be amenable to the methods of the invention. These conditions are selected from hyperproliferative conditions such as cancer, precancerous conditions, metabolic disorders (such as diabetes), skin disorders or diseases, cardiovascular diseases, hyperproliferative cell diseases or disorders, psoriasis, obesity, inflammatory airway diseases, asthma, COPD, and neurological disorders. Patients suffering from these conditions can also be tested using the methods of the invention.

[0078] One aspect of the present invention is a method for treating a subject suffering from a disease or disorder with overactive EGFR, particularly non-small cell lung cancer, comprising the steps of: i) measuring the level of phosphorylation of SHP2 at residue T59 in a sample from said subject; if the phosphorylation level indicates that the subject is more likely to respond to EGFR therapy compared to a control level, ii) administering to the subject an EGFR therapy (administering an EGFR inhibitor, such as an inhibitor of EGFR kinase activity at effective levels);

[0079] Preferably, the EGFR inhibitor is BIBX 1382; cetuximab (Erbitux); CI-1033 (canertinib); EKB-569; EMD 55900; EMD 72000; erlotinib (OSI-774; Tarceva); gefitinib (ZD1839; Iressa); GW-2016; hR3; ICR-62; lapatinib (GW-572016); lavendustin A; lavendustin B; monoclonal antibody E7.6.3; panitumumab (ABX-EGF); PD 153035; PD-168393; PKIl 66; RG-13022; RG-14620; TheraCim hR3; Tyrophostin; Tyrophostin AG 490; Tyrophostin AG 494; Tyrophostin AG 825; Tyrophostin AG 1478; Tyrophostin 1; Tyrophostin 23 (RG-50810); Tyrophostin 25 (RG-50875); Tyrophostin 46; Tyrophostin 47 (RG-50864; AG-213); Tyrophostin 51; ZD-6474; derivatives thereof; or combinations thereof.

[0080] In a preferred embodiment, the subject is a human patient suffering from NSCLC and the EGFR kinase inhibitor is selected from Iressa (gefitinib), Tarceva (erlotinib), Vizinpro (dacomitinib), Gilotrif (afatinib), or a mixture thereof.

[0081] These agents can also be used in combination with each other or with conventional agents, such as chemotherapeutic agents. Although compounds may be characterized as "EGFR inhibitors," "EGFR kinase inhibitors," or "inhibitors of the EGFR pathway," the present invention is not limited by the mechanism by which such agents achieve their therapeutic effect. For example, a subpopulation of patients selected according to the present invention may respond to EGFR therapy, but the mechanism of action may be unrelated, or completely unrelated, to modulation of the EGFR pathway.

[0082] There are many downstream proteins in the EGFR tyrosine kinase cascade that are hyperphosphorylated at specific residues in subjects with NSCLC and hyperactive EGFR (and / or cultured cells harboring this mutation).

[0083] A further aspect of the present invention is to treat a patient identified as a potential responder with a pharmaceutical composition comprising an effective amount of at least one EGFR kinase inhibitor as defined above and a pharmaceutically acceptable carrier.

[0084] Such pharmaceutical compositions can be administered to a subject with an overactive EGFR, wherein the subject has been determined by the methods of the invention to be likely to respond to an EGFR kinase inhibitor.

[0085] The inhibitors discussed herein can be formulated into various compositions, e.g., pharmaceutical compositions, for use in therapeutic treatment methods. The pharmaceutical compositions can be assembled into kits. Generally, the pharmaceutical compositions of the present invention contain an anti-cancer effective amount of the inhibitor. As used herein, an "anti-cancer effective amount" is an amount sufficient to produce at least a therapeutic response in the individual over a reasonable time frame. This can, for example, improve cancer symptoms to at least a detectable extent or inhibit tumor growth.

[0086] The composition can include a carrier, such as a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a substance that is not biologically or otherwise undesirable, i.e., a substance that can be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is included. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0087] Those skilled in the art will understand that the particular formulation will depend in part on the particular inhibitor or other chemotherapeutic agent used and the route of administration selected. Accordingly, suitable composition formulations vary widely. Suitable formulations for oral, parenteral, aerosol, transdermal, topical, or other modes of administration will be apparent to those skilled in the art.

[0088] One of ordinary skill in the art can readily determine the appropriate dosage, schedule, and method of administration of the precise composition formulation to be used to achieve the desired anti-cancer effective amount or concentration in an individual patient. One of ordinary skill in the art can also readily determine and use appropriate indicators of "effective concentrations" of the compounds of the invention by direct or indirect analysis of appropriate patient samples (e.g., blood and / or tissue).

[0089] The dose of the inhibitor or composition thereof of the present invention administered to an animal, particularly a human, should be sufficient to produce at least a therapeutic response in the individual within a reasonable time frame (an anti-cancer effective dose). The precise amount of the dose will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity or mechanism of the disease being treated, the specific agent or vehicle used, its method of administration, and the like. The dose used to achieve a desired anti-cancer concentration in the body will be determined by the potency of the specific inhibitor used, the pharmacodynamics associated with the agent in the host, the severity of the disease state of the infected individual, and, in the case of systemic administration, the weight and age of the individual. The size of the dose will also be determined by the presence of any adverse side effects that may accompany the specific inhibitor or composition thereof used. Minimizing adverse side effects whenever possible is generally desirable.

[0090] When administered in combination therapy (e.g., an EGFR kinase inhibitor in combination with one or more inhibitors of downstream proteins in the EGFR signaling pathway), the inhibitors can be administered simultaneously or, if necessary, staggered. Two or more of the above drugs can also be combined in a single composition. When used in combination, the dosage of each drug will be lower than when either drug is used alone.

[0091] A second embodiment of the present invention is a method for identifying overactive EGFR in a subject, the method comprising the steps of: i) obtaining a sample from a tumor of a subject with a cancer of epithelial origin, in particular from a tumor of a subject with non-small cell lung cancer; ii) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; iii) comparing the phosphorylation level of at least one phosphorylation site comprising said residue T59 with a control level; Here, a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to have a hyperactive EGFR.

[0092] A third embodiment of the present invention is a kit for predicting the response of a subject suffering from a disease or disorder, in particular non-small cell lung cancer, to an EGFR kinase inhibitor, optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, said phosphorylation site including residue T59.

[0093] For example, the kit may be useful for predicting the response of subjects suffering from diseases or disorders caused by overactive EGFR, particularly non-small cell lung cancer and cancers of epithelial origin, comprising reagents and / or devices for measuring the phosphorylation level of at least one phosphorylation site including residue T59.

[0094] Additionally, the kit may include reagents and / or equipment for preparing the sample, such as collecting tissue and / or excising a sample from tissue, reagents and / or equipment for spotting the test sample onto a suitable surface, such as a nitrocellulose strip or glass slide, reagents and / or equipment for performing immunoassays, such as labeled antibodies or reagents for labeling antibodies, and instructions for carrying out the methods of the invention.

[0095] Optionally, the components of the kit may be packaged in one or more containers.

[0096] Among other uses, the kits of the invention can be used in experimental applications to identify patterns of phosphorylation that can predict a subject's response to a therapeutic agent. Those skilled in the art will recognize kit components suitable for carrying out any of the methods of the invention.

[0097] Optionally, the kits of the invention include suitable buffers, one or more containers or packaging materials, and / or instructions for carrying out the method. The reagents of the kits may be contained in containers in which the reagents are stable, e.g., in lyophilized or stabilized liquid form. The reagents may be in single-use form, e.g., single-dose form.

[0098] Companion diagnostic tests and compounds to determine whether a mutation is present and whether it is associated with a particular treatment can be found in the FDA database: https: / / www.fda.gov / medical-devices / in-vitro-diagnostics / list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools.

[0099] A list of authorized or approved companion diagnostic devices (in vitro diagnostic and imaging tools) can be found on the EMA webpage (https: / / www.ema.europa.eu / en / human-regulatory / post-authorisation / data-medicines-iso-idmp-standards / public-data-article-57-database).

[0100] The following table summarizes the five FDA-approved CDx tests and their corresponding drugs. 1. Cobas EGFR Mutation Test v2 (Roche Molecular Systems, Inc.) 2. FoundationOne CDx (Foundation Medicine, Inc.) 3. ONCO / Reveal Dx Lung and Colon Cancer Assay (O / RDx-LCCA) (Pillar Biosciences, Inc.) 4. Oncomine Dx Targeted Test (Life Technologies Corporation) 5. TheraScreen EGFR RGQ PCR Kit (Qiagen Manchester, Ltd.)

[0101] A fourth embodiment of the present invention is the use of T59 on SHP2 as a marker that can predict response to inhibitors of EGFR kinase activity in subjects with overactive EGFR, including measuring amino acid residues in one or more proteins from the EGFR signaling pathway that are over- or under-phosphorylated compared to control levels, particularly measuring the phosphorylation of T59 on SHP2.

[0102] The present invention will be further described with reference to the following examples, which do not limit the scope of the particular embodiments described. The present invention includes all combinations of the described features, particularly preferred features that are not mutually exclusive. [Brief explanation of the drawings]

[0103] Over 30,000 phosphorylation sites have been measured in patients with NSCLC, with an average of approximately 22,000 phosphorylation sites per patient, including many members of the EGFR signaling pathway. Whether any combination of these phosphorylation sites could predict EGFR activity based on mutational status was tested, and thus far, the best predictor was a single phosphorylation site on SHP2.

[0104] Figure 1 shows the frequency of EGFR mutations. EGFR activity is influenced by specific genetic mutations. Somatic mutations detected in patient tumor samples can serve as a primary indicator of increased EGFR kinase activity. Figure 1 shows the frequency counts of all 82 distinct mutations reported for EGFR across samples from the NSCLC cohort, including 15 mutations that occurred in at least two samples. These plots demonstrate the representativeness of the tumor sample database used to identify promising phosphorylation sites. This is because well-known EGFR-activating exon 19 deletions (Glu746_Ala750del 23 times, Leu747_Pro753delinsSer 7 times, Leu747_Flu749del 3 times, and Leu747_Thr571del 2 times) were found to be the most frequent mutations, as predicted by prior art. This is followed by mutations at Leu858Arg (23 times) and at Gly719 (Gly719Ala 6 times, Gly719Cys 2 times).

[0105] Figure 2 shows the log2-transformed intensity of PTPN11-T59. Based on the mutations shown in Figure 1, samples can be classified based on EGFR activity. Samples with only somatic EGFR mutations affecting kinase activity can be classified as "active EGFR." Conversely, samples without any mutations other than EGFR-activating mutations can be considered "wild-type EGFR." Based on this classification, 435 wild-type EGFR samples and 54 active EGFR samples were identified. Analysis of the log2-transformed intensity of PTN11-T59 revealed significant differences between both groups. The example of a threshold t ≥ 16 (dashed horizontal line) indicates that this phosphorylation site serves as an appropriate group separator. In other words, samples with active EGFR generally have a PTN11-T59 intensity greater than 16, while wild-type EGFR is below this threshold. Additionally, samples with multiple, but only partial, EGFR-activating mutations are classified as "wild-type EGFR," but also exhibit a high PTN11-T59 intensity greater than 16. This indicates that this method can find samples with active EGFR that may exceed the pure level of kinase activity from somatic mutations.

[0106] Figure 3: Effect of ischemia time. This figure shows the log2-transformed intensity of PTN11-T59 samples as in Figure 2. The data are divided into 5-minute ischemia time intervals, i.e., intervals representing the time elapsed from tissue extraction to freezing. This method allowed us to track whether PTN11-T59 remained stable over time. The differences between samples with "wild-type EGFR" and "activated EGFR" remained constant over the entire period, and none of the groups showed a significant downward trend over time.

[0107] Figures 4a, 4b, 4c, and 4d): The PTN11-T59 biomarker for EGFR kinase activity according to the present invention can be viewed as a threshold-based binary classification, with a high score associated with elevated kinase activity. Thus, when each sample is ranked by its level of PTN11-T59 phosphorylation, samples with hyperactivated EGFR kinase activity should be enriched among the highest measured phosphorylation values. The better this enrichment, the higher the area under the curve (AUC) of the corresponding receiver operating characteristic (ROC) curve. This curve reflects the concordance between EGFR hyperactive samples, defined by genetic mutations, and samples with a high phosphorylation signal (denoted here as the true positive rate, TPR). The false positive rate here reflects the presence of EGFR wild-type samples in which a high phosphorylation signal is observed. The latter may indicate a biological EGFR activation process, regardless of EGFR genetic mutation status.

[0108] Predictions based solely on the intensity of PTN11-T59 are shown to outperform all other models by exhibiting the highest AUC (area under the curve) observable from the line closest to the upper left corner of the plot. This represents the best achievable model performance. In addition, the plot further demonstrates that using a more comprehensive set of phosphorylation sites results in poorer performance, as the specific signal provided by PTN11-T59 is diluted by other non-specific signals. Specifically, all PTN11 threonine phosphorylation sites (PTN11-T59) are predicted to be 100% phospho- and 100% phospho-, respectively. * By using the sum of the phosphorylation intensities of all p sites of PTN11 (PTN11- * ) is integrated, it further drops to 0.7763. Also, Figure 4 shows that ERBB3 (ERBB3- * ) and ERK1 and ERK2 (ERK1 / 2- * ) p-site has lower predictive power compared to the pure PTN11-T59 model according to the present invention.

[0109] As a result, the method for predicting elevated EGFR kinase activity according to the present invention relies on the specific measurement of the p site, and therefore general protein phosphorylation is not a sufficient source of information.

[0110] 5a), 5b), 5c), and 5d): In addition to the ROC curve, the information provided is the so-called precision-recall (PR) curve. This graph is constructed similarly to the ROC curve, but now compares precision (positive predictive value, PPV) (on the y-axis), which is the proportion of genetically defined EGFR-hyperactivated samples to the total number of samples predicted to be EGFR-hyperactivated using the present invention, with recall, which corresponds to the true positive rate (TPR on the x-axis).

[0111] It was found that none of the models mentioned in the prior art achieved exceptionally high AUC (area under the curve) in the PR curve plot. The highest was PTN11-T59, with an AUC of 0.6181. This indicates that the model of the present invention predicts that previously unknown samples, which are expected to show wild-type activity based on mutation information, are EGFR hyperactivated.

[0112] Figure 6: Graphical representation of markers useful for EGFR therapy. First, we define patients who are likely to be suitable for EGFR therapy from all NSCLC patients. This is currently done using mutations in EGFR. According to the present invention, a single phosphorylation site on PTN11 is used to define EGFR hyperactivation. Within this group, it is possible to further narrow down which patients will respond to treatment and which will not, and further, which of the patients who respond to treatment may eventually experience tumor recurrence. Zhang et al., 2021, list attempts to identify markers of non-responders from lung cell lines and use EGFR mutations to define EGFR activity. Li et al., 2014, define negative selection criteria for response to treatment. Both approaches use negative selection criteria that have nothing to do with EGFR activity itself.

Claims

1. 1. A method for predicting response to an inhibitor of EGFR kinase activity in a subject with non-small cell lung cancer (NSCLC), comprising: a) obtaining a sample from a tumor of a subject suffering from non-small cell lung cancer (NSCLC); b) measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59; c) comparing the phosphorylation level of at least one phosphorylation site comprising residue T59 with a control level; Including, A method in which a significantly increased phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to respond to an agent that inhibits the EGFR kinase activity.

2. The method of claim 1 , wherein the subject has an overactive EGFR.

3. 10. The method of any one of the preceding claims, wherein the overactive EGFR is caused by a mutation in or a protein interaction with EGFR, which results in a conformational change of the receptor leading to active signaling that contributes to a pathology.

4. 4. The method of claim 3, wherein the mutation alters the structure of the receptor resulting in an active signaling state that contributes to a pathology.

5. The method according to claim 3 or 4, wherein the mutation is in a nucleic acid encoding EGFR and is a mutation that alters L858, T790, G719, L861, S768, or a deletion in exon 19.

6. The method of any one of claims 1 to 3, wherein the overactivity of EGFR is caused by a protein-protein interaction with EGFR, resulting in a conformational change of the receptor leading to an active signaling state that contributes to the pathology.

7. 10. The method of any one of the preceding claims, wherein the cancer comprises primary and secondary metastatic disease.

8. 10. The method of any one of the preceding claims, wherein the subject is a human.

9. The EGFR inhibitor is selected from the group consisting of BIBX 1382; cetuximab (Erbitux); CI-1033 (canertinib); EKB-569; EMD 55900; EMD 72000; erlotinib (OSI-774; Tarceva); gefitinib (ZD1839; Iressa); GW-2016; hR3; ICR-62; lapatinib (GW-572016); lavendustin A; lavendustin B; monoclonal antibody E7.6.3; panitumumab (ABX-EGF); PD 153035; PD-168393; PKIl 66; RG-13022; RG-14620; TheraCim hR3; Tyrophostin; Tyrophostin AG 490; Tyrophostin AG 494; Tyrophostin AG 825; Tyrophostin AG 1478; Tyrophostin 1; Tyrophostin 23 (RG-50810); Tyrophostin 25 (RG-50875); Tyrophostin 46; Tyrophostin 47 (RG-50864; AG-213); Tyrophostin 51; ZD-6474; derivatives thereof; or combinations thereof.

10. 10. The method of any one of the preceding claims, wherein the subject is a human patient suffering from NSCLC and the EGFR kinase inhibitor is gefitinib, erlotinib, dacomitinib, afatinib, amivantam, or a mixture thereof.

11. 1. A method for identifying an overactive EGFR in a subject, comprising: i) obtaining a sample from a tumor of a subject with non-small cell lung cancer; ii) measuring the phosphorylation level of at least one phosphorylation site in said SHP2 protein, said phosphorylation site including residue T59; iii) comparing the phosphorylation level of at least one phosphorylation site comprising residue T59 with a control level. Including, A method wherein a significantly elevated phosphorylation level of at least one phosphorylation site including residue T59 compared to a control level indicates that the subject is likely to have a hyperactive EGFR.

12. 1. A kit for predicting the response of a subject suffering from non-small cell lung cancer (NSCLC) to an EGFR kinase inhibitor, optionally comprising, in one or more containers, means for measuring the phosphorylation level of at least one phosphorylation site in the SHP2 protein, the phosphorylation site including residue T59.

13. Use of phosphorylated T59 on SHP2 as a marker that can predict response to inhibitors of EGFR kinase activity in subjects with overactive EGFR.

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