WO2024042077A1 - Procédé de prédiction de la réponse à un inhibiteur de l'activité de la kinase egfr - Google Patents

Procédé de prédiction de la réponse à un inhibiteur de l'activité de la kinase egfr Download PDF

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WO2024042077A1
WO2024042077A1 PCT/EP2023/073029 EP2023073029W WO2024042077A1 WO 2024042077 A1 WO2024042077 A1 WO 2024042077A1 EP 2023073029 W EP2023073029 W EP 2023073029W WO 2024042077 A1 WO2024042077 A1 WO 2024042077A1
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egfr
phosphorylation
subject
level
residue
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PCT/EP2023/073029
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Jonathan WOODSMITH
Alexander Flohr
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Indivumed Gmbh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to a method to select patients that may respond to a tyrosine kinase inhibitor (TKI) treatment.
  • the invention further relates to a diagnostic kit for the prediction of a response to an inhibitor of EGFR kinase activity.
  • the invention relates to the use of phosphorylated T59 on SHP2 as a biomarker to predict EGFR kinase activity.
  • tyrosine kinases Enzymes capable of catalyzing such reactions are denoted tyrosine kinases, whereas tyrosine kinase inhibitors (TKI) are pharmaceutics that inhibit the activation of proteins by tyrosine phosphorylation.
  • Growth factor receptor tyrosine kinases play a role in the etiology and progression of a variety of disorders or diseases, including, e.g., malignant diseases in humans. These receptors are anchored by means of a transmembrane domain in the membranes of cells that express them. An extracellular domain binds to the growth factor.
  • the binding of the growth factor to the extracellular domain results in a signal being transmitted from the exterior of the cell to the intracellular kinase domain.
  • the transduction of this signal contributes to a variety of pleiotrophic responses, which are responsible, e.g., for the induction of DNA synthesis, altered gene expression, cell growth, proliferation and differentiation, etc.
  • the epidermal growth factor receptor is a tyrosine kinase receptor and a member of HER/ErbB family that consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB 4).
  • the receptor consists of three parts: a glycosylated extracellular ligand-binding domain, a transmembrane domain that consists of a single chain, and an intracellular protein-tyrosine kinase domain. Ligand binding to the extracellular domain causes the receptor to dimerize and activate the protein-tyrosine kinase activity.
  • EGFRs are widely distributed at the cell surface of the mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc.
  • the EGFR signaling pathway plays an important role in physiological processes like cell growth, proliferation and differentiation, etc.
  • the functional deficiency of protein tyrosine kinase, like EGFR, or the abnormality in the activity or cellular localization of the key factors in the related signaling pathways may all lead to occurrence of tumors, diabetes, immune deficiencies and cardiovascular diseases.
  • Several malignancies are associated with the mutation or an increased expression of members of the ErbB family.
  • glioblastomas including glioblastoma multiforme
  • cancers of the lung adenocarcinomas, including bronchoalveolar carcinoma (BAC) and non-small cell lung cancer (NSCLC)
  • BAC bronchoalveolar carcinoma
  • NSCLC non-small cell lung cancer
  • Lapatinib (Tykerb®) is an orally effective quinazoline derivative which is an EGFR/HER2 dual inhibitor, used in the treatment of ErbB2- overexpressing breast cancer.
  • the afore-mentioned TKI competitively bind to the phosphorylation site of the tyrosine kinase at the intracellular segment to block the interaction between the phosphorylation site and ATP and inhibit the phosphorylation of tyrosine and a series of downstream signal transduction, and then inhibit the growth of tumor cells.
  • the reversible EFGR inhibitors Gefitinib and Erlotinib show favorable therapeutic effects on non-small cell lung cancer patients with EGFR mutation; they can significantly prolong the progression-free survival (PFS) and overall survival (OS) of the patients.
  • Trastuzumab, Pertuzumab, Amivantamb (Rybrevant®) and Trastuzumab- Emtansine are monoclonal antibodies which are given intravenously, target the extracellular domain and are used for the treatment of ErbB2-positive breast cancer.
  • Trastuzumab-Emtansine is an antibody-drug conjugate that delivers a cytotoxic drug to cells overexpressing ErbB2.
  • Cetuximab and Panitumumab are monoclonal antibodies that target ErbB1 and are used in the treatment of colorectal cancer.
  • W02007/106432 describes a method for predicting the response of a subject having a disease or condition mediated by EGFR (e.g., a cancer of epithelial origin, such as NSCLC) to an EGFR kinase inhibitor.
  • EGFR e.g., a cancer of epithelial origin, such as NSCLC
  • the amount of phosphorylation at residues Y1068 and T1148 in EGFR is measured.
  • An elevated level of phosphorylation at the two residues is used as an indicator that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • this method has not become established in practice. There is currently no companion diagnostic test, wherein phosphosites on the EGFR are used to select responders for an anti tumor treatment.
  • a further group of enzymes that together with the tyrosine kinases play an important role in signal transduction cascades are the protein tyrosine phosphatases (PTPs) that are capable of removing phosphate groups from phosphorylated tyrosine residues on proteins.
  • PTPs protein tyrosine phosphatases
  • a member of the PTP family is SHP2 (Src homology region 2 domain-containing phosphatase-2), an enzyme also known as protein tyrosine phosphatase non-receptor type 11 (also denoted as PTPN11).
  • SHP2 contains two tandem Src homology-2 domains, which function as phospho-tyrosine binding domains and mediate the interaction of SHP2 with its substrates.
  • SHP2 can be activated by a variety of upstream activities to mediate phospho-tryosine signal transduction in various cellular pathways (see Z.Song et.al, Acta Pharmaceutica Sinica B, 11 (1 ), 2021 , 13-29.).
  • Zhang et aL Cancer Research, vol. 81 , no. 11 , 2021 , p. 3051 -3066, relates to a study of the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. It was determined whether phosphorylation changes in SHP2 are associated with PI3K/AKT and MAPK signaling in TKI resistant cells. The relative quantification of phosphorylated proteins normalized to total protein expression was performed on Western blots.
  • figures 4 and 5 of the present application demonstrate that neither the total phospho-threonine signal, nor the total phosphorylation signal on SHP2 function as good classifiers for EGFR activity.
  • referenced antibodies could be used to measure all phospho-threonine residues of SHP2 (PTN11 ) but would have worse performance as a classifier.
  • present invention defines a novel way of defining patients who have a benefit from an EGFR therapy, and does not sub-divide this group further into responders and non-responders (see fig. 6 of the present invention).
  • HER3 and ERK1/2 are known signaling hubs, and phosphorylation of these is dependent on multiple input signals, and they phosphorylate many output proteins.
  • they may be phosphorylated in EGFR active cells, they are also phosphorylated under many other conditions or in many other molecular states, and it would not be expected that they can form the basis of an accurate EGFR activity predictor. This is confirmed by the present invention (see fig 4 and 5 of the present). In particular, this data show that HER3 and ERK1/2 perform very poorly compared to pT59 on PTN11 .
  • the method is applied to patients who have a cancer of epithelial cell origin, in particular NSCLC (nonsmall cell lung cancer).
  • the method should overcome the disadvantages of the state of the art.
  • Current state of the art methods are based on mutation analysis, which has its biological limitations. Over active EGFR can be caused not only by mutations, but also through other mechanisms.
  • An IHC (immunohistochemical) based method would be a good alternative and allow to examine and identify more patients that could benefit from TKI treatment.
  • tumor activating mutations that can be used to identify patients that respond to TKI therapy.
  • EGFR mutations which are present in a subgroup of NSCLC adenocarcinoma patients (about 15%) that can be used as indicative standard of EGFR activity, and identify patients that respond to TKI treatment.
  • the phosphosite used according to the invention was identified from a NSCLC tumor data set by comparison between NSCLC cases annoated with a wild-type EGFR and mutant activated EGFR. In this analysis the phosphorylation signal of phosphosites was measured through mass spectrometry. Surprisingly, the phosphorylation of the T59 amino acid on SHP2 showed the largest statistically significant effect between the two groups, and in isolation resulted a signal strong enough to clearly distinguish samples with an activated EGFR from the bulk of the cohort.
  • the method according to the invention can determine an individual patient's drug sensitivity prior to treatment, optionally in conjunction with genomic analysis, and thus can serve as the basis for individualized targeted therapy.
  • the phosphosite (p-site) at T59 on SHP2 can act as a biomarker.
  • the method allows to identify patients with activated EGFR that would be missed by classical mutation testing.
  • An immunohistochemistry based companion diagnostic may be a useful alternative in a routine laboratory or clinical setting.
  • the stability of the phosphosite employed according to the invention is not influenced by the ischemia time. Stability of p-sites are sometimes limiting the practical use, however in this case ischemia time is no limiting factor for method of the invention.
  • the 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, comprising the steps: a) collecting a sample from a tumor of a subject that has a cancer of epithelial cell origin b) determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein; c) comparing the level of phosphorylation of the at least one phosphorylation site, comprising residue T59, to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • the 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, comprising the steps: a) collecting a sample from a tumor of a subject that having non-small cell lung cancer (NSCLC); b) determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein; c) comparing the level of phosphorylation of the at least one phosphorylation site, comprising residue T59, to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • NSCLC non-small cell lung cancer
  • the 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, comprising the steps: a) collecting a sample from a tumor of a subject that has a cancer of epithelial cell origin b) determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein; c) comparing the level of phosphorylation of the at least one phosphorylation site, comprising residue T59, to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • the 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, comprising the steps: a) collecting a sample from a tumor of a subject that has non-small cell lung cancer (NSCLC); b) determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein; c) comparing the level of phosphorylation of the at least one phosphorylation site, comprising residue T59, to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • NSCLC non-small cell lung cancer
  • the invention further relates to a method for identifying over-active EGFR in a subject, comprising the steps: i) collecting a sample from a tumor of a subject having a cancer of epithelial cell origin; ii) determining the level of phosphorylation of at least one phosphorylation site comprising residue T59 in the SHP2 protein; iii) comparing the level of phosphorylation of at least one phosphorylation site comprising residue T59 to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site comprising residue T59 compared to the control level indicates that the subject has likely an over-active EGFR.
  • the invention further relates to a method for identifying over-active EGFR in a subject, comprising the steps: i) collecting a sample from a tumor of a subject having non-small cell lung cancer (NSCLC); ii) determining the level of phosphorylation of at least one phosphorylation site comprising residue T59 in the SHP2 protein; iii) comparing the level of phosphorylation of at least one phosphorylation site comprising residue T59 to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site comprising residue T59 compared to the control level indicates that the subject has likely an over-active EGFR.
  • NSCLC non-small cell lung cancer
  • the invention also relates to a kit for predicting the response of a subject suffering from a disease or disorder to an inhibitor of EGFR kinase, comprising means for determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein, optionally in one or more containers.
  • the invention also relates to a kit for predicting the response of a subject suffering from non-small cell lung cancer (NSCLC) to an inhibitor of EGFR kinase, comprising means for determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein, optionally in one or more containers.
  • NSCLC non-small cell lung cancer
  • the invention also relates to a kit for predicting an over-active EGFR of a subject suffering from a disease or disorder to an inhibitor of EGFR kinase, comprising means for determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein, optionally in one or more containers.
  • the invention also relates to a kit for predicting an over-active EGFR of a subject suffering from non-small cell lung cancer (NSCLC)to an inhibitor of EGFR kinase, comprising means for determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein, optionally in one or more containers.
  • NSCLC non-small cell lung cancer
  • the invention also relates to a use of phosphorylated T59 on SHP2 as a marker that can predict the response of a subject having an over active-EGFR to an inhibitor of EGFR kinase activity.
  • This use comprises determining amino acid residues in one or more proteins from the EGFR signaling pathway that are over- or under-phosphorylated, compared to a control level.
  • the invention also relates to a use of phosphorylated T59 on SHP2 as a marker that can predict the response of a subject having an over active-EGFR to an inhibitor of EGFR kinase activity, comprising determining the phosphorylation of T59 on SHP2 in one or more proteins from the EGFR signaling pathway that are over- or underphosphorylated, compared to a control level.
  • Another embodiment of the invention relates to a method for predicting, whether a patient suffering from non-small cell lung cancer (NSCLC) is suitable for anti-EGFR therapy, comprising the steps: a) collecting a sample from a tumor of a subject that having non-small cell lung cancer (NSCLC); b) determining the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein; c) comparing the level of phosphorylation of the at least one phosphorylation site, comprising residue T59, to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is suitable for anti-EGFR therapy.
  • NSCLC non-small cell lung cancer
  • predicting the response of a subject suffering from a disease, in particular non-small cell lung cancer (NSCLC) can be replaced by the term “predicting, whether a patient is suitable for anti-EGFR therapy”, if nothing specific is defined.
  • NSCLC non-small cell lung cancer
  • Protein is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole proteins.
  • Phosphorylatable amino acid or phosphorylated means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.
  • Phosphorylatable peptide sequence means a peptide sequence comprising at least one phosphorylatable amino acid.
  • the term “subject” refers to any human or animal.
  • a (nonhuman) animal includes all vertebrates, e.g. mammals and non-mammals, including cows, sheep, pigs, goats, horses, poultry, dogs, cats, non-human primates, rodents etc.
  • the subject is a human subject.
  • Epidermal growth factor is a protein that stimulates cell growth and differentiation.
  • the EGF-receptor binds to EGF and forms a protein-ligand interaction.
  • a first embodiment of the invention is a method for prediction of the response of a subject suffering from a disease or disorder, in particular non-small cell lung cancer, to an inhibitor of EGFR kinase activity.
  • a special embodiment of the invention is a method for predicting if a (human) patient suffering from a cancer that makes him a candidate for treatment with an inhibitor of EGFR kinase activity is responsive to a treatment with an inhibitor of the EGFR kinase activity, comprising the steps a) to c), as defined above and in the following, wherein phosphorylation of at least one phosphorylation site, comprising the residue T59 in the SHP2 protein, indicates that the patient is likely to be responsive to a treatment with an agent that inhibits the kinase activity of EGFR.
  • step a) a sample from a tumor in a subject having a cancer of epithelial cell origin is collected.
  • Suitable samples contain tissues or cells from the tumor of a subject having a cancer of epithelial cell origin.
  • the sample preferably comprises a biopsy sample, such as a tumor biopsy, primary tissue, metastatic tissue.
  • the specimen can be obtained by needle biopsy, image-guided biopsy, surgical (excisional) biopsy, shave/punch biopsy, endoscopic biopsy, laparoscopic biopsy and combinations thereof.
  • the tumor sample can be obtained from the patient by routine measures known to the person skilled in the art, i.e., biopsy (taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material). In this way, it is possible to obtain tissue for the method of the present invention. However, the tumor will not be removed (completely) during collection of a sample. In the sense of the present invention, "collecting sample from a tumor” is not intended to be directed a therapeutic treatment method.
  • the subject has an over-active EGFR.
  • Over-activity of EGFR is not synonymous to over-expression of EGFR.
  • Over-active of epidermal growth factor receptor (EGFR) is observed in many cancers, sometimes accompanied by gene amplification.
  • over-active EGFR refers to an increase in EGFR-driven phosphorylation-activity, not in the EGFR expression itself.
  • Samples are classified as EGFR over-active based on their somatic mutation profile.
  • a sample is stated EGFR over-active if it carries at least one activating mutations as defined below.
  • the over-active EGFR is caused by a mutation in EGFR or by a protein interaction with EGFR such that the mutation or the protein interaction causes the receptor to be conformationally altered and to become engaged in an active signaling state contributing to a disease condition.
  • the over-active of EGFR is caused by a mutation in the EGFR gene.
  • the EGFR gene is present on chromosome 7p11 .2 and has 28 exons coding for a transmembrane receptor protein of 464 amino acids.
  • exons 5-7 and 13-16 code for the ligand binding domain
  • exons 18-24 code for the tyrosine kinase domain and autophosphorylation occurs in the region encoded by exons 25-28.
  • the mutation affects at least one of the afore-mentioned domains.
  • EGFR mutations are in particular observed in exons 18-21.
  • the EGFR mutation is especially selected from a mutation that alters L858, T790, G719, L861 , S768; a deletion in exon 19;
  • the mutation includes:
  • a second preferred embodiment the over-active EGFR is caused by a protein interaction with EGFR such that the receptor to be conformationally altered and to become engaged in an active signaling state contributing to a disease condition.
  • the disease or disorder from which the subject is suffering is cancer, in particular cancer of epithelial cell origin.
  • This cancer includes primary cancer and secondary (metastatic) disease.
  • the cancer is selected from glioblastoma, melanoma, and lung, breast, ovary, prostrate, stomach, pancreas, bladder, head, neck, colon, colorectal and kidney cancer.
  • Especially the disease or disorder is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • step b) of the method according to the invention the level of phosphorylation of the phosphorylation site residue T59 in the SHP2 protein, is determined.
  • the level of phosphorylation can be determined by targeted mass spectrometric analysis on tumor tissue.
  • the determination can be also done by development of an antibody and/or IHC assay specifically targeting the p-site.
  • the antibodies for use in the invention may be produced by any suitable method known in the art. Such antibodies include, but are not limited to, polyclonal, monoclonal, humanized, phage display-derived antibodies or chimeric antibodies.
  • step c) of the method according to the invention the level of phosphorylation of at least one phosphorylation site, comprising residue T59 in the SHP2 protein, is compared to a control level.
  • a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject is likely to be responsive to an agent that inhibits the kinase activity of EGFR.
  • a significantly elevated level of phosphorylation of at least one phosphorylation site, comprising residue T59, compared to the control level indicates that the subject has an over-active EGFR.
  • control level refers to the level of phosphorylation at a given amino acid residue of a protein, in particular on the amino acid side chain, in a non-cancerous tissue.
  • control level refers to the level of phosphorylation at a given amino acid residue of a protein in a normal, non-tumorous tissue.
  • a "significantly elevated" level of phosphorylation is a level whose difference from the control level is statistically significant, using statistical methods that are appropriate and well-known in the art.
  • the phosphorylation of at least one phosphorylation site, comprising residue T59 of SHP2 in a subject having an over-active EGFR.
  • Methods for measuring the level of phosphorylation at an amino acid residue are conventional and routine. In general, the measurement relies on the existence of sets of antibodies that are specific for either the non-phosphorylated or the phosphorylated forms of a particular amino acid residue of interest in the context of a protein of interest (such as EGFR). Such antibodies are commercially available or can be generated routinely, using conventional procedures.
  • a synthetic peptide comprising an amino acid of interest from a protein of interest (either in the non-phosphorylated or phosphorylated form) is used as an antigen to prepare a suitable antibody.
  • the antibody can be polyclonal or monoclonal. Antibodies are selected and verified to detect only the phosphorylated version of the protein but not the non-phosphorylated version of the native or denatured protein, and vice-versa.
  • Such antibodies can be used in a variety of ways. For example, one can prepare whole cell lysates from patient samples and spot them in an array format onto a suitable substrate, such as nitrocellulose strips or glass slides. Preferably, the proteins in the samples are denatured before spotting. In general, the cells are spotted at serial dilutions, such as two-fold serial dilutions, to provide a wide dynamic range. Suitable controls, such as positive controls or controls for control levels, can be included. Each array is then probed with a suitable detectable antibody, as described above, to determine and/or to quantitate which amino acid residue(s) in the various proteins of interest are phosphorylated. Methods for immuno- quantitation are conventional.
  • suitable assays employing such antibodies to assess the level and/or degree of phosphorylation at a residue of interest include, e.g., Western blots, ELISA assays, immunhistochemistry, mass spectroscopy, and other conventional assays.
  • Suitable methods include those that can detect the phosphoprotein in a very small sample (e.g. about 200 cells). Alternatively, methods can be used that are suitable for a large sample size (e.g. FFPE tissue sections).
  • Assays to measure the presence and/or level of phosphorylated residues can be readily adapted to high throughput formats, e.g. using robotics, if desired.
  • the inventive method can further comprise determining the activation state of the EGFR.
  • the presence or absence of an activating mutation can be determined in a patient sample.
  • a subject having both an activating mutation and hyperphosphorylation at the SHP2 residues T59 can be determined in accordance with the present invention to be a responder, and thus selected for EGFR treatment.
  • hyperproliferative conditions such as cancers, precancerous conditions, metabolic disorders (e.g., diabetes), skin disorders or diseases, cardiovascular disease, hyperproliferative cell diseases or disorders, psoriasis, obesity, inflammatory airway disease, asthma, COPD, and neurological disorders. Patients suffering from those conditions can also be assayed by a method of the invention.
  • One aspect of the invention is a method for treating a subject suffering from a disease or disorder, in particular non-small cell lung cancer, having over-active EGFR, comprising the steps i) measuring the level of phosphorylation at SHP2 residue T59 in a sample from the subject and, if the levels of phosphorylation compared to a control level suggest that the subject is likely to be responsive to EGFR therapy, ii) administering EGFR treatment to the subject (administering an effective level of an EGFR inhibitor, such as an inhibitor of EGFR kinase activity).
  • an EGFR inhibitor such as an inhibitor of EGFR kinase activity
  • the EGFR inhibitor is selected from 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;
  • Tyrphostin 23 (RG-50810); Tyrphostin 25 (RG-50875); Tyrphostin 46; Tyrphostin 47 (RG- 50864, AG-213); Tyrphostin 51 ; ZD-6474; a derivative thereof, or a combination thereof.
  • the subject is a human patient suffering from NSCLC
  • the EGFR kinase inhibitor is selected from Iressa® (gefitinib), Tarceva® (erlotinib), Vizimpro® (dacomitinib), Gilotrif® (afatinib), or mixtures thereof.
  • Combinations of these agents, with each other or with conventional agents, such as chemotherapeutic agents, can be used.
  • compounds may be characterized as being "EGFR inhibitors,” “EGFR kinase inhibitors,” or “inhibitors of the EGFR pathway,” the present invention is not limited to the mechanism by which such agents achieve therapeutic efficacy.
  • a patient subset selected in accordance with the present invention may respond to EGFR treatment, although the mechanism of action may not be related to, or completely related to, modulation of the EGFR pathway.
  • a further aspect of the invention is the treatment of a patient identified as potential responder with a pharmaceutical composition comprising at least one inhibitor of EGFR kinase as defined above in an effective amount and a pharmaceutically acceptable carrier.
  • Such a pharmaceutical composition can be administered to a subject having an overactive EGFR, wherein the subject is determined by a method according to the invention to be likely to be responsive to an EGFR kinase inhibitor.
  • 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 as a kit.
  • a pharmaceutical composition of the invention comprises an anticancer-effective amount of the inhibitor.
  • An "anticancer effective amount,” as used herein, is an amount that is sufficient to effect at least a therapeutic response in the individual over a reasonable time frame. For example, it can ameliorate, at least to a detectable degree, the symptoms of a cancer, or can inhibit the growth of a tumor, etc.
  • composition can comprise a carrier, such as a pharmaceutically acceptable carrier.
  • a carrier such as a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • 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.
  • compositions suitable for oral, parenteral, aerosol, transdermal, topical, or other forms of administration will be evident to the skilled person.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired anti-cancer effective amount or effective concentration of the agent in the individual patient.
  • One skilled in the art also can readily determine and use an appropriate indicator of the "effective concentration" of the compounds of the present invention by a direct or indirect analysis of appropriate patient samples (e.g., blood and/or tissues).
  • an anticancer effective amount The dose of an inhibitory agent of the invention, or composition thereof, administered to an animal, particularly a human, should be sufficient to effect at least a therapeutic response in the individual over a reasonable time frame (an anticancer effective amount).
  • the exact 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 any disorder being treated, the particular agent or vehicle used, its mode of administration and the like.
  • the dose used to achieve a desired anticancer concentration in vivo will be determined by the potency of the particular inhibitory agent employed, the pharmacodynamics associated with the agent in the host, the severity of the disease state of infected individuals, as well as, in the case of systemic administration, the body weight and age of the individual.
  • the size of the dose also will be determined by the existence of any adverse side effects that may accompany the particular inhibitory agent, or composition thereof, employed. It is generally desirable, whenever possible, to keep adverse side effects to a minimum.
  • the inhibitors can be given at the same time, or the dosing can be staggered as desired.
  • the two or more drugs can also can be combined in a composition. Doses of each can be less when used in combination than when either is used alone.
  • a second embodiment of the invention a method for identifying over-active EGFR in subject, comprising the steps: i) collecting a sample from a tumor of a subject having a cancer of epithelial cell origin, in particular collecting a sample from a tumor of a subject having non-small cell lung cancer; ii) determining the level of phosphorylation of at least one phosphorylation site comprising residue T59 in the SHP2 protein; iii) comparing the level of phosphorylation of at least one phosphorylation site comprising residue T59 to a control level; wherein a significantly elevated level of phosphorylation of at least one phosphorylation site comprising residue T59 compared to the control level indicates that the subject has likely an over-active EGFR.
  • a third embodiment of the 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 inhibitor of EGFR kinase, comprising means for determining the level of phosphorylation of at least one phosphorylation site comprising residue T59 in the SHP2 protein, optionally in one or more containers.
  • the kit can be useful for predicting the response of a subject suffering from a disease or disorder, in particular non-small cell lung cancer, caused by an over-active EGFR, in particular cancer of epithelial origin, comprising reagents and/or devices for measuring the level of phosphorylation of at least one phosphorylation site comprising residue T59.
  • the kit may comprise reagents and/or devices for preparing a sample such as collecting a tissue and/or excising a sample from the tissue; for spotting test samples on a suitable surface, such as nitrocellulose strips or glass slides; for performing immunoquantitation such as labeled antibodies, or reagents for labeling antibodies; instructions for performing a method of the invention; etc.
  • the components of the kit may, optionally, be packaged in one or more containers.
  • kits according to the invention can be in experimental applications to identify a phosphorylation pattern that is predictive of the response of a subject to a therapeutic agent.
  • a skilled person will recognize components of kits suitable for carrying out any of the methods of the invention.
  • the kit according to the invention comprises suitable buffers; one or more containers or packaging material; and/or instructions for performing the method.
  • the reagents of the kit may be in containers in which the reagents are stable, e.g. in lyophilized form or stabilized liquids.
  • the reagents may also be in single use form, e.g., in single dosage form.
  • Companion diagnostic tests & compounds to determine e.g., whether a mutation exists and is linked to a specific treatment can be found on the FDA database https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved- companion-diagnostic-devices-in-vitro-and-imaging-tools.
  • a fourth embodiment of the invention is the use of T59 on SHP2 as a marker that can predict the response of a subject having an over active-EGFR to an inhibitor of EGFR kinase activity.
  • This use comprises determining amino acid residues, in particular determining the phosphorylation of T59 on SHP2, in one or more proteins from the EGFR signaling pathway that are over- or under-phosphorylated, compared to a control level.
  • Figure 1 shows EGFR mutation frequencies. EGFR-activity is influenced by specific genetic mutations. The somatic variations that are detected in tumor samples from patients can be utilized as primary indicator for increased EGFR kinase-activity. Figure 1 shows those 15 mutations that arise in at least two samples from a frequency counts of all 82 distinct mutations reported for EGFR throughout the samples in a NSCLC cohort.
  • Figure 2 shows Iog2-transformed intensities for PTPN11 -T59. Based on the mutations presented in Figure 1 , it is possible to classify samples based on their EGFR activity. Samples that carry solely somatic EGFR mutations with increasing effects on kinase activity, it can be classified as “Activated EGFR”. Vice versa, samples that carry none or other mutations than EGFR-activating ones can be seen as “Wild-Type EGFR”. Based on this classification, we 435 Wild-Type EGFR samples and 54 activated EGFR samples were identified. When analyzing the Iog2 transformed intensities of PTN11-T59, a significant difference between both groups occurred.
  • An example threshold of t > 16 shows that this phosphosite acts as suitable group separator.
  • samples with activated EGFR commonly show PTN11 -T59 intensities of over 16, while Wild-Type EGFR is below this threshold.
  • samples that carry multiple but only partially EGFR-activating mutations are classified as “Wild-Type EGFR” but also show high PTN11-T59 intensities above 16, which indicates that this method is able to find samples with activated EGFR, that might exceed the pure level of somatic mutation derived kinase-activity.
  • FIG. 3 Ischemia time impact. This figure represents the sample Iog2-transformed intensities of PTN11 -T59 as Figure 2. The data is divided into ischemia time intervals of 5 minutes, i.e. intervals that describe the time that passed between tissue extraction and freezing. By this method it was possible to track if PTN11 -T59 is stable over time. The difference between samples with “Wild-Type EGFR” and “Activated EGFR” stays intact over the complete time periods and none of the groups shows a notable downwards trend with increasing time.
  • PTN11-T59 biomarker according to the invention for EGFR kinase activity can be seen as threshold based binary classification where high scores are associated with elevated kinase activity.
  • samples with overactivated EGFR kinase activity should be enriched among the highest phosphorylation measurements.
  • the better this enrichment the higher the area under the curve (AUC) of the corresponding receiver operating characteristic (ROC) curve.
  • AUC area under the curve
  • ROC receiver operating characteristic
  • the curve reflects the concordance of EGFR overactive samples as defined by genetic mutations, and those samples with a high phosphorylation signal (seen here as true positive rate TPR).
  • the false positive rate here reflects the presence of EGFR wild-type samples for which a high phosphorylation signal is observed. The latter could indicate biological EGFR activating processes independent of the genetic mutation state of EGFR.
  • figure 4 shows that p-sites of ERBB3 (ERBB3-*) and ERK1 and ERK2 (ERK1/2-*) have a low predictive power compared to the pure PTN11-T59 model according to the invention.
  • the method to predict elevated EGFR kinase activity according to the present invention relies on a p-site-specific measurement. Hence, general protein phosphorylation is not a sufficient source of information.
  • PR precision-recall
  • the graphic is constructed analogous to the ROC curve, but here one compares the precision (positive predictive value, PPV), the fraction of genetically defined EGFR-overactivated samples relative to the total number of samples predicted to be EGFR-overactivated using the current invention (on the y-axis) with the recall, which is equivalent to the true positive rate (TPR on the x-axis).
  • PR precision-recall
  • Figure 6 A graphical representation of markers that could be useful in EGFR therapy.
  • patients who appear suitable for EGFR therapy are defined from all NSCLC patients. This is currently done using mutations in EGFR.
  • a single phosphosite on PTN11 is used to define the over activation of the EGFR.
  • Zhang et aL, 2021 lists attempts to identify markers for none- responders from lung cell lines, and uses on EGFR mutations to define activity of the EGFR.
  • Li et aL, 2014 defines a negative selection criteria response to therapy. Both approaches are a negative selection criterion that has nothing to do with EGFR activity per se.

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Abstract

L'invention concerne un procédé pour sélectionner des patients qui peuvent répondre à un traitement par des inhibiteurs de tyrosine kinase (TKI). L'invention concerne en outre un kit de diagnostic pour la prédiction d'une réponse à un inhibiteur de l'activité de la kinase EGFR. De plus, l'invention concerne l'utilisation de T59 phosphorylé sur la SHP2 en tant que biomarqueur pour prédire l'activité de la kinase EGFR.
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Citations (2)

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WO2007027916A2 (fr) 2005-08-31 2007-03-08 Cell Signaling Technology, Inc. Reactifs de detection de la phosphorylation proteinique dans la voie de signalisation de carcinome
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