WO2005107803A2 - Procédés de détermination du pronostic et du traitement de sujets souffrant d'un cancer du poumon - Google Patents

Procédés de détermination du pronostic et du traitement de sujets souffrant d'un cancer du poumon Download PDF

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WO2005107803A2
WO2005107803A2 PCT/US2005/015644 US2005015644W WO2005107803A2 WO 2005107803 A2 WO2005107803 A2 WO 2005107803A2 US 2005015644 W US2005015644 W US 2005015644W WO 2005107803 A2 WO2005107803 A2 WO 2005107803A2
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egfr
subject
pinl
ppinl
level
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WO2005107803A3 (fr
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Janusz M. Sowadshi
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Vernalis Plc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • EGFR epidermal growth factor receptor
  • EGFR is abnormally expressed in many human solid tumors, including non-small cell lung cancer (NSCLC), prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancers (Salomon, et al, Crit. Rev. Oncol. Hematol. 1995; 19: 183-232). From precancerous lesions to malignant tumors, elevated EGFR tyrosine kinase activity has been detected at all stages of tumor progression (Kelloff et al. Cancer Epidemiol Biomarker Prev 1996;5:657-666; Kurei et al, Clin. Cancer Res. 1996;2:1787-1793). The EGFR pathway contributes to a number of processes involved in tumor survival and growth including cell proliferation, inhibition of apoptosis, angiogenesis, and metastasis.
  • NSCLC non-small cell lung cancer
  • prostate breast
  • colorectal head and neck
  • ovarian gastric and pancreatic cancers
  • the first-line treatment for patients with EGFR related solid tumors e.g., NSCLC
  • platinum-based combination chemotherapy e.g., carboplatin plus paclitaxel, cisplatin plus docetaxel, or cisplatin plus gemcitobine or vinorelbine.
  • these regimens have limited efficacy, with a number of combination regimens showing 2-year survival rates of less than 15% (Kelly et al, J. Clin. Oncol. 2001; 19-3210-3218).
  • EGFR-TK inhibitors there are also several small-molecule EGFR-TK inhibitors that have been approved or are in clinical development, such as STI571 (imatinib mesylate, GleevecTM; Novartis Pharmaceuticals Corporation, East Hanover, NJ); gefitinib (Iressa®; Astra- Zeneca Pharmacruticals LP; Wilmington, DE), and OSI-774 (erlotinib, Tarceva TM; OSI Pharmaceuticals; Melville, NE/Genetech; South San Francisco, CA); CI-1033 (Pfizer Inc.; New York, NY) and GW572016 (GlaxoSmithKline; Research Triangle Park, NC).
  • STI571 imatinib mesylate, GleevecTM; Novartis Pharmaceuticals Corporation, East Hanover, NJ
  • gefitinib Iressa®
  • OSI-774 erlotinib, Tarceva TM
  • OSI Pharmaceuticals Melville, NE/Genetech; South
  • PPIases The peptidyl-prolyl cis-trans isomerases (PPIases), or rotamases, are a family of ubiquitous enzymes that catalyze the cis/trans isomerization of the peptide bond on the n-terminal side of proline residues in proteins (Hunter, Cell 92:141-142, 1998). PPIases are divided into three classes, cyclophilins (Cyps), FK-506 binding proteins (FKBPs) and the Pinl/parvulin class. Pinl is a highly conserved protein that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Rananathan, R.
  • Pinl contains an N-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol Biol 65:113-32).
  • This phosphorylation-dependent interaction targets Pinl to a subset of phosphorylated substrates, including Cdc25, Wee 1, Mytl, Tau-Rad4, and the C-terminal domain of RNA polymerase II large domain (Crenshaw, D.G., et al (1998) Embo. J 17: 1315-27; Shen, M. (1998) Genes Dev. 12:706-20; Wells, NJ. (1999) J Cell. Sci. 112: 3861-71).
  • Pinl is involved in the onset and progression of many types of cell proliferative and neurodegenerative disorders (see, for example, Lu, K.P. (2003) Cancer Cell 4:175-80; Wulf, G. et al. (2003) Breast Cancer Res. 5:76-82; Ryo, A. et al. (2003) J. Cell Sci. 116: 773-83; Wulf, G.M. et al. (2001) EMBO J. 20:3459-72; Liou et al. (2003) Nature 424:556-61; Lu, P.J. et al. (1999) Nature 399:784-8).
  • Pinl activity has also been shown to restore the conformation and function of phosphorylated tau by promoting its dephosphorylation, indicating that Pinl is involved in neurodegeneration (Liou, Y.C. et al. (2003) Nature 424:556-61). Accordingly, the present invention is based on the use of for modulators of Pinl , and methods of selecting and treating subjects with disorders characterized by the misexpression of Pinl and/or EGFR, e.g., cancer.
  • the present invention is based, at least in part, on the discovery that there is a correlation between EGFR and pPinl expression and the survival of patients having lung cancer.
  • the ability of a clinician to obtain information regarding the expression of EGFR and Pinl would be advantageous in the determination of which subjects are likely to benefit from treatment with a Pinl modulator and/or an EGFR modulator, and in determining the prognosis of a subject with a cell proliferative disorder, e.g., cancer.
  • the instant invention provides methods to determine which subjects in a population would benefit from treatment with a Pinl modulator and/or an EGFR modulator.
  • the instant invention also provides methods for determining the prognosis of a subject with a cell proliferative disorder.
  • the invention further provides methods to monitor the efficacy of treatment of individuals having a cell proliferative disorder with a Pinl modulator and/or an EGFR modulator.
  • kits are provided to perform the methods of the invention.
  • the invention provides a method for treating a subject having a cell proliferative disorder that has been preselected based on increased levels of EGFR by administering a Pinl modulator and/or an EGFR modulator to the subject such that treatment of the subject occurs.
  • the invention provides a method for treating a subject having a cell proliferative disorder that has been preselected based on decreased levels of phosphorylated Pinl and increased levels of EGFR by administering a Pinl modulator and/or an EGFR modulator to the subject such that the treatment of the subject occurs.
  • the invention provides a method of treating a subject having a cell proliferative disorder that has been preselected based on the increase levels of unphosphorylated Pinl and EGFR, comprising administering a Pinl modulator and/or an EGFR modulator to the subject such that the treatment of the subject occurs.
  • the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein an increase in the level of pPinl is indicative of good prognosis.
  • the cell proliferative disorder is an EGFR related disorder.
  • the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein a decrease in the level of pPinl is indicative of poor prognosis.
  • the cell proliferative disorder is an EGFR disorder.
  • the invention provides a method of monitoring the efficacy of treatment of a subject by determining the levels of phosphorylated and/or unphosphorylated Pinl after the administration of a Pinl modulator and/or EGFR modulator to the subject; wherein a change in the level of phosphorylated Pinl and/or unphosphorylated Pinl and/or EGFR is indicative of the efficacy of treatment.
  • the level of phosporylated Pinl is increased.
  • the level of unphosphorylated Pinl is decreased.
  • the level of EGFR is decreased.
  • the level of EGFR is decreased and the level of phosphorylated Pinl is increased.
  • Embodiments of the present invention include methods in which the cell proliferative disorder is an EGFR related cancer.
  • the cell proliferative disorder is a solid tumor including, but not limited to, lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancer.
  • the cell proliferative disorder is lung cancer.
  • the lung cancer is adenocarcinoma, non-small cell or squamous cell lung cancer.
  • the tumor is non-responsive to traditional cancer therapies, e.g., surgery, radiotherapy, chemotherapy and endocrine therapy.
  • the levels of Pinl are determined by measuring the level of phosphorylated and/or unphosphorylated Pinl and/or EGFR using immunohistochemistry.
  • the immunohistochemistry is performed using antibodies specific for phosphorylated, unphosphorylated Pinl, or EGFR.
  • the pPinl levels are determined using an antibody specific for pPinl .
  • the levels of pPinl and EGFR are determined using antibodies specific for pPinl and EGFR, respectively.
  • the levels of pPinl and/or EGFR are determined using fluorescence in situ hybridization (FISH).
  • the subject is classified into one of the following groups based on the level, location and phosphorylation state of Pinl, e.g., nPinlStr, nPinlMod, nPinlweak, nPinlNeg.
  • the subject is classified based on the level or amplification of EGFR as described herein.
  • the subject is classified based on the level of EGFR.
  • the methods include optionally, and one or more other anti-cancer treatments including, but not limited to, surgery, radiotherapy, chemotherapy, and endocrine therapy.
  • the other chemotherapy is an EGFR inhibitor such as, but not limited to, STI571 (imatinib mesylate, GleevecTM; Novartis Pharmaceuticals Corporation, East Hanover, NJ); gefitinib (Iressa®; Astra-Zeneca Pharmacruticals LP; Wilmington, DE), and OSI-774 (erlotinib, Tarceva TM; OSI Pharmaceuticals; Melville, NE/Genetech; South San Francisco, CA); CI-1033 (Pfizer Inc.; New York, NY) and GW572016 (GlaxoSmithKline; Research Triangle Park, NC).
  • the subject is further classified based on the presence of a somatic mutation in EGFR.
  • the subject is treated with a Pinl modulator when the subject is classified as not having a somatic mutation, e.g., in the tyrosine kinase domain, and is non-responsive to small molecule EGFR inhibitors (e.g., gefitinib).
  • a somatic mutation e.g., in the tyrosine kinase domain
  • small molecule EGFR inhibitors e.g., gefitinib
  • the invention provides a kit for selecting a subject that would benefit from treatment with a Pinl modulator and/or an EGFR modulator including a phosphorylated Pinl specific antibody and/or an unphosphorylated Pinl specific antibody and/or an EGFR specific antibody, and directions for use.
  • the invention provides a kit for determining the prognosis of a subject with a cell proliferative disorder comprising an antibody specific for pPinl and instructions for use.
  • the antibody enclosed in the kit is a monoclonal or a polyclonal antibody.
  • the kit can contain a second antibody specific for specific for EGFR.
  • Figure 1 depicts the polypeptide sequence of Pinl (SEQ ID NO:l).
  • the serine residue identified in Lu et al. ((2002) J Biol Chem. 277: 2381-4) is underlined and in bold.
  • Figure 2 is a bar graph that depicts the fraction of samples with cytoplasmic and nuclear pPINl staining.
  • Figure 3 depicts a survival curve based on subject's expression of pPinl.
  • the graph depicts the fraction of subjects surviving as a function of time (in months) and negative, weak, moderate or strong pPINl expression.
  • Figure 4 depicts a survival curve based on the two group classification of pPinl expression.
  • Figure 5 depicts a survival curve that shows the influence of cytoplasmic pPINl expression.
  • Figures 6A and 6B is a bar graph that depicts the fraction of samples with nuclear and cytoplasmic Pinl staining as a function of pPinl staining, respectively.
  • Figure 7 A and 7B depict the fraction of samples that are positive for EGFR (IHC) or the fraction of altered EGFR samples (FISH) as a function of pPinl staining, respectively.
  • Figure 8 and 9 depict survival curves of subjects based on the expression of pPinl/EGFR co-expression and pPinl/EGFR co-alterations. The graphs depict the fraction of subjects surviving as a function of time (in months).
  • biological sample includes solid and body fluid samples.
  • the biological samples of the present invention may include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • solid biological samples include samples taken from the lung, prostate, breast, colorectal, head and neck, ovaries, gastric and pancreas.
  • body fluid samples include samples taken from the blood, serum, cerebrospinal fluid, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears.
  • Samples for use in the methods of the invention can be obtained by standard methods including venous puncture and surgical biopsy.
  • the biological sample is a tissue or organ sample obtained by surgery or by needle biopsy.
  • Pinl status indicates a classification of a subject into one of a defined series of groups based on the presence and level of phosphorylated and unphosphorylated Pinl. Exemplary classifications of Pinl status defined herein in the Examples. However, a skilled artisan could further define the categories presented herein to better define Pinl status. For example, Pinl status can be represented as ratio of phosphorylated Pinl to unphosphorylated Pinl. The presence and level of Pinl can be determined using art recognized techniques, e.g., immunohistochemistry using Pinl specific antibodies. Subjects can be treated for Pinl -associated states based on their Pinl status.
  • nuclear Pinl is intended to include Pinl polypeptide that is localized to the nucleus of a cell. In certain embodiments, nuclear Pinl is predominantly phosphorylated.
  • cytoplasmic Pinl is intended to include Pinl polypeptide that is localized to the cytoplasm of a cell. In certain embodiments, cytoplasmic Pinl is predominantly unphosphorylated.
  • phosphorylation state is intended to denote that the Pinl polypeptide can exist in either a phosphorylated or unphosphorylated state.
  • the phosphorylation state denotes whether the Pinl in a biological sample is phosphorylated or unphosphorylated, or the relative ratios of phosphorylated to unphosphorylated Pinl in a sample.
  • Lu et al. (2002) J Biol Chem. 277: 2381-4
  • Pinl -associated state or "Pinl associated disorder” includes disorders and states (e.g., a disease state) that are associated with the abnormal activity of Pinl.
  • This abnormal activity can be as a result of the misexpression or misregulation of the production, degradation, or regulation of Pinl, e.g., the phosphorylation dephosphorylation of Pinl .
  • Pinl associated disorders that are related to higher than necessary levels of Pinl can be caused by (1) an increase in the level of transcription or translation, or a decrease in the level of degradation, of Pinl such that an abnormally high amount of Pinl polypeptide is present in a cell, or (2) the amount Pinl that is present in the unphosphorylated, i.e., active form, is abnormally high due to either an increase in the dephosphorylation of Pinl or a decrease in the phosphorylation of Pinl.
  • Pinl disorders are often associated with abnormal cell growth, abnormal cell proliferation, or misexpression of Pinl (e.g., Pinl protein or nucleic acid).
  • Pinl -associated states include states resulting from an elevation in the expression of cyclin Dl and/or Pinl .
  • Pinl -associated states also include states resulting from an elevation in the phosphorylation level of c-Jun, particularly phosphorylation of c-Jun on Ser -Pro and/or from an elevation in the level of c-Jun amino terminal kinases (JNKs) present in a cell.
  • Pinl -associated states include neoplasia, cancer, undesirable cell growth, and/or tumor growth.
  • Pinl -associated states include states caused by DNA damage, an oncogenic protein (i.e.
  • Pinl -associated state is also intended to include diseases or disorders caused by, or associated with, deregulation of genes and/or gene products involved in a biological pathway that includes Pinl and/or cyclin Dl (e.g. beta-catenin, APC or WNT). Beta-catenin, APC and WNT have been linked to cancer development as demonstrated in Biocliim Biophys Acta. (2003) 1653: 1-24 and Eur J Surg Oncol. (2003) 29: 107-117.
  • Pinl associated states further include disorders and states associated with regulation or activity of Pinl in the brain, e.g., Alzheimer's disease, wherein the phosphorylation state of tau is influenced by the activity of Pinl.
  • EGFR related disorder includes cell proliferative disosrders or states associated with the abnormal activity of EGFR. This abnormal activity can be as a result of the misexpression or misregulation of the production, degradation, or regulation of EGFR.
  • misregulation of EGFR is associated with a mutation in the EGFR gene that results in increased EGFR tyrosine kinase activity, e.g., a somatic mutation such as described by Lynch et al, supra, and Paez, et al, supra.
  • misexpression includes a non-wild type pattern of gene expression.
  • Expression as used herein includes transcriptional, post transcriptional, e.g., mRNA stability, translational, and post translational stages.
  • Misexpression includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as
  • Misexpression includes any expression from a transgenic nucleic acid. Misexpression includes the lack or non-expression of a gene or transgene, e.g., that can be caused by a deletion of all or part of the gene or its control sequences.
  • the term "carcinoma” includes malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, melanomas, choriocarcinoma, and carcinomas of the cervix, lung, head and neck, colon, and ovary.
  • carcinoma also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • adenocarcinoma includes carcinomas derived from glandular tissue or a tumor in which the tumor cells form recognizable glandular structures.
  • the therapeutic methods of the present invention can be applied to cancerous cells of mesenchymal origin, such as those producing sarcomas (e.g., fibrosarcoma, myxosarcoma, liosarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosardcoma, lympangiosarcoma, synoviosarcoma or mesothelisosarcoma); leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease; sarcomas such as leiomysarcoma or rhabdomysarcoma, tumors of epithelial origin such as squamous cell carcinoma, basal cell carcinoma,
  • Additional cell types amenable to treatment according to the methods described herein include those giving rise to mammary carcinomas, gastrointestinal carcinoma, such as colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region.
  • Examples of cancers amenable to treatment according to the methods described herein include colorectal cancers, e.g., colon cancer.
  • the language "inhibiting undesirable cell growth” is intended to include the inhibition of undesirable or inappropriate cell growth.
  • the inhibition is intended to include inhibition of cell proliferation, including rapid proliferation. For example, undesirable cell growth can result in benign masses or malignant tumors.
  • Examples of benign conditions which result from inappropriate cell growth or angiogenesis are diabetic retinopathy, retrolental f ⁇ brioplasia, neovascular glaucoma, psoriasis, angiofibromas, rheumatoid arthritis, hemangiomas, Karposi's sarcoma, and other conditions or dysfunctions characterized by dysregulated endothelial cell division.
  • tumor growth or “inhibiting neoplasia” includes the prevention of the growth of a tumor in a subject or a reduction in the growth of a preexisting tumor in a subject, or can be the inhibition of the metastasis of a tumor from one site to another.
  • tumor is intended to encompass both in vitro, e.g., in cell culture, and in vivo tumors that form in any organ or body part of the subject.
  • the tumors preferably are tumors sensitive to the Pinl -modulating compounds of the present invention. .
  • cancer includes malignancies characterized by deregulated or uncontrolled cell growth, for instance carcinomas, sarcomas, leukemias, and lymphomas.
  • carcinomas for instance carcinomas, sarcomas, leukemias, and lymphomas.
  • cancer and “tumor” may be used interchangeably herein.
  • the term “cancer” includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • Pinl modulating agents may be used to treat, inhibit, and/or prevent undesirable cell growth, neoplasia, neurodegenerative diseases, and/or cancer in a subject.
  • Pinl modulating compounds may be used to inhibit Pinl activity in a subject.
  • Pinl modulating compounds may be used to inhibit cyclin Dl expression in a subject.
  • Pinl modulating compounds of the invention can be used to treat subjects having Alzheimer's disease.
  • Pinl modulators have a Kj for Pinl of less than 0.2mM, less than 0. ImM, less than 750 ⁇ M, less than 500 ⁇ M, less than 250 ⁇ M, less than 100 ⁇ M, less than 50 ⁇ M, less than 500 nM, less than 250nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 2 nM.
  • the language "Pinl modulating compound” refers to compounds that modulate, e.g., inhibit, promote, or otherwise alter, the activity of Pinl. Pinl modulating compounds include both Pinl agonists and antagonists.
  • the Pinl modulating compounds include compounds that interact with the peptidyl prolyl isomrease domain (PPI) and/or the WW domain of Pinl .
  • the Pinl modulating compound is substantially specific to Pinl.
  • the phrase "substantially specific for Pinl" is intended to include inhibitors of the invention that have a Kj or K d that is at least 2, 3, 4, 5, 10, 15, or 20 times less than the Kj or K d for other peptidyl prolyl isomerases, e.g., hCyP-A, hCyP-B, hCyP-C, NKCA, hFKBP-12, hFKBP-13, and hFKBP-25.
  • Pinl modulating compounds include compounds described in U.S. Provisional Application No. 60/537,171, filed January 16, 2004, entitled “Pinl- Modulating Compounds and Methods of Use Thereof; U.S. Application No. 10/361,206 filed March 3, 2003, entitled “Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No. 10/379,410, filed March 3, 2003, entitled “Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No: 10/379,404, filed March 3, 2003, entitled “Pinl -Modulating Compounds and Methods of Use Thereof; U.S. Application Serial No.
  • the Pinl inhibiting compounds include compounds that interact with the PPI and/or the WW domain of Pinl.
  • preselected is intended to mean that a subject has been identified based on their level and/or phosphorylation state of Pinl to be likely to benefit from treatment with a Pinl modulator. In certain embodiments, a subject is preselected based on the levels of unphosphorylated Pinl, phosphorylated Pinl, or the relative amounts of phosphorylated and unphosphorylated Pinl .
  • prognosis is intended to mean the probable course and outcome of a disease, e.g., a Pinl associated disease. In certain embodiments, the term is intended to mean the likelihood that a subject will live longer than the average length of time that a population of subjects with a similar disease will live. In related embodiments, a subject's prognosis is indicative of the aggressiveness and course of treatment that a subject will receive.
  • This invention relates to methods of diagnosis, treatment and prognosis of subjects with a cell proliferative disorder such as cancer, e.g., an EGFR related disorder.
  • Pinl is a highly conserved protein (SEQ ID NO:l) that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Rananathan, R. et al. (1997) Cell 89:875-86; Yaffe, et al. 1997, Science 278:1957-1960; Shen, et al. 1998,Genes Dev. 12:706-720; Lu, et al. 1999, Science 283:1325-1328; Crenshaw, et al. 1998, Embo J.
  • Pinl contains an N-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol. Biol. 65:113-32). This phosphorylation-dependent interaction targets Pinl to a subset of phosphorylated substrates, including Cdc25, Wee 1, Mytl, Tau-Rad4, and the C- terminal domain of RNA polymerase II large domain (Crenshaw, D.G., et al. (1998) Embo. J. 17:1315-27; Shen, M. (1998) Genes Dev. 12:706-20; Wells, NJ. (1999) J. Cell. Sci. 112: 3861-71).
  • Pinl activity is essential for cell growth; depletion or mutations of Pinl cause growth arrest, affect cell cycle checkpoints and induce premature mitotic entry, mitotic arrest and apoptosis in human tumor cells, yeast or Xenopus extracts (Lu, et al. 1996, Nature 380:544-547; Winkler, et al. 200, Science 287:1644-1647; Hani, et al. 1999. J. Biol. Chem. 274:108-116).
  • Pinl is dramatically misexpressed in human cancer samples and the total level or concentration of Pinl is correlated with the aggressiveness of tumors.
  • inhibition of Pinl by various approaches including Pinl antisense polynucleotides or genetic depletion, kills human and yeast dividing cells by inducing premature mitotic entry and apoptosis.
  • the invention further provides a method of determining the prognosis of a subject with an EGFR related cell proliferative disorder, e.g., lung cancer, comprising determining the levels of pPinl and EGFR in a biological sample.
  • the sequence of the EGFR polypeptide can be found at, for example, SwissProt Accession Number P00533.
  • the invention provides a method of detecting the presence and amount of unphosphorylated Pinl and/or phosphorylated Pinl in a biological sample.
  • the invention also provides a method of determining the amount of phosphorylated Pinl relative to the amount of unphosphorylated Pinl in a sample.
  • the methods of the invention may use antibodies that recognize phosphorylated Pinl and unphosphorylated Pinl, antibodies that are specific for phosphorylated Pinl, and antibodies that are specific for unphosphorylated Pinl .
  • Antibodies that are specific for Pinl are described in US patent 6,596,848, the entire contents of which are incorporated herein by reference.
  • Phosphorylation specific Pinl antibodies are commercially available from, for example, Cell Signaling Technology (Beverly, MA).
  • the invention also provides methods of detecting levels of Pinl in combination with EGFR.
  • Levels of EGFR can be determined using antibodies as described in the Examples set forth below, or using nucleic acid probes, specific for the EGFR coding sequence uisng methods standard to one of skill in the art.
  • the phrase "antibodies specific for phosphorylated Pinl” is intended to include antibodies that preferentially bind to an antigen of Pinl that contains a phosphorylated residue.
  • Antibodies that are specific for phosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein).
  • an antibody that is specific for phosphorylated Pinl has more affinity for phosphorylated Pinl than it does unphosphorylated Pinl .
  • the antibody specific for phosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to phosphorylated Pinl as it does unphosphorylated Pinl .
  • the antibody specific for phosphorylated Pinl recognizes a Pinl molecule that is phosphorylated on serine 16 of SEQ ID NO: 1.
  • antibodies specific for unphosphorylated Pinl is intended to include antibodies that preferentially bind a Pinl polypeptide that is not phosphorylated. Antibodies that are specific for unphosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein). Further, an antibody that is specific for unphosphorylated Pinl has more affinity for unphosphorylated Pinl than it does phosphorylated Pinl. In certain embodiments, the antibody specific for unphosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to unphosphorylated Pinl as it does phosphorylated Pinl. In at least one embodiment, the antibody specific for unphosphorylated Pinl recognizes an epitope of Pinl that comprising a residue that is capable of being phosphorylated, e.g., serine 16 of SEQ ID NO:l.
  • Polyclonal antibodies for use in the present invention can be produced by immunizing animals, usually a mammal, by multiple subcutaneous or intraperitoneal injections of an immunogen (antigen) and an adjuvant as appropriate.
  • animals are typically immunized against a protein, peptide or derivative by combining about 1 ⁇ g to 1 mg of protein capable of eliciting an immune response, along with an enhancing carrier preparation, such as Freund's complete adjuvant, or an aggregating agent such as alum, and injecting the composition intradermally at multiple sites.
  • Animals are later boosted with at least one subsequent administration of a lower amount, as 1/5 to 1/10 the original amount of immunogen in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Animals are subsequently bled, serum assayed to determine the specific antibody titer, and the animals are again boosted and assayed until the titer of antibody no longer increases (i.e., plateaus).
  • Such populations of antibody molecules are referred to as "polyclonal" because the population comprises a large set of antibodies each of which is specific for one of the many differing epitopes found in the immunogen, and each of which is characterized by a specific affinity for that epitope.
  • An epitope is the smallest determinant of antigenicity, which for a protein, comprises a peptide of six to eight residues in length (Berzofsky, J. and I. Berkower, (1993) in Paul, W., Ed., Fundamental Immunology-,
  • the polyclonal antibody fraction collected from mammalian serum is isolated by well known techniques, e.g. by chromatography with an affinity matrix that selectively binds immunoglobulin molecules such as protein A, to obtain the IgG fraction.
  • the specific antibodies may be further purified by immunoaffinity chromatography using solid phase-affixed immunogen.
  • the antibody is contacted with the solid phase-affixed immunogen for a period of time sufficient for the immunogen to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex.
  • Bound antibodies are eluted from the solid phase by standard techniques, such as by use of buffers of decreasing pH or increasing ionic strength, the eluted fractions are assayed, and those containing the specific antibodies are combined.
  • Monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies can be prepared using a technique which provides for the production of antibody molecules by continuous growth of cells in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497; see also Brown et al.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • a monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response.
  • an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response.
  • the mammal is a rodent such as a rabbit, rat or mouse.
  • the mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described.
  • a suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody.
  • the animal e.g., mouse
  • antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood.
  • Spleen cells are preferred, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art.
  • the antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line.
  • Mouse lymphocytes give a high percentage of stable fusions with mouse homologous myelomas, however rat, rabbit and frog somatic cells can also be used.
  • Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol.
  • a fusing agent such as polyethylene glycol.
  • Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, MD.
  • the fusion-product cells which include the desired hybridomas, are cultured in selective medium such as HAT medium, designed to eliminate unfused parental myeloma or lymphocyte or spleen cells.
  • selective medium such as HAT medium
  • Hybridoma cells are selected and are grown under limiting dilution conditions to obtain isolated clones.
  • the supematants of each clonal hybridoma is screened for production of antibody of desired specificity and affinity, e.g., by immunoassay techniques to determine the desired antigen such as that used for immunization.
  • Monoclonal antibody is isolated from cultures of producing cells by conventional methods, such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (Zola et al, Monoclonal Hybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press, 1982).
  • Hybridomas produced according to these methods can be propagated in culture in vitro or in vivo (in ascites fluid) using techniques well known to those with skill in the art.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No.
  • Labeled antibody as used herein includes antibodies that are labeled by a detectable means and includes enzymatically, radioactively, fluorescently, chemiluminescently, and/or bioluminescently labeled antibodies.
  • an antibody can be detectably labeled is by linking the same to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the Pinl -specific or a EGFR-specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-N- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-NI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays.
  • a description of a radioimmune assay (RIA) may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audioradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: H, I, S, C, and preferably I.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • An antibody can also be detectably labeled using fluorescence emitting metals such as Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • An antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label an antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the invention provides methods for evaluating subjects for the level and phosphorylation state of Pinl .
  • the invention provides methods for evaluating subjects for the level on Pinl and EGFR. These results can be used to preselect a subject for treatment with a Pinl modulator and/or and EGFR modulator.
  • the amount of phosphorylated and/or unphosphorylated Pinl and/or EGFR in a biological sample may be determined by a radioimmunoassay, an immunoradiometric assay, enzyme immunoassay, and /or by immunohistochemistry using antibodies specific for phosphorylated Pinl or unphosphorylated Pinl, or EGFR, respectively.
  • Radioimmunoassay is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. radioactively labeled) form of the antigen.
  • radioactive labels for antigens include 3 H, 14 C, and 125 I.
  • the concentration of phosphorylated and/or unphosphorylated Pinl and/or EGFR in a sample is measured by having the antigen in the sample compete with a labeled (i.e. radioactively) antigen for binding to an antibody to the antigen.
  • the labeled antigen is present in a concentration sufficient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.
  • the antigen-antibody complex In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen.
  • One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum.
  • Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with formalin-killed S. aureus.
  • Yet another method for separating the antigen-antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (i.e.
  • the concentration of antigen in the biological sample can be determined.
  • An “Immunoradiometric assay” is an immunoassay in which the antibody reagent is radioactively labeled.
  • An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein e.g., rabbit serum albumin (RSA).
  • the multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen.
  • the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere.
  • sample antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere.
  • the antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites.
  • the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined.
  • the amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.
  • the most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA).”
  • ELISA Enzyme-Linked Immunosorbent Assay
  • ELISA is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. enzyme linked) form of the antibody.
  • an antibody i.e. to phosphorylated and unphosphorylated Pinl
  • a solid phase i.e. a microtiter plate
  • a labeled i.e. enzyme linked
  • enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and ⁇ -galactosidase.
  • the enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be assayed for.
  • antibody is incubated with a sample containing phosphorylated and unphosphorylated Pinl .
  • the antigen-antibody mixture is then contacted with an antigen-coated solid phase (i.e. a microtiter plate).
  • an antigen-coated solid phase i.e. a microtiter plate.
  • a labeled (i.e. enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.
  • an "immunohistochemistry assay” a section of tissue for is tested for specific proteins by exposing the tissue to antibodies that are specific for the type of Pinl protein that is being assayed (e.g., phosphorylated and unphosphorylated Pinl.
  • the antibodies are then visualized by any of a number of methods to determine the presence and amount of the protein present. Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or ⁇ -galactosidase), or chemical methods (e.g.,
  • the subject can be classified based on the level of phosphorylated and/or unphosphorylated Pinl and/or EGFR. Exemplary classifications provided in the Examples. The data presented in the Examples indicates that the majority of phosphorylated Pinl localizes to the nucleus while the majority of unphosphorylated Pinl localizes to the cytoplasm.
  • subjects with high levels of unphosphorylated Pinl localized in the cytoplasm or low levels of pPin in the nucleus are preselected for treatment with a Pinl modulator.
  • subjects with high levels of EGFR and low levels of pPinl are selected for treatment with a Pinl modulator and an EGFR modulator.
  • the instant invention provides method of determining the prognosis of a subject with a Pinl associated disorder or an EGFR related cell proliferative disorder, such as cancer.
  • the cancer is a solid tumor such as, but not limited to lung, prostate, breast, colorectal, head and neck, ovarian, gastric and pancreatic cancer.
  • the cancer is lung cancer such as non-small cell carcinoma, adenocarcinoma and squamous cell carcinoma.
  • the instant invention provides for the determination of the prognosis of a subject by evaluating the levels of pPinl and/or EGFR in the subject at one or more points in time.
  • the level of pPinl and/or EGFR in a subject can be compared to the statistical mean level in a population of subjects with similar diseases and the prognosis of the subject can be determined based on the level of pPinl and/or EGFR relative to the statistical mean. For example, if the level of pPinl in a subject is lower than the mean, the prognosis of the individual is considered poor, e.g., the subject will likely not survive for as long as the mean length of survival of the population.
  • the prognosis of the individual is considered good, e.g., the subject will survive for as long as the mean length of survival of the population, or longer.
  • the term "statistical mean” is used herein in a manner consistent with the well- understood definitions in the art of statistics. The statistical mean can be determined by quantitating the level of pPinl and/or EGFR in a statistically significant number of subjects and determining the mean value of that population.
  • the prognosis of an individual can be determined by evaluating the level of pPinl and/or EGFR in biological samples isolated at different time points. If the levels of pPinl decrease from a first sample to a second sample, the prognosis of the subject is poor. If the levels of pPinl in a sample stay the same, or increase, the prognosis is good. If the levels of EGFR increase from the first sample to a second sample, the prognosis of the subject is poor. The levels of pPinl and/or EGFR can be determined and compared with a survival curve generated with data from a statistically significant number of subjects having a similar disease.
  • the comparison of the pPinl and/or EGFR levels in a subject to the survival curve will determine the prognosis of a subject, i.e., the chance the subject has to survive for a given amount of time.
  • the levels of pPinl in a biological sample can be determined and used in combination with the levels of EGFR to determine the prognosis ' of a subject. Exemplary methods to predict the prognosis of a subject in are provided in the Examples. Survival curves are provided for subjects based on the determination of pPinl levels and/or EGFR levels or alterations. One of ordinary skill in the art would understand that these markers are exemplary and this analysis could be performed with any other known cancer marker. The use of pPinl and one or more additional makers allows for a more accurate determination of a subject's prognosis.
  • the prognosis of a subject as determined by the methods disclosed herein can aide in the determination of what course of treatment to provide a subject. Further, the prognosis can indicate the aggressiveness of treatment that is required.
  • subject is intended to include living organisms, e.g., prokaryotes and eukaryotes.
  • subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Most preferably the subject is a human.
  • subject that would benefit from treatment with a Pinl modulator is intended to include subjects having a Pinl associated disorder wherein treatment of said subject with a Pinl modulator would alleviate, reduce or eliminate one or more symptoms of the Pinl disorder and/or an EGFR related cell proliferative disorder, e.g., cancer..
  • an effective amount of the compound is that amount necessary or sufficient to treat or prevent or alleviate at least one symptom of a cell proliferative morphological and/or somatic symptom of the cell proliferative disorder.
  • an effective amount of a Pinl modulator and/or EGFR modulator is the amount sufficient to inhibit undesirable cell growth in a subject.
  • an effective amount of the modulator compound(s) is the amount sufficient to reduce the size of a pre-existing benign cell mass or malignant tumor in a subject. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular Pinl compound, EGFR compound or combination thereof.
  • the choice of the Pinl modulator and/or EGFR modulator can affect what constitutes an "effective amount".
  • One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the Pinl modulating compound and/or EGFR modulating compound without undue experimentation.
  • the regimen of administration can affect what constitutes an effective amount of the compound(s).
  • a Pinl modulator compound and/or EGFR modulator can be administered to the subject either prior to or after the onset of a Pinl associated state, EGFR related disorder, e.g., cell proliferative disorder. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the modulators can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • the term “treated,” “treating” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder. While it is possible for a compounds to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
  • pharmaceutical composition includes preparations suitable for administration to mammals, e.g., humans. When the modulators are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • phrases "pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, • hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, co , germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of pharmaceutical compositions for use in the methods of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for the topical or transdermal administration of a compound include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • compositions suitable for parenteral administration comprise one or more compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be. ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and ⁇ oly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. For the methods of treatment of the instant invention, formulations may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • formulations may be given orally, parenterally, topically, or rect
  • they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a Pinl associated or EGFR related disorder, e.g., a cell proliferative disorder. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub- doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a lung cancer tissue microarray containing 1516 lung tissue samples was analyzed. The breakdown of histopathological features is presented in the results section.
  • Immunohistochemistry assay was used to detect expression of PIN1 in tissue microarrays (TMAs).
  • TMAs tissue microarrays
  • Phosphorylated Pinl was detected using a phosphor-PINl specific antibody (Cell Signaling Cat#3721)
  • total PIN1 was detected using a PIN1 polyclonal antibody (Oncogene Research Cat#PC270).
  • Freshly cut tissue microarray sections were used to avoid the possibility of reduced assay sensitivity due to age-dependent oxidation of stored slides.
  • EGFR IHC data were analyzed according the following criteria: EGFR Thresh-holding Criteria Negative No staining Weak 1 + intensity in > 50% of cells or
  • EGFR FISH data were defined as follows: EGFR normal Ratio gene/centromer ⁇ 1.0 or ⁇ 4 FISH signals
  • Nuclear pPINl expression was found in 353/391 (90.3%) of pPFNl positive samples. Cytoplasmic expression was found in only 49 tissue samples, all in samples with no or weak nuclear pPINl staining ( Figure 2). Because of the low frequency of cytoplasmic pPINl staining, the following analyses were generally limited to samples with nuclear pPINl expression, with analyses of tissues with cytoplasmic pPPNl positive staining added where appropriate. Association ofpPINl alterations with tumor phenotype
  • Example 2 Analysis of pPinl Co-expression of pPinl and EGFR pPinl levels were evaluated as described above. Immunohistochemistry and
  • FISH analyses were performed according to standard assays were also used to detect EGFR expression and amplification in the lung tissue microarrays (EGFR antibody, PharmDX, DALO Cat #1492).

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Abstract

La présente invention fournit des procédés de sélection de sujets qui tireront un avantage d'un traitement avec un modulateur de Pinl en déterminant le niveau et l'état de phosphorylation de la Pinl présente dans un échantillon biologique provenant du sujet. L'invention fournit également des procédés de détermination du pronostic d'un sujet ayant un trouble prolifératif des cellules, par exemple un cancer du côlon, en déterminant le niveau de pPinl dans un échantillon biologique. L'invention fournit en plus des procédés de traitement de sujets souffrant d'un trouble associé à la Pinl et des procédés de suivi de l'efficacité du traitement.
PCT/US2005/015644 2004-05-06 2005-05-05 Procédés de détermination du pronostic et du traitement de sujets souffrant d'un cancer du poumon WO2005107803A2 (fr)

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US7939272B2 (en) 2007-10-03 2011-05-10 Osi Pharmaceuticals, Inc. Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8048621B2 (en) 2007-10-03 2011-11-01 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8093011B2 (en) 2005-03-16 2012-01-10 Haley John D Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8377636B2 (en) 2007-04-13 2013-02-19 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to kinase inhibitors
US8383357B2 (en) 2005-03-16 2013-02-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US9896730B2 (en) 2011-04-25 2018-02-20 OSI Pharmaceuticals, LLC Use of EMT gene signatures in cancer drug discovery, diagnostics, and treatment

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8093011B2 (en) 2005-03-16 2012-01-10 Haley John D Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8383357B2 (en) 2005-03-16 2013-02-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US9244058B2 (en) 2005-03-16 2016-01-26 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to epidermal growth factor receptor kinase inhibitors
US8377636B2 (en) 2007-04-13 2013-02-19 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to kinase inhibitors
US7939272B2 (en) 2007-10-03 2011-05-10 Osi Pharmaceuticals, Inc. Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US8048621B2 (en) 2007-10-03 2011-11-01 OSI Pharmaceuticals, LLC Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors
US9896730B2 (en) 2011-04-25 2018-02-20 OSI Pharmaceuticals, LLC Use of EMT gene signatures in cancer drug discovery, diagnostics, and treatment

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