WO2013166290A1 - Dosage de biomarqueurs de p21 - Google Patents

Dosage de biomarqueurs de p21 Download PDF

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WO2013166290A1
WO2013166290A1 PCT/US2013/039276 US2013039276W WO2013166290A1 WO 2013166290 A1 WO2013166290 A1 WO 2013166290A1 US 2013039276 W US2013039276 W US 2013039276W WO 2013166290 A1 WO2013166290 A1 WO 2013166290A1
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tweakr
seq
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antibody
days
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Patricia Culp
Yongke Zhang
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Abbvie Biotherapeutics Inc.
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    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4739—Cyclin; Prad 1
    • 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

  • TWEAK Tumor Necrosis Factor-Related Weak inducer of Apoptosis
  • TWEAK Tumor Necrosis Factor-Related Weak inducer of Apoptosis
  • TNF Tumor Necrosis Factor
  • TWEAK has a number of biological activities, including inducing angiogenesis, stimulating the release of cytokines or chemokines, stimulating proliferation of human smooth muscle and endothelial cell lines, inducing TNF-a-mediated cell death, cathepsin B-dependent necrosis, and, as its name implies, apoptosis. See Han et al, 2003, Bioch. Biophys. Res.
  • TWEAK binds to and activates a member of the TNF receptor family, TWEAKR (also known as Fibroblast Growth Factor-Inducible 14 or Fnl4). See id.
  • TWEAKR is overexpressed in a wide range of human cancers, making it a good target for therapy of cancers that overexpress it. See Culp et ah, 2010, Clin. Cancer Res. 16(2):497-508; Nakayama et al, 2003, J. Immunology 170:341-348.
  • TWEAKR induces a wide array of biological responses some of which are tumorigenic, such as the induction of angiogenesis and migration, and others of which are anti- tumorigenic, such as cell growth inhibition and induction of apoptosis. See Winkles et al., 2006, Cancer Lett. 235:11-17.
  • TWEAKR positive cancers As a consequence, not all patients diagnosed with a TWEAKR positive cancer will respond to therapies that target and activate TWEAKR, such as TWEAKR agonists. It would be beneficial to identify TWEAKR positive cancers that benefit from TWEAKR agonist therapies and monitor the efficacy of TWEAKR agonist therapies in these cancers.
  • TWEAKR agonists inhibit proliferation of some, but not all, tumor cells that express TWEAKR in vitro and in vivo.
  • the ability of TWEAKR agonists to inhibit proliferation of TWEAKR positive tumors is not dependent on cancer type.
  • FIG. 1 in vitro experiments carried out in tumor cell lines derived from a variety of different types of cancers showed that an anti-TWEAKR antibody having TWEAKR agonist activity, enavatuzumab (also known as, and referred to herein as, PDL192) inhibited the proliferation and/or survival of some, but not all, TWEAKR positive cancer cell lines.
  • p21 a cyclin-dependent kinase inhibitor that regulates cell cycle progression
  • p21 expression is upregulated in certain TWEAKR positive tumor cell lines that respond to treatment with compounds having TWEAKR agonist activity.
  • the present disclosure provides methods of monitoring the efficacy of a TWEAKR agonist therapy in a subject suffering from a TWEAKR positive cancer.
  • the methods generally involve determining whether a subject being treated with a TWEAKR agonist exhibits an increase in p21 expression as a function of time as compared to a baseline level.
  • the baseline level can be obtained, for example, from a sample taken from the subject being treated prior to, or at the time of, initiation of TWEAKR agonist therapy.
  • the baseline level is obtained from a sample containing TWEAKR positive cancer cells, such as a tumor biopsy sample, taken from the subject to be treated prior to, or at the time of, initiation of TWEAKR agonist therapy.
  • the baseline level can be a specific threshold value, for example, the average level of p21 expression observed in samples taken from individuals suffering from the same type of TWEAKR positive cancer as the subject being treated, who have not been treated with TWEAKR agonist therapy.
  • Levels of p21 expression can be measured at the nucleic acid level (e.g., mRNA level) or at the protein level, as is well known in the art.
  • the present disclosure provides methods of treating a subject having a TWEAKR positive cancer.
  • the methods generally comprise administering a TWEAKR agonist, typically in an amount effective in treating the TWEAKR cancer.
  • the methods can also comprise determining whether a test sample from the subject exhibits an increase in p21 expression as compared to a baseline level, as described above and in Section 4.3.
  • the method of treating a TWEAKR positive cancer is carried out in a subject having a TWEAKR positive cancer who tests positive for an increase in the level of expression of p21 as compared to a baseline level when receiving a TWEAKR agonist.
  • the methods further comprise continuing to administer or administering one or more additional doses of a TWEAKR agonist to the subject, where an increase in the level of p21 expression compared to a baseline level is detected.
  • An exemplary method of treating a subject having a TWEAKR positive cancer comprises administering a first dose of a TWEAKR agonist to a subject with a TWEAKR positive cancer, determining whether p21 expression increases relative to a baseline level, and administering one or more additional doses of TWEAKR agonist if an increase in p21 expression is detected.
  • TWEAKR agonists also referred to herein as TWEAKR agonist compounds, useful in the methods of the present disclosure are described in Section 4.2.
  • kits useful for carrying out the monitoring and/or treatment methods described herein comprise at least one reagent for detecting p21 expression, such as p21 -specific nucleic acids, hybridization probes and/or primers, or antibodies. Additionally, kits may include one or more assay reagents and buffers. The kits may include instructional materials containing instructions (e.g., protocols) for the practice of the monitoring methods of disclosed herein.
  • FIG. 1 provides a bar chart illustrating the percent growth inhibition for 38 TWEAKR positive cancer cell lines treated with PDL192 (enavatuzumab) for 5 days;
  • FIG. 2 provides a graph showing NFicB-mediated induction of a luciferase activity, measured in luminescence units, in PDL192-treated (grey bars) or control IgGl- treated (black bars) BT549 (breast cancer) cells;
  • FIG. 3 provides a Western blot detecting phosphorylated ⁇ , indicative of NFKB activation, in 6 TWEAKR positive cancer cell lines treated with PDL192 or a control IgGl;
  • FIG. 4A-D provides a bar chart illustrating the relative binding of four NFKB subunits to an NFKB consensus sequence (as a measure of relative activation) in
  • FIG. 4A illustrates the relative activation of p65.
  • FIG. 4B illustrates the relative activation of p52.
  • FIG. 4C illustrates the relative activation of p50.
  • FIG. 4D illustrates the relative activation of Re IB;
  • FIG. 5 provides a grey-scale map of gene expression changes for NFxB-regulated genes in a TWEAKR agonist sensitive cell line as compared to a TWEAKR agonist resistant cell line, each treated with PDL192 or a control IgGl for 6 to 72 hours;
  • FIG. 6 provides a graph of tumor volume over time (days post inoculation) of xenograft tumors of H358 lung cancer cells in mice treated with PDL192, a version of PDL192 bearing a mutation in the Fc domain abolishing antibody-dependent cellular cytotoxicity, or a control IgGl ;
  • FIG. 7 provides a Western blot showing activation of NFKB subunits in H358 (lung cancer) xenograft tumor samples from mice 4 days after treatment with PDL192 or control IgGl;
  • FIG. 8 provides a bar chart of cell survival as measured by luminescence (in arbitrary units) in BT549 (breast cancer) cells treated with PDL192 (grey bars) or control IgGl (black bars), showing survival of PDL192 -treated cells following inhibition of the NFKB signaling pathway by small interfering R As (siRNA) to p65, IKKa (CHUK), or ⁇ .
  • siRNA small interfering R As
  • CHUK small interfering R As
  • FIG. 9 provides a bar chart of percent survival in two TWEAKR agonist sensitive breast cancer cell lines (BT549, MDA-MB-468) and one TWEAKR agonist sensitive cervical cancer cell line (HT3) treated with PDL192 (grey bars), IKK 16 (black bars), an inhibitor of NFKB signaling, or both PDL192 and IKK16 (striped bars). * denotes p ⁇ 0.05;
  • FIG. 10A-B provides bar chart of percent cell survival in two TWEAKR agonist sensitive breast cancer cell lines in the presence of siRNA targeting NFkB subunits. * denotes p O.05.
  • FIG. 10A illustrates growth inhibition in MDA-MB-468.
  • FIG. 10B illustrates growth inhibition in BT-549;
  • FIG. 11 provides graphs illustrating viability, as indicated by fluorescence intensity, of HT-3 cells transfected with control siRNA or siRNA targeting p65, p50, p52 or RelB, and treated with either a control IgG or PDL192.
  • control siRNA or siRNA targeting p65, p50, p52 or RelB were transfected with two different sets of siRNAs (denoted target siRNA #1 and target siRNA#2);
  • FIG. 12 provides a Western blot showing increased p21 expression in four TWEAKR agonist sensitive cancer cell lines treated with PDL192 or a control IgGl;
  • FIG. 13 provides a Western blot showing presence or absence of p21 in PDL192- or control IgGl -treated cervical cancer cells in the presence of siRNA targeting NFKB subunits;
  • FIG. 14 provides a graph showing increased viability of PDL192-treated
  • FIG. 15 provides a graph of the number of p21 positive tumor cells at different time-points from lung cancer xenografts treated with PDL192 compared to a control IgGl antibody (* denotes p O.001);
  • FIG. 16 provides Western blots showing p21 induction by PDL192, as compared to a control IgG antibody, in three TWEAKR agonist sensitive cell lines at 4, 24, 48, and 72 hours. GAPDH was detected as a positive control for the Western; and
  • FIG. 17 provides a Western blot showing p21 induction in HT3 cells, in response to TWEAKR agonists PDL192, PDL400, 136.1, 19.2.1, 18.3.3 and TWEAK, as compared to a control IgGl .
  • GAPDH was detected as a positive control for the Western.
  • TWEAKR triggers pleiotropic effects when activated, including inhibition of cell proliferation, apoptosis, release of cytokines or chemokines, antibody-dependent cellular cytotoxicity (ADCC) and stimulation of angiogenesis, migration and invasion. Reports indicated that some of these activities, such as stimulation of migration in smooth muscle cells and in glioma cells, appear to be dependent on activation of NFKB, a transcriptional activator involved in a wide array of biological processes, including inflammation and immune responses. See Tran et ah, 2006, Cancer Res. 66(19): 9535-9542; Han et ah, 2003, Bioch. Biophys. Res. Communications 305:789-796.
  • NFKB activation has been observed in many aspects of cancer growth and survival, including tumor development, inhibition of apoptosis, resistance to chemotherapy and radiation therapy, and metastasis. See Prasad et ah, 2010, Mol. Cell. Biochem. 336:25-37; Kim et ah, 2006, Cell Death and Differentiation 13:738-747; Perkins, 2004, Trends Cell. Biol. 14:64-69; Luo et ah, 2005, J. Clin. Invest. 115:2625-2632. NFKB has also been reported to act as a tumor suppressor in certain circumstances. See Perkins et ah, 2006, Cell Death and Differentiation 13:759- 772.
  • cyclin-dependent kinase inhibitor p21 also known as WAF1 or CIPl
  • expression is induced by TWEAKR agonists in many TWEAKR positive cancer cells that exhibit growth inhibition, but not in TWEAKR positive cancer cells that show no growth inhibition.
  • p21 is known to bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, and functions as a regulator of cell cycle progression at Gl stage of the cell cycle.
  • TWEAKR agonists may inhibit cancer cell proliferation through induction of NFi B-mediated signaling pathways that induce p21 expression and result in cell cycle arrest.
  • p21 expression serves as an indicator of TWEAKR agonist- induced cell growth inhibition in TWEAKR positive cancer cells.
  • p21 expression increases within hours of exposure to a TWEAKR agonist and can remain elevated for multiple days thereafter. Based on these results it is expected that an increase in p21 mR A or protein, referred to herein as an "increase in p21
  • TWEAKR agonist treatment efficacy is indicative of TWEAKR agonist treatment efficacy in subjects having TWEAKR positive cancers and is useful for determining whether or not to continue TWEAKR agonist therapy. Accordingly, the present disclosure provides methods of monitoring efficacy of TWEAKR agonist therapy, methods of treating subjects having TWEAKR positive cancers, and kits useful for carrying out these methods.
  • the present disclosure provides methods of monitoring TWEAKR agonist treatment efficacy in a subject having a TWEAKR positive cancer.
  • the method comprises determining whether treatment of the subject with a TWEAKR agonist exhibits an increase in p21 expression as compared to a baseline level.
  • TWEAKR positive cancer is a cancer in which TWEAKR is expressed, including cancers in which TWEAKR is overexpressed as compared to matched non-cancer tissue (e.g. adjacent normal tissue or normal tissue from healthy donors).
  • TWEAKR expression can be determined by detecting the presence of TWEAKR mRNA or TWEAKR protein, using standard techniques such as PCR, Northern blotting, microarray analysis (for mRNA detection) or Western blotting, ELISA, or immunohistochemistry (for protein detection). Specific assays for detection of TWEAKR expression using microarray analysis and
  • TWEAKR has been shown to be highly expressed in a number of human cancers. See Culp et al, 2010, Clin. Cancer Res. 16(2):497-508; Feng et al, 2000, Am. J. Pathol. 156:1253-1261; Tran et al, 2006, Cancer Res. 66:9535-9542; Wang et al, 2006, Oncogene 25:3346-3356.
  • TWEAKR is expressed in a wide range of cancers, both primary and metastatic, including breast cancer, lung cancer, melanoma, ovarian cancer, uterine cancer, colon cancer, renal cancer, pancreatic cancer, cervical cancer, bladder cancer, head and neck cancer, glioblastoma, esophageal cancer, sarcomas, and salivary gland cancer.
  • a TWEAKR agonist is an agent capable of activating TWEAKR signaling cascade leading to one or more of the biological activities associated with TWEAKR, such as killing TWEAKR positive cancer cells by apoptosis, inhibiting proliferation and/or survival of TWEAKR positive cancer cells, and inducing chemokine or cytokine release in TWEAKR positive cancer cells.
  • TWEAKR agonists induce apoptosis in cells expressing TWEAKR.
  • induction of apoptosis by TWEAKR agonists is postulated to occur through caspase activation.
  • Induction of apoptosis by TWEAKR agonists can be assayed by measuring activation of caspase 3/7, using methods known in the art, including commercial kits such as Caspase-Glo® 3/7 Assay Systems from Promega®. Assays for apoptosis may also be performed by terminal
  • TUNEL deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling
  • a TWEAKR agonist inhibits proliferation (i.e. cell division) and/or survival of a cell, and in particular, a TWEAKR positive cancer cell.
  • TWEAKR agonists including TWEAK and anti-TWEAKR agonist antibodies, have been shown to inhibit proliferation and/or survival of TWEAKR positive cancer cell lines in in vitro and in vivo growth inhibition assays (Culp et al., 2010, Clin. Cancer Res. 16(2): 497-508). Inhibition of proliferation and/or survival can be measured by an in vitro growth inhibition assay as described in Example 1 below.
  • a cancer cell line known to be sensitive to TWEAKR agonists such as BT549 or H358, is cultured at 500 cells per well in triplicate with 5-10 ⁇ g/ml of the proposed TWEAKR agonist for 5 days in 96 well plates. Growth inhibition is determined using CellTiter-BlueTM (Promega). Cells are incubated with the reagent and fluorescence emitted at 590 nm is measured and used to calculate the effect on viable cells of the proposed TWEAKR agonist relative to a control, such as an IgGl control.
  • a test compound is considered to be a TWEAKR agonist if it induces growth inhibition of at least about 20% relative to a control compound.
  • cells can be incubated with anti-TWEAKR antibody (10 g/ml) and an antibody (5 ⁇ g/ml) or antibody fragment (3.5 ⁇ g/ml) that specifically binds to the anti-TWEAKR antibody.
  • the antibody or antibody fragment is preferably species- and isotype-specific.
  • the anti-TWEAKR antibody is a humanized IgG antibody
  • a F(ab') 2 antibody fragment that is a goat anti- human immunoglobulin (Fey specific) can be used.
  • a full goat anti-human- IgG antibody can be used.
  • Agonist activity can be determined by measuring the release of cytokines and/or chemokines in an in vitro cell growth assay. For example, in a typical assay the cells are incubated in vitro with a TWEAKR agonist. Twenty four hours later, the cell supernatant is assessed for the presence of cytokines and/or chemokines using an ELISA assay or a commercial fluorescent bead-based multiplex assay (e.g., Luminex®, Upstate). In some embodiments, the chemokine that is measured for release is IL-8.
  • TWEAKR agonists include the natural ligand of TWEAKR, TWEAK. TWEAK forms trimers and is thought to promote activation of TWEAKR via trimerization of the receptor. See Winkles, 2008, Nature Reviews Drug Discovery 7: 411-425. Therefore, it is expected that TWEAKR agonists can also be proteins that form multimers, e.g., trimers and are capable of binding to and promoting multimerization of TWEAKR. Accordingly, TWEAKR agonists include fusions of TWEAK, or a fragment of TWEAK capable of binding TWEAKR, to polypeptides comprising an oligomerization domain.
  • Polypeptides comprising suitable oligomerization domains include CD8 (Nakayama et al., 2000, J. Exp. Med. 192:1373), collagen (Frank et al., 2001, J. Mol. Bio. 308:1081-1089), bacteriophage T4 fibritin (Yang et al., 2002, J. Virology 76(9):4634-4642), engineered leucine/isoleucine zipper polypeptides (Harbury, 1994, Nature 371(6492):80-83), macrophage scavenger receptor (U.S. Pat. No.
  • TNF tumor necrosis factor
  • Methods of making fusion proteins are well known in the art, and involve combining coding sequences of two or more polypeptides to generate a coding sequence of a fusion protein.
  • Techniques for the manipulation of nucleic acids including techniques for the synthesis, isolation, cloning, detection, and identification are well known in the art and are well described in the scientific and patent literature. See, e.g., Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols.
  • Nucleic acids comprising the expression vectors described herein or components thereof include isolated, synthetic, and recombinant nucleic acids.
  • TWEAK agonists also include polypeptides of TWEAK engineered or modified to have an increased serum half-life.
  • Suitable engineered TWEAK proteins include fusions of TWEAK, or a fragment of TWEAK capable of binding TWEAKR, to human immunoglobulin proteins or human serum albumin (HSA). Fusions of various proteins to HSA have been described, and are known to increase serum half-life, see U.S. Patents Nos. 5,876,969; 6,994,857; and 7,189,690.
  • Modified TWEAK proteins also include conjugates of TWEAK, or a fragment of TWEAK capable of binding TWEAKR, and high molecular weight dextrans or polyethylene glycol (PEG), which can be made according to methods known in the art. See U.S. Pat. No. 5,177,059 and 7,587,286. PEG has been shown to prolong half-life and reduce immunogenicity of the conjugated protein. See Walsh et al., 2003, Antimicrob Agents Chemother. 47(2): 554-558 and Abuchowski et al., 1977, J Biol Chem 252:3582- 3586. Similar increases in serum half-life and reductions in immunogenicity have been observed for dextran conjugates, see, e.g., Mehvar et al., 1992, J Pharm Sci, 81 :908-912.
  • TWEAKR agonists include antibodies.
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, chimeric, humanized, fully human, genetically engineered and otherwise modified forms of antibodies.
  • a full-length antibody contains two heavy chains and two light chains, each of which comprises complementarity determining regions (CDRs), also known as hypervariable regions, and more highly conserved framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs highly conserved framework regions
  • the amino acid position/boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art.
  • Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria.
  • One or more of these positions can also be found in extended hypervariable regions.
  • the present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions.
  • variable domains of native heavy and light chains each comprise four FR regions, largely by adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (See Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).
  • numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • an anti-TWEAKR agonist antibody can be an antibody fragment.
  • antibody fragment refers to a portion of a full-length antibody capable of multivalent binding to the target, where multivalent refers to bivalent, trivalent, tetravalent, etc.
  • suitable antibody fragments include F(ab') 2 fragments.
  • F(ab') 2 fragments are bivalent, having two antigen-binding F(ab) portions linked together by disulfide bonds, and contain portions of VH and VL chains.
  • References to "VH” refer to the variable region of an immunoglobulin heavy chain of an antibody.
  • VL refer to the variable region of an immunoglobulin light chain.
  • an F(ab') 2 fragment can be produced by pepsin cleavage of a whole antibody, removing most of the immunoglobulin constant region (Fc) portion of the antibody. Such F(ab') 2 fragments clear more rapidly from the circulation of the animal or plant, and may have less nonspecific tissue binding than an intact antibody (Wahl et al., 1983, J. Nucl. Med. 24:316). Multivalent antibody fragments can also be produced by linking CDRs using chemical couplings known to those of ordinary skill in the art.
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • the term monoclonal antibody (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as, for example, F(ab') 2 fragments) which are capable of specifically binding to a protein.
  • the anti-TWEAKR agonist antibodies of the disclosure can be chimeric antibodies.
  • the term "chimeric" antibody as used herein refers to an antibody having variable sequences derived from immunoglobulins from one species, such as rat or mouse antibody, and immunoglobulin constant regions from a second species, typically chosen from the species to be treated, e.g., where the subject is a human, the constant regions can be from a human immunoglobulin template.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, BioTechniques 4:214-221; Gillies et al, 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397.
  • the anti-TWEAKR agonist antibodies of the disclosure can be humanized.
  • Humanized forms of non-human ⁇ e.g., murine antibodies are chimeric
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an Fc, typically that of a human immunoglobulin consensus sequence.
  • the anti-TWEAKR agonist antibodies of the disclosure can be fully human antibodies.
  • Fully human anti-TWEAKR agonist antibodies can be desirable for therapeutic treatment of human patients.
  • "fully human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos.
  • Fully human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a fully human antibody recognizing the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).
  • anti-TWEAKR agonist antibodies can be of any of the recognized isotypes.
  • anti-TWEAKR agonist antibodies are one of the four human IgG isotypes, i.e., IgGl, IgG2, IgG3 and IgG4, or one of the four mouse IgG isotypes, i.e., murine IgGl, murine IgG2a, murine IgG2b, or murine IgG3.
  • the anti-TWEAKR antibodies are of the human IgGl isotype.
  • Anti-TWEAKR agonist antibodies specifically bind to TWEAKR proteins.
  • An example of a TWEAKR protein is the human TWEAKR protein encoded by the nucleotide sequence given by SEQ ID NO:l, or the protein of the amino acid sequence given by SEQ ID NO:2. See Table 4 below.
  • the antibody preferably binds the TWEAKR from the same species as the subject. For example, when treating a human, an antibody to human TWEAKR is administered.
  • a TWEAKR agonist antibody need not be a pure agonist.
  • a TWEAKR agonist antibody can also have TWEAKR antagonist activity.
  • Antagonist activity refers to the ability of test agent ⁇ e.g. a TWEAKR agonist antibody) to inhibit the induction by TWEAK of one or more biological responses associated with TWEAKR.
  • the cytokine/chemokine release assay described above can be performed by incubating cells in vitro with an anti-TWEAKR antibody +/- TWEAK.
  • An anti-TWEAKR agonist antibody can bind to TWEAKR protein with a K D of at least about ⁇ ⁇ , at least about 0.1 ⁇ or better, at least about 0.01 ⁇ , and at least about 0.001 ⁇ or better.
  • Anti-TWEAKR agonist antibodies useful in the present methods include antibodies that induce antibody-dependent cytotoxicity (ADCC) of TWEAKR-expressing cells.
  • ADCC antibody-dependent cytotoxicity
  • the ADCC of an anti-TWEAKR antibody can be improved by using antibodies that have low levels of or lack fucose.
  • Antibodies lacking fucose have been correlated with enhanced ADCC (antibody-dependent cellular cytotoxicity) activity, especially at low doses of antibody (Shields et al, 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al, 2003, J. Biol. Chem. 278:3466).
  • Methods of preparing fucose-less antibodies include growth in rat myeloma YB2/0 cells (ATCC CRL 1662).
  • YB2/0 cells express low levels of FUT8 mRNA, which encodes an enzyme (a-l,6-fucosyltransferase) necessary for fucosylation of polypeptides.
  • Alternative methods for increasing ADCC activity include mutations in the Fc portion of an anti-TWEAKR agonist antibody, particularly mutations which increase antibody affinity for an FcyR receptor.
  • a correlation between increased FcyR binding with mutated Fc has been demonstrated using targeted cytoxicity cell-based assays (Shields et al, 2001, J. Biol. Chem.
  • an anti-TWEAKR agonist antibody of the disclosure has a constant region that binds FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type constant region. Mutations in the Fc portion of an antibody that result in increased ADCC activity and methods of generating immunoglobulins with increased ADCC activity are known in the art and described in various references, e.g., U.S. Pat. No. 8,039,592, U.S. Pat. No.
  • ADCC activity can be monitored and quantified using a 51 Cr release assay.
  • Target cells are loaded with 51 Cr, which is released in culture supernatant upon damage to the plasma membrane in the presence of immune effector cells and an anti-TWEAKR agonist antibody.
  • the target cells can be derived from solid tumors, for example lung, pancreatic, breast, or renal cancer cells.
  • the anti-TWEAKR agonist antibodies induce at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 80% cytotoxicity in the target cells.
  • An example of an ADCC assay that can be used to measure ADCC of an anti-TWEAKR agonist antibody is provided in Culp et al, 2010, Clin. Cancer Res. 16(2):497-508.
  • Anti-TWEAKR agonist antibodies or fragments thereof as described herein can be antibodies or antibody fragments whose sequence has been modified to reduce at least one constant region-mediated biological effector function relative to the corresponding wild type sequence.
  • the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173: 1483-1491 ; and Lund et al., 1991, J. Immunol. 147:2657-2662).
  • Reduction in FcR binding ability of the antibody can also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis.
  • Anti-TWEAKR agonist antibodies or fragments thereof as described herein can be antibodies or antibody fragments that have been modified to increase or reduce serum half-life by altering their binding affinities to the fetal Fc receptor, FcRn, for example by mutating the immunoglobulin constant region segment at particular regions involved in FcRn interactions (see, e.g., WO 2005/123780 and U.S. Pat. No. 5,739,277).
  • anti-TWEAKR agonist antibodies and sequences of VH, VL and CDRs which are described in U.S. Pat. Pub. No. 2009/0074762 and provided in Table 4 herein, include:
  • an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, comprising a heavy chain variable region corresponding to SEQ ID NO:3 and a light chain variable region corresponding to SEQ ID NO:4 (PDL192);
  • an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, comprising a heavy chain variable region corresponding to SEQ ID NO: l 1 and a light chain variable region corresponding to SEQ ID NO:12 (PDL400);
  • an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V H CDR1 is XjYWMX 2 (SEQ ID NO:49), V H CDR2 is EIRX 3 KSX 4 NYATX S HYAESX 6 KG (SEQ ID NO:50), V H CDR3 is X7X8ADX9X10DY (SEQ ID NO:51);
  • V L CDR1 is XnASQSVSTSXi 2 YSY Xi 3 (SEQ ID NO:52);
  • V L CDR2 is YAX ]4 Xi 5 LX 16 S (SEQ ID NO:53); and
  • V L CDR3
  • V H CDR1 is SYWMS (SEQ ID NO: 13)
  • V H CDR2 is EIRLKSDNYATHYAESVKG (SEQ ID NO: 19)
  • V H CDR3 is
  • V L CDR1 is RASQS VSTS SYS YMH (SEQ ID NO:31)
  • V L CDR2 is YASNLES (SEQ ID NO:37)
  • V L CDR3 is
  • V H CDR1 is NYWMS (SEQ ID NO: 15)
  • V H CDR2 is EIRLKSDNYATHYAESVKG (SEQ ID NO:21)
  • V H CDR3 is
  • V L CDR1 is RASQSVSTSSYSYMQ (SEQ ID NO:33)
  • V L CDR2 is YATNLDS (SEQ ID NO:39)
  • V L CDR3 is
  • V H CDR1 is KYWMN (SEQ ID NO: 16)
  • V H CDR2 is EIRLKSDNYATHYAESAKG (SEQ ID NO:22)
  • V H CDR3 is
  • V L CDR1 is KASQSVSTSTYSYMQ (SEQ ID NO:34)
  • V L CDR2 is YASKLDS (SEQ ID NO:40)
  • V L CDR3 is
  • QHSWELPYT (SEQ ID NO:46); (g) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V H CDR1 is RYWMS (SEQ ID NO: 17), V H CDR2 is EIRVKSDNYATTHYAESVKG (SEQ ID NO:23), V H CDR3 is YYADAMDY (SEQ ID NO:29), V L CDR1 is RASQSVSTSSYSYMH (SEQ ID NO:35), V L CDR2 is YASKLDS (SEQ ID NO:41), and V L CDR3 is
  • V H CDR1 is NYWMN (SEQ ID NO: 17)
  • V H CDR2 is EIRLKSNNYATHYAESVKG (SEQ ID NO:24)
  • V H CDR3 is
  • V L CDR1 is RASQSVSTSTYSYMH (SEQ ID NO:36)
  • V L CDR2 is YASNLES (SEQ ID NO:42)
  • V L CDR3 is
  • Anti-TWEAKR agonist antibodies of the present disclosure also include antibodies that compete for binding to TWEAKR with a reference antibody of interest. Any of the antibodies described herein can be used as a reference antibody in a competition assay.
  • a competition assay can be carried out between a test antibody and a reference antibody. The assay is conducted by first labeling the reference antibody with a detectable label, such as, biotin, or an enzymatic, radioactive, or fluorescent label to enable detection. The unlabeled test antibody is incubated (in fixed or increasing amounts) with a known amount of TWEAKR, forming an anti-TWEAKR antibody/ TWEAKR complex. The labeled reference antibody is then added to the complex. The intensity of the complexed label is measured.
  • a detectable label such as, biotin, or an enzymatic, radioactive, or fluorescent label
  • test antibody competes with the labeled antibody by binding to an overlapping epitope, the intensity will be decreased relative to the binding of the labeled reference antibody in the absence of the test antibody.
  • An antibody is considered to compete for binding TWEAKR with a reference anti- TWEAKR agonist antibody, and thus considered to bind approximately the same or overlapping epitope of TWEAKR as the reference antibody, if it reduces binding of the reference antibody by at least 50% at a test antibody concentration in the range of 0.01 to 100 ⁇ g/mL, although higher levels of reduction, for example, 60%, 70%, 80%, 90% or even 100%, may be desirable.
  • the present disclosure provides methods of monitoring the efficacy of a
  • TWEAKR agonist therapy comprising determining whether a subject being treated with a TWEAKR agonist exhibits an increase in p21 expression.
  • p21 expression is measured in a test sample and the resulting measurement is evaluated relative to a baseline level.
  • Suitable test samples for use in measuring p21 expression include any tissue or biological material that contains TWEAKR positive cells.
  • the sample can be a tumor sample, such as from a biopsy or from a cultured tumor specimen.
  • the sample can be fresh or processed, e.g. a frozen tissue sample or a formalin-fixed paraffin-embedded sample.
  • p21 levels can also be measured in circulating tumor cells (CTCs) present in blood.
  • CTCs circulating tumor cells
  • CTCs can be isolated using an assay such as CellSearch® (Veridex) and the sample is collected in such a way to preserve nucleic acids and/or proteins within the blood cells, such as by using a Cell Save tube (Veridex).
  • CellSearch® Veridex
  • an "increase in p21 expression” means an increase in p21 mRNA or p21 protein.
  • An increase in p21 expression can be evidenced by an increase in the amount of p21 protein or p21 mRNA detected in a test sample relative to a baseline level, expressed either as a percentage or fold change relative to the baseline level or in relevant measurement units for the assay.
  • An increase in p21 expression can also be determined based on the number or percent of cells in a test sample versus a baseline sample in which p21 expression is detected, referred to herein as p21 -positive cells. The same measurement technique is used to obtain the p21 level from the test sample and to obtain the baseline level.
  • test and baseline samples can be prepared for sectioning and staining, e.g., by formalin fixation and paraffin embedding.
  • Suitable baseline samples include samples containing TWEAKR positive tumor cells obtained from the subject prior to, or at the time of, initiation of TWEAKR agonist therapy. 5 ⁇ tissue sections are then mounted onto slides, deparaffinized and ethanol rehydrated. Samples can be blocked using 0.3% H 2 0 2 Dako X0909 blocking solution and then treated with a primary antibody against p21 (p21 mlgGl, Dako Cat. No.
  • a secondary antibody bearing a detectable label AlexaFluor®488 goat anti-rabbit, InvitrogenTM/ Life TechnologiesTM.
  • Slides containing the stained tissue sections are mounted in a mounting medium, e.g., Vector Lab DAPI, and imaged on a fluorescence microscope.
  • the number of p21 positive cells in a sample is determined by scoring cells in three 40X magnification fields. The number of p21- positive cells in a test sample can be compared to the number of p21 -positive cells in a sample which provides a baseline level, to determine whether p21 levels have increased.
  • an increase in p21 expression is evidenced by an increase in the amount of p21 protein or mRNA, determined relative to a baseline level.
  • a baseline sample that is, a sample containing tumor cells taken prior to, or at the time a TWEAKR agonist is administered, is used.
  • a baseline sample can be from the subject to be treated or from individuals suffering from the same type of TWEAKR positive cancer as the subject to be treated.
  • the baseline level can be the level of p21 in a baseline sample containing TWEAKR positive cancer cells obtained from the subject being treated prior to initiation of TWEAKR agonist therapy.
  • the baseline level can be an amount or concentration of p21 protein or mRNA determined from multiple samples.
  • the baseline value could be the average level of p21 expression observed in baseline samples taken from individuals suffering from the same type of TWEAKR positive cancer as the subject to be treated.
  • An increase in p21 expression can be expressed as a percentage and can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%>, at least about 350%, at least about 400%, or greater, ranging up to 1000%» greater than a baseline level.
  • An increase in p21 expression can also be expressed as a fold increase relative to a baseline level, e.g., at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold , at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 50 fold, at least about 75 fold, or greater, ranging up to 100 fold more than a baseline level.
  • a baseline level e.g., at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold , at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at
  • TWEAKR agonist administration of a TWEAKR agonist as shown in Examples 6 and 7 below.
  • an increase in p21 expression provides an early indicator that TWEAKR agonist therapy is effective.
  • p21 expression is measured at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 1 day (or 24 hours), at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 6 days, at least 8 days, at least 9 days, after administration of a TWEAKR agonist to the subject.
  • p21 expression is measured after a first administration of a TWEAKR agonist to the subject.
  • the methods provided herein involve detection and quantification of p21. Any analytical procedure permitting accurate measurement of p21 protein or p21 nucleic acid (e.g., mRNA) may be used. Numerous techniques for detecting protein or mRNA levels in samples are available and well known in the art, including methods of detecting p21 mRNA or protein.
  • p21 protein or p21 nucleic acid e.g., mRNA
  • Numerous techniques for detecting protein or mRNA levels in samples are available and well known in the art, including methods of detecting p21 mRNA or protein.
  • RNA extraction and/or quantification begins with a step of isolating total RNA or mRNA from a sample.
  • General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., 1997, Current Protocols of Molecular Biology, John Wiley and Sons. Methods for RNA extraction from processed samples, such as paraffin embedded tissues, are also known. See, e.g., Rupp et al., 1987, Lab. Invest. 56:A67 and De Andres et al, 1995,
  • RNA isolation can be performed using commercially available purification kits, buffer sets, and proteases from vendors such as Qiagen (Valencia, CA) or Life TechnologiesTM (Carlsbad, CA).
  • Qiagen Valencia, CA
  • Life TechnologiesTM Carlsbad, CA
  • the presence and/ or level of p21 transcript in the isolated RNA is then measured using an appropriate assay.
  • a number of methods can be used to detect and/or quantify p21 mRNA levels, including but not limited to reverse transcriptase polymerase chain reaction (RT-PCR), northern blotting, in situ detection of p21 mRNA, microarray analysis, and nuclease protection assays.
  • RT-PCR is one of the most sensitive and flexible methods for measuring expression levels, and includes techniques such as quantitative RT-PCR and real-time quantitative RT-PCR.
  • RNA isolated from a sample is reverse transcribed into cDNA according to standard methods (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2 nd Ed.) and used as the template for the PCR reaction.
  • p21 expression can be detected using primers or probes that specifically hybridize to p21 mRNA.
  • p21 also called CIPl, CDNKIA and WAF1
  • CIPl CIPl
  • CDNKIA CDNKIA
  • WAF1 Several alternate transcripts for p21 are known. See, Radhakrishnan et al, 2006, Oncogene 25:1812-1815.
  • Primers for p21 can be designed to amplify a sequence common to two or more p21 transcript variants, including a sequence corresponding to GenBank accession number NM_000389 or SEQ ID NO:55, shown in Table 4.
  • primers may amplify only certain variant transcripts.
  • primer pairs for p21 are described in Radhakrishnan et al, 2006, Oncogene 25: 1812-1815 and Kasahara et al, 2005, J. Health Sci. 51(2): 185- 190, the contents of which are incorporated in their entirety.
  • Suitable primers can be designed and made using techniques that are well known to those of skill in the art, and are also commercially available (see, e.g., human p21/CIPl/CDKNl A Primer PairTM, Catalog no. RDP- 164-025, from R&D Systems, Inc.). In addition to being specific to p21, primers will also be adapted to the specific detection methodology used.
  • Probes suitable for detecting p21 expression can be deoxyribonucleic acids, such as cDNAs, or mixtures of deoxyribonucleic acids and nucleotide analogs or other detectable moieties.
  • the probes are typically obtained by PCR amplification of the sequence of interest, in this case the cDNA sequence corresponding to all or a portion of the transcribed sequence of the p21 gene, for example a sequence corresponding to all or a portion of the nucleotide sequence corresponding to GenBank Accession Number NM 000389 or SEQ ID NO:55, shown in Table 4.
  • Expression levels of p21 can be measured using quantitative RT-PCR and real time quantitative RT-PCR. See, e.g., Chen et al., 2005, Nucl. Acids Res. 33:el79.
  • PCR can be performed using a variety of thermostable DNA-dependent DNA polymerases, including Thermus aquaticus, or Taq, DNA polymerase and Pyrococcus furiosus, or Pfu, DNA polymerase.
  • RT-PCR can be carried out using the TaqMan® assay and primers sold by Applied BiosystemsTM / Life TechnologiesTM (Carlsbad, CA).
  • Other chemistries for detecting and quantifying PCR products are also possible and include, but are not limited to, molecular beacons, scorpion probes, and SYBR® Green detection.
  • RT-PCR is performed using any RT-PCR instrumentation available in the art.
  • instrumentation used in real-time RT-PCR data collection and analysis comprises a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.
  • p21 expression levels may be represented by gene transcript numbers per nanogram of cDNA.
  • RT-PCR data can be subjected to standardization and normalization against one or more housekeeping genes as has been previously described. See, e.g., Rubie et al, Mol. Cell. Probes 19(2): 101-9 (2005).
  • p21 expression levels can also be determined by using an array, such as a cDNA microarray.
  • cDNA microarrays consist of probes spotted (usually using a robotic spotting device) onto known locations on a solid support, such as a glass microscope slide.
  • Microarrays for use in the methods described herein comprise a solid substrate onto which at least one probe capable of hybridizing to a p21 mRNA is covalently or non- covalently attached.
  • PCR products suitable for production of microarrays are typically between 0.5 and 2.5 kB in length.
  • the microarray includes at least one probe comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, even at least 20, 21, 22, 23, or 24 contiguous nucleotides identically present in p21 mRNA.
  • Microarray analysis can be performed by commercially available equipment, following manufactuer's protocols, such as by using Affymetrix GeneChip® technology, Agilent Technologies cDNA microarrays, Illumina Whole-Genome DASL® array assays, or any other comparable microarray technology.
  • p21 mRNA can also be quantified using Northern blot analysis or using a nuclease protection assay. Techniques for Northern blot analysis and nuclease protection assays are well known in the art.
  • Nucleic acids for use in mRNA detection such as the p21 -specific probes and primers described herein can be coupled to a detectable substance or "label."
  • a label can be conjugated directly or indirectly to a p21 -specific probe and primer.
  • the label can itself be detectable (e.g., radioisotope labels, isotopic labels, or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition which is detectable.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
  • the detectable substance can be coupled or conjugated either directly to the probe or primer or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase ⁇ -galactosidase
  • acetylcholinesterase glucoamylase
  • lysozyme saccharide oxidases
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, dimethylamine-l-napthalenesulfonyl chloride, or phycoerythrin and the like;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable isotopic materials include 13 C, 15 N, and deuterium; and
  • suitable radioactive material include I25 1, 131 I, 11 'in or 99 Tc.
  • Presence and amount of p21 protein can be determined using antibody-based
  • Detection can be carried out in situ or on protein isolated from samples. For assay performed on isolated proteins, total protein from a sample to be analyzed can easily be isolated using techniques which are well known to those of skill in the art. Protein isolation methods include, for example, those described in Harlow and Lane (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).
  • methods for the detection of p21 protein involve detection via interaction with a protein-specific antibody. Any antibody that specifically detects p21, or an anti-p21 antibody, can be used. Antibodies can be generated utilizing standard techniques well known to those of skill in the art. See, e.g., Section 5.2 of U.S. Pub. No. 20040018200 for a more detailed discussion of antibody generation techniques.
  • Antibodies can be polyclonal or monoclonal. An intact antibody, or an antibody fragment (e.g., Fab or F(at>') 2 ) can be used. The antibody can be a human or humanized antibody. For detection purposes, antibodies to p21 can be coupled to a detectable substance or "label" as described herein above. Antibodies against p21 are available commercially from a variety of vendors, with or without detectable moieties (e.g., Abeam® Cat. no. abl8209, WAF1/CIP1 (DCS60) purchased from Cell Signaling Technology® Cat. no. 2946).
  • detectable moieties e.g., Abeam® Cat. no. abl8209, WAF1/CIP1 (DCS60) purchased from Cell Signaling Technology® Cat. no. 2946).
  • p21 expression increases after a dose of a TWEAKR agonist in cancer cells whose growth is inhibited by the same agonist.
  • the present disclosure provides a method of treating a subject having a TWEAKR positive cancer, comprising administering a dose of a TWEAKR agonist in an amount effective to treat a TWEAKR positive cancer, determining whether p21 expression increases relative to a baseline level, and continuing to administer or administering one or more additional doses of the TWEAKR agonist, if an increase in p21 expression is detected.
  • the method can be used to treat subjects who have a TWEAKR positive cancer as described above in Section 4.1.
  • the subject treated may be any animal, including a mammal, e.g., a human.
  • the methods comprise administering a TWEAKR agonist to a subject having a TWEAKR positive cancer who tests positive for an increase in a level of p21 expression as compared to a baseline level.
  • TWEAKR agonists for use in the methods of treatment are described in Section 4.2. Where the TWEAKR agonist used to treat subjects having from a
  • TWEAKR positive cancer is an anti-TWEAKR antibody, it should be specific for the species of animal being treated.
  • the TWEAKR agonist can be used alone, as monotherapy, or in combination with or adjunctive to other therapies commonly used to treat the specific type (e.g., lung, breast, colon, etc.) of TWEAKR positive cancer.
  • Other therapies include surgery, chemotherapy, radiation therapy, and targeted therapy.
  • the TWEAKR agonist and other therapy can be administered simultaneously, successively, or separately.
  • Therapies that can be combined with TWEAKR agonist therapy are described in, e.g., U.S. Pub. No. US 2009/0074762.
  • TWEAKR agonist in amount effective in treating the TWEAKR positive cancer.
  • the amount can be an amount sufficient to provide a therapeutic benefit.
  • a therapeutic benefit can be evidenced by one or more of the following: halting or slowing the growth of tumors, reducing the number and/or size of tumors within a patient, shrinking inoperable tumors to a size and location such that they can be surgically removed, decreasing the rate of progression of the cancer, and/or increasing life expectancy.
  • a complete cure, while desirable, is not required for therapeutic benefit to exist.
  • Tumor size and/or number can be measured using various scanning techniques, such as, but not limited to CT, MRI, functional MRI, SPECT and PET, as well as other methods known to those skilled in the art.
  • TWEAKR agonists will typically be administered in the form of pharmaceutical formulations or compositions. Such formulations or compositions may optionally include additional active and/or therapeutic agents, as is known in the art.
  • the formulations will typically include a pharmaceutically acceptable carrier, excipient, or diluent. The specific carriers, excipients and/or diluents used will depend upon the desired mode of administration.
  • the composition can be in any suitable form depending upon the desired method of administering it to a subject. Pharmaceutical compositions can be
  • the concentration of TWEAKR agonist in formulations can range from about 0.1 to about 100 mg/ml, from about 0.1 mg/ml to about 1 mg/ml, from about 1 mg/ml to about 10 mg/ml, or from about 10 mg/ml to about 20 mg/ml.
  • TWEAKR agonists can be administered to a subject by a variety of routes, typically parenterally, for example, via subcutaneous, intravenous, intraperitoneal or intramuscular injection. Administration can be effected as one or more bolus injections, or as one or more infusions.
  • TWEAKR agonist The appropriate amount and dosage of a TWEAKR agonist will depend on a variety of factors, including the stage of the TWEAKR positive cancer, the form, route and site of administration, the therapeutic regimen (e.g., whether TWEAKR agonist treatment is combined with another therapy), the age and condition of the particular subject being treated, and the sensitivity of the TWEAKR positive cancer to TWEAKR agonist therapy.
  • the appropriate dosage can be readily determined by a person skilled in the art. Suitable doses can be within the range of about 0.1 to about 1 mg/kg, about 1 to about 5 mg/kg, about 1 to about 10 mg/kg, about 10 to about 20 mg/kg.
  • a clinician will determine appropriate dosages to be used. This dosage can be repeated as often as appropriate. Effective dosages can be estimated initially from animal models. Frequency and duration of administration will depend on a variety of factors, such as the patient's age, weight, and disease condition.
  • Determination of an increase in p21 expression can be carried out as described above in Section 4.3. Subjects in whom an increase in p21 expression is observed following administration of a TWEAKR agonist are given one or more further doses.
  • kits useful for carrying out the monitoring methods described herein comprise detection agents suited to the particular target, e.g. p21 mRNA or p21 protein.
  • detection agents suited to the particular target e.g. p21 mRNA or p21 protein.
  • kits intended for use in detecting p21 mRNA can comprise nucleic acid reagents such as primers and probes specific to p21.
  • Kits intended for use in detecting p21 protein can comprise antibodies that specifically bind p21 protein.
  • Kits suited to detecting p21 mRNA will typically contain one more nucleic acids capable of specifically binding p21 -encoding nucleic acid, such as p21 -specific primer(s) or probe(s).
  • the nucleic acids will be suitable for the specific assay to be used.
  • Suitable nucleic acids for binding to a p21 -encoding nucleic acid include complementary nucleic acids.
  • Complementary nucleic acid may include oligonucleotides (labeled or unlabeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon primers, and the like.
  • the kits may include reagents for reverse transcription and or PCR reactions, for labeling probes or primers with a label as described herein, for detecting one or more reference or control nucleic acids.
  • Kits suited to detection of p21 protein will typically contain one or more antibodies that specifically bind p21 (i.e. an anti-p21 antibody).
  • the anti-p21 antibody or antibodies may be detectably labeled or the kit may contain additional reagents used to detect anti-p21 antibody, such as a detectably labeled secondary antibody capable of specifically binding the anti-p21 antibody.
  • kits can comprise one or more reagents for performing a detection assay (e.g., buffers, diluents, p21 positive control, negative control, calibration standards, sample preparation reagents, microarrays or other physical support), and instructions for their use.
  • a detection assay e.g., buffers, diluents, p21 positive control, negative control, calibration standards, sample preparation reagents, microarrays or other physical support
  • TWEAKR agonist PDL192 inhibits growth of a
  • PDL192 a monoclonal antibody that acts as a TWEAKR agonist
  • PDL192 a monoclonal antibody that acts as a TWEAKR agonist
  • a wide range of cancer cell lines including breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, and melanoma cancer cell lines, in vitro.
  • Antibodies. PDL192 is a humanized monoclonal antibody to TWEAKR described in US Pat. App. No. 2009/0074762 and Culp et al, 2010, Clin. Cancer Res. 16(2):497-508.
  • Human IgGl antibody to cytomegalovirus (MSL109) was used as a negative control.
  • Antibodies were used at 10 ⁇ g/ml for in vitro studies, unless otherwise stated, and crosslinked with F(ab') 2 goat anti-human IgG (Fc Y specific) from Jackson ImmunoResearch at 3.5 ⁇ g/ml. Wiley et al, 2001, Immunity 15(5):837-46.
  • Results Inhibition of proliferation by PDL192, as compared to an isotype control antibody, was tested in 102 cancer cell lines representing the majority of solid tumor types. Of these, 38 (or 37%) cell lines showed growth inhibition of at least 20% (relative to growth observed with the control antibody) in response to PDL192 (FIG. 1). Table 1 below summarizes the TWEAKR-agonist sensitive and TWEAKR-agonist resistant cell lines for each type of cancer tested.
  • Cancer Sensitive Resistant type at least 20% growth ( ⁇ 10% growth inhibition) inhibition
  • Cancer Sensitive Resistant type at least 20% growth ( ⁇ 10% growth inhibition) inhibition
  • PDL192 displayed the ability to block proliferation in some but not other cell lines from each of the following tumor types: breast cancer, lung cancer, colon cancer, renal cancer, ovarian cancer, melanoma, and pancreatic cancer. PDL192 also blocked cell proliferation in cell lines derived from bladder cancer, cervical cancer, salivary cancer and oral cancer tumors.
  • TWEAKR agonist PDL192 induces the expression of an NFicB-responsive reporter construct.
  • the Cancer 10-Pathway Reporter Luciferase Kit was purchased from SA Biosciences (Ref. no. CCA-101L (Plate Format)) and reverse transfected into cells using Lipofectamine 2000 (Invitrogen), per manufacturer's instructions. Cells were treated with PDL192 or IgGl control as described in Example 1 above for 24 h following transfection. Cells were harvested an additional 24 h later and reporter activity for NFKB was measured using the Dual-Luciferase Reporter Assay (Invitrogen).
  • phosphorylated ⁇ ( ⁇ - ⁇ (Ser32/36) (5A5) were purchased from Cell Signaling.
  • GAPDH antibodies used as a control, were obtained from Santa Cruz.
  • Mouse and rabbit secondary antibodies were obtained from GE-Amersham.
  • NFKB transcription factor ELISA TransAMTM NFKB family ELISA kit (Active Motif) was used to determine NFKB subunit DNA binding and functional activity in response to PDL192 treatment. Nuclear protein extracts were generated and the subunit ELISAs (p65, p50, RelB, p52) performed according to the manufacturer's guidelines. NFKB activation was calculated by subtracting the OD450 of the control samples from that of the PDL 192 -treated samples. [0103] Affymetrix gene chip analysis.
  • RNA from BT549 (a TWEAKR agonist sensitive cell line) or T47D (a TWEAKR agonist resistant cell line) was isolated and purified with RNeasy Microkits (Qiagen) and analyzed using an Agilent 2100 Bioanalyzer RNA 6000.
  • cDNA was prepared from the RNA template and used for in vitro transcription in the presence of biotinylated nucleoside triphosphates.
  • the biotinylated RNA targets were fragmented, and hybridized to Eos Hu03, a customized Affymetrix genechip containing 59,000 probe sets representing 46,000 genes, EST cluster, and predicted exons.
  • Genechips were performed in duplicate and stained with streptavidin phycoerythrin (SAPE) and scanned on an Affymetrix GeneChip Scanner 3000.
  • SAPE streptavidin phycoerythrin
  • Raw data files were obtained after analysis of scanned images with GCOS (GeneChip Operating Software, Affymetrix).
  • TWEAKR agonist resistant T47D cells at this time-point.
  • Many of the transcriptional changes were for genes known to be regulated by NFKB.
  • a heat map of NFi B-related gene expression in BT549 and T47D cells at 6 to 72 hours post-antibody treatment is shown in FIG. 5.
  • Many NFKB pathway members and known NFKB regulated genes were induced in BT549 cells and upregulation of NFicB-regulated genes was seen across all the time-points in the BT549 gene chip.
  • few NFKB regulated genes were induced in the T47D resistant cell line, and these differed from the NFKB-regulated genes that were activated in BT549 cells.
  • Xenograft generation ICR SCID mice (Taconic) were injected subcutaneously with 10 7 H358 cells in RPMI media. Mice were randomized into groups when the average tumor volume reached -100 mm 3 . The animals were injected with 10 mg/kg PDL192 or IgGl control 3 times per week (by intraperitoneal injection). For protein analysis of the tumors, animals were sacrificed at day 4, and tumors were harvested and flash frozen for protein extraction. All animal protocols and procedures were approved by the vivarium's Institutional Animal Care and Use Committee consistent with The U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare, National Institutes of Health).
  • Tumor growth inhibition assay Tumor growth inhibition was assayed using a tumor xenograft model as described in Culp et al, 2010, Clin, Cancer Res. 16(2):497- 508. The role of ADCC on growth inhibition was tested using a mutated version of PDL192, PDL192M, which does not elicit ADCC. PDL192M contains an alanine instead of a leucine at each of positions 234 and 235. See Culp et al, 2010, Clin. Cancer Res. 16(2):497-508.
  • siRNA transfections ON-TARGET plus® pooled siRNA or individual siRNAs against the target gene of interest (15 nM) (Thermo Scientific Dharmacon (Lafayette, CO)) or non-targeting pooled control siRNA were reverse transfected with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol.
  • ON-TARGET plus® siRNA oligonucleotides from Thermo Scientific Dharmacon (Lafayette, CO) were as follows, for each of the genes of interest: p65 (RelA)(Pooled siRNA, Catalog no. L- 003533-00-0002, or set of 4 individual siRNAs Catalog no. LQ-003533-00-0002);
  • p52/pl00 (NFKB2)(Pooled siRNA, Catalog no. L-003918-00-0002, or set of 4 individual siRNAs, Catalog no. LQ-003918-00-0002)
  • p50/pl05 (NFi Bl)(Pooled siRNA, Catalog no. L-003520-00-0002, or set of 4 individual siRNAs, Catalog no. LQ-003520-00-0002)
  • RelB Pooled siRNA, Catalog no. L-004767-00-0002, or set of 4 individual siRNAs, Catalog no. LQ-004767-00-0002)
  • IKKoc (CHUK) (Pooled siRNA, Catalog no.
  • L- 003473-00-0002 or set of 4 individual siRNAs, Catalog no. LQ-003473-00-0002); and ⁇ (Pooled siRNA, Catalog no. L-003503-00-0002, or set of 4 individual siRNAs, Catalog no. LQ-003503-00-0002).
  • Target knockdown was maximally achieved 2 days post transfection. Cells were then treated with PDL192 or IgGl control for the time indicated. The relative viability of the cells was determined, and cells were also harvested for Western blot analyses.
  • IKK16 NFKB inhibition.
  • the ⁇ kinase (IKK) selective inhibitor IKK 16 (Tocris Bioscience) was used at 160 nM. Waelchli et al, 2006, Bioorg Med Chem Lett 16(1): 108-12. The IKK16 was added to cells either alone or 1 h prior to addition of PDL192 (10 ⁇ g/ml). Cell viability was determined 5 days post treatment.
  • siRNA knockdown by siRNA of individual NFKB subunits resulted in a marked reduction in the growth inhibition observed in response to antibody treatment.
  • siRNA knockdown of p65 and p52 reduced PDL192 growth inhibition in MDA-MB-468 cells, while BT549 cells primarily showed a dependency on p50 and p65 for PDL192 activity (FIG. 10A-B).
  • Knockdown of RelB reduced PDL192 growth inhibition in some sensitive cell lines such as HT3.
  • CellTraceTM dye The dye is contained within the labeled cells and is diluted when passed to daughter cells during cell division, resulting in reduced fluorescence in the progeny. As shown in FIG. 11, HT3 cells treated with PDL192 exhibited higher fluorescence than control cells, suggesting that they divided less frequently. Inhibition of cell division by PDL192 was shown to be NFKB-dependent by using siRNA against individual NFKB subunits (p50, p65, RelB, p52). Each subunit was targeted by two different sets of siRNAs. Knockdown of these NFKB subunits allowed HT3 cells to divide more frequently, thereby overcoming inhibition of cell division by PDL192.
  • p21 a cyclin-dependent kinase inhibitor, in TWEAKR positive tumors in vitro and in vivo
  • Primary antibodies were p21 mlgGl (Dako M7202) and cytokeratinl8 rlgGl (Abeam). Secondary antibodies were AlexaFluor488 goat anti-rabbit and AlexaFluor594 goat anti- mouse (Invitrogen). Slides were mounted in Vector Lab DAPI mounting medium and imaged on a Zeiss Axioskop-2 fluorescent microscope. The number of p21 positive cells in response to PDL192 or IgGl control was determined by counting three 40x magnification fields for each xenograft tested.
  • siRNA transfections and assays Experiments to examine p21 induction in PDL192- or IgGl -treated HT3 cells transfected with siRNA targeting NFKB subunits p52 or p65 were performed as described above in Example 5. In another experiment, BT549 cells were transfected with 4 different siRNA oligonucleotides against p21 (ON- TARGET plus® siRNA, Catalog no. LQ-003471-00-0002, Thermo Scientific
  • FIG. 12 shows that p21 (WAF1/CIP1) is upregulated by treatment with PDL192, compared to treatment with an antibody control, in TWEAKR-sensitive HT3, BT549, HCC38 and MALME3 cell lines (FIG. 12).
  • FIG. 13 shows that induction of p21 protein levels by PDL192 was decreased in HT3 cells in the presence of siRNA targeting NFKB subunit p52 or p65, showing p21 upregulation by PDL192 is NFicB-dependent.
  • siRNA knockdown of p21 expression reduces the ability of PDL192 to inhibit the growth of BT549 breast cancer cells, which displayed increased viability as compared to control-RNA transfected cells, treated with PDL192.
  • TWEAKR positive cancer cell lines were tested in vitro for p21 expression in response to PDL192.
  • Indicated cell lines were treated with PDL192 or an IgG control antibody (MSL109) as described in Example 1 and Western analysis was performed as described in Example 6.
  • p21 expression is increased in cell lines in response to PDL192.
  • H358, BxPC3, and A253 cells show a detectable increase in p21 within 24 to 48 hours of treatment with PDL192, as compared to the control IgG antibody MSL109.
  • Table 3 shows the percent growth inhibition observed for cell lines tested for p21 changes, along with the observed change (i.e., increase or decrease), or lack of change (NC), in p21 expression in response to PDL192.
  • p21 expression was undetectable under both treatment and control conditions and is indicated as "nd.” With a single exception in the cell lines tested, all cell lines regardless of cancer type that exhibited at least 20% growth inhibition also showed an increase in p21 expression.
  • TWEAKR agonists including TWEAK and anti-TWEAKR antibodies PDL400, 136.1, 19.2.1, and 18.3.3.
  • p21 expression was detected by Western analysis, as described in Example 6, in HT3 cells treated with the indicated TWEAKR agonist for 24 hours.
  • Anti-TWEAKR antibodies were tested using the same conditions described in Example 1 for in vitro assays with PDL192. TWEAK was tested by incubating cells with 300 ng/mL TWEAK protein for 24 hours.
  • Table 4 below provides sequences of SEQ ID Nos: 1-56.

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Abstract

La présente invention concerne des procédés et des kits de contrôle de l'efficacité d'une thérapie par agoniste TWEAKR chez des sujets souffrant de cancers positifs pour TWEAKR, les procédés comportant les étapes consistant à déterminer si le sujet traité avec un agoniste TWEAKR présente une augmentation de l'expression de p21.
PCT/US2013/039276 2012-05-04 2013-05-02 Dosage de biomarqueurs de p21 WO2013166290A1 (fr)

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