WO2013166290A1

WO2013166290A1 - P21 biomarker assay

Info

Publication number: WO2013166290A1
Application number: PCT/US2013/039276
Authority: WO
Inventors: Patricia Culp; Yongke Zhang
Original assignee: Abbvie Biotherapeutics Inc.
Priority date: 2012-05-04
Filing date: 2013-05-02
Publication date: 2013-11-07

Abstract

The present disclosure is directed to methods and kits for monitoring efficacy of TWEAKR agonist therapy in subjects suffering from TWEAKR positive cancers, comprising determining whether the subject treated with a TWEAKR agonist exhibits an increase in p21 expression.

Description

P21 BIOMARKER ASSAY

1. BACKGROUND

[0001] Tumor Necrosis Factor-Related Weak inducer of Apoptosis, or TWEAK, is a member of the Tumor Necrosis Factor (TNF) family of cytokines that can cause cell death (in many instances via apoptosis). 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. Communications 305:789-796; Nakayama et al, 2003, J. Immunology 170:341-348. 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.

[0002] 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. However, 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. Consistent with these varied activities, studies of TWEAKR in cancer report both cancer-promoting effects and anti-cancer effects. For example, Tran et al., 2006, Cancer Res. 66(19):9535-9542, reported that increased levels of TWEAKR promote glioma cell invasion and correlate with poor patient outcomes, and Dai et al, 2009, Cancer Lett. 283(2): 159- 167, reported that signaling via TWEAKR promoted cell migration and invasion in an ovarian cancer cell line. Culp et al, 2010, Clin. Cancer Res. 16(2):497-508, reported that anti-TWEAKR agonistic antibodies inhibited growth and/or induced cell death in in vitro experiments with cancer cell lines and in vivo mouse xenograft experiments. Nakayama et al, 2003, J. Immunology 170:341-348, reported that TWEAKR agonists induced cell death via apoptosis or necrosis in a variety of cancer cell lines. Collectively, these data demonstrate that activation of TWEAKR in cancers that overexpress TWEAKR promotes positive anticancer responses in some, but not all, 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.

2. SUMMARY

[0003] As discussed in the Background Section, compounds that have TWEAKR agonist activity 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. As illustrated in 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. These data indicate that some but not all tumors that express TWEAKR may respond to treatment with compounds having TWEAKR agonist activity.

[0004] Applicants have discovered that p21, a cyclin-dependent kinase inhibitor that regulates cell cycle progression, is useful as a predictor of anti-tumorigenic efficacy of TWEAKR agonists. Specifically, applicants have discovered that p21 expression is upregulated in certain TWEAKR positive tumor cell lines that respond to treatment with compounds having TWEAKR agonist activity. As explained in more detail in the Examples section, applicants found a strong positive correlation between increased p21 expression and growth inhibition in TWEAKR positive cancer cell lines treated with a TWEAKR agonist antibody, PDL192. This discovery provides a new tool for monitoring the efficacy of subjects receiving TWEAKR agonist therapy.

[0005] Accordingly, in one aspect, 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. In a specific embodiment, 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. Alternatively, 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.

[0006] 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.

[0007] In another aspect, 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. Alternatively, 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.

[0008] TWEAKR agonists, also referred to herein as TWEAKR agonist compounds, useful in the methods of the present disclosure are described in Section 4.2.

[0009] In another aspect, the present disclosure provides kits useful for carrying out the monitoring and/or treatment methods described herein. Kits 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.

3. BRIEF DESCRIPTION OF THE FIGURES

[0010] 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;

[0011] 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;

[0012] FIG. 3 provides a Western blot detecting phosphorylated ΙκΒα, indicative of NFKB activation, in 6 TWEAKR positive cancer cell lines treated with PDL192 or a control IgGl;

[0013] 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

TWEAKR agonist sensitive (black bars) and resistant (grey bars) cell lines treated with PDL192. 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;

[0014] 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;

[0015] 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 ;

[0016] 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;

[0017] 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 ΙΚΚβ. * denotes p <0.05;

[0018] 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;

[0019] 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;

[0020] 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. For each NFkB subunit targeted -p65, p50, p52, and RelB- cells were transfected with two different sets of siRNAs (denoted target siRNA #1 and target siRNA#2);

[0021] 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;

[0022] 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;

[0023] FIG. 14 provides a graph showing increased viability of PDL192-treated

(squares) ΒΤ549 cells transfected with four different siRNAs targeting p21, relative to ΒΤ549 cells transfected with a control siRNA;

[0024] 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); [0025] 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

[0026] 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.

4. DETAILED DESCRIPTION

[0027] 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.

[0028] In an effort to understand the events associated with TWEAKR signaling in TWEAKR positive cancer cells, applicants observed that growth inhibition in these cells is mediated by NFKB, making the signaling molecule a tumor suppressor rather than tumor promoter in these cells. Applicants have now discovered that 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. Without being bound by theory, it is thought that 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. Thus, p21 expression serves as an indicator of TWEAKR agonist- induced cell growth inhibition in TWEAKR positive cancer cells. Furthermore, as shown in the Examples, in those cancer cell lines that exhibit TWEAKR agonist-induced cell growth inhibition, 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

expression," 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.

4.1. Method of Monitoring Treatment Efficacy

[0029] In one aspect, the present disclosure provides methods of monitoring TWEAKR agonist treatment efficacy in a subject having a TWEAKR positive cancer. Generally, 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.

[0030] The methods described herein are useful for monitoring subjects having a

TWEAKR positive cancer. The subject may be any animal, for example, a mammal, such as a farm animal or domesticated animal, or a human. A 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

immunohistochemistry are described in Culp et al., 2010, Clin. Cancer Res. 16(2):497- 508. Using these and other assays, 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. As detailed in Example 1, a test of 102 cancer cell lines further shows that 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.

4.2. TWEAKR Agonists

[0031] As described herein, the methods of the disclosure are useful for monitoring the progress of treatment with a TWEAKR agonist. 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.

[0032] In some embodiments, TWEAKR agonists induce apoptosis in cells expressing TWEAKR. Without being bound by theory, 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

deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al. (1994), Nature: 371 :346).

[0033] In some embodiments, 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. Generally, 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 μg/ml of the proposed TWEAKR agonist for 5 days in 96 well plates. Growth inhibition is determined using CellTiter-Blue™ (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.

[0034] In a specific exemplary assay where the proposed TWEAKR agonist is an anti- TWEAKR antibody as described below, cells can be incubated with anti-TWEAKR antibody (10 g/ml) and an antibody (5 μg/ml) or antibody fragment (3.5 μg/ml) that specifically binds to the anti-TWEAKR antibody. The antibody or antibody fragment is preferably species- and isotype-specific. For example, where 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. Alternatively, a full goat anti-human- IgG antibody can be used.

[0035] 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.

[0036] Using data generated from a cytokine/chemokine release assay, the percent agonist activity can be calculated using the formula: % agonist activity= (a-c)/(b-c) in which a= quantity of cytokine/chemokine released from cells treated with TWEAKR agonist at 10 g/ml, b- quantity of cytokine/chemokine released from cells treated with TWEAK at 300 ng/ml, and c= quantity of cytokine/chemokine released from untreated cells.

[0037] 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. 7,238,499), and members of the tumor necrosis factor (TNF) superfamily including TNF, TRAIL, LIGHT, FasL, CD40L, OX40L, RANKL, GITRL (see Gravestein et al., 1998, Immunology 10:423-434).

[0038] 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. 1-3, Cold Spring Harbor Laboratory (1989); Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1997); Tijssen, ed., Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, N.Y. (1993). Nucleic acids comprising the expression vectors described herein or components thereof include isolated, synthetic, and recombinant nucleic acids.

[0039] 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.

[0040] 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.

[0041] In addition to TWEAK, TWEAKR agonists include antibodies.

[0042] Unless indicated otherwise, the term "antibody" (Ab) 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.

[0043] 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). As is known in the art, 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. The variable domains of native heavy and light chains each comprise four FR regions, largely by adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-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). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.

[0044] An anti-TWEAKR agonist antibody can be an antibody fragment. As used herein, the term "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. Examples of 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. References to "VL" refer to the variable region of an immunoglobulin light chain. It is in this configuration that the three CDRs -typically denoted CDR1, CDR2, and CDR3- of each variable domain interact to define a target binding site on the surface of the VH-V_l dimer. Collectively, the six CDRs confer target binding specificity to the antibody. 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.

[0045] 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. Moreover, unless otherwise indicated, 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.

[0046] 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.

[0047] The anti-TWEAKR agonist antibodies of the disclosure can be humanized.

"Humanized" forms of non-human {e.g., murine) antibodies are chimeric

immunoglobulins, immunoglobulin chains or fragments thereof (such as F(ab')₂ or other target-binding subsequences of antibodies) which contain minimal sequences derived from non-human immunoglobulin. In general, 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. Methods of antibody humanization are known in the art. See, e.g., Riechmann et al, 1988, Nature 332:323-7; U.S. Pat. Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al, 1994, Prot.

Eng. 7:805-814; Roguska et a/., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

[0048] 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. As used herein, "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. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. 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).

[0049] The anti-TWEAKR agonist antibodies can be of any of the recognized isotypes. In some embodiments, 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. In other embodiments, the anti-TWEAKR antibodies are of the human IgGl isotype.

[0050] 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. For purposes of treating a subject using anti- TWEAKR agonist antibodies, 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.

[0051] 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. For example, the cytokine/chemokine release assay described above can be performed by incubating cells in vitro with an anti-TWEAKR antibody +/- TWEAK. Using the data generated from an cytokine/chemokine assay, the percent antagonist activity can be calculated using the formula: % antagonist=(b-d)/(b-a); in which a= quantity of cytokine/chemokine released from cells treated with antibody at 10 μg/ml, b= quantity of cytokine/chemokine released from cells treated with TWEAK at 300 ng/ml, c= quantity of cytokine/chemokine released from untreated cells, and d= quantity of cytokine/chemokine released from cells treated with TWEAK at 300 ng/ml and antibody at 10 μg/ml.

[0052] An anti-TWEAKR agonist antibody can bind to TWEAKR protein with a K_D of at least about Ι μΜ, at least about 0.1 μΜ or better, at least about 0.01 μΜ, and at least about 0.001 μΜ or better.

[0053] Anti-TWEAKR agonist antibodies useful in the present methods include antibodies that induce antibody-dependent cytotoxicity (ADCC) of TWEAKR-expressing cells. 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. 276:6591-6604; Presta et al, 2002, Biochem Soc. Trans. 30:487-490). In specific embodiments, 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.

7,632,497, U.S. Pat. No. 7,183,387, U.S. Pat. Pub. No. 2009/0074762, WO 00/42072.

[0054] ADCC activity can be monitored and quantified using a ⁵¹Cr release assay. Target cells are loaded with ⁵¹Cr, 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. In various embodiments, 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.

[0055] 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. To modify an antibody of the disclosure such that it exhibits reduced binding to the Fc receptor, 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.

[0056] 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).

[0057] Specific examples of 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:

(a) 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);

(b) 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); (c) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V_H CDR1 is XjYWMX₂ (SEQ ID NO:49), V_H CDR2 is EIRX₃KSX₄NYATX_SHYAESX₆KG (SEQ ID NO:50), V_H CDR3 is X7X8ADX9X10DY (SEQ ID NO:51); V_L CDR1 is XnASQSVSTSXi₂YSY Xi₃ (SEQ ID NO:52); V_L CDR2 is YAX_]4Xi₅LX₁₆S (SEQ ID NO:53); and V_L CDR3 is QHSWEXnPXisT (SEQ ID NO:54), wherein: X₁ is selected from K, N, R, and S; X₂ is selected from N and S; X₃ is selected from L and V; X₄ is selected from D and N; X₅ is T or no amino acid; X₆ is selected from A and V; X₇ is selected from A, G, T and Y; X₈ is selected from F and Y; X₉ is selected from A, T and Y; X₁₀ is selected from F and M; Xn is selected from R and K; X_i2 is selected from S and T; Xn is selected from H and Q; X₁₄ is selected from S and T; X_!5 is selected from N and K; Xi₆ is selected from E and D; and Xn is selected from I and L; and Xig is selected from W and Y;

(d) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V_H CDR1 is SYWMS (SEQ ID NO: 13), V_H CDR2 is EIRLKSDNYATHYAESVKG (SEQ ID NO: 19), V_H CDR3 is

YYADAMDY (SEQ ID NO:25), V_L CDR1 is RASQS VSTS SYS YMH (SEQ ID NO:31), V_L CDR2 is YASNLES (SEQ ID NO:37), and V_L CDR3 is

QHSWEIPYT (SEQ ID NO:43);

(e) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V_H CDR1 is NYWMS (SEQ ID NO: 15), V_H CDR2 is EIRLKSDNYATHYAESVKG (SEQ ID NO:21), V_H CDR3 is

GFADYFDY (SEQ ID NO:27), V_L CDR1 is RASQSVSTSSYSYMQ (SEQ ID NO:33), V_L CDR2 is YATNLDS (SEQ ID NO:39), and V_L CDR3 is

QHSWEIPYT (SEQ ID NO:45);

(f) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V_H CDR1 is KYWMN (SEQ ID NO: 16), V_H CDR2 is EIRLKSDNYATHYAESAKG (SEQ ID NO:22), V_H CDR3 is

TYADTMDY (SEQ ID NO:28), V_L CDR1 is KASQSVSTSTYSYMQ (SEQ ID NO:34), V_L CDR2 is YASKLDS (SEQ ID NO:40), and 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

QHSWEIPWT (SEQ ID NO:47); and

(h) an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, wherein V_H CDR1 is NYWMN (SEQ ID NO: 17), V_H CDR2 is EIRLKSNNYATHYAESVKG (SEQ ID NO:24), V_H CDR3 is

AYADYFDY (SEQ ID NO:30), V_L CDR1 is RASQSVSTSTYSYMH (SEQ ID NO:36), V_L CDR2 is YASNLES (SEQ ID NO:42), and V_L CDR3 is

QHSWEIPYT (SEQ ID NO:48).

[0058] 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. If the 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. Numerous methods for carrying out binding competition assays are known. 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 μg/mL, although higher levels of reduction, for example, 60%, 70%, 80%, 90% or even 100%, may be desirable. 4.3. p21 Detection

[0059] 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. Generally, 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. Where the sample is a blood sample from a subject suffering from a TWEAKR positive cancer, 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).

[0060] As used herein, 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.

[0061] A suitable assay for determining whether an increase in p21 expression is observed after administrating a TWEAKR agonist is described in Example 6. Briefly, 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 μπι tissue sections are then mounted onto slides, deparaffinized and ethanol rehydrated. Samples can be blocked using 0.3% H₂0₂ Dako X0909 blocking solution and then treated with a primary antibody against p21 (p21 mlgGl, Dako Cat. No. M7202) and a secondary antibody bearing a detectable label (AlexaFluor®488 goat anti-rabbit, Invitrogen™/ Life Technologies™). 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.

gs

[0062] For some applications, an increase in p21 expression is evidenced by an increase in the amount of p21 protein or mRNA, determined relative to a baseline level. For some applications, it is desirable to compare p21 expression before and after (or with and without) administration of a TWEAKR agonist. For such applications, 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. In a specific embodiment, 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. Alternatively, the baseline level can be an amount or concentration of p21 protein or mRNA determined from multiple samples. For example, 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.

[0063] 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.

[0064] An increase in p21 expression is detectable in vivo and in vitro after

administration of a TWEAKR agonist as shown in Examples 6 and 7 below.

Accordingly, an increase in p21 expression provides an early indicator that TWEAKR agonist therapy is effective. In the methods of the present disclosure, 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. In some embodiments, p21 expression is measured after a first administration of a TWEAKR agonist to the subject.

[0065] 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.

[0066] For some applications, it may be desirable to measure p21 mRNA levels.

Detection 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,

BioTechniques 18:42044. RNA isolation can be performed using commercially available purification kits, buffer sets, and proteases from vendors such as Qiagen (Valencia, CA) or Life Technologies™ (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.

[0067] 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.

[0068] p21 expression can be detected using primers or probes that specifically hybridize to p21 mRNA. In humans, p21 (also called CIPl, CDNKIA and WAF1) is encoded by a single gene, located on chromosome 6. 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. Alternatively, primers may amplify only certain variant transcripts. Examples of suitable 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 Pair™, 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.

[0069] 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. For example, RT-PCR can be carried out using the TaqMan® assay and primers sold by Applied Biosystems™ / Life Technologies™ (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.

[0070] RT-PCR is performed using any RT-PCR instrumentation available in the art. Typically, 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. To control for variability in cDNA quantity, integrity and the overall transcriptional efficiency of individual primers, 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).

[0071] 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. Generally, 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.

[0072] 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. [0073] 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. Examples of 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. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-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. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of 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; examples of suitable isotopic materials include ¹³C, ¹⁵N, and deuterium; and examples of suitable radioactive material include ^I251, ¹³¹I, ¹¹ 'in or ⁹⁹Tc.

[0074] For some applications, it may be desirable to measure p21 protein levels.

Presence and amount of p21 protein can be determined using antibody-based

immunoassays such as Western blots, ELISA, immunoprecipitation followed by SDS- PAGE, immunohistochemistry, fluorescence assisted cell sorting (FACS) and other techniques, all of which are well known to those of skill in the art. A specific example of detection of p21 protein by Western blot is provided below in Example 6. The same example provides a specific assay for in situ p21 detection and quantification. [0075] 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)).

[0076] Generally, 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).

4.4. Methods of Treating a TWEAKR Positive Cancer

[0077] Applicants have discovered that p21 expression increases after a dose of a TWEAKR agonist in cancer cells whose growth is inhibited by the same agonist.

Consequently, 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.

[0078] 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. For some applications, 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.

[0079] Suitable 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.

[0080] An increase in the level of p21 expression can be determined as described in Section 4.3.

[0081] 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. When used in combination with other treatments, 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.

[0082] In the methods of treatment provided herein, subjects are administered a

TWEAKR agonist in amount effective in treating the TWEAKR positive cancer. For example, the amount can be an amount sufficient to provide a therapeutic benefit. In the context of treating a TWEAKR positive cancer, 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.

[0083] 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

conveniently presented in unit dose forms containing a predetermined amount of a TWEAKR agonist per dose. In embodiments where the TWEAKR agonist is an anti- TWEAKR antibody, 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.

[0084] 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.

[0085] 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. Ultimately, 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.

[0086] 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.

4.5. Kits

[0087] In an aspect, the present disclosure provides a kit useful for carrying out the monitoring methods described herein. Such kits comprise detection agents suited to the particular target, e.g. p21 mRNA or p21 protein. Thus, 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.

[0088] 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 (e.g. an mRNA, a spliced mRNA, a cDNA, or the like) 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. Optionally, 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.

[0089] 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.

[0090] In addition, 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.

5. EXAMPLES

5.1 Example 1: TWEAKR agonist PDL192 inhibits growth of a

wide range of cancers in vitro

[0091] This example shows that a monoclonal antibody that acts as a TWEAKR agonist, PDL192 (used interchangeable herein with enavatuzumab), inhibits the growth of 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. [0092] 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 μg/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 μg/ml. Wiley et al, 2001, Immunity 15(5):837-46.

[0093] Cell lines. All cell lines were obtained from the American Tissue Culture Collection (ATCC) or National Cancer Institute (NCI), except HSC-3 which was kindly provided by the Japan Health Science Foundation, and the MDA-MB231 variant cell line, which was derived from the MDA-MB-231 cell line and selected for consistent growth in vivo.

[0094] Growth inhibition assay. 102 cancer cell lines were cultured at 500 to 2000 cells per well in triplicate with enavatuzumab or IgGl control in the presence of F(ab')₂ goat anti-human IgG, (Fey specific) for 5 days in 96 well plates. Relative cell viability was determined using CellTiter-Blue™ (Promega). Fluorescence emitted at 590 nm was used to calculate the growth effect relative to the IgGl control antibody treatment. Each cell line was tested twice with the average growth inhibition reported. Cell lines with at least about 20% growth inhibition were considered TWEAKR agonist sensitive.

[0095] 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.

Table 1

Cancer Sensitive Resistant type (at least 20% growth (<10% growth inhibition) inhibition)

Breast BT-549 BT-20

CAMA-1 BT-474

HCC1143 DU-4475

HCC1500 HCC-1428

HCC1569 Hs 578T

HCC1937 MCF-7

HCC38 MDA-MB-175-VII

HCC70 MDA-MB-361

MDA-MB-157 MDA-MB-435S

MDA-MB-231 MX-1

MDA-MB231 variant T47D

MDA-MB-453 ZR-75-1

MDA-MB-468 ZR-75-30

NCI-ADR-RES

SK-BR-3

Colon HT-29 COLO-205

SW480 HCC2998

HCT-116

HCT-15

KM12

LoVo

SW48

SW620

SW837

SW948

Table 1

Lung A549 Calu-3

Calu-6 DMS-79

EKVX NCI-H1155

HOP-62 NCI-H146

HOP-92 NCI-H157

NCI-H1792 NCI-H1838

NCI-H358 NCI-H187

NCI-H209

NCI-H226

NCI-H23

NCI-H292

NCI-H322M

NCI-H345

NCI-H460

NCI-H522

NCI-H596

NCI-H69

NCI-H82

Renal ACHN 786-0

CAKI-1 A498

RXF393

SN12C

TK10

U031

Melanoma A375 A2058

Malme-3M C32

SK-MEL-2 HT-144

SK-MEL-28 LOX

M14

MDA-MB-435

SK-MEL-5

UACC-257

UACC-62

WM-115

Ovarian PA-1 IGROV-1

OVCAR-3 OVCAR-4

OVCAR-5

OVCAR-8

SK-OV-3

SW626 (metastasis from colon) Table 1

Cancer Sensitive Resistant

type (at least 20% growth (<10% growth

inhibition) inhibition)

Pancreatic AsPC-1 MIA-PaCa-2

BxPC3

Bladder SW780 —

Cervical HT3 —

Salivary A253 ~

Oral HSC-3 —

[0096] 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.

5.2 Example 2: TWEAKR agonist PDL192 induces NFKB

expression in TWEAKR positive cancers in vitro

[0097] This example shows that TWEAKR agonist PDL192 induces the expression of an NFicB-responsive reporter construct.

[0098] Assay. 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).

[0099] Western blot. Whole cell protein lysates were generated using Cell Signaling protein lysis buffer containing protease inhibitors (Roche) and phosphatase inhibitor cocktail (Sigma), quantified using BCA reagent (Pierce). Protein expression was detected using ECL Plus Chemiluminescence kit (GE-Amersham). Antibodies against

phosphorylated ΙκΒα (Ρ-ΙκΒα (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.

[0100] Results. To identify potential signaling pathways related to inhibition of proliferation by PDL192, TWEAKR agonist sensitive cells were transfected with a luciferase transcriptional reporter construct designed to show NFKB signal transduction. PDL192 treatment induced a marked increase in NFKB driven luciferase expression in a breast cancer cell line, BT549 (FIG. 2). Similar increases in NFKB reporter activity was observed in other breast cancer, lung cancer, cervical cancer and melanoma cell lines (HCC38, H358, HT3, A375) (not shown). To further investigate the activation of the NFKB pathway by PDL192, phosphorylation of ΙκΒα, the cytoplasmic inhibitor of classical NFKB subunits, was assayed. Phosphorylation of ΙκΒα on Serine 32/36 has been shown to reflect NFKB pathway activation, as it allows the cytoplasmic release and migration of NFKB dimers into the nucleus to initiate transcription. Baldwin (1996) Annu Rev Immunol 14:649-83; Hacker et al, 2006 Sci STKE 2006(357); Whiteside et al, 1997, Semin Cancer Biol 8(2):75-82. As shown in FIG. 3, PDL192 induced

phosphorylation of ΙκΒα in several TWEAKR agonist sensitive cell lines, indicating activation of the NFKB pathway at 4 and 24 hours after treatment (FIG. 3).

5.3 Example 3: TWEAKR agonist sensitive cancer cells show

increased NFKB activation and transcriptional changes relative to resistant cancer cells in response to PDL192

[0101] This example shows that PDL192 induces NFKB activation and transcriptional changes in TWEAKR agonist sensitive cancer cells but not in TWEAKR agonist resistant cancer cells.

[0102] NFKB transcription factor ELISA. TransAM™ 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. Raw data files were obtained after analysis of scanned images with GCOS (GeneChip Operating Software, Affymetrix).

[0104] Results. Having established that NFKB was activated in response to PDL192 in TWEAKR sensitive cells, a functional ELISA for NFKB was performed in a series of cell lines to determine which specific NFKB subunits are activated in response to PDL192 treatment. The NFKB DNA binding ability of 6 sensitive cell lines (A375, HCC38, H358, BT549, MDA-MB-468, HT3) and 3 resistant cell lines (UACC62, T47D, BT20) were examined by capture ELISA post treatment (FIG. 4A-D). Induction of all NFKB subunits (p50, p65, p52, RelB and c-Rel) occurred in sensitive cell lines following treatment. This indicates that both classical (p50, p65) as well as non-classical (p52, RelB) NFKB pathways could be activated by PDL192 treatment. Resistant cell lines showed significantly less induction of all NFKB subunits, with little or no subunit induction occurring in UACC62, T47D and BT20.

[0105] The differential pattern of NFKB activation between TWEAKR agonist sensitive and resistant lines was further confirmed using Affymetrix gene chip microarrays. As shown in Table 2, below, the number of genes up- or down-regulated >2-fold was greater in sensitive lines (BT549, MDA-MB-468) compared to those in resistant lines (T47D, BT20). TABLE 2

No. of genes Up or Down > 2 fold

Sensitive Resistant

MDA-468 BT-549 T47D BT-20

6hr 51 126 1 NA

24hr 50 408 9 NA

48hr 329 648 15 3

72hr 445 766 13 NA

[0106] For example, 408 genes were up or down regulated in TWEAKR agonist sensitive BT549 cells after 24 h of treatment, while expression of only 9 genes changed in

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. In contrast, 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.

5.4 Example 4 : TWEAKR agonist PDL192 activates NFKB and

inhibits growth in an in vivo cancer xenograft model

[0107] This example shows that PDL192 activates signaling via NFKB and inhibits growth of H358 tumor xenografts in mice.

[0108] Xenograft generation: ICR SCID mice (Taconic) were injected subcutaneously with 10⁷ H358 cells in RPMI media. Mice were randomized into groups when the average tumor volume reached -100 mm³. 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).

[0109] 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.

[0110] Western blot. Western blot analysis was performed on H358 xenograft tumors as described in Example 2 to detect activation of NFKB subunits. Mice were treated with PDL192 or an IgG control antibody at day 0 and day 3 and tumors excised at day 4. Three mice were tested per treatment. Antibodies against NFKB subunits p65, pl00/p52, pl05/p50 and RelB were purchased from Cell Signaling. GAPDH antibodies, used as a control, were from Santa Cruz. Mouse and rabbit secondary antibodies were from GE- Amersham.

[0111] Results. To determine whether NFKB was activated in vivo in response to PDL192 treatment, the TWEAKR agonist sensitive H358 cell line was tested in a mouse xenograft model. PDL192 inhibited tumor growth (measured in terms of tumor volume), compared to the control antibody treated tumors, by 70% in H358 tumors in vivo.

Response to PDL192 was dependent on signaling through TWEAKR: As shown in FIG. 6, a modified antibody based on PDL192 that does not trigger antibody-dependent cellular cytotoxicity blocks tumor growth to the same extent as PDL192. An increase in NFKB subunits RelB, pl05/p50 and p52 levels was observed in H358 tumors harvested from PDL192-treated mice (FIG. 7), indicating activation of components in the NFKB pathway in vivo, consistent with the observations made in vitro. No change in p65 expression was observed following PDL192 treatment in this model. GAPDH was used as a positive control and showed no difference between the PDL192-treatment and the IgG 1 -treatment. 5.5 Example 5: Inhibition of components in the NFKB signaling pathway reduces TWEAKR agonist-induced growth inhibition

[0112] This example shows that targeted knockdown of components in the NFKB pathway using small interfering RNAs blocks PDL192-induced inhibition of cell growth.

[0113] 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 ΙΚΚβ (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.

[0114] IKK16 NFKB inhibition. The ΙκΒ 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 μg/ml). Cell viability was determined 5 days post treatment.

[0115] Cell division analyses. HT3 cells (30,000 cells per 6-well) were siRNA transfected as described above. 2 days post transfection, CellTrace™ violet (Invitrogen) was added to the cells at 5 uM for 30 mins, cells were washed according to the manufacturer's instructions, and treated with PDL192 or IgGl control for 5 days. Cells were trypsinized and then fixed in 2% paraformaldehyde. The amount of CellTrace™ dye within the cells was analyzed by flow cytometry using a violet laser (CyAn, Dako).

[0116] Results. To determine if NFKB signaling was important for PDL192 inhibition of cancer cell growth, siR A experiments were performed targeting components of the NFKB pathway. Transfection with siRNAs against NFKB pathway members IKKa (CHUK), ΙΚΚβ, and p65 resulted in a statistically significant increase in survival of PDL192-treated cells relative to mock-transfected cells (see light grey bars in FIG. 8). To confirm the role of the NFKB pathway in growth inhibition by PDL192, a small molecule inhibitor (IKK16) of the NFKB upstream kinases ΙΚΚ /β was tested and found to reduce growth inhibition by PDL192 in breast cancer cell lines and a cervical cancer cell line (FIG. 9). Knockdown by siRNA of individual NFKB subunits (p65, p52, RelB and p50) 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.

[0117] The rate of cell proliferation in PDL192-treated cells was assessed using

CellTrace™ 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.

5.6 Example 6: TWEAKR agonist PDL192 induces expression of

p21, a cyclin-dependent kinase inhibitor, in TWEAKR positive tumors in vitro and in vivo

[0118] This example shows that p21, a cyclin-dependent kinase inhibitor, is activated by PDL192 both in vitro and in vivo in TWEAKR sensitive cancer cells. [0119] Immunohistochemistry for p21. Xenograft samples were generated as described in Example 4 except mice received a single dose of antibody at 10 mg/kg. Tumors were harvested at the various times indicated post-dose and were formalin fixed and paraffin embedded. Tissue sections (5 μιη) were cut, mounted on slides, deparaffinized and ethanol rehydrated. Antigen retrieval was performed using BORG Decloaker RTU (Biocare Medical). Samples were blocked in 0.3% H₂0₂ Dako X0909 blocking solution. 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.

[0120] Western blot analysis. Western blots for p21 in HT3, BT549, HCC38, and MALME3 cells were performed as described above in Example 2, using antibodies against p21 WAF1/CIP1 (DCS60) purchased from Cell Signaling.

[0121] 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

Dharmacon, Lafayette, CO). Two days after transfection, cells were treated with PDL192 or IgGl control antibody for 5 days and cell viability was determined using CellTiter Glo® (Promega, Madison, WI) used according to manufacturer's guidelines. Viability of siRNA-transfected cells treated with PDL192 or control antibody is relative to the cell viability of control RNA-transfected cells, which was set at 1. Numbers above 1 denote an increase in cell viability which numbers below 1 denote a decrease in cell viability.

[0122] Results. In searching for the mechanism by which PDL192 inhibits cell growth, cell cycle regulators were investigated. 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). As shown in FIG. 13, 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. Furthermore, as shown in FIG. 14, 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.

[0123] Induction of p21 by PDL192 also occurred in vivo in H358 xenografts, as determined by immunohistochemistry. An increase in the number of p21 positive cells was observed 3 days post dose, and was still maintained one week after a single i.p. dose of PDL192 (FIG. 15).

5.7 Example 7: Increase in p21 expression correlates with growth

inhibition in PDL192-treated cell lines

[0124] This example shows that almost all TWEAKR positive cell lines tested that exhibited at least 20% growth inhibition also exhibited an increase in p21 expression in response to PDL192.

[0125] In vitro growth inhibition assay. Growth inhibition of TWEAKR positive cancer cell lines by PDL192 was assayed as described in Example 1. The cell lines tested are listed in Table 3 and included cell lines derived from the following types of cancers: breast, cervical, colon, lung, bladder, salivary gland, melanoma, ovarian, pancreatic, glioblastoma, and renal. Data were collected from at least 2 different experiments and the mean percent growth inhibition for each cell line was calculated.

[0126] p21 expression. 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.

[0127] Results. p21 expression is increased in cell lines in response to PDL192. As shown in FIG. 16, 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 below 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. For some cell lines, 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.

5.8 Example 8: TWEAKR agonists induce p21 expression in vitro

[0128] This example shows that p21 expression is increased in response to a variety of TWEAKR agonists, including TWEAK and anti-TWEAKR antibodies PDL400, 136.1, 19.2.1, and 18.3.3.

[0129] p21 expression. 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.

[0130] Results. As shown in FIG. 17, each of the anti-TWEAKR antibodies induced p21 expression, consistent with the response observed in HT3 cells with PDL192. TWEAK, the natural ligand also induced expression of p21 albeit to a lesser extent. This may be attributable to internalization of the ligand abrogating signaling from TWEAKR.

6. SEQUENCE LISTING

[0131] Table 4 below provides sequences of SEQ ID NOs: 1-56.

TABLE 4

SEQ ID Description SEQUENCE

NO:

1 Human TWEAK-R TCGACCCACG CGTCCGCCCA CGCGTCCGCC CACGCGTCCG GGCGCAGGAC GTGCACTATG

nucleic acid GCTCGGGGCT CGCTGCGCCG GTTGCTGCGG CTCCTCGTGC TGGGGCTCTG GCTGGCGTTG

CTGCGCTCCG TGGCCGGGGA GCAAGCGCCA GGCACCGCCC CCTGCTCCCG CGGCAGCTCC TGGAGCGCGG ACCTGGACAA GTGCATGGAC TGCGCGTCTT GCAGGGCGCG ACCGCACAGC GACTTCTGCC TGGGCTGCGC TGCAGCACCT CCTGCCCCCT TCCGGCTGCT TTGGCCCATC CTTGGGGGCG CTCTGAGCCT GACCTTCGTG CTGGGGCTGC TTTCTGGCTT TTTGGTCTGG AGACGATGCC GCAGGAGAGA GAAGTTCACC ACCCCCATAG AGGAGACCGG CGGAGAGGGC TGCCCAGCTG TGGCGCTGAT CCAGTGACAA TGTGCCCCCT GCCAGCCGGG GCTCGCCCAC TCATCATTCA TTCATCCATT CTAGAGCCAG TCTCTGCCTC CCAGACGCGG CGGGAGCCAA GCTCCTCCAA CCACAAGGGG GGTGGGGGGC GGTGAATCAC CTCTGAGGCC TGGGCCCAGG GTTCAGGGGA ACCTTCCAAG GTGTCTGGTT GCCCTGCCTC TGGCTCCAGA ACAGAAAGGG AGCCTCACGC TGGCTCACAC AAAACAGCTG ACACTGACTA AGGAACTGCA GCATTTGCAC AGGGGAGGGG GGTGCCCTCC TTCCTAGAGG CCCTGGGGGC CAGGCTGACT TGGGGGGCAG ACTTGACACT AGGCCCCACT CACTCAGATG TCCTGAAATT CCACCACGGG GGTCACCCTG GGGGGTTAGG GACCTATTTT TAACACTAGG GGGCTGGCCC ACTAGGAGGG CTGGCCCTAA GATACAGACC CCCCCAACTC CCCAAAGCGG GGAGGAGATA TTTATTTTGG GGAGAGTTTG GAGGGGAGGG AGAATTTATT AATAAAAGAA TCTTTAACTT TAAAAAAAAA AAAAAAAAAG GGCGGCCGCT CTAGAGGATC CCTC

Human TWEAK-R MARGSLRRLL RLLVLGLWLA LLRSVAGEQA PGTAPCSRGS SWSADLDKCM DCASCRARPH protein SDFCLGCAAA PPAPFRLLWP ILGGALSLTF VLGLLSGFLV WRRCRRREKF TTPIEETGGE

GCPAVALIQ

3 PDL192 V_H EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMSWVRQA PGKGLEWVAE IRLKSDNYAT

HYAESVKGRF TISRDDSK S LYLQMNSLRA EDTAVYYCTG YYADAMDYWG QGTLVTVSS

4 PDL192 V_L DIQMTQSPSS LSASVGDRVT ITCRASQSVS TSSYSYMHWY QQKPGKAPKL LIKYASNLES

GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQHSWEIPY TFGGGTKVEI KR

Ί 19.2.1 V_H EVKLEESGGG LVQPGGSMKL SCVASGFTFS SYWMSWVRQS PEKGLEWVAE IRLKSDNYAT

HYAESVKGKF TISRDDSKSR LYLQMNSLRA EDTGIYYCTG YYADAMDYWG QGTSVTVSS

6 19.2.1 V_L DIVLTQSPAS LAVSLGQRAT ISCRASQSVS TSSYSYMHWY QQKPGQPPKL LIKYASNLES

GVPARFSGSG SGTDFTLNIH PVEEEDTATY YCQHSWEIPY TFGGGTKLEI KR

1 18.3.3 V_H EVKLGESGGG LVQPGGSMKL SCVASGFPFT KYWMNWVRQS PEKGLEWVAE IRLKSDNYAT

HYAESAKGRF TISRDDSRSS VYLQMNNLRA EDTAIYYCSP TYADTMDYWG QGTSVTVSS

Ϊ 18.3.3 V_L DIVLTQSPAS LAVSLGQRAT ISCKASQSVS TSTYSYMQWY QQRPGQSPKL LIKYASKLDS

[0132] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0133] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

Claims

WHAT IS CLAIMED IS:

1. A method of monitoring efficacy of a TWEAKR agonist therapy, comprising:

determining whether a subject with a TWEAKR positive cancer being treated with a TWEAKR agonist exhibits an increase in p21 expression as compared to a baseline level.

2. The method of claim 1, wherein the increase in p21 expression is an increase in the number of p21 -positive cells in a test sample from the subject relative to a baseline level.

3. The method of claim 2, wherein the test sample has 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% more p21 -positive cells relative to a baseline level.

4. The method of claim 2, wherein the baseline level is a level from a baseline sample taken from the subject.

5. The method of claim 1, wherein the increase in p21 expression is an increase in p21 protein in a test sample from the subject relative to a baseline level.

6. The method of claim 5, wherein p21 protein in the test sample is 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.

7. The method of claim 5, the baseline level is a level from a baseline sample taken from the subject.

8. The method of claim 5, wherein the baseline level is an average level from baseline samples from a population of individuals suffering from the same type of TWEAKR positive cancer as the subject.

9. The method of claim 1, wherein the increase in p21 expression is an increase in p21 mRNA in a test sample from the subject relative to a baseline level.

10. The method of claim 9, wherein the p21 mRNA in a test sample is 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.

11. The method of claim 9, wherein the baseline level is a level from a baseline

sample taken from the subject.

12. The method of claim 9, wherein the baseline level is an average level from

baseline samples from a population of individuals suffering from the same type of TWEAKR positive cancer as the subject.

13. The method of any one of the preceding claims, wherein the increase in 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.

14. The method of any one of the preceding claims, wherein the TWEAKR agonist is an anti-TWEAKR agonist antibody.

15. The method of claim 14, wherein the anti-TWEAKR agonist antibody is a monoclonal antibody.

16. The method of claim 14, wherein the anti-TWEAKR agonist antibody is a

humanized antibody.

17. The method of claim 14, wherein the anti-TWEAKR agonist antibody is a

monoclonal antibody or anti-TWEAKR antigen binding fragment, comprising the following six CDRs:

V_H CDR 1 is: XJ Y W M X₂ (SEQ ID NO:49);

V_H CDR 2is:EIRX₃KSX₄NYATX₅HYAESX₆KG (SEQ ID NO:50);

V_H CDR3 is: X₇ X₈ A D X₉ Xio D Y (SEQ ID NO:51);

V_LCDR1 is: X„ A S Q S V S T S X₁₂ Y S Y M X₁₃ (SEQ ID NO:52);

V_L CDR2 is: Y A X₁₄ Xi₅ L Xj₆ S (SEQ ID NO:53); and

V_L CDR3 is: Q H S W E X₁₇ P Xig T (SEQ ID NO:54), wherein:

Xi is selected from K, N, R, and S; X₂ is selected from N and S; X₃ is selected from L and V; X₄ is selected from D and N; X₅ is T or no amino acid; X₆ is selected from A and V; X₇ is selected from A, G, T and Y; X₈ is selected from F and Y; X9 is selected from A, T and Y; Xio is selected from F and M; Xn is selected from R and K; Xi₂ is selected from S and T; Xj₃ is selected from H and Q; X₁₄ is selected from S and T; X15 is selected from N and K; X₁₆ is selected from E and D; and X_]7 is selected from I and L; and Xi₈ is selected from W and Y.

18. The method of claim 17, wherein the anti-TWEAKR agonist antibody is a

V_H CDR 1 is: S Y W M S (SEQ ID NO: 13);

V_H CDR 2is:EIRLKSDNYATHYAESVKG (SEQ ID NO: 19); V_H CDR3 is: Y YA D AMD Y (SEQ ID NO:25);

VL CDRI is: AA S Q S V S T S S Y S Y M H (SEQ ID NO:31);

V_L CDR2 is: Y A S N L E S (SEQ ID NO:37); and

V_L CDR3 is: Q H S W E IP Y T (SEQ ID NO:43).

19. The method of claim 17, wherein the anti-TWEAKR agonist antibody which comprises a VH region having a sequence selected from SEQ ID NO:3 and SEQ ID NO:l 1 and a V_L region having a sequence selected from SEQ ID NO:4 and SEQ ID NO: 12.

20. The method of claim 17, wherein the anti-TWEAKR agonist antibody is selected from the group consisting of: PDL192 and PDL400.

21. A method of treating cancer in a subject, comprising:

(a) determining in a subject suffering from a TWEAKR positive cancer who has received at least a first dose of a TWEAKR agonist whether p21 expression increases relative to a baseline level; and

(b) continuing treatment with the TWEAKR agonist if an increase in p21 expression is detected.

22. The method of claim 21, wherein the increase in p21 expression is an increase in p21 mRNA or p21 protein in a test sample from the subject relative to a baseline level.

23. The method of claim 22, wherein the p21 mRNA in a test sample is 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 to a baseline level.

24. The method of claim 22, wherein the baseline level is a level from a baseline sample taken from the subject.

25. The method of any one of claims 22, wherein the baseline level is an average level from baseline samples from a population of individuals suffering from the same type of TWEAKR positive cancer as the subject.

26. The method of any one of claims 21 to 25, wherein the increase in 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 said dose of

TWEAKR agonist to the subject.

27. A method of treating a human subject having a TWEAKR positive cancer

comprising: administering to the subject a TWEAKR agonist in an amount effective in treating the TWEAKR positive cancer, determining whether a test sample from the subject exhibits an increase in p21 expression as compared to a baseline level, and if so, continuing to administer the TWEAKR agonist compound to the subject.

28. The method of claim 27, wherein the increase in p21 expression is an increase in the number of p21 -positive cells in a test sample from the subject as compared to a baseline level.

29. The method of claim 27, wherein the increase in p21 expression is an increase in p21 mRNA or p21 protein in a test sample from the subject as compared to a baseline level.

30. The method of claim 29, wherein the p21 mRNA in a test sample is 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 to a baseline level.

31. The method of claim 30, wherein the baseline level is a level from a baseline sample taken from the subject.

32. A method of treating a TWEAKR positive cancer, comprising administering to a subject who has a TWEAKR positive cancer and who tests positive for an increase in the level of expression of p21 as compared to a baseline level when receiving a TWEAKR agonist, a TWEAKR agonist in an amount effective to treat the TWEAKR positive cancer.

33. The method of claim 32, wherein the increase in p21 expression is an increase in the number of p21 -positive cells in a test sample from the subject relative to a baseline level.

34. The method of claim 32, wherein the increase in p21 expression is an increase in p21 mRNA or p21 protein in a test sample from the subject as compared to a baseline level.

35. The method of claim 34, wherein the p21 mRNA in a test sample is 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 to a baseline level.

36. The method of claim 35, wherein the baseline level is a level from a baseline sample taken from the subject.

37. The method of any one of claims 21 to 36, wherein the increase in 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 said dose of TWEAKR agonist to the subject.

38. The method of any one of claims 21 to 36, wherein the TWEAKR agonist is an anti-TWEAKR antibody.

39. The method of claim 38, wherein the anti-TWEAKR agonist antibody is a

monoclonal antibody.

40. The method of claim 38, wherein the anti-TWEAKR agonist antibody is a

humanized antibody.

41. The method of claim 38, wherein the anti-TWEAKR agonist antibody is a

V_H CDR 1 is: X_I Y W M X₂ (SEQ ID NO:49);

V_H CDR 2 is: E I R X₃ K S ¾N Y A T X₅ H Y A E S X₆K G (SEQ ID NO:50);

V_H CDR3 is: X₇ X₈ A D X₉ Xio D Y (SEQ ID NO:51);

V_L CDRl is: X„ A S Q S V S T S X,₂ Y S Y M X_J3 (SEQ ID NO:52);

V_L CDR2 is: Y A X₁₄ X_]5 L X₁₆ S (SEQ ID NO:53); and

V_L CDR3 is: Q H S W E Xi_? P X₁₈ T (SEQ ID NO:54), wherein:

Xi is selected from K, N, R, and S; X₂ is selected from N and S; X₃ is selected from L and V; X₄ is selected from D and N; X₅ is T or no amino acid; X₆ is selected from A and V; X₇ is selected from A, G, T and Y; X₈ is selected from F and Y; X9 is selected from A, T and Y; Xio is selected from F and M; Xn is selected from R and K; X₁₂ is selected from S and T; Xi₃ is selected from H and Q; X₁₄ is selected from S and T; X15 is selected from N and K; X₁₆ is selected from E and D; and X₁₇ is selected from I and L; and Xi₈ is selected from W and Y.

42. The method of claim 41, wherein the anti-TWEAKR agonist antibody is a

V_H CDR 1 is: S Y W M S (SEQ ID NO: 13);

V_H CDR 2is:EIRLKSDNYATHYAESVKG (SEQ ID NO: 19);

V_H CDR3 is: YYA D AMD Y (SEQ ID NO:25);

V_LCDR1 is: A A S Q S V S T S S Y S Y M H (SEQ ID NO:31);

V_L CDR2 is: Y A S N L E S (SEQ ID NO:37); and

V_L CDR3 is:QHSWEIPYT (SEQ ID NO:43).

43. The method of claim 41, wherein the anti-TWEAKR agonist antibody which comprises a V_H region having a sequence selected from SEQ ID NO: 3 and SEQ ID NO:l 1 and a V_L region having a sequence selected from SEQ ID NO:4 and SEQ ID NO: 12.

44. The method of claim 41, wherein the anti-TWEAKR agonist antibody is selected from the group consisting of: PDL192 and PDL400.