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WO2013177386A1 - Biomarqueurs destinés à prédire la réponse à une thérapie par un agoniste du récepteur tweak (tweakr) - Google Patents

Biomarqueurs destinés à prédire la réponse à une thérapie par un agoniste du récepteur tweak (tweakr) Download PDF

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WO2013177386A1
WO2013177386A1 PCT/US2013/042409 US2013042409W WO2013177386A1 WO 2013177386 A1 WO2013177386 A1 WO 2013177386A1 US 2013042409 W US2013042409 W US 2013042409W WO 2013177386 A1 WO2013177386 A1 WO 2013177386A1
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tweakr
seq
cancer
antibody
mutation
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Patricia Culp
Keith Wilson
Shiming Ye
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Abbvie Biotherapeutics Inc.
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • TWEAK RECEPTOR AGONIST THERAPY
  • TWEAK Tumor Necrosis Factor-Related Weak inducer of Apoptosis
  • TWEAK Tumor Necrosis Factor-Related Weak inducer of Apoptosis
  • TNF Tumor Necrosis Factor
  • TWEAK has a number of biological activities, including inducing angiogenesis, stimulating the release of cytokines or chemokines, stimulating proliferation of human smooth muscle and endothelial cell lines, inducing TNF-a-mediated cell death, cathepsin B-dependent necrosis, and, as its name implies, apoptosis. See Han et al, 2003, Bioch. Biophys. Res.
  • TWEAK binds to and activates a member of the TNF receptor family, TWEAKR (also known as Fibroblast Growth Factor-Inducible 14 or Fnl4). See id.
  • TWEAKR agonists induced cell death via apoptosis or necrosis in a variety of cancer cell lines.
  • Phosphoinositide 3-kinases are lipid kinases that function as signal transducers downstream of cell surface receptors and mediate pathways important for cell growth, proliferation, adhesion, survival and motility. Mutations in PI3K genes result in deregulation of one or more of these biological processes and have been associated with cancer pathogenesis (Samuels et al, Science 304:554; Bachman et al, Cancer Biol. Ther. 3:772; Campbell, et al, Cancer Res. 64:7678; Broderick et al, Cancer Res 64:5048; Lee et al, Oncogene 24:1477; Levin et al, Clin. Cancer Res.
  • PDKs are classified based on primary structure, regulation, and in vitro lipid substrate specificity. Studies of proliferation and tumorigenesis have focused on Class I A POKs, which are heterodimeric molecules composed of a catalytic subunit, referred to as pi 10a, and an associated regulatory subunit, referred to as p85.
  • Class IA PDKs are responsible for the production of phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) from phosphatidylinositol 4,5-bisphopshate (PIP 2 ), while phosphatases, in particular the PTEN phosphatase, carry out the reverse reaction, dephosphorylating PIP 3 .
  • PIP 3 phosphatidylinositol 3,4,5-triphosphate
  • PTEN phosphatases in particular the PTEN phosphatase
  • Both pi 10a and PTEN have been linked to cancer: the gene encoding pi 10a, called PIK3CA, has been identified as an oncogene (Samuels, et al, Science 304:443) and PTEN has been classified as a tumor suppressor (Deane and Fruman, Annu. Rev. Immunol. 22:563; Okkenhaug et al, Sci. STKE 65:PE1).
  • TWEAKR agonists inhibit proliferation of some, but not all, tumor cells that express TWEAKR.
  • the ability of TWEAKR agonists to inhibit proliferation of TWEAKR positive tumors does not correlate with cancer type.
  • Table 1 and FIG. 1 in in vitro experiments carried out in tumor cell lines derived from a variety of different types of cancers, 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 cell lines derived from the same type of cancer.
  • PIK3CA which encodes pi 10a, a PI3K lipid kinase, and in PTEN, a phosphatase
  • PIK3CA activating mutations mutations that result in increased activity of pi 10a
  • PTEN null mutations mutations that result in reduced activity of PTEN
  • the present disclosure provides methods of treating a subject having a TWEAKR-positive cancer comprising administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PIK3CA activating mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the TWEAKR agonist can be an anti-TWEAKR agonist antibody, including monoclonal and humanized antibodies.
  • the methods further comprise the step of testing the subject for one or more PIK3CA activating mutations.
  • PIK3CA activating mutations Exemplary methods for detecting a PIK3CA activating mutation are described in Section 5.3, below.
  • the testing of the subject is conducted prior to administration of the TWEAKR agonist, and can be conducted simultaneously with testing the subject for TWEAKR expression.
  • the present disclosure provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by testing a subject for one or more PIK3CA activating mutations, preferably concurrently with or after testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PIK3CA activating mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the present disclosure further provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by (i) testing a subject for one or more PIK3CA activating mutations and (ii) prior to or concurrently with testing the subject for one or more PIK3CA activating mutations, testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PIK3CA activating mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the present disclosure provides methods of treating a subject having a TWEAKR-positive cancer comprising administering to a subject who has tested negative for a PTEN null mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the TWEAKR agonist can be an anti-TWEAKR agonist antibody, including monoclonal and humanized antibodies.
  • the methods further comprise the step of testing the subject for a PTEN null mutation.
  • exemplary methods for detecting a PTEN null mutation are described in Section 5.3, below.
  • the testing of the subject is conducted prior to administration of the TWEAKR agonist, and can be conducted simultaneously with testing the subject for TWEAKR expression.
  • the present disclosure provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by testing a subject for a PTEN null mutation, preferably concurrently with or after testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR- positive cancer who has tested negative for a PTEN null mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the present disclosure further provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by (i) testing a subject for a PTEN null mutation and (ii) prior to or concurrently with testing the subject for a PTEN null mutation, testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PTEN null mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the present disclosure provides methods of treating a subject having a TWEAKR-positive cancer comprising administering to a subject who has tested negative for a PTEN null mutation and for one or more PIK3CA activating mutations an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the methods of the disclosure further comprise the step of testing the subject for a PTEN null mutation and the one or more PIK3CA activating mutations, as described herein.
  • the testing of the subject is conducted prior to administration of the TWEAKR agonist, and can be conducted simultaneously with testing the subject for TWEAKR expression.
  • the present disclosure provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by testing a subject for a PTEN null mutation and one or more PIK3CA activating mutations, preferably concurrently with or after testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PTEN null mutation and one or more PIK3CA activating mutations an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the present disclosure further provides methods comprising (a) identifying a subject suitable for therapy with a TWEAKR agonist by (i) testing a subject for a PTEN null mutation and one or more PIK3CA activating mutations and (ii) prior to or concurrently with testing the subject for a PTEN null mutation and one or more PIK3CA activating mutations, testing the subject for TWEAKR expression, and (b) administering to a subject suffering from a TWEAKR-positive cancer who has tested negative for a PTEN null mutation and a PIK3CA activating mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • the PIK3CA activating mutation can be any type of mutation that leads to increased kinase activity relative to wild type human PIK3CA, including a substitution mutation, deletion mutation, or insertion mutation.
  • the PIK3CA activating mutation is a substitution mutation, e.g., a mutation selected from the group consisting of: K111N, E542K, E545K, E545G, E545D, Q546K, Q546R, Ml 0431, M1043V, H1047R, H1047L, H1047Y, G1049R and G1049S.
  • substitution mutations are provided in Section 5.1, below.
  • the PIK3CA activating mutation can be detected at the nucleic acid level (e.g., DNA or mRNA) or at the protein level.
  • the subject is tested for one, two, three, four or more (or all) of Kl 1 IN, E542K, E545K, E545G, E545D, Q546K, Q546R, M1043I, M1043V, H1047R, H1047L, H1047Y, G1049R and G1049S, optionally in addition to further PIK3CA activating mutations, such as those provided in Section 5.1.
  • the PTEN null mutation can be any type of mutation that leads to decreased phosphatase activity compared to wild type human PTEN, including a substitution mutation, deletion mutation, or insertion mutation.
  • the PTEN null mutation can be detected at the nucleic acid level (e.g., DNA or mRNA) or at the protein level. Further PTEN null mutations are provided in Section 5.2, below.
  • TWEAKR agonists also referred to herein as TWEAKR agonist compounds, useful in the methods of the present disclosure are described in Section 5.4.
  • FIG. 1 provides a bar chart illustrating the percent growth inhibition for 38
  • FIG. 2 provides results of 91 cell lines treated with TWEAKR agonist PDL192 classified based on growth inhibition and presence (mutation) or absence (wild type) of a PIK3CA activating mutation. Cell lines that exhibited growth inhibition (responders) or lack of growth inhibition (nonresponders) when treated with PDL192 are indicated on the left.
  • FIGS. 3A-B provide the results of in vivo experiments in which mice bearing xenograft tumors derived from cell lines were treated with TWEAKR agonist PDL192.
  • the cell lines were classified based on growth inhibition response (responders or non-responders) and PIK3CA and PTEN genotype (mutation or wild type) (FIG. 3A).
  • a subset of cell lines were classified as being responsive via a non-ADCC mechanism or nonresponders and PIK3CA and PTEN genotype (mutation or wild type) (FIG. 3B).
  • FIG. 4A illustrates phospho-Akt (S473) levels in cell lines treated with PDL192 in vitro. Cell lines that were treated with PDL192 are indicated on the right hand side.
  • FIG. 5 provides the relative cell viability of cells transfected with control vector, wild type PIK3CA, or PIK3CA containing an activating mutation, as indicated, that were treated with TWEAKR agonist (PDL192, square data points) or control compound (MSL109, round data points).
  • FIG. 6 provides the results of treatment with TWEAKR agonist (PDL192, black bars) or control compound (MSL109, white bars) for DLD-1 and HCTl 16 cells.
  • DLD-1 cells have an E545K mutation in PIK3CA
  • HCTl 16 cells have an H1047R mutation in PIK3CA.
  • the cell lines were subjected to siRNA depletion using control or PIK3CA siR A.
  • TWEAKR also known as Fnl4 or TNFRSF12A
  • Fnl4 or TNFRSF12A tumor necrosis factor
  • TNF tumor necrosis factor
  • TWEAKR agonists are potentially useful in the treatment of some TWEAKR positive cancers, particularly if it is possible to determine whether the TWEAKR positive cancer is likely to be a responder or non-responder to TWEAKR agonist therapy.
  • the present disclosure is based in part on applicants' discovery that PIK3CA activating mutations and PTEN null mutations are indicative of non-responsiveness to TWEAKR agonist therapy in treating a TWEAKR positive cancer. As shown in the
  • the methods of treating a subject having a TWEAKR positive cancer comprise administering to a subject who has tested negative for a PIK3CA activating mutation or PTEN null mutation an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • methods of the disclosure may be employed to classify subjects as responders or non-responders to a TWEAKR agonist therapy. In some embodiments, the methods may be also used to predict whether or not a subject suffering from a TWEAKR positive cancer will respond to a TWEAKR agonist therapy.
  • PIK3CA encodes the catalytic subunit of a Class 1 A PI3 kinase, a class of enzymes that catalyzes the phosphorylation of inositol-containing lipids.
  • PI3 kinases PI3Ks
  • PI3CA sequence refers to a polynucleotide encoding the pi 10a kinase subunit.
  • a PIK3CA sequence can be a gene sequence (with or without introns), a cDNA, or an mRNA.
  • a representative example of a wild type human PIK3CA sequence is SEQ ID NO:55.
  • the corresponding polypeptide sequence for a human pi 10a is SEQ ID NO: 56.
  • Mutations in PIK3CA that correlate with proliferative disorders such as cancer are known to cluster in exon 9 (SEQ ID NO:57) and exon 20 (SEQ ID NO:58). See U.S. Patent Publication Nos. 2009/0208505, paragraph [0017] and Table 2 herein.
  • Exon 9 corresponds to the helical domain of PIK3CA
  • exon 20 corresponds to its kinase domain.
  • PIK3CA activating mutations cluster in these regions, known as mutational hotspots. Additional mutations found in human cancers are described in Table 3 of U.S. Patent Publication No. 2009/0208505.
  • PIK3CA activating mutations are PIK3CA activating mutations, a number of which have been described, including those described in Bader, et al. Nat. Rev. Cancer (2005) 5:921-929, Kang et al, PNAS (2005), 102:802, Samuels et al, Science (2004) 304:554, Zhang et al. , Breast Cancer Res. Treat. (2008) 112:217-227, and U.S. Patent Publication No. 2011/0060605.
  • a "PIK3CA activating mutation” can be any type of mutation, including a substitution mutation, a deletion mutation, or an insertion mutation, which results in increased kinase activity compared to wild type PIK3CA.
  • Increased kinase activity can be the result of increased enzymatic activity or increased levels of the pi 10a polypeptide.
  • Numerous assays for kinase activity are known, which can be used to determine whether a PIK3CA sequence results in a pi 10a with increased kinase activity relative to wild type pi 10a.
  • pi 10a kinase activity can be determined using an in vitro kinase assay.
  • PI3 kinase assays have been described (see Kang et ah, 2005, PNAS 102:802-807).
  • complexes of pi 10a and an anti- pi 10a antibody are prepared by incubating 400 ⁇ g of pi 10a with 5 ⁇ of anti-pl 10a antibody for 2 h at 4°C, followed by 2 h of incubation with Protein A-agarose. Protein A-agarose beads are washed three times with the lysis buffer and once with TNE (10 mM Tris, Ph 7.5/100 mM NaCl/1 mM EDTA).
  • the reaction is terminated by adding 80 ⁇ of 1 N HC1, and the phosphorylated lipids are extracted with 160 ⁇ of chloroform/methanol (1 : 1). After samples are dried down, they are dissolved in chloroform and spotted onto Silica Gel 60 TLC plates (Merck).
  • an increased kinase activity refers to a greater detection of radioactivity (indicating more incorporation of radiolabeled phosphate) per ⁇ g of pi 10a test protein, compared to incorporation per ⁇ g of pi 10a wild type protein, e.g., a protein having the sequence of SEQ ID NO-.56.
  • Mutations in the coding region of PIK3CA can be synonymous or non-synonymous mutations. Mutations in the noncoding or coding regions of a PIK3CA sequence can lead to altered RNA transcripts and/or altered protein as a result of differential post-transcriptional processing ⁇ e.g., mRNA splicing, nonsense-mediated decay, post-translational modification, etc.) as compared to wild type PIK3CA mRNA or pi 10a.
  • a PIK3CA sequence from a test sample e.g., a. sample containing cancer cells
  • a PIK3CA sequence from a different tissue in the same individual can be compared to a PIK3CA sequence from a different tissue in the same individual. A difference in the sequences from the two tissues indicates a mutation.
  • the PIK3CA sequence from a test sample can be compared to SEQ ID NO:55, with one or more differences between these two sequences indicative of a mutation.
  • PIK3CA activating mutations can be identified by reference to a wild type polypeptide or polynucleotide sequence where the number indicates the position within the polypeptide or polynucleotide sequence, the first letter indicates the identity of the residue or nucleotide in the wild type sequence and the second letter indicates the identity of the residue or nucleotide in the mutant sequence.
  • PIK3CA activating mutations are identified by reference to the wild type polypeptide sequence of SEQ ID NO: 56.
  • E545K refers to a polypeptide sequence for which the glutamate residue at position 545 has been mutated to lysine.
  • a PIK3CA activating mutation is selected from the group consisting of: Kl 1 IN, E542K, E545K, E545G, E545D, Q546K, Q546R, Ml 0431, Ml 043V, H1047R, H1047L, H1047Y, G1049R and G1049S. Subjects can be tested for one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or all fourteen of these mutations.
  • a PIK3CA activating mutation can also be identified by reference to a wild type polynucleotide sequence where the number indicates the position within the polynucleotide sequence, the first letter indicates the identity of the wild-type nucleotide, and the second letter indicates the identity of the mutant nucleotide.
  • the ">" character is placed between the first and second letter to denote that the change occurs from the nucleotide represented by the first letter to that of the second.
  • the underscore character indicates a range of nucleotides, "ins” indicates an insertion, and "del” indicates a deletion.
  • the number following "ins” or “del” indicates the number of nucleotides that are inserted or deleted, respectively.
  • a first number followed by a plus sign ("+”) or minus sign ("-"), followed by a second number indicates the presence of a mutation within an intron that is a number of positions downstream (for plus sign) or upstream (for minus sign) given by the second number with respect to the position in the coding sequence given by the first number.
  • a PIK3CA activating mutation is a mutation identified as PIK3CA (2188-7delT), with reference to SEQ ID NO:55, where the designation indicates that subjects bearing this mutation possess a PIK3CA gene in which the T nucleotide that occurs 7 positions upstream from position 2188 in the coding sequence is deleted.
  • a PIK3CA activating mutation is a mutation that gives rise to a higher expression level of pi 10a compared to wild type levels. Methods for detection and quantitation of pi 10a expression levels are known and have been described, for example, in U.S. Application Publication No. 2009/0192110.
  • Phosphatase and tensin homolog is encoded by the PTEN gene, and, like PIK3CA, is known to be mutated in the development of certain cancers.
  • PTEN has been identified as the phosphatase that catalyzes the reverse reaction of the reaction catalyzed by the PIK3CA-encoded protein (pi 10a), specifically the dephosphorylation of inositol polyphosphates.
  • a "PTEN polynucleotide sequence” refers to a
  • a PTEN polynucleotide encoding the PTEN protein can be a gene sequence (with or without introns), a cDNA, or an mRNA.
  • a polynucleotide sequence of a representative wild type human PTEN gene is SEQ ID NO:59.
  • the corresponding wild type PTEN polypeptide sequence is SEQ ID NO:60.
  • PTEN null mutation refers to any mutation or set of mutations within PTEN that leads to a decreased phosphatase activity compared to wild type PTEN, as a result of decreased activity or levels of the PTEN protein.
  • assays for determining phosphatase activity are known in the art.
  • PTEN phosphatase activity can be determined using an in vitro phosphatase assay. Such assays are known and described, for example, in U.S. Application Publication No. 2011/0150868, U.S. Patent No. US7745485, and Georgescu et al, PNAS (1999), 96(18): 10182-10187.
  • PTEN phosphatase activity can be measured with each assay performed in 50 ⁇ buffer containing 100 mM Tris-HCl (pH 8.0), 10 mM DTT, and 100 ⁇ water-soluble diC8-PIP3 (Echelon, Salt Lake City, Utah). Reactions containing PTEN can be
  • lysates can be precleared with protein G-agarose beads. After immunoprecipitation, the beads can be washed once in lysis buffer; five times in low stringency buffer containing 20 mM Hepes (pH 7.7), 50 mM NaCl, 0.1 mM EDTA, and 2.5 mM MgC12; and once in phosphatase assay buffer lacking PIP 3 . Reactions can be incubated for 40 min at 37° C and transferred to a 96- well plate. Release of phosphate from substrate may be measured using Biomol Green Reagent (Biomol Research Laboratories, Inc., Plymouth Meeting, Pa.).
  • PTEN phosphatase activity can be measured without
  • An exemplary reaction mixture for this assay includes 100 mM TrisHCl (pH 8), 10 mM dithiothreitol, 0.5 mM diC16 phosphatidylserine (PS), 25 ⁇ PIP 3 - diC16, BIOMOL PH-107 (Biomol Research Laboratories, Inc., Plymouth Meeting, Pa.) and 50 ⁇ g/ml purified recombinant wild type or mutant PTEN.
  • Lipids can be prepared in organic solvents and dispensed into 1.5 ml microfuge tubes followed by solvent removal under reduced pressure. Buffer can then be added and a lipid suspension formed by sonication.
  • PTEN phosphatase assays are initiated by the addition of PTEN and carried out at 37° C. At different time points, 15 ⁇ of 100 ⁇ NEM (N-ethylmaleimide) is added to 10 ⁇ of reaction mixture followed by rapid centrifugation at 18,000xg for 15 minutes at 4° C. Liberated inorganic phosphate is detected in twenty microliters of supernatant using the Malachite green assay with reference to an inorganic phosphate standard curve. Malachite green reaction with inorganic phosphate is detected spectrophotometrically at 620 nm wavelength.
  • NEM N-ethylmaleimide
  • a PTEN null mutation gives rise to a lowered expression level of PTEN compared to wild type levels.
  • Methods for detection and quantitation of PTEN expression levels are known and have been described, for example, in U.S. Application Publication No. 2010/0303809.
  • a PTEN null mutation can be any type of mutation, including substitution mutations, deletion mutations, and insertion mutations. Mutations in the coding region of PTEN can be synonymous or non-synonymous mutations. Mutations in the noncoding or coding regions of PTEN can lead to mutant RNA transcripts and/or mutant protein as a result of differential post-transcriptional processing ⁇ e.g., mRNA splicing, nonsense-mediated decay, post- translational modification, etc.) as compared to wild type PTEN. PTEN null mutations are known in the art, see Jhawer et al, Cancer Res (2008), 68:1953-1961, incorporated herein by reference in its entirety.
  • PTEN null mutations can be identified by reference to a wild type polypeptide sequence where the number indicates the position within the polypeptide sequence, the first letter indicates the identity of the residue in the wild type sequence and the second letter indicates the identity of the residue in the mutant sequence.
  • PTEN null mutations are identified by reference to the wild type polypeptide sequence of SEQ ID NO: 60.
  • "LI 12Q" refers to a polypeptide sequence for which the leucine residue at position 112 has been mutated to glutamine.
  • a PTEN null mutation is a mutation in which an amino acid is mutated to a stop codon, identified by reference to a wild type polypeptide sequence in which the number indicates the position within the sequence, the first letter indicates the identity of the wild type amino acid, and "*" indicates substitution of a stop codon at this position. For example, "Q149*” indicates that the glutamine at position 149 is mutated to a stop codon, resulting in a truncated polypeptide.
  • a PTEN null mutation is a frameshift mutation that introduces a stop codon, resulting in a truncated sequence.
  • Such mutations can be identified by reference to a wild type polypeptide where the number indicates the position where the frameshift occurs, "fs" indicates a frameshift mutation, "*” indicates introduction of a stop codon, and the final number indicates the position of the stop from the first amino acid affected by the frameshift.
  • a PTEN null mutation is selected, with reference to SEQ ID NO:60, from the group consisting of: Q149*, LI 12Q, F90fs*9, K267fs*9, V175fs*3, and P248fs*5. Subjects can be tested for one or more, two or more, three or more, four or more, five or more, or all six of these mutations.
  • a PTEN null mutation can also be identified by reference to a wild type
  • polynucleotide sequence where the number indicates the position within the polynucleotide sequence, the first letter indicates the identity of the wild-type nucleotide, and the second letter indicates the identity of the mutant nucleotide.
  • the ">" character is placed between the first and second letter to denote that the change occurs from the nucleotide represented by the first letter to that of the second.
  • the underscore character indicates a range of nucleotides, "ins” indicates an insertion, and “del” indicates a deletion. The number following "ins” or “del” indicates the number of nucleotides that are inserted or deleted, respectively.
  • 165_209del45 refers to a mutant in which the 45 nucleotides spanning from position 165 to position 209 are deleted.
  • a first number followed by a plus sign ("+”) or minus sign ("-"), followed by a second number indicates the presence of a mutation within an intron that is a number of positions downstream (for plus sign) or upstream (for minus sign) given by the second number with respect to the position in the coding sequence given by the first number.
  • a PTEN null mutation is selected, with reference to SEQ ID NO:59, from the group consisting of: l_164dell64, 165_209del45, 253+lOT, 270delT, 335T>A, 4450T, 524_558del35, 741_742insA, 800delA, and 1026+1 G>T. Subjects can be tested for one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten of these mutations.
  • a PTEN polynucleotide sequence from a test sample e.g., a sample containing cancer cells
  • a PTEN polynucleotide sequence in a different tissue from the same individual can be compared to a PTEN polynucleotide sequence in a different tissue from the same individual. A difference in the sequences from the two tissues indicates a mutation.
  • the PTEN polynucleotide sequence determined in a PTEN gene in a test sample can be compared to SEQ ID NO:59.
  • a difference between the test sample sequence and SEQ ID NO:59 can be identified as a mutation.
  • Methods described herein for the detection of PIK3CA activating mutations also can be applied using the appropriate primers and probes to detect PTEN null mutations, and vice versa.
  • the Sanger Wellcome database contains information about PTEN deletions and truncations, and therefore may also be useful in determining a PTEN null mutation (see Wang et al, PNAS (2009) 106(15):6279-6284). 5.3. Detecting PIK3CA Activating and PTEN Null Mutations
  • the presence or absence of a PIK3CA activating mutation or a PTEN null mutation can be determined in a subject using a variety of methods known in the art.
  • a subject is tested for a PIK3CA activating mutation and/or a PTEN null mutation.
  • the testing can be performed in conjunction with T WEAKR agonist therapy in order to treat a T WEAKR-positive cancer.
  • the testing is preferably performed prior to treatment with the TWEAKR agonist, and can be carried out at the same time the subject's cancer is tested for TWEAKR expression, for example as described in Section 5.5.
  • the determination of a PIK3CA activating mutation or a PTEN null mutation can be made at the nucleic acid level and/or at the protein level.
  • the nucleic acid and/or proteins used for the determination can be obtained from biological samples, including, but not limited to tissues, cells, biological fluids and isolates thereof, including those isolated from a subject, as well as those present within a subject.
  • biological samples comprise cells, most preferably tumor cells, that are isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid, urine and feces, or tissue which has been removed from organs, such as breast, lung, pancreas, intestine, skin, cervix, prostate, and stomach.
  • Tissues suspected of being cancerous can be tested for a PIK3CA activating mutation or a PTEN null mutation, as can body samples that may be expected to contain sloughed-off cells from tumors or cells of cancers.
  • An allele containing a PIK3CA activating mutation is referred to as a PIK3CA mutant allele, and the corresponding wild type region of DNA is referred to as a PIK3CA wild type allele.
  • An allele containing a PTEN null mutation is referred to as a PTEN mutant allele, and the corresponding wild type region of DNA is referred to as a PTEN wild type allele.
  • Methods for detecting mutations in DNA include genotyping, microarrays, direct sequencing, restriction mapping, Restriction Fragment Length Polymorphism,
  • primers flanking a PIK3CA or PTEN allele are selected and used to amplify that region.
  • the amplified region is then sequenced using DNA sequencing techniques known in the art and analyzed for the presence of a PIK3CA activating or PTEN null mutation.
  • a PIK3CA activating or PTEN null mutation in a nucleic acid can be detected using a probe.
  • an amplified region comprising a wild type or mutant allele is hybridized using a composition comprising a probe specific for the mutation under stringent hybridization conditions.
  • isolated nucleic acids that bind to mutant alleles at high stringency may be used as probes to determine the presence of the mutation.
  • Nucleic acids may be labeled with a detectable marker.
  • the marker or label is typically capable of producing, either directly or indirectly, a detectable signal.
  • the label may be radioopaque or a radioisotope, such as H, ' , "P, "S, " ⁇ , a fluorescent
  • fluorescein fluorescein
  • a PIK3CA activating or PTEN null mutation can also be detected using restriction enzymes.
  • amplified products can be digested with a restriction enzyme that specifically recognizes sequence comprising a mutant allele, but does not recognize the corresponding wild type allele or vice versa.
  • PCR may be used to amplify DNA comprising a PIK3CA or PTEN allele, and amplified PCR products are subjected to restriction enzyme digestion under suitable conditions and restriction products are assessed. If, for example, a PIK3CA or PTEN mutant allele corresponds to a sequence digested by the restriction enzyme, digestion is indicative of detecting that particular PIK3CA or PTEN mutant allele. Restriction products may be assayed electrophoretically as is common is the art.
  • PIK3CA and PTEN mutant alleles may also be detected by a variety of other methods known in the art.
  • PCR and RT-PCR and primers flanking a PIK3CA or PTEN allele can be employed to amplify sequences and transcripts, respectively, in a sample comprising DNA (for PCR) or RNA (for RT-PCR).
  • the amplified products are optionally sequenced to determine whether the PIK3CA or PTEN allele in the sample is mutant or wild type.
  • a PIK3CA or PTEN allele in a sample can be detected by a variety of other techniques known in the art including microarrays, hybridization assays, PCR based assays, molecular beacons, dynamic allele-specific hybridization (DASH) and/or
  • Mutant PIK3CA or PTEN polynucleotide sequences may also be identified by methods which detect particular mRNAs in cells. These include hybridization assays using complementary DNA probes (such as in situ hybridization using labeled mutant or wild type PIK3CA or PTEN riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for PIK3CA or PTEN polynucleotide sequences, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • complementary DNA probes such as in situ hybridization using labeled mutant or wild type PIK3CA or PTEN riboprobes, Northern blot and related techniques
  • nucleic acid amplification assays such as RT-PCR using complementary primers specific for PIK3CA or PTEN polynucleotide sequences, and other amplification type detection methods, such as,
  • Mutant pi 10a or PTEN protein bearing a PIK3CA activating mutation or PTEN null mutation can be detected using a specific agent, most preferably an antibody, that is itself detectably labeled, or using an unlabeled antibody specific for mutant pi 10a or PTEN protein and a second antibody that is detectably labeled and recognizes the unlabeled antibody specific for mutant pi 10a or PTEN protein.
  • a specific agent most preferably an antibody, that is itself detectably labeled, or using an unlabeled antibody specific for mutant pi 10a or PTEN protein and a second antibody that is detectably labeled and recognizes the unlabeled antibody specific for mutant pi 10a or PTEN protein.
  • any molecule that can be detectably labeled and that specifically binds to mutant pi 10a or PTEN protein can be used in the practice of the methods of the disclosure.
  • a two-component immunohistochemical staining system may be used to differentially stain mutant pi 10a or PTEN protein and the tissue or cell sample so that the stained mutant pi 10a or PTEN protein can be more readily distinguished from the counterstained tissue or cell sample.
  • pi 10a or PTEN protein can also be enriched by affinity purification using, for example, an anti-pl 10a or anti-PTEN antibody, accordingly, and the enriched protein can be sequenced by mass spectrometric methods known in the art to determine the presence of a PIK3CA activating or PTEN null mutation.
  • PIK3CA activating mutation or a PTEN null mutation in a given cell line can also be determined by reference to the Sanger Wellcome database of somatic mutations related to human cancers (see Wang et al, PNAS (2009) 106(15):6279-6284).
  • downstream pathway members can also be used as an indicator of PI3K pathway activation status as a result of upregulation of PIK3CA protein levels and/or the presence of activating mutations in PIK3CA.
  • downstream pathway members phosphorylated when the PI3K pathway is activated include PDK1 (S241, Y373 Y376, or S396), Akt (S473 or T308), FoxOl (T24, S249, S256, or S319), Fox04 (SI 93 or S262), Fox03a (T32, S253, S318, S321, or S644). Reagents specific for phosphorylated forms of these proteins are readily available.
  • a TWEAKR agonist is an agent capable of activating a
  • TWEAKR signaling cascade leading to one or more of the biological activities associated with TWEAKR such as killing TWEAKR positive cancer cells by apoptosis, inhibiting proliferation and/or survival of TWEAKR positive cancer cells, and inducing chemokine or cytokine release in TWEAKR positive cancer cells.
  • TWEAKR agonists induce apoptosis in cells expressing TWEAKR. 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).
  • TUNEL terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling
  • a TWEAKR agonist inhibits proliferation ⁇ i.e. cell division) and/or survival of a cell, and in particular, a TWEAKR positive cancer cell.
  • TWEAKR agonists including TWEAK and anti-TWEAKR agonist antibodies, have been shown to inhibit proliferation and/or survival of TWEAKR positive cancer cell lines in in vitro and in vivo growth inhibition assays (Culp et al, 2010, Clin. Cancer Res. 16(2): 497-508).
  • Inhibition of proliferation and/or survival can be measured by an in vitro growth inhibition assay as described in Example 1 below.
  • a cancer cell line known to be sensitive to TWEAKR agonists such as BT549 or H358, is cultured at 500 cells per well in triplicate with 5-10 ⁇ g/ml of the proposed TWEAKR agonist for 5 days in 96 well plates.
  • Growth inhibition is determined using CellTiter-BlueTM (Promega): cells are incubated with the reagent and fluorescence emitted at 590 nm is measured and used to calculate the effect on viable cells of the proposed TWEAKR agonist relative to a control, such as an IgGl control.
  • a test compound is considered to be a TWEAKR agonist if it induces growth inhibition of at least about 20% relative to control treatment.
  • cells can be incubated with anti-TWEAKR antibody (10 ⁇ g/ml) and an antibody (5 ⁇ g/ml) or antibody fragment (3.5 ⁇ g/ml) that specifically binds to the anti-TWEAKR antibody.
  • the antibody or antibody fragment is preferably species- and isotype-specific.
  • the anti-TWEAKR antibody is a humanized IgG antibody
  • a F(ab') 2 antibody fragment that is a goat anti-human
  • Agonist activity can be determined by measuring the release of cytokines and/or chemokines in an in vitro cell growth assay. For example, in a typical assay the cells are incubated in vitro with a TWEAKR agonist. Twenty four hours later, the cell supernatant is assessed for the presence of cytokines and/or chemokines using an ELISA assay or a commercial fluorescent bead-based multiplex assay (e.g., Luminex®, Upstate). In some embodiments, the chemokine that is measured for release is IL-8.
  • TWEAKR agonists include the natural ligand of TWEAKR, TWEAK. TWEAK forms trimers and is thought to promote activation of TWEAKR via trimerization of the receptor. See Winkles, 2008, Nature Reviews Drug Discovery 7: 411-425. Therefore, it is expected that TWEAKR agonists can also be proteins that form multimers, e.g., trimers and are capable of binding to and promoting multimerization of TWEAKR. Accordingly, TWEAKR agonists include fusions of TWEAK, or a fragment of TWEAK capable of binding TWEAKR, to polypeptides comprising an oligomerization domain.
  • Polypeptides comprising suitable oligomerization domains include CD8 (Nakayama et al., 2000, J. Exp. Med. 192:1373), collagen (Frank et al., 2001, J. Mol. Bio. 308:1081-1089), bacteriophage T4 fibritin (Yang et al., 2002, J. Virology 76(9):4634-4642), engineered leucine/isoleucine zipper polypeptides (Harbury, 1994, Nature 371(6492):80-83), macrophage scavenger receptor (U.S. Pat. No.
  • TNF tumor necrosis factor
  • Methods of making fusion proteins are well known in the art, and involve combining coding sequences of two or more polypeptides to generate a coding sequence of a fusion protein.
  • Techniques for the manipulation of nucleic acids including techniques for the synthesis, isolation, cloning, detection, and identification are well known in the art and are well described in the scientific and patent literature. See, e.g., Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor
  • TWEAKR 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
  • HSA human serum albumin
  • 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
  • a specific embodiment of a TWEAKR agonist is an anti-TWEAKR agonist antibody.
  • 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 2 below.
  • the antibody preferably binds the TWEAKR from the same species as the subject.
  • an antibody to human TWEAKR is administered.
  • a TWEAKR agonist antibody need not be a pure agonist.
  • a TWEAKR agonist antibody can also have TWEAKR antagonist activity.
  • Antagonist activity refers to the ability of test agent (e.g. a TWEAKR agonist antibody) to inhibit the induction by TWEAK of one or more biological responses associated with TWEAKR.
  • test agent e.g. a TWEAKR agonist antibody
  • cytokine/chemokine release assay described above can be performed by incubating cells in vitro with an anti-TWEAKR antibody +/- TWEAK.
  • An anti-TWEAKR agonist antibody can bind to TWEAKR protein with a K D of at least about ⁇ ⁇ , at least about 0.1 ⁇ or better, at least about 0.01 ⁇ , and at least about 0.001 ⁇ or better.
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, chimeric, humanized, fully human, genetically engineered and otherwise modified forms of antibodies.
  • a full-length antibody contains two heavy chains and two light chains, each of which comprises complementarity determining regions (CDRs), also known as hypervariable regions, and more highly conserved framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs highly conserved framework regions
  • the amino acid position/boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art.
  • Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria.
  • One or more of these positions can also be found in extended hypervariable regions.
  • the present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions.
  • variable domains of native heavy and light chains each comprise four FR regions, largely by adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (See Kabat et al. ,
  • an anti-TWEAKR agonist antibody can be an antibody fragment.
  • antibody fragment refers to a portion of a full-length antibody capable of multivalent binding to the target, where multivalent refers to bivalent, trivalent, tetravalent, etc.
  • F(ab') 2 fragments examples 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-VL 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 non-specific tissue binding than an intact antibody (Wahl et al. , 1983, J. Nucl. Med. 24:316). Multivalent antibody fragments can also be produced by linking CDRs using chemical couplings known to those of ordinary skill in the art.
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • the term monoclonal antibody (mAb) is meant to include both intact molecules, as well as, antibody fragments (such as, for example, F(ab') 2 fragments) which are capable of specifically binding to a protein.
  • the anti-TWEAKR agonist antibodies of the disclosure can be chimeric antibodies.
  • the term "chimeric" antibody as used herein refers to an antibody having variable sequences derived from a non-human immunoglobulins, such as rat or mouse antibody, and human immunoglobulins constant regions, typically chosen 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;
  • 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.
  • 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
  • the anti-TWEAKR agonist antibodies of the disclosure can be fully human antibodies. Fully human anti-TWEAKR agonist antibodies can be desirable for therapeutic treatment of human patients.
  • "fully human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 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.
  • Fully human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • is used to guide the selection of a fully human antibody recognizing the same epitope Jespers et al, 1988, Biotechnology 12:899-903.
  • anti-TWEAKR agonist antibodies can be of any of the recognized isotypes.
  • anti-TWEAKR agonist antibodies are one of the four human IgG isotypes, i.e., IgGl, IgG2, IgG3 and IgG4, or one of the four mouse IgG isotypes, i.e., murine IgGl, murine IgG2a, murine IgG2b, or murine IgG3.
  • the anti- TWEAKR antibodies are of the human IgGl isotype.
  • Anti-TWEAKR agonist antibodies useful in the present methods include antibodies that induce antibody-dependent cytotoxicity (ADCC) of TWEAKR-expressing cells.
  • ADCC antibody-dependent cytotoxicity
  • the ADCC of an anti-TWEAKR antibody can be improved by using antibodies that have low levels of or lack fucose.
  • Antibodies lacking fucose have been correlated with enhanced ADCC (antibody-dependent cellular cytotoxicity) activity, especially at low doses of antibody (Shields et al, 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al, 2003, J. Biol. Chem. 278:3466).
  • Methods of preparing fucose-less antibodies include growth in rat myeloma YB2/0 cells (ATCC CRL 1662).
  • YB2/0 cells express low levels of FUT8 mRNA, which encodes an enzyme (a-l,6-fucosyltransferase) necessary for fucosylation of polypeptides.
  • Alternative methods for increasing ADCC activity include mutations in the Fc portion of an anti-TWEAKR agonist antibody, particularly mutations which increase antibody affinity for an FcyR receptor.
  • a correlation between increased FcyR binding with mutated Fc has been demonstrated using targeted cytoxicity cell-based assays (Shields et al, 2001, J. Biol. Chem.
  • an anti-TWEAKR agonist antibody of the disclosure has a constant region that binds FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type constant region. Mutations in the Fc portion of an antibody that result in increased ADCC activity and methods of generating immunoglobulins with increased ADCC activity are known in the art and described in various references, e.g., U.S. Pat. No. 8,039,592, U.S. Pat. No. 7,632,497, U.S. Pat. No. 7,183,387, U.S. Pat. Pub. No. 2009/0074762, WO 00/42072.
  • ADCC activity can be monitored and quantified using a 51 Cr release assay.
  • Target cells are loaded with 5I Cr, which is released in culture supernatant upon damage to the plasma membrane in the presence of immune effector cells and an anti-TWEAKR agonist antibody.
  • the target cells can be derived from solid tumors, for example lung, pancreatic, breast, or renal cancer cells.
  • the anti-TWEAKR agonist antibodies induce at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 80% cytotoxicity in the target cells.
  • An example of an ADCC assay that can be used to measure ADCC of an anti-TWEAKR agonist antibody is provided in Culp et al, 2010, Clin. Cancer Res. 16(2):497-508.
  • Anti-TWEAKR agonist antibodies or fragments thereof as described herein can be antibodies or antibody fragments whose sequence has been modified to reduce at least one constant region-mediated biological effector function relative to the corresponding wild type sequence.
  • the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al, 1991, J. Immunol.
  • Reduction in FcR binding ability of the antibody can also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis.
  • Anti-TWEAKR agonist antibodies or fragments thereof as described herein can be antibodies or antibody fragments that have been modified to increase or reduce their binding affinities to the fetal Fc receptor, FcRn, or serum half-life, for example by mutating the immunoglobulin constant region segment at particular regions involved in FcRn interactions (see, e.g., WO 2005/123780 and U.S. Pat. No. 5,739,277).
  • anti-TWEAKR agonist antibodies and sequences of VH, VL and CDRs which are described in U.S. Pat. Pub. No. 2009/0074762 and provided in Table 2 herein, include:
  • an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, comprising a heavy chain variable region corresponding to SEQ ID NO: 3 and a light chain variable region corresponding to SEQ ID NO: 4 (PDL192);
  • an anti-TWEAKR antibody that is a monoclonal antibody or anti-TWEAKR antigen binding fragment, comprising a heavy chain variable region corresponding to SEQ ID NO: 11 and a light chain variable region corresponding to SEQ ID NO: 12 (PDL400);
  • V H CDR1 is XiYWMX 2 (SEQ ID NO:49)
  • V H CDR2 is EIRX 3 KSX 4 NYATX 5 HYAESX 6 KG (SEQ ID NO:50)
  • V H CDR3 is X 7 X 8 ADX 9 X 10 DY (SEQ ID NO:51)
  • V L CDR1 is X n ASQSVSTSX 12 YSYMX 13 (SEQ ID NO:52);
  • V L CDR2 is YAX 14 X 15 LX 16 S (SEQ ID NO:53);
  • V L CDR3 is QHSWEX 17 PX 18 T (SEQ ID NO:54), wherein: Xj is selected from K, N, R, and S; X 2 is selected from N and S; X 3 is selected from L and V; j is selected from D and N; X 5 is T or no amino acid;
  • V H CDRl is SYWMS (SEQ ID NO: 13)
  • V H CDR2 is EIRLKSDNYATHYAESVKG (SEQ ID NO: 19)
  • V H CDR3 is
  • V L CDRl is RASQSVSTSSYSYMH (SEQ ID NO:31)
  • V L CDR2 is YASNLES (SEQ ID NO:37)
  • V L CDR3 is QHSWEIPYT (SEQ ID NO:43);
  • V H CDRl 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 CDRl is RASQSVSTSSYSYMQ (SEQ ID NO:33)
  • V L CDR2 is YATNLDS (SEQ ID NO:39)
  • V L CDR3 is QHSWEIPYT (SEQ ID NO:45);
  • V H CDRl is KYWMN (SEQ ID NO: 16)
  • V H CDR2 is EIRLKSDNYATHYAESAKG (SEQ ID NO:22)
  • V H CDR3 is
  • V L CDRl is KASQSVSTSTYSYMQ (SEQ ID NO:34)
  • V L CDR2 is YASKLDS (SEQ ID NO:40)
  • V L CDR3 is QHSWELPYT (SEQ ID NO:46);
  • V H CDRl is RYWMS (SEQ ID NO: 17)
  • V H CDR2 is EIRVKSDNYATTHYAESVKG (SEQ ID NO:23)
  • V H CDR3 is
  • V L CDRl is RASQSVSTSSYSYMH (SEQ ID NO:35)
  • V L CDR2 is YASKLDS (SEQ ID NO:41)
  • V L CDR3 is QHSWEIPWT (SEQ ID NO:47);
  • V H CDR1 is NYWMN (SEQ ID NO: 17)
  • V H CDR2 is EIRLKSNNYATHYAESVKG (SEQ ID NO:24)
  • V H CDR3 is
  • V L CDR1 is RASQSVSTSTYSYMH (SEQ ID NO:36)
  • V L CDR2 is YASNLES (SEQ ID NO:42)
  • V L CDR3 is QHSWEIPYT (SEQ ID NO:48).
  • 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 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 ⁇ g/mL, although higher levels of reduction, for example, 60%, 70%, 80%, 90% or even 100%, may be desirable.
  • TWEAKR positive cancer is a cancer in which TWEAKR is overexpressed as compared to matched non-cancer tissue.
  • 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).
  • TWEAKR has been shown to be overexpressed 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; Han et al, 2002, Cancer Res. 62:2890-2896; Wang et al, 2006, Oncogene 25:3346-3356.
  • TWEAKR positive cancers include 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.
  • the present disclosure provides methods of treating a TWEAKR positive cancer.
  • a subject who has tested negative for at least one PIK3CA activating mutation is treated with an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • a subject who has tested negative for at least one PTEN null mutation is treated with an amount of a TWEAKR agonist effective to promote a therapeutic benefit.
  • a subject who has tested negative for at least one PIK3CA activating mutation has also tested negative for at least one PTEN null mutation.
  • a subject who has tested negative for at least one PTEN null mutation may also have tested negative for at least one PIK3CA activating mutation.
  • the TWEAKR agonists are administered to a subject in an amount effective to promote a therapeutic benefit.
  • the amount effective to promote a therapeutic benefit refers to the amount of a pharmaceutical formulation or composition that is sufficient to cure, alleviate, attenuate or at least partially arrest the cancer and/or its symptoms, and/or complications.
  • Clinical methods for determining a therapeutic benefit are well known to those of ordinary skill in the art and may be determined empirically using routine
  • an amount effective to promote a therapeutic benefit is an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (2) reduction in the number of cancer cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell metastasis; (6) enhancement of anti-cancer immune response, which may, but does not have to, result in the regression or rejection of a tumor; and/or (7) relief, to some extent, of one or more symptoms associated with the disorder.
  • 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.
  • 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, radiation therapy, and treatment with conventional therapeutic agents, such as targeted agents, conventional chemotherapy agents, and hormonal therapy agents.
  • the conventional standard of care for a particular cancer is known to those skilled in the art and thus, one of skill in the art will be able to select suitable other therapy to combine with the TWEAKR agonist described herein.
  • 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
  • the TWEAKR agonist can be used in combination with conventional therapeutic agents used to treat a particular cancer.
  • therapeutic agents that are presently used to treat cancer, and thus can be used in combination with the TWEAKR agonist described herein, include, but are not limited to, targeted agents, conventional chemotherapy agents, and hormonal therapy agents.
  • the TWEAKR agonist described herein can be used in combination with targeted agents.
  • Targeted agents include, but are not limited to, antiangiogenic agents (such as bevacizumab, sunitinib, sorafenib, temsirolimus, 2- methoxyestradiol or 2ME2, finasunate, PTK787, and vandetanib), EGFR inhibitors (such as erlotinib, cetuximab, panitumumab, gefinitib, lapatinib, and trastuzumab),
  • antiangiogenic agents such as bevacizumab, sunitinib, sorafenib, temsirolimus, 2- methoxyestradiol or 2ME2, finasunate, PTK787, and vandetanib
  • EGFR inhibitors such as erlotinib, cetuximab, panitumumab, gefinitib, lapatini
  • immunomodulators such as rituximab, alemtuzumab, and aldesleukine
  • proteasome inhibitors such as bortezomib, PR-171, and NPI-052
  • integral inhibitors such as natalizumab, volociximab, etaracizumab, and cilengitide
  • pro-apoptotic agents such as mapatumumab, lexatumumab, AMG951, ABT-737, oblimersen, and plitidepsin
  • agents with other mechanisms of action such as imatinib, dasatinib, lenalidomide, thalidomide, aldesleukin, and interferon alpha).
  • the TWEAKR agonist described herein can be used in combination with conventional chemotherapy agents.
  • Conventional chemotherapy agents include, but are not limited to, alkylating agents (such as oxaliplatin, carboplatin, cisplatin, cyclophosphamide, melphalan, ifosfamide, uramustine, chlorambucil, mechloethamine, thiotepa, busulfan, temozolomide, and dacarbazine), anti-metabolites (gemcitabine, cytosine arabinoside, Ara-C, capecitabine, 5FU (5-fluorouracil), azathioprinc, mercaplopurine (6- MP), 6-thioguanine, aminopterin, pemetrexed, and methotrexate), plant alkaloids and terpenoids (such as docetaxel, paclitaxel, protein-bound paclitaxel, vincristine, vinblastin,
  • the TWEAKR agonist described herein can be used in combination with hormonal therapy agents including, but not limited to, anastrozole, letrozole, goserelin, and tamoxifen.
  • concentration of a TWEAKR agonist in formulations administered by methods of the disclosure varies widely from about 0.1 to 100 mg/ml, but is often in the range 1 to 20 mg/ml.
  • the appropriate dosage of the antibody will depend on the severity and course of disease, the patient's clinical history and response, the toxicity of the antibodies, and the discretion of the attending physician.
  • the dose can range from about 0.1 to about 1 mg/kg, about 1 to about 5 mg/kg, about 1 to about 10 mg/kg, and about 10 to about 20 mg/kg.
  • the antibodies are suitably administered to the patient at one time or over a series of treatments.
  • the proper dosage and treatment regimen can be established by monitoring the progress of therapy using conventional techniques known to the people skilled of the art.
  • the methods described herein provide for administration of a TWEAKR agonist to a patient intravenously as a bolus or by continuous infusion over a period of time; or by intramuscular,
  • compositions to be administered are formulated for pharmaceutical use.
  • the compositions optionally further comprise a carrier.
  • compositions described herein typically comprise a TWEAKR agonist and a pharmaceutical carrier, and, commonly they comprise a solution of a TWEAKR agonist, or a cocktail thereof, dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water for injection (WFI), or water buffered with phosphate, citrate, acetate, etc. to a pH typically of 5.0 to 8.0, most often 6.0 to 7.0, and/or containing salts such as sodium chloride, potassium chloride, etc. to make the composition isotonic.
  • WFI water for injection
  • the carrier can also contain excipients such as human serum albumin, polysorbate 80, sugars or amino acids to protect the agonist.
  • Administration of a TWEAKR agonist can trigger killing of cancer cells.
  • the cancer cells can be present within a solid tumor, the lymph system, or in the bloodstream.
  • the therapeutic methods described herein are usually applied to human patients, but can be applied to other mammals.
  • TWEAKR agonist can induce apoptosis or cyto lysis of cells expressing TWEAKR.
  • induction of cytolysis is achieved via antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • the TWEAKR agonist can induce between 10% to greater than 80% cytotoxicity of cells expressing TWEAKR.
  • administration of the TWEAKR agonists induces at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% or more cytotoxicity of cells expressing
  • TWEAKR administration of the TWEAKR agonist can reduce the size of a solid tumor by targeting TWEAKR on the cancer cell's surface with one or more of the antibodies described herein.
  • the tumor can be a primary tumor or a secondary tumor.
  • administration of the TWEAKR agonists can reduce the size of a solid tumor by at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% or more.
  • administration of the TWEAKR agonists can completely inhibit or prevent the growth of a solid tumor. It will be appreciated that a range of different cytotoxicities and a range of reductions in tumor size are described for the TWEAKR agonists disclosed herein. The skilled person will appreciate that the TWEAKR agonists can have any one of the described cytotoxicities and any one of the described reductions in tumor size.
  • responder in the context of the present disclosure includes persons where the cancer/tumor(s) is eradicated, reduced or improved, or stabilized such that the disease is not progressing after treatment.
  • the period of stabilization is such that the quality of life and/or patients' life expectancy is increased (for example stable disease for more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months) in comparison to a patient that does not receive treatment.
  • a non-responder includes persons for whom the cancer/tumor(s) does not show reduction or improvement after treatment.
  • the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set.
  • the standard may be the profile of a person/patient who is known to be a responder or nonresponder or alternatively may be a numerical value.
  • Such pre-determined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media.
  • Example 1 TWEAKR agonist PDL192 inhibits growth of a wide range of cancers in vitro
  • 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, pancreatic cancer, cervical cancer, colon cancer, bladder cancer, lung cancer and melanoma cancer cell lines, in vitro.
  • PDL192 used interchangeable herein with enavatuzumab
  • PDL192 is a humanized monoclonal antibody to TWEAKR described in US Pat. App. No. 2009/0074762 and Culp et al, 2010, Clin. Cancer Res. 16(2):497-508.
  • Human IgGl antibody to cytomegalovirus (MSL109) was used as a negative control.
  • Antibodies were used at 10 ⁇ g ml for in vitro studies, unless otherwise stated, and crosslinked with F(ab') 2 goat anti-human IgG (Fe y specific) from Jackson ImmunoResearch at 3.5 ⁇ g/ml.
  • [0104] Growth inhibition assay 102 cancer cell lines were cultured at 500 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-BlueTM (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.
  • Cancer Sensitive Resistant type at least 20% growth ( ⁇ 20% growth inhibition) inhibition
  • PDL192 displayed the ability to block proliferation in some but not other cell lines from each of the following tumor types: breast cancer, lung cancer, colon cancer, renal cancer, ovarian cancer, melanoma, and pancreatic cancer. PDL192 also blocked cell proliferation in cell lines derived from bladder cancer, cervical cancer, salivary cancer and oral cancer tumors.
  • Example 2 In vitro correlation of PIK3CA activating mutations with responsiveness to PDL192
  • This example shows a correlation between mutations activating the PI3K pathway and non-responsiveness to PDL192 treatment in vivo. Somatic mutation analysis was performed on cells from 26 tumors to determine if a PIK3CA activating mutation or a PTEN null mutation was present. The results are shown in FIG. 3A. Of the tumors that contained either a PIK3CA activating mutation or a PTEN null mutation, three of sixteen were responsive to PDL192 while thirteen were non-responsive. Response was defined as statistically significant decrease in tumor volumes in the PDL192-treated animals compared to control-treated tumors. In contrast, for tumors whose cells bore wild type PIK3CA and wild type PTEN, eleven of fourteen were responsive to PDL192 and three were non- responsive.
  • PDL192M had no anti-tumor activity, suggesting that ADCC is the predominant mechanism of action for PDL192 in these models.
  • PDL192M had activity equivalent to PDL192; thus, direct growth inhibitory activity is presumed to be the predominant mechanism of action for PDL192 in these lines.
  • FIG. 3B none of the 3 cell lines responding to PDL192 via direct growth inhibition in vivo harbored activating mutations in PIK3CA or PTEN null mutations; in contrast, 13 of 16 nonresponding cell lines carried such PIK3CA or PTEN mutations.
  • Wild type A375 cells were transfected with vector alone, wild type PIK3CA,
  • PIK3CA E545K mutant or PIK3CA H1047R mutant.
  • E545K and H1047R have been shown to be PIK3CA activating mutations.
  • the cells were treated with PDL 192 or control MSL109 compounds, and response was analyzed using the growth inhibition assay of Example 1.
  • FIG. 5 cells transfected with vector alone or with wild type PIK3CA showed a dose-dependent response to PDL 192.
  • cells that received vectors bearing PIK3CA activating mutations showed a decreased response to PDL 192.
  • This study showed that activation of PIK3CA results in decreased sensitivity to PDL 192 treatment.
  • Example 6 PIK3CA depletion studies
  • Two cell lines bearing PIK3CA activating mutations were subjected to siRNA knockdown of PIK3CA or control, and subsequently tested for PDL192 responsiveness.
  • the DLD-1 cell line bears an E545K PIK3CA activating mutation
  • the HCT116 cell line bears an H1047R PIK3CA activating mutation.
  • Each of the two cell lines was treated with 10 nM control siRNA or 10 nM PIK3CA siRNA (for DLD-1) or 2.5 nM PIK3CA siRNA (for HCT116).
  • FIG. 6 cell lines receiving control siRNA did not respond to PDL192 (compared to control compound, MSL109) while cell lines that had been subjected to PIK3CA siRNA depletion showed a dose-dependent response to PDL192.
  • Table 2 below provides sequences of SEQ ID Nos:l-60.

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Abstract

La présente invention concerne des procédés de traitement de sujets présentant un cancer positif pour TWEAKR comprenant l'administration à un sujet, qui a été testé négatif pour une mutation d'activation de PIK3CA ou une mutation nulle de PTEN, d'une quantité d'un agoniste de TWEAKR efficace pour promouvoir un bénéfice thérapeutique.
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