Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

• TWEAK receptor also known as Fnl4 or TNFRSF12A
• Fnl4 or TNFRSF12A is a member of the tumor necrosis factor receptor superfamily. It is expressed on the surface of cancer cells derived from a variety of solid tumors. Expression of TweakR can be detected on some normal tissues, including the kidney (Bowman's capsule), liver (hepatocytes), and on proliferating endothelial cells and fibroblasts (Meighan-Mantha, et al., 1999, J. Biol. Chem. 1999; 274:33166-33176; and Jakubowsld, et al., 2002, J. Cell Sci. 2002; 115:267- 274).
• TweakR TweakR
• compositions comprising monoclonal and humanized antibodies or antigen binding fragments thereof that bind human TweakR, and methods for their use in therapy, such as in treating cancer.
• the anti-TweakR monoclonal or humanized antibodies can be generated from nucleic acids encoding polypeptides of monoclonal or humanized anti-TweakR antibodies, having 65%, 75%, 85%, 90%, 95%, 97% or 99% or more sequence identity to one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 3-12. Jn other embodiments, the nucleic acid encodes a polypeptide of a monoclonal or humanized anti-TweakR antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-12.
• anti-TweakR monoclonal or humanized antibodies can be derived from polypeptides having 65%, 75%, 85%, 90%, 95%, 97% or 99% or more sequence identity to one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 3 12.
• the polypeptide comprises one or more of the amino acid sequences selected from tbc group consisting of SEQ ID NOs: 3- J 2.
• compositions comprise anti-TweakR mouse monoclonal antibodies or antibody fragments (hereof, wherein said antibody comprises a mature heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, or 9 and a mature light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8 or 10,
• compositions comprise anti-TweakR humanized antibodies oi antibody fragments thereof, wherein said antibody comprises a mature heavy chain variable region comprising an amino acid sequence selected from tbc group consisting of SEQ ID NOs: 3 and 11 and a mature light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 12.
• the compositions comprise anti-TweakR monoclonal or humanized antibodies or antibody fragments thereof, wherein said antibody comprises a heavy chain complementarity determining region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-30 and 120-122 and a light chain complementarity region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-83 and 127-129.
• the compositions can comprise a heavy chain framework region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-65 and 123-126 and/or a light chain framework region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 84-119 and 130-133.
• the antibodies can comprise all of the complementarity determining regions of the heavy and/or light chain variable domain of an anti-TweakR antibody disclosed herein, optionally, wherein one or more of the framework regions surrounding the complementarity determining regions are also present. In some embodiments, the complementarity determining regions of both heavy and light chain variable regions are present.
• the antibody or antigen binding fragment binds to a human TweakR epitope recognized by the and- TweakR humanized antibody, PDLl 92, wherein the epitopes comprises an R at position 56 of SEQ TD NO: 2.
• PDLl 92 and other antibodies that are capable of binding a TweakR epilope comprising an R at position 56 are capable of killing TweakK expressing cells,
• TweakR expressing cells include cancer cells from, bladder cancer, breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, stomach cancer, uterine cancer, cervical cancer, esophageal cancer, renal cell carcinoma, glioblastoma, and sarcomas.
• compositions described herein can be used to inhibit the proliferation and/or kill cells expressing TweakR.
• the compositions described herein can be used to treat an individual suffering from cancer, including, but not limited to, bladder cancer, breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, ovarian cancer, renal cancer, head and neck cancer, esophageal cancer, uterine cancer, stomach cancer, cervical cancer, glioblastoma, and sarcomas.
• FIG. IA provides the nucleic acid (SEQ ID NO: 1) and FIG. IB provides the amino acid sequence of TweakR (SEQ ID NO: 2);
• FIG. 2A provides the amino acid sequence of the heavy chain variable region (SEQ ID NO: 3) and light chain variable region (SEQ ID NO: 4) of the humanized anti- TweakR antibody, PDLl 92;
• FIG. 2B provides the amino acid sequence of the heavy chain variable region (SEQ ID NO: 5) and light chain variable region (SEQ ID NO: 6) of the murine anti- TwcakR antibody 19.2.1 ;
• FIG. 2C provides the amino acid sequence of the heavy chain variable region (SEQ ⁇ D NO: 7) and light chain variable region (SHQ ID NO. 8) of the murine anti- TweakR antibody 18,3.3;
• FIG. 2T provides the amino acid sequence of the heavy chain variable region (SEQ ID NO- 9) and light chain variable region (SEQ Tf) NO: J 0) of the murine anli TweakR antibody 136.1;
• FlG. 2E provides the amino acid sequence of the heavy chain variable region (SEQ ID NO: 1 J ) and light chain variable region (SEQ ID NO: ] 2) of the humanized anti-TweakR antibody, PDL400;
• FIG. 3 ⁇ provides ihc amino acid sequences of the heavy chain complcnicnta ⁇ ly determining region 1 (CDRl) for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 13), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 14), murine anti TweakR antibody, 136.1 (SEQ ID NO: 15), murine anti TweakR antibody, 18.3.3 (SEQ ID NO: 16), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 17), and murine anti- TweakR antibody, ITEM-I (SEQ ID NO: 18);
• FIG. 3 B provides the amino acid sequences of the heavy chain CDR2 for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 19), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 20), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 21), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 22), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 23), and murine anti-TweakR antibody, ITEM-I (SEQ ID NO: 24);
• FIG. 3C provides the amino acid sequences of the heavy chain CDR3 for the humanized anti-TweakR antibody, PDL192 (SEQ ID NO: 25), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 26), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 27), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 28), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 29), and murine anti-TweakR antibody, ITEM-I (SEQ ID NO: 30);
• FIG. 3D provides the amino acid sequences of the heavy chain framework region J (FRl) for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 31), humanized anli -TweakR antibody, PULl 92-2 (SEQ ID NO: 32), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 33), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 34), murine anti-TweakR antibody, J 8.3.3 (SIiQ ID NO: 35), humanized anti TweakR antibody, PDLl 83 (SEQ fD NO: 36), humanized anti-TweakR antibody, PDLI 83 -2 (SEQ ID NO; 37), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 38), and murine anti-TweakR antibody, ITFiIVI I (SEQ Q.) NO: 39);
• FIG. 3E provides the amino acid sequences of the heavy chain FR2 for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 40), humanized anti-TweakR antibody, PDU 92 -2 (SEQ ID NO: 41 ), murine anti-TweakR antibody, 19,2, i (SEQ ID NO: 42), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 43), murine anti-TweakR antibody, 18.3,3 (SEQ ID NO: 44), humanized anti-TweakR antibody, PDLl 83 (SEQ ID NO: 45), humanized anti TweakR antibody, PDLI 83 -2 (SEQ ID NO: 46), murine anti- TweakR antibody, ITEM-3 (SEQ ID NO: 47), and murine anli-TweakR antibody, ITEM- 1 (SEQ ID NO: 48); [0026] FIG.
• 3F provides the amino acid sequences of the heavy chain FR3 for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 49), humanized anti-TweakR antibody, PDL192-2 (SEQ ID NO: 50), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 51), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 52), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 53), humanized anti-TweakR antibody, PDL183 (SEQ ID NO: 54), humanized anti-TweakR antibody, PDL183-2 (SEQ ID NO: 55), murine anti- TweakR antibody, ITEM-3 (SEQ ID NO: 56), and murine anti-TweakR antibody, ITEM- 1 (SEQ ID NO: 57);
• FIG. 3 G provides the amino acid sequences of the heavy chain FR4 for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 58), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 59), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 60), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 61), humanized anti-TweakR antibody, PDLl 83 (SEQ ID NO: 62), humanized anti-TweakR antibody, PDLl 83-2 (SEQ ID NO: 63), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 64), and murine anti- TweakR antibody, ITEM-I (SEQ ID NO: 65);
• FIG. 4A provides the amino acid sequences of the light chain CDRl for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 66), murine anti -TweakR antibody, J 9.2.1 (SEQ ID NO: 67), murine anti TweakR antibody, 136.1 (SEQ ID NO 68), murine ant) TweakR antibody, 18 3.3 (SKQ ID NO: 69), mu ⁇ ne anti-TweakR antibody, TTRM 3 (SEQ ID NC)- 70), and murine aoli-TweakR antibody, ITFM 1 (SEQ ID NO. 71),
• FlG 4B provides the amino acid sequences of Ihe light chain CDR2 for the humanized anti TweakR antibody, Pl)Ll 92 (SEQ ID NO: 72), murine ant] - FwcakR antibody, 19.2.1 (SEQ ID NO: 73), murine an ti -Tw cakR antibody, 136. ] (SEQ ID NO 74), murine anti TweakR antibody, 18.3.3 (SRQ ID NO: 75), murine anti TweakR antibody, ITEM 3 (SEQ ID NO: 76), and murine anti 1 weakR antibody, ITEM-I (SKQ ID NO: 77);
• FlG 4C provides the amino acid sequences of the Sight chain CDR3 for the humanized anti-TweakR antibody, PDLl 92 (SEQ ID NO: 78), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 79), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 80), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 8] ), murine anti-TweakR antibody, ITEM-3 (SEQ ID NO: 82), and murine anti-TweakR antibody, ITEM-I (SEQ ID NO: 83);
• FIG. 4D provides the amino acid sequences of the light chain FRl for the humanized anti-TweakR antibody, PDL192 (SEQ ID NO: 84), humanized anti-TweakR antibody, PDL192-2 (SEQ ID NO: 85), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 86), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 87), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 88), humanized anti-TweakR antibody, PDL183 (SEQ ID NO: 89), humanized anti-TweakR antibody, PDLl 83-2 (SEQ ID NO: 90), murine anti- TweakR antibody, ITEM-3 (SEQ ID NO: 91), and murine anti-TweakR antibody, ITEM- 1 (SEQ ID NO: 92);
• FIG. 4E provides the amino acid sequences of the light chain FR2 for the humanized anti-TweakR antibody, PDL192 (SEQ ID NO: 93), humanized anti-TweakR antibody, PDLl 92-2 (SEQ ID NO: 94), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 95), murine anti-TweakR antibody, 136.1 (SEQ ID NO: 96), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 97), humanized anti-TweakR antibody, PDLl 83 (SEQ ID NO: 98), humanized anti-TweakR antibody, PDLl 83 »2 (SEQ ID NO: 99), murine anti- TweakR antibody, ITEM-3 (SEQ ID NO: 100), and murine anti-TweakR antibody, TTEM- I (SEQ ID NO: 101);
• FIG. 4F provides the amino acid sequences of the light chain FR3 for the humanized anti TwcakR antibody, PDLl 9? (STiQ ID NO: 102), humanized anti TweakR antibody, PDLJ 92-2 (SKQ ID NO' J 03), murine anti TwcakR antibody, 19.2.
• FIG. 4G provides the amino acid sequences of the light chain FR4 for the humanized anli-TweakR antibody, PDI, 192 (SEQ ID NO: 11 1), humanized anti-TweakR antibody, PDLl 92 2 (SEQ ID NO: 1 12), murine anti-TweakR antibody, 19.2.1 (SEQ ID NO: 113), murine anti-TweakR antibody, 136.1 (SEQ ID NO: J 14), murine anti-TweakR antibody, 18.3.3 (SEQ ID NO: 1 J 5), humanized anti-TweakR antibody, PDLl 83 (SEQ ID NO: 116), humanized anti-TweakR antibody, PDL183-2 (SEQ ID NO: 117), murine anti- TweakR antibody, ITEM-3 (SEQ ID NO: 118), and murine anti-TweakR antibody, ITEM-I (SEQ ID NO: 119);
• FIG. 5 provides the CDR and FR consensus sequences for anti-TweakR antibodies
• FIG. 6 provides an exemplary gene expression profile of TweakR gene expression in a variety of solid tumors.
• the X-axis represents individual samples assessed on the gene chip, and the Y-axis indicates the average intensity of TweakR expression for each sample;
• FIG. 7 depicts an immunohistological staining of TweakR-expressing tumor cells with the anti-TweakR antibody 29.Tl 0;
• FIG. 8A depicts the release of IL-8 by the anti-Tweak R antibodies ITEMl, ITEM2, ITEM3, ITEM4 and isotype controls;
• FIG. 8B depicts the release of IL -8 by the anti-Tweak R antibody PDL192
• FIG. 8C depicts the release of TL- 8 by the anti -Tweak R antibody 18.3.3
• FIG. 8D depicts the release of JL-8 by the anti-Tweak R antibody 136.1
• FIG. 8F depicts the release of IL 8 by the anti -Tweak R antibody PDL400;
• FIGS. 10/v 1OB provide exempiaj ⁇ examples by which the anti -TweakK antibodies PDL 192 (FIG 1 OA) and J 36.1 (lOB) inhibit cell growth by inducing apoptosis through caspase activation;
• FIG. 1 J depicts anti-tumor activity of FDIJ 92 in the SN 12C renal cell xenograft model
• FIG. 12 depicts a dose-range finding study with J 9.2, 1 in the ⁇ 253 xenograft model of head and neck cancer
• FlG, 13 depicts anti-tumor activity using 19.2.1 in a chronic dosing regimen in the A253 model of head and neck cancer;
• FIG. 14 depicts anti-tumor activity of PDL192 in the HT1376 urinary bladder xenograft model;
• FIG. 15 depicts anti-rumor activity in a dose-range finding study with PDL 192 in the A375 melanoma xenograft model
• FIG. 16 depicts the anti-tumor activity of PDLl 92 in the CSOC ovarian xenograft model
• FIG. 17 depicts the anti-tumor activity of PDL192 ⁇ gemcitabin ⁇ in the Panel xenograft model
• FIGS 18A-18D depict the anti-tumor activity of PDLl 92 ⁇ irinotecan ⁇ cetuximab in the HT29 xenograft model
• FIGS. 19A-19B depict the anti-tumor and anti-metastatic activity of PDLl 92 in a model of breast cancer using the MDA-MB-231 variant xenograft;
• FIGS. 20A-20B depict the ADCC activity of the anti-TweakR antibody PDLl 92 on TweakR transf ⁇ ctants (FIG. 20A) and SNl 2C renal cancer cells (FIG. 20B) using human PBMCs as effector cells;
• FIG, 2OC depicts the A ⁇ CC activity of the anti-TweakR antibody PDLl 92 on TweakR transfectants using mouse splenocytes as cffectoj cells;
• FfGS. 21 A 21B depict the anti-tumor activity of a murine IgGl isotype (19.2 I xGJ ) and murine TgG2a isotype (J 9.2.1 ) in the SN 12C renaj xenograft model
• FIGS 22 ⁇ 22B depict the binding of anti TweakR antibodies to human TweakR, human. TweakR (R56P) and mouse TweakR .
• antibody includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
• the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies).
• the term "antibody” also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab').sub.2, Fab, Fv and rlgG). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IU.).
• antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148: 1547, Pack and Phickthun (1992) Biochemistry 31 : 1579, Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci 6:781 , Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
• the term "monoclonal antibody” as used herein is not limited to antibodies produced through bybridoma technology.
• the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaiyotic, prokaryotic, or phage clone, and not the method by which it is produced.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the an including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma lecbniqucs including those known in the art and taught, for example, in Harlow and Lane, “Antibodies: A Laboraloiy Manual,” Cold Spring Harbor Laboratoiy Press, New York (1988); Hammerling et al., in: “Monoclonal Antibodies and T Cell Hybridomas,” Elsevier, N. Y. (1981 ), pp. 563 681 (boih of which are incorporated herein by reference in their
• An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al, Science 246: 1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen.
• the mouse spleen is harvested and splenocytes isolated.
• the splenocytes are then fused by well-known techniques to any suitable myeloma cells.
• Hybridomas are selected and cloned by limiting dilution.
• the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen.
• Ascites fluid which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.
• an immunoglobulin typically has a heavy and light chain.
• Each heavy and light chain contains a constant region and a variable region , (the regions are also known as “domains").
• Light and heavy chain variable regions contain four "framework” regions interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs".
• CDRs complementarity-determining regions
• the sequences of the framework regions of different light or heavy chains arc relatively conserved within a species.
• the framework region of an antibody that is the combined fiamcwork regions of the constituent light and heavy chains, serves to position and align the Cl)Rs in three dimensional space.
• the CDRs are primarily responsible for binding to an epitope of an antigen.
• the CDRs of each chain are typically referred to as CDR i, CDR2, and CDR3, numbered sequentially starting from the N terminus, and arc also typically identified by the chain in which the particular CDS is located.
• CDR i CDR2
• CDR3 CDR3
• Vn CDBl is located in the variable domain of the heavy chain of the antibody in which it is found
• a Vj CDRJ is the CDRl from the variable domain of the light chain of the antibody in which it is found.
• references to "Vn” refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv , or Fab
• References to "V L” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
• the phrase “single chain Fv” or “scFv” refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
• a "chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drag, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
• Any of the anti-TweakR antibodies described herein can be chimeric.
• humanized antibody or “humanized immunoglobulin” refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85%, at least 90%, and at least 95% identical.
• CDRs complementarity determining regions
• all parts of a humanized immunoglobulin, except possibly the CDRs arc substantially identical to corresponding parts of one or more native human immunoglobulin sequences.
• framework residues in Ihe human framework regions will be substituted, with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• framework substitutions arc identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Queen et a),, U.S. Pat. Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,1 80,170 (each of which is incorporated by reference in its entirety).
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-graftrog (EP 239,400; PCT publication WO 91/09967; U.S. Pat, Nos.
• the anti-TweakR antibodies described herein include humanized antibodies, such as mouse humanized antibodies, fully human antibodies, and mouse antibodies.
• Epitopes refers to a site on an antigen to which an antibody binds.
• Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).
• the determination of whether two antibodies bind substantially to the same epitope is accomplished by the methods known in the art, such as a competition assay.
• a control antibody for example, one of the anti-TweakR antibodies described herein
• any test antibody one may first label the control antibody with a detectable label, such as, biolin, enzymatic, radioactive label, or fluorescent labeJ to enable the subsequent identification.
• the intensity of the bound label is measured, If the labeled antibody competes with the unlabeled antibody by binding to an overlapping epitope, the intensity will be decreased re] alive Io the binding by negative control unlabeled antibody.
• isolated refers Io material that is substantially or essentially free from components thai normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyaciylamide gel electrophoresis or high performance liquid chromatography. ⁇ protein or nucleic acid thai is the predominant species present in a preparation is substantially purified, In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene.
• purified in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
• nucleic acid or protein is at least 85% pure, at least 95% pure, and at least 99% pure.
• “Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
• polypeptide peptide
• protein protein
• amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refers Io compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to 9 hydrogen, a carboxyl group, an amino group, and an R group, e.g., bomosorine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical montciure as a naturally occurring amino acid.
• Amnio acid mimetics refers to chemical compounds that have a structure thai is different from the general chemical deviscturc of an amino acid, but that functions Similarly to a naturally occurring amino acid.
• Amino acids may be referred to herein by their commonly known three letter symbols or by the one -letter symbols recommended by the JUPAC -TUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
• variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
• Eveiy nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule.
• amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
• Conservative substitution tables providing functionally similar amino acids arc well known in the art.
• Such conseivatively modified variants are in addition Io and do not exclude polymorphic variants, interspecies hotnoJogs, and alleles of the invention.
• Conservative substitutions for one another typically include e.g.: I ) Alanine (A), GJycroo (G); 2) ⁇ spartie acid (D), Glutamic acid (R); 3) Asparaginc (N), Glutamine (Q); 4) ⁇ rgir ⁇ ne (R), Lysine (K); 5) lsolcucinc (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (I 1' ), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (Vl) (see, e.g., Creigbton, Proteins (1984)).
• label or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
• useful labels include fluorescent dyes, electron -dense reagents, enzymes (e.g., as commonly used in an ELIS ⁇ ), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide
• radioisotope may be, for example, 3H, 14C, 32P, 35S, or 1251. In some embodiments, radioisotopes are used as toxic moieties.
• the labels may be incorporated into the antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al, Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
• the lifetime of radiolabeled peptides or radiolabeled antibody compositions can be extended by the addition of substances that stablize the radiolabeled peptide or antibody and protect it from degradation. Any substance or combination of substances that stablize the radiolabeled peptide or antibody may be used including those substances disclosed in U.S. Pat. No. S Qfi1 c
• recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or thai the cell is derived from a cell so modified.
• recombinant cells express genes that are not found within the native (non- iccombmant) form of the cell or express native genes that are otheiwisc abnoimally expressed, undei expressed or not expressed at aJI.
• nucleic acid By the term “recombinant nucleic acid” herein is meant ⁇ ucleic acid, originally foinicd in vitro, in general, by the manipulation of nucleic acid, c g., using polymerases and endo ⁇ cleases, in a foim not normally found in nature In this mannci, opes ably linkage of different sequences is achieved.
• nucleic acid in a linear form, or an expression vectoi formed in vitro by ligatmg DN ⁇ molecules that arc not not mall y joined, arc both considered recombinant for the memeposcs of this invention.
• a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non recombinanlly, i.e., using the in vivo cellular machineiy of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced rccombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
• a "recombinant protein” is a protein made using
• heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature.
• the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
• a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
• a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
• a promoter includes necerney nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
• a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
• An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
• operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wtoein the expression control sequence directs transcription of the nucleic acid coi responding to the second sequence.
• a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
• An "expression vector” is a nucleic acid constiuct, generated reconibinanily or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
• the expression vector can be part of a plasmid, vims, or nucleic acid fragment, Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
• the specified antibodies bind to a particular protein sequences at least two times the background and more typically more than 10 to 100 times background.
• compositions described herein are based upon the identification of monoclonal and humanized anti-TweakR antibodies capable of triggering apoptosis and/or stimulating ADCC in cells expressing TweakR.
• these antibodies find use in pharmaceutical compositions used to treat cancer.
• the anti-TweakR antibodies described herein specifically bind to TweakR proteins.
• specifically bind herein is meant that the antibodies bind to the protein with a K D of at least about 1 ⁇ M, at least about 0.1 ⁇ M or better, at least about 0.01 ⁇ M, and at least about 0.001 ⁇ M or better.
• the antibodies described herein are capable of selectively binding to the specific target and not to related sequences.
• TweakR proteins herein is meant that the anti-TweakR antibodies can bind a protein comprising an amino acid sequence encoded by SEQ ID NO; 1, or encoded by a nucleic acid sequence which can hybridize to SEQ ID NO: J , or which comprises the amino acid sequence of SEQ ID NO: 2,
• the anti TweakR antibodies described herein are capable of modulating the activity of a TweakR or ligand gene.
• Tn general, lbc antibodies exhibit at least one of the propci ties associated with agonists and antagonists of TweakR.
• agonisi herein is meant an antibody that is capable of promoting one or more biological activities associated with TweakR
• antagonistic herein is meant an tints body that is capable of inhibiting one or more biological activities associated with TweakR and/or TweakR ligand interaction.
• Biological properties associated with the TweakR include, but are not limited to, pro apoptosis, pro-proliferation, pro migration and pro- inflammatory activity (see, e.g., WiJey, ct a)., 2001, Immunity, 15:837-846, Wiley and Winkles, 2003, Cytokine & Growth Factor Review, 14 :24 J -249; and Winkles, el ai, 2006, Cancer Letters, 235(1): 11 -7). Accordingly, the anti TweakR antibodies described here can exhibit between 0% to 100% agonist activity.
• the anti 'TweakR antibodies can exhibit at least about 0% agonist activity, at least about 10% agonist activity, at least about 20% agonist activity, at least about 30% agonist activity, at least about 40% agonist activity, at least about 50% agonist activity, at least about 60% agonist activity, at least about 70% agonist activity, at least about 80% agonist activity, at least about 90% agonist activity, at least about 100 % agonist activity.
• the anti-TweakR antibodies described herein can exhibit greater than 100% agonist activity.
• Agonist/antagonist activity can be determined by measuring the release of cytokines and/or chemokines in an in vitro cell growth assay. For example, as set forth in the EXAMPLES, in a typical assay the cells are incubated in vitro with an anti-TweakR antibody ⁇ TWEAK ligand. 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).
• an IL- 8 release assay is used to characterize the agonist/antagonistic activity of the various anti-TweakR antibodies described herein (see e.g., FIGS. 8A-8E and Example 2).
• fTEMl exhibits approximately 25% agonist activity
• ITEM2 exhibits approximately 28% agonist activity
• JTEM3 exhibits approximately 37% agonist activity
• ITTCM4 exhibits approximately 12% agonist activity
• PDL 192 exhibits approximately 25% agonist activity
• 19.2.1 exhibits approximately 39% agonist activity
• 18,3.3 exhibits approximately 14% agonist activity
• 136.1 exhibits approximately 278% agonist activity
• PDL400 exhibits approximately 8% agonist activity.
• anti-TweakR antibodies induce apoptosis in cells expressing TweakR.
• Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al., Nature: 371, 346 (1994).
• TUNEL terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling
• anti-TweakR antibodies are capable of inhibiting the growth of tumor cells via the induction of apoptosis.
• induction of apoptosis is postulated to occur through caspase activation.
• anti-TweakR antibodies stimulate antibody-dependent cellular cytotoxicity (ADCC) in cells expressing TweakR.
• ADCC antibody-dependent cellular cytotoxicity
• anti-TweakR antibodies bind antigens on the surface of target cells (cells that express TweakR) in the presence of effector cells (such as natural killer cells or macrophages).
• effector cells such as natural killer cells or macrophages.
• Fc receptors on effector cells recognize bound antibodies.
• the cross-linking of Fc receptors signals the effector cells to kill the target cells by cytolysis or apoptosis, Cytolysis can be detected via detection of the release of label or lactate dehydrogenase from the lysed cells. Cytotoxicity can be detected directly by detection kits known in the art, such as Cytotoxicity Detection Kit from Roche Applied Science (Indianapolis, IN),
• antibody -associated ADCC activity JS monitored and quantified through measurement of lactate dehydrogenase (TJ)H) release in Ihe supernatant, which is rapidly released upon damage to the plasma tnembranc ' 1 o dctcitnine the percentage of cell mediated cytotoxicity, the average absorbancc of a sample JS calculated and background controls subtracted using the following equation.
• TJ lactate dehydrogenase
• SR refers to spontaneous release and MR refers to maximum release Sec also, the methods disclosed in US Patent Application Pub. No. 2005/0075763, ihe disclosure of which is incorporated herein ID its entirety
• the anti Tweak R antibodies induce al least 10%, 20%, 30%,, 40%, 50%, 60%, or 80% cytotoxicity of the target cells.
• the anti TweakR antibodies described herein can have any one of the defined agonist and/or antagonist activities and any one of the defined cytotoxicities.
• the anti-TweakR antibody, PDL 192 induces ADCC activity in SN12C renal cells, as well as TweakR transfectant cell lines using either human peripheral blood mononuclear cells or mouse splenocytes as effector cells. Induction of ADCC by anti-TweakR antibodies was not observed in cells that did not express TweakR (data not shown).
• Biological pathways associated with the death of tumor cells in vivo include apoptosis and cytolysis of cells via ADCC.
• the contributions of apoptosis and ADCC to the anti -tumor activity of anti-Tweak R antibodies was measured in vivo using two anti- TweakR murine antibodies with differing ADCC activities in vitro (data not shown).
• Murine anti-TweakR 19.2.1, an ⁇ gG2a antibody exhibiting strong ADCC activity in vitro had significant anti-tumor activity in the SN12C renal xenograft model (FIG. 21A) and in the A375 melanoma xenograft model (FIG. 21B).
• the murine anti-TweakR The murine anti-TweakR.
• the anti -TweakR antibodies described herein are capable of reducing or inhibiting tumor cell growth via the induction of one or more biological pathways associated with the killing of cancer cells in vivo.
• Antibodies useful in the compositions and methods described herein include and TweakR antibodies that specifically bind to the same epitope or epitopes as PDLI 92, J 8,3.3, or 136J .
• the epitope bound by the antibodies described herein can be the same, overlapping, or distinct.
• the specific binding of PD L J 92 and 19.2.1 to the human TweakR protein is eliminated by changing a single amino acid at position 56, i.e., R56P, of its amino acid sequence (SEQ ID NG:2; see FIGS.
• the anti-TweakR antibodies 18.3.3, ITEM] and ITEM3 antibodies aie still capable of binding the human TweakR sequence (sec, e.g., FIG. 22A).
• anti-TweakR antibodies 18.3.3, ITEMl , and ITEM3 bind the mouse TweakR protein, which comprises a proline at position 56 of its amino acid sequence.
• the anti TweakR antibodies PDLl 92 and 19.2.1 do not bind the mouse TweakR protein (see, e.g. FIGS. 22A-22B).
• variable regions, framework regions and CDRs are well-known to those of ordinary skill in the art (see e.g., Kabat, et al., "Sequences of Proteins of Immunological Interest", 5.sup.th ed., National Institutes of Health, Bethesda, Md. (1991)).
• anti-TweakR antibodies capable of specifically binding TweakR can comprise various combinations of heavy and/or light chain CDRs and/or framework regions.
• the anti-TweakR antibodies comprise a heavy and/or a light chain variable region.
• the anti-TweakR antibodies described herein comprise one, two, or all three CDRs of a heavy and/or light chain variable.
• the an ti- TweakR antibodies described herein comprise one, two, three, or all four framework regions.
• the anti -TweakR antibodies comprise CDRs of both the heavy and light chain variable regions.
• the anti-TweakR antibodies comprise CDRs and framework regions of both the heavy and light chain variable regions. ⁇ s is apparent from the foregoing description, anti TweakR antibodies usefu) in the compositions and methods described herein can comprise any combination of Cl)R and framework regions provided that the resulting antibodies arc capable of specifically binding TweakR and killing cancer cells,
• the anti TweakR antibodies can be monoclonal, chimeric, humanized, or fully human.
• the aoti -TweakR antibodies can comprise one or more CDRs of a heavy and/or light chain variable region comprising a consensus sequence selected from the group consisting of SEQ ID NOs: 120-123 and 127 129 and various combinations of one or more framework regions of a heavy and/or light chair) variable region comprising a consensus sequence selected from the group consisting of SEQ ID NOs: 123- 126 and SEQ ID NOs: 130433.
• an anti-TweakR antibody or an antigen binding fragment thereof can be selected from the group consisting of: a heavy chain CDRl encoded by an amino acid sequence comprising the formula XaaYWMXaa (SEQ ID NO 120), wherein Xaa at position 1 is S, N, or K and Xaa at position 5 is S or N; a heavy chain CDR2 encoded by an amino acid sequence comprising the formula EIRLKSDNY ATHY AESXaaKG (SEQ ID NO 121), wherein Xaa at position 17 is A or V; a heavy chain CDR3 encoded by an amino acid sequence comprising the formula XaaXaaADXaaXaaDY (SEQ ID NO 122), wherein Xaa at position 1 is G, T, or Y, Xaa at position 2 is F or Y, Xaa at position 5 is A, T, or Y, and Xaa position 6 is F or M; a light chain CDRl encoded by an amino acid
• WVRQXaaPXaaKGLEWXaa ⁇ (SKQ ID NO 124), wherein Xaa at position 5 is A or S, Xaa at position 7 is E or G, Xaa at position 13 is V or L; a heavy chain FR3 encoded by an amino acid sequence composing the formula
• Xaa FnSRDXaaXaaXaaXaaXaaXaaXaaYLQMNXaaLRAEXaaTXaaXaaYYCXaaXaa (SEQ ID NO 125), wherein Xaa at position ] is R or K, Xaa at position 3 is D or N, Xaa at postion 9 is S or ⁇ , Xaa at position J 0 is K or R, Xaa at position 11 is N or S, Xaa at position J 2 is S, R, or T, Xaa at position 13 is L or V, Xaa at position 19 is S or N, Xaa at position 24 is D or N, Xap at position 26 is A or G, Xaa at position 27 is I or V, Xaa at position 31 is T, S or A, and Xaa at position 32 is G, P, or R; a heavy chain FR4 encoded by an amino acid sequence
• an anli-TweakR antibody can comprise a heavy chain CI)Rl encoded by an amino acid sequence comprising the formula XaaYWMXaa (SEQ ID NO 120), wherein Xaa at position 1 is S, N, or K and Xaa at posilion 5 is S or N, a heavy chain CDR2 encoded by an amino acid sequence comprising the formula ElFLKSDNYATHY ⁇ KSXaaKO (SEQ IO NO 121), wherein Xaa at posilion I / is ⁇ or V; and, a heavy chain C ⁇ R.3 encoded by an amino acid sequence comprising the formula XaaXaaADXaaXaaDY (SIiQ TD NO 122), wherein Xaa at position ! is G, T, or Y, Xaa at position ? is F or V, Xaa at position 5 is A, '! ' , or Y, and Xaa position
• ao anti-TweakR antibody can comprise a light chain CDRl encoded by an amino acid sequence comprising the formula Xaa ASQS VSTSXaaYSYMXaa (SEQ ID NO 127), wherein Xaa at position is 1 is R or K, Xaa at position 10 is S or T and Xaa at position Ib is H or Q, a light chain CDR2 encoded by an amino acid sequence comprising the formula YAXaaXaaLXaaS (SEQ ID NO J 28), wherein Xaa at position 3 is S or T, Xaa at position 4 is N or K, Xaa at position 6 is E or D, and, a light chain CDR3 encoded by an amino acid sequence comprising the formula QHSWEXaaPYT (SEQ ID NO 129), wherein Xaa at position 6 is I or L.
• an anti-TweakR antibody can comprise a heavy chain CDRl encoded by an amino acid sequence comprising the formula XaaYWMXaa (SEQ ID NO 120), wherein Xaa at position 1 is S, N, or K and Xaa at position 5 is S or N, a heavy chain CDR2 encoded by an amino acid sequence comprising the formula EIRLKSDNY ATHY AESXaaKG (SEQ ID NO 121), wherein Xaa at position 17 is A or V; a heavy chain CDR3 encoded by an amino acid sequence comprising the formula XaaXaaADXaaXaaDY (SEQ ID NO 122), wherein Xaa at position 1 is G, T, or Y, Xaa at position 2 is F or Y, Xaa at position 5 is A, T, or Y, and Xaa position 6 is F or M, a light chain CDRl encoded by an amino acid sequence comprising the formula XaaYWMXaa (
• an aim TweakR antibody can comprise a heavy chain CDRl encoded by an amino acid sequence composing the formula XaaYWMXaa (SEQ ID NO 120), wherein Xaa at position 1 is S, N, or K and Xaa ai position 5 is S or N, a heavy chain CDR2 encoded by an amino acid sequence comprising the formula EIRLKSDNY ⁇ THY AESXaaKG (SEQ ID NO 121 ), wherein Xaa at position 17 is A oi V; a heavy chain CDR3 encoded by an ammo acid sequence comprising the foimula XaaXaaADXaaXaaDY (SEQ ID NO 122), wherein Xaa at position 1 JS G, T, or Y, Xaa at position 2 is F or Y, Xaa at position 5 is A, T, or Y, and Xaa position 6 is F or M, a light chain CDRl encoded by an amino acid sequence composing the formula
• STSXaaYSYMXaa (SEQ ID NO 127), wherein Xaa at position is 1 is R or K, Xaa at position 10 is S or T and Xaa at position 15 is H or Q, a light chain CDR2 encoded by an amino acid sequence comprising the formula YAXaaXaaLXaaS (SEQ ID NO 128), wherein Xaa at position 3 is S or T, Xaa at position 4 is N or K, Xaa at position 6 is E or D, a light chain CDR3 encoded by an amino acid sequence comprising the formula QHSWEXaaPYT (SEQ ID NO 129), wherein Xaa at position 6 is I or L, a heavy chain FRl encoded by an amino acid sequence comprising the formula EVXaaLXaaESGGGLVQPGGSXaaXaaLSCXaaASGFXaaFXaa (SEQ ID NO 123), wherein Xaa at
• XaaFTISRDXaaXaaXaaXaaXaaXaaXaaYLQMNXaaLRAEXaaTXaaXaaYYCXaaXaa (SEQ TD NO 125), wherein Xaa at position 1 is R or K, Xaa at position 8 is D or N, Xaa at postion 9 is S or A, Xaa at position 10 is K or R, Xaa at position 11 is N or S, Xaa at position 12 is S, R, or T, Xaa at position 13 is L or V, Xaa at position 19 is S or N, Xaa at position 24 is D or N, Xaa at position 26 is ⁇ or G, Xaa at posiiion 27 is 1 or V, Xaa at position 31 is T, S or A, and Xaa at position 32 is G, P, or R, and a heavy chain FR 4 encoded by an amino acid
• an anli -TweaicR antibody can comprise a heavy chain CDRi encoded by an amino acid sequence comprising the formula XaaYWMXaa (SEQ ID NO 120), wherein Xaa at position 1 is S, N, or K and Xaa at position 5 is S or N, a heavy chain CDR2 encoded by an amino acid sequence comprising the formula EIRLK SDN Y ⁇ THYAESXaaKG (SEQ ID NO 12J), wbeiejn Xaa at position 17 is A or V; a heavy chain CDR3 encoded by an amino acid sequence comprising the formula XaaXaaADXaaXaaDY (SEQ ID NO 122), wherein Xaa at position J is G, T, or Y, Xaa at position 2 is F or Y, Xaa at position 5 is A, T, or Y, and Xaa position 6 is F or M, a light chain CDRl encoded by an amino acid sequence comprising the formula
• XaaFTISRDXaaXaaXaaXaaXaaXaaXaaYLQMNXaaLRAEXaaTXaaXaaYYCXaaXaa (SEQ ID NO 125), wherein Xaa at position 1 is R or K, Xaa at position 8 is D or N, Xaa at postion 9 is S or A, Xaa at position 10 is K or R, Xaa at position 11 is N or S, Xaa at position 12 is S, R, or T, Xaa at position 13 is L or V, Xaa at position 19 is S or N, Xaa at position 24 is Tl or N, Xaa at position 26 is ⁇ or G, Xaa at position 27 is I or V, Xaa at position 3 ] is T, S or A, and Xaa at position 32 is G, P, or R, a heavy chain FR4 encoded by an amino acid sequence comprising
• Xaa al position is 3 is Q or V
• Xaa at position 4 is M or L
• Xaa at position 9 is G
• S or ⁇ Xaa at position 12 is S, A or T
• Xaa at position 13 is A, L, or V
• Xaa at position 15 is P, V, or L
• Xaa at position 17 is D, 11, or Q
• Xaa at position 19 is V or A
• Xaa at position 22 is T or S
• the anti-TweakR antibodies described herein can comprise various combinations of heavy and/or light chain CDRs and framework regions selected from the group consisting of SEQ ID NOs: 13, 19, 25, 66, 72, 78, 31, 40, 49, 58, 84, 93, 102 and 111.
• an anti-TweakR antibody can comprise a heavy chain CDRl encoded by the amino acid sequence comprising SYWMS (SEQ ID NO: 13), a heavy chain CDR2 encoded by the amino acid sequence comprising EIRLKSDNYATHYAESVKG (SEQ ID NO: 19), and a heavy chain CDR3 encoded by the amino acid sequence comprising the formula YYADAMDY (SEQ ID NO: 25), f0109
• an anti-TweakR antibody can comprise a light chain CDR] encoded by the ammo acid sequence comprising RASQSVSTSSYSYMH (SEQ ID NO 66), a light chain CDR2 encoded by the ammo acid sequence comprising YASNLES (SEQ ID NO 72), and a light chain CDR3 encoded by the ammo acid sequence comprising the formula QHSWEfPYT (SEQ ID NO 78).
• &a anti TweakR anybody can comprise all three CIlRs from boih the heavy sad light chain, e.g., a heavy chain CDR I encoded by the ammo acid sequence comprising SYWMS (SEQ ID NO: 13), a heavy chain CDR2 encoded by the amino acid sequence comprising ETRLKSDNY ATHY AES VKG (SEQ ID NO: 19), a heavy chain CDR3 encoded by the amino acid sequence comp ⁇ sing the formula Y YADAMDY (SEQ ID NO: 25), a light chain CDR] encoded by the ammo acid sequence composing RASQSVSTSSYSYMIf (SEQ ID NO 66), a light chain CDR2 encoded by the amino acid sequence comprising YASNLES (SEQ ID NO 72), and a light chain CDR3 encoded by the amino acid sequence comprising the formula QHSWEIPYT (SEQ ID NO 78).
• an anti-TweakR antibody can comprise a heavy chain FRl encoded by the amino acid sequence comprising
• EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO 31), a heavy chain FR2 encoded by the amino acid sequence comprising WVRQAPGKGLEWVA (SEQ ID NO 40), a heavy chain FR3 encoded by the amino acid sequence comprising RFTISRDDSKNSLYLQMNSLRAEDTAVYYCTG (SEQ ID NO 49), and a heavy chain FR4 encoded by the amino acid sequence comprising WGQGTLVTVSS (SEQ ID NO 58).
• the anti-TweakR antibodies described herein can comprise one or more heavy chain CDR sequences, e.g., SEQ ID NOs: 13, 19 and 25 and/or one more light chain CDR sequences, e.g., SEQ ID NOs: 66, 72, and 78.
• the resulting antibodies are capable of binding TweakR and killing cancer cells.
• an anti-TweakR antibody can comprise a light chain FRl encoded by the amino acid sequence comprising DIQMTQSPSSLS ⁇ SVG))RVTITC (SEQ ID NO 84), a light chain FR2 encoded by the amino acid sequence comprising WYQQKPGKAPKLIJK (SEQ ID NO 93), a light chain PR3 encoded by the amino acid sequence comprising GVPSRFSG SGSGTDFTLT ⁇ SSLQPfiDFATYYC (SEQ ID MO 102), and a light chain PR4 encoded by the amino acid sequence comprising FGGGTKVEIKR (SEQ ID NO 1 11).
• the anli- TweakR antibodies described herein can comprise one or more heavy chain CDR sequences, e.g., SEQ ID NOs: 13, 19 and 25 and/or one more light chain CDR sequences, e.g., SEQ ID NOs: 66, 72, and 78, and/or one or more heavy chain framework regions encoded by SEQ ID NOs; 31, 40, 49 and 58.
• the anti-TweakR antibodies can comprise other combinations of heavy and/or light chain CDRs and framework regions, provided that the resulting antibodies are capable of binding TweakR and killing cancer cells.
• variable, constant, framework region, or CDR refers to an antibody region wherein at least about 85-90%, and preferably at least 95% of the amino acid sequence is identical to a natural or unaltered antibody variable or constant region.
• identical or percent “identity,” in the context of two or more amino acid or nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., description of BLAST at the National Center for Biotechnology Information (NCBI) web site located at www.ncbi.nlm.nih.gov).
• NCBI National Center for Biotechnology Information
• Identical or substantially identical sequences include sequences having deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants such as conservative) y modified variants.
• the well-known algorithms for measuring sequence identity can account for gaps and the like.
• sequence identity exists over a region that is at least about 25 amino acids or nucleotides m length, or more preferably over a region that js SO 100 amino acids or nucleotides in length.
• amino acid sequences of the anti-TweakR antibodies useful in the methods described herein arc not confined to the sequences found in natural antibodies; antibodies can be redesigned to obtain desired characteristics using well known recombinant DN ⁇ techniques.
• Such "genetically altered antibodies” include those where the amino acid sequence has been varied from that of a parent (i.e., unaltered) antibody. The possible variations range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region.
• Changes, by site- directed mutation, in the constant region may be made in order to improve or alter the functional characteristics of a therapeutic antibody such as immunogenicity, pharmacokinetic characteristics (e.g., serum half-life), complement fixation, interaction with membranes and other effector functions.
• changes to the antibody variable region may be made in order to improve the antigen binding characteristics.
• nucleic acids that encode the anti-TweakR antibodies and amino acid sequences described herein, as well as molecules which hybridize to said nucleic acid sequences and which encode anti-TweakR antibodies, such antibodies having the functional properties described herein can be used in the methods disclosed herein.
• anti-TweakR antibodies can be of any of the recognized isotypes.
• anti-TweakR 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.
• the antibodies have low levels or lack fucose.
• Antibodies lacking fucose have been correlated with enhanced ADCC (antibody-dependent cellular cytotoxicity) activity, especially at low doses of antibody. Shields, R.L., et al, (2002) J. Biol. Chero. 277:26733-26740; Shinkawa, T. 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 (ex 1 ,6 fucosyltransfcrase) necessary foi fucosylation of polypeptides.
• Alternative methods for increasing ⁇ DCC activity include mutations in the Fc portion of an anti -TweakR antibody, particularly mutations which increase antibody affinity for an JPc ⁇ R receptor. ⁇ correlation between increased Fc ⁇ R binding with mutated Fc has been demonstrated using targeted cyloxicity cell-based assays, Shields, R. L. et al. (2001) J Biol. Chem 276:6591 -6604; Presta et al. (2002), Biochem Soc. Trans. 30:487 490.
• Other methods for increasing ADCC activity include the generation of specific Fc region mutations as described in US Patent ⁇ ppi. Pub. No. 2005/0025763; the disclosure of which is incoiporated herein in its entirety.
• antibodies that can be used in the compositions and methods described herein include, but are not limited to, any immunoglobulin molecule that binds (preferably, jmmunospecifically, i.e., competes off non-specific binding, as determined by immunoassays well known in the art for assaying specific antigen-antibody binding) an antigen.
• Such antibodies include, but are not limited to, polyclonal, monoclonal, bi- specific, multi-specific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, disulfide-linked Fvs, and fragments containing either a V L or V H domain or even a complementary determining region (CDR) that specifically binds an antigen, in certain cases, engineered to contain or fused to an Fc binding domain.
• CDR complementary determining region
• TweakR sequences for diagnostic applications.
• Expression levels of genes in normal tissue (e.g., not undergoing a disorder) and in diseased tissue (and in some cases, for varying severities of disorders that relate to prognosis, as outlined below) are evaluated to provide expression profiles.
• a gene expression profile of a particular cell state or point of development is essentially a "fingerprint" of the state of the cell. While two states may have a particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is reflective of the state of the cell.
• diagnosis may be performed or confirmed to determine whether a tissue sample has the gene expression profile of normal or diseased tissue. This will provide for molecular diagnosis of related conditions.
• Differential expression refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among ceJls and tissue.
• a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus diseased tissue.
• Genes may be turned on or tinned off in a parti cula? state, reJativc to another state thus permitting comparison o f two of more states.
• ⁇ qualitatively regulated ecne will exhibit an expression pattern wilbin a state or cell type which is detectable by standard techniques, Some genes will be expressed in one state or cell type, but not in both.
• the difference in expression may be quantitative, e.g., in that expression is increased or decreased; e.g., gene expression is cither upreguJatcd, resulting in an increased amount of transciipt, or downregulated, resulting in a decreased amount of transcript.
• the degree to which expression differs need only be large enough to quantify via Standard characterization techniques as outlined below, such as by use of Affymetrix GENECHIP ® (DNA microchip array) expression arrays. See, Lockhart (1996) Nature Biotechnology 14: 1675-1680.
• Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, northern analysis, and RNase protection.
• the change in expression is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, with from 300 to at least 1000% being especially preferred.
• Evaluation may be at the gene transcript or the protein level.
• the amount of gene expression may be monitored using nucleic acid probes to the RNA or DNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, e.g., with antibodies to TweakR protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc.
• Proteins corresponding to TweakR e.g., those identified as being important in a disease phenotype, can be evaluated in a disease diagnostic lest.
• gene expression monitoring is performed simultaneously on a number of genes. Multiple protein expression monitoring can be performed as well.
• TweakR nucleic acid probes are attached to biochips for the detection and quantification of TweakR sequences in a particular cell.
• the assays arc further described below m the example. PCR techniques can be used Io provide greater sensitivity.
• nucleic acids encoding TweakR are detected Although J)NA or RNA encoding TweakR protein may be detected, of particular interest arc methods wherein an inRNA encoding a TweakR protein is detected.
• Probes to detect mRNA can be a nucleotide/deoxynucleolide probe that is complementary to and hybridizes with the mRNA and includes, but is not limited to, oligonucleotides, cDNA, or RNA. Probes also should contain a detectable label, as defined herein, in one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample.
• RNA probe a digoxygenin labeled riboprobe that is complementary to the mRNA encoding a myelomaprotein is detected by binding the digoxygenin with an anti-digoxygenin secondaiy antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
• TweakR proteins, antibodies, nucleic acids, modified proteins, and cells containing TweakR sequences are used in diagnostic assays. These assays can be performed on an individual gene or corresponding polypeptide level.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or corresponding polypeptides.
• TweakR protein finds use as a disease marker of cancerous conditions, including, but are not limited to, bladder cancer, breast cancer, colorectal cancer, lung cancer, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, stomach cancer, uterine cancer, cervical cancer, esophageal cancer, renal cell carcinoma, glioblastoma, and sarcomas. Additionally, TweakR finds use as a marker for prognostic or diagnostic purposes.
• TweakR antibodies are used to delect TweakR.
• a preferred method separates proteins from a sample by electrophoresis on a gel (typically a denatuiing and reducing protein gel, but may be another type of gel, including isoelectric focusing geJs and the like). Following separation of proteins, TweakR is detected, e.g., by immunoblotting with antibodies raised against TweakR.
• antibodies to TweakR find use in in situ imaging techniques, e.g., in histology. Sec, e.g., Asai, et al. (eds. S 993) Methods in Cell Biology: Antibodies in Cell Biology (vol. 37) Academic Press.
• cells are contacted with from one to many antibodies to TweakR protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected.
• the antibody is detected by incubating with a secondaiy antibody that contains a detectable label.
• the primary antibody to TweakR contains a detectable label, e.g., an enzyme marker that can act on a substrate.
• each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for TweakR along with other markers of the aforementioned conditions. Many other histological imaging techniques are also provided by the invention.
• the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths.
• a fluorescence activated cell sorter FACS
• FACS fluorescence activated cell sorter
• antibodies find use in diagnosing cancer from blood, serum, plasma, stool, and other samples.
• Antibodies can be used to detect TweakR by previously described immunoassay techniques including FXISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like.
• in situ hybridization of labeled TweakR nucleic acid probes to tissue arrays is done. For example, arrays of tissue samples, including diseased tissue and/or normal tissue, are made. In situ hybridization (sec, e.g., ⁇ usubel, supra) is then performed. When comparing the fingerprints between an individual and a standard, a diagnosis, a prognosis, or a prediction may be based on the findings. It is further understood thai the genes which indicate (he diagnosis may differ from ihose which indicate the prognosis and molecular profiling of the condiiion of the cells may lead Io distinctions between responsive or refractory conditions or may be predictive of outcomes.
• TweakR proteins, antibodies, nucleic acids, modified proteins, and cells containing TweakR sequences arc used in prognosis assays.
• gene expression profiles can be generated that correlate to a disease state, clinical, pathological, or other information, in terms of long term prognosis. Again, this may be done on cither a protein or gene level, with the use of genes being preferred. Single or multiple genes may be useful in various combinations.
• TweakR probes may be attached to biochips for the detection and quantification of TweakR sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. PCR method may provide more sensitive and accurate quantification.
• abnormal cell proliferation can be manifested as tumors or as metastases.
• tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• metastasis is used herein in the broadest sense and refers to the spread of tumor, e.g. cancer from one part of the body to another. Tumors formed from cells that have spread are called secondary tumors, and contain the same type of cells as the original (primary) tumor.
• prostate cancer that has metastasized to liver or bone is not liver or bone cancer, rather metastasized prostate cancer, as it still contains prostate cancer cells, regardless of their location.
• cancer cells can be present within a solid tumor, the lymph system, or in the bloodstream.
• cancer includes all malignant neoplasms, including, but not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
• cancers examples include, but arc not limited to, bladder cancer, breast cancel, colon cancer, lung cancer, melanoma, pancreatic cancer, ovarian cancer, renal cancer, head and neck cancer, esophageal cancer, uterine cancer, siomacb cancer, cervical cancer, glioblastoma, and sarcomas.
• the therapeutic methods described herein are usually applied to human patients, but can be applied Io other mammals.
• administration of anti -TweakR antibodies induces apoptosis or cytolysis of cells exptcssing TweakR.
• induction of cytolysis is achieved via antibody -dependent cellular cytotoxicity (ADCC).
• ADCC antibody -dependent cellular cytotoxicity
• the anti -Tweak R antibodies can induce between 10% to greater than 80% cytotoxicity of cells expressing TweakR.
• administration of the anti-TweakR antibodies induces at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% or more cytotoxicity of cells expressing TweakR.
• administration of the anti-TweakR antibodies 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 secondaiy tumor.
• administration of the anti-TweakR antibodies can reduce the size of a solid tumor by at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% or more.
• administration of the anti-TweakR antibodies can completely inhibit or prevent the growth of a solid tumor.
• the methods can employ an anti-TweakR antibody conjugated to an effector moiety.
• the effector moiety may be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or preferably may be a therapeutic moiety.
• the effector moiety (or "effector component") may be bound (or linked, or conjugated), to the anti-TweakR antibody either covalently, through a linker or a chemical bond, or noncovalcntly, through ionic, van der Waals, electrostatic, or hydrogen bonds.
• the therapeutic moiely can be a small molecule that modulates the activity TweakR.
• Ihe therapeutic moiety affects the activity of molecules or cells associated with or in close proximity to TweakR.
• the therapeutic moiety may be a cytotoxic agent.
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes (e.g., I 131 , 1 125 , Y 90 and Re 186 ), cbeinotherapcutic agents, and toxins sucb as enzymatically active toxins of bacteria], fungal, plant or animal origin, or fragments thereof.
• Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin ⁇ chain, curcin, crotin, phcnomycin, enomycin, auristatins (e.g., auristatin E, or auri statin F), and the like.
• Targeting the therapeutic moiety to the TweakR expressed on the surface of a cancer cell not only serves to increase the local concentration of therapeutic moiety in the afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
• the anti-TweakR antibodies can be used in combination with conventional therapeutic agents used to treat a particular cancer.
• 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 one or more therapeutic agents to combine with the anti-TweakR antibodies described herein.
• pancreatic cancer is currently treated using the therapeutic agent gemcitabine with or without erlotinib (Tarceva®).
• the anti-TweakR antibodies can be used in combination with gemcitabine and/or erlotinib to treat pancreatic cancer.
• Examples of other therapeutic agents that are presently used to treat cancer, and thus can be used in combination with the anti-TweakR antibodies described herein, include, but are not limited to, targeted agents, conventional chemotherapy agents, and hormonal therapy agents.
• the anti-TweakR antibodies described herein can be ⁇ in combination with targeted agents.
• Targeted agents include, but are not limited to, angiogenic agents (such as bevacizumab, sunitinib, sorafen ⁇ b, temsirolimus, 2 ⁇ mcthoxyesrradiol or 2ME2, finasunate, PTK787, and vandetanib), KGFR inhibitors (such as erlotinib, cetuximab, panitum ⁇ mab, gefinitib, lapatinib, and trastuzumab), imrounomodulators (such as riruximab, alemtuzumab, and aJdesleukine), proteasomc inhibitors (sucb as bortczomib, PK-I ' / 1, and NPI 052), intogrin inhibitors (such as nalaShzumab, voloc
• the anli-TweakR antibodies 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, carbopJatin, cisplatin, cyclophosphamide, mclphaJan, ifosfamide, uramustine, chlorambucil, mechloethamine, thiotcpa, busulfan, temozolomide, and dacarbazine), anti-metabolites (gemcitabine, cytosine arabinoside, Ara-C, capecitabine, 5FU (5-fluorouracil), azathioprine, mercaptopurine (6-MP), 6-thioguanine, aminopterin, pemetrexed, and methotrexate), plant alkaloids and terpenoids (such as docetaxel, paclitaxel, protein- bound paclitaxel, vin
• the anti-TweakR antibodies described herein can be used in combination with hormonal therapy agents (such as anastrozole, letrozoJe, goserelin, and tamoxifen).
• hormonal therapy agents such as anastrozole, letrozoJe, goserelin, and tamoxifen.
• the anti-TweakR antibodies described herein can be used as a monotherapy or in combination with other therapeutic agents.
• the anti-TweakR antibodies described herein are useful as monotherapeutic agents or as part of a combination therapy regime for the treatment of cancer patients that have become resistant to anti-EGFR antibodies.
• Resistance to anti-EGFR antibodies has been hypothesized to be associated with mutations that result in constitutive activation of EGFR-mediated signaling (see, e.g., Jhawer, et aJ., 2008, Cancer Res., 68(6): 1953-1961).
• the anti-TweakR antibody PDL 192 was effective at reducing and/or inhibiting the growth of the tumor (see Table 2), suggesting that anti-TweakR antibodies can be used to treat cancer patients that do not respond to, or have become resistant to, EGFR inhibitors, such as anti-EGFR antibodies.
• anti-TweakR antibodies are administered to cancer patients that do not respond to, or become resistant to treatment with anti-epidermal growth factor receptor (EGFR) antibodies such as cetuximab and panitumumab.
• EGFR anti-epidermal growth factor receptor
• the anti-TweakR antibodies can be administered alone or in combination with other therapeutic agents, including, but not limited to, targeted agents, conventional chemotherapy agents and hormonal therapy agents.
• cancer patients that are resistant to or do not respond to anti-EGFR antibodies are screened for BRAF, KRAS, PTEN, and PIK3CA mutations as described in Benvemiti, ct al. 2007, Cancer Res., 67(6):2643-2648.
• Patients that have mutations in one or more of these genes are identified and treated with anti-TweakR antibodies a ⁇ ooc or in combinaiion with other therapeutic agents, including, but not limited to, targeted agents, conventional chemotherapy agents and hormonal tbciapy agents.
• One or more of the above therapeutic agents can be administered concurrently, prior to, or following administration of an anti TweakR antibody.
• the agents can be administered separately or combined in a cocktail and administered together as a single composition,
• the composition(s) can be administered by any means known in the ait.
• the anti-TweakR antibodies can be formulaled into a pharmaceutical composition that is administered to a subject in a therapeutically effective amount.
• therapeutically effective amount 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 the therapeutically effective amount of an anti-TweakR antibody for treatment of cancer are well-known to those of ordinary skill in the art and may be determined empirically using routine experimentation.
• a "therapeutically effective amount” 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 anticancer 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.
• the concentration of an antibody in these formulations varies widely from about 0.1 to 100 mg/ml, but is often in the range 1 to 20 rng/m).
• the appropriate dosage of the antibody will depend on the severity and course of disease, the patient's clinical histoiy and response, the toxicity of the antibodies, and the discretion of the attending physician.
• the antibodies arc suitably administered to the patient at ooc time oi over a series of treatments.
• the proper dosage and treatment iegiroen can be established by monitoring the progress of therapy using conventional techniques known to the people skilled of the ait.
• compositions described herein are administered to a parent intravenously as a bolus or by continuous infusion over a period of time; or by intramuscular, subcutaneous, intraperitoneal, or intra -cerebrospinal routes.
• parent intravenously as a bolus or by continuous infusion over a period of time; or by intramuscular, subcutaneous, intraperitoneal, or intra -cerebrospinal routes.
• Methods for preparing parentally administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (J 5th Ed., Mack Publishing Company, Easton, Pa., 1980), which is incorporated herein by reference.
• compositions formulated for pharmaceutical use comprise one or more of the anti-TweakR antibodies described herein.
• the compositions optionally further comprise a carrier.
• the pharmaceutical compositions described herein typically comprise an anti-TweakR antibody and a pharmaceutical carrier, and, commonly they comprise a solution of an anti-TweakR antibody, or a cocktail thereof, dissolved in an acceptable carrier, preferably an aqueous earner.
• 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 isotonic.
• the carrier can also contain excipients such as human serum albumin, polysorbate 80, sugars or amino acids to protect the active protein.
• kits are also provided herein.
• such kits may include at least one of the following: assay reagents, buffers, TweakR-specific nucleic acids or antibodies, hybridization probes and/or primers, antisense polynucleotides, ribozymes, dominant negative TweakR polypeptides or polynucleotides, and/or small molecule inhibitors of TweakR-associated sequences.
• kits may include instructional materials containing instructions
• Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
• Such media may include addresses to internet sites that piovide sucii instructional materials
• the anti TweakR antibodies and antigen binding fragments disclosed herein, and the compositions and kits comprising these antibodies and antigen binding fragments can be used for therapy, and in particular for treatment of any of the diseases described herein.
• Example 1 TweakR. Expression in Primary Tumors
• TweakR mRNA was used to detect the expression of TweakR mRNA m a variety of primary solid tumors, including lung, pancreatic, renal, breast and head i neck cancers. mRNA was isolated from various cancers and 347 normal adult tissues and was hybridized to Eos HuO3, a customized Affymetrix gene chip containing approximately 59,000 probesets representing 46,000 genes, EST clusters, and predicted exons.
• cDNA array expression levels of TweakR in pancreatic, lung (adenocarcinoma and squamous cell carcinoma), and renal cancers are compared to a range of normal body tissues.
• the samples arc displayed along on the X ⁇ axis, with the height of each sample reflecting the average intensity or expression level of TweakR. Samples with expression levels above Ihe honzontaJ dashed line at 100 are considered positive for TweakR expression.
• TweakR (29. flO, 374 2, and 349 2)
• TweakR protein was detected on lung adenocarcinomas, lung squamous cell carcinomas, pancrealjc adenocarcinomas, renal eel! carcinomas, breast ductal carcinomas, colorectal adenocarcinomas, ovarian carcinomas, stomach adenocarcinomas, bladder cancers, and cancers of head and neck origin cancers.
• FIG. 7 is a typical example of TweakR protein staining using antibody 29.Tl 0 on solid tumors.
• Monoclonal antibodies were generated by immunizing Balb/c mice intraperitoneally with mouse 3T12 cells overexpressing human TweakR. Spleens were harvested, and splenocytes were fused with the multiple myeloma cell line, NSO. Hybridomas were selected using aminopterin. Hybridomas expressing anti-TweakR antibodies of interest were subcloned several times to isolate individual clones.
• Humanization of 19.2.1 was carried out essentially according to the procedure of Queen, C. et al. (Proc. Natl. Acad. Sci. USA 86: 1002940033 (1989)). First, human VH and VL segments with high homology to the 19.2.1 VH and VL amino acid sequences, respectively, were identified. Next, the CDR sequences together with framework amino acids important for maintaining the structures of the CDRs were grafted into the selected human framework sequences. The resulting humanized monoclonal antibody (PDL] 92) was expressed in the mouse myeloma oelJ line NSO.
• PCR polymerase chain read ion
• VH and VL c ⁇ NAs were subcloned into the pCR4Blunt TOPO vector (Invitrogen Corporation, Carlsbad, CA) foi sequence determination.
• DN ⁇ sequencing was carried out by PCR cycle sequencing read ions with fluorescent dideoxy chain terminators (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Multiple plasmid clones were sequenced for each of the heavy and light chains variable regions.
• DNA segments encoding each of PDIJ 92 VH and VL were designed as a mini exons including a signal peptide, a splice donor signal, and appropriate restriction enzyme sites foi subsequent cloning into a mammalian expression vector.
• the 19.2.1 VII ami VL mini cxons were constructed by extension of overlapping synthetic oligonucleotides ranging m length from 33 to 43 bases and PCR amplification (Sternraor ct al, Gene 164:49-53 (1995)).
• PCR amplified fragments were purified by Qiaquick PCR purification kit (Qiagen), digested with appropriate iesiriction enzymes, cloned into a vector for expression containing human IgG 1 and kappa constant domains, and expressed in mouse NSO cells.
• TWLAK the Jigand for TwcakK activates multiple signaling pathways, resulting in pJeiotropjr effects on cells, including increasing the expression of cytokines and chemokines, stimulation of ang ⁇ ogenesis, and induction of apoptosis m cancel cells
• the antt TweakR antibodies were assessed for their abilities to exert biological activities attributed Io TWEAK using several in vitro assays designed to measure the effects of the anti TweakR antibodies on cell growth, stimulation of angiogenesis and expression of cytokines and chemokines.
• human umbilical vein endothelial cells (HUVEC), coronary artery smooth muscle cells, lung fibroblasts, or hepatocytes were incubated for 3 days in the presence of 100 ng/ml TWEAK or 10 ⁇ g/ml isotype control antibody or the anti-TweakR antibody, 19.2.1.
• Cell viability was assessed using alamar blue.
• HUVEC growth was stimulated by TWEAK or 19.2.1 (data not shown).
• TWEAK enhanced growth by 2.4- fold, while 19.2.1 stimulated a 1.6-fold increase in growth.
• Neither TWEAK nor 19.2.1 had any effect on growth of coronary artery smooth muscle cells, aortic smooth muscle cells, lung fibroblasts, or hepatocytes.
• Anti-TweakR antibodies were assessed for their abilities to promote the formation of endothelial tubes in vitro. HUVEC cells were incubated in a fibrin matrix in the presence of 200 ng/ml TWEAK, 10 ⁇ g/ml isotype control antibody or 10 ⁇ g/ml 19.2.1. After 6 days, the length of the tubes generated was quantified. Both TWEAK and 19.2.1 stimulated endothelial tube formation, with TWEAK showing an approximately 3.5-fold increase in total tube length, and 19.2.1 stimulating a 2.5 ⁇ fold increase (data not shown).
• Each cell type released a unique set of cytokines/chemokines in response to TWEAK.
• Some of the anti TweakR antibodies, such as 19.2,1 and PDl ,192 induced secretion of the same subset of cytokines/chemokjnes (e.g., IT, -8, II- 6, GM-CSF, MCP-I, RANTIiS 5 and IP 10) as TWEAK, but at lower concentrations.
• TWEAK, 19.2.1, and PDL192 did not stimulate the release of IFN ⁇ , TNF ⁇ , TNf ⁇ , IL -2, IL-4, 11 ,-5, IL-10, IL 12, or IL 13 from any cell type examined (data not shown).
• FIG. 8 depicts the release of IL-8 by ITEMl, ITEM2, ITEM3, and ITEM 4 (all used at 10 ⁇ g/ml), as well as various isotype controls. As illustrated in FIG.
• ITEM antibodies have agonist activity, in that they induce some IL-8 expression when incubated with A375 cells.
• all of the ITEM antibodies have some antagonist activity, in that when A375 cells are incubated with TWEAK ligand (300 ng/ml) plus the antibodies, the amount of IL-8 released is less than that observed with TWEAK treatment alone.
• TWEAK ligand 300 ng/ml
• TweakR-Fc soluble form of TweakR extracellular domain
• neutralizing anti-TWEAK antibody are shown for comparison.
• FIGS. 8B, 8C, 8D, and 8E Representative IL ⁇ 8 release assays for the anti-TweakR antibodies PDLl 92, 18.3.3, 136.1 and PDL 400 are shown in FIGS. 8B, 8C, 8D, and 8E, respectively.
• the agonist/antagonist activities differ between the antibodies described herein.
• PDLl 92 (FIG. 8B) and 19.2.1 (data not shown) have virtually no antagonistic activity, i.e., they do not block IL-8 release induced by TWEAK.
• PDL 400 (FIG. 8E) and 18.3.3 (FIG. 8C) have agonist and antagonist activities.
• the anti- TweakR antibody 136.1 has veiy high agoD ⁇ st activity, which precludes assessment of its antagonist activity in this assay.
• the anti-TweakR antibodies, PDLl 92, 19.2. ⁇ , and 136.1 were tested for the ability to decrease or inhibit the growth of the cancer cells by inducing apoptosis through caspasc activation.
• IFN ⁇ -treated HT29 colon cancers cells were incubated with titrations of the three antibodies. Within 1 -2 days of antibody treatment, the cells were assessed foi activation of caspase 3/7.
• PDL192 and 136.1 were capable of inducing apoptosis through caspase activation in HT29. 19.2.1 had a similar effect (data not shown).
• apoptosis does not appear to be the mechanism by which the anti-TweakR antibodies inhibits proliferation; in these cell lines, the antibodies are postulated to mediate activity through an alternate mechanism(s) that causes a reduction in proliferation in a longer-term 7-10 day growth assay (data not shown).
• Example 3 In vivo Tumor Activity of Anti-TweakR Antibodies [0181] Treatment with anti-TweakR antibodies in vivo was performed on xenograft models generated from a number of cancer cell lines. As shown in Table 2, anti-TweakR antibodies were effective in reducing and/or inhibiting the growth of a number of different cancers.
• Peiocnt inhibition was calculated by companng the a ⁇ eiage turaoi volume of control animals with tieated animals using measujcmcnts taken the day of saciifice of the contiol animal gjoup (when the majoiity of tumois weie at the allowable linut) 2 nt: not tested.
• Severe-combined immunodeficient (SCID) mice were inoculated subcutaneously with SN12C renal cancer cells. When the tumors reached approximately 100 mm 3 , the animals were dosed intraperitoneally with PDLl 92, PDL400, or 19.2.1, or an isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks. A dose-range finding study was also used to determine the range of efficacious dose levels with PDLl 92 in the SNl 2C model.
• mice were dosed with PDLl 92 at 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 1 mg/kg, 0.3 mg/kg, or 0.1 mg/kg, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.
• PDLl 92 causes tumor regression in the SNl 2C renal cancer xenograft model. Similar results were observed with PDL400 and 19.2.1 (data not shown). In the dose- range finding study, shown in FIG.l 1, tumor regression is observed with PDLl 92 at doses as low as 1 mg/kg. At a lower dose of 0.3 mg/kg, regression is not achieved, but PDLI 92 significant anti-tumor activity is observed.
• Severe -combined immunodeficient (SCID) mice were inoculated subcutaneousJy with A253 saliva) y gland cancer cells. When the tumors ieached approximately 100 mm , the animals were dosed intraperitoneally with 19.7. 1, PDL 192, 18.3.3, and PDL400, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks. Dose range finding studies in the A253 model of head and neck cancer were also used with J 9.2. i to determine the minimum concentration of Circulating antibody that shows biological activity (minimum effective serum conoentiation).
• Severe-combined jmmunodeficient (SCID) mice were inoculated subcutaneously with A253 salivaiy gland cancer cells. When the tumors reached approximately 100 mm 3 , the animals were dosed intraperitoneally with 19.2.1 at 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 1 mg/kg, or 0.5 mg/kg, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.
• SCID mice Severe-combined immunodeficient mice were inoculated subcutaneously with A253 salivary gland carcinoma cells. When the tumors reached approximately 100 mm 3 , the animals were dosed intraperitoneally with 19.2.1 at 5 mg/kg or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks (9 doses) or for 7 weeks (22 doses) (19.2.1 only).
• mice Severe combined immunodeficicnt (SCfD) mice were inoculated subcutaneous Iy with UT) 376 ij/inaiy bJaddei carcinoma ceils. V/hcn the tumors readied approximately 100 mm 3 , the animals were dosed inirapc ⁇ toneally with PDL192, 19.2 1 , and PDI 400, or jsotype control antibody at 10 mg/kg, 3 times per week for 7 doses.
• SCfD Severe combined immunodeficicnt mice were inoculated subcutaneous Iy with UT) 376 ij/inaiy bJaddei carcinoma ceils. V/hcn the tumors readied approximately 100 mm 3 , the animals were dosed inirapc ⁇ toneally with PDL192, 19.2 1 , and PDI 400, or jsotype control antibody at 10 mg/kg, 3 times per week for 7 doses.
• Severe- combined immunodeficient mice woe inoculated subcutaneously with ⁇ 375 melanoma cells.
• the animals were dosed intraperitoneally with PDLl 92, 19.2.1, and 18.3.3, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.
• a dose-range finding study was also used to determine the range of efficacious dose levels with PDL192 in the A375 model. In this study, animals were dosed with PDL192 at 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 1 mg/kg, 0.3 mg/kg, or 0.1 mg/kg, or isotype control antibody at 10 mg/kg, 3 times per week (3qw) for 3 weeks.
• FIG. 15 depicts the reduction in tumor growth following treatment with PDLl 92 in the dose-range finding study, with significant anti-tumor activity observed at all dose levels, even as low as 0.1 mg/kg.
• a separate dose-range finding study was conducted in the A375 xenograft model to con-elate the minimal and optimal biological activities of PDLl 92 with circulating antibody concentrations.
• Severe-combined immunodeficient (SCID) mice were inoculated subcutaneously with A375 melanoma cells. When the tumors reached approximately 100 mm 3 , the animals were dosed inlraperiloneally with PDL 192 at 5 mg/kg, 1 mg/kg, 0.6 mg/kg, 0.3 mg/kg, or 0.1 mg/kg, or isotype control antibody at 5 mg/kg, every three days (q3d) for a total of 8 doses. Animals were bled at various tiroes throughout the study to measure PDL 192 concentrations in the scram.
• Minimal -to- moderate anti- tumor activity was obsctved at the 0.1, 0.3, and 0.6 mg/kg dose levels (dala not shown), where trough serum concentrations of PDlJ 92 were 0.97 ng/ral, 4.1 ng/ml, and 27 ng/ml, respectively, and peak sejum concentrations were 0,087, I A, and 3 ⁇ g/ral, respectively.
• This dose range cbaracteiizcs the potential therapeutic window for anti TweakR antibodies
• mice Severe -combined immunodeficient (SCID) mice were inoculated subcutaneously with CSOC ovarian cancer carcinoma cells. When the tumors reached approximately 100 mm , the animals were dosed intraperitoneally with PDLl 92 or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks. [0195] In the CSOC ovarian cancer xenograft model, tumor growth was strongly inhibited by PDL 192. FIG. 16 depicts the reduction in tumor growth following treatment with PDLl 92.
• Severe-combined immunodeficient (SCID) mice were inoculated subcutaneously with Panel pancreatic cancer cells. When the tumors reached approximately 100 mm 3 , the animals were dosed intraperitoneally with PDLl 92 at 10 mg/kg, or with isotype control antibody at 10 mg/kg, or with PDL 192 at 10 mg/kg and gemcitabine at 60 mg/kg, or with isotype control antibody at 10 mg/kg and gemcitabine at 60 mg/kg. Animals received doses of antibody 3 times per week for 3 weeks and/or gemcitabine 2 times per week for 5 doses.
• FIG. 17 depicts the reduction in tumor growth following treatment with PDLl 92 ⁇ gemcitabine.
• Severe-combined immunodeficienl (SClD) mice were inoculated subcutaneously with HT29 colorectal cancer cells.
• the animals were dosed intraperitoneally with PDLl 92 at 10 mg/kg or 0.5 mg/kg, with isotype control antibody at 10 mg/kg, with cetoxirnab at 10 mg/kg, with irinotecan at 25 mg/kg and isotype contiol antibody at 10 mg/kg, with irinotecan at 25 mg/kg and cetuximab at 10 mg/kg, with irinotecan at 25 mg/kg and PDIJ 92 at 0.5 mg/kg, ⁇ vitb PDLl 92 at 0.5 mg/kg and cetuximab at 10 mg/kg, or with PDl, 192 ai 0.5 mg/kg and cetuxiroab at lOmg/kg and irinotecan at 25 mg/kg.
• FIG. 18A depicts the inhibition of tumor growth by PDIJ 92 at 10 mg/kg. At a lower dose of 0.5 mg/kg, PDLl 92 did ⁇ oi inhibit tumor growth alone, but did significantly enhance the anti tumor activity of irinotecan.
• Figure 18B depicts the reduction in tumor growth following treatment with PDL 192 A irinotecan.
• IT29 colorectal cancer cells are mutant for B-raf.
• cetuximab at 10 mg/kg showed no inhibition of tumor growth.
• combining cetuximab at 10 mg/kg with a low dose of PDL192 at 0.5 mg/kg resulted in statistically significant tumor growth inhibition.
• Figure 18C depicts the lack of anti-tumor activity of cetuximab alone in the HT29 xenograft model and the tumor growth inhibition of the cetuximab/PDL192 combination.
• mice Severe-combined immunodeficient (SCID) mice were inoculated orthotopically in the mammary fat pad with a variant of the MDA-MB-231 breast cancer cell line.
• the animals were dosed intraperitoneally with PDLl 92 or 19.2.1, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.
• PDL192 PDL 192
• animals were dosed with PDL 192 at 10 mg/kg, 3 mg/kg, 1 mg/kg, or 0.3 mg/kg, or isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.
• FIG. J 9 ⁇ depjcls the > eduction in primary tumor size following treatment with PDLl 92. Similar results were obtained using 19.2.1 (data not shown).
• the MDA MB 23 J variant model exhibits metastatic growth in ihe lungs of tumor bearing mice. Quantification of the lung metastases revealed thai PDL192 significantly reduced the establishment and growth of lung metastases in treated mice.
• FIG. 19B depicts the inhibition of lung metastases. Metastatic growth was more sensitive to PDL 192 treatment than was the growth of primary tumors.
• the in vivo anti-tumor activities of the anti Tweakll antibodies is likely due, in part, to the ability of the antibodies to stimulate anti body-dependent cellular cytotoxicity (ADCC) through the Fc portion of the antibody rnoiecuJe.
• ADCC body- dependent cellular cytotoxicity
• the antibodies were incubated in vitro with: 1) TweakR transfectant cells in the presence of healthy human peripheral blood mononuclear cells (PBMCs), 2) cancer cell lines in the presence of PBMCs, 3) TweakR transfectant cells in the presence of mouse splenocytes, or 4) cancer cell lines in the presence of mouse splenocytes.
• PBMCs peripheral blood mononuclear cells
• a TweakR transfectant cell line was labeled with 51 Cr and incubated with human PBMCs at a 50: 1 ratio of effector cells:target cells in the presence of a titration of PDLl 92, 19.2.1, PDL400, and 18.3.3, or isotype-control antibodies. ADCC activity was observed with all four antibodies on the TweakR transfectant with human PBMCs.
• FIG. 2OA depicts a typical experiment using PDL 192. Similar results were obtained with the other three antibodies (data not shown).
• the SN12C renal cancer cell line was labeled with Cr and incubated with human PBMCs at a 50: 1 ratio of effector cells: target cells in the presence of a titration of PDLl 92 and PDL400, or isotype-control antibodies. ADCC activity was observed with both antibodies against SN12C cells with human PBMCs.
• FIG. 2OB depicts a typical experiment using PDLl 92. Similar results were obtained with PDL400 (data not shown).
• TweakR transfectant cell line was labeled with ' 1 Cr and incubated with mouse splenocytes at a 50: 1 ratio of effector cells: target cells in tbc presence of a titration of PDL192, 19.2.1 , PDJ /400, 136, 1 and 18.3.3, or isotype control antibodies.
• ADCC activity was observed with all five antibodies on the TweakR Irarisfeclani with mouse splenocytes.
• FlG. 2OC depicts a typical experiment using PDL 192. Similar iosulls were obtained with the other four antibodies (data not shown).
• the Fc region of 19.2.1 was changed from a murine IgG2a to murine IgGl , generating 19.2. IxGl .
• 19.2.1 arid 19.2.IxGl exhibited similar binding to TweakR -expressing cells, and both antibodies killed HT29 colon cancer cells in a dose- dcpcndent manner.
• 19.2.1 potently induced ADCC on a TweakR transfectant 19.2. IxGl exhibited weak ADCC activity.
• Severe combined immunodeficient mice were inoculated subcutaneously with SN 12C renal cancer cells or A375 melanoma cells. When the tumors reached approximately 100 mm , the animals were dosed intraperitoneally with 19.2.1, 19.2.IxGl, or a murine IgGl isotype control antibody at 10 mg/kg, 3 times per week for 3 weeks.

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