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WO2003042367A2 - Anticorps se liant specifiquement au niveau immunitaire a des recepteurs trail - Google Patents

Anticorps se liant specifiquement au niveau immunitaire a des recepteurs trail Download PDF

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Publication number
WO2003042367A2
WO2003042367A2 PCT/US2002/036431 US0236431W WO03042367A2 WO 2003042367 A2 WO2003042367 A2 WO 2003042367A2 US 0236431 W US0236431 W US 0236431W WO 03042367 A2 WO03042367 A2 WO 03042367A2
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Prior art keywords
antibody
replaced
polypeptide
antibodies
amino acid
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PCT/US2002/036431
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English (en)
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WO2003042367A3 (fr
Inventor
Theodora Salcedo
Victor Roschke
Steven M. Ruben
Craig A. Rosen
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Human Genome Sciences, Inc.
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Priority to AU2002352676A priority Critical patent/AU2002352676A1/en
Publication of WO2003042367A2 publication Critical patent/WO2003042367A2/fr
Publication of WO2003042367A3 publication Critical patent/WO2003042367A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • the present invention relates to antibodies and related molecules that immunospecifically bind to TRAIL receptors. Such antibodies, have uses, for example, in the prevention and treatment of cancers and other proliferative disorders.
  • the invention also relates to nucleic acid molecules encoding anti-TRAIL receptor antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same.
  • the present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder, especially cancer and other hyperproliferative disorders, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TRAIL receptor.
  • TNF tumor necrosis factors
  • TNF-ligand superfamily
  • TNF-receptor cytokines which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease.
  • the TNF molecules belong to the "TNF-ligand” superfamily, and act together with their receptors or counter- ligands, the "TNF-receptor” superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.
  • TNF- lymphotoxin- ⁇
  • LT- lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • FasL CD40L
  • CD27L CD30L
  • 4-1BBL 4-1BBL
  • OX40L nerve growth factor
  • NGF nerve growth factor
  • the superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-lBB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).
  • Many members of the TNF-ligand superfamily are expressed by activated T- cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).
  • TNF and LT- ⁇ are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors).
  • TNF and LT- ⁇ are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel, C, Science 264:661-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection.
  • Apoptosis or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H.
  • Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science 267, 1456-1462 (1995)).
  • Fas/APO-1 and TNFR-1 Two cell surface death receptors, Fas/APO-1 and TNFR-1 (J.L. Cleveland, et ah, Cell 81, 479-482 (1995); A. Fraser, et ah, Cell 85, 781-784 (1996); S. Nagata, et ah, Science 267, 1449-56 (1995)).
  • Both are members of the TNF receptor family which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (C.A. Smith, et ah, Science 248, 1019-23 (1990); M. Tewari, et ah, in Modular Texts in Molecular and Cell Biology M.
  • Fas/ APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the "death domain", which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et ah, Cell 81, 185-6 (1995); K. White et ah, Science 264, 677-83 (1994)).
  • This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified.
  • Fas/ APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A.M. Chinnaiyan, et ah, Cell 81, 505-12 (1995); M. P. Boldin, et ah, J. Biol Chem 270, 7795-8 (1995); F.C. Kischkel, et ah, EMBO 14, 5579-5588 (1995)), which in turn binds and presumably activates FLICE/MACHl, a member of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio et ah, Cell 85, 817-827 (1996); M.P.
  • TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L.A. Tartaglia, et ah, Immunol Today 13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu, et ah, Cell 81, 495-504 (1995); H. Hsu, et ah, Cell 84, 299-308 (1996)).
  • TRADD can signal both apoptosis and NF-kB activation (H. Hsu, et ah, Cell 84, 299-308 (1996); H. Hsu, et ah, Immunity 4, 387- 396 (1996)).
  • Apoptosis Inducing Molecule I Apoptosis Inducing Molecule I
  • TRAIL TNF-related apoptosis-inducing ligand or
  • TRAIL acts independently from FAS ligand (Wiley, S.R., et al. (1995)), supra). Studies by Marsters, S.A. et al., have indicated that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signalling by FAS/Apo-IL but much faster than TNF-induced apoptosis (Current Biology, 6:150-152 (1996)).
  • TRAIL receptor 1 TRAIL receptor 1
  • TR4 death receptor 4
  • DR4 death receptor 4
  • TRAIL receptor 2 also referred to as TR7, DR5, and KILLER, Pan et al, Science 277:815-8 (1997), Sheridan et al., Science 277:818-21 (1997), Chaudhury et al., Immunity 7:821-30 (1997), International Patent Application Nos.
  • WO 98/46643 WO 99/09165, WO 99/11791, WO 98/41629, WO00/66156, and WO 98/35986, SEQ ID NO:3); TRAIL receptor 3 (TRAIL-R3, also referred to as TR5, decoy receptor 1 (DcRl) and TRID) (Degli-Esposti et al., J. Exp. Med. 186:1165-70 (1997), International Patent Application Nos.
  • WO98/30693 WO0071150, WO 99/00423, EP867509, WO 98/58062, SEQ ID NO:2); and TRAIL Receptor 4 (TRAIL-R4, also referred to as TRIO, DcR2, and TRUNDD, Pan et al., FEBS Lett. 424:41-5 (1998), Degli-Eposti et al., Immunity 7:813-20 (1997), International Patent Application Nos. WO 98/54202, WO00/73321, WO 2000/08155, WO 99/03992, WO 2000/34355 and WO9910484, SEQ ID NO:4).
  • TRAIL receptors 1 and 2 contain death domains in their cytoplasmic tails and the triggering of these receptors results in apoptosis.
  • TRAIL receptor 3 and 4 inhibit apoptosis induced by the cytotoxic ligand TRAIL in part because of their absent or truncated cytoplasmic death domains, respectively.
  • TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system.
  • compositions such as antibodies
  • TNF receptors both normally and in disease states.
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TRAIL receptor polypeptide or polypeptide fragment or variant of a TRAIL receptor.
  • the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment or variant of human TRAIL receptors such as those of SEQ ID NOS: 1-4.
  • the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment or variant of human TR4 such as that of SEQ ID NO:l.
  • an antibody of the invention that immunospecifically bind to a TR4 polypeptide also immunospecifically bind TR7 (e.g., SEQ ID NO:3), but not other proteins, including (TR5, and TRIO (SEQ ID NOS:2 and 4.)
  • the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to a TRAIL receptor or a fragment or variant thereof.
  • the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with TRAIL receptor function or TRAIL receptor ligand function or aberrant TRAIL receptor or TRAIL receptor ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to a TRAIL receptor or a fragment or variant thereof.
  • the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma).
  • diseases and disorders which can be treated, prevented or ameliorated with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes viral infections, and other viral infections) and proliferative disorders.
  • neurodegenerative disorders e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease
  • immune disorders e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome
  • inflammatory disorders e.g., asthma, allergic disorders,
  • the present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TRAIL receptor or a fragment or variant thereof.
  • the present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with TRAIL receptor function or TRAIL receptor ligand function or aberrant TRAIL receptor or TRAIL receptor ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TRAIL receptor or a fragment or variant thereof.
  • the present invention relates to antibody-based methods and compositions for detecting, diagnosing, or prognosing cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma).
  • diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes viral infections, and other viral infections), and proliferative disorders.
  • neurodegenerative disorders e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease
  • immune disorders e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome
  • inflammatory disorders e.g., asthma, allergic disorders
  • Another embodiment of the present invention includes the use of the antibodies of the invention as a diagnostic tool to monitor the expression of TRAIL receptor expression on cells.
  • the present inventors have generated hybridoma cell lines that express antibodies that immunospecifically bind one or more TRAIL receptor polypeptides (e.g., SEQ ID NOs:l-4).
  • the invention encompases these cell lines, listed in Table 1 below which were deposited with the American Type Culture Collection ("ATCC") on the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1
  • ATCC American Type Culture Collection
  • the ATCC is located at 10801 University Boulevard, Manassas, NA 20110-2209, USA.
  • the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • the present invention encompasses the polynucleotides encoding the antibodies expressed by these cell lines, as well as the amino acid sequences encoding the antibodies expressed by these cell lines.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., heavy chains, NH domains, VH CDRs, light chains, NL domains, or NL CDRs having an amino acid sequence of any one of those expressed by one or more cell lines referred to in Table 1), that immunospecifically bind to one or more TRAIL receptors or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules.
  • the present invention encompasses antibodies, or fragments or variants thereof, that bind to the extracellular regions/domains of one or more TRAIL receptors or fragments and variants thereof.
  • the present invention also provides antibodies that bind one or more TRAIL receptor polypeptides which are coupled to a detectable label, such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.
  • the present invention also provides antibodies that bind one or more TRAIL receptor polypeptides which are coupled to a therapeutic or cytotoxic agent.
  • the present invention also provides antibodies that bind one or more TRAIL receptor polypeptides which are coupled to a radioactive material.
  • the present invention also provides antibodies that bind one or more TRAIL receptor polypeptides that act as either TRAIL receptor agonists or TRAIL receptor antagonists.
  • the antibodies of the invention stimulate apoptosis of TRAIL receptor expressing cells.
  • the antibodies of the invention inhibit TRAIL binding to a TRAIL receptor.
  • the antibodies of the invention upregulate TRAIL receptor expression.
  • the present invention also provides antibodies that inhibit apoptosis of TRAIL receptor expressing cells.
  • the antibodies of the invention downregulate TRAIL receptor expression.
  • the antibodies of the invention have a dissociation constant (K D ) of 10 "7 M or less. In preferred embodiments, the antibodies of the invention have a dissociation constant (K D ) of 10 "9 M or less.
  • the present invention further provides antibodies that stimulate apoptosis of TRAIL receptor expressing cells better than an equal concentration of TRAIL polypeptide stimulates apoptosis of TRAIL receptor expressing cells.
  • the present invention further provides antibodies that stimulate apoptosis of TRAIL receptor expressing cells equally well in the presence or absence of antibody cross-linking reagents; and/or stimulate apoptosis with equal or greater potency as an equal concentration of TRAIL in the absence of a cross-linking antibody or other cross- linking agent.
  • antibodies of the invention have an off rate (k o ) of 10 " 3 /sec or less. In preferred embodiments, antibodies of the invention have an off rate (k o f ) of 10 "4 /sec or less. In other preferred embodiments, antibodies of the invention have an off rate (k 0ff ) of 10 " /sec or less.
  • the present invention also provides for antibodies that preferentially bind one or more of the TRAIL receptors selected from the group of TR4, TR5, TR7, and TRIO. [0028] In certain embodiments, properties of the antibodies of the present invention, as detailed in the Examples below, make the antibodies better therapeutic agents than previously described TRAIL receptor binding antibodies.
  • the present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs).
  • antibodies including molecules comprising, or alternatively consisting of, antibody fragments or variants
  • the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs).
  • the present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab') 2 fragments, Fd fragments, disulfide- linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)).
  • the present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof).
  • a composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof.
  • a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.
  • the present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
  • a composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.
  • a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.
  • the present invention also provides for a nucleic acid molecule(s), generally isolated, encoding an antibody (including molecules, such as scFvs, NH domains, or NL domains, that comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention.
  • the present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof.
  • the present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention.
  • the present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule.
  • FIG. 1 Flow cytometric staining of Hela, SW480 and HT1080 cells for TR4 (TRAEL-R1) expression using monoclonal antibody 7.3. Cells were incubated with 1 microgram/ml monoclonal antibody 7.3 for 45 minutes, washed and stained with anti- human IgG2-FITC detector antibody. Reactivity of the 7.3 monoclonal antibody with the cells is shown in the histogram with the dark line; isotype control staining is shown in the shaded histogram.
  • Figure 2 Sensitivity of HeLa cells to killing mediated by TRAIL (A), monoclonal antibody 7.3 (B) or monoclonal antibody 7.12 (C).
  • Sensitivity of HeLa cells to anti-TRAIL receptor monoclonal antibodies was tested in the presence of cycloheximide either with or without an equivalent amount of secondary goat anti-human Ig Fc specific antibody.
  • Use of an equivalent amount of secondary goat anti-human Ig Fc specific antibody means that the secondary goat anti-human Ig Fc specific antibody concentration was equal to the concentration of the test antibodies, 7.3 and 7.12.
  • FIG. 3 Sensitivity of SW480 cells to killing mediated by TRAIL (A), monoclonal antibody 7.3 (B) monoclonal antibody 7.12 (C). Sensitivity of SW480 cells to monoclonal antibodies was tested in the presence of cycloheximide either with or without an equivalent amount of secondary goat anti-human Ig Fc specific antibody.
  • FIG. 4 Sensitivity of HT1080 cells to killing mediated by TRAIL (A), monoclonal antibody 7.3 (B) or monoclonal antibody 7.12 (C). Sensitivity of HT1080 cells to monoclonal antibodies was tested in the presence of cycloheximide either with or without an equivalent amount of secondary goat anti-human Ig Fc specific antibody.
  • FIG. 5 Sensitivity of HeLa, SW480 and HT1080 cells to killing mediated by anti- monoclonal antibody 7.12. Sensitivity of cells to monoclonal antibodies were tested in the absence of either cycloheximide or additional crosslinking with a secondary goat anti-human Ig Fc specific antibody.
  • Figure 6 Sensitivity of HeLa and SW480 to TRAIL-Receptor mediated killing mediated induced by monoclonal antibody 7.12 in the presence of TOPOTECAN. A comparison is shown for sensitization of cells to TRAIL-R1 monoclonal antibody killing using either cycloheximide or topotecan.
  • Figure 7 Sensitivity of SW480 cells to killing mediated by anti-TRAIL receptor monoclonal antibodies 7.3, 7.3.1, 7.3.2 or 7.3.3
  • A monoclonal antibodies 7.12, 7.12.1, 7.12.2, or 7.12.3
  • B monoclonal antibodies 7.10, 7.10.1, 7.10.2, or 7.10.3, or monoclonal antibodies 7.1.3, 7.2, 7.8, 8.3.1, or 8.3.2.
  • Sensitivity of SW480 cells to monoclonal antibodies was tested in the presence of cycloheximide
  • Figure 8 Effect of 7.12.2 treatment on tumor growth in Swiss nu/nu mice.
  • Figure 9 Effect of 7.12.2 treatment on tumor growth in Swiss nu/nu mice-II.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments.
  • molecules which are described by the term “antibody” herein include, but are not limited to: single chain Fvs (scFvs),
  • Fab fragments fragments, Fab' fragments, F(ab') 2 , disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • the term "single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody.
  • Antibodies that immunospecifically bind to a TRAIL receptor may have cross-reactivity with other antigens, e.g., another TRAIL
  • antibodies that immunospecifically bind to a TRAIL receptor do not cross-react with other antigens (e.g., other TRAIL receptors or other members of the
  • Antibodies that immunospecifically bind to a TRAIL receptor can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.
  • Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments,
  • immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG ls
  • an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof.
  • the immunoglobulin is an IgGl isotype.
  • the immunoglobulin is an IgG4 isotype.
  • Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
  • Antibodies of the invention may also include multimeric forms of antibodies.
  • antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab') 2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent.
  • Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers.
  • individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art.
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-l-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, BL)) can be used to form antibody multimers.
  • antibody homodimers can be converted to Fab' 2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.
  • antibodies can be made to multimerize through recombinant D ⁇ A techniques.
  • IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide.
  • Non-IgA or non-IgM molecules such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules, (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31.
  • ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety.
  • Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.
  • specific binding or immunospecifc binding by an anti-TR4 antibody means that the anti-TR4 antibody binds TR4 but does not significantly bind to (i.e., cross react with) proteins other than TR4, such as other proteins in the same family of proteins).
  • An antibody that binds TR4 protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the TR4-specific antibody of the invention preferentially binds TR4 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment.
  • An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein).
  • TR4-specific antibodies bind to epitopes of TR4
  • an antibody that specifically binds TR4 may or may not bind fragments of TR4 and/or variants of TR4 (e.g., proteins that are at least 90% identical to TR4) depending on the presence or absence of the epitope bound by a given TR4-specific antibody in the TR4 fragment or variant.
  • TR4-specific antibodies of the invention may bind species orthologues of TR4 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue.
  • TR4-specific antibodies of the invention may bind modified forms of TR4, for example, TR4 fusion proteins.
  • the antibody when antibodies of the invention bind TR4 fusion proteins, the antibody must make binding contact with the TR4 moiety of the fusion protein in order for the binding to be specific.
  • Antibodies that specifically bind to TR4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below. [0046] In some embodiments the present invention encompasses antibodies that immunospecifcally or specifically bind both TR4 and TR7.
  • Specific binding or immunospecifc binding by an antibody that immunospecifically binds TR4 and TR7 means that the antibody binds TR4 and TR7 but does not significantly bind to (i.e., cross react with) proteins other than TR4 or TR7, such as other proteins in the same family of proteins).
  • an antibody that binds TR4 and TR7 proteins and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the antibody that immunospcifically or specifically binds both TR4 and TR7 preferentially binds TR4 and TR7 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope.
  • An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein).
  • an antibody that specifically binds TR4 and TR7 may or may not bind fragments of TR4, TR7 and/or variants of TR4 or TR7 (e.g., proteins that are at least 90% identical to TR4 or TR7, respectively) depending on the presence or absence of the epitope bound by a given antibody in the TR4 or TR7 fragment or variant.
  • antibodies of the invention that immunospecifically bind TR4 and TR7 may bind species orthologues of TR4 and/or TR7 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologues.
  • antibodies of the invention that immunospecifically bind TR4 and TR7 may bind modified forms of TR4 or TR7, for example, TR4 or TR7 fusion proteins. In such a case when antibodies of the invention bind fusion proteins, the antibody must make binding contact with the TR4 or TR7 moiety of the fusion protein in order for the binding to be specific.
  • Antibodies that specifically bind to TR4 or TR7 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.
  • variant refers to a polypeptide that possesses a similar or identical function as a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti-TRAIL receptor antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti-TRAIL receptor antibody or antibody fragment thereof, or possess a similar or identical structure of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti- TRAIL receptor antibody or antibody fragment thereof.
  • a variant having a similar amino acid refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a TRAIL receptor polypeptide, a fragment of , an anti-TRAIL receptor antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or NLCDR having an amino acid sequence of any one of those expressed by one or more cell lines referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding a TRAIL receptor polypeptid
  • a polypeptide with similar structure to a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti-TRAIL receptor antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an anti-TRAIL receptor antibody, or antibody fragment thereof, described herein.
  • the structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art.
  • An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Nath Acad. Sci. USA 87:2264-2268(1990), modified as in Karlin and Altschul Proc. Nath Acad. Sci. USA 90:5873-5877(1993).
  • the BLASTn and BLASTx programs of Altschul, et al. J. Moh Bioh 215:403-410(1990) have incorporated such an alogrithm.
  • Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402(1997).
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • derivative refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an antibody of the invention that immunospecifically binds to a TRAIL receptor polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
  • derivative as used herein also refers to a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, an antibody that immunospecifically binds to a TRAIL receptor polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide.
  • a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti-TRAIL receptor antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • a derivative of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti- TRAIL receptor antibody may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti- TRAIL receptor antibody, may contain one or more non-classical amino acids.
  • a polypeptide derivative possesses a similar or identical function as a TRAIL receptor polypeptide, a fragment of a TRAIL receptor polypeptide, or an anti-TRAIL receptor antibody, described herein.
  • epitopes refers to portions of TRAIL receptor having antigenic or immunogenic activity in an animal, preferably a mammal.
  • An epitope having immunogenic activity is a portion of TRAIL receptor that elicits an antibody response in an animal.
  • An eptiope having antigenic activity is a portion of TRAIL receptor to which an antibody immunospecifically binds as determined by any method known in the art, for example, by the immunoassays described herein.
  • Antigenic epitopes need not necessarily be immunogenic.
  • fragment refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of a TRAIL receptor, or an anti-TRAIL receptor antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to TRAIL receptor.
  • an anti-TRAIL receptor antibody including molecules such
  • fusion protein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-TRAIL receptor antibody of the invention and an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain).
  • the term "host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • isolated antibody is intended an antibody removed from its native environment. Thus, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the heavy and the ligt chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Moh Bioh 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547 1553 (1992).
  • bispecific antibodies may be formed as "diabodies” (Holliger et al. "'Diabodies': small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448 (1993)) or "Janusins" (Traunecker et al.
  • bispecific single chain molecules target cytotoxic lymphocytes on HTV infected cells
  • EMBO J 10:3655-3659 (1991) and Traunecker et al. "Janusin: new molecular design for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992)).
  • Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies.
  • Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).
  • the present invention is directed to fully human antibodies, generally isolated, that immunospecifically bind one or more TRAIL receptor polypeptides.
  • XenoMouse lines of mice from Abgenix, Inc. (Fremont, CA) expressing human antibodies were immunized with TRAIL receptor polypeptides, lymphatic cells (such as B-cells) were recovered from the mice that had high titers of anti-TRAIL receptor antibodies, and such recovered cells were fused with a myeloid-type cell line to prepare immortal hybridoma cell lines.
  • Hybridoma cell lines were screened to select and identify hybridoma cell lines that produced antibodies specific to the immunogen.
  • the IgG2 expressing strain was used to make the cell lines and antibodies of the present invention, thus each of the antibodies produced by cell lines are fully human IgG2 heavy chains with human kappa light chains.
  • These hybridoma cell lines were deposited with the American Type Culture Collection ("ATCC") on the date listed in Table 1, and given ATCC Deposit Numbers listed in Table 1. The ATCC is located at 10801 University Boulevard, Manassas, NA 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • hybridoma cell lines that have a numeric designation that contains one period indicates a primary hybridoma isolate.
  • the number preceding the period indicates the fusion panel a hybridoma came from and the number after the period designates the primary hybridoma isolate number.
  • primary hybridoma isolate is meant a hybridoma obtained by fusing spleen cells of immunized mice with a fusion partner, plating the cells at limiting dilution in 96 well plates, and selecting a hybridoma cell line, that by visual inspection appeared to have only a single colony, i.e., that appeared to have grown up from a single cell.
  • hybridoma cell line is most likely a monoclonal cell line.
  • Primary hybridoma isolates were also subcloned. In a subcloning procedure, cells corresponding to a single primary hybridoma isolate are plated out at limiting dilution, and hybridoma sublones, that by visual inspection appeared to have only a single colony, i.e., that appeared to have grown up from a single cell, are selected. In this application, hybridoma subclones have designations containing two periods.
  • the number preceding the first period indicates which fusion panel a hybridoma came from; the number immediately after the first period designates the primary hybridoma isolate number; and the number following the second period indicates the number of a particular subclone derived from the primary hybridoma isolate with the designation indicated by the number immediately following the first period.
  • Subcloned cell lines are monoclonal and are typically more stable with respect to antibody expression.
  • hybridoma cell lines deposited at the American Type Culture Collection contain equal proportions of three sublones of a particular hybridoma isolate.
  • Hybridomas 7.3.1, 7.3.2, and 7.3.3 were collectively deposited at the ATCC on November 16, 2000 and given ATCC Deposit Number PTA-2687.
  • Hybridomas 7.8.1, 7.8.2, and 7.8.3 were collectively deposited at the ATCC on November 27, 2000 and given ATCC Deposit Number PTA-2730.
  • Hybridomas 7.10.1, 7.10.2, and 7.10.3 were collectively deposited at the ATCC on November 27, 2000 and given ATCC Deposit Number PTA-2729.
  • Hybridomas 7.12.1, 7.12.2, and 7.12.3 were collectively deposited at the ATCC on November 27, 2000 and given ATCC Deposit Number PTA-2728.
  • Hybridomas 8.3.1 and 8.3.2 were collectively deposited at the ATCC on November 27, 2000 and given ATCC Deposit Number PTA-2731.
  • the ATCC is located at 10801 University Boulevard, Manassas, VA 20110- 2209, USA. Each of the ATCC deposits described herein was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The ATCC Deposit Numbers and the hybridoma designations are also presented in Table 1.
  • an antibody expressed by one (or two) of the sublcones has a property that is distinct from the remaining sublone(s) of a given primary hybridoma isolate
  • a cell line expressing the antibody with that property can be retrieved from the pooled ATCC deposit using methods that are routine in the art. Briefly, retrieval of such a clone would require plating cells of the ATCC deposit at limiting dilution, growing the cells in culture, selecting monoclonal cell lines, and testing the antibodies expressed by the monoclonal cell lines for the presence or absence of the property using methods that are routine in the art.
  • reference to an antibody herein by the primary hybridoma designation references not only to the primary hybridoma isolate but also each of its subclones deposited at the ATCC.
  • hybridoma cell line 7.3 references hybridoma cell lines 7.3, 7.3.1, 7.3.2, and 7.3.3.
  • the only exceptions to this policy are the recitation of hybridoma designations in the Figures, Figure Legends, Examples, and Claims.
  • reference to a particular hybridoma designation references only the hybridoma defined by that designation.
  • the antibody secreted by a hybridoma cell lines has the same designation as the cell line itself.
  • the term 7.3 may also reference the antibody expressed by hybridoma cell lines 7.3, 7.3.1, 7.3.2, and 7.3.3.
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TRAIL receptor polypeptide or a fragment, variant, or fusion protein thereof.
  • a TRAIL receptor polypeptide includes, but is not limited to, TR4 (SEQ ID NO:l) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in ATCC Deposit 97853 deposited Jan 21, 1997; TR5 (SEQ ID NO:2) or the polypeptide encoded by the cDNA in clone HPRCB54 contained in ATCC Deposit 97798 deposited Nov.
  • TR7 SEQ ID NO:3 or the polypeptide encoded by the cDNA in clone HLYBX88 contained in ATCC Deposit 97920 deposited Mar. 7, 1997, and/or TRIO (SEQ [0068] If an antibody expressed by one (or two) of the sublcones has a property that is distinct from the remaining sublone(s) of a given primary hybridoma isolate, a cell line expressing the antibody with that property can be retrieved from the pooled ATCC deposit using methods that are routine in the art.
  • retrieval of such a clone would require plating cells of the ATCC deposit at limiting dilution, growing the cells in culture, selecting monoclonal cell lines, and testing the antibodies expressed by the monoclonal cell lines for the presence or absence of the property using methods that are routine in the art.
  • one of the subclones expressed an antibody with a superior affinity for a TRAIL receptor polypeptide of the invention one would test the affinities of the antibodies expressed by the monoclonal cell lines derived from the ATCC deposit.
  • a monoclonal cell line that expressed an antibody with an affinity matching or resembling the desired affinity would be equivalent to the specific subclone from the ATCC deposit sought after.
  • reference to an antibody herein by the primary hybridoma designation references not only to the primary hybridoma isolate but also each of its subclones deposited at the ATCC.
  • hybridoma cell line 7.3 references hybridoma cell lines 7.3, 7.3.1, 7.3.2, and 7.3.3.
  • the only exceptions to this policy are the recitation of hybridoma designations in the Figures, Figure Legends, Examples, and Claims.
  • reference to a particular hybridoma designation references only the hybridoma defined by that designation.
  • the antibody secreted by a hybridoma cell lines has the same designation as the cell line itself.
  • the term 7.3 may also reference the antibody expressed by hybridoma cell lines 7.3, 7.3.1, 7.3.2, and 7.3.3.
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TRAIL receptor polypeptide or a fragment, variant, or fusion protein thereof.
  • a TRAIL receptor polypeptide includes, but is not limited to, TR4 (SEQ ID NO:l) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in ATCC Deposit 97853 deposited Jan 21, 1997; TR5 (SEQ ID NO:2) or the polypeptide encoded by the cDNA in clone HPRCB54 contained in ATCC Deposit 97798 deposited Nov. 20, 1996; TR7 (SEQ ID NO:3) or the polypeptide encoded by the cDNA in clone HLYBX88 contained in ATCC Deposit 97920 deposited Mar. 7, 1997, and or TRIO (SEQ ID NO:l) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in ATCC Deposit 97853 deposited
  • the antibodies of the present invention bind TR4 polypeptide, or fragments or variants thereof.
  • TR4 polypeptides, fragments and variants that may be bound by the antibodies of the invention in more detail.
  • the TR4 polypeptides, fragments and variants which may be bound by the antibodies of the invention are also described in International Publication Numbers, for example, WO98/32856 and WO00/67793 which are herein incorporated by reference in their entireties.
  • the antibodies of the present invention immunospecifically bind TR4 polypeptide.
  • An antibody that immunospecifically binds TR4 may, in some embodiments, bind fragments, variants (including species orthologs of TR4), multimers or modified forms of TR4.
  • an antibody immunospecific for TR4 may bind the TR4 moiety of a fusion protein comprising all or a portion of TR4.
  • TR4 proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to antibodies that bind TR4 proteins found as monomers or as part of multimers.
  • antibodies of the invention bind TR4 monomers, dimers, trimers or tetramers. In additional embodiments, antibodies of the invention bind at least dimers, at least trimers, or at least tetramers containing one or more TR4 polypeptides.
  • Antibodies of the invention may bind TR4 homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only TR4 proteins of the invention (including TR4 fragments, variants, and fusion proteins, as described herein). These homomers may contain TR4 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR4 proteins having an identical polypeptide sequence.
  • antibodies of the invention bind TR4 homomers containing TR4 proteins having different polypeptide sequences.
  • antibodies of the invention bind a TR4 homodimer (e.g., containing TR4 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR4 proteins having identical or different polypeptide sequences).
  • antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer of TR4.
  • heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR4 gene) in addition to the TR4 proteins of the invention.
  • antibodies of the invention bind a heterodimer, a heterotrimer, or a heterotetramer.
  • the antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer containing one or more TR4 polypeptides.
  • Multimers bound by one or more antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers bound by one or more antibodies of the invention such as, for example, homodimers or homotrimers
  • heteromultimers bound by one or more antibodies of the invention such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the TR4 polypeptides (including antibodies to the heterologous polypeptide sequence in a fusion protein) in solution.
  • multimers bound by one or more antibodies of the invention are formed by covalent associations with and/or between the TR4 proteins of the invention.
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:l or the polypeptide encoded by the deposited cDNA clone of ATCC Deposit 97853).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR4 fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a TR4-Fc fusion protein (as described herein).
  • covalent associations of fusion proteins are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No.
  • the multimers that may be bound by one or more antibodies of the invention may be generated using chemical techniques known in the art.
  • proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers that may be bound by one or more antibodies of the invention may be generated using techniques known in the art to form one or more inter- molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • proteins that may be bound by one or more antibodies of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer that may be bound by one or more antibodies of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers that may be bound by one or more antibodies of the invention may be generated using genetic engineering techniques known in the art.
  • proteins contained in multimers that may be bound by one or more antibodies of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer that may be bound by one or more antibodies of the invention are generated by ligating a polynucleotide sequence encoding a TR4 polypeptide to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • TR4 polypeptides which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • two or more TR4 polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR4 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • antibodies of the invention bind proteins comprising multiple TR4 polypeptides separated by peptide linkers.
  • Another method for preparing multimer TR4 polypeptides involves use of TR4 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
  • Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins.
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric TR4 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble TR4 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR4 is recovered from the culture supernatant using techniques known in the art.
  • antibodies of the invention bind TR4-leucine zipper fusion protein monomers and/or TR4-leucine zipper fusion protein multimers.
  • trimeric TR4 may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
  • antibodies of the invention bind TR4-leucine zipper fusion protein trimers.
  • antibodies of the invention bind TR4- fusion protein monomers and/or TR4 fusion protein trimers.
  • Antibodies of the invention that bind TR4 polypeptides may bind them in as isolated polypeptides, in their naturally occurring state and/or their native conformation.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • Also intended as an "isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • antibodies of the present invention may bind recombinantly produced TR4 polypeptides.
  • Antibodies of the present invention may also bind TR4 expressed on the surface of a cell, wherein said TR4 polypeptide is encoded by a polynucleotide encoding amino acids 1 to 468 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide.
  • Antibodies of the present invention may bind TR4 polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:l, encoded by the cDNA contained in ATCC deposit Number 97853, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in ATCC deposit Number 97853, or the complementary strand thereto.
  • Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Antibodies of the present invention may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 23, 24 to 43, 44 to 63, 64 to 83, 84 to 103, 104 to 123, 124 to 143, 144 to 163, 164 to 183, 184 to 203, 204 to 223, 224 to 238, 239 to 264, 265 to 284, 285 to 304, 305 to 324, 325 to 345, 346 to 366, 367 to 387, 388 to 418, 419 to 439, and/or 440 to 468 of SEQ ID NO:l.
  • polypeptide fragments bound by the antibodies of the invention can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.
  • about includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • antibodies of the present invention bind polypeptide fragments selected from the group: a polypeptide comprising or alternatively, consisting of, the TR4 receptor extracellular domain (predicted to constitute amino acid residues from about 24 to about 238 in SEQ ID NO:l); a polypeptide comprising or alternatively, consisting of, both TR4 cysteine rich domains (both of which may be found in the protein fragment consisting of amino acid residues from about 131 to about 229 in SEQ ID NO:l); a polypeptide comprising or alternatively, consisting of, the TR4 cysteine rich domain consisting of amino acid residues from about 131 to about 183 in SEQ ID NO:l); a polypeptide comprising or alternatively, consisting of, the TR4 cysteine rich domain consisting of amino acid residues from about 184 to about 229 in SEQ ID NO:l); a polypeptide comprising or alternatively, consisting of, the TR4 receptor transmembrane domain (predicted to constitute amino acid
  • polypeptide fragments of the invention comprise, or alternatively, consist of, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members.
  • the amino acid residues constituting the TR4 receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, as one of ordinary skill would appreciate, the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • TR4 extracellular cysteine rich motifs of TR4 is important for interactions between TR4 and its ligands (e.g., TRAIL). Accordingly, in highly preferred embodiments, antibodies of the present invention bind TR4 polypeptide fragments comprising, or alternatively consisting of amino acid residues 131 to 183, and/or 184 to 229 of SEQ ID NO:l.
  • antibodies of the present invention bind TR4 polypeptides comprising, or alternatively consisting of both of the extracellular cysteine rich motifs (amino acid residues 131 to 229 of SEQ ID NO:l.) In another preferred embodiment, antibodies of the present invention bind TR4 polypeptides comprising, or alternatively consisting the extracellular soluble domain of TR4 (amino acid residues 24-238 of SEQ ID NO:l.) In highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 (e.g., one or both cysteine rich domains) prevent TRAIL ligand from binding to TR4.
  • the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 agonize the TR4 receptor.
  • the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR4 induce cell death of the cell expressing the TR4 receptor.
  • Antibodies of the invention may also bind fragments comprising, or alternatively, consisting of structural or functional attributes of TR4.
  • fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson- Wolf program) of complete (i.e., full-length) TR4.
  • Certain preferred regions are those set out in Table 2 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in (SEQ ID NO:l), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson- Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.
  • the data presented in columns VEH, XII, and XIII of Table 2 can be used to determine regions of TR4 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID ⁇ O:l.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson- Wolf regions of high antigenic index and Emini surface-forming regions.
  • polypeptide fragmnents bound by one or more antibodies of the invention are those that comprise regions of TR4 that combine several stmctural features, such as several (e.g., 1, 2, 3 , or 4) of the same or different region features set out above and in Table 2.
  • Lys 414 A A B -0.16 * -0.60 0.47
  • the invention provides an antibody that binds a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide described herein.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et ah, Proc. Nath Acad. Sci. USA 67:3998- 4002 (1983).
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides are therefore useful to raise antibodies, including monoclonal antibodies, that bind to a TR4 polypeptide of the invention. See, for instance, Wilson et ah, Cell 37:161-11 (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of SEQ ID NO:l.
  • Antibodies of the invention may bind one or more antigenic TR4 polypeptides or peptides including, but not limited to: a polypeptide comprising amino acid residues from about 35 to about 92 of SEQ ID NO:l; a polypeptide comprising amino acid residues from about 114 to about 160 of SEQ ID NO:l; a polypeptide comprising amino acid residues from about 169 to about 240 of SEQ ID NO:l; a polypeptide comprising amino acid residues from about 267 to about 298 of SEQ ID NO:l; a polypeptide comprising amino acid residues from about 330 to about 364 of SEQ ID NO:l; a polypeptide comprising amino acid residues from about 391 to about 404 of SEQ ID NO:l; and/or a polypeptide comprising amino acid residues from about 418 to about 465 of SEQ ID NO:l.
  • TR4 polypeptides and the epitope-bearing fragments thereof described herein e.g., corresponding to a portion of the extracellular domain such as, for example, amino acid residues 1 to 240 of SEQ ID NO:l can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • antibodies of the invention may bind fusion proteins that comprise all or a portion of a TRAIL receptor polypeptide such as TR4.
  • TR4 polypeptides such as TR4
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • Antibodies of the present invention may also bind such modified TR4 polypeptides or TR4 polypeptide fragments or variants.
  • TR4 is a member of the death domain containing receptor (DDCR) polypeptide family
  • deletions of N-terminal amino acids up to the cysteine residue at position 109 in SEQ ID NO:l may retain some biological activity such as the ability to induce apoptosis.
  • Polypeptides having further N- terminal deletions including the cysteine residue at position 109 (C-109) in SEQ ID NO:l would not be expected to retain such biological activities because this residue is conserved among family members and may be required for forming a disulfide bridge to provide stmctural stability which is needed for ligand binding.
  • TR4 ligand e.g., TRAIL
  • the ability of shortened TR4 polypeptides to induce and/or bind to antibodies which recognize the complete or mature forms of the TR4 polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the TR4 amino acid sequence of SEQ ID NO: 1 up to the serine residue at position number 463 and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n 1 -468 of SEQ ID NO:l, where n 1 is an integer from 2 to 463 corresponding to the position of the amino acid residue in SEQ ID NO:l.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of A-2 to E-468; P-3 to E-468; P-4 to E-468; P-5 to E-468; A-6 to E-468; R-7 to E-468; N-8 to E- 468; H-9 to E-468; L-10 to E-468; G-l 1 to E-468; A-12 to E-468; F-13 to E-468; L-14 to E-468; A-15 to E-468; N-16 to E-468; T-17 to E-468; P-18 to E-468; ⁇ -19 to E-468; P-20 to E-468; G-21 to E-468; S-22 to E-468; A-23 to E-468; A-24 to E-468; S-25 to E-468; G- 26 to E-468; T-27 to E-468; E-28 to E-468; A-29 to E-468; A-30 to E-468; A-31 to E-4
  • N-terminal deletions of the TR4 polypeptide can be described by the general formula n 2 to 238 where n 2 is a number from 2 to 238 corresponding to the amino acid sequence identified of SEQ ID NO:l.
  • antibodies of the invention bind N terminal deletions of the TR4 comprising, or alternatively consisting of, the amino acid sequence of residues: A-2 to H- 238; P-3 to H-238; P-4 to H-238; P-5 to H-238; A-6 to H-238; R-7 to H-238; V-8 to H- 238; H-9 to H-238; L-10 to H-238; G-11 to H-238; A-12 to H-238; F-13 to H-238; L-14 to H-238; A-15 to H-238; V-16 to H-238; T-17 to H-238; P-18 to H-238; N-19 to H-238; P- 20 to H-238; G-21 to H-238; S-22 to H-238; A-23 to H
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR4 polypeptide sequence of SEQ ID NO: 1 up to the alanine residue at position number 30, and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 24-m 1 of SEQ ID NO:l, where m 1 is an integer from 30 to 467 corresponding to the position of the amino acid residue in SEQ ID NO: 1.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues A-24 to L- 467; A-24 to S-466; A-24 to V-465; A-24 to A-464; A-24 to S-463; A-24 to G-462; A-24 to T-461; A-24 to G-460; A-24 to D-459; A-24 to E-458; A-24 to L-457; A-24 to Y-456; A-24 to 1-455; A-24 to F-454; A-24 to K-453; A-24 to G-452; A-24 to S-451; A-24 to D- 450; A-24 to V-449; A-24 to L-448; A-24 to L-447; A-24 to D-446; A-24 to Q-445; A-24 to 1-444; A-24 to K-443; A-24 to E-442; A-24 to K-441; A-24 to A-440; A-24 to H-439; A-24 to R-438; A-24 to E-437
  • antibodies of the invention bind C-terminal deletions of the TR4 polypeptide that can be described by the general formula 24-m 2 where m 2 is a number from 30 to 238 corresponding to the amino acid sequence identified of SEQ ID NO:l.
  • the invention provides antibodies that bind TR4 polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: A-24 to G-237; A-24 to N-236; A-24 to G-235; A-24 to S-234; A-24 to E-233; A-24 to K-232; A-24 to H-231; A-24 to V-230; A-24 to C-229; A-24 to E-228; A-24 to I- 227; A-24 to D-226; A-24 to S-225; A-24 to W-224; A-24 to P-223; A-24 to T-222; A-24 to C-221; A-24 to D-220; A-24 to K-219; A-24 to V-218; A-24 to K-217; A-24 to V-216; A-24 to M-215; A-24 to G-214; A-24 to R-213; A-24 to P-212; A-24 to C-211; A-24 to G- 210; A-24 to T-209; A-24 to S-208; A-24 to C-207;
  • the present invention further provides antibodies that bind polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the TR4 polypeptide of SEQ ID NO:l, up to C-221 of SEQ ID NO:l.
  • the present invention provides antibodies that bind polypeptides having the amino acid sequence of residues 1-m 9 of the amino acid sequence in SEQ ID NO:l, where m 9 is any integer in the range of 221-468 and residue C-221 is the position of the first residue from the C- terminus of the complete TR4 polypeptide (shown in SEQ ID NO:l) believed to be required for receptor binding activity of the TR4 protein.
  • the invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR4
  • 1 1 9 9 polypeptide which may be described generally as having residues n - m and/or n - m of 1 9 ' 1 9
  • antibodies of the present invention bind fragments of TR4 comprising a portion of the extracellular domain; i.e., within residues 24-238 of SEQ ID NO:l, since any portion therein is expected to be soluble.
  • TR4 amino acid sequence of TR4 can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Such areas will usually comprise residues which make up the ligand binding site or the death domain, or which form tertiary structures which affect these domains.
  • the invention further includes antibodies that bind variations of the TR4 protein which show substantial TR4 protein activity or which include regions of TR4 such as the protein fragments discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitution.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U. et ah, Science 247:1306-1310 (1990).
  • antibodies of the present invention may bind a fragment, derivative, or analog of the polypeptide of SEQ ID NO: 1, or that encoded by the cDNA in ATCC deposit 97853.
  • Such fragments, variants or derivatives may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or
  • the replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et ah, Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors.
  • the antibodies of the present invention may bind a TR4 receptor that contains one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 3).
  • the number of substitutions, additions or deletions in the amino acid sequence of SEQ ID NO:l and/or any of the polypeptide fragments described herein is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.
  • the antibodies of the invention bind TR4 polypeptides or fragments or variants thereof (especially a fragment comprising or alternatively consisting of, the extracellular soluble domain of TR4), that contains any one or more of the following conservative mutations in TR4: Ml replaced with A, G, I, L, S, T, or V; A2 replaced with G, I, L, S, T, M, or V; A6 replaced with G, I, L, S, T, M, or V; R7 replaced with H, or K; N8 replaced with A, G, I, L, S, T, or M; H9 replaced with K, or R; L10 replaced with A, G, I, S, T, M, or V; Gil replaced with A, I, L, S, T, M, or V; A12 replaced with G, I, L, S, T, M, or V; F13 replaced with W, or Y; L14 replaced with A, G, I, S, T, M, or V; A15 replaced with G, I, I, S, T, M
  • the antibodies of the invention bind TR4 polypeptides or fragments or variants thereof (especially a fragment comprising or alternatively consisting of, the extracellular soluble domain of TR4), that contains any one or more of the following non-conservative mutations in TR4: Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A2 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P3 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P4 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P5 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or
  • E, H, K, R, ⁇ , Q, F, W, Y, P, or C substituted with D, E, H, K, R, A, G, I, L, S, T, M, N, ⁇ , Q, F, W, Y, or C; ⁇ 19 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, N, ⁇ , Q, F, W, Y, or C; G21 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; S22 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; A23 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A24 replaced with D,
  • S139 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C
  • E140 replaced with H, K, R, A, G, I, L, S, T, M, N, ⁇ , Q, F, W, Y, P, or C
  • R141 replaced with D, E, A,
  • D, E, H, K, R, N, Q, F, W, Y, P, or C D304 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S305 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L306 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S307 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T308 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F309 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V310 replaced with D,
  • E, H, K, R, N, Q, F, W, Y, P, or C S311 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E312 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q313 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q314 replaced with
  • P331 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C
  • G332 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • E333 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • A334 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • Q335 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C
  • C336 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P
  • L337 replaced with D
  • F, W, Y, P, or C F371 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A372 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N373 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; 1374 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V375 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P376 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F377 replaced with D, E, H, K, R, N, Q, A, G
  • D, E, H, K, R, N, Q, F, W, Y, P, or C D395 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V396 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V397 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R398 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A399 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T401 replaced with D, E, H, K, R, N, Q, F, W,
  • E, H, K, R, N, Q, F, W, Y, P, or C L412 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M413 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K414 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W415 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V416 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N417 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K418 replaced with
  • Amino acids in the TR4 protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by stmctural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et ah, J.
  • antibodies of the present invention bind regions of TR4 that are essential for TR4 function. In other preferred embodiments, antibodies of the present invention bind regions of TR4 that are essential for TR4 function and inhibit or abolish TR4 function. In other preferred embodiments, antibodies of the present invention bind regions of TR4 that are essential for TR4 function and enhance TR4 function.
  • TR4 polypeptides may be employed to improve or alter the characteristics of TR4 polypeptides.
  • Recombinant D ⁇ A technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • Antibodies of the present invention may bind such modified TR4 polypeptides.
  • ⁇ on-naturally occurring variants of TR4 may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et ah, Nuch Acids Res. 13:4331 (1986); and Zoller et ah, Nuch Acids Res. 10:6481 (1982)), cassette mutagenesis (see e.g., Wells et ah, Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et ah, Philos. Traits. R. Soc. London SerA 377:415 (1986)).
  • art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e
  • the invention also encompasses antibodies that bind TR4 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TR4 polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognition sequences in the TR4 polypeptides and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the TR4 at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193- 1197).
  • one or more of the amino acid residues of TR4 polypeptides e.g., arginine and lysine residues
  • the antibodies of the present invention also include antibodies that bind a polypeptide comprising, or alternatively, consisting of the polypeptide encoded by the deposited cDNA (the deposit having ATCC Accession Number 97853) including the leader; a polypeptide comprising, or alternatively, consisting of the mature polypeptide encoded by the deposited the cDNA minus the leader (i.e., the mature protein); a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:l including the leader; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:l minus the amino terminal methionine; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:l minus the leader; a polypeptide comprising, or alternatively, consisting of the TR4 extracellular domain; a polypeptide comprising, or alternatively, consisting of the TR4 cysteine rich domain; a polypeptide comprising
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TR4 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TR4 polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:l or to the amino acid sequence encoded by deposited cDNA clones can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Bmtlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • the present application is also directed to antibodies that bind proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to the TR4
  • antibodies of the invention bind TR4 fusion proteins as described above wherein the TR4 portion of the fusion protein are those described as n ⁇ m 1 , and/or n 2 -m 2 herein.
  • the antibodies of the present invention bind TR5 polypeptide, or fragments or variants thereof.
  • the following section describes the TR5 polypeptides, fragments and variants that may be bound by the antibodies of the invention in more detail.
  • the TR5 polypeptides, fragments and variants which may be bound by the antibodies of the invention are also described in International Publication Numbers, for example, WO98/30693 and WO00/71150 which are herein incorporated by reference in their entireties. Amino acids 41-299 of SEQ ID NO: 2 are identical to the TR5 protein disclosed in WO98/30693 and WOOO/71150.
  • the antibodies of the present invention immunospecifically bind TR5 polypeptide.
  • TR5 may, in some embodiments, bind fragments, variants (including species orthologs of TR5), multimers or modified forms of TR5.
  • an antibody immunospecific for TR5 may bind the TR5 moiety of a fusion protein comprising all or a portion of TR5.
  • TR5 proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to antibodies that bind TR5 proteins found as monomers or as part of multimers. In specific embodiments, antibodies of the invention bind TR5 monomers, dimers, trimers or tetramers.
  • antibodies of the invention bind at least dimers, at least trimers, or at least tetramers containing one or more TR5 polypeptides.
  • Antibodies of the invention may bind TR5 homomers or heteromers.
  • the term homomer refers to a multimer containing only TR5 proteins of the invention (including TR5 fragments, variants, and fusion proteins, as described herein). These homomers may contain TR5 proteins having identical or different polypeptide sequences.
  • a homomer of the invention is a multimer containing only TR5 proteins having an identical polypeptide sequence.
  • antibodies of the invention bind TR5 homomers containing TR5 proteins having different polypeptide sequences.
  • antibodies of the invention bind a TR5 homodimer (e.g., containing TR5 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR5 proteins having identical or different polypeptide sequences). In additional embodiments, antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer of TR5.
  • heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR5 gene) in addition to the TR5 proteins of the invention.
  • antibodies of the invention bind a heterodimer, a heterotrimer, or a heterotetramer.
  • the antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer containing one or more TR5 polypeptides.
  • Multimers bound by one or more antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • multimers bound by one or more antibodies of the invention such as, for example, homodimers or homotrimers
  • heteromultimers bound by one or more antibodies of the invention such as, for example, heterotrimers or heterotetramers, are formed when TR5 proteins contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein) in solution.
  • multimers bound by one or more antibodies of the invention are formed by covalent associations with and/or between the TR5 proteins of the invention.
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:2 or the polypeptide encoded by the deposited cDNA clone of ATCC Deposit 97798).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR5 fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a TR5-Fc fusion protein (as described herein).
  • covalent associations of fusion proteins are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
  • the multimers that may be bound by one or more antibodies of the invention may be generated using chemical techniques known in the art.
  • proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers that may be bound by one or more antibodies of the invention may be generated using techniques known in the art to form one or more inter- molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • proteins that may be bound by one or more antibodies of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer that may be bound by one or more antibodies of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • multimers that may be bound by one or more antibodies of the invention may be generated using genetic engineering techniques known in the art.
  • proteins contained in multimers that may be bound by one or more antibodies of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • polynucleotides coding for a homodimer that may be bound by one or more antibodies of the invention are generated by ligating a polynucleotide sequence encoding a TR5 polypeptide to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • TR5 polypeptides which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • two or more TR5 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR5 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • antibodies of the invention bind proteins comprising multiple TR5 polypeptides separated by peptide linkers.
  • TR5 polypeptides of the invention involves use of TR5 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
  • Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins.
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric TR5 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble TR5 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR5 is recovered from the culture supernatant using techniques known in the art.
  • antibodies of the invention bind TR5-leucine zipper fusion protein monomers and/or TR5-leucine zipper fusion protein multimers.
  • trimeric TR5 may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
  • antibodies of the invention bind TR5-leucine zipper fusion protein trimers.
  • TR5 polypeptides are preferably provided in an isolated form, and preferably are substantially purified.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the TR5 polypeptide is substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • Antibodies of the present invention may bind TR5 polypeptides or polypeptide fragments including polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2, encoded by the cDNA contained in ATCC deposit Number 97798, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the cDNA contained in ATCC deposit Number 97798 or to SEQ ID NO:l or the complementary strand thereto.
  • Protein fragments may be "freestanding,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Antibodies of the present invention may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 40, 41 to 66, 67 to 90, 91 to 140, 191 to 240, 241 to 280, and/or 281 to 299, of SEQ ID NO:2.
  • polypeptide fragments bound by the antibodies of the invention can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length.
  • antibodies of the present invention bind polypeptide fragments comprising, or alternatively consisting of, amino acid residues: 41-299, 67-299, 67-280, 93-193, and/or 281-299, of TR5 (SEQ ID NO:2).
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • antibodies of the present invention bind TR5 polypeptide fragments comprising, or alternatively consisting, of one or more TR5 domains.
  • antibodies of the present invention bind TR5 polypeptides or polypeptide fragments selected from the group consisting of: (a) a polypeptide comprising or alternatively, consisting of, the TR5 transmembrane domain (predicted to constitute amino acid residues from about 281 to about 299 of SEQ ID NO:2); (b) a polypeptide comprising or alternatively, consisting of, the TR5 receptor extracellular domain (predicted to constitute amino acid residues from about 67 to about 280 of SEQ ID NO:2); (c)a polypeptide comprising or alternatively, consisting of, both TR5 cysteine rich domains (both of which may be found in the protein fragment consisting of amino acid residues from about 93 to about 193 in SEQ ID NO:2); (d) a polypeptide comprising or alternatively, consisting of, the group consisting of: (a) a
  • antibodies of the present invention bind TR5 polypeptide fragments comprising, or alternatively consisting of, amino acid residues 93 to 150 and/or 151-193 of SEQ ID NO:2.
  • antibodies of the present invention bind TR5 polypeptides comprising, or alternatively consisting of, both of the extracellular cysteine rich motifs (amino acid residues 93 to 193 SEQ ID NO:2.) In another preferred embodiment, antibodies of the present invention bind TR5 polypeptides comprising, or alternatively consisting of the extracellular soluble domain of TR5 (amino acid residues 67-280 of SEQ ID NO:2.)
  • Antibodies of the invention may also bind fragments comprising, or alternatively, consisting of stmctural or functional attributes of TR5.
  • Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) TR5.
  • Certain preferred regions are those set out in Table 4 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, tum-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and tum-regions; Kyte-Doolittle predicted hydrophilic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson- Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.
  • the data presented in columns NIH, IX, XIH, and XIV of Table 4 can be used to determine regions of TR5 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns N ⁇ i, IX, XIII, and or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Table 4 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID ⁇ O:2.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and tum-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
  • antibodies of the present invention bind TR5 polypeptides or TR5 polypeptide fragments and variants comprising regions of TR5 that combine several stmctural features, such as several (e.g., 1, 2, 3 , or 4) of the same or different region features set out above and in Table 4.
  • the invention provides an antibody that binds a peptide or polypeptide comprising, or alternatively, consisting of, one, two, three, four, five or more, epitope-bearing portions of TR5.
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical mles, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind to a TR5 polypeptide. See, for instance, Wilson et ah, Cell 37:161-118 (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention.
  • Antibodies of the invention may bind one or more antigenic TR5 polypeptides or peptides including, but not limited to: a polypeptide comprising amino acid residues from about Gln-82 to about Glu-92 SEQ ID NO:2; a polypeptide comprising amino acid residues from about His-98 to about Cys-106 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Gln-82 to about Thr-Cys-106 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Pro- 108 to about Thr-116 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Ser-119 to about Cys- 125 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Cys-131 to about Thr-142 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about Gln-150 to about Pro-162 in SEQ ID NO:
  • the epitope-bearing TR5 peptides and polypeptides may be produced by any conventional means (See, e.g., Houghten, R. A. "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.” Proc. Nath Acad. Sci.
  • Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et ah, supra; Wilson et ah, supra; Chow, M. et ah, Proc. Nath Acad. Sci. USA 82:910-914; and Bittle, F. J. et ah, J. Gen. Virol. 66:2341-2354 (1985).
  • Immunogenic epitope-bearing peptides of the invention i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art.
  • U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S.
  • Patent No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear Cl-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest.
  • non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.
  • TR5 receptor polypeptides of the present invention and the epitope-bearing fragments thereof described herein above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • antibodies of the invention may bind fusion proteins that comprise all or a portion of a TRAIL receptor polypeptide such as TR5.
  • TR5 TRAIL receptor polypeptide
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • Antibodies of the present invention may also bind such modified TR5 polypeptides or TR5 polypeptide fragments or variants.
  • the proteins of the invention are members of the TNFR polypeptide family
  • deletions of N-terminal amino acids up to the cysteine at position C-93 of SEQ ID NO:2 may retain some biological activity such as regulation of proliferation and apoptosis of lymphoid cells.
  • Polypeptides having further N-terminal deletions including the C-93 residue of SEQ ID NO:2 would not be expected to retain such biological activities because it is known that these residues in a TR5-related polypeptide are required for forming a disulfide bridge to provide stmctural stability which is needed for ligand binding.
  • antibodies of the present invention bind N-terminally deleted protein fragments which retain biological activity.
  • the present invention further provides antibodies that bind TR5 polypeptides having one or more residues deleted from the amino terminus of TR5 (SEQ ID NO:2), up to the cysteine residue in each which is at position number 93, and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind TR5 polypeptides comprising the amino acid sequence of residues n 3 -299 of SEQ ID NO:2 where n 3 is an integer in the range of 1-93 where 93 is the position of the first cysteine residue from the N-terminus of the complete TR5 polypeptide believed to be required for activity of the TR5 protein.
  • the invention provides antibodies that bind polypeptides having the amino acid sequence of residues: Q-2 to V-299; G-3 to V-299; V-4 to V-299; K-5 to V-299; E-6 to V-299; R-7 to V-299; F-8 to V-299; L-9 to V-299; P-10 to V-299; L- 11 to V-299; G-12 to V-299; N-13 to V-299; S-14 to V-299; G-15 to V-299; D-16 to V- 299; R-17 to V-299; A-18 to V-299; P-19 to V-299; R-20 to V-299; P-21 to V-299; P-22 to V-299; D-23 to V-299; G-24 to V-299; R-25 to V-299; G-26 to V-299; R-27 to V-299; V-28 to V-299; R-29 to V-299; P-30 to V-299; R-31 to V-299; T-32 to V-299; Q
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the TR5 amino acid sequence shown in SEQ ID NO:2, up to the leucine residue at position number 295.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n 4 -299 of SEQ ID NO:2, where n 4 is an integer from 2 to 294 corresponding to the position of the amino acid residue in SEQ ID NO:2.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: Q-2 to V- 299; G-3 to N-299; N-4 to N-299; K-5 to V-299; E-6 to V-299; R-7 to V-299; F-8 to V- 299; L-9 to V-299; P-10 to V-299; L-ll to V-299; G-12 to V-299; ⁇ -13 to V-299; S-14 to V-299; G-15 to V-299; D-16 to V-299; R-17 to V-299; A-18 to V-299; P-19 to V-299; R- 20 to V-299; P-21 to V-299; P-22 to V-299; D-23 to V-299; G-24 to V-299; R-25 to V- 299; G-26 to V-299; R-27 to V-299; V-28 to V-299; R-29 to V-299; P-30 to V-299; R-31 to
  • Polypeptides having further C-terminal deletions including the cysteine at position 189 of SEQ ID NO:2 would not be expected to retain such biological activities because it is known that this residue in TNF receptor-related polypeptides is required for forming a disulfide bridge to provide stmctural stability which is needed for ligand binding.
  • deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR5 ligand (e.g., TRAIL)) may still be retained.
  • the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature form of the protein generally will be retained when less than the majority of the residues of the complete or mature form protein are removed from the C-terminus.
  • Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR5 polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR5 amino acid residues may often evoke an immune response.
  • the present invention further provides antibodies that bind polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of TR5 shown in SEQ ID NO:2 up to the cysteine at position 189 of SEQ ID NO: 2.
  • the present invention provides antibodies that bind polypeptides having the amino acid sequence of residues 1-x of the amino acid sequence in SEQ ID NO:2, where x is any integer in the range of 189-299.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR5 polypeptide shown in SEQ ID NO: 2 up to the glutamine residue at position number at position number 6.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m 3 of SEQ ID NO:3, where m 3 is an integer from 6 to 298 corresponding to the position of the amino acid residue in SEQ ID NO:3.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-l to F- 298; M-l to V-297; M-l to 1-296; M-l to L-295; M-l to L-294; M-l to V-293; M-l to I- 292; M-l to L-291; M-l to V-290; M-l to 1-289; M-l to 1-288; M-l to G-287; M-l to V- 286; M-l to 1-285; M-l to T-284; M-l to C-283; M-l to S-282; M-l to L-281; M-l to Y- 280; M-l to H-279; M-l to S-278; M-l to S-277; M-l to A-276; M-l to P-275; M-l to T- 274; M-l to G-273; M-l to
  • antibodies of the invention bind C-terminal deletions of the extracellular domain of the TR5 polypeptide that can be described by the general formula 67 -m 4 where m 4 is a number from 73 to 299 corresponding to the amino acid sequence identified in SEQ ID NO:2.
  • antibodies of the invention bind C terminal deletions of the TR5 polypeptide comprising, or alternatively, consisting of, amino acid residues: T-67 to F-298; T-67 to V-297; T-67 to 1-296; T-67 to L-295; T-67 to L-294; T-67 to V-293; T-67 to 1-292; T-67 to L-291; T-67 to V-290; T-67 to 1-289; T-67 to 1-288; T-67 to G-287; T-67 to V-286; T-67 to 1-285; T-67 to T-284; T-67 to C-283; T-67 to S-282; T-67 to L-281; T-67 to Y-280; T-67 to H-279; T-67 to S-278; T- 67 to S-277; T-67 to A-276; T-67 to P-275; T-67 to T-274; T-67 to G-273; T-67 to P-272
  • the invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n 4 to m 3 of SEQ ID NO:2, where n 4 and m 3 are integers as described above.
  • antibodies that bind a polypeptide consisting of a portion of the complete TR5 amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97798, where this portion excludes from 1 to about 89 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798, or from 1 to about 110 amino acids from the carboxy terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798.
  • the invention further includes variations of the TR5 polypeptide, which show substantial TR5 polypeptide activity or which include regions of TR5 protein such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions
  • antibodies of the present invention may bind a fragment, derivative, or analog of the polypeptide of SEQ ID NO:2, or that encoded by the cDNA in ATCC deposit 97798.
  • Such fragments, variants or derivatives may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residue(s)), and such substituted amino acid residue(s) may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the mature or soluble extracellular polypeptide is fused with another compound, such as a compound to increase the half -life of the polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an Ig

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Abstract

La présente invention concerne des anticorps et des molécules associées qui se lient spécifiquement au niveau immunitaire à des récepteurs TRAIL. On utilise ces anticorps, par exemple, dans la prévention et le traitement de cancers et d'autres troubles prolifératifs. Cette invention a également trait à des molécules d'acides nucléiques codant des anticorps du récepteur anti-TRAIL, à des vecteurs et à des cellules hôtes renfermant ces acides nucléiques, et à des méthodes d'élaboration correspondantes. Ladite invention a aussi pour objet des méthodes et des compositions de prévention, de détection, de diagnostic, de traitement ou d'amélioration d'une maladie ou d'un trouble, notamment le cancer et d'autres troubles hyperprolifératifs, lesdites méthodes consistant à administrer à un animal, de préférence à un être humain, une quantité efficace d'au moins un anticorps ou un fragment ou un variant associé ou une molécule correspondante qui se lie spécifiquement au niveau immunitaire à des récepteurs TRAIL.
PCT/US2002/036431 2001-11-14 2002-11-13 Anticorps se liant specifiquement au niveau immunitaire a des recepteurs trail WO2003042367A2 (fr)

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WO2005000894A3 (fr) * 2003-06-25 2005-07-07 Crucell Holland Bv Molecules de liaison pour traiter les malignites de cellules myeloides
US7115717B2 (en) 2001-05-18 2006-10-03 Kirin Beer Kabushiki Kaisha Anti-TRAIL-R antibodies
US7252994B2 (en) 2001-07-03 2007-08-07 Genentech, Inc. Human DR4 antibodies and uses thereof
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