WO2006055704A2 - Anticorps diriges contre des proteines ten-m et utilisations associees - Google Patents

Anticorps diriges contre des proteines ten-m et utilisations associees Download PDF

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WO2006055704A2
WO2006055704A2 PCT/US2005/041673 US2005041673W WO2006055704A2 WO 2006055704 A2 WO2006055704 A2 WO 2006055704A2 US 2005041673 W US2005041673 W US 2005041673W WO 2006055704 A2 WO2006055704 A2 WO 2006055704A2
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Prior art keywords
antibody
antibodies
sequence
protein
amino acid
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PCT/US2005/041673
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WO2006055704A3 (fr
Inventor
Jaspal Kang
Seth Ettenberg
John Herrmann
Luca Rastelli
Michael E. Jeffers
William Larochelle
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Curagen Corporation
Abgenix, Inc.
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Publication of WO2006055704A2 publication Critical patent/WO2006055704A2/fr
Publication of WO2006055704A3 publication Critical patent/WO2006055704A3/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates generally to antibodies that bind to Ten-M proteins. More specifically, the invention relates to fully human antibodies that specifically bind to Ten-M4 proteins.
  • the human Ten-M family of genes also known as teneurins or hOdz, are a class of type II transmembrane proteins containing a short intracellular N-terminus followed by a transmembrane region, which is followed by eight EGF-like repeats, which are followed by a large globular domain on the extracellular side.
  • the EGF-like repeats of Ten-M proteins are thought to mediate dimerization.
  • Ten-M proteins as well as in vitro models of cell migration, such as neurite outgrowth, have suggested a role in neural development. This may also involve binding to extracellular matrix proteins such as heparin, indicating a role as a cell adhesion molecule.
  • the structure and function of the Ten-M protein has previously been examined (e.g., Oohashi et al., J. Cell Biol, 145:563-577 (1999)).
  • the various forms of the protein e.g., ml, m2, m3, and m4 are generally 2700 to 2800 amino acids in length.
  • Ten-M4 protein appears to lack a signal peptide at the NH 2 terminus.
  • Ten-M4 contains a short hydrophobic domain, similar to those in transmembrane proteins, of 300-400 amino acids after the NH 2 terminus. This section of the protein is then followed by eight consecutive EGF-like domains.
  • a fully human antibody that selectively binds to a Ten-M4 protein on a cell is provided.
  • the fully human antibody when administered to a patient, reduces the metastasis of a cancer in a patient.
  • the fully human antibody binds to a Ten-M4 protein at least five, ten, thirty, or forty fold more strongly than the antibody binds to a Ten-M2 protein on a cell.
  • a fully human antibody that selectively binds to a Ten-M4 protein that is not connected to a cell is provided.
  • the antibody binds to a Ten-M4 protein at least five fold more strongly than the antibody binds to a Ten-M2 protein.
  • the antibody that binds to Ten-M4 comprises a human heavy chain immunoglobulin molecule having an amino acid sequence shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 38.
  • the antibody that binds to Ten-M4 comprises a human kappa light chain immunoglobulin molecule having an amino acid sequence of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40.
  • the antibody molecules are formed by combinations comprising the above recited heavy chain immunoglobulin molecules with the above recited light chain immunoglobulin molecules, such as the kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.
  • the antibody has a sequence from the heavy chain CDRl, CDR2, CDR3, FRl, FR2, FR3, and/or FR4 or any of the sequences listed in FIG. 12. In some embodiments, the antibody has a sequence from the light chain CDRl, CDR2, CDR3, FRl, FR2, FR3, and/or FR4 or any of the sequences listed FIG. 13.
  • Yet another aspect of the invention is a fully human antibody that binds to Ten-M4 and has a heavy chain variable gene derived from the VH3-33 germline.
  • a conjugated fully human antibody that binds to a Ten-M4 protein is provided. Attached to the antibody is an agent, and the binding of the antibody to a cell results in the delivery of the agent to the cell.
  • the above conjugated fully human antibody binds to an extracellular section of the Ten-M4 protein.
  • the antibody binds to an EGF-like repeat of the Ten-M4 protein.
  • the antibody and conjugated toxin are internalized by a cell that expresses a Ten- M4 protein.
  • the agent is a cytotoxic agent.
  • the agent is saporin.
  • the conjugated fully human antibody has an IC 50 of no more than about 1300 pM for PC-3 cells.
  • the conjugated fully human antibody has an IC 5O of no more than about 250 or 121 pM for PC-3 cells.
  • composition comprising a monoclonal antibody or antigen-binding portion described herein and a pharmaceutically acceptable carrier is provided.
  • a kit for treating Ten-M4 related disorders comprising a Ten-M4 antibody and instructions for administering the Ten-M4 antibody to a subject is provided.
  • a method of reducing the metastasis of a cancer in a patient comprises administering a fully human antibody to a patient.
  • the antibody binds to a Ten-M4 protein so as to prevent the Ten-M4 protein from binding to and thereby forming a duplex with a second Ten-M4 protein.
  • the antibody thereby reduces the metastasis of a cancer in a patient.
  • a method of reducing the risk of metastasis of a cancer in a patient is provided.
  • the method comprises administering a fully human antibody to a patient.
  • the antibody binds to a first Ten-M4 protein in a manner so as to prevent the Ten-M4 protein from forming an active Ten-M4/Ten-M4 duplex with a second Ten-M4 protein.
  • the antibody binds so as to still allow the first Ten-M4 protein to bind to the second Ten-M4 protein.
  • the antibody thereby reduces the risk of metastasis of a cancer in a patient.
  • a method of selectively killing a cancerous cell in a patient comprises administering a fully human antibody conjugate to a patient.
  • the fully human antibody conjugate comprises an antibody that can bind to a Ten-M4 protein and an agent.
  • the agent is either a toxin or another substance that will kill a cancer cell.
  • the antibody conjugate thereby selectively kills the cancer cell.
  • the agent can be saporin.
  • the antibody conjugate has an IC 50 of no more than about 130 pM.
  • a method of diagnosing a risk of cancer metastasis in a patient comprises administering to a patient a fully human antibody conjugate that selectively binds to a ten-M4 protein on a cell.
  • the antibody conjugate comprises an antibody that selectively binds to ten-M4 and a label.
  • the method further comprises observing the presence of the label in the patient. A relatively high amount of the label will indicate a relatively high risk of cancer metastasis and a relatively low amount of the label will indicate a relatively low risk of cancer metastasis.
  • the label is a green fluorescent protein.
  • Further embodiments include an isolated antibody, or fragment thereof, that comprises a heavy chain amino acid sequence. Other embodiments include an isolated antibody, or fragment thereof, that comprises a heavy chain nucleic acid sequence.
  • the isolated antibodies can be monoclonal antibodies, chimeric antibodies and/or human or humanized antibodies.
  • the antibodies are human antibodies.
  • embodiments of the invention are not limited to any particular form of an antibody.
  • the antibodies provided may be a full length antibody (e.g. having an intact human Fc region) or an antibody fragment (e.g. a Fab, Fab' or F(ab') 2 ).
  • the antibodies may be manufactured from a hybridoma that secretes the antibody, or from a recombinantly produced cell that has been transformed or transfected with a gene or genes encoding the antibody.
  • the invention provides an isolated polynucleotide molecule described herein.
  • FIG. IA is an amino acid sequence of the Ten-m4 protein.
  • FIG. IB is a nucleic acid sequence encoding the Ten-M4 protein.
  • FIG. 1C is an amino acid sequence of the antigen from the Ten-M4 protein that was used to create the discussed antibodies.
  • FIG. ID is a diagram of the various structures in the Ten-M4 protein and the antigen that was used to create the current antibodies.
  • FIG. 2A is the nucleotide sequence of the heavy chain variable region of antibody 1.16.
  • FIG. 2B is the amino acid sequence of the heavy chain variable region of antibody 1.16.
  • FIG. 2C is the nucleotide sequence of the light chain variable region of antibody 1.16.
  • FIG. 2D is the amino acid sequence of the light chain variable region of antibody 1.16.
  • FIG. 3A is the nucleotide sequence of the heavy chain variable region of antibody 1.20.
  • FIG. 3B is the 1.20 amino acid sequence of the heavy chain variable region of antibody 1.20.
  • FIG. 3 C is the nucleotide sequence of the light chain variable region of antibody 1.20.
  • FIG. 3D is the amino acid sequence of the light chain variable region of antibody 1.20.
  • FIG. 4A is the nucleotide sequence of the heavy chain variable region of antibody 1.44.
  • FIG. 4B is the amino acid sequence of the heavy chain variable region of antibody 1.44.
  • FIG. 4C is the nucleotide sequence of the light chain variable region of antibody 1.44.
  • FIG. 4D is the amino acid sequence of the light chain variable region of antibody 1.44.
  • FIG. 5A is the nucleotide sequence of the heavy chain variable region of antibody 1.47.
  • FIG. 5B is the amino acid sequence of the heavy chain variable region of antibody 1.47.
  • FIG. 5 C is the nucleotide sequence of the light chain variable region of antibody 1.47.
  • FIG. 5D is the amino acid sequence of the light chain variable region of antibody 1.47.
  • FIG. 6A is the nucleotide sequence of the heavy chain variable region of antibody 1.48.
  • FIG. 6B is the amino acid sequence of the heavy chain variable region of antibody 1.48.
  • FIG. 6C is the nucleotide sequence of the light chain variable region of antibody 1.48.
  • FIG. 6D is the amino acid sequence of the light chain variable region of antibody 1.48.
  • FIG. 7A is the nucleotide sequence of the heavy chain variable region of antibody 1.58.
  • FIG. 7B is the amino acid sequence of the heavy chain variable region of antibody 1.58.
  • FIG. 7C is the nucleotide sequence of the light chain variable region of antibody 1.58.
  • FIG. 7D is the amino acid sequence of the light chain variable region of antibody 1.58.
  • FIG. 8A is the nucleotide sequence of the heavy chain variable region of antibody 2.13.
  • FIG. 8B is the amino acid sequence of the heavy chain variable region of antibody 2.13.
  • FIG. 8C is the nucleotide sequence of the light chain variable region of antibody 2.13.
  • FIG. 8D is the amino acid sequence of the light chain variable region of antibody 2.13.
  • FIG. 9A is the nucleotide sequence of the heavy chain variable region of antibody 2.15.
  • FIG. 9B is the amino acid sequence of the heavy chain variable region of antibody 2.15.
  • FIG. 9C is the nucleotide sequence of the light chain variable region of antibody 2.15.
  • FIG. 9D is the amino acid sequence of the light chain variable region of antibody 2.15.
  • FIG. 1OA is the nucleotide sequence of the heavy chain variable region of antibody 2.36.
  • FIG. 1OB is the amino acid sequence of the heavy chain variable region of antibody 2.36.
  • FIG. 1OC is the nucleotide sequence of the light chain variable region of antibody 2.36.
  • FIG. 1OD is the amino acid sequence of the light chain variable region of antibody 2.36.
  • FIG. 1 IA is the nucleotide sequence of the heavy chain variable region of antibody 2.59.
  • FIG. HB is the amino acid sequence of the heavy chain variable region of antibody 2.59.
  • FIG. 1 1 C is the nucleotide sequence of the light chain variable region of antibody 2.59.
  • FIG. 1 ID is the amino acid sequence of the light chain variable region of antibody 2.59.
  • FIG. 12 is a comparison of the heavy chain amino acid sequences of the various antibodies generated towards Ten-M4.
  • FIG. 13 is a comparison of the light chain amino acid sequences of the various antibodies generated towards Ten-M4.
  • FIG. 14 is a graph displaying the effectiveness of the various antibodies conjugated to a toxin on the inhibition of cancer cell proliferation.
  • the Ten-M proteins appear to be involved in neuron growth and guidance.
  • the present compositions and methods can be applied to promote or inhibit neuron growth and development in situations where such needs arise.
  • the presently disclosed antibodies that promote neuron growth through the binding of the antibody to the Ten-
  • M protein can be used in situations such as nerve regeneration in damaged tissues.
  • Some embodiments of the invention relate to the generation and identification of isolated, preferably fully human, monoclonal antibodies that bind to the Ten-M4 protein.
  • these antibodies can bind to Ten-M4 with high affinity, high potency, or both.
  • these antibodies are associated with a toxin or similar compound and be used to associate the toxin to a particular location or cell type to promote the killing of the cell type or cells in a desired location.
  • the antibodies are internalized with a high degree of efficiency.
  • the antibody will prevent effective functioning (e.g., signaling) of the Ten-M4 protein.
  • the antibody may prevent dimerization of the Ten-M4 protein with another Ten-M4 protein by binding to a dimerization domain (e.g., EGF- like repeats) of the protein.
  • a dimerization domain e.g., EGF- like repeats
  • some of the present embodiments provide isolated antibodies, or fragments of those antibodies, that bind to the EGF-like repeat domain of the ten- M4 protein.
  • Embodiments of the invention also provide cells for producing these antibodies.
  • embodiments provide for using these antibodies as a diagnostic or treatment for diseases related to the under or over expression of the Ten-m4 protein.
  • nucleic acids described herein, and fragments and variants thereof may be used, by way of nonlimiting example, (a) to direct the biosynthesis of the corresponding encoded proteins, polypeptides, fragments and variants as recombinant or heterologous gene products, (b) as probes for detection and quantification of the nucleic acids disclosed herein, (c) as sequence templates. Such uses are described more fully below.
  • Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. Standard techniques are also used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • PCR Polymerase chain reaction
  • sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • the 5' terminal nucleotides of the two primers can coincide with the ends of the amplified material.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al, Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987); Erlich, ed., PCR Technology (Stockton Pres, NY, 1989).
  • a used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid.
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Chothia et al. J. MoI. Biol. 186:651 (1985; Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985); Chothia et al, Nature 342:877-883 (1989)).
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies ⁇ e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments including Fab and F(ab)'2, so long as they exhibit the desired biological activity.
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called K and ⁇ , based on the amino acid sequences of their constant domains. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies, as described in more detail below.
  • An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical.
  • intact antibodies can be assigned to different "classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et at, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et at, J. MoI. Biol, 222:581-597 (1991), for example.
  • an "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • a “neutralizing antibody” is an antibody molecule that is able to eliminate or significantly reduce an effector function of a target antigen to which it binds. Accordingly, a “neutralizing" Ten-M4 antibody is capable of eliminating or significantly reducing an effector function, such as ten-M4 activity. In one embodiment, a neutralizing antibody will reduce an effector function by 1-10, 10-20, 20-30, 30-50, 50-70, 70-80, 80-90, 90-95, 95-99, 99-100%. In one embodiment, the Ten-M4 antibody inhibits function by inhibiting, to some extent, the dimerization of two Ten-M4 proteins.
  • the Ten-M4 antibody inhibits function by inhibiting, to some extent, the association of the dimerized Ten-M4 protein duplex with another protein.
  • the neutralizing antibody inhibits dimmer formation by directly binding to the location on the Ten-M4 protein that binds to a second ten-M4 protein.
  • the neutralizing antibody binds to one part of the Ten-M4 protein, while a part of the antibody, or something associated with the antibody, blocks the dimerzation of the Ten-M4 proteins.
  • the antibody binds to the Ten-M4 protein and induces a conformational change in the protein which prevents dimerization from occurring.
  • the Ten-M4 antibody actually increases the likelihood of dimerization occurring.
  • the antibodies are activating antibodies and binding of the antibody functions to effectively cause the Ten-M4 protein to act as if it had dimerized with another ten-M4 protein.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell- mediated reaction in which non-specific cytotoxic cells that express Ig Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Ig Fc receptors
  • the primary cells for mediating ADCC, NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcRs expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US Patent No. 5,500,362, or 5,821,337.
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1988).
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the Ig light-chain and heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Digestion of antibodies with the enzyme, papain results in two identical antigen-binding fragments, known also as "Fab” fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize.
  • F(ab') 2 fragment Digestion of antibodies with the enzyme, pepsin, results in the a F(ab') 2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites.
  • the F(ab') 2 fragment has the ability to crosslink antigen.
  • Fv when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites.
  • Fab when used herein refers to a fragment of an antibody which comprises the constant domain of the light chain and the CHl domain of the heavy chain.
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" ⁇ e.g. residues 24-34 (Ll), 50-62 (L2), and 89-97 (L3) in the light chain variable domain and 31-55 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • CDRs complementarity determining regions
  • the CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to bind an antigen when the dissociation constant is ⁇ 1 ⁇ M, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • An increased or greater dissociation constant ("K D ") means that there is less affinity between the epitope and the antibody. In other words, that the antibody and the epitope are less favorable to bind or stay bound together.
  • a decrease of lower dissociation constant means that there is a higher affinity between the epitope and the antibody.
  • An antibody with a K D of "no more than" a certain amount means that the antibody will bind to the epitope with the given affinity, or more strongly (or tightly).
  • K D describes the binding characteristics of an epitope and an antibody
  • potency describes the effectiveness of the antibody itself for a function of the antibody.
  • a relatively low K D does not automatically mean a high potency.
  • antibodies can have a relatively low K D and a high potency (e.g., they bind well and alter the function strongly), a relatively high K D and a high potency (e.g., they don't bind well but have a strong impact on function), a relatively low K D and a low potency (e.g., they bind well, but not in a manner effective to alter a particular function) or a relatively high K D and a low potency (e.g., they simply do not bind to the target well).
  • high potency means that there is a high level of inhibition with a low concentration of antibody.
  • an antibody is potent or has a high potency when its ICs 0 is a small value, for example, 1300-600, 600-200, 200-130, 130-120, 12-50, 50-10, 10-1 or less pM.
  • substantially unless otherwise specified in conjunction with another term, means that the value can vary within the any amount that is contributable to errors in measurement that may occur during the creation or practice of the embodiments. "Significant” means that the value can vary as long as it is sufficient to allow the claimed invention to function for its intended use. [0098]
  • the term "selectively bind" in reference to an antibody does not mean that the antibody only binds to a single substance. Rather, it denotes that the K D of the antibody to a first substance is less than the K D of the antibody to a second substance. Antibodies that exclusively bind to an epitope only bind to that single epitope.
  • amino acid or “amino acid residue,” as used herein, refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants.
  • amino acids are used herein
  • mAb refers to monoclonal antibody.
  • XENOMOUSE® refers to strains of mice which have been engineered to contain 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, as described in Green et al. Nature Genetics 7:13-21 (1994), incorporated herein by reference. The XENOMOUSE ® strains are available from Abgenix, Inc. (Fremont, CA).
  • XENOMAX ® refers use of to the use of the "Selected
  • Lymphocyte Antibody Method (Babcook et al., Proc. Natl. Acad. Sci. USA, i93:7843-7848 (1996)), when used with XENOMOUSE ® animals.
  • SLAM ® refers to the "Selected Lymphocyte Antibody Method” (Babcook et al., Proc. Natl. Acad. Sci. USA, i93:7843-7848 (1996), and Schrader, US Patent No. 5,627,052), both of which are incorporated by reference in their entireties.
  • disease refers to a physiological state of a cell or of a whole mammal in which an interruption, cessation, or disorder of cellular or body functions, systems, or organs has occurred.
  • symptom means any physical or observable manifestation of a disorder, whether it is generally characteristic of that disorder or not.
  • symptoms can mean all such manifestations or any subset thereof.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized ⁇ i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the term "inhibit,” when used in conjunction with a disease or symptom can mean that the antibody can reduce or eliminate the disease or symptom.
  • administering means to deliver to a patient.
  • such delivery can be intravenous, intraperitoneal, by inhalation, intramuscular, subcutaneous, oral, topical, transdermal, or surgical.
  • “Therapeutically effective amount,” for purposes of treatment, means an amount such that an observable change in the patient's condition and/or symptoms could result from its administration, either alone or in combination with other treatment.
  • a "pharmaceutically acceptable vehicle,” for the purposes of treatment, is a physical embodiment that can be administered to a patient.
  • Pharmaceutically acceptable vehicles can be, but are not limited to, pills, capsules, caplets, tablets, orally administered fluids, injectable fluids, sprays, aerosols, lozenges, neutraceuticals, creams, lotions, oils, solutions, pastes, powders, vapors, or liquids.
  • a pharmaceutically acceptable vehicle is a buffered isotonic solution, such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Down-regulate for purposes of treatment, means to lower the level of a particular target composition.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as monkeys, dogs, horses, cats, cows, etc.
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms ofDNA.
  • isolated polynucleotide shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • oligonucleotide includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages.
  • Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides can be either sense or antisense oligonucleotides.
  • nucleotide as used herein includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides referred to herein includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res.
  • oligonucleotide can include a label for detection, if desired.
  • the term "selectively hybridize” referred to herein means to detectably and specifically bind.
  • Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • the nucleic acid sequence homology between the polynucleotides, oligonucleotides, or antibody fragments and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are connected. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • operably linked refers to positions of components so described that are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus.
  • Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules represented by SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, and 38, for example, and the human kappa light chain immunoglobulin molecules represented by SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40, for example, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.
  • the antibody has a sequence from the heavy chain CDRl, CDR2, CDR3, FRl, FR2, FR3, and/or FR4 or any of the sequences listed in FIG. 12. In some embodiments, the antibody has a sequence from the light chain CDRl, CDR2, CDR3, FRl, FR2, FR3, and/or FR4 or any of the sequences listed FIG. 13.
  • the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ - carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserme, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the lefthand direction is the amino terminal direction and the righthand direction is the carboxy- terminal direction, in accordance with standard usage and convention.
  • polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.
  • the following terms are among those used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, “substantial identity”, and “homology.”
  • a “reference sequence” is a defined sequence used as a basis for a sequence comparison.
  • a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence.
  • a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length.
  • two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences
  • sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least about 18 contiguous nucleotide positions or about 6 amino acids wherein the polynucleotide sequence or amino acid sequence is compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may include additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.
  • sequence identity means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • substantially identical denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window.
  • the reference sequence may be a subset of a larger sequence.
  • Two amino acid sequences or polynucleotide sequences are "homologous" if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10.
  • the two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
  • minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%.
  • conservative amino acid replacements are contemplated.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • serine and threonine are aliphatic-hydroxy family
  • asparagine and glutamine are an amide-containing family
  • alanine, valine, leucine and isoleucine are an aliphatic family
  • phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). The foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physiocochemical or functional properties of such analogs.
  • Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), which are each incorporated herein by reference.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long. In other embodiments polypeptide fragments are at least 25 amino acids long, more preferably at least 50 amino acids long, and even more preferably at least 70 amino acids long.
  • Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non- peptide compound are termed "peptide mimetics" or "peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • a paradigm polypeptide i.e., a polypeptide that has a biochemical property or pharmacological activity
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, " Tc, ⁇ l In 5 125 1, 131 1), fluorescent labels (e.g., FITC 5 rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • radioisotopes or radionuclides e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, " Tc, ⁇ l In 5 125 1, 131 1
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporated herein by reference).
  • an "agent” refers to a substance that is useful in the treatment of active disease, prophylactic treatment, or diagnosis of a mammal including, but not restricted to, a human, bovine, equine, porcine, murine, canine, feline, or any other warm-blooded animal.
  • the agent is selected from the group of radioisotope, toxin, pharmaceutical agent, oligonucleotide, cytotoxic agents, recombinant protein, antibody fragment, anti-cancer agents, anti-adhesion agents, anti-thrombosis agents, anti-restenosis agents, anti-autoimmune agents, anti-aggregation agents, anti-bacterial agents, anti-viral agents, and anti-inflammatory agents.
  • anti-viral agents including acyclovir, ganciclovir, and zidovudine
  • anti-thrombosis/resteno- sis agents including cilostazol, dalteparin sodium, reviparin sodium, and aspirin
  • anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib, and nimesulid
  • anti- autoimmune agents including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide
  • anti-adhesion/anti-aggregation agents including limaprost, clorcromene
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • the Ten-M proteins are a class of type IItransmembrane proteins containing a short intracellular N-terminus, which is followed by a transmembrane region, which is followed by 8 EGF-like repeats (Epidermal Growth Factor-like repeats), which is followed by a large globular domain on the extracellular side.
  • Ten-M4 denotes a particular member of that family of proteins. Ten-M4 is also known as CR105 or curagen 105. Ten-M4 cDNA can be highly similar if not identical to DOC4 cDNA.
  • the amino acid sequence of a human Ten-M4 protein is shown in FIG. IA, SEQ ID NO: 41. The highlighted section represents the antigen used for the present antibodies.
  • a nucleic acid sequence is displayed in FIG. IB, SEQ ID NO: 42.
  • the isolated section of the Ten-M4 protein used in the present application is displayed in FIG. 1C, SEQ ID NO: 43.
  • FIG. ID The general layout of the Ten-M4 protein and its various components (e.g., TM, intracellular, extracellular, EGF-like repeat, and C terminal globular region) are shown in FIG. ID.
  • FIG. ID also depicts the actual modified (V5- 6xHis) peptide used as the antigen to generate the present antibodies.
  • V5- 6xHis modified modified peptide used as the antigen to generate the present antibodies.
  • the present antibodies can prevent the formation of a ten-M4/Ten-M4 duplex on a cancerous cell and thereby reduce the likelihood that a cancer will spread to another location.
  • the antibody can prevent or reduce the formation of the Ten-M4/Ten-M4 duplex formation in a number of ways.
  • the antibody can directly bind to the section of the Ten-M4 protein that is involved in binding for Ten-M4/Ten-M4 duplex formation (e.g., an EGF-like repeat) and thus prevent the two Ten-M4 proteins from effectively binding together.
  • the antibody directly binds to the EGF-like repeat or repeats that are involved in duplex formation.
  • the antibody can be created to bind to any one or multiple EGF-like repeats, including, for example, the 1 st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , and 8 th repeat.
  • the antibody can bind to the second and fourth EGF-like repeats.
  • Fully human antibodies that bind to these particular sections can be generated by the methods disclosed herein and the knowledge of one of skill in the art.
  • the antibody can bind at another location and the nonbonding section of the antibody can sterically interfere with the binding of the two halves of the protein.
  • the antibody can bind to a location on the Ten-M4 proteins and induce a conformational change in the protein that will prevent duplex formation.
  • the antibody binds in such a way as to allow for the binding of the two Ten-M4 proteins, but so as to prevent any functional signaling from occurring. In some embodiments, this involves the antibody binding to only a part of the section of the Ten-M4 protein directly involved in duplex formation (e.g., one EGF-like repeat), while the antibody does not interfere with the binding of the two Ten-M4 proteins otherwise (e.g., the other EGF-like repeat will still bind together).
  • This particular antibody has the advantage of reducing the number of Ten-M4 proteins available to form duplexes, as each antibody can bind to two Ten-M4 proteins. Similarly, antibodies that bind to two Ten-M4 proteins at once can also have this advantage in some embodiments.
  • the antibody actually promotes the dissociation of the Ten-M4/Ten-M4 duplex. In other embodiments, the antibody promotes the formation of the Ten-M4/Ten-M4 duplex.
  • Such antibodies can be created by raising antibodies against particular locations of the Ten-M4 protein or duplex thereof, so that the antibody, when bound to the Ten- M4 protein or duplex thereof, will help stabilize the other state (individual or duplexed) of the Ten-M4 protein.
  • the antibodies to Ten-M4 are selective for various forms of Ten-M and various forms of Ten-M4.
  • the antibody to Ten-M4 will bind to Ten-M4 more tightly than it will to other forms of Ten-M (e.g., Ten-M2, Ten-Mi, and Ten-M3).
  • the antibody can bind 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 fold more tightly to Ten-M4 than one of or any combination of the other Ten-M proteins.
  • the antibodies are selective for cell associated and cell dissociated Ten-M proteins and Ten-M4 in particular.
  • the antibodies can bind more tightly to a Ten-M4 protein that is attached to the cell surface.
  • the antibodies can bind more tightly to a Ten-M4 protein that has been shed or that is secreted or cleaved from a cell. In some embodiments, the antibody can bind to both forms equally as strongly. In other embodiments, the antibody can effectively bind to only one form of the Ten-M4 protein.
  • the selectivity can be any amount, for example, from 2-10, 10-20, 20-30, 30-40, 40-50, fold more selective, or more, for one form compared to the other form. An example of how to generate such selective antibodies and determine such selectivity is presented below in the examples. [0147] In other embodiments, the antibodies that bind to Ten-M4 are associated with an agent or compound of some type.
  • the association of the agent with the antibody allows for the delivery of the agent or compound to cells that express Ten-M4.
  • Ten-M4 is expressed in cancerous cells; therefore, this combination allows the delivery of an agent, such as a cytotoxic agent or therapeutic agent, to a cancer cell.
  • the agent can be associated with the antibody in a variety of ways, for example, it can be directly linked to the antibody, attached via a linker (which can be a cleavable linker), or associated via a secondary antibody.
  • the antibodies comprise epitopes so as to allow binding to the antibodies by other antibodies or agents.
  • the exact manner by which the agent is associated with the toxin is not critical to the device or method. This, and other issues associated with these compositions and methods of using them are discussed in more detail below, with a particular emphasis in the section entitled "Design and Generation of Other Therapeutics.”
  • 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 1 10 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, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. 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 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 two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FRl, CDRl, 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. MoI Biol. 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). 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).
  • Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.
  • XENOMOUSE ® strains of mice which have been engineered to contain 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus. See Green et al. Nature Genetics 7:13-21 (1994).
  • the XENOMOUSE ® strains are available from Abgenix, Inc. (Fremont, CA).
  • minilocus In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V H genes, one or more D H genes, one or more J H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Patent No. 5,545,807 to Surani et al. and U.S. Patent Nos.
  • Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See European Patent Application Nos. 773 288 and 843 961, the disclosures of which are hereby incorporated by reference in their entireties.
  • HAMA Human anti-mouse antibody
  • HACA human anti-chimeric antibody
  • mice were prepared using the XENOMOUSE ® technology, as described below. Such mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Technologies utilized for achieving the same are disclosed in the patents, applications, and references referred to herein. In particular, however, a preferred embodiment of transgenic production of mice and antibodies therefrom is disclosed in U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000, the disclosures of which are hereby incorporated by reference. See also Mendez et al. Nature Genetics 15:146-156 (1997), the disclosure of which is hereby incorporated by reference.
  • XENOMOUSE ® lines of mice are immunized with an antigen of interest ⁇ e.g., human Ten-M4), lymphatic cells (such as B-cells) are recovered from mice that expressed antibodies, and the recovered cell lines are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines.
  • lymphatic cells such as B-cells
  • myeloid-type cell line to prepare immortal hybridoma cell lines.
  • These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest.
  • methods for the production of multiple hybridoma cell lines that produce antibodies specific to the desired multimeric enzyme subunit oligomerization domain are provided herein.
  • characterization of the antibodies produced by such cell lines including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies.
  • the recovered cells are screened further for reactivity against the initial antigen, preferably human Ten-M4.
  • the initial antigen preferably human Ten-M4.
  • Such screening includes ELISA with the desired Ten-M4 protein and functional assays such as TenM4 mediated antibody internalization.
  • Single B cells secreting antibodies of interest are then isolated using a desired Ten-M4-specific hemolytic plaque assay (Babcook et al., Proc. Natl. Acad. Sci. USA, i93:7843-7848 (1996)).
  • Cells targeted for lysis are preferably sheep red blood cells (SRBCs) coated with the desired ten-M4 antigen.
  • SRBCs sheep red blood cells coated with the desired ten-M4 antigen.
  • B cell culture secreting the immunoglobulin of interest and complement the formation of a plaque indicates specific Ten-M4-mediated lysis of the target cells.
  • the single antigen-specific plasma cell in the center of the plaque can be isolated and the genetic information that encodes the specificity of the antibody is isolated from the single plasma cell.
  • the DNA encoding the variable region of the antibody secreted can be cloned.
  • Such cloned DNA can then be further inserted into a suitable expression vector, preferably a vector cassette such as a pcDNA, more preferably such a pcDNA vector containing the constant domains of immunoglobulin heavy and light chain.
  • the generated vector can then be transfected into host cells, preferably CHO cells, and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • host cells preferably CHO cells
  • conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • antibodies produced by the above-mentioned cell lines possessed fully human IgGl or IgG2 heavy chains with human kappa light chains.
  • the antibodies possessed high affinities, typically possessing Kd's of from about 10 "9 through about 10 "13 M, when measured by either solid phase and solution phase.
  • anti-Ten-M4 antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference).
  • the transformation procedure used depends upon the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • ATCC American Type Culture Collection
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with Ten-M4 binding properties.
  • antibodies generated from the antigen will bind to the full protein in its native environment.
  • antibodies are tested for and selected for binding to the native protein, rather than variants of the original protein or antigen.
  • Anti-Ten-M4 antibodies can have therapeutic effects in treating symptoms and conditions related to Ten-M4 activity.
  • the antibodies can inhibit the formation of the ten-M4/ten-M4 duplex, thereby inhibiting cancer metastasis, or the antibodies can be associated with an agent and deliver a lethal toxin to a targeted cell.
  • the anti-Ten- M4 antibodies are useful as diagnostics for the disease states, especially cancer and the metastasis of cancer.
  • the isotype of an anti-Ten-M4 antibody can be switched, for example to take advantage of a biological property of a different isotype.
  • the antibodies in some circumstances it may be desirable in connection with the generation of antibodies as therapeutic antibodies against Ten-M4 that the antibodies be capable of fixing complement and participating in complement-dependent cytotoxicity (CDC).
  • CDC complement-dependent cytotoxicity
  • isotypes of antibodies that are capable of the same, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGl, and human IgG3.
  • antibodies that are generated need not initially possess such an isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art.
  • Such techniques include the use of direct recombinant techniques (see e.g., U.S. Patent No. 4,816,397), cell-cell fusion techniques ⁇ see e.g., U.S. Patent Nos. 5,916,771 and 6,207,418), among others.
  • the anti-ten-M4 antibodies discussed herein are human antibodies. If an antibody possessed desired binding to Ten-M4, it could be readily isotype switched to generate a human IgM, human IgGl, or human IgG3 isotype, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule would then be capable of fixing complement and participating in CDC.
  • a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the light chain.
  • Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated.
  • Biologically active antibodies that bind Ten-M4 are preferably used in a sterile pharmaceutical preparation or formulation to reduce the activity of Ten-M4.
  • Anti-Ten- M4 antibodies preferably possess adequate affinity to potently suppress Ten-M4 activity to within the target therapeutic range. The suppression can result from the ability of the antibody to interfere with the binding of Ten-M4 to another Ten-M4 protein. Additionally, the antibodies can alter the conformation or the Ten-M4 proteins so that Ten-M4 signaling events do not generally occur.
  • the antibody formulation is preferably sterile. This is readily accomplished by any method know in the art, for example by filtration through sterile filtration membranes.
  • the antibody ordinarily will be stored in lyophilized form or in solution. Sterile filtration may be performed prior to or following lyophilization and reconstitution.
  • Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • the route of antibody administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, or by sustained release systems as noted below.
  • the antibody is preferably administered by infusion or by bolus injection.
  • a therapeutic composition comprising the antibody can be administered through the nose or lung, preferably as a liquid or powder aerosol (lyophilized).
  • the composition may also be administered intravenously, parenterally or subcutaneously as desired.
  • the therapeutic composition should be sterile, pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability. These conditions are known to those skilled in the art.
  • Antibodies for therapeutic use are typically prepared with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. Briefly, dosage formulations of the antibodies described herein are prepared for storage or administration by mixing the antibody having the desired degree of purity with one or more physiologically acceptable carriers, excipients, or stabilizers.
  • formulations may include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LipofectinTM
  • the formulation may include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol.
  • buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts
  • antioxidants such as ascorbic
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20 th ed, Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
  • the antibodies can also be administered in and released over time from sustained-release preparations.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide.
  • the matrices may be in the form of shaped articles, films or microcapsules.
  • sustained- release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. B ⁇ omed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days
  • certain hydrogels release proteins for shorter time periods.
  • encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved.
  • stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • Sustained-released compositions also include preparations of crystals of the antibody suspended in suitable formulations capable of maintaining crystals in suspension. These preparations when injected subcutaneously or intraperitonealy can produce a sustained release effect.
  • Other compositions also include liposomally entrapped antibodies. Liposomes containing such antibodies are prepared by methods known per se: U.S. Pat. No. DE 3,218,121; Epstein et al, Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692; Hwang et al, Proc. Natl. Acad. Sci.
  • the dosage of the antibody formulation for a given patient may be determined by the attending physician. In determining the appropriate dosage the physician may take into consideration various factors known to modify the action of therapeutics, including, for example, severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Therapeutically effective dosages may be determined by either in vitro or in vivo methods.
  • an effective amount of the antibodies, described herein, to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it is preferred for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • a typical daily dosage might range from about 0.001 mg/kg to up to 100 mg/kg or more, depending on the factors mentioned above.
  • the clinician will administer the therapeutic antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.
  • the antibodies described herein will have therapeutic effect in treatment of symptoms and conditions resulting from or related to the activity of Ten-M4.
  • Such modalities include, without limitation, advanced antibody therapeutics, such as bispecif ⁇ e antibodies, immunotoxins, and radiolabeled therapeutics, generation of peptide therapeutics, gene therapies, particularly intrabodies, antisense therapeutics, and small molecules.
  • advanced antibody therapeutics such as bispecif ⁇ e antibodies, immunotoxins, and radiolabeled therapeutics
  • generation of peptide therapeutics gene therapies, particularly intrabodies, antisense therapeutics, and small molecules.
  • bispecific antibodies can be generated that comprise (i) two antibodies, one with a specificity to Ten-M4 and another to a second molecule, that are conjugated together, (ii) a single antibody that has one chain specific to Ten-M4 and a second chain specific to a second molecule, or (iii) a single chain antibody that has specificity to both Ten-M4 and the other molecule.
  • Such bispecific antibodies can be generated using techniques that are well known; for example, in connection with (i) and (ii) see e.g., Fanger et al. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra, and in connection with (iii) see e.g., Traunecker et al.
  • the second specificity can be made as desired.
  • the second specificity can be made to the heavy chain activation receptors, including, without limitation, CD 16 or CD64 ⁇ see e.g., Deo et al. 18:127 (1997)) or CD89 ⁇ see e.g., Valerius et al. Blood 90:4485-4492 (1997)).
  • the antibodies are designed so as to bind to two Ten-M4 proteins.
  • the antibodies are designed so as to bind to two Ten-M4 proteins, and to further prevent the two Ten- M4 proteins from actually contacting one another in a manner so as to allow signaling to occur.
  • the result can be beneficial in that the Ten-M4 is being prevented from signaling by the antibody, and each antibody can stop two Ten-M4 molecules.
  • Antibodies can also be modified to act as immunotoxins utilizing techniques that are well known in the art. See e.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Patent No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared utilizing techniques that are well known in the art. See e.g., Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafher and Longo, eds., Lippincott Raven (1996)). See also U.S. Patent Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902.
  • a pharmaceutical composition comprising an effective amount of the antibody in association with a pharmaceutically acceptable carrier or diluent is provided.
  • an anti-Ten-M4 antibody is linked to an agent ⁇ e.g., radioisotope, pharmaceutical composition, or a toxin).
  • agents e.g., radioisotope, pharmaceutical composition, or a toxin.
  • such antibodies can be used for the treatment of diseases, such diseases can relate to the over or under expression of ten-M proteins and ten-M4 in particular.
  • the drug possesses the pharmaceutical property selected from the group of antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic, alkaloid, COX-2, and antibiotic agents and combinations thereof.
  • the drug can be selected from the group of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antimetabolites, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, oxaliplatin, doxorubicins and their analogs, and a combination thereof.
  • toxins further include gelonin, Pseudomonas exotoxin (PE),
  • radioisotopes examples include gamma-emitters, positron-emitters, and x- ray emitters that may be used for localization and/or therapy, and beta-emitters and alpha- emitters that may be used for therapy.
  • the radioisotopes described previously as useful for diagnostics, prognostics and staging are also useful for therapeutics.
  • Non-limiting examples of anti-cancer or anti-leukemia agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, and morpholino and substituted derivatives, combinations and modifications thereof.
  • anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, and morpholino and substituted derivatives, combinations and modifications thereof.
  • Exemplary pharmaceutical agents include cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, and bleomycin, and derivatives, combinations and modifications thereof.
  • the anti-cancer or anti-leukemia is doxorubicin, morpholinodoxorubicin, or mo ⁇ holinodaunorubicin.
  • affinity values can be important, other factors can be as important or more so, depending upon the particular function of the antibody.
  • an immunotoxin toxin associated with an antibody
  • the act of binding of the antibody to the target can be useful; however, in some embodiments, it is the internalization of the toxin into the cell that is the desired end result.
  • antibodies with a high percent internalization can be desirable in these situations. However, they need not be desirable if the antibody is to prevent duplex formation of the Ten- M4 protein with another Ten-M4 protein.
  • antibodies with a high efficiency in internalization are contemplated.
  • a high efficiency of internalization can be measured as a percent internalized antibody, and can be from a low value to 100%.
  • 0.1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-99, and 99-100% can be a high efficiency.
  • the desirable efficiency can be different in different embodiments, depending upon, for example, the associated agent, the amount of antibody that can be administered to an area, the side effects of the antibody-agent complex, the type ⁇ e.g., cancer type) and severity of the problem to be treated.
  • the antibodies disclosed herein provide an assay kit for the detection of Ten-M4 protein in mammalian tissues or cells in order to screen for a disease or disorder associated with changes in levels of Ten-M4.
  • the kit comprises an antibody that binds the antigen protein and means for indicating the reaction of the antibody with the antigen, if present.
  • an article of manufacture comprising a container, comprising a composition containing an anti-Ten-M4 antibody, and a package insert or label indicating that the composition can be used to treat disease mediated by Ten-M4.
  • a mammal and, more preferably, a human receives the anti-Ten-M4 antibody.
  • EXAMPLE 1 Antibody Generation Immunization of Animals.
  • Monoclonal antibodies against ten-M4 were developed by immunizing XenoMouse® mice (IgG2 Kappa XenoMouse Strain), Abgenix, Inc. Fremont, CA) using an antigen with the sequence depicted in FIG. 1C.
  • the antigen also had a V5-6xHis tag added, as shown in FIG. ID.
  • the antigen that was used to immunize the mice was amino acids 371-830, followed by the V5-HIS tag, as shown in FIG. 1C and FIG. ID.
  • Hybridomas produced from the above immunized mouse were screened for ten-M4 specific monoclonal antibodies.
  • the ELISA plates were coated with soluble ten-M4 (truncated ECD) and incubated at 4°C overnight. After the incubation, the plates were washed with Washing Buffer (0.05% Tween 20 in PBS) 3 times. Blocking Buffer (200 ⁇ L/well, 0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in Ix PBS) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed with Washing Buffer three times. Hybridoma supernatant (50 ⁇ L/well), positive and negative controls were added and the plates were incubated at room temperature for 2 hours [0197] After incubation, the plates were washed three times with Washing Buffer.
  • Goat anti-hulgGfc-HRP detection antibody 100 ⁇ L/well was added and the plates were incubated at room temperature for 1 hour. After the incubation, the plates were washed three times with Washing Buffer. TMB substrate (100 ⁇ L/well) was added and the plates allowed to develop for about 10 minutes (until negative control wells barely started to show color), then 50 ⁇ L/well of stop solution was added and the plates read on an ELISA plate reader at wavelength 450nm.
  • Hybridoma supernatants that were identified as positive for binding in the above ELISA were then assayed for the ability to bind to endogenously expressed TenM4 using the PC3 cell line which naturally expresses the antigen.
  • a FMAT based fluorescence assay was performed for 208 samples identified in the screen above. Briefly, PC3 cells were seeded at 10,000 cells per well in a 96- well microtiter dish. After the cells had adhered, the media was removed and replaced with hybridoma supernatant. After an one hour incubation, the cells were washed and the bound antibody was detected via a Cy5-co ⁇ jugated anti-Human IgGFc specific polyclonal antibody. Positive wells were imaged using the FMAT reader. Table 2, below, summarizes the number of hybridoma lines that bound to the soluble Ten-M4 (Curl 05) truncated ECD and the number of lines which then also bound to the PC3 cell line.
  • variable heavy chains and the variable light chains for the antibodies shown in Table 1 were sequenced to determine their DNA sequences.
  • the complete sequence information for examples of the anti-Ten-M4 antibodies are shown in the sequence listing submitted herewith, including nucleotide and amino acid sequences.
  • the sequences are also displayed in FIG. 2 through FIG. 13.
  • FIG. 12 displays a sequence comparison of the various hybridoma derived antibody heavy chain regions to a particular germ line heavy chain region.
  • FIG. 13 displays a sequence comparison of the various hybridoma derived antibody light chain regions to a particular germ line light chain region.
  • ELISA plates were coated with a truncated form of soluble Ten-M4 corresponding to the extracellular domain (ECD) having amino acids 370-830.
  • the plates also had soluble Ten-M2 and a V5-HIS peptide and incubated 4°C overnight. After incubation, the plates were washed with Washing Buffer (0.05% Tween 20 in PBS) 3 times. Blocking Buffer (1.0% milk, Ix PBS, 200 ⁇ L/well) was added and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed with Washing Buffer three times.
  • Hybridoma supernatants 50 ⁇ L/well
  • positive and negative controls were added and the plates incubated at room temperature for 2 hours. After incubation, the plates were washed three times with Washing Buffer. Goat anti-hulgGfc-HRP detection antibody (50 ⁇ L/well) was added and the plates were incubated at room temperature for 1 hour. After the incubation, the plates were washed three times with Washing Buffer. TMB substrate (100 ⁇ L/well) was added and the plates allowed to develop for about 30 minutes and then 50 ⁇ L/well of stop solution was added and the plates read on an ELISA plate reader at wavelength 450nm.
  • the described antibodies demonstrated an increase in binding relative to the background level. These results demonstrated that the antibodies can be relatively selective or specific for binding to Ten-M4 over other, closely related antigens and proteins.
  • mAbl.44 demonstrated an increase in selectivity for Ten-M4 compared to Ten-M2
  • mAbl.20 demonstrated an increase in activity towards Ten-M4 over Ten-M2.
  • EXAMPLE 4 INTERNALIZATION ASSAYS QF VARIOUS TEN-M4 ANTIBODIES
  • PC3 cells were removed from culture dishes using Cell Dissociation Media (Sigma), counted, and transferred (100,000 cells) to a 96-well VEE bottom plate. The cells were spun down, the media removed, and the cells resuspended with lOOuL of hybridoma supernatant and incubated for 30 min on ice. The incubation cells were spun down, and bound antibody was detected using a secondary antibody which had been linked to Alex647 dye via a disulphide linkage (anti-Hu IgG Fc-SS-Alexa 647 or anti-Hu IgG Fab-SS-Alexa647 @ 1 ⁇ g/ml). The secondary antibody was incubated for 7 min on ice. After the incubation, cells were washed and resuspended with ice cold 10% FCS/PBS. The sample was then split into three samples, the cells were spun down, and the supernatant removed.
  • the third buffer was 250 ⁇ L of cold 10% FCS/PBS, which was added to the other 4 degree sample. This sample was a control to show the maximum fluorescence. [0209] The samples were then incubated on ice for 30 minutes, spun down, and resuspended with 300 ⁇ L of ice cold 10% FCS/PBS and analyzed by Flow Cytometry. [0210] The results are displayed in Table 4.
  • This example demonstrates how an antibody conjugated to a toxin was used as an effective composition to prevent cancer cells from proliferating.
  • a clonogenic assay was used to determine whether primary antibodies could induce cancer cell death when the antibody was conjugated with a saporin toxin conjugated secondary antibody reagent.
  • a saporin toxin conjugated secondary antibody reagent for example, as described in Kohls and Lappi, "Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin," Biotechniques, 28(l):162-5 (Jan. 2000), hereby incorporated by reference in its entirety).
  • V genes that are associated with the particular antibodies characterized above.
  • the V genes in many of the antibodies were analyzed to determine which genes had been employed in the particular antibodies.
  • the V genes involved in both the heavy and the light chains of the antibodies are presented in Table 6 below.
  • This example demonstrates one method for generating an antibody and determining if a given Ten-M4 antibody can effectively prevent the formation of the Ten- M4/Ten-M4 duplex.
  • Various sections of the Ten-M4 protein are generated as a selection of antigens.
  • the sections are approximately 10-20 amino acids in length and comprise overlapping sections of SEQ ID NO: 41 or 43.
  • amino acids in the EGF-like repeats especially those in the EGF-like repeats responsible for duplex formation, can be used to generate antibodies.
  • Antibodies created from such antigens are then incubated with Ten-M4 proteins that are expressed on 786-0 cells.
  • the 786-0 cells have an additional marker on or in them so that they may readily be followed.
  • the 786-0 cells are then placed on an experimental medium similar to that discussed in Example 10. Antibodies that result in cells that are no longer able to migrate in the medium can be effective in preventing duplex formation.
  • Duplex formation can also be tested and confirmed in vivo. Additionally, binding can be detected in devices such as a BIACORE, in which a Ten-M4 is attached to the chip surface, various antibodies are flowed over the chip, binding of the antibody detected, followed by the flowing the above 786-0 cells, and binding (or absence thereof) of the cells being detected.
  • devices such as a BIACORE, in which a Ten-M4 is attached to the chip surface, various antibodies are flowed over the chip, binding of the antibody detected, followed by the flowing the above 786-0 cells, and binding (or absence thereof) of the cells being detected.
  • a cell migration assay identifies proteins that promote or inhibit cell motility.
  • Cells expressing Ten-M4 proteins are placed in a chamber (1x10 4 - 3x10 4 cells/well) suspended above a membrane and a lower chamber.
  • the lower chamber contains purified Ten-M4 protein.
  • the cells are incubated for 6 to 24 h and then assayed quantitatively for migratory activity.
  • Collagen I coated inserts (Becton Dickinson) are prepared by rehydration for at least 30 minutes at room temperature with 300 ⁇ L of basal media. Approximately 90% confluent Tl 75 cells are treated with 5 mL trypsin, neutralized with 10 mL neutralization solution (complete media), and centrifuged for 7 minutes at 800 RPM. Cells are resuspended in 10 mL basal media containing 0.1%BSA (diluent) and spun again. Cells are resuspended in diluent, counted, and adjusted to an optimized cell concentration.
  • a patient suffering from cancer is identified.
  • a dosage of 5 mg/kg of a Ten- M4 antibody that prevents Ten-M4 duplex formation is administered by intravenous injection to the patient.
  • a booster administration is given three weeks later, and every three weeks thereafter.
  • the Ten-M4 antibody causes a partial or complete inhibition of the metastasis of the cancer.
  • a patient suffering from prostate cancer is identified.
  • a dosage of 5 mg/kg of the Ten-M4 antibody conjugated to saporin is administered by intravenous injection to the patient.
  • a booster administration is given three weeks later, and every three weeks thereafter.
  • the Ten-M4 antibody conjugated to saporin causes a partial or complete destruction of the cancer cells in the prostate.

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Abstract

L'invention concerne des anticorps entièrement humains dirigés contre Ten-M4 et des utilisations de ces anticorps. La présente invention porte également sur des anticorps monoclonaux humains dirigés contre Ten-M4, sur des séquences polynucléotidiques isolées codant pour des molécules d'immunoglobuline à chaînes légères et lourdes, sur des séquences d'acide aminé comprenant des molécules d'immunoglobuline à chaînes légères et lourdes, en particulier sur des séquences correspondant à des séquences contiguës à chaînes légères et lourdes couvrant les régions structurales (FR) et/ou les régions déterminant la complémentarité (CDR). L'invention concerne aussi des hybridomes et d'autres lignées cellulaires exprimant ces molécules d'immunoglobuline et ces anticorps monoclonaux. Certains anticorps peuvent inhiber les métastases de tumeurs, et certains peuvent servir à libérer un agent, tel qu'une toxine, dirigé vers une cible particulière.
PCT/US2005/041673 2004-11-17 2005-11-17 Anticorps diriges contre des proteines ten-m et utilisations associees WO2006055704A2 (fr)

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WO2010052288A1 (fr) * 2008-11-07 2010-05-14 Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research Teneurine et cancer
US8361473B2 (en) 2007-03-29 2013-01-29 Technion Research & Development Foundation Ltd. Antibodies and their uses for diagnosis and treatment of cytomegalovirus infection and associated diseases
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US9783618B2 (en) 2013-05-01 2017-10-10 Kymab Limited Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics
US9783593B2 (en) 2013-05-02 2017-10-10 Kymab Limited Antibodies, variable domains and chains tailored for human use
US9788534B2 (en) 2013-03-18 2017-10-17 Kymab Limited Animal models and therapeutic molecules
US9924705B2 (en) 2012-03-28 2018-03-27 Kymab Limited Animal models and therapeutic molecules
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US10251377B2 (en) 2012-03-28 2019-04-09 Kymab Limited Transgenic non-human vertebrate for the expression of class-switched, fully human, antibodies
US10667501B2 (en) 2012-05-17 2020-06-02 Kymab Limited Transgenic non-human vertebrate for the in vivo production of dual specificity immunoglobulins or hypermutated heavy chain only immunoglobulins
US11051497B2 (en) 2011-09-19 2021-07-06 Kymab Limited Manipulation of immunoglobulin gene diversity and multi-antibody therapeutics
US11564380B2 (en) 2009-07-08 2023-01-31 Kymab Limited Animal models and therapeutic molecules
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EP2514766A3 (fr) * 2007-03-29 2013-06-05 Technion Research & Development Foundation Ltd. Anticorps, procédés et kits pour diagnostiquer et traiter un mélanome
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US8642280B2 (en) 2008-11-07 2014-02-04 Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Teneurin and cancer
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