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WO2011053630A1 - Utilisations d'une cytokine de mammifère - Google Patents

Utilisations d'une cytokine de mammifère Download PDF

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Publication number
WO2011053630A1
WO2011053630A1 PCT/US2010/054239 US2010054239W WO2011053630A1 WO 2011053630 A1 WO2011053630 A1 WO 2011053630A1 US 2010054239 W US2010054239 W US 2010054239W WO 2011053630 A1 WO2011053630 A1 WO 2011053630A1
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Prior art keywords
antibody
mdl
antibodies
cell
binding
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English (en)
Inventor
Barbara Joyce-Shaikh
Daniel J. Cua
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Merck Sharp and Dohme LLC
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention provides agonists or antagonists of MDL-1 to modulate metabolic disorders, e.g., obesity.
  • Obesity is a complex, chronic disease characterized by excessive
  • Obesity increases the risk of illness from about 30 serious medical conditions, including osteoarthritis, Type II diabetes, hypertension, cancer, and cardiovascular disease, and is associated with increases in deaths from all causes. Additionally, obesity is associated with depression, and can further affect the quality of life through limited mobility and decreased physical endurance.
  • cardiovascular problems including hypertension, chronic heart disease and stroke
  • conditions associated with insulin resistance namely Non-Insulin Dependent Diabetes Mellitus (NIDDM)
  • NIDDM Non-Insulin Dependent Diabetes Mellitus
  • cancers mainly the hormonally related and large-bowel cancers
  • gallbladder disease mainly the hormonally related and large-bowel cancers
  • Other problems associated with obesity include respiratory difficulties, chronic musculo-skeletal problems, skin problems and infertility.
  • DAP 12 is a disulfide -bonded, homodimeric type I transmembrane
  • ITAM immunoreceptor tyrosine -based activation motif
  • the DAP 12 cytoplasmic domain constitutes the signaling subunit of these receptor complexes.
  • the DAP 12 cytoplasmic IT AM is phosphorylated by Src kinases.
  • the IT AM of DAP 12 then interacts with Syk cytoplasmic tyrosine kinases, which initiates a cascade of events that leads to activation (Lanier et al. (1998) Nature 391 :703-707; Campbell and Colonna (1999) Int. J. Biochem. Cell Biol. 31 :631-636; Lanier and Bakker (2000) Immunol. Today 21 :611-614).
  • DAP12 is expressed in monocytes, macrophages, natural killer (NK) cells, granulocytes, dendritic cells and mast cells, where it provides signaling function for at least eight distinct receptors (Gingras et al. (2001) Mol. Immun. 38:817-824; Lanier and Bakker, (2000) Immunol. Today 21 :611-614).
  • the myeloid receptor of the C-type lectin superfamily associated with DAP 12 is Myeloid DAP12-associating Lectin- 1 (MDL-1), a type II transmembrane protein. MDL-1 was the first DAP 12 associating molecule to be identified and cloned (Bakker et al.
  • DAP 12 a negatively charged residue in the transmembrane domain of DAP 12 precludes its cell surface expression in the absence of a partner receptor, such as MDL-1, which has a positively charged residue in its transmembrane domain.
  • DAP 12 alone is not sufficient for its expression and function at the cell surface.
  • DAP12-associating molecule such as MDL-1
  • DAP 12 may account for transmitting a particular physiological signal via DAP 12 (Nochi et al. (2003) Am. J. of Pathology 162: 1191-1201).
  • the current invention provides use of MDL-1 agonists or antagonists to modulate metabolic disorders.
  • the present invention is based, in part, upon the discovery that MDL-1 KO mice had significant weight gain on normal and high fat diets as compared to a control wild type (WT) mouse.
  • the present invention provides a method of treating a metabolic disorder in a subject comprising administering to the subject an effective amount of an antibody or antibody fragment thereof that specifically binds MDL-1.
  • the antibody is humanized.
  • the antibody is fully human or chimeric,
  • the antibody fragment is a Fab, Fab2, or Fv antibody fragment.
  • the antibody or antibody fragment can be conjugated to another chemical moiety.
  • the chemical moiety is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the antibody or antibody fragment thereof inhibits a metabolic disorder selected from the group consisting of obesity, insulin tolerance, and weight related diabetes.
  • the present invention provides a method of treating a metabolic disorder in a subject comprising administering to the subject an effective amount of an MDL-1 agonist.
  • the MDL-1 agonist is an MDL-1 fusion protein in particular,an MDL-1 extracellular domain linked to a heterologous protein.
  • the metabolic disorder is selected from the group consisting of anorexia, cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, and bulimia.
  • DAP12-associated lectin- 1 DAP-12
  • DAP 12 DAP 12
  • DNAX Activation Protein, 12 kD are well known in the art.
  • polypeptide sequences are disclosed in WO 99/06557.
  • the human MDL-1 nucleotide and amino acid sequences are defined by SEQ ID NO: 11 and SEQ ID NO: 12 of WO 99/06557, respectively.
  • GenBank ® deposits of the human MDL-1 nucleic acid sequence (AR217548) and mouse MDL-1 nucleic and amino acid sequences (AR217549 and AAN21593, respectively) are also available.
  • Soluble forms of MDL-1 are also within the scope of the invention.
  • a structural feature of the MDL-1 protein is the extracellular domain, which is defined by amino acid residues 26 to 188 of a human MDL-1 protein, and amino acid residues 26 to 190 a mouse MDL-1 protein.
  • Soluble MDL-1 protein can be fused to heterologous proteins, e.g., the Fc portion of antibody, or conjugated to chemical moieties, e.g., PEG or human serum albumin (HSA).
  • heterologous proteins e.g., the Fc portion of antibody
  • chemical moieties e.g., PEG or human serum albumin (HSA).
  • Soluble MDL-1 polypeptides may be used as therapeutics or diagnostics similar to the use of MDL-1 antibodies or antigen-binding fragments thereof.
  • the cell surface expression of MDL-1 indicates that this molecule is an attractive target for antibody- based therapeutic strategies.
  • MDL-1 antibodies may be introduced into a patient such that the antibody binds to MDL-1
  • Activity of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity, to the ability to stimulate gene expression, to antigenic activity, to the modulation of activities of other molecules, and the like.
  • Activity of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton.
  • Activity may also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], or the like.
  • administering refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell.
  • administering also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.
  • Treatment refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications.
  • Treatment as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid.
  • Treatment of a cell also encompasses situations where the agent contacts MDL-1 (MDL-1 /DAP 12 heterodimer), e.g., in the fluid phase or colloidal phase, but also situations where the agonist or antagonist does not contact the cell or the receptor.
  • MDL-1 MDL-1 /DAP 12 heterodimer
  • antibody refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies ⁇ e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, etc. so long as they exhibit the desired biological activity.
  • MDL-1 binding fragment encompass a fragment or a derivative of an antibody that still substantially retains its biological activity of inhibiting DAP 12 signaling mediated by MDL-1, such inhibition being referred to herein as "MDL-1 inhibitory activity.” Because antagonists of MDL-1 will have the biological activity of inhibiting DAP 12 signaling, such antagonists are said (interchangeably) to inhibit DAP 12, inhibit MDL-1, or inhibit both MDL-1/DAP12.
  • antibody fragment or MDL-1 binding fragment refers to a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments;
  • a binding fragment or derivative retains at least 10% of its MDL-1 inhibitory activity.
  • a binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its MDL-1 inhibitory activity, although any binding fragment with sufficient affinity to exert the desired biological effect will be useful.
  • a MDL-1 binding fragment can include variants having conservative amino acid substitutions that do not substantially alter its biologic activity.
  • 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 epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. 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 al. (1975) Nature 256: 495, or may be made by recombinant DNA methods ⁇ see, e.g., U.S. Pat. No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581- 597, for example.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies
  • immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • a “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more V H regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two V H regions of a bivalent domain antibody may target the same or different antigens.
  • a “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
  • single-chain Fv or "scFv” antibody refers to antibody fragments comprising the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • the monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231 :25; WO 94/04678; WO 94/25591 ; U.S. Pat. No.
  • the present invention provides single domain antibodies comprising two V H domains with modifications such that single domain antibodies are formed.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L or V L - V H ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP
  • humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the prefix "hum”, "hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies.
  • the humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.
  • the antibodies of the present invention also include antibodies with modified
  • Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol.116:731 at 734-35.
  • the antibodies of the present invention also include antibodies with intact Fc regions that provide full effector functions, e.g. antibodies of isotype IgGl, which induce complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC) in the a targeted cell.
  • antibodies of isotype IgGl which induce complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC) in the a targeted cell.
  • the antibodies of the present invention also include antibodies conjugated to cytotoxic payloads, such as cytotoxic agents or radionuclides.
  • cytotoxic payloads such as cytotoxic agents or radionuclides.
  • cytotoxic agents include ricin, vinca alkaloid, methotrexate, Psuedomonas exotoxin, saporin, diphtheria toxin, cisplatin, doxorubicin, abrin toxin, gelonin and pokeweed antiviral protein.
  • radionuclides for use in
  • immunotherapy with the antibodies of the present invention include 125 I, 131 I, 90 Y, 67 " Cu, 211 At, 177 Lu, 143 Pr and 213 Bi. See, e.g., U.S. Patent Application Publication No. 2006/0014225.
  • Fully human antibody refers to an antibody that comprises human immunoglobulin protein sequences only.
  • a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell.
  • mouse antibody or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.
  • a fully human antibody may be generated in a human being, in a transgenic animal having human immunoglobulin germline sequences, by phage display or other molecular biological methods.
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a "complementarity determining region” or "CDR” (e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and/or those residues from a "hypervariable loop" (i.e.
  • Binding compound refers to a molecule, small molecule, macromolecule, polypeptide, antibody or fragment or analogue thereof, or soluble receptor, capable of binding to a target.
  • Binding compound also may refer to a complex of molecules, e.g., a non-covalent complex, to an ionized molecule, and to a covalently or non-covalently modified molecule, e.g., modified by phosphorylation, acylation, cross-linking, cyclization, or limited cleavage, that is capable of binding to a target.
  • binding compound refers to both antibodies and antigen binding fragments thereof.
  • Binding refers to an association of the binding composition with a target where the association results in reduction in the normal Brownian motion of the binding composition, in cases where the binding composition can be dissolved or suspended in solution.
  • Binding composition refers to a molecule, e.g. a binding compound, in combination with a stabilizer, excipient, salt, buffer, solvent, or additive, capable of binding to a target.
  • a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound.
  • Effective amount encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530.
  • An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.
  • the effect will result in an improvement of a diagnostic measure or parameter by at least 5%, usually by at least 10%, more usually at least 20%, most usually at least 30%>, preferably at least 40%>, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%>, and most ideally at least 90%>, where 100%) is defined as the diagnostic parameter shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of SOPs or Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.
  • Immunode condition encompasses, e.g., pathological inflammation, an inflammatory disorder, and an autoimmune disorder or disease.
  • Immuno condition also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist eradication by the immune system.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples without the agent.
  • Control samples i.e., not treated with agent, are assigned a relative activity value of 100%.
  • Inhibition is achieved when the activity value relative to the control is about 90%o or less, typically 85% or less, more typically 80%> or less, most typically 75% or less, generally 70%> or less, more generally 65%> or less, most generally 60%> or less, typically 55%o or less, usually 50%> or less, more usually 45% or less, most usually 40%> or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
  • Activation is achieved when the activity value relative to the control is about 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
  • Endpoints in activation or inhibition can be monitored as follows. Activation, inhibition, and response to treatment, e.g., of a cell, physiological fluid, tissue, organ, and animal or human subject, can be monitored by an endpoint.
  • the endpoint may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine, toxic oxygen, or a protease.
  • the endpoint may comprise, e.g., a predetermined quantity of ion flux or transport; cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30: 145- 158; Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme et al. (2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med. Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev. Genomics Hum. Genet. 3: 101-128; Bauer, et al. (2001) Glia 36:235-243; Stanimirovic and Satoh (2000) Brain Pathol. 10: 113-126).
  • An endpoint of inhibition is generally 75% of the control or less, preferably
  • an endpoint of activation is at least 150% the control, preferably at least two times the control, more preferably at least four times the control, and most preferably at least 10 times the control.
  • Small molecule is defined as a molecule with a molecular weight that is less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules comprising a radioactive atom, synthetic molecules, peptide mimetics, and antibody mimetics.
  • a small molecule may be more permeable to cells, less susceptible to degradation, and less apt to elicit an immune response than large molecules.
  • Small molecules, such as peptide mimetics of antibodies and cytokines, as well as small molecule toxins are described.
  • a specified ligand binds to a particular receptor and does not bind in a significant amount to other proteins present in the sample.
  • an antibody is said to bind specifically to a polypeptide comprising a given sequence (in this case MDL-1) if it binds to polypeptides comprising the sequence of MDL-1 but does not bind to proteins lacking the sequence of MDL-1.
  • MDL-1 a polypeptide comprising a given sequence
  • an antibody that specifically binds to a polypeptide comprising MDL-1 may bind to a FLAG ® -tagged form of MDL-1 but will not bind to other FLAG ® -tagged proteins.
  • the antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with unrelated antigens.
  • the antibody will have an affinity that is greater than about 10 9 liters/mol, as determined, e.g., by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239.
  • metabolic disorder includes obesity, diabetes, hyperlipidemia and hyperinsulinemia diabetes, hyperlipidemia, hyperinsulinemia, insulin tolerance or resistance, . Also included are anorexia, cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, and bulimia.
  • biological activity include, but are not limited to, free fatty acid level lowering activity, glucose level lowering activity, triglyceride level lowering activity, stimulating adipose lipolysis, stimulating muscle lipid or free fatty acid oxidation, increasing leptin uptake in a liver cell line, significantly reducing the postprandial increase in plasma free fatty acids or glucose due to a high fat meal, significantly reducing or eliminate ketone body production as the result of a high fat meal, increasing glucose uptake in skeletal muscle cells, adipose cells, red blood cells or the brain, increasing insulin sensitivity, inhibiting the progression from impaired glucose tolerance to insulin resistance, reducing body mass, decreasing fat mass, increasing lean muscle mass, preventing or treating an metabolic-related disease or disorder, controlling blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus or Noninsulin Dependent Diabetes Mellitus, treating insulin resistance, preventing the development of insulin resistance and other activities as described
  • the term "obesity" as used herein is defined in the WHO classifications of weight (Kopelman (2000) Nature 404:635643). Underweight is less than 18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is 25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade 3 overweight is greater than or equal to 40.0 BMI.
  • BMI body mass index (morbid obesity) and is kg/m 2 .
  • Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk.
  • greater than or equal to 88 cm indicates an increased risk
  • greater than or equal to 88 cm indicates a substantially increased risk.
  • the waist circumference is measured in cm at midpoint between lower border of ribs and upper border of the pelvis.
  • Other measures of obesity include, but are not limited to, skinfold thickness which is a measurement in cm of skinfold thickness using calipers, and bioimpedance, which is based on the principle that lean mass conducts current better than fat mass because it is primarily an electrolyte solution; measurement of resistance to a weak current (impedance) applied across extremities provides an estimate of body fat using an empirically derived equation.
  • diabetes as used herein is intended to encompass the usual diagnosis of diabetes made from any of the methods included, but not limited to, the following list: symptoms of diabetes (e.g. polyurea, polydipsia, polyphagia) plus casual plasma glucose levels of greater than or equal to 200 mg/dl, wherein casual plasma glucose is defined any time of the day regardless of the timing of meal or drink consumption; 8 hour fasting plasma glucose levels of less than or equal to 126 mg/dl; and plasma glucose levels of greater than or equal to 200 mg/dl 2 hours following oral administration of 75 g anhydrous glucose dissolved in water.
  • symptoms of diabetes e.g. polyurea, polydipsia, polyphagia
  • casual plasma glucose e.g. polyurea, polydipsia, polyphagia
  • IGT equivalent glucose tolerance
  • a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter.
  • glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly.
  • the blood glucose levels are higher and the drop-off level is at a slower rate.
  • Insulin-Resistance Syndrome is intended to encompass the cluster of abnormalities resulting from an attempt to compensate for insulin resistance that sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.
  • CAD coronary artery disease
  • the invention provides methods for reducing or preventing the appearance of insulin-resistance syndrome.
  • insulin resistance is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, including but not restricted to: the intravenous glucose tolerance test or measurement of the fasting insulin level. It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance. Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance. Another way to do this is to follow the approach as disclosed in The New England Journal of Medicine, No. 3, pp. 1188 (1995), i.e. to select obese subjects as an initial criteria for entry into the treatment group.
  • the target of the treatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.
  • a diagnosis of insulin resistance can also be made using the euglycemic glucose clamp test.
  • This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion. During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged. Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body. The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.
  • the present invention includes recombinant versions of the MDL-1 antibody or antigen-binding fragment of the invention.
  • the present invention includes a nucleic acid, which encodes an anti-MDL-1 antibody, an anti-MDL-1 antibody heavy or light chain, an anti- MDL-1 antibody heavy or light chain variable region, an anti-MDL-1 antibody heavy or light chain constant region or anti-MDL-1 antibody CDR (e.g., CDR- LI, CDR-L2, CDR-L3, CDR-H1, CDR-H2 or CDR-H3), which may be amplified by PCR.
  • CDR- LI CDR-L2, CDR-L3, CDR-H1, CDR-H2 or CDR-H3
  • sequence of any nucleic acid may be sequenced by any method known in the art (e.g., chemical sequencing or enzymatic sequencing).
  • “Chemical sequencing” of DNA may denote methods such as that of Maxam and Gilbert (1977) (Proc. Natl. Acad. Sci. USA 74:560), in which DNA is randomly cleaved using individual base-specific reactions.
  • Enzymatic sequencing of DNA may denote methods such as that of Sanger (Sanger et al., (1977) Proc. Natl. Acad. Sci. USA 74:5463).
  • nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • promoters include promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
  • IVS internal ribosome entry sites
  • Promoters which may be used to control gene expression, include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and
  • a coding sequence is "under the control of, “functionally associated with” or
  • transcriptional and translational control sequences in a cell when the sequences direct R A polymerase mediated transcription of the coding sequence into R A, preferably m NA, which then may be trans-RNA spliced (if it contains introns) and, optionally, translated into a protein encoded by the coding sequence.
  • the present invention contemplates modifications, especially any superficial or slight modification, to the amino acid or nucleotide sequences that correspond to the proteins e.g., anti-MDL-1 antibodies.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule may anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • Typical low stringency hybridization conditions may be 55°C, 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%> formamide, 5X SSC, 0.5%> SDS.
  • Typical, moderate stringency hybridization conditions are similar to the low stringency conditions except the hybridization is carried out in 40% formamide, with 5X or 6X SSC.
  • High stringency hybridization conditions are similar to low stringency conditions except the hybridization conditions are carried out in 50%> formamide, 5X or 6X SSC and, optionally, at a higher temperature (e.g., 57 °C, 59 °C, 60 °C, 62 °C, 63 °C, 65°C or 68 °C).
  • SSC is 0.15M NaCl and 0.015M Na-citrate.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although, depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency under which the nucleic acids may hybridize. For hybrids of greater than 100 nucleotides in length, equations for calculating the melting temperature have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook, et al., supra, 11.7-11.8).
  • nucleic acids comprising nucleotide sequences and polypeptides comprising amino acid sequences that are at least 70% identical, at least 80% identical, at least 90% identical e.g., 91%, 92%, 93%, 94%, and at least 95% identical e.g., 95%, 96%, 97%, 98%, 99%, 100%, to the reference nucleotide and amino acid sequences of Table 1 when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences.
  • Polypeptides comprising amino acid sequences which are at least 70% similar, at least 80% similar, at least 90% similar e.g., 91%, 92%, 93%, 94%, and at least 95% similar e.g., 95%, 96%, 97%, 98%, 99%, 100%, when the comparison is performed with a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences, are also included in the present invention.
  • Sequence identity refers to exact matches between the nucleotides or amino acids of two sequences which are being compared.
  • Sequence similarity refers to both exact matches between the amino acids of two polypeptides which are being compared in addition to matches between nonidentical, biochemically related amino acids. Biochemically related amino acids which share similar properties and may be interchangeable are discussed above.
  • BLAST ALGORITHMS Altschul et al., (1990) J. Mol. Biol. 215:403-410; Gish et al, (1993) Nature Genet. 3:266-272; Madden et al, (1996) Meth.
  • the chimeric proteins can be synthesized (e.g., in mammalian cells) using conventional methods for protein expression using recombinant DNA technology. Because many of the polypeptides used to create the chimeric proteins have been previously purified, many of the previously-described methods of protein purification should be useful, along with other conventional methods, for purifying the chimeric proteins of the invention. If desired, the chimeric protein can be affinity-purified according to standard protocols with antibodies directed against the cytokine. Antibodies directed against the enzymatically inactive protein are also useful for purifying the chimeric protein by conventional
  • the activity of the chimeric protein can be assayed with methods that are commonly used to test the activity of the protein alone. It is not necessary that the activity of the chimeric protein be identical to the activity of the protein alone.
  • the present invention also includes fusions which include the polypeptides and polynucleotides of the present invention and a second polypeptide or polynucleotide moiety, which may be referred to as a "tag".
  • the fused polypeptides of the invention may be conveniently constructed, for example, by insertion of a polynucleotide of the invention or fragment thereof into an expression vector as described above.
  • the fusions of the invention may include tags which facilitate purification or detection. Such tags include glutathione- S- transferase (GST), hexahistidine (His6) tags, maltose binding protein (MBP) tags, haemagglutinin (HA) tags, cellulose binding protein (CBP) tags and myc tags.
  • Detectable labels or tags such as 32 P, 35 S, 14 C, 3 H, 99m Tc, m In, 68 Ga, 18 F, 125 I, 131 I, 113m In, 76 Br, 67 Ga, 99m Tc, 123 I, m In and 68 Ga may also be used to label the polypeptides of the invention.
  • Modifications e.g., post-translational modifications
  • a polypeptide often will be a function of how it is made.
  • the nature and extent of the modifications, in large part will be determined by the host cell's post-translational modification capacity and the modification signals present in the polypeptide amino acid sequence.
  • glycosylation often does not occur in bacterial hosts such as E. coli.
  • a polypeptide when glycosylation is desired, a polypeptide may be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out post-translational glycosylations which are similar to those of mammalian cells. For this reason, insect cell expression systems have been developed to express, efficiently, mammalian proteins having native patterns of glycosylation. Alternatively, deglycosylation enzymes may be used to remove carbohydrates attached during production in eukaryotic expression systems.
  • Analogs of the MDL-1 antibody proteins of the invention may be prepared by chemical synthesis or by using site-directed mutagenesis, Gillman et al, (1979) Gene 8:81; Roberts et al, (1987) Nature, 328:731 or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY or the polymerase chain reaction method PCR; Saiki et al, (1988) Science 239:487, as exemplified by Daugherty et al, (1991) (Nucleic Acids Res. 19:2471) to modify nucleic acids encoding the peptides. Adding epitope tags for purification or detection of recombinant products is envisioned.
  • Still other analogs are prepared by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.
  • Preferred derivatization sites with cross-linking agents are free amino or carboxy groups, carbohydrate moieties and cysteine residues.
  • the peptides of the invention may be produced by expressing a nucleic acid which encodes the polypeptide in a host cell which is grown in a culture (e.g., liquid culture such as Luria broth).
  • a culture e.g., liquid culture such as Luria broth
  • the nucleic acid may be part of a vector (e.g., a plasmid) which is present in the host cell.
  • the peptides of the invention may be isolated from the cultured cells.
  • the peptides of this invention may be purified by standard methods, including, but not limited to, salt or alcohol precipitation, affinity chromatography (e.g., used in conjunction with a purification tagged peptide as discussed above), preparative disc-gel electrophoresis, isoelectric focusing, high pressure liquid chromatography (HPLC), reversed-phase HPLC, gel filtration, cation and anion exchange and partition chromatography, and countercurrent distribution.
  • affinity chromatography e.g., used in conjunction with a purification tagged peptide as discussed above
  • HPLC high pressure liquid chromatography
  • reversed-phase HPLC gel filtration
  • anion exchange and partition chromatography e.g., Amberlite chromatography
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer includes 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 may include a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain may define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • 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.
  • variable regions of each light/heavy chain pair may form the antibody binding site.
  • an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are usually aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of
  • the anti-MDL-1 antibody molecules of the invention preferably recognize human MDL-1.
  • the present invention includes antibody molecules which recognize mouse MDL-1, and MDL-1 from other species, preferably mammals ⁇ e.g., rat, rabbit, sheep or dog).
  • the present invention also includes anti-MDL-1 antibodies or fragments thereof which are complexed with MDL-1 or any fragment thereof or with any cell which is expressing MDL-1 or any portion or fragment thereof on the cell surface. Such complexes may be made by contacting the antibody or antibody fragment with MDL-1 or the MDL-1 fragment. Examples of MDL-1 agonist antibodies are provided in WO 2008/133857.
  • transgenic mice carrying parts of the human immune system rather than the mouse system.
  • These transgenic mice which may be referred to, herein, as "HuMAb” mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (Lonberg, N., et al., (1994) Nature 368(6474):856-859). These antibodies are also referred to as fully human antibodies.
  • mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N., et al., (1994), supra; reviewed in Lonberg, N. (1994) Handbook of
  • HuMab mice The preparation of HuMab mice is commonly known in the art and is described, for example, in Taylor et al, (1992) Nucleic Acids Research 20:6287-6295; Chen et al, (1993) International Immunology 5:647- 656; Tuaillon et al, (1993) Proc. Natl. Acad.
  • HuMab mice may be immunized with an antigenic MDL-1 polypeptide as described by Lonberg et al., (1994) Nature 368(6474):856-859; Fishwild et al., (1996) Nature Biotechnology 14:845-851 and WO 98/24884.
  • the mice will be 6-16 weeks of age upon the first immunization.
  • a purified preparation of MDL-1 may be used to immunize the HuMab mice intraperitoneally.
  • the mice may also be immunized with whole cells which are stably transformed or transfected with an MDL-1 gene.
  • HuMAb transgenic mice respond well when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (usually, up to a total of 6) with antigen in incomplete Freund's adjuvant.
  • IP intraperitoneally
  • Mice may be immunized, first, with cells expressing MDL-1, then with a soluble fragment of MDL-1 and continually receive alternating immunizations with the two antigens. The immune response may be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds.
  • the plasma may be screened for the presence of anti-MDL-1 antibodies, for example by ELISA, and mice with sufficient titers of immunoglobulin may be used for fusions. Mice may be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice may be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains may be immunized.
  • Hybridoma cells which produce the monoclonal anti-MDL-1 antibodies may be produced by methods which are commonly known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler, et al., (1975) (Nature 256:495-497), as well as the trioma technique (Hering et al., (1988) Biomed.
  • mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols.
  • the resulting hybridomas may then be screened for the production of antigen-specific antibodies.
  • single cell suspensions of splenic lymphocytes from immunized mice may by fused to one-sixth the number of P3X63- Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • Cells may be plated at approximately 2 x 10 5 cells/mL in a flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells may be cultured in medium in which the HAT is replaced with HT.
  • selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L- glutamine, 1 mM sodium pyruvate, 5mM
  • Individual wells may then be screened by ELISA for human anti-MDL-1 monoclonal IgG antibodies. Once extensive hybridoma growth occurs, medium may be observed usually after 10-14 days.
  • the antibody secreting hybridomas may be replated, screened again, and if still positive for human IgG, anti-MDL-1 monoclonal antibodies, may be subcloned at least twice by limiting dilution.
  • the stable subclones may then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.
  • the anti-MDL-1 antibody molecules of the present invention may also be produced recombinantly ⁇ e.g., in an E.coli/ ⁇ expression system as discussed above).
  • nucleic acids encoding the antibody molecules of the invention e.g., V H or V L
  • V H or V L may be inserted into a pET-based plasmid and expressed in the E.coli/ ⁇ system.
  • Transformation may be by any known method for introducing polynucleotides into a host cell.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei.
  • nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.
  • 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). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells ⁇ e.g., Hep G2), A549 cells, 3T3 cells, and a number of other cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels.
  • insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, 5 secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies may be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines may be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4.
  • Antibody fragments preferably antigen-binding antibody fragments, fall within the scope of the present invention also include F(ab) 2 fragments which may be produced by enzymatic cleavage of an IgG by, for example, pepsin.
  • Fab fragments may be produced by, for example, reduction of F(ab) 2 with dithiothreitol or mercaptoethylamine.
  • a Fab fragment is a V L -C L chain appended to a V H -C HI chain by a disulfide bridge.
  • a F(ab) 2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges.
  • the Fab portion of an F(ab) 2 molecule includes a portion of the F c region between which disulfide bridges are located.
  • Fv the minimum antibody fragment which contains a complete antigen recognition and binding site, consists of a dimer of one heavy and one light chain variable domain (V H -V L ) in non-covalent association.
  • V H -V L variable domain
  • CDRs complementarity determining regions
  • the constant domains are not required for binding function, but may aid in stabilizing V H -V L interaction. 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 usually at a lower affinity than an entire binding site (Painter, Biochem. 11 (1972), 1327-1337).
  • said domain of the binding site of the antibody construct as defined and described in the present invention may be a pair of V H -V L , V H - V H or V L - V L domains of different immunoglobulins.
  • V H and V L domains within the polypeptide chain is not decisive for the present invention, the order of domains given hereinabove may be reversed usually without any loss of function. It is important, however, that the V H and V L domains are arranged so that the antigen binding site may properly fold.
  • An F v fragment is a V L or V H region.
  • immunoglobulins may be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.
  • the anti-MDL-1 antibody molecules or the MDL-1 soluble proteins of the invention may also be conjugated to a chemical moiety.
  • the chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor.
  • the chemical moiety is a polymer which increases the half- life of the antibody molecule in the body of a subject.
  • Suitable polymers include, but are not limited to, polyethylene glycol (PEG) ⁇ e.g., PEG with a molecular weight of 2kDa, 5 kDa, 10 kDa, 12kDa, 20 kDa, 30kDa or 40kDa), dextran and monomethoxypoly ethylene glycol (mPEG).
  • PEG polyethylene glycol
  • mPEG monomethoxypoly ethylene glycol
  • DTP A diethylenetriaminpentaacetic acid
  • the antibodies and antibody fragments or the MDL-1 soluble proteins or fragments thereof of the invention may also be conjugated with labels such as 99 Tc, 90 Y, m In, 32 P, 14 C, 125 1, 3 H, 131 I, U C, 15 0, 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 Eu, 67 CU, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K, 157 Gd, 55 Mn, 52 Tr and 56 Fe.
  • labels such as 99 Tc, 90 Y, m In, 32 P, 14 C, 125 1, 3 H, 131 I, U C, 15 0, 13 N, 18 F, 35 S, 51 Cr, 57 To, 226 Ra, 60 Co, 59 Fe, 57 Se, 152 Eu, 67 CU, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K,
  • the antibodies and antibody fragments, the MDL-1 soluble proteins, MDL-1 fusion proteins, or fragments thereof of the invention may also be conjugated with
  • fluorescent or chemilluminescent labels including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152 Eu, dansyl,
  • fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152 Eu, dansyl,
  • umbelliferone luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3- dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.
  • the antibody molecules or soluble MDL-1 proteins may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytolacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
  • a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g
  • Antigenic (i.e., immunogenic) fragments of the MDL-1 peptides of the invention are within the scope of the present invention. Antigenic fragments may be joined to other materials, such as fused or covalently joined polypeptides, to be used as
  • the antigenic peptides may be useful for preparing antibody molecules which recognize MDL-1 or any fragment thereof.
  • An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin (Coligan et al. (1994) Current Protocols in Immunol, Vol. 2, 9.3-9.4, John Wiley and Sons, New York, NY).
  • Peptides of suitable antigenicity may be selected from the polypeptide target, using an algorithm, see, e.g., Parker et al. (1986) Biochemistry 25:5425-5432; Jameson and Wolf (1988) Cabios 4: 181-186; Hopp and Woods (1983) Mol. Immunol. 20:483-489.
  • MDL-1 peptides are used as antigens to elicit antibody production in an immunologically competent host
  • smaller antigenic fragments are preferably first rendered more immunogenic by cross-linking or concatenation, or by coupling to an immunogenic carrier molecule (i.e., a macromolecule having the property of independently eliciting an immunological response in a host animal, such as diptheria toxin or tetanus).
  • an immunogenic carrier molecule i.e., a macromolecule having the property of independently eliciting an immunological response in a host animal, such as diptheria toxin or tetanus.
  • Cross-linking or conjugation to a carrier molecule may be required because small polypeptide fragments sometimes act as haptens (molecules which are capable of specifically binding to an antibody but incapable of eliciting antibody production, i.e., they are not immunogenic). Conjugation of such fragments to an immunogenic carrier molecule renders them more immunogenic through what is commonly known as
  • Carrier molecules include, e.g., proteins and natural or synthetic polymeric compounds such as polypeptides, polysaccharides, lipopolysaccharides, etc. Protein carrier molecules are especially preferred, including, but not limited to, keyhole limpet hemocyanin and mammalian serum proteins such as human or bovine gammaglobulin, human, bovine or rabbit serum albumin, or methylated or other derivatives of such proteins. Other protein carriers will be apparent to those skilled in the art. Preferably, the protein carrier will be foreign to the host animal in which antibodies against the fragments are to be elicited.
  • Covalent coupling to the carrier molecule may be achieved using methods well known in the art; the exact choice of which will be dictated by the nature of the carrier molecule used.
  • the immunogenic carrier molecule is a protein
  • the fragments of the invention may be coupled, e.g., using water-soluble carbodiimides such as
  • Coupling agents such as these, may also be used to cross-link the fragments to themselves without the use of a separate carrier molecule. Such cross-linking into aggregates may also increase immunogenicity. Immunogenicity may also be increased by the use of known adjuvants, alone or in combination with coupling or aggregation.
  • Adjuvants for the vaccination of animals include, but are not limited to,
  • Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N',N'-bis(2- hydroxymethyl) propanediamine, methoxyhexadecylglycerol and pluronic polyols;
  • polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides such as muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.
  • the polypeptides could also be administered following incorporation into liposomes or other microcarriers.
  • the anti-MDL-1 "antibody molecules" of the invention include, but are by no means not limited to, anti-MDL-1 antibodies ⁇ e.g., monoclonal antibodies, polyclonal antibodies, bispecific antibodies and anti-idiotypic antibodies) and fragments, preferably antigen-binding fragments, thereof, such as Fab antibody fragments, F(ab) 2 antibody fragments, Fv antibody fragments ⁇ e.g., V H or V L ), single chain Fv antibody fragments and dsFv antibody fragments.
  • the antibody molecules of the invention may be fully human antibodies, mouse antibodies, rabbit antibodies, chicken antibodies, human/mouse chimeric antibodies or humanized antibodies.
  • the anti-MDL-1 antibody molecules of the invention preferably recognize human or mouse MDL-1 peptides of the invention; however, the present invention includes antibody molecules which recognize MDL-1 peptides from different species, preferably mammals ⁇ e.g., pig, rat, rabbit, sheep or dog).
  • the present invention also includes complexes comprising the MDL-1 peptides of the invention and one or more antibody molecules, e.g., bifunctional antibodies. Such complexes may be made by simply contacting the antibody molecule with its cognate peptide.
  • Various methods may be used to make the antibody molecules of the invention. In preferred embodiments, the antibodies of the invention are produced by methods which are similar to those disclosed in U.S. Patent Nos. 5,625,126; 5,877,397;
  • Hybridoma cells which produce monoclonal, fully human anti-MDL-1 peptide antibodies may then be produced by methods which are commonly known in the art. These methods include, but are not limited to, the hybridoma technique originally developed by Kohler et al., (1975) ⁇ Nature 256:495-497), as well as the trioma technique (Hering et al., (1988) Biomed. Biochim. Acta. 47:211-216 and Hagiwara et al., (1993) Hum. Antibod.
  • Hybridomas 4: 15 the human B-cell hybridoma technique (Kozbor et al., (1983) Immunology Today 4:72 and Cote et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030), and the EBV-hybridoma technique (Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).
  • ELISA may be used to determine if hybridoma cells are expressing anti-MDL-1 peptide antibodies.
  • Immunization may be performed by DNA vector immunization, see, e.g., Wang, et al. (1997) Virology 228:278-284.
  • animals may be immunized with cells bearing the antigen of interest.
  • Splenocytes may then be isolated from the immunized animals, and the splenocytes may be fused with a myeloma cell line to produce a hybridoma (Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al. (1997) Eur. J. Immunol. 27: 1911-1918).
  • Resultant hybridomas may be screened for production of the desired antibody by functional assays or biological assays, that is, assays not dependent on possession of the purified antigen. Immunization with cells may prove superior for antibody generation than immunization with purified antigen (Kaithamana et al. (1999) J. Immunol. 163:5157-5164).
  • Antibody to antigen and ligand to receptor binding properties may be measured, e.g., by surface plasmon resonance (Karlsson et al. (1991) J. Immunol. Methods 145:229-240; Neri et al. (1997) Nat. Biotechnol. 15: 1271-1275; Jonsson et al. (1991) Biotechniques 11 :620-627) or by competition ELISA (Friguet et al. (1985) J. Immunol.
  • Antibodies may be used for affinity purification to isolate the antibody's target antigen and associated bound proteins, see, e.g., Wilchek et al. (1984) Meth. Enzymol. 104:3-55.
  • Antibodies that specifically bind to variants of MDL-1, where the variant has substantially the same nucleic acid and amino acid sequence as those recited herein, but possessing substitutions that do not substantially affect the functional aspects of the nucleic acid or amino acid sequence, are within the definition of the contemplated methods.
  • Variants with truncations, deletions, additions, and substitutions of regions which do not substantially change the biological functions of these nucleic acids and polypeptides are within the definition of the contemplated methods.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of MDL-1. Alternatively, bispecific MDL-1 antibodies can bind to another antigen, e.g., DC- SIGN, CD20, RANK-L, etc.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light- chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy-chain-light-chain pair (providing a second binding specificity) in the other arm.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al. Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • E. coli which can be chemically coupled to form bispecific antibodies.
  • Shalaby et al. (1992) J. Exp. Med., 175:217-225 describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re -oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the "diabody” technology described by Hollinger et al. (1993) Proc. Natl Acad. Sci.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light- chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. (1991) J. Immunol. 147: 60. Therapeutic Uses
  • MDL-1 agonists of the invention could be used to treat or prevent include, but are not limited to, obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin
  • NIDDM Insulin Dependent Diabetes Mellitus
  • IDDM Insulin Dependent Diabetes Mellitus
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • Antagonists e.g., antagonist antibodies or soluble MDL-1 protein can be used to treat other metabolic-related diseases or disorders including, cachexia, wasting, AIDS- related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • MDL-1 agonists of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.
  • Other compounds useful for the treatment of obesity and other diseases and disorders are currently well-known in the art.
  • the MDL-1 agonists are useful for, and used in, the treatment of insulin resistance and diabetes using methods described herein and known in the art. More particularly, a preferred embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of treatment (alternatively on a timed daily basis) MDL-1 agonists, e.g., agonist antibodies, in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.
  • MDL-1 agonists e.g., agonist antibodies
  • Further preferred embodiments relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of treatment (alternatively on a timed daily basis) an MDL-1 agonist in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject
  • an effective amount of therapeutic will decrease the symptoms typically by at least 10%; usually by at least 20%>; preferably at least 30%>; more preferably at least 40%>, and most preferably by at least 50%.
  • Formulations of therapeutic agents may be prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions, see, e.g., Hardman, et al.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. When in combination, an effective amount is in ratio to a combination of components and the effect is not limited to individual components alone. Guidance for methods of treatment and diagnosis is available (Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).
  • the MDL agonists, e.g., agonist antibodies, of the invention may be administered, preferably for therapeutic purposes, to a subject, preferably in a pharmaceutical composition.
  • a pharmaceutical composition includes a pharmaceutically acceptable carrier.
  • the antibody molecules may be used therapeutically ⁇ e.g., in a pharmaceutical composition) to target the MDL-1 receptor and, thereby, to treat any medical condition mediated by the receptor.
  • Pharmaceutically acceptable carriers are conventional and very well known in the art. Examples include aqueous and nonaqueous carriers, stabilizers, antioxidants, solvents, dispersion media, coatings, antimicrobial agents, buffers, serum proteins, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for injection into a subject's body.
  • compositions useful for parenteral administration of such drugs are well known; e.g. , Remington 's Pharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, PA, 1990).
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions of the invention may be administered in conjunction with a second pharmaceutical composition or substance.
  • both compositions may be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions ⁇ e.g., a kit).
  • Analgesics may include aspirin, acetominophen, codein, morphine, aponorphine, normorphine, etorphine, buprenorphine, hydrocodone, racemorphan, levorphanol, butorphand, methadone, demerol, ibuprofen or oxycodone.
  • compositions of the invention may also include other types of substances, including small organic molecules and inhibitory ligand analogs, which may be identified using the assays described herein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al. (eds.) (1990),_73 ⁇ 4e Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, supra, Easton, Penn.; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York.
  • a further formulation and delivery method herein involves that described, for example, in WO 2004/078140, including the ENHANZETM drug delivery technology (Halozyme Inc.). This technology is based on a recombinant human hyaluronidase
  • rHuPH20 is a recombinant form of the naturally occurring human enzyme approved by the FDA that temporarily clears space in the matrix of tissues such as skin. That is, the enzyme has the ability to break down hyaluronic acid (HA), the space-filling "gel”-like substance that is a major component of tissues throughout the body. This clearing activity is expected to allow rHuPH20 to improve drug delivery and bioavailability of the therapeutic by enhancing the entry of therapeutic molecules through the subcutaneous space.
  • HA hyaluronic acid
  • this technology can act as a "molecular machete” to facilitate the penetration and dispersion of these drugs by temporarily opening flow channels under the skin. Molecules as large as 200 nanometers may pass freely through the perforated extracellular matrix, which recovers its normal density within approximately 24 hours, leading to a drug delivery platform that does not permanently alter the architecture of the skin.
  • the present invention includes a method of delivering the MDL-1 antibody or soluble MDL-1 protein herein to a tissue containing excess amounts of glycosaminoglycan, comprising administering a hyaluronidase glycoprotein (sHASEGP) (this protein comprising a neutral active soluble hyaluronidase polypeptide and at least one N-linked sugar moiety, wherein the N-linked sugar moiety is covalently attached to an asparagine residue of the polypeptide) to the tissue in an amount sufficient to degrade glycosaminoglycans sufficiently to open channels less than about 500 nanometers in diameter; and administering the antibody or soluble protein to the tissue comprising the degraded glycosaminoglycans.
  • sHASEGP hyaluronidase glycoprotein
  • the invention includes a method for increasing the diffusion of an antibody or soluble protein herein that is administered to a subject comprising administering to the subject a sHASEGP polypeptide in an amount sufficient to open or to form channels smaller than the diameter of the antibody and administering the antibody, whereby the diffusion of the therapeutic substance is increased.
  • the sHASEGP and antibody may be administered separately or simultaneously in one formulation, and consecutively in either order or at the same time.
  • the dosage regimen involved in a therapeutic application may be determined by a physician, considering various factors which may modify the action of the therapeutic substance, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration, and other clinical factors. [0139] Often, treatment dosages are titrated upward from a low level to optimize safety and efficacy. Dosages may be adjusted to account for the smaller molecular sizes and possibly decreased half-lives (clearance times) following administration.
  • compositions of the invention may be administered, for example, by parenteral routes (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intratumoral injection or by infusion) or by a non-parenteral route
  • parenteral routes e.g., intravenous injection, intramuscular injection, subcutaneous injection, intratumoral injection or by infusion
  • non-parenteral route e.g., intravenous injection, intramuscular injection, subcutaneous injection, intratumoral injection or by infusion
  • compositions may be administered with medical devices known in the art.
  • a pharmaceutical composition of the invention may be administered by injection with a hypodermic needle.
  • compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements);
  • recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
  • appropriate segments e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
  • Hochuli (1990) Purification of Recombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: The High Level Expression & Protein Purification System, Qiagen, Inc., Chatsworth, CA.
  • Anti-mouse MDL-1 agonist antibodies e.g., DX163, mouse IgGl
  • antagonist antibodies DX192
  • mice on the C57BL/6 background were fed a normal diet containing 5% fat (Harlan, Madison WI). After 4 and 5.5 months both mice weights were compared (see Table 1 below). On average, MDL-1 KO mice were 5 grams heavier at 4 months, and 7 grams heavier at 5.5 months. Table 1 : Weight of MDL-1 KO and WT mice on normal (5% fat) diet. Weights are in grams.
  • MDL-1 KO and WT mice were fed low fat (LF) and high fat (HF) diets
  • the low fat diets contained 10% fat, while the high fat diet had 60% fat. Weights (grams) were measured at 11 weeks. MDL-1 KO mice gained 15% more weight on either diet as compared to the WT counterparts (see Table 2).
  • mice Serum leptin levels were measured in 10-week old WT and MDL-1 KO mice, by Millinex assay (Millipore,Billerica, MA) following manufacturers protocols. WT mice has average leptin levels of 4000 pg/mL, while MDL-1 KO mice had average leptin levels of 9000 mg/mL (see Table 3). Weights of the mice did not significantly differ at 10 weeks.
  • Insulin tolerance test was performed on day 49 of diet-induced obesity
  • mice were fasted 16 hours and resting glucose levels were measured. Mice were given a 1.5 mg/kg dose of insulin and glucose was monitored at defined time points. MDL-1 KO mice were consistently less sensitive to insulin compared to WT controls indicating greater insulin resistance (see Table 4) ⁇
  • Table 4 Insulin tolerance in MDL-1 KO mice after 49 days on a high fat (60% fat) diet; average glucose levels (mg/dL).
  • MDL-1 agonist antibody or an IgGl isotype control antibody
  • mice fed a high fat (60%) diet.
  • Mice weights were monitored for 18 days.
  • DX163 treated mice had an average weight gain of 10% while isotype control mice gained 23% over the same time period (see Table 5.)
  • Table 5 - Treatment with MDL-1 agonist antibody inhibits weight gain in diet-induced obesity model; values are percent weight gain.
  • mice Male CD57BL/6 mice were fed either a HF or LF diet for 77 days. Peri-renal fat was harvested from the adipose tissue adjacent to the kidney and samples were snap frozen in liquid nitrogen. RNA was isolated by Stat60 purification process and equal sample quantities were converted into cDNA. Gene expression of various metabolic genes were measured and values were reported relative to ubiquitin. Several genes were significantly upregulated in MDL-1 KO mice. Table 6 - Metabolic Gene Expression

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Abstract

La présente invention concerne des procédés de traitement de troubles métaboliques. En particulier, des anticorps anti-MDL-1 ou des protéines de fusion de MDL-1 solubles sont utilisées.
PCT/US2010/054239 2009-10-30 2010-10-27 Utilisations d'une cytokine de mammifère Ceased WO2011053630A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6953843B2 (en) * 1997-08-01 2005-10-11 Schering Corporation Antibodies and fragments thereof to MDL-1 protein
WO2009006112A1 (fr) * 2007-06-29 2009-01-08 Schering Corporation Utilisation de mdl-1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6953843B2 (en) * 1997-08-01 2005-10-11 Schering Corporation Antibodies and fragments thereof to MDL-1 protein
WO2009006112A1 (fr) * 2007-06-29 2009-01-08 Schering Corporation Utilisation de mdl-1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BHOWMICK ET AL.: "The prevalence of Elevated Serum Thyroid-Stimulating Hormone in Childhood/Adolescent Obesity and of Autoimmune Thyroid Diseases in a Subgroup", JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, vol. 99, no. 7, 2007, pages 773 - 776 *

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