Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
  • C07K16/2839—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 integrin superfamily
  • C07K16/2848—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 integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61

Definitions

• the reconstitution step itself requires certain specific procedures: (1) a sterile diluent (i.e., water of intravenous administration and 5% dextrose in water for intramuscular administration) is added to the vial containing lyophilized antibody, slowly and aseptically, and the vial must be swirled very gently for 30 seconds to avoid foaming; (2) the reconstituted antibody may need to stand at room temperature for a minimum of 20 minutes until the solution clarifies; and (3) the reconstituted preparation must be administered within six (6) hours after the reconstitution.
• a sterile diluent i.e., water of intravenous administration and 5% dextrose in water for intramuscular administration
• liquid formulations of the present invention enable a healthcare professional to quickly administer a sterile dosage of an antibody or an antibody fragment that immunospecifically binds to integrin cvft without having to accurately and sterilely reconstitute the antibody or antibody fragment prior to administration.
• the present invention provides liquid formulations substantially free of surfactant and/or inorganic salts, said formulations comprising histidine and a concentration of 50 mg/ml or higher of antibodies or fragments thereof that immunospecifically bind to integrin o ft.
• the present invention also provides liquid formulations substantially free of surfactant and/or inorganic salt, said formulations having a pH ranging from about 5.0 to about 7.0, preferably about pH 6.0, and comprising histidine, and a concentration of 50 mg/ml or higher of antibodies or antibody fragments that immunospecifically bind to integrin O 1 S 3 .
• the present invention encompasses liquid formulations of antibodies or antibody fragments that immunospecifically bind to integrin c-v/-» 3 , said formulations having stability at 38-42°C as assessed by high performance size exclusion chromatography (HPSEC).
• HPSEC high performance size exclusion chromatography
• the liquid formulations of the present invention exhibit stability, as assessed by HPSEC, at temperature ranges of 38-42°C for at least 15 days but no more than 25 days; at temperature ranges of 20-24°C for at least 6 months but not more than 1.5 years; and at temperature ranges of 2-8°C (especially at 4°C) for at least 1.5 years, at least 2 years, at least 2.5 years, or at least 3 years.
• the present invention also encompasses liquid formulations of antibodies or fragments thereof that immunospecifically bind to integrin c-vj-. 3 , said formulations having low to undetectable levels of antibody aggregation as measured by HPSEC.
• the liquid formulations of the present invention exhibit stability at 38-42°C for at least 15 days and exhibit low to undetectable levels of antibody aggregation as measured by HPSEC and, further, exhibit very little to no loss of the biological activity of the antibodies or antibody fragments of the formulation compared to the reference antibodies as measured by antibody binding assays such as, e.g., ELISAs.
• the present invention provides methods for preparing liquid formulations of an antibody or antibody fragment that immunospecifically binds to integrin s / ⁇ , said methods comprising concentrating a fraction containing the purified antibody to a final antibody concentration ranging from about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 150 mg/ml, about 175 mg/ml, or about 200 mg/ml using a semipermeable membrane with an appropriate molecular weight (MW) cutoff (e.g., a 30 kD cutoff for whole antibody molecules and F(ab') 2 fragments; and a 10 kD cutoff for antibody fragments such as Fab fragments) and diafiltering the concentrated antibody fraction into the formulation buffer using the same membrane.
• MW molecular weight
• the liquid formulations of the present invention may be sterilized by sterile filtration using a 0.2 ⁇ filter. Sterilized liquid formulations of the present invention may be administered to a subject to prevent, treat, manage or ameliorate inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abno ⁇ nal angiogenesis, disorders associated with aberrant expression and/or activity of integrin o ft, cancer, or one or more symptoms thereof.
• liquid formulations of the present invention can also be used to diagnose, detect or monitor disorders associated with abnormal expression of integrin c-vft, inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis and cancer.
• the term "about” in the context of a given numerate value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.
• abnormal angiogenesis refers to the growth of new blood vessels.
• abnormal angiogenesis or “aberrant angiogenesis” refers to altered (e.g., increased or decreased) activity of angiogenesis, i.e., any angiogenesis that deviates from the normal process of angiogenesis, such as but not limited to, increased angiogenesis activity in a body, and angiogenesis at an abnormal location of the body.
• a disease or disorder may be completely caused by abnormal angiogenesis or may be exacerbated by abnormal angiogenesis.
• a proteinaceous agent with similar structure to a second proteinaceous agent refers to a proteinaceous agent that has a similar secondary, tertiary or quaternary structure to the second proteinaceous agent.
• the structure of a proteinaceous agent can be determined by methods known to those skilled in the art, including but not limited to, peptide sequencing, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
• the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• the term "effective amount" in the context of detecting the expression of integrin c-v/33 refers to the amount of antibodies, antibody fragments, or a formulation comprising antibodies or antibody fragments which is sufficient to detect integrin ⁇ v/33 expression.
• a disease or disorder e.g., an inflammatory disease, an autoimmune disease, a disorder associated with abnormal bone metabolism, a disorder associated with abnormal angiogenesis, a disorder associated with aberrant expression and/or activity of integrin ⁇ rv/33, or cancer.
• the term “isolated” or “purified” in the context of a proteinaceous agent refers to a proteinaceous agent which is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• substantially free of cellular material includes preparations of a proteinaceous agent in which the proteinaceous agent is separated from cellular components of the cells from which it is isolated or recombinantly produced.
• polyol refers to a sugar that contains many -OH groups compared to a normal saccharide.
• Side effects from administration of REMICADETM include, but are not limited to, risk of serious infection and hypersensitivity reactions. Other side effects range from nonspecific symptoms such as fever or chills, pruritus or urticaria, and cardiopulmonary reactions such as chest pain, hypotension, hypertension or dyspnea, to effects such as myalgia and/or arthralgia, rash, facial, hand or lip edema, dysphagia, sore throat, and headache.
• Side effects from chemotherapy include, but are not limited to, gastrointestinal toxicity such as, but not limited to, early and late-forming diarrhea and flatulence; nausea; vomiting; anorexia; leukopenia; anemia; neutropenia; asthenia; abdominal cramping; fever; pain; loss of body weight; dehydration; alopecia; dyspnea; insomnia; dizziness, mucositis, xerostomia, and kidney failure, as well as constipation, nerve and muscle effects, temporary or permanent damage to kidneys and bladder, flu-like symptoms, fluid retention, and temporary or permanent infertility.
• Side effects from radiation therapy include but are not limited to fatigue, dry mouth, and loss of appetite.
• the antibody or antibody fragment that immunospecifically binds to integrin ⁇ v/3 3 which is included in the liquid formulations of the invention is NITAXIN ® or an antigen-binding fragment thereof
• the antibody or antibody fragment that immunospecifically binds to integrin o-v/3 3 which is included in the liquid formulations of the invention is not NITAXIN ® or an antigen-binding fragment thereof.
• the antibody or antibody fragment that immunospecifically binds to integrin ofyf ⁇ which is included in the liquid formulations of the invention is an antibody or antibody fragment comprising one or more NH CDRs and/or one or more NL CDRs listed in Table 1, supra.
• the concentration of an antibody or antibody fragment that immunospecifically binds to integrin otyft which is included in the liquid formulations of the invention is at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, at least 105 mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml, at least 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, at least 140 mg/ml, at least
• the concentration of an antibody or antibody fragment that immunospecifically binds to integrin c-vft which is included in the liquid formulation of the invention is about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml.
• the concentration of histidine which is included in the liquid formulations of the invention ranges from 1 mM to 100 mM, preferably 5 mM to 50 mM, and more preferably 10 mM to about 25 mM.
• the concentration of histidine which is included in the liquid formulations of the invention is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.
• Histidine can be in the form of L-histidine, D-histidine, or a mixture thereof, but L-histidine is the most preferable. Histidine can be also in the form of hydrates.
• Histidine may be used in a form of pharmaceutically acceptable salt, such as hydrochloride (e.g., monohydrochloride and dihydrochloride), hydrobromide, sulfate, acetate, etc.
• the purity of histidine should be at least 98%), preferably at least 99%, and most preferably at least 99.5%.
• the term "purity" in the context of histidine refers to chemical purity of histidine as understood in the art, e.g., as described in The Merck Index, 13* ed., O'Neil et al. ed. (Merck & Co., 2001).
• the formulations of the present invention may further comprise glycine at a concentration of less than 150 mM, less than 100 mM, less than 75 mM, less than 50 mM, less than 25 mM, less than 10 mM, less than 5.0 mM, or less than 2.0 mM.
• the formulations of the present invention further comprise glycine at a concentration of 1-150 mM, 1-100 mM, 1-75 mM, 1-50 mM, 1-25 mM, 1-10 mM, 1-5.0 mM, or 1-2.0 mM.
• the amount of glycine in the formulation should not cause a significant buffering effect so that antibody precipitation at its isoelectric point can be avoided.
• Glycine may be also used in a form of pharmaceutically acceptable salt, such as hydrochloride, hydrobromide, sulfate, acetate, etc.
• the purity of glycine should be at least 98%, preferably at least 99%, and most preferably 99.5%>.
• the term "purity" in the context of glycine refers to chemical purity of glycine as understood in the art, e.g., as described in The Merck Index, 13 th ed., O'Neil et al ed. (Merck & Co., 2001). In a specific embodiment, glycine is not included in the formulations of the present invention.
• the liquid formulations of the present invention exhibit stability at the temperature range of 38°C-42°C for at least 15 days and, in some embodiments, not more than 25 days, at the temperature range of 20°C-24°C for at least 6 months, at the temperature range of 2°C-8°C (in particular, at 4°C) for at least 6 months, at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years or at least 4 years, and at the temperature of -20°C for at least 2 years, at least 3 years, at least 4 years, or at least 5 years, as assessed by high performance size exclusion chromatography (HPSEC).
• HPSEC high performance size exclusion chromatography
• the antibodies that immunospecifically bind to integrin c-v/3 3 may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of integrin c-v/3 3 or may be specific for both an integrin ⁇ v/3 3 epitope as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Patent Publication Nos WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt, et al, J. Immunol. 147:60-69(1991); U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992).
• an antibody that immunospecifically binds to integrin o ⁇ v/3 3 is NITAXIN ® or an antigen-binding fragment thereof (e.g., one or more CDRs of NITAXIN ® ).
• NITAXIN ® is disclosed, e.g., in International Publication Nos. WO 98/33919, WO 00/78815, and WO 02/070007, and U.S. application Serial No. 09/339,222, each of which is incorporated herein by reference in its entirety.
• an antibody that immunospecifically binds to integrin o. v /3 3 is not NITAXIN® or an antigen-binding fragment of NITAXIN ® .
• antibodies or antibody fragments that immunospecifically bind to integrin c- v /3 3 comprise a NL CDR2 having the amino acid sequence of SEQ ID ⁇ O:5.
• antibodies or antibody fragments that immunospecifically bind to integrin ⁇ v /3 3 comprise a NL CDR3 having the amino acid sequence of SEQ ID ⁇ O:6.
• antibodies or antibody fragments that immunospecifically bind to integrin a v ⁇ _ comprise a NL CDRl having the amino acid sequence of SEQ ID ⁇ O:4 and a NL CDR2 having the amino acid sequence of SEQ ID ⁇ O:5.
• antibodies or antibody fragments that immunospecifically bind to integrin c v /3 3 comprise a NL CDRl having the amino acid sequence of SEQ ID ⁇ O:4, a NL CDR2 having the amino acid sequence of SEQ ID ⁇ O:5, and a NL CDR3 having the amino acid sequence of SEQ ID ⁇ O:6.
• the present invention also encompasses antibodies and antibody fragments that immunospecifically bind to integrin 0! v /3 , said antibodies comprising a NH domain disclosed herein combined with a NL domain disclosed herein, or other NL domain.
• the present invention further provides antibodies and antibody fragments that immunospecifically bind to integrin a _, said antibodies and antibody fragments comprising a NL domain disclosed herein combined with a NH domain disclosed herein, or other NH domain.
• an antibody or antibody fragment that immunospecifically binds to integrin c- v ft comprises a VH CDR2 having the amino acid sequence of SEQ ID NO:2 and a VL CDRl having the amino acid sequence of SEQ ID NO:4.
• an antibody or antibody fragment that immunospecifically binds to integrin o- v /3 3 comprises a VH CDR2 having the amino acid sequence of SEQ ID NO:2 and a VL CDR2 having the amino acid sequence of SEQ ID NO:5.
• an isolated nucleic acid molecule encodes an antibody or antibody fragment that immunospecifically binds to integrin c- v j8 3 , said antibody or antibody fragment comprising a VH domain having the amino acid sequence of the VH domain of the monoclonal antibody produced by the cell line deposited with the ATCC® as Accession Number HB 9537.
• an isolated nucleic acid molecule encodes an antibody or antibody fragment that immunospecifically binds to integrin c-vj-. 3 , said antibody or antibody fragment comprising a NL domain having the amino acid sequence of the NL domain of LM609 or NITAXIN®.
• the present invention encompasses antibodies and antibody fragments that immunospecifically bind to integrin a v ⁇ 3 , said antibodies and antibody fragments comprising the amino acid sequence of LM609 or NITAXIN® with one or more amino acid residue substitutions in the variable light (VL) domain and/or variable heavy (VH) domain.
• the present invention also encompasses antibodies and antibody fragments that immunospecifically bind to integrin G.
• an antibody that immunospecifically binds to integrin c-v/3 3 comprises the framework region of VITAXIN ® .
• the antibodies or antibody fragments can be fused to marker sequences, such as a peptide to facilitate purification.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• VEGF vascular endothelial growth factor
• an anti-angiogenic agent e.g., angiostatin, endostatin or a component of the coagulation pathway (e.g., tissue factor); or, a biological response modifier such as, for example, a lymphokine (e.g., interferon gamma ("IFN- ⁇ "), interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-5 (“IL-5"), interleukin-6 (“IL-6”), interleuking-7 (“IL-7”), interleukin-10 ("IL-10”), interleukin-12 (“IL-12”), interleukin-15 (“IL-15”), interleukin-23 (“IL-23”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., IFN- ⁇ "), interleukin-1 ("IL-1"), interleukin-2 (“IL-2
• an antibody or antibody fragment can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alpha-emitters such as Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 131 hi, 131 LU, 131 Y, 131 Ho, 131 Sm, to polypeptides.
• the macrocyclic chelator is l,4,7,10-tefraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
• Antibodies and antibody fragments may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
• the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with a non-murine antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind to the antigen.
• Antibody fragments which recognize specific particular epitopes may be generated by any teclinique known to those of skill in the art.
• the vectors for expressing the VH or VL domains comprise an EF-lc- promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
• the VH and VL domains may also cloned into one vector expressing the necessary constant regions.
• the heavy chain conversion vectors and light chain conversion vectors are then co- transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
• Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al, 1986, BioTechniques 4:214; Gillies et al, 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,8 16397, and 6,331,415, which are incorporated herein by reference in their entirety.
• a humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immuoglobulin.
• a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') 2 , Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
• a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
• the antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
• the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG 2 , IgG 3 and lgG 4 .
• the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically IgGi. Where such cytotoxic activity is not desirable, the constant domain may be of the IgG 2 class.
• the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
• the framework and CDR regions of a humanized antibody need not conespond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not conespond to either the consensus or the import antibody. Such mutations, however, will not be extensive.
• Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR- grafting (European Patent No. EP 239,400; hitemational publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos.
• framework residues in the framework regions will be substituted with the conesponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Patent No. 5,585,089; and Riechmann et al, 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)
• the antibodies that immunospecifically bind to an antigen can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" an antigen using teclmiques well known to those skilled in the art.
• an antigen e.g., integrin GV/33
• teclmiques well known to those skilled in the art.
• the invention provides polynucleotides comprising a nucleotide sequence encoding an antibody or antibody fragment that immunospecifically binds to an antigen.
• the invention also encompasses polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody of the invention.
• Such a polynucleotide encoding the antibody or antibody fragment maybe assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• a polynucleotide encoding an antibody or antibody fragment may be generated from nucleic acid from a suitable source.
• an antibody of the invention e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention
• an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well-known in the art. See, e.g., U.S. Patent No.
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such an antibody is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E.
• Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al, 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the amino glycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87- 95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. hi such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• the challenged filter is aseptically fransfened to 100 ml of sterile soybean-casein digest medium or fluid thioglycollate medium.
• the media are incubated at appropriate temperatures and observed three times over a 14 day period for evidence of bacterial or fungal growth. 5.5. Prophylactic And Therapeutic Utility Of The Antibody Formulations
• the formulations of the present invention may also be advantageously utilized in combination with one or more other therapies (e.g., one or more other prophylactic or therapeutic agents), preferably therapies useful in the treatment, prevention, management or amelioration of an inflammatory disorder, an autoimmune disorder, a disorder associated with abenant expression and/or activity of integrin ovft, a disorder associated with abnonnal bone metabolism, a disorder associated with abenant angiogenesis, cancer or one or more symptoms thereof.
• therapies e.g., prophylactic or therapeutic agents
• they can be administered separately, in any appropriate form and by any suitable route.
• a liquid formulation of the invention may be administered to a mammal, preferably a human, concunently with one or more other therapies (e.g., one or more other prophylactic or therapeutic agents), preferably therapies useful for the prevention, treatment, management or amelioration of an inflammatory disorder, an autoimmune disorder, a disorder associated with abenant expression and/or activity of integrin Gyft, a disorder associated with abnormal bone metabolism, a disorder associated with abenant angiogenesis, cancer or one or more symptoms thereof.
• therapies e.g., one or more other prophylactic or therapeutic agents
• a liquid formulation of the invention and one or more other prophylactic or therapeutic agents useful for prevention, treatment, management or amelioration of an inflammatory disorder, an autoimmune disorder, a disorder associated with abenant expression and/or activity of integrin ovft, a disorder associated with abnonnal bone metabolism, a disorder associated with abenant angiogenesis or cancer may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
• a liquid formulation of the invention and one or more other therapies e.g., one or more other prophylactic or therapeutic agents
• therapies useful for prevention, treatment, management or amelioration of an inflammatory disorder, an autoimmune disorder, a disorder associated with abenant expression and/or activity of integrin ovft, a disorder associated with abnormal bone metabolism, a disorder associated with abenant angiogenesis or cancer are administered within the same patient visit.
• the invention provides methods of preventing, managing, treating or ameliorating a disorder associated with abenant angiogenesis or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention
• the invention provides methods of preventing, managing, treating or ameliorating a disorder associated with abenant angiogenesis or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin c- v ft.
• therapies e.g., prophylactic or therapeutic agents
• Diseases or disorders that are associated with abenant bone metabolism include, but not limited to, rickets and osteomalacia; hypercalcemia which can be caused by, but not limited to, primary hype arathyroidism (e.g., solitary adenomas, multiple endocrine neoplasia), lithium therapy, familial hypocalciuric hypercalcemia, solid tumor with metastases (e.g., breast cancer), solid tumor with humoral mediation of hypercalcemia (e.g., lung or kidney cancer), hematologic malignancies (e.g., multiple myeloma, lymphoma, leukemia), vitamin D intoxication, sarcoidosis and other granulomatous diseases, idopathic hypercalcemia of infancy, hyperthyroidism, vitamin A intoxication, aluminum intoxication, milk-alkali syndrome, and renal failure; hypocalcemia which can be caused by, but not limited to, hereditary hypoparathyroidism, acquired hypoparathyroidis
• the liquid formulations of the invention may be used as first, second, third or fourth line of treatment for a periodontal disease.
• the invention provides methods for managing, treating or ameliorating a periodontal disease or one or more symptoms thereof in a subject refractory to conventional therapies for such a disease, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention.
• the invention also provides methods for managing, treating or ameliorating a periodontal disease or one or more symptoms thereof in a subject refractory to existing single agent therapies for such a disease, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• the invention also provides alternative methods for the management or treatment of a periodontal disease where conventional therapies have proven or may prove too toxic, i.e., results in unacceptable or unbearable side effects, for the subject being treated.
• the invention provides methods for preventing the recunence of a periodontal disease in patients that have been treated and have no disease activity by administering a liquid formulation.
• the invention provides methods of preventing, managing, treating or ameliorating aseptic loosening of a joint replacement or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• the liquid formulations of the invention may be used as first, second, third or fourth line of freatment for aseptic loosening of a joint replacement.
• the invention provides methods for managing, treating or ameliorating aseptic loosening of a joint replacement or one or more symptoms thereof in a subject refractory to conventional therapies for such a disease, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention.
• the liquid formulation of the invention may be administered to a subject in need thereof to prevent, treat, manage or ameliorate a disorder associated with abnormal bone reso ⁇ tion or one or more symptoms thereof.
• the liquid formulations of the invention may also be administered in combination with one or more therapies (e.g., prophylactic or therapeutic agents) to a subject in need thereof to prevent, manage, treat or ameliorate a disorder associated with abnom al bone reso ⁇ tion or one or more symptoms thereof.
• therapies e.g., prophylactic or therapeutic agents
• the invention provides methods of preventing, managing, treating or ameliorating a disorder associated with abnormal bone reso ⁇ tion or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention, hi another embodiment, the invention provides methods of preventing, managing, treating or ameliorating a disorder associated with abnormal bone reso ⁇ tion or one or more symptoms thereof, said method comprising administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• the invention also provides methods for managing, treating or ameliorating a disorder associated with abnormal bone reso ⁇ tion or one or more symptoms thereof in a subject refractory to existing single agent therapies for such a disease, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or fragments thereof that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• the present invention provides methods of preventing, treating, managing or ameliorating diseases or disorders associated with abnormal bone metabolism or one or more symptoms thereof, said methods comprising administering to a subject in need thereof a liquid formulation of the invention and one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments thereof that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drags, inorganic molecules, and organic molecules.
• a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context, h a specific embodiment, a cancer is refractory where the number of cancer cells has not been significantly reduced, or has increased.
• Additional cancers include, but are not limited to, the following: leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erytlroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodg in's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma;
• cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B.
• cancers caused by abenations in apoptosis can also be treated by the methods and compositions of the invention.
• Such cancers may include, but not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
• the cancer that is being prevented, managed, freated or ameliorated in accordance with the method of the invention is prostate cancer, breast cancer, bone cancer, melanoma, lung cancer and ovarian cancer
• the cancer that is being prevented, managed, treated or ameliorated in accordance with the methods of the invention are metastatic tumors including, but not limited to, tumors that have or may metastasize to the bone (non-limiting examples are prostate, breast and lung cancers that have metastasized or have the potential to metastasize to the bone), tumors that have or may metastasize to the lung, tumors that have or may metastasize to the brain, and tumors that have or may metastasize to other organs or tissues of a subject.
• agent or therapy e.g., chemotherapies, radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies
• agent or therapy which is known to be useful, or which has been used or is cunently being used for the prevention, treatment, management or amelioration of cancer or one or more symptoms thereof
• chemotherapies e.g., radiation therapies, hormonal therapies, and/or biological therapies/immunotherapies
• anti-cancer agents include, but are not limited to, angiogenesis inhibitors, topoisomerase inhibitors and immunomodulatory agents (such as chemotherapeutic agents and non-therapeutic immunomodulatory agents, including but not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (-DEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (-DEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies
• anti-T cell receptor antibodies e
• Angiogenesis inhibitors include, but are not limited to, angiostatin (plasminogen fragment); antiangiogenic antitlirombin III; angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; combretastatin A-4; endostatin (collagen XVIII fragment); fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); IM- 862; Interferon alpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin- 12; Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinase inhibitors (TIMP
• anti-cancer agents which can be used in accordance with the methods of the invention include, but not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; alfretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochlor
• anti-cancer drags include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarabicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing mo ⁇ hogenetic protein- 1; antiandrogen, prostatic carcinoma; antiesfrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL
• the invention also encompasses administration of a liquid fonnulation of the invention in combination with radiation therapy comprising the use of x-rays, gamma rays and other sources of radiation to destroy the cancer cells, h prefened embodiments, the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other prefened embodiments, the radiation treatment is administered as internal therapy or brachytherapy wherein a radiaoactive source is placed inside the body close to cancer cells or a tumor mass.
• patients with prostate cancer are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with the administration of a prophylactically or therapeutically effective amount of one or more other agents useful for prostate cancer therapy including but not limited to: external-beam radiation therapy, interstitial implantation of radioisotopes (i.e., I , palladium, Iridium), leuprolide or other LHRH agonists, non-steroidal antiandrogens (flutamide, nilutamide, bicalutamide), steroidal antiandrogens (cyproterone acetate), the combination of leuprolide and flutamide, estrogens such as DES, chlorotrianisene, ethinyl esfradiol, conjugated estrogens U.S.P., DES-diphosphate, radioisotopes, such as strontium- 89, the combination of extemal-beam radiation therapy and strontium
• radioisotopes
• immunomodulatory agents which can be administered in combination with a liquid formulation of the invention to a subject with an inflammatory disorder include, but are not limited to, methothrexate, leflunomide, cyclophosphamide, cytoxan, hnmuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (-DEC and SKB), mAB 4162W94, Orthoclone and OK
• T ⁇ F-c- antagonists which can be administered in combination with a liquid formulation of the invention to a subject with an inflammatory disorder include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab) 2 fragments, and antigen-binding fragments thereof) such as antibodies that immunospecifically bind to T ⁇ F-o; nucleic acid molecules (e.g., antisense molecules or triple helices), organic molecules, inorganic molecules, and small molecules that blocks, reduces, inhibits or neutralizes the function, activity and/or expression of T ⁇ F-c..
• proteins polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab) 2 fragments, and antigen-bind
• the present invention also encompasses the use of antibodies that immunospecifically bind to TNF-c. disclosed in the following U.S. Patents in the compositions and methods of the invention: 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380; 5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272; 5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and 6,171,787; each of which are herein inco ⁇ orated by reference in their entirety.
• TNF-c- antagonists encompassed by the invention include, but are not limited to, IL-10, which is known to block TNF-o. production via interferon ⁇ - activated macrophages (Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.
• patients with osteoarthritis are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with other agents or therapies useful for osteoarthritis prevention, treatment, management or amelioration including but not limited to: analgesics (non-limiting examples are acetaminophen, in a dose up to 4000mg/d; phenacetin; and tramadol, in a daily dose in the range of 200 to 300mg); NSAJDs (non-limiting examples include but not limited to, aspirin, diflunisal, diclofenac, etodolac, fenamates, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methylsalicylate, nebumetone, naproxin, oxaprazin, phenylbutazone, piroxicam, sulindac, and tolmetin.
• analgesics non-
• patients with chronic obstructive pulmonary disease are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of COPD including but not limited to: bronchodilators including but not limited to, short- and long- acting ft-adrenergic agonists (examples of short-acting ft agonist include but not limited to, albuterol, pirbuterol, terbutaline, and metaproterenol; examples of long-acting ft agonist include but not limited to, oral sustained-release albuterol and inhaled salmeterol), anticholinergics (examples include but not limited to iprafropium bromide), and theophylline and its derivatives (therapeutic range for theophylline is preferably 10 - 20 ⁇ ,g/mL); glucocorticoids; exogenous GiiAT (e
• patients with pulmonary fibrosis are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with an effective amount of one or more other agents useful for pulmonary fibrosis therapy including but not limited to: oxygen; corticosteroids (a non- limiting example is to administer daily prednisone beginning at 1-1.5 mg/kg/d (up to 100 mg/d) for six weeks and tapering slowly over 3 - 6 months to a minimum maintenance dose of 0.25 mg/kg/d); cytotoxic drugs (non-limiting examples are cyclophosphamide at 100 - 120 mg orally once daily, and azathioprine at 3 mg/kg up to 200 mg orally once daily); bronchodilators (non-limiting examples are short- and long- acting ft-adrenergic agonists, anticholinergics, and theophylline and its derivatives); and antihistamines (non-limiting examples are diphenhydramine and doxyl
• Inhalation is the prefened route of adminisfration for adrenergic stimulants); methylxanthines including but not limited to theophylline and its various salts; anticholinergics including but not limited to, afropine sulfate, atropine methylnitrate, and ipratropium bromide; glucocorticoids (examples including but not limited to systemic or oral steroids, and inhaled glucocorticoids); mast cell stabilizing agents (examples include but not limited to, cromolyn sodium and nedocromil sodium); leukotriene modifiers (examples include but not limited to, Zileuton, zafirlukast and montelukast); immunosuppressant agents (examples include but not limited to, methotrexate and gold salts); and mucolytic agents (examples include but not limited to acetylcysteine).
• the invention provides methods for managing, treating or ameliorating an autoimmune disorder or one or more symptoms thereof in a subject refractory to conventional therapies for such an autoimmune disorder, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention.
• the invention also provides methods for managing, treating or ameliorating an autoimmune disorder or one or more symptoms thereof in a subject refractory to existing single agent therapies for such an autoimmune disorder, said methods comprising administering to said subject a dose of a prophylactically or therapeutically effective amount of a liquid formulation of the invention and a dose of a prophylactically or therapeutically effective amount of one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin ovft.
• the invention also provides methods for managing, treating or ameliorating an autoimmune disorder or one or more symptoms thereof by administering a liquid formulation of the invention in combination with any other treatment to patients who have proven refractory to other treatments but are no longer on these treatments.
• the invention also provides alternative methods for the management or treatment of an autoimmune disorder where another therapy has proven or may prove too toxic, i.e., results in unacceptable or unbearable side effects, for the subject being treated.
• the invention provides alternative methods for the management or treatment of an autoimmune disorder where the patient is refractory to other therapies.
• the invention provides methods for preventing the recunence of an autoimmune disorder in patients that have been treated and have no disease activity by administering a liquid formulation of the invention.
• autoimmune disorders the immune system triggers an immune response when there are no foreign substances to fight and the body's normally protective immune system causes damage to its own tissues by mistakenly attacking self.
• autoimmune disorders which affect the body in different ways.
• the brain is affected in individuals with multiple sclerosis
• the gut is affected in individuals with Crohn's disease
• the synovium, bone and cartilage of various joints are affected in individuals with rheumatoid arthritis.
• the autoimmune disorder may affect only one organ or tissue type or may affect multiple organs and tissues.
• Organs and tissues commonly affected by autoimmune disorders include red blood cells, blood vessels, connective tissues, endocrine glands (e.g., the thyroid or pancreas), muscles, joints, and skin.
• autoimmune disorders that can be freated by the methods of the invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid
• the present invention provides methods of preventing, managing, treating or ameliorating an autoimmune disorder or one or more symptoms thereof, said methods comprising administering to a subject in need thereof a liquid formulation of the invention and one or more therapies (e.g., prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin ovft.
• therapies e.g., prophylactic or therapeutic agents
• Any agent or therapy which is known to be useful, or which has been used or is cunently being used for the prevention, management, treatment or amelioration of an autoimmune disorder or one or more symptoms thereof can be used in combination with a liquid formulation of the invention in accordance with the invention described herein.
• agents include, but are not limited to, immunomodulatory agents, anti-inflammatory agents and TNF-c- antagonists.
• Specific examples of immunomodulatory agents, anti-inflammatory agents and TNF-c- antagonists which can be used in combination with a liquid formulation of the invention for the prevention, management, treatment or amelioration of an autoimmune disorder are disclosed
• patients with multiple sclerosis are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with other agents or therapies useful in prevention, freatment, management and amelioration of MS including but not limited to: IFN-/31b (Betaseron) (e.g., 8.0 million international unites (MIU) is administered by subcutaneous injection every other day); IFN-/31a (Avonex) (e.g., 6.0 MIU is administered by intramuscular injection once every week); glatiramer acetate (Copaxone) (e.g., 20 mg is administered by subcutaneous injection every day); mitoxanfrone (e.g., 12 mg/m is administered by intravenous infusion every third month); azathioprine (e.g., 2-3 mg/kg body weight is administered orally each day); methotrexate (e.g., 7.5 mg is administered orally once each week); cyclophosphamide
• IFN-/31b Betas
• patients with psoriasis are administered a prophylactically or therapeutically effective amount of a liquid fonnulation of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of psoriasis including but not limited to: topical steroid cream or ointment; tar (examples including but not limited to, Estar, Psorigel, Fototar cream, and LCD 10% in Nufraderm lotion or mixed directly with triamcinolone 0.1% cream); occlusion; topical vitamin D analogue (a non-limiting example is calcipotriene ointment); ultraviolet light; PUVA (psoralen plus ultraviolet A); methotrexate (e.g., up to 25 mg once weekly or in divided doses every 12 hours for three doses once a week); synthetic retinoid (a non- limiting examples is efretinate, e.g., in dosage of 0.5-1 mg/kg/d); immuno
• patients with Crohn's disease are admimstered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of Crohn's disease including but not limited to: antidianheals (non- limiting examples are loperamide 2-4 mg up to 4 times a day, diphenoxylate with afropine 1 tablet up to 4 times a day, tincture of opium 8-15 drops up to 4 times a day, cholestyramine 2-4 g or colestipol 5 g once or twice daily); antispasmodics (non-limiting examples are propantheline 15 mg, dicyclomine 10 -20 mg, or hyoscyamine 0.125 mg given before meals); 5-aminosalicylic acid agents (non-limiting examples are sulfasalazine 1.5-2 g twice daily, mesalamine (Asacol) and its slow release form (Pentas
• patients with lupus erythematosus are administered a prophylactically or therapeutically effective amount of a liquid formulation of the invention in combination with other agents or therapies useful in prevention, treatment, management and amelioration of lupus erythematosus including but not limited to: antimalarials (including but not limited to, hydroxychloroquine); glucocorticoids (e.g., low dose, high dose, or high-dose intravenous pulse therapy can be used); immunosuppressive agents (including but not limited to, cyclophosphamide, chlorambucil, and azanthioprine); cytotoxic agents (including but not limited to methotrexate and mycophenolate mofetil); androgenic steroids (including but not limited to danazol); and anticoagulants (including but not limited to warfarin). 5.6.
• Methods of administering antibody liquid formulations of the present invention or a therapy include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, infraperitoneal, intravenous and subcutaneous), epidural administration, topical administration, and mucosal administration (e.g., intranasal and oral routes).
• parenteral administration e.g., intradermal, intramuscular, infraperitoneal, intravenous and subcutaneous
• epidural administration e.g., epidural administration
• topical administration e.g., intranasal and oral routes
• mucosal administration e.g., intranasal and oral routes.
• liquid formulations of the present invention are administered intramuscularly, intravenously, or subcutaneously.
• the liquid formulations of the invention are administered subcutaneously.
• the antibodies or antibody fragments that immunospecifically bind to integrin ovft contained in the liquid formulations of the invention are derived from a subject that is of the same species origin or species reactivity as recipient of the liquid formulations of the invention.
• liquid fomiulations of the invention comprising human or humanized antibodies that immunospecifically bind to integrin ovft contained in the liquid fomiulations of the invention are administered to a human patient for therapy or prophylaxis.
• the invention also provides that a liquid formulation of the present invention is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of antibody or antibody fragment.
• a hermetically sealed container such as an ampoule or sachette indicating the quantity of antibody or antibody fragment.
• the liquid formulations of the present invention are in a hermetically sealed container indicating the quantity and concentration of the antibody or antibody fragment.
• the liquid fonnulation of the present invention is supplied in a hermetically sealed container and comprises at least 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml of an antibody or fragment thereof that immunospecifically binds to integrin ovft, in a quantity of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml and, most preferably, 1.2 ml.
• the amount of a liquid formulation of the present invention which will be effective in the treatment, management, prevention or amelioration of an inflammatory disorder, an autoimmune disorder, a disease associated with abenant expression and/or activity of integrin ovft, a disease or disorder associated with abenant bone metabolism, a disease or disorder associated with abenant angiogenesis, cancer or one or more symptoms thereof can be determined by standard clinical techniques well-known in the art or described herein.
• the precise dose to be employed in the formulation will also depend on the route of adminisfration, and the seriousness of the inflammatory disorder, autoimmune disorder or cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight.
• the dosage administered to a patient is between 15 mg/kg and 50 mg/kg of the patient's body weight, more preferably 5 to 25 mg/kg or 5 to 15 mg/kg of the patient's body weight.
• the dosage administered to a patient is 5 mg/kg, 8 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg or 50 mg/kg of the patient's body weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides.
• lower dosages of human antibodies and less frequent administration is often possible.
• the dosage, volume and frequency of administration of liquid formulations of the present invention maybe reduced by increasing the concentration of an antibody or a fragment thereof in the formulations, increasing affinity and/or avidity of the antibody or a fragment thereof, and/or increasing the half-life of the antibody or a fragment thereof.
• Exemplary doses of a small molecule include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).
• a liquid formulation of the invention is administered in a dosing regimen that maintains the plasma concentration of the antibody immunospecific for ⁇ vft at a desirable level (e.g., about 0.1 to about 100 ⁇ g/ml), which continuously blocks the integrin ovft activity.
• a desirable level e.g., about 0.1 to about 100 ⁇ g/ml
• a liquid formulation of the invention is administered to a subject with a disease or disorder that associated with abnormal bone metabolism using a dosing regimen that maintains the plasma concentration of the antibody or antibody fragment that immunospecifically binds to integrin ovft at a level that blocks at least 40%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% » at least 85%, at least 90% or at least 95% of bone reso ⁇ tion.
• the plasma concentration of the antibody or antibody fragment that immunospecifically binds to integrin ovft is maintained at about 0.1 ⁇ g/ml to about 100 ⁇ g/ml in a subject with a disease or disorder that associated with abnormal bone metabolism.
• a liquid formulation of the invention comprising a conjugated antibody or antibody fragment immunospecific for ovft is admimstered intermittently.
• a conjugated antibody or antibody fragment refers to an antibody or antibody fragment that is conjugated or fused to another moiety, including but not limited to, a heterologous peptide, polypeptide, another antibody or antibody fragment, a marker sequence, a diagnostic agent, a therapeutic moiety, a therapeutic drag, a radioactive metal ion, a polymer, albumin, and a solid support.
• a subject preferably a human, is administered one or more doses of a prophylactically or therapeutically effective amount of an antibody or antibody fragment that immunospecifically binds to integrin ovft (e.g., VITAXIN® or a fragment thereof) in a liquid formulation of the invention, wherein the dose of a prophylactically or therapeutically effective amount of the antibody or antibody fragment in the liquid formulation of the invention administered to said subject is increased by, e.g., 0.01 ⁇ g/kg, 0.02 ⁇ g/kg, 0.04 ⁇ g/kg, 0.05 ⁇ g/kg, 0.06 ⁇ g/kg, 0.08 ⁇ g/kg, 0.1 ⁇ g/kg, 0.2 ⁇ g/kg, 0.25 ⁇ g/kg, 0.5 ⁇ g/kg, 0.75 ⁇ g/kg, 1 ⁇ g/kg, 1.5 ⁇ g/kg, 2 ⁇ g/kg, 4 ⁇ g/kg, 5 ⁇ g/kg, 10 ⁇
• liquid formulations of the invention are preferably tested in vitro, in a cell culture system, and in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans.
• assays which can be used to determine whether administration of a specific liquid formulation of the invention is indicated, include cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise contacted with a liquid formulation of the invention, and the effect of such composition upon the tissue sample is observed.
• the tissue sample can be obtained by biopsy from the patient.
• in vitro assays can be carried out with representative cells of cell types involved in an autoimmune disorder, an inflammatory disorder, a disorder associated with abenant expression and/or activity of integrin o. v ft, a disorder associated with abnormal bone metabolism, a disorder associated with abenant angiogenesis, or cancer (e.g., endothelial cells, activated T cells, osteoclasts and B cells), to detennine if a liquid formulation of the invention has a desired effect upon such cell types.
• a lower level of proliferation or survival of the contacted cells indicates that the liquid formulation of the invention is effective to treat the condition in the patient.
• a liquid formulation of the invention may be screened using cells of a tumor or malignant cell line or an endothelial cell line.
• Many assays standard in the art can be used to assess such survival and/or growth; for example, cell proliferation can be assayed by measuring 3 H-thymidine inco ⁇ oration, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g., fos and myc) or cell cycle markers; cell viability can be assessed by trypan blue staining, differentiation can be assessed visually based on changes in mo ⁇ hology, etc.
• the binding specificity, affinity and functional activity of the antibody or antibody fragment in the liquid formulations of the invention can be characterized in various in vitro binding and cell adhesion assays known in the art, including but limited to, those that are disclosed in International Publication Nos. WO 00/78815 and WO 02/070007, U.S. Patent No. 6,248,326, U.S. Patent No. 6,472,403, Pecheur et al, The FASEB J. 16(10):1266-8 (2002), Ahmed et al, The Journal of Histochemisfry & Cytochemistry 50:1371-1379 (2002), all of which are inco ⁇ orated herein by reference.
• v ft and ovft can be isolated by know techniques in the art, e.g. , by affinity chromatography as described in Cheresh, Proc. Natl. Acad. Sci. USA 84:6471-6475 (1987), and Cheresh and Spiro, J. Biol. Chem. 262:17703-17711 (1987).
• an anti-ovft antibody affinity column is used to isolate ovft from an octylglucoside human placental lysate, whereas an anti-ov affinity column is used to isolate ovft from the ovft.depleted column flow through.
• Antibody binding activity is assessed by ELISA using a goat anti-human IgG-alkaline phosphatase conjugate. A purified human IgGi antibody can be used as a control.
• the binding affinity and specificity are assessed in a competitive binding assay with the parental anti-integrin ovft antibody or antibody fragment against integrin ovft.
• Competitive binding is measured in an ELISA assay. Binding of the antibody is determined in the presence of increasing concentrations of antibody competitor.
• the control competitor antibody is again a human IgGi.
• binding affinity and specificity are assessed by measuring the inhibitory activity of the antibody or antibody fragment on integrin ovft binding to fibrinogen. Briefly, integrin ovft is plated onto ELISA plates. Inhibitory activity of the antibody or antibody fragment is determined by measuring the amount of bound biotinylated fibrinogen in the presence of increasing concentrations of antibody or control antibody. Streptavidin alkaline phosphatase is used to detect the bound fibrinogen.
• the specificity of the antibody or antibody fragment binding is assessed by the inhibition of integrin ovft binding in cell adhesion assays.
• Endothelial cell adhesion events are an important component in the angiogenic process and inhibition of ovft is known to reduce the neovascularization of tumors and thereby reduce the rate of tumor growth.
• the inhibition of ovft-mediated cell attachment by anti-integrin ovft antibody in these assays is indicative of the inhibitory activity expected when this antibody is used in situ or in vivo. Briefly, integrin ovft-positive M21 melanoma cells grown in RPMI containing 10% FBS are used for these cell binding assays.
• Cells are released from the culture dish by trypsimzation and re-suspended in adhesion buffer at a concentration of 4 x 10 5 cells/ml.
• the antibody and the control antibody are diluted to the desired concentration in 250 ⁇ l adhesion buffer (10 mM Hepes, 2 mM MgCl 2 , 2 mM CaCl 2 , 0.2 mM MnCl 2 , and 1% BSA in Hepes buffered saline at pH 7.4) and added to wells of a 48-well plate precoated with fibrinogen. Each well is coated with 200 ⁇ l fibrinogen at a concentration of 10 ⁇ g/ml for 1 hour at 37 °C.
• the inhibitory activity of an antibody or antibody fragment that immunospecifically binds to integrin ovft is also tested in an endothelial cell migration assay.
• the Transwell cell migration assay is used to assess the ability of Vitaxin to inhibit endothelial cell migration (Choi et al, J. Vascular Surg., 19:125-134(1994) and Leavesly et al, J. Cell Biol., 121:163-170 (1993).
• human umbilical vein endothelial cells in log phase and at low passage number are harvested by gentle trypsinization, wash and resuspend at a concentration of 2 x 10 6 cells/ml in 37°C HBS containing 1% BSA (20 mM Hepes, 150 mM NaCI, 1.8 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM KCI, and 5 mM glucose, pH 7.4).
• Antibodies are diluted to 10 ⁇ l/ml from stock solutions.
• Visualization of cell migration is performed by first removing the remaining cells in the upper compartment with a cotton swab. Cells that had migrated to the lower side of insert are stained with crystal violet for 30 minutes, followed by solubilization in acetic acid and the absorbance of the dye is measure at a wavelength of 550 nm. The amount of absorbance is directly proportional to the number of cells that have migrated from the upper to the lower chamber.
• BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies or antibody fragments to integrin ovft.
• BIAcore kinetic analysis comprises analyzing the binding and dissociation of integrin ovft from chips with immobilized antibodies or fragments thereof on their surface.
• in vitro assays e.g., Western blotting analysis, flow cytometric analysis, cell adhesion assay to cortical bone and exfracellular matrix proteins, cell migration assay, cell invasion assay, and cell proliferation assay, can be found in Pecheur et al, The FASEB J. 16(10):1266-8 (2002), of which the entire text is inco ⁇ orated herein by reference.
• the liquid formulations of the invention can be tested in suitable animal model systems prior to use in humans.
• animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used.
• the liquid formulations of the invention are tested in a mouse model system.
• Such model systems are widely used and well-known to the skilled artisan such as the SOD mouse model or transgenic mice where a mouse integrin ovft is replaced with the human integrin ovft, nude mice with human xenografts, animal models wherein an antibody or fragment thereof that immunospecifically binds to integrin ovft recognizes the same target as VITAXIN,® such as hamsters, rabbits, etc. known in the art and described in Relevance of Tumor Models for Anticancer Drug Development (1999, eds. Fiebig and Burger); Contributions to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, eds. Boven and Winograd); and Anticancer Drug Development Guide (1997 ed. Teicher), herein inco ⁇ orated by reference in their entireties.
• the liquid formulations of the invention can be administered repeatedly. Several aspects of the procedure may vary.
• a targeted disease or disorder e.g., inflammatory diseases, autoimmune diseases, diseases or disorders associated with abenant bone metabolism and/or abenant angiogenesis, or cancer
• a targeted disease or disorder e.g., inflammatory diseases, autoimmune diseases, diseases or disorders associated with abenant bone metabolism and/or abenant angiogenesis, or cancer
• WO 00/78815 U.S. Patent No. 6,248,326, U.S. Patent No. 6,472,403, Pecheur et al, The FASEB J. 16(10):1266-8 (2002), Ahmed et al, The Journal of Histochemisfry & Cytochemistry 50:1371-1379 (2002), all of which are inco ⁇ orated herein by reference.
• inhibition of tumor growth by a liquid formulation of the invention is tested in two animal models.
• the first model measures angiogenesis in the chick chorioallantoic membrane (CAM).
• CAM chick chorioallantoic membrane
• This assay is a well recognized model for in vivo angiogenesis because the neovascularization of whole tissue is occurring. Specifically, the assay measures growth factor induced angiogenesis of chicken CAM vessels growing toward the growth factor-impregnated filter disk or into the tissue grown on the CAM. hihibition of neovascularization is based on the amount and extent of new vessel growth or on the growth inhibition of tissue on the CAM.
• the assay has been described in detail by others and has been used to measure neovascularization as well as the neovascularization of tumor tissue (Ausprank et al, Am. J. Pathol., 79:597-618 (1975); Ossonski et al, Cancer Res., 40:2300-2309 (1980); Brooks et al, Science, 264:569-571 (1994a) and Brooks et al, Cell, 79:1157-1164 (1994b). Briefly, for growth factor induced angiogenesis filter disks are punched from #1 Whatman Qualitative Circles using a skin biopsy punch.
• Disks are first sterilized by exposure to UV light and then saturated with varying concentrations of TNF-c- of HBSS as a negative control (for at least 1 hour) under sterile conditions.
• Angiogenesis is induced by placing the saturated filter disks on the CAMs.
• Inhibition of angiogenesis is performed by treating the embryos with various amounts of Vitaxin and controls (antibody or purified human IgGi). The treatments are performed by intravenous injection approximately 24 hours after disk placement. After 48 hours, CAMs are dissected and angiogenesis is scored on a scale of 1-4.
• HBSS saturated filter disks are used as the negative control, representing angiogenesis that may occur in response to tissue injury in preparing CAMs, and, values for these CAMs are subtracted out as background.
• Purified human IgGi is used as the negative control for injections since Vitaxin is of the human IgGi subclass.
• CAM assay using growth factor-induced neovascularization additional assays can be performed utilizing tumor-induced neovascularization.
• angiogenesis is induced by transplanting of ⁇ vft- negative tumor fragments into the CAMs.
• the use of ovft-negative tumor fragments ensures that any inhibition of tumor growth is due to the inhibition of ovft -mediated neovascularization by CAM-derived endothelial cells and not to adhesion events mediated by ovft present on the tumor cells.
• Inhibition of tumor growth is assessed by placing a single cell suspension of FG (8 x 10 6 cells, pancreatic carcinoma) and Hep-3 cells (5 x 10 5 cells, laryngeal carcinoma) onto CAMs in 30 ⁇ l. One week later, tumors are removed and cut into approximately 50 mg fragments at which time they are placed onto new CAMs. After 24 hours of this second placement, embryos are injected intravenously with Vitaxin or human IgGi as a negative confrol. The tumors are allowed to grow for about 7 days following which they are removed and weighed.
• This repair mechanism is characterized by the formation of new connective tissue and the production of new capillaries.
• the newly formed capillaries are restricted to the repair zone at day 4, however, by day 8 they have extended to the outer region of the tumor.
• BALB/c nu/nu mice are used as animal models to study different diseases, especially those associated with abenant bone metabolism and/or abenant angiogenesis.
• Different cell lines e.g., CHO, or a type of cancer cells such as ' breast cancer cells
• ⁇ vft in various forms can be injected intravenously into the nude mice.
• CHO cells are fransfected with various cD ⁇ A constructs of ovft (e.g., wild-type, mutated forms) and injected intravenously into nude mice.
• the effects of ovft (with various level of activity because of the mutations) and anti-Qvft antibodies on bone metastases can be assessed by, e.g., radiograph, histological examination of bone tissue or statistical analysis.
• the anti-inflammatory activity of the liquid formulations of the invention can be assessed using a canageenan-induced arthritis rat model.
• Canageenan-induced arthritis has also been used in rabbit, dog and pig in studies of chronic arthritis or inflammation. Quantitative histomo ⁇ hometric assessment is used to determine therapeutic efficacy.
• the methods for using such a canageenan-induced arthritis model is described in Hansra P. et al, "Canageenan-hiduced Arthritis in the Rat," Inflammation, 24(2): 141-155, (2000). Also commonly used are zymosan-induced inflammation animal models as known and described in the art.
• body weight can be measured relative to a control group to determine the anti-inflammatory activity of the liquid formulations of the invention.
• the efficacy of the liquid formulations of the invention can be assessed using assays that determine bone loss.
• Animal models such as ovariectomy- induced bone reso ⁇ tion mice, rat and rabbit models are known in the art for obtaining dynamic parameters for bone formation. Using methods such as those described by Yositake et al. or Yamamoto et al, bone volume is measured in vivo by microcomputed tomography analysis and bone histomo ⁇ hometry analysis.
• animal models for inflammatory bowel disease can also be used to assess the efficacy of the liquid fonnulations of the invention (Kim et a 1., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985, Dig. Dis. Sci. 30(12 Suppl):3S-10S). Ulcerative cholitis and Crohn's disease are human inflammatory bowel diseases that can be induced in animals.
• Sulfated polysaccharides including, but not limited to amylopectin, canageen, amylopectin sulfate, and dexfran sulfate or chemical irritants including but not limited to trinitrobenzenesulphonic acid (TNBS) and acetic acid can be administered to animals orally to induce inflammatory bowel diseases.
• TNBS trinitrobenzenesulphonic acid
• Animal models for asthma can also be used to assess the efficacy of the liquid formulations of the invention.
• Animal models for autoimmune disorders can also be used to assess the efficacy of the liquid formulations of the invention.
• Animal models for autoimmune disorders such as type 1 diabetes, thyroid autoimmunity, systemic lupus erathematosus, and glomerulonephritis have been developed (Flanders et al, 1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).
• any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the liquid formulations of the inventions disclosed herein for autoimmune disorders, inflammatory diseases, diseases or disorders associated with abenant bone metabolism or abenant angiogenesis, and/or cancers.
• Assays known in the art e.g., assays described above
• Peripheral blood T-cell counts in subject can be detennined by, e.g., separating the lymphocytes from other components of peripheral blood such as plasma using, e.g., a use of Ficoll-Hypaque (Pharmacia) gradient centrifugation, labeling the T-cells with an antibody directed to a T-cell antigen such as CD3, CD4, and CD8 which is conjugated to FITC or phycoerytlirin, and measuring the number of T-cells by FACS.
• a T-cell antigen such as CD3, CD4, and CD8 which is conjugated to FITC or phycoerytlirin
• the toxicity and/or efficacy of the prophylactic and/or therapeutic protocols of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 5 0 (the dose lethal to 50%) of the population) and the ED 5 o (the dose therapeutically effective in 50%o of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
• the liquid formulations of the invention that exhibit large therapeutic indices are prefened. While liquid formulations of the invention that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
• a liquid formulation of the invention may be used to evaluate the metastatic potential of a cancer (e.g., lung cancer, breast cancer, prostate cancer, or ovarian cancer) by determining the expression and/or activity level of integrin ovft in cells or cell lines, and in tissue sections and biopsies.
• a cancer e.g., lung cancer, breast cancer, prostate cancer, or ovarian cancer
• antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 h ⁇ ), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine ( I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 h ⁇ ), and technetium ( 99 Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluor
• the detection or diagnosis of a disease or disorder can be conducted utilizing an effective amount (i.e., an amount effective to be able to detect the expression of integrin ⁇ v ft) of a liquid formulation of the invention in an in vitro and/or in vivo assay using techniques well-known to one of skilled in the art.
• an effective amount i.e., an amount effective to be able to detect the expression of integrin ⁇ v ft
• a disease or disorder is detected in the subject, preferably a mammalian subject and most preferably a human subject utilizing an effective amount of a liquid formulation of the invention in a standard imaging technique known to one of skilled in the art.
• the invention provides methods of detecting or diagnosing a disease or disorder, said methods comprising: a) administering to a subject an effective amount of a liquid formulation of the invention comprising a labeled antibody or antibody fragment that immunospecifically binds to integrin ⁇ v ft; b) waiting for a time interval following the administering for permitting the labeled antibody or antibody fragment to preferentially concentrate or localize at any desired site, e.g., cancerous site, in the subject (and for unbound labeled antibody or antibody fragment to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled antibody or antibody fragment above the background level indicates the presence of the disease.
• the invention provides methods of detecting or diagnosing a disease or disorder, said methods comprising: a) administering to a subject an effective amount of a liquid formulation of the invention comprising an antibody or antibody fragment that immunospecifically binds to integrin ⁇ v ft; b) administering to said subject a second labeled antibody or antibody fragment that recognizing the antibody or antibody fragment of the liquid formulation of the invention; c) waiting for a time interval following the administering for permitting the labeled antibody or antibody fragment to preferentially concentrate or localize at any desired site, e.g., cancerous site, in the subject (and for unbound labeled antibody or antibody fragment to be cleared to background level); d) determining background level; and e) detecting the labeled antibody or antibody fragment in the subject, such that detection of labeled antibody or antibody fragment above the background level indicates the presence of the disease.
• the tissue analyzed in accordance with methods of the invention in some embodiments are tissues from cancer patients obtained during surgery. See -Ahmed et al, The Journal of Histochemistry & Cytochemistry 50:1371-1379 (2002).
• the tissues from patient with ovary cancer presented for surgery are divided and frozen in cylinders of frozen section embedding medium (OCT) by immersion in isopentane cooled in dry ice. Frozen sections of the tissue are cut at 5 ⁇ m thickness and, if not used immediately stored at -20°C. For staining, sections are fixed in cold acetone for 15 minutes and held in Tris buffer (100 mM, pH 7.6).
• Endogenous peroxidase activity is removed using 3% H O 2 in methanol and endogenous biotin activity is blocked using a sequence of diluted egg white (5% in distilled water) and skim milk powder (5%> in distilled water), all for 10 minutes.
• the sections are incubated for 1 hour with ⁇ v ft Mab in Tris buffer (100 mM, pH 7.6).
• Antibody binding is amplified using biotin and streptavidin HRP for 15 minutes each and the complex is visualized using diaminobenzidine (DAB). Nuclei are lightly stained with Mayer's hematoxylin and the sections mounted and cover-slipped. An isotype IgGl, suitably diluted, is substituted for the antibody as a negative control. Sections are assessed microscopically for positive DAB staining by trained pathologists, and the degree of staining of ⁇ v ft expression is scored in a blind fashion.
• monitoring of a disease or disorder is carried out by repeating the method for diagnosing the disease or disorder, for example, one month after initial diagnosis, six month after initial diagnosis, and one year after initial diagnosis.
• the density of a tumor facilitates the detection of said tumor using anti-integrin ⁇ v ft antibodies in accordance with the method of the invention.
• Presence of labeled antibody or antibody fragment can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include but are not limited to: computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the antibody or antibody fragment is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al, U.S. Patent No. 5,441,050).
• the invention provides a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation of the invention.
• the liquid formulations of the invention comprise antibodies or antibody fragments recombinantly fused or chemically conjugated to another moiety, including but not limited to, a heterologous protein, a heterologous polypeptide, a heterologous peptide, a large molecule, a small molecule, a marker sequence, a diagnostic or detectable agent, a therapeutic moiety, a drug moiety, a radioactive metal ion, a second antibody, and a solid support.
• the antibody or antibody fragment included in said liquid formulations is not NITAXIN® or an antigen-binding fragment thereof.
• the kit further comprises instructions for preventing, treating, managing or ameliorating a disorder (e.g., using the liquid formulations of the invention alone or in combination with another prophylactic or therapeutic agent), as well as side effects and dosage information for method of administration.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Immunology (AREA)
• Physical Education & Sports Medicine (AREA)
• Rheumatology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Cardiology (AREA)
• Pain & Pain Management (AREA)
• Heart & Thoracic Surgery (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (5)

Publications (2)

Family

ID=32829839

Family Applications (1)

Country Status (5)

Families Citing this family (31)

Citations (2)

Family Cites Families (1)

• 2004
  • 2004-01-30 CA CA002512174A patent/CA2512174A1/en not_active Abandoned
  • 2004-01-30 WO PCT/US2004/002701 patent/WO2004066957A2/en active Application Filing
  • 2004-01-30 AU AU2004207003A patent/AU2004207003A1/en not_active Abandoned
  • 2004-01-30 JP JP2006503199A patent/JP2006516636A/ja not_active Withdrawn
  • 2004-01-30 EP EP04707034A patent/EP1589996A4/de not_active Withdrawn

Patent Citations (2)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events