Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • 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
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention in some embodiments, relates generally to the field of molecular biology, cancer biology and rheumatology. More particularly, it concerns connexin (Cx)43 hemi channel-binding antibodies and their use for the treatment and detection of disease, such as cancer, neurological injury and osteoarthritis.
• Cx connexin
• Bone tissues are a preferred site of breast and prostate cancer metastasis. Bone metastasis occurs in up to 75% of patients with advanced cancers. Currently, there is no cure for metastatic breast cancer and no reliable intervention drug for treating bone metastasis that has minimal side effects.
• Osteoarthritis (OA) is a prevalent disease that affects approximately 20% of U.S. adults. This disease causes the degeneration of joints including articular cartilage and subchondral bone. The pathology of OA is characterized by a loss of articular cartilage leading to narrowing of joint space, increased joint friction and potential structure remodeling. Current treatment includes exercise, lifestyle change and analgesics. If symptom becomes severe, joint replacement surgery is normally performed. Thus far, there is no specific pharmaceutical intervention available for the treatment of OA.
• Connexin hemichannels play important roles in the cell and tissue function, and abnormal function of connexin hemichannels may be involved various pathological conditions, such as those described above.
• pathological conditions associated with hemichannels activity e.g., inflammation, SCI, TBI, bone metastasis
• the invention provides a method of treating or preventing cancer or bone metastasis in a subject having a cancer comprising administering to the subject an effective amount of an antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening or an expression vector encoding the antibody (such as the Ab2 antibodies detailed herein).
• a method of treating or preventing osteoporosis or osteopenia in a subject comprising administering to the subject an effective amount of an antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening or an expression vector encoding the antibody (such as the Ab2 antibodies detailed herein).
• the method comprises administering an effective amount of the antibody to the subject. In further aspects, the method comprises administering an effective amount of an expression vector encoding the antibody to the subject.
• the cancer is breast cancer, prostate cancer (e.g., with bone metastasis), or osteosarcoma. In further aspects, the cancer is a cancer having bone metastases.
• the expression vector encoding the antibody may be administered in a pharmaceutically acceptable composition.
• the antibody may be administered systemically.
• the antibody may be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• the antibody may comprise a first VH CDR identical to SEQ ID NO: 19, a second VH CDR identical to SEQ ID NO: 20, a third VH CDR identical to SEQ ID NO: 21, a first VL CDR identical to SEQ ID NO: 49, a second VL CDR identical to SEQ ID NO: 50, and a third VL CDR identical to SEQ ID NO: 51.
• the antibody is a humanized antibody.
• the antibody may comprise a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 and/or a VL amino acid sequence at least 90% identical to SEQ ID NO: 63.
• the antibody comprises a VH amino acid sequence according to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 63.
• the method may additionally comprise administering at least a second anticancer therapy to the subject.
• the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.
• the invention provides a method of treating or preventing neurodegenerative disease or a neurological injury in a subject comprising administering to the subject an effective amount of an antibody that binds to a connexin 43 (Cx43) hemichannel and inhibits channel opening or an expression vector encoding the antibody (such as the Abl antibodies detailed herein).
• the method comprises administering an effective amount of the antibody to the subject.
• the method may comprise administering an effective amount of an expression vector encoding the antibody to the subject.
• the method may additionally be defined as a method for treating or preventing a neurodegenerative disease.
• the neurodegenerative disease may be multiple sclerosis or Alzheimer's disease.
• the method may additionally be defined as a method for treating or preventing a neurological injury.
• the neurological injury comprises a spinal cord injury (SCI), stroke or traumatic brain injury (TBI).
• the subject has or has been diagnosed with a neurological injury.
• the expression vector encoding the antibody is administered in a pharmaceutically acceptable composition.
• the antibody may be administered systemically.
• the antibody is administered intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• the antibody comprises a first VH CDR identical to SEQ ID NO: 19, a second VH CDR identical to SEQ ID NO: 20, a third VH CDR identical to SEQ ID NO: 21, a first VL CDR identical to SEQ ID NO: 31, a second VL CDR identical to SEQ ID NO: 32, and a third VL CDR identical to SEQ ID NO: 33.
• the antibody is a humanized antibody.
• the antibody comprises a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 and/or a VL amino acid sequence at least 90% identical to SEQ ID NO: 60.
• the antibody comprises a VH amino acid sequence according to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 60.
• the antibody comprises a first VH CDR identical to SEQ ID NO: 19, a second VH CDR identical to SEQ ID NO: 20, a third VH CDR identical to SEQ ID NO: 21, a first VL CDR identical to SEQ ID NO: 49, a second VL CDR identical to SEQ ID NO: 50, and a third VL CDR identical to SEQ ID NO: 51.
• the antibody is a humanized antibody.
• the antibody comprises a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 and/or a VL amino acid sequence at least 90% identical to SEQ ID NO: 63.
• the antibody may comprise a VH amino acid sequence according to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 63. [0015] In several aspects, the antibody may comprise a first VH CDR identical to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 63. [0015] In several aspects, the antibody may comprise a first VH CDR identical to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 63. [0015] In several aspects, the antibody may comprise a first VH CDR identical to SEQ
• the antibody is a humanized antibody.
• the antibody comprises a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 and/or a VL amino acid sequence at least 90% identical to SEQ ID NO: 60.
• the antibody comprises a VH amino acid sequence according to SEQ ID NO: 58 and/or a VL amino acid sequence according to SEQ ID NO: 60.
• the invention provides a method of treating cancer in a subject comprising administering an effective amount of a pharmaceutical composition comprising an antibody according to the embodiments and aspects described above or an expression vector encoding an antibody according to the embodiments and aspects described above to the subject.
• the pharmaceutical composition comprises an expression vector encoding an antibody according to the embodiments and aspects described above to the subject.
• the pharmaceutical composition comprises an antibody according to the embodiments and aspects described above to the subject.
• the method may further be defined as a method for inhibiting or preventing cancer bone metastasis in the subject.
• the pharmaceutical composition may be administered systemically.
• the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• the pharmaceutical composition may comprise a first VH CDR identical to SEQ ID NO: 19, a second VH CDR identical to SEQ ID NO: 20, a third VH CDR identical to SEQ ID NO: 21, a first VL CDR identical to SEQ ID NO: 31, a second VL CDR identical to SEQ ID NO: 32, and a third VL CDR identical to SEQ ID NO: 33.
• the method may further comprise administering at least a second anticancer therapy to the subject.
• the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy.
• the invention provides a method of treating an inflammatory disease, a neurodegenerative disease or a neurological injury in a subject comprising administering an effective amount of a pharmaceutical composition comprising an antibody according to the embodiments and aspects described above or an expression vector encoding an antibody according to the embodiments and aspects described above to the subject (an antibody that binds to a Cx43 hemi channel and inhibits channel opening, such as the Abl antibodies detailed herein).
• a pharmaceutical composition comprising an antibody according to the embodiments and aspects described above or an expression vector encoding an antibody according to the embodiments and aspects described above to the subject (an antibody that binds to a Cx43 hemi channel and inhibits channel opening, such as the Abl antibodies detailed herein).
• the pharmaceutical composition comprises an expression vector encoding an antibody according to the embodiments and aspects described above to the subject.
• the pharmaceutical composition comprises an antibody according to the embodiments and aspects described above to the subject.
• the method may additionally be defined as a method for treating or preventing an inflammatory disease comprising administering to the subject an effective amount of an antibody that binds to a Cx43 hemi channel and inhibits channel opening or an expression vector encoding the antibody (such as the Abl antibodies detailed herein).
• the inflammatory disease is osteoarthritis.
• a method for promoting wound healing, such as skin or cornea wound healing comprising administering to a subject an effective amount of an antibody that binds to a connexin 43 (Cx43) hemichannel and inhibits channel opening or an expression vector encoding the antibody (such as the Abl antibodies detailed herein).
• the method may additionally be defined as a method for treating or preventing a neurodegenerative disease.
• the neurodegenerative disease is multiple sclerosis or Alzheimer's.
• the method may further be defined as a method for treating or preventing a neurological injury.
• the neurological injury comprises a spinal cord injury (SCI), traumatic brain injury (TBI), or stroke.
• the subject has or has been diagnosed with a neurological injury.
• the pharmaceutical composition may be administered systemically.
• the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• an antibody of the embodiments binds an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13), KRDPCPHQVD (SEQ ID NO: 14), or LSAVYTCKR (SEQ ID NO: 15).
• an antibody binds an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13).
• the antibodies for use according to the embodiments can be any of those described in international (PCT) patent publication no. WO 2015,027120, which is incorporated herein by reference.
• the invention provides an isolated antibody which specifically binds to hemichannels, comprising a heavy chain having an amino acid sequence of SEQ ID NO:2 and a light chain having an amino acid sequence of SEQ ID NO:4.
• a first heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 13 to 37 of SEQ ID NO:2; a second heavy chain region having an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO:2; and a third heavy chain region comprising an amino acid sequence having an amino acid sequence of residues 97 to 116 of SEQ ID NO:2.
• a first light chain region comprises an amino acid sequence having an amino acid sequence of residues 9 to 40 of SEQ ID NO:4; a second light chain region having an amino acid sequence corresponding to residues 49 to 58 of SEQ ID NO:4; and a third light chain region comprising an amino acid sequence having an amino acid sequence of residues 64 to 108 of SEQ ID NO:4.
• the invention provides an isolated antibody which specifically binds to hemi channels and gap junctions, comprising a heavy chain having an amino acid sequence of SEQ ID NO:6 and a light chain having an amino acid sequence of SEQ ID NO: 8.
• a first heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 13 to 37 of SEQ ID NO:6; a second heavy chain region having an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO:6; and a third heavy chain region comprising an amino acid sequence having an amino acid sequence of residues 97 to 116 of SEQ ID NO:6.
• a first light chain region comprises an amino acid sequence having an amino acid sequence of residues 9 to 42 of SEQ ID NO: 8; a second light chain region having an amino acid sequence corresponding to residues 51 to 60 of SEQ ID NO: 8; and a third light chain region comprising an amino acid sequence having an amino acid sequence of residues 66 to 125 of SEQ ID NO: 8.
• the invention provides an isolated antibody which specifically binds to gap junctions, comprising a heavy chain having an amino acid sequence of SEQ ID NO: 10 and a light chain having an amino acid sequence of SEQ ID NO: 12.
• a first heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 10 to 34 of SEQ ID NO: 10; a second heavy chain region having an amino acid sequence corresponding to residues 43 to 59 of SEQ ID NO: 10; and a third heavy chain region comprising an amino acid sequence having an amino acid sequence of residues 94 to 109 of SEQ ID NO: 10.
• a first light chain region comprises an amino acid sequence having an amino acid sequence of residues 9 to 40 of SEQ ID NO: 12; a second light chain region having an amino acid sequence corresponding to residues 49 to 58 of SEQ ID NO: 12; and a third light chain region comprising an amino acid sequence having an amino acid sequence of residues 64 to 108 of SEQ ID NO: 12.
• antibodies include full length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies and antibody fusions, and fragments thereof.
• a further embodiment provides a pharmaceutical composition comprising an antibody as described herein with a pharmaceutically acceptable carrier. Also provided is an antibody or a pharmaceutical composition of the invention for use as a medicament or for use in therapy for cancer and to inhibit cancer metastasis. [0033] A further embodiment provides a method of treating or preventing cancer metastasis. A method of treating can comprise administering to a subject in need thereof an effective amount of an isolated antibody described herein. Also provided is the use of an antibody as described herein in the manufacture of a medicament for the treatment or prevention of cancer metastasis. [0034] Certain aspects are directed to in vitro methods of using an antibody, compounds or reagents to suppress inflamatory reactions in chondrocytes.
• methods are directed to determining the effect on inhibition of Cx43 hemichannel opening in chondrocytes by (i) determining hemichannel opening by dye uptake assay, using Lucifer yellow or Alexa dyes, (ii) assessing inhibitory effects on hemichannels opening by IL- ⁇ , (iii) test inhibitory effects of the reagents on hemichannels opening by mechanical loading in the form of fluid flow shear stress.
• Certain aspects are directed to methods of determining the effect of an antibody, compounds or reagents on suppressing of inflammatory responses evoked by IL-1 ⁇ and mechanical loading by (i) determining the inhibition of activation of nuclear factor-kappaB (NF-KB) induced by IL- ⁇ , (ii) determining the inhibition of activation of NF- ⁇ induced by fluid flow shear stress.
• NF-KB nuclear factor-kappaB
• Other aspects are directed to an in vivo method of using a monoclonal antibody, compounds or reagents to treat OA or identify the same comprising (i) injecting antibody, compound or reagent into knee cap cavity, (ii) assessing the inhibition of activation of NF-KB induced by IL- ⁇ , (iii) assessing OA development by X-ray, histological analysis and physical movement.
• the term "antigen" is a molecule capable of being bound by an antibody or T-cell receptor.
• binding moieties other than antibodies and be engineered to specifically bind to an antigen, e.g., aptamers, avimers, and the like.
• antibody or “immunoglobulin” is used to include intact antibodies and binding fragments/segments thereof.
• the term “antibody” is intended to refer broadly to any immunologic binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG as well as polypeptides comprising antibody CDR domains that retain antigen binding activity.
• the antibody may be selected from the group consisting of a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment or a natural or synthetic ligand.
• fragments compete with the intact antibody from which they were derived for specific binding to an antigen.
• Fragments include separate heavy chains, light chains, Fab, Fab' F(ab')2, Fabc, and Fv. Fragments/segments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.
• the term "antibody” also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins.
• antibody also includes bispecific antibodies.
• a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites.
• Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol. 148: 1547-53, 1992.
• isolated can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized).
• an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel.
• an "isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state.
• Moieties of the invention such as polypeptides, peptides, antigens, or immunogens, may be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
• conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
• conjugated or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical "conjugation.”
• the term "providing” is used according to its ordinary meaning “to supply or furnish for use.”
• the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid that encodes the protein.
• the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and/or epitopes.
• the phrase "specifically binds" or "specifically immunoreactive" to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologies.
• a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologies present in the sample.
• Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target.
• a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
• FIG. 1 - Cx43 is normally localized into gap junctions between cells or as hemichannels on the plasma membrane.
• side A Pathological opening of Cx43 hemichannels result in propagation of secondary injury, activation of astro/microglia, and inflammation.
• Side B illustrates the proposal that preventing pathological opening of Cx43 hemichannels prevents release of molecules, enabling astrocytes to act as caretaker cells and prevent further spread of secondary injury.
• FIG. 2 The activation of Cx43 hemichannels by IL- ⁇ in human primary astrocytes was inhibited by both Cx43 hemi channel blocking mouse monoclonal antibody (Ml) and mouse-human chimeric antibody HMAbl (these antibodies comprise the same murine variable domains and CDRs).
• Ml Cx43 hemi channel blocking mouse monoclonal antibody
• HMAbl mouse-human chimeric antibody
• FIGS. 3A-3G - Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control IgG, or HMAbl (25 mg/kg). Glial scarring was measured at 14 and 56 days after injury.
• A-F Representative images of spinal cords in mice treated with (A-C) control IgG or (D-F) HMAbl that were subjected to immunohistochemistry for the astrocyte marker GFAP. The lesion boundary is indicated with a dotted white line.
• FIGS. 5A-5B - HMAbl treatment improved the recovery of physical activity and coordination after SCI.
• Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control IgG, or human-mouse chimeric anti-Cx43 antibody (HMAbl) (25 mg/kg). Behavioral measurements are in mice with a BMS score of 0-3 at the 6 hour time point after injury.
• Rotarod Mice were tested for the ability to remain on an accelerating rotarod for up to 300 seconds to measure motor coordination. Results are averages with SEM.
• FIGS. 6A-6G - Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control Igg, or HMAbl (25 mg/kg). Neuronal dendrites were measured by immune-labeling against the neuronal marker MAP2.
• A-F Representative images of spinal cords in mice treated with (A-C) control IgG or (D-F) HMAbl that were subjected to immunohistochemistry. The lesion boundary is indicated with a dotted white line.
• FIG. 7A-7G Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control Igg, or HMAbl (25 mg/kg). Neuronal nuclei were measured by immunolabeling against the neuronal marker NeuN.
• A-F Representative images of spinal cords in mice treated with (A-C) control IgG or (D-F) HMAbl that were subjected to immunohistochemistry. The lesion boundary is indicated with a dotted white line.
• G NeuN immunolabeling was quantified as the mean intensity multiplied by area of positive stain. Results are expressed as a percentage of Sham surgery, IgG treated mice.
• FIGS. 8A-8C Breast cancer growth in bone was suppressed by human- mouse chimeric anti-Cx43 antibody HMAb2 (this antibody comprises the same murine variable domains and CDRs as the "M2" antibody).
• Py8119-Luc cells were injected into right tibias of control and cKO female mice. The left tibias were injected with PBS as controls.
• B Representative images of Cx43 cKO mice with tumor spread to the lungs and to the brain shown with white arrowheads.
• C Representative X-ray radiographs with tibia injected with Py8119 cells indicate where the tumor cells were injected and osteolytic lesions occurred (arrowheads). The left tibias injected with PBS showed no osteolytic lesions.
• FIGS. 9A-9B - Cx43 hemichannels in MLO-Y4 osteocytes (A) or primary mouse osteocytes (B) were activated by HMAb2, but blocked by HMAbl.
• the cells were incubated with E2 (polyclonal), HMAbl and HMAb2 antibody or carbenoxolone (CBX), a connexin channel blocker.
• E2 polyclonal
• CBX carbenoxolone
• Ethium bromide (EtBr) dye uptake assay was performed. Data presented as SEM. Compared to basal control, ***, P ⁇ 0.001.
• FIGS. 10A-10B Activation of Hemichannels by MHAb2 in Osteocytes in vivo.
• Evans blue dye was injected into tail vein of WT mice and 25 ⁇ g/ml MHAb2 was IP injected. Mice were sacrificed two hours after injection and perfused with PBS. Tibias were isolated and fixed tibial bone tissue sections were prepared.
• A Presence of antibodies was detected with rhodamine-conjugated anti-human IgG. Bar, 50 ⁇ .
• B Dye uptake was measured in cortical and trabecular bones by Evans blue (EB) fluorescence and quantified. *,
• FIGS. 11A-11C - HMAb2 suppresses osteolytic growth of breast cancer cells and protects bone from fractures.
• A Py8119-Luc breast cancer cells were injected into tibias of female mice.
• C The MHAb2 or saline injected mice were imaged by X-ray. *, PO.05.
• FIG. 12 - Cx43 is abundantly expressed in chondrocytes.
• Primary chondrocytes isolated from mouse bone were immunostained with anti-Cx43 antibody against C-terminal domain (Total) in permeable cells and Cx43E2 antibody in non-permeable cells.
• FIG. 13 - HMAbl blocked Cx43 hemichannels in chondrocytes.
• Primary chondrocytes isolated from mouse bone were pre-treated with carbenoxolone (connexin channel blocker) or HMAbl antibody and then treated with or without IL- ⁇ . Ethidium bromide dye uptake assay was performed to determine hemichannel activity.
• FIG. 14 - HMAbl blocked hemichannel activity in mouse chondrocytes in vivo.
• Evans blue dye was injected into tail vein of WT mice.
• Cx43(Ml) mAb 25 mg/kg was i.p. injected 2 hrs before dye injection. 30 min after dye injection, left tibias were mechanically loaded once for 10 min.
• FIG. 15 Both HMAb2 and HAb2 antibodies recognize Cx43 and bind Cx43 on osteocyte cell surface.
• A Parental HeLa or HeLa cells expressing Cx43 were immunolabeled with HMAb2 (MHC2) or HAb2 (HC2) antibody.
• B Non-permeable osteocyte MLO-Y4 cells were immunofluoresently labeled with anti-HMAb2 (MHC2) or HAb2 (HC2) antibody.
• FIG. 16 Dose-dependent inhibition of osteolytic breast cancer growth by MHAb2. Py8119-Luc breast cancer cells were injected into tibias of female mice. HMAb2 at 5, 15 and 25 mg/kg was i.p.
• FIGS. 17A-17D - HMAb2 increases trabecular bone mass, volume and thickness.
• 4 month-old mice were i.p. injected with 25 mg/kg HMAb2 antibody or saline once a week for two weeks.
• FIGS. 18A-18B Inhibition of osteolytic human breast cancer growth by MHAb2.
• Mice were sacrificed after 7 weeks and tumors were isolated.
• FIG. 19 - MHAbl suppresses inflammatory response by inhibiting nuclear translocation of NF-kB.
• Primary mouse chondrocytes were treated with or without interleukin (IL) 1 ⁇ and MHAbl, fixed and immunolabeled with antibody NF-kB antibody and countered labeled with FITC-WGA. The merged images are shown in right panels.
• IL interleukin
• Various cells are able to communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by the protein connexin.
• Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins make up one hemichannel, and 2 hemichannels make up 1 gap junction channel.
• Gap junctions are a cluster of channels that are located in the plasma membrane between adjoining cells and they mediate intercellular communication.
• Hemichannels are a separate entity from gap junction channels. Hemichannels permit the exchange of molecules between the intracellular compartments and the extracellular environment.
• Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels.
• osteocyte hemichannels are normally closed and can be opened when exposed to mechano-stimulation, which leads to the release of various factors into the bone microenvironment.
• the factors released by hemichannel opening can mediate other processes that can decrease tumor cell migration and bone metastasis.
• Certain embodiments are directed to methods of identifying reagents that modulate the opening of connexin hemichannels.
• the methods identify compounds or drugs that positively modulate the opening of connexin hemichannels.
• Other embodiments are directed to methods of treating cancer by administering a compound that open hemichannels to a patient having cancer, such as breast cancer or prostate cancer.
• the patient has a primary tumor.
• compounds that open Cx43 hemichannels can be used to inhibit or reduce metastasis to the bone.
• compounds that open Cx43 channels are used to treat osteoporosis, osteopenia, or osteosarcoma.
• Cancer metastasis occurs when a cancer spreads from the part of the body where it originated (e.g., breast or prostate) to other parts of the body (e.g., liver or bone) and establishes a secondary tumor.
• the bone is one of the most common sites of cancer metastasis.
• Cancers that metastasize to bone include, but are not limited to breast cancer, prostate cancer, lung cancer, and skin cancers (e.g., melanoma). Bone metastasis can be identified in up to 75% of patients with advanced breast and prostate cancers.
• Bone metastasis are associated with many significant clinical and quality of life consequences, such as, but not limited to intractable pain, pathological fractures, spinal cord and nerve compression, bone marrow infiltration, and impaired motility. In many cases the systemic presence of a cancer can also make the cancer incurable.
• Normal bone is made up of three major cell types: bone-forming osteoblasts, bone-resorbing osteoclasts, and osteocytes. Osteocytes make up approximately 95% of bone cells and maintain the bone remodeling process by coordinating osteolytic and osteoblastic activities. When cancer cells invade the bone, many of the normal bone functions are affected. Cancer cells interact with the local microenvironment to promote cancer cell survival via bone destruction and vascularization.
• Cx43 hemichannels in osteocytes have been shown to open by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug.
• Bisphosphonates are a class of drugs known for treating many bone disorders including bone metastasis.
• Powles et al. have shown administration of bisphosphonates to be associated with a decrease in the incidence of bone metastasis and a decrease in death rate in patients with breast cancer.
• AD has been associated with decreased tumor growth as well as reduced bone destruction and pain.
• AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in osteocytes (Plotkin et al, 2002).
• AD administration is accompanied by multiple, severe side- effects. I. ANTIBODIES
• Certain aspects of the invention are directed to antibodies that modulate, positively or negatively, the function of hemichannels.
• An example of identifying and isolating a monoclonal antibody is described below.
• CDR refers to a Complementarity Determining Region of an antibody variable domain. Systematic identification of residues included in the CDRs have been developed by Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda).
• VL Variable light chain
• VH Variable heavy chain
• CDR1 residues at positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).
• the CDRs disclosed herein may also include variants.
• the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% .
• a "variant CDR" is one with the specified identity to the parent CDR of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of the parent CDR.
• the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activity.
• Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, Ml 3 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of antigen binding protein activities as described herein.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about one (1) to about twenty (20) amino acid residues, although considerably larger insertions may be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases deletions may be much larger.
• Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative or variant. Generally these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances.
• Fab or "Fab region” as used herein is meant the polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein, or any other antibody embodiments as outlined herein.
• Fv or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.
• frame as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs.
• Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).
• antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hemi channel). It has been shown that the antigen- binding function of an antibody can be performed by fragments of a full-length antibody.
• binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL/VK, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed, 3rd ed.
• the term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule. Suitably there is no significant cross-reaction or cross- binding with undesired substances.
• the affinity of the antibody will, for example, be at least about 5 fold, such as 10 fold, such as 25-fold, especially 50-fold, and particularly 100-fold or more, greater for a target molecule than its affinity for a non-target molecule.
• specific binding between an antibody or other binding agent and an antigen means a binding affinity of at least 10 6 M _1 .
• Antibodies may, for example, bind with affinities of at least about 10 7 M _1 , such as between about 10 8 M _1 to about 10 9 M _1 , about 10 9 M _1 to about 10 10 M _1 , or about 10 10 M _1 to about 10 11 M _1 .
• Antibodies may, for example, bind with an EC50 of 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 pM or less.
• an antibody or a fragment thereof that binds to at least a portion of Cx43 protein and inhibits Cx43 signaling and cancer cell proliferation are contemplated.
• the anti-Cx43 antibody is a monoclonal antibody or a humanized antibody.
• polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs may be created that are specific to Cx43 protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.
• antibody fragments suitable for the present embodiments include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL, and CHI domains; (ii) the "Fd” fragment consisting of the VH and CHI domains; (iii) the "Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the "dAb” fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv”), wherein a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers (see U.S.
• Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains.
• Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, 1996).
• Antibody -like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
• ABSiPs antibody like binding peptidomimetics
• Animals may be inoculated with an antigen, such as a Cx43 extracellular domain protein, in order to produce antibodies specific for Cx43 protein. Frequently an antigen is bound or conjugated to another molecule to enhance the immune response.
• a conjugate is any peptide, polypeptide, protein, or non-proteinaceous substance bound to an antigen that is used to elicit an immune response in an animal.
• Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) made from a variety of individual antibody producing B lymphocytes.
• a polyclonal antibody is a mixed population of antibody species, each of which may recognize a different epitope on the same antigen.
• a monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B-lymphocyte cell line.
• the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
• rodents such as mice and rats are used in generating monoclonal antibodies.
• rabbit, sheep, or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may provide certain advantages.
• Mice e.g. , BALB/c mice) are routinely used and generally give a high percentage of stable fusions.
• Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a Cx43 antigen with an immortal myeloma cell (usually mouse myeloma).
• This technology provides a method to propagate a single antibody- producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced.
• Plasma B cells may be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for Cx43 binding cells. After enrichment of antibody producing B cells, total RNA may be isolated and cDNA synthesized.
• DNA sequences of antibody variable regions from both heavy chains and light chains may be amplified, constructed into a phage display Fab expression vector, and transformed into E. coli.
• Cx43 specific binding Fab may be selected out through multiple rounds enrichment panning and sequenced.
• Selected Cx43 binding hits may be expressed as full length IgG in rabbit and rabbit/human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and purified using a protein G resin with a fast protein liquid chromatography (FPLC) separation unit.
• HEK293 human embryonic kidney
• FPLC fast protein liquid chromatography
• the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non-human, human, or humanized sequence (e.g., framework and/or constant domain sequences).
• a heterologous non-human, human, or humanized sequence e.g., framework and/or constant domain sequences.
• Methods have been developed to replace light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact.
• "fully human" monoclonal antibodies are produced in mice transgenic for human immunoglobulin genes.
• Methods have also been developed to convert variable domains of monoclonal antibodies to more human form by recombinantly constructing antibody variable domains having both rodent, for example, mouse, and human amino acid sequences.
• Antibodies may be produced from any animal source, including birds and mammals.
• the antibodies are ovine, murine (e.g. , mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken.
• newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries.
• bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by reference. These techniques are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).
• antibodies to Cx43 will have the ability to neutralize or counteract the effects of Cx43 regardless of the animal species, monoclonal cell line, or other source of the antibody.
• Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the "Fc" portion of the antibody.
• whole antibodies may be enzymatically digested into "Fc” (complement binding) fragment, and into antibody fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immunological response, and thus, antibodies without Fc may be preferential for prophylactic or therapeutic treatments.
• antibodies may also be constructed so as to be chimeric or partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.
• Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
• Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
• substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
• Non- conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
• Proteins may be recombinant, or synthesized in vitro.
• a non- recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
• compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
• concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
• about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that binds Cx43.
• An antibody or preferably an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
• a fusion protein with other proteins.
• all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.
• Embodiments provide antibodies and antibody-like molecules against
• Cx43 polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload.
• it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
• a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
• Effector molecules comprise molecules having a desired activity, e.g. , cytotoxic activity.
• Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like.
• a reporter molecule is defined as any moiety that may be detected using an assay.
• Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
• a metal chelate complex employing, for example, an organic chelating agent such as a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody.
• DTP A diethylenetriaminepentaacetic acid anhydride
• ethylenetriaminetetraacetic acid ethylenetriaminetetraacetic acid
• N- chloro-p-toluenesulfonamide and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody.
• Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
• Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothio
• Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder associated with Cx43 signaling.
• Signaling of Cx43 may be reduced by any suitable drugs to prevent cancer cell proliferation.
• such substances would be an anti-Cx43 antibody.
• Treatment refers to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
• a treatment may include administration of a pharmaceutically effective amount of an antibody that inhibits the Cx43 signaling.
• Subject and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subj ect is a human.
• therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
• treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
• compositions e.g., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof formulated with a pharmaceutically acceptable carrier.
• Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates described herein.
• a pharmaceutical composition of the invention can comprise a combination of antibodies that bind to different epitopes on the target antigen or that have complementary activities.
• Pharmaceutical compositions of the invention also can be administered as combination therapy, i.e., combined with other agents.
• the combination therapy can include an anti-hemichannel antibody combined with at least one other anti-cancer agent.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier is suitable for intravenous, intramuscular, subcutaneous, or parenteral administration (e.g., by injection or infusion).
• the active compound i.e., antibody, or immunoconjugate
• the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
• aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions typically must be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
• this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
• Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
• the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
• dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1 -10 mg/kg.
• An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
• Preferred dosage regimens for an anti-hemichannel antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. [00117] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g/ml and in some methods about 25-300 ng/ml.
• compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• a "therapeutically effective dosage" of an anti-hemichannel antibody results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
• a therapeutically effective amount of a therapeutic compound or antibody can decrease tumor metastasis, or otherwise ameliorate symptoms in a subject.
• One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
• a composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
• routes and/or mode of administration will vary depending upon the desired results.
• Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or other parenteral routes of administration, for example by injection or infusion.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular injection and infusion.
• compositions and methods of the present embodiments involve an antibody or an antibody fragment against Cx43 to inhibit its activity in cancer cell proliferation, in combination with a second or additional therapy.
• Such therapy can be applied in the treatment of any disease that is associated with Cx43-mediated cell proliferation.
• the disease may be cancer.
• Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation.
• This process may involve contacting the cells with both an antibody or antibody fragment and a second therapy.
• a tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., antibody or antibody fragment or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) both an antibody or antibody fragment and an anti-cancer agent.
• a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
• contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
• both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
• An inhibitory antibody may be administered before, during, after, or in various combinations relative to an anti-cancer treatment.
• the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
• the antibody or antibody fragment is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
• a course of treatment will last 1 -90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered.
• This time period may last 1-7 days, and/or 1 -5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
• an antibody therapy is "A”
• an anti-cancer therapy is "B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A/A/A/A B/B/A/A/A/A/A B/B/A/A/A/A/A B/B/A/A/A/A/A B/B/A/A/A/A/A/A B/B/A/A/A/A/A/A/B
• chemotherapeutic agents may be used in accordance with the present embodiments.
• the term "chemotherapy” refers to the use of drugs to treat cancer.
• a "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
• agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
• an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
• chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); do
• DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
• Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV- irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
• Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
• Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. iii. Immunotherapy
• immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
• Rituximab (RITUXAN®) is such an example.
• the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
• the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
• the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent.
• the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
• Various effector cells include cytotoxic T cells and NK cells
• the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
• Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55.
• An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
• Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
• cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
• chemokines such as MIP-1, MCP-1, IL-8
• growth factors such as FLT3 ligand.
• immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S.
• cytokine therapy e.g., interferons ⁇ , ⁇ , and y, IL-1, GM-CSF, and TNF
• gene therapy e.g., TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S.
• Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
• Tumor resection refers to physical removal of at least part of a tumor.
• treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
• a cavity may be formed in the body.
• Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. v. Other Agents
• agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
• additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
• cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments.
• Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
• Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
• kits are envisioned containing therapeutic agents and/or other therapeutic and delivery agents.
• the present embodiments contemplates a kit for preparing and/or administering a therapy of the embodiments.
• the kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments.
• the kit may include, for example, at least one Cx43antibody as well as reagents to prepare, formulate, and/or administer the components of the embodiments or perform one or more steps of the inventive methods.
• the kit may also comprise a suitable container, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• a suitable container which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• the container may be made from sterilizable materials such as plastic or glass.
• the kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art.
• the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent. IV. Examples
• Anti-Cx43 monoclonal antibodies were generated and clones were identified that produced Cx43-binding monoclonal antibodies. CDR sequences of both DNA and amino acids for all antibody sequences are shown in the tables below along with the correct pairing for each of the characterized antibodies. [00141] Table 1 : Pairing of heavy chain and light chain for two functional antibodies.
• Table 2 Sequence of antibody chains from the hybridomas.
• Cx43 is normally localized into gap junctions between cells or as hemichannels on the plasma membrane.
• Pathological opening of Cx43 hemichannels result in propagation of secondary injury, activation of astro/microglia, and inflammation. It is proposed that preventing pathological opening of Cx43 hemichannels prevents release of molecules, enabling astrocytes to act as caretaker cells and prevent further spread of secondary injury.
• the activation of Cx43 hemi channels by IL- ⁇ in human primary astrocytes was inhibited by both Cx43 hemichannel blocking mouse monoclonal antibody (Ml) and mouse-human chimeric antibody HMAbl.
• mice treated with HMAbl had decreased glial scarring. Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control Igg, or HMAbl (25 mg/kg). Glial scarring was measured at 14 and 56 days after injury. Spinal cord tissue sections were subjected to immunohistochemistry for the astrocyte marker GFAP (red). Representative images of spinal cords in mice treated with the control IgG are shown in FIGS. 3A-C and those treated with HMAbl are shown in FIGS. 3D-F. The lesion boundary is indicated with a dotted white line.
• mice were subjected to SCI and treated with IgG or anti-Cx43 antibody (Ml) at 30 minutes post injury. Two weeks after injury, tissue sections were analyzed for expression of the astrocyte marker GFAP. Respresentative images are shown in FIGS. 4A and 4B. The results are quantified in FIG. 4C. [00150] Mice with SCI recover hind limb function after treatment with HMAbl
• FIGS. 5A-5B Mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control IgG, or human-mouse chimeric anti-Cx43 antibody (HMAbl) (25 mg/kg).
• HMAbl human-mouse chimeric anti-Cx43 antibody
• mice treated with HMAbl were found to have more neuronal dendrites in the perilesional area 14 days post SCI. As described above, mice were subjected to a single SCI and treated 30 minutes after injury with IP saline, control Igg, or HMAbl (25 mg/kg). Neuronal dendrites were measured by immunolabeling against the neuronal marker MAP2. Immunohistochemistry representative images of spinal cords in mice treated with control IgG are shown in FIGS. 6A-C and those treated with HMAbl are shown in 6D-F. The lesion boundary is indicated with a dotted white line. MAP2 immunolabeling was quantified as the mean intensity multiplied by area of positive stain, illustrated in FIG.
• mice treated with HMAbl also were observed to have more neuronal nuclei in the perilesional area 14 days post SCI. Mice were again subjected to a single SCI and treated 30 minutes after injury with IP saline, control IgG, or HMAbl (25 mg/kg). Neuronal nuclei were measured by immunolabeling against the neuronal marker NeuN. Immunohistochemistry representative images of spinal cords in mice treated with control IgG are shown in FIGS. 7A-C and those treated with HMAbl are shown in 7D-F. The lesion boundary is indicated with a dotted white line. NeuN immunolabeling was quantified as the mean intensity multiplied by area of positive stain, shown in FIG. 7G. Results are expressed as a percentage of Sham surgery, IgG treated mice. Example 3 - Diagnostic and Cancer Therapeutic Use
• E2 polyclonal
• HMAb 1 and HMAb2 antibody or carbenoxolone (CBX), a connexin channel blocker.
• CBX carbenoxolone
• EtBr Ethium bromide
• Cx43(Ml) antibody was delivered to osteocytes in vivo and found to block Evans blue uptake induced by tibial loading.
• Evans blue dye was injected into tail vein of WT, osteocyte-specific Cx43 KO.
• Mouse IgG or Cx43(Ml) mAb (25 mg/kg) was i.p. injected 2 hrs before dye injection. 30 min after dye injection, left tibias were mechanically loaded once for 10 min. Mice were sacrified and perfused with PBS. Tibias were isolated and fixed tibial bone tissue sections were prepared. The results are shown in FIGS. lOA-lOC. [00157] The inhibition of osteolytic tumor growth by HMAb2 was also observed.
• Evans blue uptake induced by tibial loading was blocked by Cx43 hemi channel blocking antibody in vivo.
• Evans blue dye was injected into tail vein of WT mice.
• Cx43(Ml) mAb 25 mg/kg was i.p. injected 2 hrs before dye injection. 30 min after dye injection, left tibias were mechanically loaded once for 10 min.
• Dye uptake was measured by Evans blue (EB) fluorescence and quantified (FIG. 14). *

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