HK1262261B - Connexin (cx) 43 hemichannel-binding antibodies and uses thereof - Google Patents

Description

Connexin (Cx)43 hemichannel binding antibodies and uses thereof

Description

This application claims the benefit of U.S. provisional patent application No. 62/300,492 filed on 26/2/2016, which is incorporated herein by reference in its entirety.

Background

1. Field of the invention

In some embodiments, the present invention relates generally to the fields of molecular biology, cancer biology and rheumatism. More particularly, it relates to connexin (Cx)43 hemichannel binding antibodies and their use for the treatment and detection of diseases such as cancer, nerve damage and osteoarthritis.

2. Description of the related Art

Traumatic Spinal Cord Injury (SCI) and Traumatic Brain Injury (TBI) are serious health problems worldwide, and over 150 million patients are diagnosed with traumatic brain injury and spinal cord injury each year. SCI and TBI patients may not only lose neurological function, but also have a greater risk of neuropathic pain and other complications associated with loss of nerve control. Secondary injury is responsible for significant post-traumatic nerve function loss. Part of the process of neuroinflammation following injury is the activation of astrocytes and the formation of glial scars, resulting in an impermeable environment for axon regeneration. Therapeutic goals include limiting lesion size and axonal loss by innovative methods of targeting astrocytes, a class of supporting cells that play a major role in supporting neuronal function and glial scarring. However, there remains a need for compositions that can be used to successfully limit colloidal scarring.

Bone tissue is the preferred site for metastasis of breast and prostate cancer. Up to 75% of patients with advanced cancer develop bone metastases. Currently, there is no cure for metastatic breast cancer and no reliable intervention drugs for treating bone metastases with minimal side effects.

Osteoarthritis (OA) is a common disease affecting approximately 20% of the adults in the united states. The disease causes the degeneration of joints, including articular cartilage and subchondral bone. OA is characterized pathologically by the loss of articular cartilage, resulting in narrowing of the joint space, increased joint friction and potential structural remodeling. Current treatments include exercise, lifestyle changes and analgesics. If the symptoms become severe, joint replacement surgery is often performed. To date, there is no specific pharmaceutical intervention available for the treatment of OA.

Connexin hemichannels play an important role in cellular and tissue function, and dysfunction of connexin hemichannels may involve various pathological conditions, such as those described above. Thus, there remains a need for additional therapies for treating pathological conditions associated with hemichannel activity (e.g., inflammation, SCI, TBI, bone metastasis), and methods for identifying such therapies.

Disclosure of Invention

In a first embodiment, the invention provides a method of treating or preventing cancer or bone metastasis in a subject having cancer, the method comprising administering to the subject an effective amount of an antibody (such as the Ab2 antibody detailed herein) or an expression vector encoding the antibody that binds to a connexin 43(Cx43) hemichannel and enhances channel opening. In a further embodiment, there is provided a method of treating or preventing osteoporosis or osteopenia in a subject, the method comprising administering to the subject an effective amount of an antibody (such as Ab2 antibody detailed herein) or an expression vector encoding the antibody that binds to connexin 43(Cx43) hemichannel and enhances channel opening. In certain aspects, the method comprises administering to the subject an effective amount of the antibody. In a further aspect, the method comprises administering to the subject an effective amount of an expression vector encoding the antibody. In some aspects, the cancer is breast cancer, prostate cancer (e.g., with bone metastases), or osteosarcoma. In a further aspect, the cancer is a cancer with bone metastasis.

In a further aspect, the expression vector encoding the antibody can be administered in a pharmaceutically acceptable composition. In certain aspects, the antibody may be administered systemically. In other aspects, the antibody can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In several aspects, the antibody can comprise a first V identical to SEQ ID NO 19 _H CDR, second V identical to SEQ ID NO 20 _H CDR, third V identical to SEQ ID NO 21 _H CDR, first V identical to SEQ ID NO. 49 _L CDR, second V identical to SEQ ID NO 50 _L CDR, and a third V identical to SEQ ID NO 51 _L And (5) CDR. In some aspects, the antibody is a humanized antibody. In certain aspects, 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. In a further aspect, 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.

In still further aspects, the method can further comprise administering at least a second anti-cancer therapy to the subject. In certain aspects, the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

In a further embodiment, the invention provides a method of treating or preventing a neurodegenerative disease or nerve injury in a subject, the method comprising administering to the subject an effective amount of an antibody (such as Ab1 antibody detailed herein) or an expression vector encoding the antibody that binds to a connexin 43(Cx43) hemichannel and inhibits channel opening. In several aspects, the method comprises administering to the subject an effective amount of the antibody. In other aspects, the method can comprise administering to the subject an effective amount of an expression vector encoding the antibody.

In some aspects, the methods may be further defined as methods for treating or preventing a neurodegenerative disease. In a further aspect, the neurodegenerative disease can be multiple sclerosis or alzheimer's disease. In other aspects, the method may be further defined as a method for treating or preventing nerve damage. In certain aspects, the nerve injury comprises Spinal Cord Injury (SCI), stroke, or Traumatic Brain Injury (TBI). In some particular aspects, the subject has or has been diagnosed with nerve damage. In several aspects, the expression vector encoding the antibody can be administered in a pharmaceutically acceptable composition. In certain aspects, the antibody may be administered systemically. In a further aspect, the antibody is administered intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In several aspects, the antibody comprises a first V identical to SEQ ID NO 19 _H CDR, second V identical to SEQ ID NO 20 _H CDR, third V identical to SEQ ID NO 21 _H CDR, first V identical to SEQ ID NO 31 _L CDR, second V identical to SEQ ID NO 32 _L CDR, and a third V identical to SEQ ID NO 33 _L And (5) CDR. In some aspects, the antibody is a humanized antibody. In certain aspects, 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. In some particular aspects, 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.

In yet further embodiments, recombinant connexin 43(Cx43) hemichannel binding antibodies are provided. In certain aspects, the antibody comprises a first V identical to SEQ ID NO 19 _H CDR, second V identical to SEQ ID NO 20 _H CDR, third V identical to SEQ ID NO 21 _H CDR, first V identical to SEQ ID NO. 49 _L CDR, second V identical to SEQ ID NO 50 _L CDR, and a third V identical to SEQ ID NO 51 _L And (5) CDR. In some aspects, the antibody is a humanized antibody. In certain particular aspects, 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. In particular aspects, 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.

In several aspects, the antibody can comprise a first V identical to SEQ ID NO 19 _H CDR, second V identical to SEQ ID NO 20 _H CDR and SEQThird V with ID NO. 21 being identical _H CDR, first V identical to SEQ ID NO. 31 _L CDR, second V identical to SEQ ID NO 32 _L CDR, and a third V identical to SEQ ID NO 33 _L And (5) CDR. In certain aspects, the antibody is a humanized antibody. In some aspects, 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. In certain particular aspects, 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.

In a still further embodiment, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody according to the above embodiments and aspects or an expression vector encoding an antibody according to the above embodiments and aspects. In some aspects, the pharmaceutical composition to the subject comprises an expression vector encoding an antibody according to the above embodiments and aspects. In other aspects, a pharmaceutical composition to a subject comprises an antibody according to the above embodiments and aspects. In several aspects, the method may also be defined as a method for inhibiting or preventing cancer bone metastasis in a subject. In certain aspects, the pharmaceutical composition may be administered systemically. In particular aspects, the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In some aspects, the pharmaceutical composition can comprise a first V identical to SEQ ID NO 19 _H CDR, second V identical to SEQ ID NO 20 _H CDR, third V identical to SEQ ID NO 21 _H CDR, first V identical to SEQ ID NO 31 _L CDR, second V identical to SEQ ID NO 32 _L CDR, and a third V identical to SEQ ID NO 33 _L And (5) CDR. In several aspects, the method can further comprise administering at least a second anti-cancer therapy to the subject. In a further aspect, the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

In a further aspect, the invention provides a method of treating an inflammatory disease, a neurodegenerative disease, or a nerve injury in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody (an antibody that binds to a Cx43 hemichannel and inhibits channel opening, e.g., Ab1 antibody detailed herein) or an expression vector encoding an antibody according to the above embodiments and aspects. In certain aspects, the pharmaceutical composition to the subject comprises an expression vector encoding an antibody according to the above embodiments and aspects. In a particular aspect, the pharmaceutical composition to the subject comprises an antibody according to the above embodiments and aspects.

In a further aspect, the method can be further defined as a method for treating or preventing an inflammatory disease, the method comprising administering to a subject an effective amount of an antibody that binds to a Cx43 hemichannel and inhibits channel opening (such as the Ab1 antibody detailed herein) or an expression vector encoding the antibody. In some particular aspects, the inflammatory disease is osteoarthritis. In some aspects, methods are provided for promoting wound healing, such as skin or cornea wound healing, comprising administering to a subject an effective amount of an antibody (such as Ab1 antibody detailed herein) or an expression vector encoding the antibody that binds to a connexin 43(Cx43) hemichannel and inhibits channel opening. In other aspects, the method may be further defined as a method for treating or preventing a neurodegenerative disease. In certain particular aspects, the neurodegenerative disease is multiple sclerosis or alzheimer's disease. In several aspects, the methods can also be defined as methods for treating or preventing nerve damage. In some particular aspects, the neural injury comprises Spinal Cord Injury (SCI), Traumatic Brain Injury (TBI), or stroke. In certain aspects, the subject has suffered from or has been diagnosed with nerve damage.

In some aspects, the pharmaceutical composition may be administered systemically. In particular aspects, the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

In certain embodiments, antibodies to hemichannel polypeptides, and nucleic acid molecules encoding such antibodies, are also provided. In certain aspects, the antibodies of the embodiments bind to an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO:13), KRDPCPHQVD (SEQ ID NO:14), or LSAVYTCKR (SEQ ID NO: 15). In a particular aspect, the antibody binds to an epitope having the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13).

In a further embodiment, the antibody used according to the embodiments may be any of those described in international (PCT) patent publication No. WO 2015,027120, which is incorporated by reference. In one embodiment, the invention provides an isolated antibody that specifically binds to a hemichannel, comprising a heavy chain having the amino acid sequence of SEQ ID NO. 2 and a light chain having the amino acid sequence of SEQ ID NO. 4.

In certain aspects, the first heavy chain region comprises an amino acid sequence having the amino acid sequence of residues 13 to 37 of SEQ ID No. 2; the second heavy chain region has an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO 2; and the third heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 97 to 116 of SEQ ID NO. 2.

In another aspect, the first light chain region comprises an amino acid sequence having the amino acid sequence of residues 9 to 40 of SEQ ID NO 4; the second light chain region has an amino acid sequence corresponding to residues 49 to 58 of SEQ ID NO 4; and the third light chain region comprises an amino acid sequence having the amino acid sequence of residues 64 to 108 of SEQ ID NO. 4.

In one embodiment, the invention provides an isolated antibody that specifically binds to a hemichannel and gap junction, comprising a heavy chain having the amino acid sequence of SEQ ID NO. 6 and a light chain having the amino acid sequence of SEQ ID NO. 8.

In certain aspects, the first heavy chain region comprises an amino acid sequence having the amino acid sequence of residues 13 to 37 of SEQ ID No. 6; the second heavy chain region has an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO 6; and the third heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 97 to 116 of SEQ ID NO 6.

In another aspect, the first light chain region comprises an amino acid sequence having the amino acid sequence of residues 9 to 42 of SEQ ID NO 8; the second light chain region has an amino acid sequence corresponding to residues 51 to 60 of SEQ ID NO 8; and the third light chain region comprises an amino acid sequence having the amino acid sequence of residues 66 to 125 of SEQ ID NO 8.

In one embodiment, the invention provides an isolated antibody that specifically binds to a gap junction, comprising a heavy chain having the amino acid sequence of SEQ ID NO. 10 and a light chain having the amino acid sequence of SEQ ID NO. 12.

In certain aspects, the first heavy chain region comprises an amino acid sequence having the amino acid sequence of residues 10 to 34 of SEQ ID No. 10; the second heavy chain region has an amino acid sequence corresponding to residues 43 to 59 of SEQ ID NO 10; and the third heavy chain region comprises an amino acid sequence having an amino acid sequence of residues 94 to 109 of SEQ ID NO 10.

In another aspect, the first light chain region comprises an amino acid sequence having the amino acid sequence of residues 9 to 40 of SEQ ID NO 12; the second light chain region has an amino acid sequence corresponding to residues 49 to 58 of SEQ ID NO 12; and the third light chain region comprises an amino acid sequence having the amino acid sequence of residues 64 to 108 of SEQ ID NO 12.

In certain aspects, 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 described herein and a pharmaceutically acceptable carrier. Also provided are antibodies or pharmaceutical compositions of the invention for use as a medicament or for use in cancer therapy and for inhibiting cancer metastasis.

Further embodiments provide methods of treating or preventing cancer metastasis. The method of treatment 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.

Certain aspects relate to in vitro methods of inhibiting inflammatory responses in chondrocytes using antibodies, compounds, or agents. In certain aspects, the methods involve determining the effect on inhibition of Cx43 hemichannel opening in chondrocytes by: (i) determining the opening of the hemichannel by a dye uptake assay using a fluoroyellow or Alexa dye, (ii) assessing the inhibition of IL-1 β on the opening of the hemichannel, (iii) testing the inhibition of the opening of the hemichannel by the agent by mechanical stress in the form of shear stress of fluid flow.

Certain aspects relate to methods of determining the effect of an antibody, compound or agent on inhibiting IL-1 β and mechanical load-induced inflammatory responses by: (i) determining inhibition of IL-1 β -induced activation of nuclear factor- κ B (NF- κ B), and (ii) determining inhibition of in vivo flow shear stress-induced activation of NF- κ B.

Other aspects relate to in vivo methods of treating or identifying OA using a monoclonal antibody, compound or agent, the method comprising: (i) injecting an antibody, compound or agent into the patellar cavity, (ii) assessing inhibition of IL-1 β -induced NF- κ B activation, (iii) assessing OA development by X-ray, histological analysis and physical movement.

As used herein, the term "antigen" is a molecule capable of being bound by an antibody or a T cell receptor. In certain embodiments, binding moieties other than antibodies can be engineered to specifically bind to antigens, e.g., aptamers, avimers, and the like.

The term "antibody" or "immunoglobulin" is used to include whole antibodies and binding fragments/segments thereof. As used herein, the term "antibody" is intended to broadly refer to any immunobinder, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides comprising CDR domains of antibodies that retain antigen binding activity. The antibody may be selected from the group consisting of: chimeric antibodies, affinity matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies or antigen binding antibody fragments or natural or synthetic ligands. Typically, the fragment competes with the intact antibody from which it was derived for specific binding to the antigen. Fragments include the individual heavy, light chains, Fab 'F (ab')2, Fabc, and Fv. Fragments/segments are generated by recombinant DNA techniques or by enzymatic or chemical isolation of intact immunoglobulins. The term "antibody" also includes one or more immunoglobulin chains chemically conjugated to other proteins or expressed as fusion proteins. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with 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 ligation of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79: 315-; kostelny et al, J.Immunol.148:1547-53, 1992.

The term "isolated" may 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) or chemical precursors or other chemicals (when chemically synthesized) in its source. Furthermore, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, a compound is not simply considered "isolated" if it is attached to a column or embedded in an agarose gel. In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally occur as a fragment and/or is not normally in a functional state.

A moiety of the invention (such as a polypeptide, peptide, antigen or immunogen) may be conjugated or covalently or non-covalently linked to another moiety (such as an adjuvant, protein, peptide, support, fluorescent moiety or label). The terms "conjugate" or "immunoconjugate" are used broadly to define the operative association of one moiety with another agent, and are not intended to refer to any type of operative association alone, and are not particularly limited to chemical "conjugation".

The term "provide" is used according to its ordinary meaning "supply or provision. In some embodiments, the protein is provided directly by administering the protein, while in other embodiments the protein is effectively provided by administering a nucleic acid encoding the protein. In certain aspects, the invention encompasses compositions comprising various combinations of nucleic acids, antigens, peptides, and/or epitopes.

The phrase "specifically binds" or "specifically immunoreactive with" a target refers to a binding reaction that determines the presence of a molecule in the presence of a heterogeneous population of other biomolecules. Thus, under the specified immunoassay conditions, the specified molecule preferentially binds to a particular target, but does not bind in large amounts to other biomolecules present in the sample. Specific binding of an antibody to a target under such conditions requires selection of the antibody for specificity with the target. A variety of immunoassay formats can 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. A description of immunoassay formats and conditions that may be used to determine specific immunoreactivity is described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention is also applicable to other aspects of the invention and vice versa. Each embodiment described herein is to be understood as an embodiment of the invention applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein may be practiced with respect to any method or composition of the present invention, and vice versa. In addition, the compositions and kits of the invention can be used to practice the methods of the invention.

The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "a" or "an", but it also conforms to the meaning of "one or more", "at least one", and "one or more".

Throughout this application, the term "about" is used to indicate that the standard deviation of error of the device or method used to determine the value is included.

The term "or" as used in the claims is intended to mean "and/or" unless specified otherwise than as specifically indicated, but only to the exclusion of alternatives or alternatives, but the disclosure supports definitions that refer only to alternatives and "and/or".

As used in this specification and claims, the word "comprising" (and any form of comprising such as "comprises" and "comprising"), "having" (and any form of having such as "has" and "has"), "including" (and any form of including such as "includes" and "includes") or "including" (and any property of including such as "includes" and "includes") is inclusive or open-ended, and does not exclude other unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of illustrative embodiments presented herein.

FIGS. 1-Cx43 are generally localized in the gap junctions between cells or as hemichannels on the plasma membrane. On the A) side: pathological opening of Cx43 hemichannels leads to the spread of secondary injury, activation of astrocytes and inflammation. B) The side illustrates the proposal to prevent pathological opening of Cx43 hemichannels from blocking molecular release, thereby allowing astrocytes to act as managing (caretaker) cells and preventing further spread of secondary injury.

Figure 2-inhibition of IL-1 β activation of Cx43 hemichannel in human primary astrocytes by Cx43 hemichannel blocking mouse monoclonal antibody (M1) and mouse-human chimeric antibody HMAb1 (these antibodies comprise the same murine variable domains and CDRs). Hemichannel activity was determined by ethidium bromide uptake.

FIG. 3A-FIG. 3G-mice were subjected to a single SCI and treated 30 min post-injury with IP saline, control IgG or HMAb1(25 mg/kg). Glial scarring was measured 14 and 56 days after injury. (A-F) representative image of spinal cord in mice treated with (A-C) control IgG or (D-F) HMAb1, where spinal cord was immunohistochemically directed to the astrocyte marker GFAP. The lesion boundaries are indicated by dashed white lines. (G) GFAP immune markers were quantified as mean intensity multiplied by the area of positive staining. Results are expressed as percentage of sham-operated, IgG-treated mice. Results are mean ± SEM. P <0.05, w/dukels (Tukey's) HSD n 3-4 compared to Igg.

FIG. 4A-FIG. 4C-mice were SCI and treated 30 min after injury with IgG or anti-Cx 43 antibody (M1). Two weeks after injury, tissue sections were analyzed for expression of the astrocytic marker GFAP. (A) Representative images of spinal cord in IgG-treated or (B) M1-treated mice. White dashed lines mark the lesion area. C) Quantification of images from n-3-5 mice shows mean ± SEM of GFAP immune markers in sections. ^* Significance was tested using two-way ANOVA followed by dukeley HSD.

Fig. 5A-fig. 5B-HMAb1 treatment improved recovery of physical activity and coordination after SCI. Mice were subjected to a single SCI and treated 30 min post injury with IP saline, control IgG, or human-mouse chimeric anti-Cx 43 antibody (HMAb1) (25 mg/kg). Behavioral measures were performed in mice at 6 hour time points after injury with BMS scores of 0-3. (A) BMS: hind limb function; 0-no hindlimb function and 9-full normal hindlimb function. (B) Rotating rod method (Rotarod): the ability of the mice to remain on the accelerated rotating bar for up to 300 seconds was tested to measure motor coordination. Results are mean ± SEM. P <0.05, p <0.01, p <0.001, w/dukel HSD compared to Igg.

FIG. 6A-FIG. 6G-mice were subjected to a single SCI and treated 30 min post-injury with IP saline, control Igg or HMAb1(25 mg/kg). Neuronal dendrites were measured by immunolabeling against the neuronal marker MAP 2. (A-F) representative image of spinal cord in mice treated with (A-C) control IgG or (D-F) HMAb1, where spinal cord was immunohistochemically performed. Lesion boundaries are indicated by dashed white lines. (G) MAP2 immunolabeling was quantified as the average intensity multiplied by the area of positive staining. Results are expressed as percentage of sham-operated, IgG-treated mice. Results are mean ± SEM. P <0.05, p <0.001, using dukel HSD, n-3-4.

FIG. 7A-FIG. 7G-mice were subjected to a single SCI and treated 30 min post-injury with IP saline, control Igg or HMAb1(25 mg/kg). Neuronal nuclei were measured by immunolabeling against the neuronal marker NeuN. (A-F) representative image of spinal cord in mice treated with (A-C) control IgG or (D-F) HMAb1, where spinal cord was immunohistochemically performed. The lesion boundaries are indicated by dashed white lines. (G) NeuN immunolabeling was quantified as the mean intensity multiplied by the area of positive staining. Results are expressed as percentage of sham-operated, IgG-treated mice.

Figure 8A-figure 8C-inhibition of breast cancer growth in bone by the human-mouse chimeric anti-Cx 43 antibody HMAb2 (this antibody comprises the same murine variable domains and CDRs as the "M2" antibody). Py8119-Luc cells were injected into the right tibia of control and cKO female mice. The left tibia was injected with PBS as a control. (A) Tumor growth was recorded once a week by bioluminescence imaging for 4 weeks and quantified. Data are presented as mean ± SEM. P < 0.01. n is 7/group. (B) Representative images of Cx43 cKO mice, where the tumor spread to the lungs and brain, are shown by white arrows. (C) Representative radiographs of tibias injected with Py8119 cells indicated the location of the injected tumor cells and indicated the development of osteolytic lesions (arrows). The left tibia injected with PBS showed no osteolytic lesions.

Fig. 9A-fig. 9B-MLO-Y4 half-channels Cx43 in bone cells (a) or primary mouse bone cells (B) were activated by HMAb2, but blocked by HMAb 1. Cells were incubated with E2 (polyclonal), HMAb1 and HMAb2 antibodies or Carbenoxolone (CBX) (connexin channel blocker). Ethidium bromide (EtBr) dye uptake assays were performed. Data are presented as SEM. P <0.001 compared to basal control.

FIG. 10A-FIG. 10B-activation of hemichannels by MHAb2 in bone cells in vivo. Evans blue dye was injected into the tail vein of WT mice and IP injected with 25 μ g/ml MHAb 2. Mice were sacrificed 2 hours after injection and perfused with PBS. The tibia was isolated and fixed tibial tissue sections were prepared. (A) The presence of antibodies was detected with rhodamine conjugated anti-human IgG. Scale bar, 50 μm. (B) Dye uptake in cortex and trabecular bone was measured and quantified by Evans Blue (EB) fluorescence. P < 0.05; p < 0.001.

Fig. 11A-fig. 11C-HMAb2 inhibit osteolytic growth of breast cancer cells and protect bone from fractures. (A) Py8119-Luc breast cancer cells were injected into the tibia of female mice. (B) Once or twice weekly intraperitoneal injections of 25mg/kg HMAb2 were given for four weeks. Saline was injected twice weekly in control mice. Tumor growth was recorded once a week by bioluminescence imaging for 4 weeks and quantified (lower panel). Data are presented as mean ± SEM. For HMAb2 and brine, n ═ 6. (C) Mice injected with MHAb2 or saline were imaged by X-ray. P < 0.05.

FIG. 12-Cx43 was abundantly expressed in chondrocytes. Primary chondrocytes isolated from mouse bone were immunostained with anti-Cx 43 antibody against the C-terminal domain (total) in permeable cells and Cx43E2 antibody in impermeable cells.

Fig. 13-HMAb1 blocked Cx43 hemichannels in chondrocytes. Primary chondrocytes isolated from mouse bone were pre-treated with carbenoxolone (connexin channel blocker) or HMAb1 antibody, with or without IL-1 β treatment. Ethidium bromide dye uptake assays were performed to determine hemichannel activity.

Figure 14-HMAb1 blocks hemichannel activity in mouse chondrocytes in vivo. Evans blue dye was injected into the tail vein of WT mice. Cx43(M1) mAb (25mg/kg) was injected intraperitoneally 2 hours prior to dye injection. The left tibia was mechanically loaded once for 10 minutes 30 minutes after dye injection. Dye uptake was measured by Evans Blue (EB) fluorescence and quantified. P < 0.001. n is 3.

Fig. 15-HMAb2 and HAb2 antibodies both recognize Cx43 on the surface of osteocytes and bind Cx 43. (A) Parental HeLa or HeLa cells expressing Cx43 were immunolabeled with HMAb2(MHC2) or HAb2(HC2) antibodies. (B) Non-permeable bone cell MLO-Y4 cells were immunofluorescent labeled with anti-HMAb 2(MHC2) or HAb2(HC2) antibodies.

Figure 16-dose-dependent inhibition of osteolytic breast cancer growth by MHAb 2. Py8119-Luc breast cancer cells were injected into the tibia of female mice. Once a week, 5mg/kg, 15mg/kg and 25mg/kg of HMAb2 were injected intraperitoneally for 4 weeks. Saline was injected weekly in control mice. Tumor growth was recorded once a week by bioluminescence imaging for 4 weeks and quantified. Data are presented as mean ± SEM. For HMAb2 and saline, n ═ 6. P < 0.05.

Fig. 17A-fig. 17D-HMAb2 increase bone mass, volume, and thickness of trabeculae. 4-month-old mice were injected intraperitoneally with 25mg/kg HMAb2 antibody or saline once a week for two weeks. Bone parameters, (a) bone volume; (B) trabecular bone thickness; (C) trabecular number of bones; and (D) Bone Mineral Density (BMD) was determined by mciroCT imaging and quantified. Data are presented as mean ± SEM. n is 6; p < 0.05; p < 0.01.

Figure 18A-figure 18B-inhibition of osteolytic human breast cancer growth by MHAb 2. (A) MDA-MB231 human breast cancer cells were injected into the tibia of female immunocompromised nude mice. Once a week, 25mg/kg HMAb2 was injected intraperitoneally for 7 weeks. Saline or human IgG was injected weekly in control mice. Tumor growth was recorded once a week by bioluminescence imaging for 7 weeks and quantified. Data are presented as mean ± SEM. n is 6. P < 0.05. (B) Mice were sacrificed 7 weeks later and tumors were isolated.

Figure 19-MHAb1 inhibited the inflammatory response by inhibiting nuclear translocation of NF-kB. Primary mouse chondrocytes were treated with or without Interleukin (IL)1 β and MHAb1, fixed and immunolabeled with antibody NF-kB antibody, and back-labeled with FITC-WGA. The merged image is shown in the right image.

Detailed Description

Various cells are able to communicate with each other and the extracellular environment by hemichannels and gap junctions formed by connexins. Connexins are ubiquitously expressed throughout the body. Six connexins constitute one hemichannel, and 2 hemichannels constitute 1 gap junction channel. Gap junctions are a cluster of channels in the plasma membrane located between adjacent cells, and they mediate inter-cellular communication. The half-channels are separate entities from the gap-link channel. Hemichannels allow molecular exchange between the intracellular compartment and the extracellular environment.

Bone cells express a hemichannel known as the connexin (Cx)43 hemichannel. These bone cell hemichannels are normally closed and can open upon exposure to mechanical stimuli, which results in the release of various factors into the bone microenvironment. Factors released by hemichannel opening may mediate other processes that may reduce tumor cell migration and bone metastasis.

Certain embodiments relate to methods of identifying agents that modulate the opening of connexin hemichannels. In certain aspects, the methods identify compounds or drugs that positively modulate the opening of connexin hemichannels. Other embodiments relate to methods of treating cancer by administering a hemichannel-opening compound to a patient having cancer. In certain aspects, the patient has a primary tumor. In certain aspects, compounds that open Cx43 hemichannels can be used to inhibit or reduce metastasis to bone. In other aspects, compounds that open Cx43 hemichannels are useful for treating osteoporosis, osteopenia, or osteosarcoma.

Cancer metastasis occurs when cancer spreads from the body part from which it originates (e.g., breast or prostate) to other body parts (e.g., liver or bone) and secondary tumors are established. 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 cancer (e.g., melanoma). Bone metastases can be identified in up to 75% of patients with advanced breast and prostate cancer. Bone metastases (mets) are associated with a number of serious clinical and quality of life consequences, such as, but not limited to, intractable pain, pathological fractures, spinal and nerve compression, bone marrow infiltration, and impaired motility. In many cases, the systemic presence of cancer can also render the cancer incurable.

Normal bone is composed of three large cell types: osteoblasts that form bone, osteoclasts and osteocytes that resorb bone. Osteocytes constitute approximately 95% of osteoblasts and maintain the bone remodeling process by coordinating osteolytic and osteogenic activities. When cancer cells invade bone, many normal bone functions are affected. Cancer cells interact with the local microenvironment to promote cancer cell survival via bone destruction and angiogenesis.

Cx43 hemichannels in bone cells have been shown to be opened by treatment with Alendronate (AD), an effective and commonly used bisphosphonate drug. Bisphosphonates are a class of drugs known for the treatment of many bone conditions, including bone metastases. Polles et al have shown that administration of bisphosphonates is associated with a reduced incidence of bone metastasis and a reduced mortality in breast cancer patients. AD is associated with reduced tumor growth as well as reduced bone destruction and pain. AD inhibits osteoclast activity and induces opening of Cx43 hemichannels in osteocytes (Plotkin et al, 2002). AD administration, however, is associated with a number of serious side effects.

I. Antibodies

Certain aspects of the invention relate to antibodies that positively or negatively modulate hemichannel function. Examples of identification and isolation of monoclonal antibodies are described below.

As used herein, the term "CDR" refers to the complementarity determining regions of an antibody variable domain. Systematic identification of residues included in CDRs has been developed by Kabat et al (1991, Sequences of Proteins of Immunological Interest, 5 th edition, United States Public Health Service, National Institutes of Health, Bethesda). The variable light chain (VL) CDRs are defined herein to include residues at positions 27-32(CDR1), 50-56(CDR2), and 91-97(CDR 3). The variable heavy chain (VH) CDRs are defined herein to include residues at positions 27-33(CDR1), 52-56(CDR2), and 95-102(CDR 3).

As recognized by one of skill in the art, the CDRs disclosed herein can also include variants. Generally, the amino acid identity between the individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a "variant CDR" is a CDR that has a specified identity to a parent CDR of the invention and shares a 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% specificity and/or activity of the parent CDR.

Although the site or region for introducing amino acid sequence variation is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of mutations at a given site, random mutagenesis is performed at the target codon or target region and the expressed antigen binding protein CDR variants are screened for the optimal combination of desired activities. Techniques for generating substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of mutants was performed using the antigen binding protein activity assay as described herein.

Amino acid substitutions are typically single residues; insertions will typically be on the order of about one (1) to about twenty (20) amino acid residues, but significantly larger insertions may also be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, but in some cases the deletion may be much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at the final derivative or variant. Generally, these changes are made on a few amino acids in order to minimize changes to the molecule (particularly the immunogenicity and specificity of the antigen binding protein). However, larger changes may be tolerated in some cases.

As used herein, "Fab" or "Fab region" means a polypeptide comprising VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to such regions in the context of isolation, or in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody embodiment as outlined herein.

As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL and VH domains of a single antibody.

As used herein, "framework" means regions of an antibody variable domain that do not include those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions separated by CDRs (FR1, FR2, FR3 and FR 4).

As used herein, the term "antigen-binding portion (or simply" antibody 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., a hemichannel). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL/VK, VH, CL and CH1 domains; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) fab' fragment, which is essentially a Fab with a partial hinge region (Fundaminatal IMMUNOLOGY (Paul eds., 3 rd edition 1993)); (iv) an Fd fragment consisting of the VH and CH1 domains; (v) fv fragments consisting of the VL and VH domains of a single arm of an antibody; (vi) dAb fragments (Ward et al, (1989) Nature341:544-546) which consist of a VH domain; (vii) an isolated Complementarity Determining Region (CDR); and (viii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains.

The term "specific binding" ("or immunospecific binding") 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 than its affinity for non-target molecules. Suitably, there is no significant cross-reaction or cross-binding with the undesired species. The affinity of the antibody for the target molecule will be, for example, at least about 5-fold, such as 10-fold, such as 25-fold, especially 50-fold, especially 100-fold or more, greater than its affinity for non-target molecules. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10 ⁶ M ^-1 Binding affinity of (4). The antibody can be, for example, at least about 10 ⁷ M ^-1 Such as 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 With an affinity therebetween. The antibody can, for example, have an EC of 50nM or less, 10nM or less, 1nM or less, 100pM or less, or more preferably 10pM or less ₅₀ And (4) carrying out combination.

In certain embodiments, antibodies or fragments thereof that bind to at least a portion of a Cx43 protein and inhibit signaling of Cx43 and cancer cell proliferation are contemplated. Preferably, the anti-Cx 43 antibody is a monoclonal or humanized antibody. Thus, by known methods and as described herein, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including any of the aforementioned engineered forms) specific for a Cx43 protein, one or more of its corresponding epitopes, or any of the aforementioned conjugates, can be generated, whether such antigen or epitope is isolated from a natural source or a synthetic derivative or variant of a natural compound.

Examples of antibody fragments suitable for use in embodiments of the invention include, but are not limited to: (i) fab fragment consisting of V _L 、V _H 、C _L And C _H1 Domain composition; (ii) fragment "Fd" consisting of V _H And C _H1 Domain composition; (iii) "Fv" fragments consisting of the V of a single antibody _L And V _H Domain composition; (iv) "dAb" fragment consisting of V _H Domain composition; (v) an isolated CDR region; (vi) a F (ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules ("scFv"), wherein V _H Domains and V _L The domains are connected by a peptide linker that allows association of the two domains to form a binding domain; (viii) bispecific single chain Fv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, i.e., multivalent or multispecific fragments constructed by gene fusion (U.S. patent application publication 20050214860). Fv, scFv or diabody molecules can be linked to V by incorporation _H And V _L The disulfide bridges of the domains are stabilized. Minibodies comprising scFv linked to the CH3 domain can also be made (Hu et al, 1996).

Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al (2003) describe "antibody-like binding peptide mimetics" (ABiP), which are peptides that act as simplified antibodies and have a longer serum half-life and certain advantageous advantages of less cumbersome synthetic methods.

Animals can be inoculated with antigens, such as Cx43 extracellular domain proteins, to generate antibodies specific for Cx43 proteins. Typically, an antigen is bound or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non-protein substance that binds to an antigen used to elicit an immune response in an animal. Antibodies produced in animals in response to antigen vaccination include a variety of non-identical molecules (polyclonal antibodies) produced by B lymphocytes producing a variety of individual antibodies. Polyclonal antibodies are a mixed population of antibody species, each of which can recognize a different epitope on the same antigen. Given the correct conditions for the production of polyclonal antibodies in an animal, most antibodies in the serum of an animal will recognize a collective epitope on the antigenic compound by which the animal has been immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the antigen or epitope of interest.

A monoclonal antibody is a single antibody in which each antibody molecule recognizes the same epitope, since all antibody-producing cells are derived from a single B lymphocyte cell line. Methods for producing monoclonal antibodies (mabs) generally begin along the same routes as those used to make polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In some embodiments, cells of rabbits, sheep, or frogs are used to produce monoclonal antibodies. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB/c mice) are routinely used and typically provide 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 immortalized myeloma cell, usually a mouse myeloma. This technology provides a means to propagate a single antibody-producing cell for an infinite number of generations so that an unlimited number of structurally identical antibodies (monoclonal antibodies) of the same antigen or epitope specificity can be produced.

Plasma B cells can be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for Cx 43-binding cells. After enrichment of antibody-producing B cells, total RNA can be isolated and cDNA synthesized. The DNA sequences from the antibody variable regions of the heavy and light chains can be amplified, constructed into phage display Fab expression vectors, and transformed into e. Fab binding specifically to Cx43 can be selected by multiple rounds of enrichment panning and sequenced. Selected Cx43 binding hits can be expressed in rabbits as full length iggs and in human embryonic (HEK293) cells (Invitrogen) as rabbit/human chimeric forms using a mammalian expression vector system and purified using a protein G resin using a Fast Protein Liquid Chromatography (FPLC) separation unit.

In one embodiment, the antibody is a chimeric antibody, e.g., an antibody comprising an antigen binding sequence from a non-human donor grafted to a heterologous non-human, human or humanized sequence (e.g., a framework and/or constant domain sequence). Methods have been developed to replace the light and heavy chain constant domains of monoclonal antibodies with human-derived analogous domains, thereby leaving the variable regions of the foreign antibody intact. Alternatively, "fully human" monoclonal antibodies are produced in mice transgenic for human immunoglobulin genes. Methods have also been developed to convert the variable domains of monoclonal antibodies to a more human form by recombinantly constructing antibody variable domains having rodent, e.g., mouse and human, amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDRs are derived from a mouse monoclonal antibody and the framework and constant regions are derived from human amino acid sequences (see U.S. Pat. nos. 5,091,513 and 6,881,557). It is believed that the substitution of the amino acid sequence in the antibody characteristic of rodents with the amino acid sequence found in the corresponding position of a human antibody will reduce the likelihood of an adverse immune response during therapeutic use. Hybridomas or other cells producing antibodies can also be subjected to genetic mutations or other changes, which may or may not alter the binding specificity of the antibodies produced by the hybridomas.

Methods for producing polyclonal antibodies in various animal species, as well as for producing monoclonal antibodies of different types (including humanized, chimeric, and fully human) are well known in the art and are highly predictable. For example, the following U.S. patents and patent applications provide enabling descriptions of such methods: U.S. patent application nos. 2004/0126828 and 2002/0172677; and U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797, respectively; 4,472,509; 4,606,855; 4,703,003, respectively; 4,742,159, respectively; 4,767,720, respectively; 4,816,567; 4,867,973, respectively; 4,938,948, respectively; 4,946,778; 5,021,236, respectively; 5,164,296, respectively; 5,196,066, respectively; 5,223,409; 5,403,484; 5,420,253, respectively; 5,565,332; 5,571,698; 5,627,052; 5,656,434, respectively; 5,770,376, respectively; 5,789,208; 5,821,337; 5,844,091, respectively; 5,858,657, respectively; 5,861,155, respectively; 5,871,907, respectively; 5,969,108; 6,054,297; 6,165,464, respectively; 6,365,157, respectively; 6,406,867, respectively; 6,709,659, respectively; 6,709,873, respectively; 6,753,407, respectively; 6,814,965, respectively; 6,849,259, respectively; 6,861,572, respectively; 6,875,434, respectively; and 6,891,024. All patents, patent application publications, and other publications cited herein and in the text of this application are hereby incorporated by reference.

Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibody is of ovine, murine (e.g. mouse and rat), rabbit, goat, guinea pig, camel, horse or chicken origin. Furthermore, newer technologies allow the development of human antibodies and the screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression techniques allow the production of specific antibodies in the absence of animal immunization, as described in U.S. patent No. 6,946,546 (incorporated herein by reference). These techniques are described in Marks (1992); stemmer (1994); gram et al (1992); barbas et al (1994); and Schier et al (1996) further.

It is fully expected that antibodies directed against Cx43 will have the ability to neutralize or counteract the effects of Cx43 regardless of the animal species, monoclonal cell line, or other antibody source. Certain animal species may be less preferred for the production of therapeutic antibodies because they may more readily elicit an allergic response due to activation of the complement system (via the "Fc" portion). However, intact antibodies can be enzymatically digested into "Fc" (complement-binding) fragments, and antibody fragments with binding domains or CDRs. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immune response, and therefore, Fc-free antibodies may be preferred for prophylactic or therapeutic treatment. As described above, antibodies can also be constructed to be chimeric antibodies or partially or fully human antibodies to reduce or eliminate adverse immune consequences resulting from administration to an animal of antibodies that have been produced in or have sequences from other species.

A substitution variant typically contains an amino acid exchange for another at one or more positions within the protein and may be designed to modulate one or more characteristics of the polypeptide (with or without loss of other function or property). Substitutions may be conservative, i.e., one amino acid is replaced by an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine to serine, glutamine to asparagine, glutamic acid to aspartic acid, 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. Alternatively, the substitutions may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the substitution of residues with chemically different residues, such as the substitution of a non-polar or uncharged amino acid with a polar or charged amino acid, and vice versa.

The protein may be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be implemented in the compositions and methods. Therefore, there is no need to isolate the protein.

It is contemplated that between about 0.001mg and about 10mg of total polypeptide, peptide, and/or protein is present per ml in the composition. Thus, the concentration of protein in the composition may 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, 10.0mg/ml or more (or any range derivable therein). Among these, 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% can be an antibody that binds Cx 43.

The antibody, or preferably the immunological portion of the antibody, may be chemically conjugated to or expressed as a fusion protein with other proteins. For the purposes of the present specification and appended claims, all such fusion proteins are included in the definition of antibody or immunological portion of an antibody.

Embodiments provide antibodies or antibody-like molecules against Cx43, polypeptides and peptides linked to at least one agent to form antibody conjugates or payloads. In order to increase the efficacy of an antibody molecule as a diagnostic or therapeutic agent, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such 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, such as cytotoxic activity. Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter is defined as any moiety that can 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.

Several methods for linking or conjugating antibodies to their conjugate moieties are known in the art. Some ligation methods involve the use of metal chelates, for example, with organic chelators attached to antibodies, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylene triamine tetraacetic acid; n-chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycoluril-3. Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

Treatment of diseases

Certain aspects of the embodiments of the invention are useful for preventing or treating diseases or disorders associated with Cx43 signaling. Cx43 signaling can be attenuated by any suitable drug to prevent cancer cell proliferation. Preferably, such substances may be anti-Cx 43 antibodies.

"Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a surgical procedure or physical therapy on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, treatment can include administering a pharmaceutically effective amount of an antibody that inhibits Cx43 signaling.

"subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In a specific embodiment, the subject is a human.

As used throughout this application, the term "therapeutic benefit" or "therapeutically effective" refers to any effect that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, the frequency or severity of signs or symptoms of the disease. For example, treatment of cancer may include, for example, reducing tumor size, reducing tumor invasiveness, reducing the growth rate of cancer, or preventing metastasis. Treatment of cancer may also refer to prolonging the survival time of a cancer subject.

A. Pharmaceutical composition

Certain aspects include compositions, e.g., pharmaceutical compositions, comprising one or a combination of monoclonal antibodies, or antigen-binding portions thereof, formulated with a pharmaceutically acceptable carrier. Such compositions can comprise one or a combination of (e.g., two or more different) antibodies or immunoconjugates described herein. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies that bind different epitopes on a target antigen or have complementary activity.

The pharmaceutical compositions of the present invention may also be administered as a combination therapy, i.e., in combination with other agents. For example, the combination therapy may include an anti-hemichannel antibody in combination with at least one other anti-cancer agent.

As used herein, "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. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous or parenteral administration (e.g. by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody or immunoconjugate) may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the invention 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). Proper fluidity can be maintained, for example, by the use of a coating material (such as lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for higher drug concentrations. 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. Proper fluidity can be maintained, for example, by the use of a coating material (such as lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include 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 which 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 by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, 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 that can be combined with the carrier material to produce a single dosage form will vary with the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Generally, the amount ranges from about 0.01% to about 99% of the active ingredient in one hundred parts, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adjusted to provide the optimum desired response (e.g., 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 particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a pre-calculated, determined amount of active compound that can be combined with the desired pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the unit dosage form of the present invention are determined by and directly depend on the following factors: (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding such active compounds for treating sensitivity in individuals.

For antibody administration, the dosage range is about 0.0001 to 100mg/kg, and more usually 0.01 to 5mg/kg of host body weight. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight or 10mg/kg body weight or in the range of 1-10mg/kg body weight. Exemplary treatment regimens entail administering once a week, once every two weeks, once every three weeks, once every four weeks, once every month, once every three months, or once every three to six months. A preferred dosage regimen for an anti-hemichannel antibody of the invention comprises administration of 1mg/kg body weight or 3mg/kg body weight intravenously, wherein the antibody is administered using one of the following dosing schedules: (i) six doses were continued once every four weeks, then once every three months; (ii) once every three weeks; (iii) 3mg/kg body weight and then 1mg/kg body weight once every three weeks.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated range. The antibody is typically administered multiple times. The interval between single doses may be, for example, weekly, monthly, every three months, or yearly. The intervals may also be irregular, as indicated by measuring blood levels of antibodies to the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μ g/ml, and in some methods 25-300 μ g/ml.

The actual dosage level of the active ingredient in the pharmaceutical compositions of this invention can 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 toxicity to the patient. The selected dosage level depends on a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, body 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 dose" of an anti-hemichannel antibody results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of injury or disability resulting from the affliction with the disease. A therapeutically effective amount of a therapeutic compound or antibody can reduce tumor metastasis or otherwise alleviate symptoms in a subject. One skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result. Preferred routes of administration of the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or other parenteral routes of administration, e.g., by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular injection and infusion.

B. Combination therapy

In certain embodiments, the compositions and methods of the invention relate to antibodies or antibody fragments against Cx43 that inhibit the activity of Cx43 in cancer cell proliferation in combination with a second or additional therapy. Such therapies may be useful for treating any disease associated with Cx 43-mediated cell proliferation. For example, the disease may be cancer.

The methods and compositions (including combination therapies) enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect, such as killing cancer cells and/or inhibiting cell hyperproliferation. The method can include contacting the cell with the antibody or antibody fragment and a second therapy. The tissue, tumor, or cell can be contacted with one or more compositions or pharmaceutical preparations comprising one or more agents (i.e., antibodies or antibody fragments or anti-cancer agents), or by contacting the tissue, tumor, and/or cell with two or more different compositions or preparations, wherein one composition provides 1) an antibody or antibody fragment, 2) an anti-cancer agent, or 3) an antibody or antibody fragment and an anti-cancer agent. In addition, it is contemplated that such combination therapy may be used in combination with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

The terms "contacted" and "exposed," when used with respect to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing, for example, the two agents are delivered to the cells in a combined amount effective to kill the cells or prevent their division.

The inhibitory antibodies can be administered before, during, after, or in various combinations with the anti-cancer treatment. Administration may have intervals ranging from simultaneous to minutes to days to weeks. In embodiments in which the antibody or antibody fragment and anti-cancer agent are provided to the patient separately, it is generally ensured that a significant period of time does not expire between the time of each delivery, so that the two compounds are still capable of producing an advantageously combined effect on the patient. In such cases, it is contemplated that the patient may be provided with antibody therapy and anti-cancer therapy within about 12 to 24 or 72 hours of each other, and more specifically within about 6-12 hours of each other. In some cases, it may be desirable to significantly extend the treatment period, with days (2, 3,4, 5,6, or 7 days) to weeks (1, 2, 3,4, 5,6, 7, or 8 weeks) between administrations.

In certain embodiments, the course of treatment will last from 1 to 90 days or longer (such range includes the number of days in between). It is contemplated that one agent may be administered on any day from day 1 to day 90 (such range includes intervening days), or any combination thereof, and the other agent may be administered on any day from day 1 to day 90 (such range includes intervening days), or any combination thereof. The agent may be administered to the patient one or more times during a day (24 hour period). Furthermore, after the course of treatment, it is envisaged that there will be a period of time during which the anti-cancer treatment is not administered. Depending on the condition of the patient, such as their prognosis, resistance, health, etc., this period may last from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months or longer (such ranges include the number of days in between). It is contemplated that the treatment cycle will be repeated as needed.

Various combinations may be employed. For the following examples, the antibody therapy is "a" and the anti-cancer therapy is "B":

administration of any compound or therapy of the present embodiments to a patient will follow the general protocol for administering such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

i. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance with embodiments of the present invention. The term "chemotherapy" refers to the treatment of cancer with drugs. "chemotherapeutic agent" is used to mean a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell (e.g., whether they affect the cell cycle and at what stage). Alternatively, agents can be characterized based on their ability to directly cross-link DNA, intercalate DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzotepa (benzodopa), carboquone (carboquone), meturedpa (meturedpa), and uredepa (uredpa); ethyleneimine and methylmelamine, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphamide), and trimethylolmelamine (trimetylomelamine); annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (including the synthetic analog topotecan); bryostatin; kelistatin (callystatin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (duocarmycins) (including the synthetic analogs KW-2189 and CB1-TM 1); shogaol (eleutherobin); pancratistatin; sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards such as chlorambucil, chlorambucil (chlorenaphazine), cholorophosphamide (cholophosphamide), estramustine, ifosfamide (ifosfamide), dichloromethyl diethylamine (mechleretamine), dichloromethyl diethylamine oxide hydrochloride, melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenesterine), prednimustine (trofosfamide), and uracil mustard; nitrosoureas such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranirnustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ lI and calicheamicin ω I1); daptomycin (dynemicin), including daptomycin a; diphosphates (bisphosphatates), such as clodronate; esperamicin (esperamicin); and neocarcinostatin (neomycin) chromophores and related chromoproteins enediyne antibiotics chromophores, aclacinomycin (aclacinomycin), actinomycin (actinomycin), amphenicol (aurramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cacinomycin), karabixin (carabicin), carminomycin (carminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detroribicin), 6-aryldiazo-5-oxo-L-norleucine, doxorubicin (doxorubicin) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolostatin and doxorubicin), epirubicin (epirubicin), such as doxorubicin (mitomycin), mitomycin (mitomycin), and doxorubicin (mitomycin), such as doxorubicin (mitomycin), streptomycin (azamycin (clarithromycin), and doxorubicin (mitomycin), Mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (olivomycin), pelomomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (queamycin), roxobicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), and zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin and trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, azathioprine, and azathioprine; pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), and floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), and testolactone (testolactone); anti-adrenal agents such as mitotane (mitotane) and trilostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acegulonone (acegultone); (ii) an aldophosphamide glycoside; (ii) aminolevulinic acid; eniluracil (eniluracil); amsacrine (amsacrine); doubly-branched betuzucil; bisantrene; edatrexate (edatraxate); deflazafamine (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); iloxanel (elformithine); ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (lentinan); lonidanine (lonidainine); maytansinoids such as maytansine and ansamitocin (ansamitocin)); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK polysaccharide complex; razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecene toxins (trichothecenes), especially T-2 toxin, veracurin a, bacillocin a and serpentine (anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; arabinoside ("Ara-C"); cyclophosphamide; taxanes (taxoids), such as paclitaxel and docetaxel; gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin (oxaplatin), and carboplatin; vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); norfloxacin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin; (xiloda); ibandronate (ibandronate); irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine (capecitabine), carboplatin, procarbazine, plicamycin (plicomycin), gemcitabine (gemcitabine), vinorelbine (navelbine), farnesyl protein transferase inhibitors (famesyl-protein transferase inhibitors), antiplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Radiotherapy

Other factors that cause DNA damage and have been widely used include the direct delivery of what are commonly referred to as gamma rays, X-rays, and/or radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and UV-irradiation. Most likely all of these factors cause extensive damage to DNA, to the precursors of DNA, to the replication and repair of DNA, and to the assembly and maintenance of chromosomes. The dose of X-rays ranges from a daily dose of 50 to 200 roentgens for a long period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

immunotherapy

One skilled in the art will appreciate that additional immunotherapies may be combined or used in conjunction with the methods of the embodiments. In the context of cancer therapy, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. RituximabIs one such 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 act as an effector of the treatment or it may recruit other cells to actually achieve cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin a chain, cholera toxin, pertussis toxin, etc.) and act as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells including cytotoxic T cells and NK cells

In one aspect of immunotherapy, tumor cells must have some markers suitable for targeting, i.e., not present on most other cells. Many tumor markers exist and any of these markers may be suitable for targeting in the context of embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p 155. An alternative aspect of immunotherapy is the combination of anti-cancer effects with immunostimulating effects. Immunostimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ -IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use are immunological adjuvants, such as Mycobacterium bovis (Mycobacterium bovis), Plasmodium falciparum (Plasmodium falciparum), dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapies such as interferon alpha, beta and gamma, IL-1, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, for example, TNF, IL-1, IL-2 and p53(Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD 20, anti-ganglioside GM2, and anti-p 185(Hollander, 2012; Hanibuchi et al, 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

Surgery

About 60% of people with cancer will undergo the same type of surgery, including preventative, diagnostic or staged, curative and palliative surgery. Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, resected, and or destroyed, and may be used in conjunction with other therapies, such as the treatments of embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or replacement therapy. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment with surgery includes laser surgery, cryosurgery, electrosurgery, and surgery controlled with a microscope (morse surgery).

After partial or complete resection of the cancerous cell, tissue, or tumor, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection or topical application of the area with additional anti-cancer therapies. 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 also have different dosages.

v. other Agents

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of the treatment. These additional agents include agents that effect upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in cell signaling by increasing the number of GAP junctions may increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents may 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 embodiments of the invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the present embodiments to improve therapeutic efficacy.

Kits and diagnostic agents

In various aspects of embodiments, kits are envisioned containing therapeutic and/or other therapeutic agents and delivery agents. In some embodiments, the present embodiments encompass kits for making and/or administering the therapies of the embodiments. The kit may include one or more sealed vials containing any of the pharmaceutical compositions of the embodiments of the present invention. Kits can include, for example, at least one Cx43 antibody and agents to make, formulate, and/or administer components of embodiments or to perform one or more steps of the methods of the invention. In some embodiments, the kit may further comprise a suitable container, which is a container that does not react with the components of the kit, such as an eppendorf tube, assay plate, syringe, bottle, or tube. The container may be made of a sterilizable material such as plastic or glass.

The kit may also include instructions summarizing the procedural steps of the methods described herein and will follow substantially the same methods described herein or known to one of ordinary skill in the art. The instruction information may be present in a computer readable medium containing machine readable instructions which, when executed using a computer, cause a display of an actual or real program for delivering a pharmaceutically effective amount of a therapeutic agent.

Example IV

The following embodiments are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 anti Cx43 monoclonal antibody

anti-Cx 43 monoclonal antibody was generated and clones producing monoclonal antibodies that bound Cx43 were identified. The CDR sequences of DNA and amino acids for all antibody sequences and the correct pairing for each characterized antibody are shown in the table below.

Table 1: pairing of heavy and light chains of two functional antibodies.

Table 2: sequences of antibody chains from hybridomas.

The cloned variable domains are shown in the following chart.

Table 1.DNA sequence:

table 2. amino acid sequence:

example 2 spinal cord and neuronal injury (SCI) therapeutic uses

As shown in fig. 1, Cx43 is typically localized in the gap junctions between cells or as hemichannels on the plasma membrane. Pathological opening of Cx43 hemichannels leads to the spread of secondary injury, activation of astrocytes and inflammation. It has been proposed that preventing the pathological opening of Cx43 hemichannels prevents the release of molecules, thereby allowing astrocytes to act as managing (caretaker) cells and preventing further spread of secondary injury.

The activation of Cx43 hemichannels by IL-1 β in human primary astrocytes was inhibited by both the Cx43 hemichannel blocking mouse monoclonal antibody (M1) and the mouse-human chimeric antibody HMAb 1. Hemichannel activity was determined by ethidium bromide uptake. The results are shown in fig. 2.

Mice treated with HMAb1 had reduced glial scarring. Mice were subjected to a single SCI and treated 30 min post-injury with IP saline, control Igg or HMAb1(25 mg/kg). Glial scarring was measured 14 and 56 days after injury. Spinal cord tissue sections were immunohistochemically (red) for the astrocyte marker GFAP. Representative images of spinal cord in mice treated with control IgG are shown in fig. 3A-C and representative images of spinal cord in mice treated with HMAb1 are shown in fig. 3D-F. The lesion boundaries are indicated by dashed white lines. GFAP immune markers were quantified as the mean intensity multiplied by the area of positive staining in fig. 3G. Results are expressed as percentage of sham-operated, IgG-treated mice. Results are mean ± SEM. Compared to Igg, p <0.05, p <0.001, w/dukel HSD n-3-4.

Glial scar formation was also reduced in mice treated with anti-Cx 43 after SCI. Mice were SCI and treated 30 min after injury with IgG or anti-Cx 43 antibody (M1). Two weeks after injury, tissue sections were analyzed for expression of the astrocytic marker GFAP. Representative images are shown in fig. 4A and 4B. The results are quantified in fig. 4C.

Mice with SCI recovered hindlimb function after treatment with HMAb1 (fig. 5A-5B). Mice were subjected to a single SCI and treated 30 min post injury with IP saline, control IgG, or human-mouse chimeric anti-Cx 43 antibody (HMAb1) (25 mg/kg).

14 days after SCI, mice treated with HMAb1 were found to have more neuronal dendrites in the perilesional area. Mice were subjected to a single SCI and treated 30 min post-injury with IP saline, control Igg or HMAb1(25mg/kg) as described above. Neuronal dendrites were measured by immunolabeling against the neuronal marker MAP 2. Immunohistochemical representative images of spinal cord in mice treated with control IgG are shown in fig. 6A-C and immunohistochemical representative images of spinal cord in mice treated with HMAb1 are shown in fig. 6D-F. The lesion boundaries are indicated by dashed white lines. MAP2 immunolabeling was quantified as the mean intensity multiplied by the area of positive staining shown in figure 6G. Results are expressed as percentage of sham-operated, IgG-treated mice.

14 days after SCI, mice treated with HMAb1 were also observed to have more neuronal nuclei in perilesional areas. Mice were again given a single SCI and treated 30 min after injury with IP saline, control IgG or HMAb1(25 mg/kg). Neuronal nuclei were measured by immunolabeling against the neuronal marker NeuN. Immunohistochemical representative images of spinal cord in mice treated with control IgG are shown in fig. 7A-C and immunohistochemical representative images of spinal cord in mice treated with HMAb1 are shown in fig. 7D-F. The lesion boundaries are indicated by dashed white lines. NeuN immunolabeling was quantified as the mean intensity multiplied by the area of positive staining shown in figure 7G. Results are expressed as percentage of sham-operated, IgG-treated mice.

Example 3 diagnostic and cancer therapeutic uses

Approximately 40,000 deaths occur in the united states each year due to metastatic breast cancer. About 70% -80% of patients with advanced breast cancer develop bone metastases. Bone metastasis alone accounts for two-thirds of the cost of breast cancer treatment.

Enhanced growth of osteolytic tumors was found in osteocyte-specific Cx43 knockout mice. Py8119-Luc cells were injected into the right tibia of control and cKO female mice. The left tibia was injected with PBS as a control. Tumor growth was recorded once a week by bioluminescence imaging for 4 weeks and quantified (fig. 8A-8C).

MLO-Y4 bone cells and primary mouse bone cells were incubated with E2 (polyclonal), HMAb1 and HMAb2 antibodies or Carbenoxolone (CBX) (connexin channel blocker). Ethidium bromide (EtBr) dye uptake assays were performed (FIGS. 9A-9B). The Cx43 HMAb2 antibody was found to activate hemichannels.

In addition, Cx43(M1) antibody was delivered to bone cells in vivo and was found to block evans blue uptake induced by tibial load. Evans blue dye was injected into the tail vein of WT osteocyte specific Cx43 KO. Mouse IgG or Cx43(M1) mAb (25mg/kg) was injected intraperitoneally 2 hours prior to dye injection. The left tibia was mechanically loaded 30 minutes after dye injection, once for 10 min. Mice were sacrificed and perfused with PBS. The tibia was isolated and fixed tibial tissue sections were prepared. The results are shown in fig. 10A-10B.

Inhibition of osteolytic tumor growth by HMAb2 was also observed. Py8119-Luc cells were injected into the right tibia of female mice (FIG. 11A). The left tibia was injected with PBS as a control. Once or twice weekly intraperitoneal injections of 25mg/kg HMAb2 were given for four weeks. Saline was injected twice weekly in control mice. Tumor growth was recorded once a week by bioluminescence imaging for 4 weeks and quantified (fig. 11B).

Example 4 osteoarthritis treatment

Primary chondrocytes isolated from mouse bone were immunostained with anti-Cx 43 antibody against the C-terminal domain (total) in permeable cells and Cx43E2 antibody in impermeable cells (fig. 12). Cx43 expression was observed on the cell surface of primary chondrocytes.

In other studies, primary chondrocytes isolated from mouse bone were pre-treated with carbenoxolone (connexin channel blocker) or HMAb1 antibody, and then treated with or without IL-1 β. Ethidium bromide dye uptake assays were performed to determine hemichannel activity (figure 13). It was observed that HMAb1 antibody inhibited hemichannel opening by IL-1 β in primary chondrocytes.

As in the above examples, evans blue uptake induced by tibial loading was blocked by Cx43 hemichannel blocking antibodies in vivo. Evans blue dye was injected into the tail vein of WT mice. Cx43(M1) mAb (25mg/kg) was injected intraperitoneally 2 hours prior to dye injection. The left tibia was mechanically loaded once for 10min 30 min after dye injection. Dye uptake was measured by Evans Blue (EB) fluorescence and quantified (fig. 14).

***

In light of the present disclosure, all methods disclosed and claimed herein can be performed and carried out without undue experimentation. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Sequence listing

<110> board of directors of university of Texas

<120> connexin (Cx)43 hemichannel binding antibodies and uses thereof

<130> 21105.0052U2

<150> 62/300,492

<151> 2016-02-26

<150> PCT/US2017/019605

<151> 2017-02-27

<160> 63

<170> SIPOSequenceListing 1.0

<210> 1

<211> 384

<212> DNA

<213> mouse (Mus musculus)

<400> 1

gaggttcagc tggagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggggga attaatccta gcaatggtgg tactaacttc 180

aatgagaagt tcaagaacaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac aagagagggt 300

aacccctact atactatgaa ctactggggt caaggaacct cagtcaccgt ctcctcagcc 360

aaaacgacac ccccatctgt ctat 384

<210> 2

<211> 128

<212> PRT

<213> mouse (Mus musculus)

<400> 2

Glu Val Gln Leu Glu Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Asn Pro Tyr Tyr Thr Met Asn Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

<210> 3

<211> 324

<212> DNA

<213> mouse (Mus musculus)

<400> 3

gatattgtga tgacacagac tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gaaa 324

<210> 4

<211> 108

<212> PRT

<213> mouse (Mus musculus)

<400> 4

Asp Ile Val Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys

100 105

<210> 5

<211> 384

<212> DNA

<213> mouse (Mus musculus)

<400> 5

gaggttcagc tggagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggggga attaatccta gcaatggtgg tactaacttc 180

aatgagaagt tcaagaacaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac aagagagggt 300

aacccctact atactatgaa ctactggggt caaggaacct cagtcaccgt ctcctcagcc 360

aaaacgacac ccccatctgt ctat 384

<210> 6

<211> 128

<212> PRT

<213> mouse (Mus musculus)

<400> 6

Glu Val Gln Leu Glu Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Asn Pro Tyr Tyr Thr Met Asn Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

115 120 125

<210> 7

<211> 375

<212> DNA

<213> mouse (Mus musculus)

<400> 7

gatattgtga tgacccagac tccatcctcc ctgagtgtgt cagcaggaga gaaggtcact 60

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagac ctacttggcc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctacggggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaaccgattt cactcttacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga tcatagttat 300

ccattcacgt tcggctcggg gacaaagttg gaaataaaac gggctgatgc tgcaccaact 360

gtatccgcat gcacc 375

<210> 8

<211> 122

<212> PRT

<213> mouse (Mus musculus)

<400> 8

Asp Ile Val Met Thr Gln Thr Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp His Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Ala Asp Ala Ala Pro Thr Val Ser

115 120

<210> 9

<211> 405

<212> DNA

<213> mouse (Mus musculus)

<400> 9

ctggagcagc ctggggctga actggtgagg cctggggctt cagtaatgct gtcctgcaag 60

gcttctggct acatcttcac cacctactgg atgcactggc tgaagcagag gcctggacaa 120

ggccttgact ggattggaga gattagtcct agcaacggtc gttctaatta caataagaag 180

ttcaagagca aggccacact gactgtagac aaatcctcca gcacagccta catgcaactc 240

agcagcctga catctgagga ctctgcggtc tattactgtg cacgattcga cgagggggac 300

ttctggggcc aaggcaccac tctcatagtc tcctcagcca aaacaacagc cccatcggtc 360

tatccactgg cccctgtgtg tggagataca actggctcct cggtg 405

<210> 10

<211> 135

<212> PRT

<213> mouse (Mus musculus)

<400> 10

Leu Glu Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Met

1 5 10 15

Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Thr Tyr Trp Met His

20 25 30

Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile Gly Glu Ile

35 40 45

Ser Pro Ser Asn Gly Arg Ser Asn Tyr Asn Lys Lys Phe Lys Ser Lys

50 55 60

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

65 70 75 80

Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Phe

85 90 95

Asp Glu Gly Asp Phe Trp Gly Gln Gly Thr Thr Leu Ile Val Ser Ser

100 105 110

Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly

115 120 125

Asp Thr Thr Gly Ser Ser Val

130 135

<210> 11

<211> 324

<212> DNA

<213> mouse (Mus musculus)

<400> 11

gatattgtga tgacacagac tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gaaa 324

<210> 12

<211> 108

<212> PRT

<213> mouse (Mus musculus)

<400> 12

Asp Ile Val Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys

100 105

<210> 13

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile

1 5 10

<210> 14

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Lys Arg Asp Pro Cys Pro His Gln Val Asp

1 5 10

<210> 15

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Leu Ser Ala Val Tyr Thr Cys Lys Arg

1 5

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggctacacct tcaccagcta ctat 24

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

attaatccta gcaatggtgg tact 24

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

acaagagagg gtaaccccta ctatactatg aactac 36

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Tyr Thr Phe Thr Ser Tyr Tyr

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Ile Asn Pro Ser Asn Gly Gly Thr

1 5

<210> 21

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Thr Arg Glu Gly Asn Pro Tyr Tyr Thr Met Asn Tyr

1 5 10

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ggctacatct tcaccaccta ctgg 24

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

attagtccta gcaacggtcg ttct 24

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gcacgattcg acgaggggga cttc 24

<210> 25

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gly Tyr Ile Phe Thr Thr Tyr Trp

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Ile Ser Pro Ser Asn Gly Arg Ser

1 5

<210> 27

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Ala Arg Phe Asp Glu Gly Asp Phe

1 5

<210> 28

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cagagtctgt taaacagtgg aaatcaaaag acctac 36

<210> 29

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ggggcatcc 9

<210> 30

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cagaatgatc atagttatcc attcacg 27

<210> 31

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Thr Tyr

1 5 10

<210> 32

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Gly Ala Ser

1

<210> 33

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Gln Asn Asp His Ser Tyr Pro Phe Thr

1 5

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

aaaagtgtca gtacatctgg ctatagttat 30

<210> 35

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cttgtatcc 9

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

cagcacatta gggagcttac acg 23

<210> 37

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

1 5 10

<210> 38

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Leu Val Ser

1

<210> 39

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Gln His Ile Arg Glu Leu Thr

1 5

<210> 40

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aaaagtgtca gtacatctgg ctatagttat 30

<210> 41

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

cttgtatcc 9

<210> 42

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

cagcacatta gggagcttac acgt 24

<210> 43

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

1 5 10

<210> 44

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Leu Val Ser

1

<210> 45

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

Gln His Ile Arg Glu Leu Thr Arg

1 5

<210> 46

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gagcctctta gaaagcgatg gaaagacata t 31

<210> 47

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

ctggtgtct 9

<210> 48

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

tggcaaggta cacattttcc gtggacg 27

<210> 49

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

Gln Ser Leu Leu Glu Ser Asp Gly Lys Thr Tyr

1 5 10

<210> 50

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

Leu Val Ser

1

<210> 51

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

Trp Gln Gly Thr His Phe Pro Trp Thr

1 5

<210> 52

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

gaggtccaac tccagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagttg 60

tcctgcaagg cttctggcta caccttcacc agctactata tgtactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggggga attaatccta gcaatggtgg tactaacttc 180

aatgagaagt tcaagaacaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtac aagagagggt 300

aacccctact atactatgaa ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 53

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

gaggtccaac tccagcaacc tggggctgaa ctggtgaggc ctggggcttc agtaatgctg 60

tcctgcaagg cttctggcta catcttcacc acctactgga tgcactggct gaagcagagg 120

cctggacaag gccttgactg gattggagag attagtccta gcaacggtcg ttctaattac 180

aataagaagt tcaagagcaa ggccacactg actgtagaca aatcctccag cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc acgattcgac 300

gagggggact tctggggcca aggcaccact ctcatagtct cctca 345

<210> 54

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

gacattgtga tgacgcagtc tccatcctcc ctgagtgtgt cagcaggaga gaaggtcact 60

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagac ctacttggcc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctacggggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaaccgattt cactcttacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga tcatagttat 300

ccattcacgt tcggctcggg gacaaagttg gaaataaaa 339

<210> 55

<211> 330

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

gacattgtgt tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gaaatcaaac 330

<210> 56

<211> 330

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

gatattgtga tgacccagtc tcccgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg ggaccaagct ggaaatcaaa 330

<210> 57

<211> 337

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

kgacgttgtg atgacccaga ctccactcac tttgtcggtt accattggac aaccagcctc 60

catctcttgc aagtcaagtc agagcctctt agaaagcgat ggaaagacat atttgaattg 120

gttgttacag aggccaggcc agtctccaaa gcgcctaatc tatctggtgt ctaaactgga 180

ctctggagtc cctgacaggt tcactggcag tggatcaggg acagatttca cactgaaaat 240

cagcagagtg gaggctgagg atttgggagt ttattattgc tggcaaggta cacattttcc 300

gtggacgttc ggtggaggca ccaagctgga aatcaaa 337

<210> 58

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Glu Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Asn Pro Tyr Tyr Thr Met Asn Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 59

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Glu Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Met Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Asp Trp Ile

35 40 45

Gly Glu Ile Ser Pro Ser Asn Gly Arg Ser Asn Tyr Asn Lys Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Asp Glu Gly Asp Phe Trp Gly Gln Gly Thr Thr Leu Ile

100 105 110

Val Ser Ser

115

<210> 60

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp His Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 61

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Ser Asn

100 105 110

<210> 62

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 63

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Glu Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Claims (19)

1. Use of an antibody that binds to connexin 43(Cx43) hemichannel and enhances channel opening, or an expression vector comprising a gene encoding said antibody, in the manufacture of a medicament for treating osteolytic breast cancer in a subject, wherein said antibody comprises:

(a) first V identical to SEQ ID NO 19 _H CDR；

(b) Second V identical to SEQ ID NO 20 _H CDR；

(c) Third V identical to SEQ ID NO 21 _H CDR；

(d) First V identical to SEQ ID NO 49 _L CDR；

(e) Second V identical to SEQ ID NO 50 _L A CDR; and

(f) third V identical to SEQ ID NO 51 _L CDR。

2. The use of claim 1, comprising administering to the subject an effective amount of the antibody.

3. The use of claim 1, comprising administering to the subject an effective amount of an expression vector comprising a gene encoding the antibody.

4. The use of claim 1, wherein the antibody or the expression vector comprising a gene encoding the antibody is administered in a pharmaceutically acceptable composition.

5. The use of claim 1, wherein the antibody is administered systemically or locally.

6. The use of claim 1, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, or subcutaneously.

7. The use of claim 1, wherein the antibody is a humanized antibody.

8. The use of claim 1, further comprising administering at least a second anti-cancer therapy to the subject.

9. The use of claim 8, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, or immunotherapy.

10. The use of claim 9, wherein the second anti-cancer therapy is cytokine therapy.

11. A recombinant connexin 43(Cx43) hemichannel binding antibody, wherein the antibody comprises:

(a) first V identical to SEQ ID NO 19 _H CDR；

(b) Second V identical to SEQ ID NO 20 _H CDR；

(c) Third V identical to SEQ ID NO 21 _H CDR；

(d) And SEQ IDNO 49 same first V _L CDR；

(e) Second V identical to SEQ ID NO 50 _L A CDR; and

(f) third V identical to SEQ ID NO 51 _L CDR。

12. The antibody of claim 11, wherein the antibody is a humanized antibody.

13. The antibody of claim 11, wherein the antibody comprises a V that is at least 90% identical to SEQ ID NO 58 _H Amino acid sequence and V at least 90% identical to SEQ ID NO 63 _L An amino acid sequence.

14. The antibody of claim 13, wherein the antibody comprises a V as set forth in SEQ ID NO:58 _H Amino acid sequence and V as shown in SEQ ID NO 63 _L An amino acid sequence.

15. Use of a pharmaceutical composition in the manufacture of a medicament for treating osteolytic breast cancer in a subject, wherein the pharmaceutical composition comprises an antibody according to any one of claims 11, 12, 13 or 14 or an expression vector containing a gene encoding an antibody according to any one of claims 11, 12, 13 or 14.

16. The use of claim 15, wherein the pharmaceutical composition comprises an expression vector comprising a gene encoding the antibody of any one of claims 11, 12, 13, or 14.

17. The use of claim 15, wherein the pharmaceutical composition comprises an antibody according to any one of claims 11, 12, 13 or 14.

18. The use of claim 15, wherein the pharmaceutical composition is administered systemically or locally.

19. The use of claim 15, wherein the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, or subcutaneously.