Compositions and Methods for Targeting Connexin Hemichannels

Abstract

Compositions and methods for targeting connexin hemichannels are provided. Specifically, a monoclonal antibody is provided that comprises the heavy chain amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10 and the light chain amino acid sequence of SEQ ID NO:4. Also provided are methods for detecting the presence, levels, and activity of total and cell surface Cx43 hemichannels using the monoclonal antibody, and methods for identifying compounds, conditions, and / or treatments that open connexin hemichannels.

Description

[0001] Priority Paragraph

[0002] This application is an international application claiming priority to U.S. Provisional Patent Application Serial No. 61 / 868,112, filed Aug. 21, 2013, which is incorporated herein by reference in its entirety. Field of the Invention

[0003] This application pertains to the biomedical field. Specifically, it relates to a monoclonal antibody comprising the heavy chain amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10 and the light chain amino acid sequence of SEQ ID NO:4. Also provided are methods for detecting the presence, level, and activity of total and cell surface Cx43 hemichannels using the monoclonal antibody, as well as various methods related to hemichannels, including methods for identifying compounds, conditions, and / or treatments that open connexin hemichannels. Background of the Invention

[0004] Bone tissue is a preferred site for breast and prostate cancer metastasis. Bone metastases occur in up to 75% of patients with advanced cancer. Currently, metastatic breast cancer is incurable, and there are no reliable intervention drugs with extremely low side effects for treating bone metastases.

[0005] Osteoarthritis (OA) is a prevalent disease affecting approximately 20% of U.S. adults. The disease causes degeneration of joints, including articular cartilage and subchondral bone. The pathology of OA is characterized by the loss of articular cartilage, leading to narrowing of the joint space, increased joint friction, and possible structural remodeling. Current treatments include exercise, lifestyle changes, and painkillers. If symptoms become severe, joint replacement surgery is usually performed. To date, there are no specific drug intervention measures available for treating OA.

[0006] Connexin hemichannels play important roles in cell and tissue function, and it is known that abnormal function of connexin hemichannels can lead to various pathological conditions. Therefore, there is still a need for additional therapies for treating pathological conditions related to hemichannel activity, such as inflammation, osteoarthritis, or bone metastasis, and methods for determining such therapies. Summary of the Invention

[0007] The present inventors have discovered that open hemichannels in cells that have metastasized have an inhibitory effect on cancer growth, migration, and metastasis. Certain embodiments provide tools and / or methods for identifying compounds that modulate the opening of hemichannels for the treatment of cancer metastasis. In certain aspects, drugs can be used to inhibit or alleviate the metastasis of cancer to bone, brain, or liver. In certain aspects, hemichannels can be expressed in bone, brain, or liver cells. In yet another aspect, the hemichannels can be osteocyte hemichannels, hepatocyte hemichannels, or astrocyte hemichannels. In certain aspects, the hemichannels can be connexin Cx43, Cx32, Cx46, Cx37, Cx40, Cx50, Cx59, Cx62, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45, Cx47, Cx30.2, Cx36, Cx31.9, Cx39, Cx40.1, Cx23, or Cx29 hemichannels. In one aspect, the hemichannel is a Cx43 or Cx32 hemichannel. For example, the opening of connexin 43 (Cx43) hemichannels in osteocytes has an inhibitory effect on metastasis to bone and can inhibit bone metastasis.

[0008] Hemichannel opening can be detected by dye uptake assays using fluorescent dyes such as Lucifer yellow, ethidium bromide, Alexa 350, Alexa 485, Alexa 594 dyes, etc. The specificity of hemichannel opening can be verified by using connexin-specific antibodies that inhibit hemichannel opening and thus inhibit the activity of the targeting reagent. Thus, the tools and / or methods can be used to screen, test, and identify reagents that open hemichannels and inhibit metastasis.

[0009] Certain embodiments relate to methods for identifying compounds that open hemichannels. Other embodiments relate to methods for positively regulating the opening of hemichannels to inhibit or alleviate bone metastasis.

[0010] The present invention provides antibodies against hemichannels, nucleic acids encoding such antibodies and therapeutic proteins, methods for preparing anti-hemichannel monoclonal antibodies and other therapeutic proteins, and methods for treating diseases such as metastatic cancer. In certain aspects, the antibody binds to an epitope having the amino acid sequence 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 FLSRPTEKTI (SEQ ID NO:13).

[0011] In one embodiment, the present invention provides an isolated antibody that specifically binds to a hemichannel, the antibody 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.

[0012] 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 the amino acid sequence of residues 97 to 116 of SEQ ID NO:2.

[0013] 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.

[0014] In one embodiment, the present invention provides an isolated antibody that specifically binds to a hemichannel and a gap junction, the antibody 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.

[0015] 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 the amino acid sequence of residues 97 to 116 of SEQ ID NO:6.

[0016] 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.

[0017] In one embodiment, the present invention provides an isolated antibody that specifically binds to a gap junction, the antibody 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.

[0018] In some 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 the amino acid sequence of residues 94 to 109 of SEQ ID NO: 10.

[0019] 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.

[0020] In some aspects, the antibody includes full-length antibodies, antibody fragments, single-chain antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies, and antibody fusions and fragments thereof.

[0021] Another embodiment provides a pharmaceutical composition comprising an antibody as described herein and a pharmaceutically acceptable carrier. Also provided is the antibody or pharmaceutical composition of the present invention for use as a therapeutic agent for treating cancer and inhibiting cancer metastasis.

[0022] Another embodiment provides a method for treating or preventing cancer metastasis. The treatment method may include administering to a subject in need thereof an effective amount of the isolated antibody described herein. Also provided is the use of the antibody as described herein in the manufacture of a medicament for treating or preventing cancer metastasis.

[0023] Certain aspects relate to an in vitro method of using an antibody, compound, or reagent to inhibit an inflammatory response in chondrocytes. In certain aspects, the method relates to determining the effect of inhibition of Cx43 hemichannel opening in chondrocytes by: (i) measuring hemichannel opening using Lucifer yellow or Alexa dyes by a dye uptake assay, (ii) evaluating the inhibitory effect of IL-1β on hemichannel opening, and (iii) testing the inhibitory effect of the reagent on hemichannel opening by mechanical loading in the form of fluid flow shear stress.

[0024] Certain aspects relate to a method for determining the effect of an antibody, compound, or reagent on inhibiting an inflammatory response induced by IL-1β and mechanical loading by: (i) measuring the inhibition of IL-1β-induced nuclear factor-κB (NF-κB) activation and (ii) measuring the inhibition of NF-κB activation induced by body fluid flow shear stress.

[0025] Other aspects relate to in vivo methods of treating OA or determining OA using monoclonal antibodies, compounds, or reagents, the method comprising: (i) injecting the antibody, compound, or reagent into the patellar cavity, (ii) assessing inhibition of IL-1β-induced NF-κB activation, and (iii) assessing OA progression by X-ray, histological analysis, and body movement.

[0026] 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 are engineered to specifically bind to an antigen, such as aptamers, avimers, etc.

[0027] The term "antibody" or "immunoglobulin" is used to include intact antibodies and their binding fragments / regions. Generally, a fragment competes with the intact antibody from which it is derived for specific binding to an antigen. Fragments include single heavy chains, light chains, Fab, Fab', F(ab')2, Fabc, and Fv. Fragments / regions are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The term "antibody" also includes one or more immunoglobulin chains chemically conjugated to other proteins or expressed as a fusion protein. The term "antibody" also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol. 148:1547–53, 1992.

[0028] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or medium (when produced by recombinant DNA techniques) or chemical precursors or other chemicals (when chemically synthesized) from its source. Additionally, an isolated compound is 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 adsorbed to a column or entrapped in an agarose gel. Additionally, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally exist as a fragment and / or is not normally in a functional state.

[0029] Portions of the invention (such as polypeptides, peptides, antigens or immunogens) can be conjugated or covalently or non-covalently linked to other portions (such as adjuvants, proteins, peptides, supports, fluorescent moieties or labels). The term "conjugate" or "immunoconjugate" is used broadly to define an operable linkage of one portion to another reagent and is not intended to refer solely to any type of operable linkage, and is particularly not limited to chemical "conjugation".

[0030] The term "provide" is used in its ordinary sense to mean "make available or equip for use". 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 compositions contemplated by the present invention include various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0031] The phrase "specifically binds" or "has specific immunoreactivity" to 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 specified immunoassay conditions, a specified molecule preferentially binds to a specific target and does not bind significantly 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 to the target. A variety of immunoassays can be used to select antibodies that have specific immunoreactivity with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to select monoclonal antibodies that have specific immunoreactivity with a protein. For a description of immunoassays and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

[0032] Other embodiments of the invention are discussed in this application. Any embodiment discussed with respect to one aspect of the invention is applicable to other aspects of the invention, and vice versa. Each embodiment described herein should be understood to be an embodiment of the invention applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be practiced with respect to any method or composition of the invention, and vice versa. In addition, the compositions and kits of the invention can be used to implement the methods of the invention.

[0033] The word "a" when used in conjunction with the term "comprising" in the claims and / or the specification can mean "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0034] In this application, the term "about" is used to indicate that a numerical value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0035] Unless expressly stated to the contrary or that the alternative forms are mutually exclusive, the term "or" as used in a claim means "and / or", but the disclosure also supports a definition that only refers to certain alternative forms and "and / or".

[0036] As used in this specification and the claims, the words "comprising" (and any form of comprising), "having" (and any form of having), "including" (and any form of including) or "containing" (and any form of containing) are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0037] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the illustrative embodiments presented herein.

[0039] Figure 1 Shows an embodiment of a fluid flow circuit device.

[0040] Figure 2 Shows the results of treating MLO-Y4 osteocytes with 20 μM AD for 30 minutes in the absence or presence of 1 μg / ml Cx43(E2) antibody. An ethidium bromide dye uptake assay was performed and quantified relative to the untreated basal uptake level. Studies were conducted under calcium conditions. Low calcium conditions were used as a control (opening hemichannels). Also shown are the results of treating MLO-A5 osteoblasts with AD or AD and Cx43(E2) antibody.

[0041] Figure 3 Shows a model system for studying the role of Cx43 hemichannels in osteocytes in regulating the effect of AD on cancer cell migration. Cx43 hemichannels in osteocytes are opened by AD or FFSS. Factors released in the CM treated with AD or FFSS reduce cancer cell migration. Cancer cells treated with control CM exhibit normal migration.

[0042] Figure 4A and 4BStudies using the soft agar non - adherent - dependent growth assay are shown, which is different from adherent - dependent growth. Only cancer cells can grow on soft agar, and their growth on this matrix indicates the degree of cancer cell proliferation. CM - AD from osteocytes reduces MDA - MB - 231 colony formation.

[0043] Figure 5A and 5B Results of studies using the wound - healing migration assay are shown. CM - AD from osteocytes inhibits MDA - MB - 231 cell migration.

[0044] Figure 6 Results of another wound - healing migration assay are shown. AD has no direct effect on the migration of MDA - MB - 231 cells.

[0045] Figure 7A and 7B Results from the transwell migration assay are shown. (B) dots represent cells that have migrated to the opposite end of the insert and have been stained. The smaller dots are the pores through which the cells have migrated. In this assay, Protein A was used to remove E2 Ab from the CM, and this resulted in the same migration effect as before the addition of E2 Ab.

[0046] Figure 8 Results from another transwell migration assay are shown. MDA - MB - 231 migration is not affected by CM - AD from MLO - A5 osteoblasts.

[0047] Figure 9 Results from another transwell migration assay are shown. MDA - MB - 231 migration is reduced by CM from osteocytes stimulated by mechanical loading.

[0048] Figure 10A and 10B Results from Py8119 breast cancer cells injected into Cx43 conditional knockout (cKO) mice are shown. Py8199 tumor growth and metastasis are increased in Cx43 cKO mice. (B) Tumors spread to other tissues in Cx43 cKO mice.

[0049] Figure 11 Results from Py8119 injected into Cx43 cKO mice are shown. Py8119 tumor growth is increased in Cx43 cKO mice.

[0050] Figure 12Immunolabeling of Cx43 by three mAbs is shown. Osteocyte MLO-Y4 cells were fixed with 70% ethanol at -20 °C for 20 min, blocked overnight, and incubated with mAbs at the concentrations shown above at room temperature for 3 h. Goat anti-mouse FITC secondary antibody was used to evaluate the activity of the three mAbs. WGA as a cell marker was labeled in red.

[0051] Figure 13 Hybridoma supernatants affinity-purified by passing through a Cx43 E2 column at pH 7.4 are shown. Western blotting was performed at a 1:100 dilution for each monoclonal antibody. The polyclonal Cx43 Ab dilution was 1:300.

[0052] Figure 14 It is shown that M1 blocks hemichannels but not gap junctions; M2 blocks gap junctions but not hemichannels; while M3 blocks both. (A) MLO-Y4 cells were incubated with a medium containing low Ca 2+ and Mg 2+ This condition induces the opening of Cx43 hemichannels. Dye uptake assays were performed for 20 min in the presence of EtBr with or without mAbs. (B) HeLa cells transfected with Cx43 were incubated with mAbs for 3 h, and signal cells were microinjected with AlexaFluor 488 to evaluate the degree of dye transfer measured for gap junctions.

[0053] Figure 15 The parachuting dye transfer assay is shown, showing that M2 and M7 but not M1 block gap junction channels. HeLa cells transfected with Cx43 were used for the parachuting dye transfer experiment. Donor cells were incubated with 5 μM calcein red-orange-AM (790 Da) permeable to gap junctions and 5 μM Oregon Green 488 BAPTA-2-AM (1752 Da) impermeable to gap junctions at 37 °C for 40 min. Donor cells were treated with trypsin, and the isolated pre-loaded cells were layered ("parachuted") on top of unlabeled recipient cells at a donor-to-recipient ratio of 1:4. The cells were allowed to adhere for 90 min. The cells were examined under a fluorescence microscope.

[0054] Figure 16 . It is shown that M1 and M7 but not M2 block the opening of hemichannels induced by mechanical loading. MLO-Y4 cells were pretreated with or without mAbs for 20 min. The cells were subjected to a fluid flow shear stress of 8 dyn / cm 2 for 10 min and a dye uptake with 100 μM EtBr for 5 min. The cells were rinsed, fixed, and images were acquired under a fluorescence microscope.

[0055] Figure 17 . It shows that in mouse osteocytes, M1 blocks the opening of hemichannels induced by mechanical loading. Mouse IgG or Cx43 (M1) mAb (25 mg / kg) was administered and Evans blue dye was injected into WT and Cx43 cKO mice 2 hours later. Thirty minutes after dye injection, a single 10-minute mechanical load was applied to the left (L) tibia. The mice were euthanized and perfused with 50 ml of PBS. The tibias were isolated, fixed, and bone tissue sections were made. Bar, 40 mm. Arrows indicate dye uptake.

[0056] Figure 18 . It shows the expression of Cx43 on the surface of chondrocytes. (A) Expression of Cx43 on the surface of primary chondrocytes. (B) Fluid flow shear (16 dyn / cm2) (FSS) opened the hemichannels, and this opening was significantly blocked by a Cx43-specific antibody. Compared with all other conditions, ***, P < 0.001 for FSS. Detailed implementation

[0057] Various cells can communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by connexins. Connexins are ubiquitously expressed throughout the body. Six connexins form a hemichannel, and 2 hemichannels form 1 gap junction channel. Gap junctions are clusters of channels that are located in the plasma membrane between adjacent cells and mediate intercellular communication. Hemichannels are separate entities from gap junction channels. Hemichannels allow molecules to exchange between the intracellular compartment and the extracellular environment.

[0058] Osteocytes express hemichannels called connexin (Cx)43 hemichannels. These osteocyte hemichannels are normally closed and can open upon exposure to mechanical stimuli, which leads to the release of various factors into the bone microenvironment. The factors released upon hemichannel opening can mediate other processes that can reduce tumor cell migration and bone metastasis.

[0059] Certain embodiments relate to methods of identifying reagents that regulate the opening of connexins. In some aspects, the method identifies compounds or drugs that positively regulate the opening of connexin hemichannels. Other embodiments relate to methods of treating cancer by administering to a patient having cancer a compound that opens hemichannels. In some aspects, the patient has a primary tumor. In some aspects, compounds that open Cx43 hemichannels can be used to inhibit or reduce metastasis to bone.

[0060] Cancer metastasis occurs when cancer spreads from the body part where it originated (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 (mets) are associated with many serious clinical and quality of life consequences, such as, but not limited to, refractory pain, pathologic fractures, spinal cord and nerve compression, bone marrow infiltration, and impaired mobility. In many cases, the systemic presence of cancer can also render the cancer incurable.

[0061] Normal bone is composed of three major cell types: osteoblasts that form bone, osteoclasts that resorb bone, and osteocytes. Osteocytes account for approximately 95% of bone cells 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.

[0062] It has been demonstrated that Cx43 hemichannels in osteocytes are opened by treatment with alendronate (AD), an effective and commonly used bisphosphonate drug. Bisphosphonates are a class of drugs known to be used in the treatment of many bone diseases, including bone metastasis. Powles et al. have shown that the administration of bisphosphonates is associated with a reduced incidence of bone metastasis and reduced mortality in breast cancer patients. AD has been associated with reduced tumor growth as well as reduced bone destruction and pain. AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in osteocytes (Plotkin et al., 2002). However, AD administration is accompanied by a variety of serious side effects.

[0063] I. Methods related to screening of candidate drugs as bone metastasis inhibitors

[0064] A. In vitro assays

[0065] Certain embodiments involve detecting the opening of hemichannels in vitro using a dye uptake assay. In some aspects, the dye is a fluorescent tracer dye (e.g., ethidium bromide or fluorescein).

[0066] In one example of an in vitro assay for detecting hemichannel opening, a fluid flow loop apparatus (FFLA) (parallel plate flow chamber) or a modified form thereof can be used. An example of an FFLA device is schematically shown in Figure 1 . The FFLA mimics the dynamic fluid microenvironment in bone to generate fluid flow shear stress (FFSS). Cells are cultured in a parallel plate flow chamber such that the cells are exposed to steady-state laminar fluid flow.

[0067] Osteocytes sense the mechanical strain generated by FFSS in the osteocyte lacuno-canalicular network. It has been proposed that bone fluid flow is driven by extravascular pressure and cyclic mechanical loading applied by osteocytes, and that peak physiological loads are 8 to 30 dynes / cm 2 . In some aspects, the FFSS levels are within the physiological values reported in previous studies that measured fluid flow within bone. The magnitude of fluid shear stress can be altered by adjusting the column height of the flow circuit.

[0068] Assays for evaluating hemichannel function can use fluorescent tracer molecules small enough to pass through the pores of the hemichannels. If the hemichannels are closed, the molecules cannot pass through. If the hemichannels are open, the dye can pass through and cause the cell to fluoresce, allowing quantification of the fluorescence. Ethidium bromide becomes fluorescent when it binds to DNA. Lucifer yellow fluoresces after being located inside the cell.

[0069] The dye transfer method can include exposing the cells to an extracellular fluorescent permeant tracer. An extracellular permeant tracer is a molecule that remains outside the cell unless some condition increases the permeability of the cell membrane to allow entry of the molecule. In some aspects, the mass of the tracer is less than 1, 2, or 3 kDa. In other aspects, the tracer will have a net charge. Such permeant tracers include, but are not limited to, the anionic dye Lucifer Yellow (LY; net charge = -1) and the cationic probes Ethidium Bromide (Etd; net charge = +1), Propidium Iodide (PI; net charge = +2). The fluorescence of EtBr is enhanced when it binds to DNA, increasing the contrast pair and making it easier to identify. In some aspects, the extracellular dye is removed at different times or after applying a stimulus to open the hemichannels, and the fluorescence intensity retained by each cell is quantified. In some aspects, the fluorescence intensity is quantified in snapshot pictures.

[0070] Figure 2 Results from an example of an in vitro dye transfer assay are shown. MLO-Y4 osteocytes were treated with 20 μM AD for 30 minutes in the absence or presence of 1 μg / ml Cx43 (E2) antibody. An ethidium bromide dye uptake assay was performed and quantified relative to the untreated basal uptake level. The assay was performed in the presence of calcium. Low calcium conditions were used as a control (open hemichannels). Additionally, MLO-A5 osteoblasts were treated with AD or AD + Cx43 (E2) antibody as a negative control – AD does not open Cx43 hemichannels in osteoblasts, and the opening of osteocyte hemichannels induced by AD is blocked by the Cx43 (E2) antibody.

[0071] Materials used in some aspects of in vitro assays for identifying positive regulators of hemichannels include:

[0072] Hemichannel-expressing cells or cell lines. Cells or cell lines expressing various connexin hemichannels can be obtained, isolated, or engineered using methods and / or expression vectors known in the art.

[0073] Osteocytes: Primary osteocytes isolated from animals (including mice, rats, rabbits, chickens) or osteocyte cell lines, including but not limited to MLO-Y4 cells and other cells.

[0074] Cancer cells: Breast cancer cell lines, including ER, PR, HER, and TP53 positive / negative cells (e.g., MD-MBA-231, MCF7, T47D, or ZR751). MDA-MB-231 is ductal breast cancer. The Py8119 breast tumor cell line was established from spontaneous breast tumors generated in C57Bl / 6 MMTV-PyMT female (mouse mammary tumor virus promoter-driven polyoma middle T transgenic) mice. The expression of the oncogene (polyoma middle T transgenic) is driven by the mouse mammary tumor virus promoter.

[0075] Prostate cancer cell lines: Include androgen receptor and 5α-reductase positive / negative and androgen-sensitive / insensitive cell lines (e.g., LNCaP-Rf, BM18, pRNA-1-1 / ras, RC58T / hTERT, PPC-1, etc.).

[0076] Osteoblasts: MLO-A5 osteoblasts were used as controls because they express connexin 43, but they do not seem to open when stimulated by alendronate.

[0077] The "reagents" to be tested include compounds, peptides, proteins, antisense oligomers, and / or microRNAs.

[0078] Tracer molecules include but are not limited to Lucifer Yellow, ethidium bromide, Evans Blue, Alexa350, Alexa488, and Alexa594.

[0079] Cx43(E2): The Cx43(E2) antibody is specific for the Cx43 hemichannel. Cx43E2 binds to the second extracellular loop of the Cx43 hemichannel and prevents the hemichannel from opening.

[0080] Methods for determining whether a reagent opens a hemichannel include one or more of the following steps:

[0081] (a) Isolate, obtain, or generate connexin-expressing cells or cell lines. For example, isolate primary osteocytes from calvaria. Other cell types can be isolated using other methods known in the art. In some aspects, isolate calvarial osteocytes from an animal (e.g., 16-day embryonic chicken calvaria or neonatal mouse). Decapitate the animal, dissect the calvaria, and quickly immerse in 70% ethanol. Then place the calvaria in αMEM and wash several times with PBS. Place the washed bone in fresh αMEM. Chop the bone into pieces about 1.5 mm in size. The bone pieces can be treated with collagenase to remove soft tissue and osteoid, and then decalcified using EDTA. Finally, release the osteocytes from the bone fragments by treating with collagenase and vigorous stirring.

[0082] (b) Isolate primary osteocytes from long bones. Isolate long bone osteocytes from 2-3-week-old mice or rats. For example, administer an overdose of anesthesia to the mouse, dislocate the cervical vertebrae, decapitate, and immerse in 70% ethanol. Isolate the femurs and tibias with the joint ends still intact. Quickly immerse the legs in 70% alcohol and then place in αMEM. Wash the legs in αMEM with PBS. Remove most of the muscle and detach from the tendon / ligament. Place the washed bone in fresh αMEM. After washing all the bones, cut off the ends of each bone with a scalpel and immediately flush out the marrow with PBS. Cut the bone into pieces 1.5 to 2 mm in length and treat with collagenase. In one example, the bone pieces are treated with collagenase successively 9 times to remove all tissue and osteoid, and then decalcified using EDTA.

[0083] (c) Culture the cells or cell lines. For example, culture primary and / or osteocyte cell lines on collagen-coated plates and then immerse in a recording medium (HEPES-buffered α-MEM medium without HCO 3 ) containing a permeable tracer in a bath.

[0084] (d) Administer a test reagent. Expose the cultured cells to the test reagent for the desired duration.

[0085] (e) Determine the permeable tracer uptake. Determine the permeable tracer uptake by detecting the amount of tracer within the cells. In some aspects, use time-lapse recording. Fluorescence can be recorded in different cells in the region of interest, where the eclipse filter on the microscope is based on the fluorescence wavelength of the tracer or other probe used. In some aspects, acquire images every 2 minutes with a rapidly cooled digital camera and perform image processing using ImageJ software. The data collected can be presented as the initial fluorescence and the fold difference in fluorescence at the time of interest compared to the basal fluorescence.

[0086] For snapshot pictures, cells can be exposed to a permeable tracer for 5 - 10 minutes, rinsed several times with PBS, and then fixed with formaldehyde. In some aspects, at least three micrographs of the fluorescence field are obtained using a microscope. Image analysis is performed using ImageJ software. The average pixel density of random cells is measured.

[0087] In some aspects, the opening of connexin hemichannels is confirmed. This can be done, for example, by incubating osteocytes with a Cx43(E2) antibody (a polyclonal antibody that specifically inhibits Cx43 hemichannels) together with a test reagent. If the reagent opens the Cx43 hemichannel, the opening of this channel will be blocked by the Cx43(E2) antibody. To control the opening of Cx43 hemichannels, osteocytes are treated with fluid flow shear stress and / or AD, both of which are known to open hemichannels in osteocytes.

[0088] In a specific example, MLO - Y4 osteocytes were treated with 20 μM AD for 30 minutes in the absence or presence of 1 μg / ml Cx43(E2) antibody. An ethidium bromide dye uptake assay was performed and quantified relative to the untreated basal uptake level. The assay was performed in the presence of calcium. Low calcium conditions were used as a control (to open hemichannels). The opening of AD - induced osteocyte hemichannels was blocked by the Cx43(E2) antibody.

[0089] The opening of osteocyte Cx43 hemichannels mediates a negative effect on cancer cell migration. The Cx43 hemichannels in osteocytes are opened by the application of AD or FFSS. The opened hemichannels allow various factors to be released into the medium that generates conditioned medium (CM). Factors released in the CM treated with AD or FFSS reduce cancer cell migration, as determined by soft agar and wound healing assays. An example of the soft agar assay is schematically shown in Figure 3 As shown in Figure 4, cancer cells treated with control CM exhibit normal migration. The soft agar assay is an anchorage - independent growth assay, as opposed to anchorage - dependent growth. Only cancer cells can grow on soft agar, and their growth on this matrix indicates the degree of cancer cell proliferation.

[0090] In some aspects, the method includes incubating primary osteocytes or an osteocyte cell line with a test reagent in α - MEM for various time periods and collecting the supernatant (conditioned medium) at each time point. In some aspects, breast cancer or prostate cancer cells are incubated with CM, and cancer cell proliferation, migration, and invasion are measured.

[0091] Cancer cell growth and viability can be measured using the WST-1 (water-soluble tetrazolium salt) assay, trypan blue live cell counting, and BrdU DNA binding and cell proliferation assays. For the WST-1 assay, cell proliferation was measured at an emission wavelength of 450 nm using a Synergy HT multimode microplate reader (Biotek).

[0092] Cell migration assays were typically performed in transwell membrane filter inserts in 24-well tissue culture plates (BD Biosciences). The transwell membrane filter inserts can be, for example, polycarbonate membranes with a diameter of 6.5 mm, a pore size of 8 μm, and a thickness of 10 nm.

[0093] Invasion assays were performed in BD Biocoat low growth factor Matrigel invasion chambers (BD Biosciences). Cancer cell lines were harvested and resuspended in CM obtained from osteocytes with or without the test reagent. The cancer cell suspension was added to the upper side of the insert. The cells were incubated at 37 °C for various time periods. Cells that did not migrate through the filter were removed, and cells that migrated through the insert were fixed and stained with Hema 3 Stat Pack (Fisher Scientific). The number of migrating cells in five fields of view per insert was counted under an optical microscope.

[0094] In some aspects, breast cancer migration was reduced when incubated in CM from osteocytes treated with AD or FFSS to stimulate Cx43 hemichannel opening. This inhibitory effect on cancer cell migration was attenuated when the osteocyte Cx43 hemichannel was blocked with an E2 antibody. This reduction in cancer cell migration was not seen when incubated with CM collected from osteoblasts or treated directly with AD. Opening of the Cx43 hemichannel prevents breast cancer cell growth and migration.

[0095] B. In Vivo Assays

[0096] Other embodiments relate to detecting the opening of connexin hemichannels in vivo. An example of an in vivo assay for discovering reagents useful for treating cancer metastasis to bone involves determining the effect of a candidate reagent on Cx43 hemichannels in osteocytes and on cancer metastasis to bone in vivo. In some aspects, Cx43 regulation in osteocytes was measured by injecting a candidate reagent into the long bone and in situ detecting the opening of hemichannels in osteocytes using a fluorescent tracer dye (e.g., calcein or Evans blue).

[0097] In vivo assay of the effect of a compound on the opening of hemichannels in osteocytes of bone tissue. An example of an in vivo assay for analyzing hemichannels in osteocytes uses mice or rats that are 3 - 4 months old. Weigh the animals. Introduce the test reagent into the animals by intraperitoneal (IP) injection. After 2 - 4 hours, inject a fluorescent tracer dye (i.e., Evans blue, Alexa 594) into the lateral tail vein of the animals or perform an intraperitoneal injection. Note: The maximum 1% (volume) of the animal's body weight can be injected. In some aspects, warm the animals before the tail vein injection to dilate the tail vein. After 2 - 4 hours, sacrifice the animals, dissect the tibia and femur without muscle tissue, and wash them several times with PBS. Fix the bones in paraformaldehyde and decalcify them in a 14% EDTA solution at 4°C for two weeks or at room temperature with constant stirring for 3 - 5 days. Wash the bones in PBS, soak them overnight in a PBS solution containing 30% sucrose, and then embed them in OCT compound. Usually adjust the position of the bones in the mold as needed. Cut 5 - micron - thick frozen sections using a cryostat, rinse the sections in PBS, and then fix them using a PBS solution containing 50% glycerol. Examine the bone sections under a fluorescence microscope and quantify the extent of tracer dye uptake by osteocytes in the bone using Image J.

[0098] The opening of Cx43 hemichannels in osteocytes can be confirmed by applying mechanical load to the tibia to open the Cx43 hemichannels in osteocytes. This can be used as a positive control for the opening of hemichannels in vivo in osteocytes. For the negative control, use mice with a defect in Cx43 in osteocytes. Such mice are generated by crossing 10 - kb DMP - 1Cre with Cx43 flox mice.

[0099] Use the intratibial injection bone metastasis model and / or the intracardiac injection cancer metastasis assay to determine the effect of the test reagent on bone metastasis in vivo.

[0100] Intratibial injection bone metastasis model. The method includes anesthetizing normal or immunocompromised mice at 1 month old with isoflurane. Buprenorphine hydrochloride (0.3 mg / ml) is administered to the mice as an analgesic. Intratibial injection is performed using cancer cells expressing a fluorescent or chemiluminescent marker (e.g., Py8119 cells expressing Luc-GFP for normal mice, or MD-MBA-231 expressing Luc-GFP for immunocompromised mice). The cancer cells are inoculated into the bone marrow region of the right tibia through a hole pre-generated by a Hamilton syringe equipped with a 30-gauge needle. PBS is injected into the left tibia as a control. The test reagent or saline is administered intraperitoneally twice a week for 5 weeks. Starting from 3 days after tumor cell inoculation, intratibial tumor growth is monitored weekly by bioluminescence imaging or fluorescence. At the end of the study after sufficient bioluminescence imaging, X-ray images are obtained to test bone quality and observe labeled metastatic cancer cell colonies, and counted by fluorescence microscopy.

[0101] Intracardiac injection bone metastasis model. Normal or immunocompromised mice at 2 months old are anesthetized with isoflurane and also given buprenorphine hydrochloride (0.3 mg / ml) as an analgesic. Cancer cells expressing a fluorescent or chemiluminescent marker (e.g., Py8119 cells expressing Luc-GFP for normal mice, or Luc-GFP-MD-MBA-231 for immunocompromised mice) are injected into the left ventricle of the mice. The procedure includes: holding the needle at an angle to the operator and to the right, inserting it into the second intercostal space, approximately 3 mm to the left of the sternum. Advance approximately 5 mm and gently rotate the needle until a pulsatile flow of bright red arterial blood is observed entering the needle hub. Inject the cell suspension over 30 seconds. Remove the needle and apply pressure with an alcohol swab at the injection site for 30 seconds. Place the mouse on a warm surface until it has fully recovered from the anesthesia. Starting from 3 days after tumor cell inoculation, bioluminescence or fluorescence imaging is performed weekly after intracardiac injection to verify the distribution of tumor cells. At the end of the study after sufficient bioluminescence imaging, X-ray images are obtained to evaluate bone quality and observe labeled metastatic cancer cell colonies, and counted by fluorescence microscopy.

[0102] Cx43 conditional knockout (cKO) mice. Since homozygous global knockout of Cx43 is lethal and also because the inventors wanted to study the role of Cx43 expressed in osteocytes, osteocyte-specific Cx43 knockout mice were generated. Crossing mice homozygous for the floxed Cx43 gene with global Cx43 heterozygous mice facilitated complete deletion of Cx43 in osteocytes. Then, Cx43fl / − mice (50% of the offspring) were crossed with mice expressing Cre recombinase driven by the human DMP-1 promoter. This produced mice that were Cx43 fl / −, DMP1 Cre+ or Cx43 fl / −, DMP1 Cre− (a small percentage were Cx43fl / fl or Cx43− / −). Cx43-deficient osteocytes were confirmed by immunohistochemistry.

[0103] The study may include 4 groups of mice: WT treated with alendronate (AD), KO treated with alendronate, WT without AD, and KO without alendronate. AD was administered to the mice at 150 μg / kg body weight. For AD treatment, an increase in bone metastasis in the KO compared to WT mice was expected. In the absence of AD, bone metastasis should be similar between WT and knockout mice.

[0104] II. Methods of Treating Conditions Associated with Connexin Hemichannels

[0105] In certain embodiments, modulators of connexin hemichannels can be used to treat disorders associated with connexin hemichannels, including inflammatory disorders such as osteoarthritis (OA) and spinal cord injury. The methods and compositions described herein can also be used to treat wounds such as corneal and skin wounds.

[0106] A. Osteoarthritis (OA)

[0107] Osteoarthritis is a prevalent disease that affects approximately 20% of American adults. The disease results in the degeneration of joints, including articular cartilage and subchondral bone. The pathology of OA is characterized by the loss of articular cartilage, leading to joint space narrowing, increased joint friction, and possible structural remodeling. Current treatments include exercise, lifestyle modifications, and painkillers. If symptoms become severe, joint replacement surgery is usually performed. To date, there are no specific pharmaceutical interventions available for the treatment of OA.

[0108] Chondrocytes express connexin (Cx) 43 hemichannels, and these channels mediate the transfer of small molecules (less than 1 kDa) between the intra- and extracellular compartments. Under normal conditions, the Cx43 hemichannels in chondrocytes remain closed; however, these channels open under inflammatory conditions and release small molecules such as pro-inflammatory factors. Mechanical loading and interleukin-β1 induce the opening of Cx43 hemichannels in chondrocytes, promoting the inflammatory response and releasing pro-inflammatory factors such as prostaglandin E2 (PGE2) and ATP.

[0109] Inhibition of the opening of Cx43 hemichannels in chondrocytes (e.g., by chemical agents, etc.) can inhibit the inflammation and development of osteoarthritis. The opening of hemichannels in chondrocytes can be detected using the methods described herein. The pro-inflammatory factors (PGE2 and ATP) released by Cx43 hemichannels are measured using ELISA assays. Agents that block the opening of hemichannels can be used as therapeutic agents for inflammatory diseases such as OA.

[0110] Elevated interleukin 1β (IL-1β) is an inducer of OA. Abnormal joint loading is also known to increase the risk of OA. Exposure to IL-1β leads to the release of prostaglandins and NO by chondrocytes. The released PGE 2 exerts a catabolic effect by inhibiting proteoglycan synthesis and inducing collagen degradation. It has been demonstrated that mechanical loading opens Cx43 hemichannels. Cx43 serves as an entry for PGE 2 released into osteocytes. Cx43 is expressed on the surface of chondrocytes (see 18) as well as in articular cartilage. IL-1β and mechanical loading lead to the opening of Cx43 hemichannels in chondrocytes ( Figure 18 ), and this opening is inhibited by a hemichannel-specific Cx43 antibody. When the hemichannels are blocked by the Cx43 (E2) antibody, the inflammatory response induced by IL-1β is inhibited.

[0111] Based on the above evidence, the Cx43 hemichannels in chondrocytes are opened by IL-1β or mechanical loading, and the PGE 2 released by the hemichannels leads to the development of OA. Specific blockade of Cx43 hemichannels in chondrocytes can be used in therapeutic strategies to treat OA caused by elevated IL-1β or trauma (abnormal loading).

[0112] An in vitro cell model for evaluating Cx43 channel activity. Primary chondrocytes are isolated from the leg joints of mice. The effects of agents on the opening of Cx43 hemichannels and gap junction coupling in chondrocytes can be detected and evaluated for time- and dose-dependent effects. For example, the opening of hemichannels is evaluated by a dye uptake assay using lucifer yellow or Alexa dyes. The downstream effects are measured by detecting the release of PGE 2 and ATP using ELISA assays.

[0113] For spinal cord injuries, local inflammation and swelling are typically caused by local injury, trauma, or infection, and these events can also be the cause of systemic inflammation. Inflammation is typically characterized by increased redness, swelling, temperature, pain, and some loss of function in the affected area. In some aspects, agents that inhibit hemichannel opening can be used to alleviate inflammation associated with central nervous system inflammation and / or spinal cord injury.

[0114] Wound healing represents another important health issue and involves complex biological processes regardless of the cause. Generally speaking, during the associated inflammatory response, the wound is cleansed by infiltrating cells and body fluids. This initial inflammatory phase is followed by a proliferative phase, in which different cell types provide the factors and tissue environment necessary for wound healing or are filled in by appropriate cells such as fibroblasts, keratinocytes, and many other cells. As epithelial cells gradually fill in the wound, additional events such as angiogenesis and wound contraction also occur. This phase typically lasts about 7 - 10 days, depending on the severity of the wound and the efficiency of the inflammatory phase. Conditions and other circumstantial factors such as greater age, immunodeficiency, and stress can affect wound healing. Prolonged exposure of the wound leads to an increased likelihood of infection, adverse inflammatory effects, and scarring and possible chronic wounds. Generally speaking, the wound healing process ends with a maturation and remodeling phase. Collagen is replaced, remodeled, and cross-linked to increase the strength of the newly formed tissue, and unnecessary blood vessels, cells, and tissues are slowly removed from the wound site. As the body moves towards the final healing phase, this final phase can last for several years.

[0115] The treatment of wounds typically involves the application of antibiotics and agents that protect the external environment, such as bandages, sutures, second skin, sealants, or other creams and ointments. Additionally, many compounds can also be used to treat inflammation in the early stages of wound healing, typically in combination with steroidal anti-inflammatory compounds or drugs. The agents described herein or identified by the methods described herein can be used to modulate the inflammatory processes associated with wound healing.

[0116] III. Antibodies

[0117] Certain aspects of the invention relate to antibodies that positively or negatively regulate hemichannel function. Examples of the identification and isolation of monoclonal antibodies are described below.

[0118] As used herein, the term "CDR" refers to the complementarity determining regions of the variable domains of an antibody. The systematic identification of the residues included in the CDRs has been developed by Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda). The variable light chain (VL) CDRs are defined herein as including the residues at positions 27 - 32 (CDR1), 50 - 56 (CDR2), and 91 - 97 (CDR3). The variable heavy chain (VH) CDRs are defined herein as including the residues at positions 27 - 33 (CDR1), 52 - 56 (CDR2), and 95 - 102 (CDR3).

[0119] As will be recognized by those skilled in the art, the CDRs disclosed herein may also include variants. Generally, the amino acid identity between 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 with the parental CDR of the invention and shares biological function, including but not limited to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parental CDR.

[0120] Although the sites or regions for introducing amino acid sequence variation need to be predetermined, the mutations themselves do not need to be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be carried out at the target codon or target region, and the expressed antigen-binding protein CDR variants can be screened for the optimal combination of desired activities. Techniques for generating substitution mutations at predetermined sites in DNA of known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. The mutants are screened using the antigen-binding protein activity assays described herein.

[0121] Amino acid substitutions are generally 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 deletions can be much larger.

[0122] Substitutions, deletions, insertions, or any combination thereof can be used to obtain the final derivatives or variants. Generally, these changes are made on a few amino acids in order to minimize the changes to the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, in certain cases, larger changes may be tolerated.

[0123] As used herein, "Fab" or "Fab region" means a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to such a region in isolation or in the context of a full-length antibody, an antibody fragment, or a Fab fusion protein or any other antibody embodiment as outlined herein.

[0124] As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL and VH domains of a single antibody.

[0125] As used herein, "framework" means the 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 (FR1, FR2, FR3, and FR4) separated by CDRs.

[0126] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more antibody fragments 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 by the term "antigen-binding portion" of an antibody include, (i) Fab fragments, a monovalent fragment consisting of VL / VK, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fab' fragments, which are essentially Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul, ed., 3rd ed., 1993)); (iv) Fd fragments consisting of VH and CH1 domains; (v) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (vi) dAb fragments consisting of a VH domain (Ward et al. (1989) Nature 341:544-546); (vii) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, a heavy-chain variable region containing a single variable domain and two constant domains.

[0127] The term "specifically binds" (or "immunologically specifically binds") does not mean that an antibody binds exclusively to its intended target. Instead, an antibody "specifically binds" if its affinity for its intended target is approximately 5-fold higher than its affinity for non-target molecules. Suitably, there is no significant cross-reactivity or cross-binding with undesired substances. The affinity of the antibody for the target molecule will be at least about 5-fold higher, such as 10-fold, such as 25-fold, especially 50-fold, particularly 100-fold or higher, 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. An antibody can bind, for example, with an affinity of at least about 10 7 M -1 , such as between about 10 8 M -1 and 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 An antibody can bind with an EC 50 of 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 pM or less.

[0128] Mouse mAb purification protocol. Protein G rather than protein A is the column selected for purifying mouse IgG because mouse IgG1 binds much better to protein G. The supernatant from the hybridoma culture without fetal bovine serum is collected after 15 days to produce IgG.

[0129] In the experiment, GammaBind Plus TM Sepharose Fast Flow column was used. The column was washed and then equilibrated with binding buffer. Each of these steps used approximately 30 ml of buffer solution. Then the diluted hybridoma supernatant with binding buffer was loaded onto the column. 1.5 ml fractions (eluate) were collected. 150 μl of 1 M Tris buffer (pH 8) was used to neutralize the pH. The column was re-equilibrated with binding buffer (30 - 50 ml). Finally, 20 μl of sodium azide was added for storage. The column binding capacity was 18 mg / ml of mouse IgG. A flow rate of 1 ml / min was used. 50 mM sodium phosphate buffered saline (pH 7) was used as the binding buffer, and 0.1 M glycine (pH 2.7) was used as the elution buffer. The supernatant was mixed with the binding buffer at a 1:1 ratio.

[0130] In certain embodiments, a mouse monoclonal antibody (M1) is used to study the function of connexin Cx43 that forms gap junctions and / or hemichannels, wherein the heavy chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO:2, and the light chain of the monoclonal antibody M1 has the amino acid sequence shown in SEQ ID NO:4.

[0131] In certain embodiments, a mouse monoclonal antibody (M2) is used to study the function of connexin Cx43 that forms gap junctions and / or hemichannels, wherein the heavy chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO:6, and the light chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO:8.

[0132] In certain embodiments, a mouse monoclonal antibody (M7) is used to study the function of connexin Cx43 that forms gap junctions and / or hemichannels, wherein the heavy chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO:10, and the light chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO:12.

[0133] Immunoblotting. MLO-Y4 cells were seeded at 3x10 5 cells per 60 mm dish and maintained for 48 h. Mouse heart tissues were harvested in lysis buffer (5 mM Tris, 5 mM EDTA, 5 mM EGTA + protease inhibitors, 20 μl / ml phenylmethylsulfonyl fluoride (PMSF), 20 μl / ml N-ethylmaleimide, 10 μl / ml NaVO 4 and 10 μl / ml leupeptin), homogenized and centrifuged at 100,000 x g for 30 minutes at 4 °C and then resuspended in lysis buffer. The crude membrane proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blotted with anti-Cx43 CT (1:300 dilution) that recognizes the C-terminus of Cx43 or anti-Cx43 E2 (1:500 dilution) that recognizes the second extracellular loop of Cx43 or a monoclonal antibody against the second extracellular loop of Cx43 (1:100 dilution). The fluorescence of the secondary antibody, IR 800 anti-rabbit IgG (1:15000) (LI-COR, Lincoln, NE, USA) was detected by the Odyssey infrared detection system (LI-COR, Lincoln, NE, USA).

[0134] Immunofluorescence: MLO-Y4 cells were cultured on collagen-coated coverslips. The cells were rinsed twice with PBS and incubated with 70% cold ethanol at -20 °C for 20 minutes. PFA was used to disrupt the epitope, which is rich in lysine and thus not recommended. Then, the cells were rinsed twice with PBS to remove the ethanol. After that, the cells were blocked overnight with a blocking solution (PBS solution containing 2% goat serum, 2% fish skin gelatin, and 1% bovine serum albumin). Then, the cells were labeled with monoclonal antibodies at different concentrations in PBS, and then labeled with FITC-conjugated goat anti-mouse antibody and WGA-alexa594 (Invitrogen) (diluted at 1:400 and 1:1500 in the blocking solution, respectively). The cells were observed by an Olympus BH-2 fluorescence microscope, and the images were processed offline by NIH ImageJ software.

[0135] Dye uptake of hemichannel activity. The dye uptake measurements were evaluated using snapshot photographs. MLO-Y4 cells were plated onto collagen-coated 35 mm dishes and incubated with the following recording medium: saline medium without HCO 3 – buffered with the following HEPES salt composition at 10 mM or less (in mM): 154 NaCl, 5.4 KCl, 1.8 CaCl 2 、1.0 MgCl 2 、5 glucose. 0.5 mM EGTA was added to the medium with low divalent cations (low [X 2 ) without adding CaCl 2 and MgCl 2 . The recording medium or low [X 2 contained 50 μM EtBr for snapshot recording. The cells were exposed to 100 μM EtBr for 5 minutes, then rinsed three times with PBS and fixed with 2% formamide. At least three micrographs of the fluorescence field were obtained through a 10x dry objective in an inverted microscope (Carl Zeiss) with a rhodamine filter. The images were analyzed offline by image J software. The average pixel density of 30 random cells was measured.

[0136] Dye coupling assay of gap junctions. MLO-Y4 cells were plated onto collagen-coated 35 mm dishes and incubated with the recording medium (without HCO 3 –αMEM medium, incubated with 10 mM HEPES buffer. Microinjection of cells was performed at 37 °C using an InjectMan NI 2 and Femtojet, both obtained from Eppendorf (Eppendorf), with alexafluor350 (Invitrogen, Eugene, Oregon, USA) (a PBS solution of 10 mM). Dye transfer was measured 2 minutes after alexafluor 350 injection. The number of cells with dye transfer was counted to record the dye coupling index. Dye coupling was observed under an inverted microscope equipped with a xenon arc lamp illumination and a Nikon eclipse (Nikon, Japan) (excitation wavelength 330 - 380 nm, emission wavelength above 420 nm).

[0137] Cell-drop dye transfer assay for gap junctions. MLO-Y4 cells were grown to confluence in a 12-well plate. Donor cells were incubated with 5 μM calcein red-orange-AM (790 Da) and 5 μM Oregon Green 488 BAPTA-2-AM (1752 Da) at 37 °C for 40 minutes. Intercellular communication through gap junctions can be traced by simultaneously labeling cells with the gap junction-permeable tracer dye calcein red-orange and the gap junction-impermeable Oregon Green 488 BAPTA-2. Pre-loaded donor cells were removed from the plate by trypsinization. The pre-loaded cells were layered ("dropped") on top of unlabeled recipient cells cultured at a donor-to-recipient ratio of 1:4. The cells were allowed to adhere for a 1-hour period, then carefully washed 3 times, fixed in fresh 2% PFA for 10 minutes at room temperature, and then rinsed 3 times. Cells were examined by fluorescence microscopy. For calcein red-orange transfer, the threshold was adjusted to clearly distinguish the dye transfer boundary. The criterion for positive dye transfer was the detection of calcein red-orange / Oregon Green 488 BAPTA-2, where the contacting cells were calcein red-orange positive and Oregon Green 488 BAPTA-2 negative. Dye transfer was barely detectable (<1%). Images were acquired where we found Oregon Green 488 BAPTA-2 green-positive cells.

[0138] Fluid flow shear stress opens hemichannels. A parallel plate flow chamber separated by a washer of defined thickness was used to form a fluid flow with a gravity-driven fluid stream using a peristaltic pump. The washer thickness determines the channel height, which was adjusted together with the flow rate to generate a stress level of 16 dynes / cm 2 The circulating medium was α-MEM buffered with 10 mM HEPES.

[0139] The provided examples and figures are included to illustrate preferred embodiments of the present invention. Those skilled in the art should recognize that the techniques disclosed in the examples or figures represent techniques that the inventors have found to function well in the practice of the present invention and can thus be considered to constitute preferred modes of practicing the present invention. However, based on this disclosure, those skilled in the art will also recognize that various modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0140] IV. Pharmaceutical Compositions

[0141] Certain aspects include compositions, such as pharmaceutical compositions, that comprise 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 the antibodies or immunoconjugates described herein (e.g., two or more different ones). For example, the pharmaceutical compositions of the present invention can comprise a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0142] The pharmaceutical compositions of the present invention can also be administered as combination therapies, i.e., in combination with other agents. For example, a combination therapy can include an anti-half-channel antibody in combination with at least one other anti-cancer agent.

[0143] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, 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., the antibody or immunoconjugate) can be coated with a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0144] Examples of suitable aqueous and non-aqueous carriers for the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size (in the case of dispersions), and by using surfactants.

[0145] 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 reagents for pharmaceutically active substances is known in the art. Unless any conventional media or reagents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0146] Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size (in the case of dispersions) and by using surfactants. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride. Delayed absorption of injectable compositions can be achieved by including in the composition agents that delay absorption such as monostearates and gelatin.

[0147] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the ingredients enumerated above, followed by sterile 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 method of preparation is vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient and any additional required ingredients from a previously sterile-filtered solution.

[0148] The amount of active ingredient that can be combined with the carrier materials to produce a single-dose form will vary depending on the subject to be treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single-dose form will generally be the amount of the composition that produces a therapeutic effect. Generally speaking, out of 100%, the amount will be about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0149] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single large dose can be administered, several divided doses can be administered over time, or the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable as a single dose for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention is controlled by and directly depends on (a) the unique properties of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of individual sensitivities.

[0150] For the administration of an antibody, the dose ranges from about 0.0001 to 100 mg / kg, more typically 0.01 to 5 mg / kg of the host body weight. For example, the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or in the range of 1 - 10 mg / kg. Exemplary treatment regimens employ once weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every 3 to 6 months. Preferred dose regimens of the anti - hemichannel antibodies of the present invention include intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight, wherein the antibody is given using the following dosing schedules: (i) once every four weeks for six doses, then once every three months; (ii) once every three weeks; (iii) 3 mg / kg body weight once every three weeks, then 1 mg / kg body weight.

[0151] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the specified range. Antibodies are typically administered multiple times. The interval between individual doses can be, for example, weekly, monthly, every three months or annually. The interval can also be irregular, as indicated by measuring the blood level of antibodies against the target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1 - 1000 μg / ml and in some methods to achieve a plasma antibody concentration of about 25 - 300 μg / ml.

[0152] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective for the particular patient, composition, and mode of administration to achieve the desired therapeutic response without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition employed of the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0153] A "therapeutically effective dose" of an anti - hemichannel antibody results in a reduction in the severity of the disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of injury or disability due to the affliction of the disease. A therapeutically effective amount of a therapeutic compound or antibody can reduce tumor metastasis in a subject or otherwise alleviate symptoms. Those of ordinary skill in the art will be able to determine such an amount 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.

[0154] The compositions of the present invention can be administered by one or more routes of administration using one or more 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 outcome. Preferred routes of administration of the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration (usually by injection) and includes, but is not limited to, intravenous, intramuscular, intraarterial, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular injection and infusion. Sequence Listing <110> Jiang, Jean X. Riquelme, Manuel A. Gu, Sumin <120> Compositions and Methods for Targeting Connexin Hemichannels <130> 161172F1 1PWCN <150> 61 / 868,112 <151> 2013-08-21 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 384 <212> DNA <213> 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> 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> 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> 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> 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> 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> 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> 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> 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> 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> 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> 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 <220> <223> Synthetic Peptide <400> 13 Phe Leu Ser Arg Pro Thr Glu Lys Thr Ile 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 14 Lys Arg Asp Pro Cys Pro His Gln Val Asp 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 15 Leu Ser Ala Val Tyr Thr Cys Lys Arg 1 5

Claims

1. Use of an antibody or an antigen-binding portion thereof in the preparation of a product for inhibiting an inflammatory response in chondrocytes of a subject at risk of developing pathological inflammatory osteoarthritis or spinal cord injury or diagnosed with pathological inflammatory osteoarthritis or spinal cord injury in vitro, wherein, the antibody or an antigen-binding portion thereof comprises: a) a heavy chain variable domain comprising an amino acid sequence as shown in the amino acid sequence of SEQ ID NO: 2, wherein the heavy chain variable domain comprises three heavy chain CDRs located at amino acid positions 27-33, 52-56, and 95-102 of the heavy chain amino acid sequence of SEQ ID NO: 2 according to the Kabat numbering system; and b) a light chain variable domain comprising an amino acid sequence as shown in the amino acid sequence of SEQ ID NO: 4, wherein the light chain variable domain comprises three light chain CDRs located at amino acid positions 27-32, 50-56, and 91-97 of the light chain amino acid sequence of SEQ ID NO: 4 according to the Kabat numbering system.

2. The use according to claim 1, wherein the subject is at risk of developing pathological inflammatory osteoarthritis or is diagnosed with pathological inflammatory osteoarthritis.

3. The use according to claim 1, wherein the subject is at risk of spinal cord injury or is diagnosed with spinal cord injury.

4. Use of an effective amount of an antibody or an antigen-binding portion thereof capable of binding to connexin 43 (Cx43) hemichannels in the preparation of a medicament for treating cancer metastasis in a subject in need thereof, wherein the antibody or an antigen-binding portion thereof comprises: first, second, and third heavy chain CDR sequences, and first, second, and third light chain CDR sequences; wherein, numbered according to the Kabat numbering system, the first, second, and third heavy chain CDR sequences are respectively composed of amino acid positions 27-33, 52-56, and 95-102 of the heavy chain amino acid sequence of SEQ ID NO: 6; and, numbered according to the Kabat numbering system, the first, second, and third light chain CDR sequences are respectively composed of amino acid positions 27-32, 50-56, and 91-97 of the light chain amino acid sequence of SEQ ID NO: 8, wherein the cancer metastasis is selected from breast cancer metastasis, prostate cancer metastasis, lung cancer metastasis, and skin cancer metastasis, or wherein the cancer metastasis is bone metastasis.

5. The use according to claim 4, wherein the cancer metastasis is bone metastasis.

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