Connexin 43 antibodies and use thereof

Abstract

To provide compositions and methods for treating a disease or condition associated with opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury.SOLUTION: Provided herein are compositions and methods for treating a disease or condition associated with opening (e.g., excessive or prolonged opening) of Cx43 hemichannels, such as inflammatory diseases or conditions and neurodegenerative diseases, e.g., spinal cord injury, Alzheimer's disease, osteoarthritis.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and benefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 800,869, filed on February 4, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure generally relates to anti - connexin (Cx) 43 antibodies and their use in the treatment of diseases or conditions associated with the opening of Cx43 hemichannels in cells such as, for example, astrocytes and osteocytes.

Background Art

[0003] Background In the United States alone, it is estimated that there are approximately 400,000 people with spinal cord injury (SCI), and more than 14,000 new cases occur each year. The acute inflammatory response is a defense mechanism aimed at preserving tissue integrity and defining the boundaries of traumatic lesions, but an excessive response can limit the potential for successful recovery. Tissue swelling, especially within the rigid closed space of the spinal canal, can reduce tissue perfusion and cause secondary ischemia. The delayed loss of tissue affects functional recovery in most patients, and there are currently no effective treatment options.

[0004] It was shown 50 years ago that injection of ATP is sufficient to induce an acute inflammatory response in the absence of injury. An important finding linking purinergic signaling to inflammatory mediators was that activation of the purinergic receptor P2RX7 induces the maturation of microglial cells and the secretion of IL - 1β from microglial cells. Adenine nucleotides (i.e., ATP and its metabolites) are recognized as inflammatory mediators, but the role of purinergic signaling in spinal cord injury is relatively poorly understood. Spinal cord injury results in excessive and persistent ATP release in the area surrounding the trauma, and inhibition of P2RX7 reduces the inflammatory response and improves functional recovery.

[0005] Previous studies have shown that astrocytes release ATP, at least in part, through the opening of connexin 43 (Cx43) hemichannels. Connexins are a family of proteins with two channel functions. The conventional role is to form gap junctions, which are composed of two coupled hemichannels that connect the cytosol of two adjacent cells. Gap junctions allow the intercellular passage of ions and small molecules, including Ca 2+ , cAMP, IP3, ATP, glutamate, and glucose. It is recognized that unopposed hemichannels constitute a pathway for regulated gliotransmitter release. Due to their relatively large inner pore diameter (about 10 Å), open hemichannels facilitate the efflux of small cytosolic compounds, and many of these, including ATP and glutamate, act as transmitters after being released. Since the persistent opening of many hemichannels is incompatible with cell survival, the opening of hemichannels is usually tightly regulated.

[0006] Cx43 is also involved in sterile and infectious inflammation, such as other inflammatory disorders including inflammatory lung disease, osteoarthritis, and spinal cord injury.

[0007] Therefore, there is a need for effective methods and compositions for treating spinal cord injury, for example, by inhibiting the opening of Cx43 hemichannels. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0008] Abstract Compositions and methods are provided herein for treating diseases or conditions associated with the opening of Cx43 hemichannels (e.g., excessive or persistent opening), such as inflammatory diseases or conditions and neurodegenerative diseases, such as spinal cord injury, Alzheimer's disease, osteoarthritis, etc.

[0009] In one aspect, herein, The first, second, and third heavy chain complementarity determining region (CDR) sequences, each having the amino acid sequences of SEQ ID NOs: 1, 2, and 3; and The first, second, and third light chain CDR sequences, each having the amino acid sequences of SEQ ID NOs: 4, 5, and 6 Provided is an anti-Cx43 antibody or an antigen-binding fragment thereof that includes the above.

[0010] In certain embodiments, the antibody or fragment thereof may have a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 8.

[0011] A further aspect relates to an anti-Cx43 antibody or an antigen-binding fragment thereof that includes a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17 and a light chain having the amino acid sequence of SEQ ID NO: 18.

[0012] In another aspect, provided herein are antibodies that bind to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may include one or more amino acids selected from the group consisting of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. In one embodiment, the epitope consists of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. In some embodiments, the epitope may include all 10 amino acids of SEQ ID NO: 19. In certain embodiments, the epitope consists of all 10 amino acids of SEQ ID NO: 19.

[0013] Another aspect relates to an isolated anti-Cx43 antibody or an antigen-binding fragment thereof that cross-competes with any antibody or fragment thereof disclosed herein with respect to binding to Cx43. In various embodiments, the antibody or fragment thereof can inhibit or reduce or block the opening of Cx43 hemichannels in cells.

[0014] A further aspect relates to an isolated anti-Cx43 antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof cross-competes with an antibody or fragment thereof disclosed herein having one or more of SEQ ID NOs: 1-18 with respect to binding to Cx43, preferably the antibody or fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19), more preferably the epitope comprises one or more amino acids selected from the group consisting of R4, P5, E7, K8, and I10 of SEQ ID NO: 19, and even more preferably the epitope comprises all 10 amino acids of SEQ ID NO: 19.

[0015] In various embodiments, the antibody or fragment thereof disclosed herein can inhibit or reduce or prevent the opening of Cx43 hemichannels in cells. In some embodiments, the opening is an excessive opening or a long-term opening.

[0016] Similarly, disclosed herein is a pharmaceutical composition for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, the pharmaceutical composition comprising one or more of the antibodies or fragments thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the opening is an excessive opening or a long-term opening.

[0017] A further aspect relates to the use of an antibody or fragment thereof disclosed herein for the manufacture of a medicament for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury. In some embodiments, the opening is an excessive opening or a long-term opening.

[0018] Another aspect relates to a method of inhibiting the opening of Cx43 hemichannels in cells for treating preferably an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, the method comprising contacting the cells with an effective amount of an antibody or fragment thereof disclosed herein. In some embodiments, the opening is an excessive opening or a prolonged opening.

[0019] A further aspect relates to a method of treating a disease or condition associated with the opening of Cx43 hemichannels in astrocytes or osteocytes for treating preferably an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or fragment thereof disclosed herein. In some embodiments, the opening is an excessive opening or a prolonged opening. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

Figure 1

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Figure 2

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Figure 3

[0023] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods of the present disclosure.

[0024] Disclosed herein are compositions and methods related to anti-Cx43 antibodies or antigen-binding fragments thereof. In some embodiments, the compositions disclosed herein exhibit unexpectedly superior activity, drugability (e.g., reduced toxicity), stability, and / or developability (e.g., reduced production costs) compared to the compositions disclosed in PCT Publication Nos. WO2015 / 027120 and WO2017 / 147561, which are hereby incorporated by reference in their entirety. In certain embodiments, the advantages are unexpected. **Definitions**

[0025] For convenience, certain terms used in the specification, examples, and appended claims are collected herein. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0026] As used herein, the following terms and phrases are intended to have the following meanings.

[0027] The articles "a" and "an" as used herein refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0028] As used herein, the term "about" means an acceptable variation within 20%, more preferably within 10%, and most preferably within 5% of the recited value.

[0029] An "anti-Cx43 antibody" is an antibody that immunospecifically binds to Cx43 (e.g., its extracellular domain). The antibody can be an isolated antibody. Such binding to Cx43 has a K D value that shows a value of, for example, 1 μM or less, 100 nM or less, or 50 nM or less. K D can be measured by any method known to those skilled in the art, such as a surface plasmon resonance assay or a cell binding assay. The anti-Cx43 antibody can be a monoclonal antibody or an antigen-binding fragment thereof.

[0030] An "antibody" as used herein is a protein that contains a binding domain that binds to a target epitope. The term antibody includes its variants and derivatives, including monoclonal antibodies, single heavy chain variable domain antibodies, and chimeric variants of monoclonal antibodies and single heavy chain variable domain antibodies, which include immunoglobulin heavy and light chain molecules. The binding domain is substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene such that the protein immunospecifically binds to an antigen. The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which respectively define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE. For most vertebrate organisms, including human and mouse species, a typical immunoglobulin structural unit includes a tetramer composed of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kD) and one "heavy chain" (about 50 - 70 kD). "V L " and "V H " respectively refer to the variable domains of these light and heavy chains. "C L」and「C H 」refers to the constant domains of the light and heavy chains. V L and V H There are three loops each on the β chains, which are involved in binding to the antigen and are called "complementary determining regions" or "CDRs". The "Fab" (fragment, antigen-binding) region consists of one constant domain and one variable domain from each heavy and light chain of the antibody, i.e., V L , C L , V H , and C H 1.

[0031] Antibodies include intact immunoglobulins as well as antigen-binding fragments thereof. The term "antigen-binding fragment" refers to a polypeptide fragment of an antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) with respect to antigen binding (i.e., specific binding). Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab’, (Fab’)2, CDR, paratope, and a single-chain Fv antibody (scFv) in which the V H and V L chains are joined together (either directly or through a peptide linker) to form a continuous polypeptide.

[0032] Antibodies also include variants, chimeric antibodies, and humanized antibodies. As used herein, the term "antibody variant" refers to an antibody having one or more mutations in the heavy and / or light chains. In some embodiments, the mutations are present in the variable region. In some embodiments, the mutations are present in the constant region. A "chimeric antibody" refers to an antibody in which one portion of the amino acid sequence of each of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequences in an antibody derived from another species or belonging to another class. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of an antibody derived from one species of mammal, while the constant portions are homologous to the sequences in an antibody derived from another species. A distinct advantage of such chimeric types is that the variable regions can be readily derived from currently known sources, for example, by using readily available hybridomas or B cells from non-human host organisms and combining them with constant regions derived from, for example, human cell preparations. The variable regions have the advantage of ease of preparation, and the specificity is not affected by their origin, while the constant regions that are human are less likely to induce an immune response from a human subject when the antibody is injected than constant regions from non-human origins. However, the definition is not limited to this particular example. A "humanized" antibody refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, as well as the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can include either a complete variable domain fused to a constant domain or only the complementarity-determining regions (CDRs) grafted into an appropriate framework region in the variable domain. The antigen-binding site can be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble a human immunoglobulin. Some types of humanized antibodies conserve all of the CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody).Other types of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, or 6) that are changed with respect to the original antibody, and these are also called one or more CDRs "derived from" one or more CDRs.

[0033] As used herein, the amino acid residues of an antibody can be numbered according to Kabat's general numbering (Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, NIH, Bethesda, MD).

[0034] The term "binds," as used herein in the context of the binding between an antibody and an epitope of Cx43 as a target, refers to the process of intermolecular non-covalent binding interactions. Preferably, said binding is specific. The specificity of an antibody can be determined based on affinity. A specific antibody has a binding affinity or dissociation constant K -7 of less than 10 -8 M, preferably less than 10 D M and may have a dissociation constant K

[0035] The term "affinity" refers to the strength of the binding reaction between the binding domain of an antibody and an epitope. This is the sum of the attractive and repulsive forces acting between the binding domain and the epitope. The term affinity, as used herein, refers to the dissociation constant K D as referred to.

[0036] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binding agent such as an antibody and can also be used in an animal to produce an antibody that can bind to an epitope of that antigen. An antigen can have one or more epitopes.

[0037] The term "epitope" includes any determinant, preferably a polypeptide determinant capable of specifically binding to an immunoglobulin or a T cell receptor. In certain embodiments, an epitope determinant includes molecules of chemically active surface groups such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. In one embodiment, an epitope is the region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to specifically bind to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Methods of epitope mapping, such as X-ray crystallography, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange, are well known in the art. Epitopes can be formed from both contiguous amino acids or discontinuous amino acids that are adjacent due to the three-dimensional folding of the protein. Epitopes formed from contiguous amino acids are typically retained even when exposed to denaturing solvents, whereas epitopes formed by three-dimensional folding are typically lost by treatment with denaturing solvents. Epitopes typically contain at least 3, more usually at least 5 or 8-10 amino acids in their own spatial conformation.

[0038] The site on the antibody that binds to the epitope is called the "paratope", which typically includes amino acid residues that are in close proximity to the epitope upon binding. See Sela-Culang et al., Front Immunol. 2013; 4: 302.

[0039] "Immunohistochemistry" or "IHC" refers to the process of detecting antigens in cells of tissue sections that enables the binding of antibodies that immunospecifically recognize the antigen of interest in biological tissue and subsequent detection. For an overview of IHC techniques, see, for example, Ramos-Vara et al., Veterinary Pathology January 2014 vol. 51 no. 1, 42-87, which is hereby incorporated by reference in its entirety. To evaluate IHC results, different qualitative and semi-quantitative scoring systems have been developed. See, for example, Fedchenko et al., Diagnostic Pathology, 2014; 9: 221, which is hereby incorporated by reference in its entirety. One example is the H score, which is determined by adding the results of multiplying the percentage of cells by an ordinal value of staining intensity (a score from 0 for "no signal" to 3 for "strong signal") having possible values of 300.

[0040] "Immunospecific" or "immunologically specifically" (sometimes used interchangeably with "specific") refers to an antibody that binds to one or more epitopes of the protein of interest via a domain substantially encoded by an immunoglobulin gene or fragment of an immunoglobulin gene, but does not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic molecules. Typically, the antibody binds immunospecifically to a cognate antigen with a K D value of 50 nM or less as measured, for example, by a real-time label-free biolayer interferometry assay such as an Octet® HTX biosensor, or by surface plasmon resonance such as BIACORE®, or by solution-affinity ELISA. The use of such assays is well known in the art.

[0041] The term "surface plasmon resonance" refers to an optical phenomenon that enables the analysis of real-time biomolecular interactions, for example, by detecting changes in protein concentration within a biosensor matrix using, for example, a BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0042] Biolayer interferometry is a label-free technique for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces, namely, a layer of immobilized protein on a biosensor chip and an internal reference layer. Any change in the number of molecules bound to the biosensor chip causes a shift in the interference pattern that can be measured in real time (Abdiche, Y. N., et al. Analytical Biochemistry, (2008), 377(2), 209-217). In certain embodiments, the binding characteristics of certain anti-Cx43 antibodies disclosed herein were evaluated using a "real-time biolayer interferometry-based biosensor (Octet HTX assay)".

[0043] The terms "cross-compete", "cross-competition", "cross-block", "cross-blocked", and "cross-blocking" are used interchangeably herein and mean the ability of an antibody or fragment thereof to interfere with direct or indirect binding to a target Cx43 through allosteric modulation of the anti-Cx43 antibodies of the present disclosure. The degree to which an antibody or fragment thereof can interfere with the binding of another antibody to a target, and thus whether it can be said to cross-block or cross-compete according to the present disclosure, can be determined using a competitive binding assay. One particularly suitable quantitative cross-competition assay uses a FACS-based or AlphaScreen-based approach to measure the competition between a labeled (e.g., His-tagged, biotinylated, or radioactively labeled) antibody or fragment thereof for binding to a target and another antibody or fragment thereof. Generally, a cross-competing antibody or fragment thereof is an antibody or fragment thereof that can bind to a target in a cross-competition assay such that, for example, in the presence of a single variable domain of an immunoglobulin or a polypeptide with a recorded substitution according to the present disclosure at a predetermined amount during the assay and in the presence of a secondary antibody or fragment thereof, the maximum theoretical substitution (e.g., substitution by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked) is up to 100% (e.g., for a FACS-based competition assay). Preferably, the cross-competing antibody or fragment thereof has a recorded substitution between 10% and 100%, more preferably between 50% and 100%.

[0044] Cross-competition between antibodies can also be determined by a real-time label-free biolayer interferometry assay. Cross-competition between two antibodies can be expressed as the binding of the second antibody being lower than the background signal due to self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as the % binding of the second antibody being less than the baseline self-self background binding (where the first and second antibodies are the same antibody).

[0045] The terms "inhibit", "block", and "reduce" are used interchangeably herein and refer to a statistically significant decrease in biological activity (e.g., hemichannel opening). For example, "inhibit" or "block" can refer to a decrease in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0046] The term "subject" or "patient" includes a human or other mammal that undergoes either a prophylactic or therapeutic treatment.

[0047] The terms "treat", "treating", and "treatment", as used herein, refer to therapeutic or preventive means such as those described herein. The method of "treatment" is for preventing, curing, delaying, reducing the severity of, or improving one or more symptoms of an inflammatory disease or condition, or a neurodegenerative disease, or for extending the survival of a patient beyond what would be expected in the absence of such treatment, and involves administering to a patient, e.g., a patient having an inflammatory disease or condition or a neurodegenerative disease, a Cx43 ligand provided herein. The method of "treatment" also involves administering to a patient a Cx43 ligand (e.g., an antibody) provided herein for providing treatment to the patient beyond what would be expected in the absence of such treatment.

[0048] As used herein, the term "effective amount" refers to an amount of an agent, such as a Cx43 ligand, e.g., an anti-Cx43 antibody, that is sufficient to effect the treatment, prognosis, or diagnosis of a disease when administered to a patient. The therapeutically effective amount will vary depending on the patient being treated and the disease state, the patient's weight and age, the severity of the disease state, the mode of administration, etc., and these can be readily determined by one of ordinary skill in the art. The dosage of administration can be, for example, from about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg, about 10 μg to about 2,600 mg, about 20 μg to about 2,575 mg, about 30 μg to about 2,550 mg, about 40 μg to about 2,500 mg, about 50 μg to about 2,475 mg, about 100 μg to about 2,450 mg, about 200 μg to about 2,425 mg, about 300 μg to about 2,000, about 400 μg to about 1,175 mg, about 500 μg to about 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500 mg, or in the range of about 525 mg to about 625 mg of the antibody or antigen-binding portion thereof provided herein, for example. The dosing can be, for example, weekly, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. The dosing regimen may be adjusted to provide an optimal therapeutic response.An effective amount is a dosage at which any toxicity or adverse effects (side effects) of the agent are minimized and / or the beneficial effects exceed it. Administration can be intravenous administration at exactly or approximately 6 mg / kg or 12 mg / kg weekly, or 12 mg / kg or 24 mg / kg once every two weeks. Additional dosing regimens are described below.

[0049] Other terms used herein in the fields of recombinant nucleic acid technology, microbiology, immunology, antibody manipulation, and molecular cell biology are generally understood by those of ordinary skill in the art of the present application. For example, conventional techniques may be used to prepare recombinant DNA, to perform oligonucleotide synthesis, and to practice tissue culture and transformation (e.g., electroporation, transfection, or lipofection). Enzyme reactions and purification techniques can be carried out according to the manufacturer's specifications, or as generally accomplished in the art, or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and described throughout this specification. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless otherwise specifically defined, the nomenclature utilized in connection with, and the experimental procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, as well as for the treatment of patients.

[0050] As used herein, the term "comprising" or "comprises" is used in connection with a composition, method, and respective components thereof that exist in a given embodiment, but there is no limitation to including elements that are not specified.

[0051] As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional characteristics of that embodiment of the present disclosure.

[0052] The term "consisting of" refers to the compositions, methods, and respective components described herein, and excludes any element not recited in that description of the embodiment.

[0053] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, a reference to "the method" includes one or more methods of the type described herein and / or that are apparent to one of ordinary skill in the art upon reading the present disclosure and / or steps.

[0054] Various aspects and embodiments are described in more detail in the following subsections. Cx43

[0055] Various cells can communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by the protein connexin. Connexin proteins are widely expressed throughout the body. Six connexin proteins constitute one hemichannel, and two hemichannels constitute one gap junction channel. Gap junctions are aggregates of channels located in the cell membranes between adjacent cells, and they mediate intercellular communication. Hemichannels are distinct entities from gap junction channels. Hemichannels enable the exchange of molecules between the intracellular compartment and the extracellular environment.

[0056] Many cells express hemichannels known as connexin (Cx)43 hemichannels. Connexin-43 is also known as gap junction alpha-1 protein (GJA1), which is a 43.0 kDa protein composed of 382 amino acids (NCBI Reference Sequence: NP_000156.1). GJA1 contains a long C-terminal tail, an N-terminal domain, and multiple transmembrane domains. The protein passes through the phospholipid bilayer four times, exposing its C-terminal and N-terminal ends to the cytoplasm. The C-terminal tail is composed of 50 amino acids and contains post-translational modification sites as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. As a result, the C-terminal tail is central to functions such as pH-dependent opening and closing and regulation of channel assembly. In particular, the DNA region of the GJA1 gene (NCBI Gene ID: 2697) encoding this tail is highly conserved, indicating that it is either resistant to mutations or lethal when mutated. On the other hand, the N-terminal domain is involved in channel opening and closing and oligomer formation, and thus can control the switch between the open and closed states of the channel. The transmembrane domains form gap junction channels, while the extracellular loops facilitate proper channel binding. Moreover, the two extracellular loops form disulfide bonds that interact with two hexamers to form a complete gap junction channel.

[0057] Cx43 plays an important role in the regulation of various immune processes. Recent reports have shown a pathogenic role of Cx43 hemichannels in sterile injury as well as infectious inflammatory diseases (Li et al., Scientific Reports (2018) 8:166, doi:10.1038 / s41598-017-18452-1). Sterile inflammation is a common event induced by physical, chemical, or metabolic noxiae. Various noxiae cause cellular stress and thus trigger a stress response. There are many types of stress responses (e.g., unfolded protein response, integrated stress response, oxidative stress), and these often intertwine with each other. The stress response induces inflammation. When the noxiae persist, the inflammation does not resolve, leading to a vicious cycle that plays an important role in the pathophysiology of many human disorders, including cancer, metabolic diseases, and genetic diseases.

[0058] Examples of acute conditions caused by sterile inflammation include ischemia-reperfusion injury (IRI), trauma (e.g., spinal cord injury, traumatic brain injury, peripheral nerve injury), crystal-induced inflammation, and toxin exposure. Acute myocardial infarction, cerebral infarction, acute kidney injury, and solid organ transplantation are all conditions in which IRI occurs. Deposition of crystals within joints leads to gouty arthritis, inducing classical clinical symptoms including erythema, pain, heat, swelling, and loss of function. Toxins, such as acetaminophen or cobra toxin, induce liver and muscle damage, respectively. Trauma, including crush injury, induces a sudden inflammatory response, and endogenous and microbial triggers (by exposure to bacteria) can contribute to inflammation in this context.

[0059] Examples of chronic conditions induced by or resulting from sterile inflammation include particle-induced lung diseases, such as asbestosis and silicosis, chronic lung diseases, such as cystic fibrosis and idiopathic pulmonary fibrosis, cardiovascular diseases, such as atherosclerosis, some causes of chronic heart failure, tumors, certain cases of arthritis (e.g., osteoarthritis and rheumatoid arthritis (RA)), and autoimmune diseases.

[0060] Infectious inflammation can be caused by various pathogens such as bacteria and fungi in multiple tissues.

[0061] In various embodiments, regardless of the cause, an inflammatory disease can be treated with the anti-Cx43 antibodies disclosed herein. An inflammatory disease, as used herein, refers to a wide range of disorders and conditions characterized by inflammation. Examples include arthritis, allergies, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), reperfusion injury, and transplant rejection. An autoimmune disease is a disease in which the immune system attacks its own proteins, cells, and tissues, or immune effector T cells are autoreactive against endogenous self-peptides, causing tissue destruction. Thus, the immune response is initiated against the subject's own antigens, called autoantigens. An exhaustive list and overview of autoimmune diseases can be found in The Autoimmune Diseases (Rose and Mackay, 2014, Academic Press). Autoimmune diseases include, but are not limited to, rheumatoid arthritis, Crohn's disease, type I diabetes, alopecia areata, multiple sclerosis, lupus, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-related infertility, glomerulonephritis (e.g., crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulin resistance, and autoimmune type 1 diabetes.

[0062] Excessive or chronic Cx43 hemichannel opening among chronic neurodegenerative diseases has also been shown to promote disease progression and induce cell death by disrupting the metabolic gradient and excessive release of toxic molecules. See, for example, Orellana et al., (2011), Neurodegenerative Diseases - Processes, Prevention, Protection and Monitoring, Chapter 10, Role of Connexin Hemichannels in Neurodegeneration, doi: 10.5772 / 28054; Bosch et al., Front Cell Neurosci. 2014; 8: 242; and Vicario et al., Front. Physiol., 2017, Vol. 8, Article 1060, doi: 10.3389 / fphys.2017.01060, all of which are incorporated herein by reference in their entirety. Thus, inhibition or reduction of Cx43 hemichannel opening using the compositions disclosed herein can be used to treat various neurodegenerative diseases such as Alzheimer's disease (AD), lysosomal storage disorders, bacterial meningitis, amyotrophic lateral sclerosis, hypoxia, ischemia, glaucoma, schizophrenia, major depressive disorder, bipolar disorder, epilepsy, traumatic brain injury, post-traumatic stress disorder, Parkinson's disease, Down syndrome, spinocerebellar ataxia, Huntington's disease, radiation therapy-induced neurodegeneration, chronic stress-induced neurodegeneration, and neurodegeneration associated with normal aging or abuse of neuroactive drugs (e.g., alcohol, opioiates, methamphetamine, fencyclidine, and cocaine). Anti-Cx43 antibody

[0063] Inhibition or reduction of Cx43 hemichannel opening can reduce or inhibit the opening of Cx43 hemichannels in cells, thereby treating inflammatory diseases or conditions or neurodegenerative diseases. Thus, anti-Cx43 antibodies can be used as effective agents in anti-inflammatory and / or neuroprotective therapies.

[0064] In certain embodiments, the anti-Cx43 antibody can be a monoclonal antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-Cx43 antibody can be a modified antibody, such as a chimeric or humanized antibody derived from a mouse anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody is an antibody or an antigen-binding fragment thereof that binds to an epitope present in the human Cx43 protein, such as an extracellular loop or a portion thereof.

[0065] Exemplary anti-Cx43 antibodies can have one or more of the following CDR sequences: Heavy chain: CDR1 (SEQ ID NO: 1): GYTFTSYY CDR2 (SEQ ID NO: 2): INPSNAGT CDR3 (SEQ ID NO: 3): TREGNPYYTMNY Light chain: CDR1 (SEQ ID NO: 4): QSLLNSGNQKTY CDR2 (SEQ ID NO: 5): GAS CDR3 (SEQ ID NO: 6): QNDHSYPFT

[0066] In some embodiments, unexpectedly, antibodies having the above CDR sequences have been found to exhibit improved binding affinity and / or antibody stability compared to the antibodies disclosed in PCT Publication Nos. WO2015 / 027120 and WO2017 / 147561. Without wishing to be bound by any theory, the mutation from "NG" to "NA" in the heavy chain CDR2 is thought to be able to reduce deamidation. In particular, deamidation of antibodies in the CDR region can cause changes in binding affinity, antibody degradation, and changes in charge variants, which can affect antibody function and increase the production cost of the antibody. Therefore, the CDRs disclosed herein provide improved binding affinity and antibody stability, resulting in advantageous technical effects compared to the CDRs disclosed in PCT Publication Nos. WO2015 / 027120 and WO2017 / 147561.

[0067] Monoclonal antibodies can be humanized and optimized to increase antibody drugability and / or developability, for example, using CDR grafting, germline modeling, and 3-D structure analysis. In some embodiments, after humanization, the anti-Cx43 antibody can have one or both of the following variable domains: Heavy chain variable domain (SEQ ID NO: 7): EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGGINPSNAGTNFNEKFKNRATLTVDKSTSTAYMELSSLRSEDTAVYYCTREGNPYYTMNYWGQGTLVTVSS Light chain variable domain (SEQ ID NO: 8): DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKTYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPFTFGQGTKLEIK

[0068] In selected embodiments, the anti-Cx43 antibody can have a variable domain fused to the constant region of human IgG1, IgG2, or IgG4, which can include one or more mutations as needed, for example. In some embodiments, the mutations can be designed to reduce or minimize the cytotoxic effector function of the antibody while maintaining binding affinity and antibody stability. For example, the anti-Cx43 antibody can have one or more of the following heavy chain sequences (the bolded portions correspond to the variable domain and the non-bolded portions correspond to the constant region):

Chemical formula

Chemical formula

Chemical formula

[0069] In some embodiments, the anti-Cx43 antibody may have the following light chain sequence (the bolded portions correspond to the variable domains and the non-bolded portions correspond to the constant regions):

Chemical formula

[0070] In yet another embodiment, the anti-Cx43 antibody may comprise a mixture or cocktail of two or more anti-Cx43 antibodies, each of which binds to the same or different epitopes on Cx43.

[0071] In some embodiments, bispecific antibodies can be made in which at least one of the specificities is an anti-Cx43 antibody or an antigen-binding fragment thereof as disclosed herein. The other specificity can be directed to another target associated with the inflammatory or neurodegenerative disease to be treated.

[0072] In one aspect, there is provided the use of a Cx43 ligand for the manufacture of a medicament in the treatment of an inflammatory or neurodegenerative disease. In another aspect, there is provided a method of suppressing inflammation or neurodegeneration in a patient, the method comprising administering to the patient an effective amount of a Cx43 ligand. Preparation of anti-Cx43 antibody

[0073] Anti-Cx43 antibodies can be made using a variety of methods generally known in the art. For example, phage display technology can be used to screen a human antibody library to produce fully human monoclonal antibodies for therapy. High-affinity conjugates can be considered candidates for neutralization assays. Alternatively, conventional monoclonal approaches can be used in which mice or rabbits are immunized with human proteins, candidate conjugates are identified and tested, and the heavy and light chain combination sites are grafted onto human antibody coding sequences to ultimately produce humanized antibodies.

[0074] Antibodies typically contain two identical pairs of polypeptide chains, with each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each chain typically contains about 100-110 or more variable regions of amino acids that are typically involved in antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that is typically involved in effector functions. Each variable region of the heavy and light chains typically exhibits the same general structure, including four relatively conserved framework regions (FRs) linked to three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, and the alignment can enable binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain typically follows the definitions of Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.), Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917, or Chothia et al., 1989, Nature 342:878-883.

[0075] Antibodies have become useful and interesting as pharmaceuticals through the development of monoclonal antibodies. Monoclonal antibodies are produced using any method that produces antibody molecules by a continuous cell line in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma method of Kohler et al. (1975, Nature 256:495-497), and the human B cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63).

[0076] Monoclonal antibodies can be modified for use as therapeutic agents. One example is a "chimeric" antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain is derived from another species or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass. Another example is fragments of such antibodies as long as they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; and Morrison et al. (1985), Proc. Natl. Acad. Sci. USA 81:6851-6855. A related development is the "CDR grafted" antibody, which contains one or more complementarity determining regions (CDRs) derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the antibody chain is derived from another species or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass.

[0077] Another development is the "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art (see U.S. Pat. Nos. 5,585,089, and 5,693,762; see also Cecile Vincke et al. J. Biol. Chem. 2009;284:3273-3284 for humanization of llama antibodies). Generally, a humanized antibody is produced by a non-human animal and then typically certain amino acid residues from the non-antigen recognition portion of the antibody are modified to be homologous to the residues in the corresponding human antibody of the same isotype. Humanization can be carried out, for example, using methods described in the art (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536) by substituting at least a portion of the rodent variable region with the corresponding region of a human antibody.

[0078] In recent years, human antibodies (“fully human antibodies”) have been produced without exposing the antigen to humans. Such antibodies are produced by immunizing transgenic animals (e.g., mice) capable of producing a human antibody repertoire in the absence of endogenous mouse immunoglobulin production with an antigen conjugated to a carrier as needed (typically having at least 6 contiguous amino acids). See, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of these methods, transgenic animals are produced by disabling the endogenous mouse immunoglobulin locus encoding the mouse heavy and light chain immunoglobulin chains therein and inserting into their genome a locus encoding human heavy and light chain proteins. Next, partially modified animals with incomplete modification complementation are crossed to obtain animals with all of the desired modifications of the immune system. When immunogens are administered, these transgenic animals produce antibodies that are immunospecific for these antigens and have human (not mouse) amino acid sequences that include variable regions. See PCT Publication Nos. WO96 / 33735 and WO94 / 02602, which are incorporated by reference. Additional methods are described in U.S. Patent No. 5,545,807, PCT Publication Nos. WO91 / 10741, WO90 / 04036, and European Patent Nos. 546073 B1 and 546073 A1, which are incorporated by reference. Human antibodies can also be produced by expression of recombinant DNA in host cells or expression in hybridoma cells described herein.

[0079] In some embodiments, phage display technology may be used to screen for therapeutic antibodies. In phage display, an antibody repertoire can be displayed on the surface of filamentous bacteriophage, and the constructed library may be screened for phage that bind to an immunogen. Antibody phage is based on genetic manipulation of bacteriophage and repeated rounds of antigen-guided selection and phage propagation. This technique enables in vitro selection of Cx43 monoclonal antibodies. Phage display begins with antibody-library preparation, then ligation of variable heavy chain (VH) and variable light chain (VL) PCR products into a phage display vector, and ends with analysis of clones of monoclonal antibodies. VH and VL PCR products representing the antibody repertoire are ligated into a phage display vector (e.g., phagemid pComb3X), which is engineered to express VH and VL as a single-chain Fv (scFv) fused to the pIII minor capsid protein of filamentous bacteriophage originally derived from M13 bacteriophage in Escherichia coli. However, the phage display vector pComb3X does not have all the other genes necessary to encode a complete bacteriophage in E. coli. For those genes, helper phage is added to E. coli and transformed with the phage display vector library. The result is a phage library having vectors each expressing a Cx43 monoclonal antibody on its surface and having their respective nucleotide sequences therein. Phage display can also be used to produce the Cx43 monoclonal antibody itself (not bound to the phage capsid protein) in a particular strain of E. coli. Additional cDNA is engineered after the VL and VH sequences in the phage display vector to enable characterization and purification of the produced mAb. Specifically, the recombinant antibody may have a hemagglutinin (HA) epitope tag and polyhistidine to enable easy purification from solution.

[0080] Diverse antibody phage libraries are produced from approximately 10 8 independent E. coli transformants infected with helper phage. Using biopanning, the library is screened for phage that bind to the above immunogenic sequence or fragments thereof through the expressed surface of monoclonal antibodies. Cyclic panning enables the isolation of potentially very rare antigen-binding clones and consists of multiple rounds of phage binding to antigen (fixed on ELISA plates or in solution on cell surfaces), washing, elution, and reamplification of the phage conjugates in E. coli. At each round, specific conjugates are selected from the pool by washing away unbound conjugates and selectively eluting the bound phage clones. After 3 or 4 rounds, very specific binding of phage clones through the Cx43 monoclonal antibody on their surface is characteristic of the directional selection for the immobilized immunogen.

[0081] Another method is to add a C-terminal His tag suitable for purification by affinity chromatography to the above immunogenic sequence. The purified protein can be inoculated into mice together with a suitable adjuvant. Monoclonal antibodies produced in hybridomas can be tested for binding to the immunogen and positive conjugates can be screened as described in the assays herein.

[0082] Fully human antibodies can also be produced from phage display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). These processes mimic immunoselection through the display of an antibody repertoire on the surface of filamentous bacteriophage, followed by selection of the phage by its binding to the selected antigen. One such technique is described in PCT Publication No. WO99 / 10494, which is incorporated by reference and describes the isolation of agonistic antibodies that are functionally high affinity for MPL- and msk-receptors using such an approach.

[0083] The nucleotide sequences encoding the antibodies described above can be determined. Thereafter, chimeric, CDR grafted, humanized and fully human antibodies can also be produced by recombinant methods. Nucleic acids encoding the antibodies can be introduced into host cells and expressed using materials and procedures generally known in the art.

[0084] The present disclosure provides antibodies against Cx43. Preferably, the antibody binds to Cx43. In a preferred embodiment, the present disclosure provides nucleotide sequences encoding heavy and light chain immunoglobulin molecules, as well as amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly sequences corresponding to their variable regions. In a preferred embodiment, sequences corresponding to the CDRs, particularly sequences corresponding to CDR1 through CDR3 are provided. In additional embodiments, the present disclosure provides hybridoma cell lines expressing such immunoglobulin molecules, monoclonal antibodies produced therefrom, preferably purified human monoclonal antibodies against human Cx43.

[0085] The CDRs of the light and heavy chain variable regions of the anti-Cx43 antibodies of the present disclosure can be grafted onto framework regions (FRs) from the same or a different species. In certain embodiments, the CDRs of the light and heavy chain variable regions of the anti-Cx43 antibodies may be grafted onto consensus human FRs. To generate the consensus human FRs, the FRs from several human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. The FRs of the anti-Cx43 heavy or light chain can be exchanged with FRs from different heavy or light chains. Rare amino acids in the FRs of the anti-Cx43 antibody heavy and light chains typically are not exchanged, but the remaining FR amino acids can be exchanged. Rare amino acids are specific amino acids that are at positions where they are not normally found in the FRs. The grafted variable regions from the anti-Cx43 antibodies of the present disclosure can be used with a constant region different from the constant region of the anti-Cx43 antibody. Alternatively, the grafted variable regions are part of a single-chain Fv antibody. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are hereby incorporated by reference for any purpose.

[0086] In some embodiments, the antibodies of the present disclosure can be produced by a hybridoma strain. In these embodiments, the antibodies of the present disclosure bind to Cx43 with a dissociation constant (K D ) of approximately 4 pM to 1 μM. In certain embodiments of the present disclosure, the antibody binds to Cx43 with a K D of less than about 100 nM, less than about 50 nM, or less than about 10 nM.

[0087] In embodiments, the antibodies of the present disclosure are antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype, such as an antibody of the IgG1 isotype. In certain embodiments, the antibody comprises a human kappa or lambda light chain and a human IgG1, IgG2, or IgG4 heavy chain. In embodiments, the variable region of the antibody is ligated to the constant region of an IgG1, IgG2, or IgG4 isotype. In certain embodiments, the variable region of the antibody is ligated to a constant region other than the constant region of an IgG1, IgG2, or IgG4 isotype. In certain embodiments, the antibodies of the present disclosure are cloned for expression in mammalian cells.

[0088] In alternative embodiments, the antibodies of the present disclosure can be expressed in cell lines other than hybridoma cell lines. In these embodiments, the sequences encoding the particular antibody can be used for transformation of a suitable host cell, such as a mammalian cell. According to these embodiments, the transformation can be achieved using any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide into a virus (or viral vector), transducing the virus (or vector) into the host cell, or by transfection procedures known in the art. Such procedures are exemplified in U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, all of which are hereby incorporated by reference herein for any purpose. Generally, the transformation procedures used can depend on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, and direct microinjection of DNA into the nucleus.

[0089] According to certain embodiments of the methods of the present disclosure, a nucleic acid molecule encoding the amino acid sequence of the heavy chain constant region, heavy chain variable region, light chain constant region, or light chain variable region of a Cx43 antibody of the present disclosure is inserted into an appropriate expression vector using standard ligation techniques. In preferred embodiments, the Cx43 antibody heavy chain or light chain constant region is added to the C-terminus of the appropriate variable region and ligated into the expression vector. The vector is typically selected to be functional in the particular host cell used (i.e., the vector is compatible with the host cell machinery such that gene amplification and / or gene expression can occur). For an overview of expression vectors, see Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press. N.Y.

[0090] Typically, an expression vector used in any host cell may contain sequences for plasmid maintenance and for the cloning and expression of exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and one or more selectable marker elements. These sequences are well known in the art.

[0091] The expression vectors of the present disclosure may be constructed from starting vectors such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they may be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those of skill in the art.

[0092] After a vector is constructed and a nucleic acid molecule encoding the light chain or the heavy chain or both the light and heavy chains constituting the anti-Cx43 antibody is inserted into an appropriate site of the vector, the complete vector may be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the selected host cell with the expression vector for the anti-Cx43 antibody can be achieved by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected is, in part, a function of the type of host cell used. These methods and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., supra.

[0093] When cultured under appropriate conditions, the host cell can synthesize the anti-Cx43 antibody, which can then be recovered from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of an appropriate host cell depends on various factors such as the desired expression level, modification of the polypeptide desirable or necessary for activity (e.g., glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

[0094] Mammalian cell lines that can be used as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, human fetal kidney cells (HEK), monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a plurality of other cell lines, but are not limited thereto. In certain embodiments, the cell line may be selected by determining which cell line has a high expression level and produces an antibody having constitutive Cx43 binding properties. In another embodiment, a cell line from the B cell lineage that does not produce its own antibody but has the ability to produce and secrete a heterologous antibody (e.g., mouse myeloma cell lines NS0 and SP2 / 0) may be selected. Epitope mapping and related techniques

[0095] The present disclosure provides anti-Cx43 antibodies that interact with one or more domains of the Cx43 molecule, such as one or more amino acids found within the extracellular loops. The epitope to which the antibody binds can include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) consecutive sequences of one or more amino acids located within one or more extracellular loops. Alternatively or in addition, the epitope can include one or more non-contiguous amino acids (or amino acid sequences) located within one or more extracellular loops (e.g., conformational epitope).

[0096] Using various techniques known to those skilled in the art, it can be determined whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross - blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Other methods include alanine - scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443 - 63), peptide cleavage analysis, crystallization studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Prot. Sci. 9: 487 - 496).

[0097] Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium, followed by binding of the antibody to the deuterium - labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo back - exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids that form part of the protein / antibody interface can retain deuterium and thus exhibit a relatively high mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, which reveals the deuterium - labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267: 252 - 259; Engen and Smith (2001) Anal. Chem. 73: 256A - 265A.

[0098] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method of classifying multiple monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see U.S. Patent Application Publication No. 2004 / 0101920, which is specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is clearly different from or partially overlapping with the epitope represented by another category. This technique enables rapid filtering of genetically identical antibodies so that the characterization focus can be on genetically different antibodies. When applied to hybridoma screening, MAP may facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. The antibodies of the present invention may be sorted using MAP into groups of antibodies that bind to different epitopes.

[0099] The present disclosure provides anti-Cx43 antibodies that bind to the same epitope or a portion of an epitope. Similarly, the present disclosure also includes anti-Cx43 antibodies that compete with the specific exemplary antibodies described herein with respect to binding to Cx43 or a fragment thereof. For example, the present disclosure includes anti-Cx43 antibodies that cross-compete with one or more antibodies obtained from the antibodies described herein with respect to binding to Cx43.

[0100] Whether an antibody binds to the same epitope as or competes with respect to binding to the reference anti-Cx43 antibody can be readily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the reference anti-Cx43 antibody of the present invention, the reference antibody can be bound to Cx43 or a peptide under saturation conditions. Next, the ability of the test antibody to bind to the Cx43 molecule is evaluated. If the test antibody can bind to Cx43 after saturation binding by the reference anti-Cx43 antibody, it can be concluded that the test antibody binds to an epitope different from that of the reference anti-Cx43 antibody. On the other hand, if the test antibody cannot bind to Cx43 after saturation binding by the reference anti-Cx43 antibody, the test antibody may bind to the same epitope as the epitope to which the reference anti-Cx43 antibody of the present disclosure binds.

[0101] To determine whether an antibody competes with respect to binding to the reference anti-Cx43 antibody, the above binding methodology can be carried out in two directions. In the first direction, the reference antibody is bound to Cx43 under saturation conditions, and then the binding of the test antibody to the Cx43 molecule is evaluated. In the second direction, the test antibody is bound to the Cx43 molecule under saturation conditions, and then the binding of the reference antibody to the Cx43 molecule is evaluated. In either direction, if only the first (saturated) antibody can bind to the Cx43 molecule, it is concluded that the test antibody and the reference antibody compete with respect to binding to Cx43. As will be appreciated by those skilled in the art, an antibody that competes with respect to binding to a reference antibody does not necessarily bind to the same epitope as the reference antibody, but can sterically hinder the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0102] When each competitively inhibits (blocks) the other's binding to an antigen, the two antibodies bind to the same or overlapping epitopes. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the other's binding by at least 50%, but preferably 75%, 90%, or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0103] Next, additional routine experiments (e.g., peptide mutagenesis and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0104] In various embodiments, provided herein are antibodies that bind to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope can comprise one or more amino acids selected from the group consisting of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. In one embodiment, the epitope consists of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. In some embodiments, the epitope can comprise all 10 amino acids of SEQ ID NO: 19. In certain embodiments, the epitope consists of all 10 amino acids of SEQ ID NO: 19. Pharmaceutical Compositions and Their Use

[0105] In another aspect, provided is a pharmaceutical composition that can be used in the methods disclosed herein, i.e., a pharmaceutical composition for inhibiting the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating an inflammatory disease or condition, or a neurodegenerative disease.

[0106] In some embodiments, the pharmaceutical composition comprises a Cx43 ligand and a pharmaceutically acceptable carrier. The Cx43 ligand can be formulated into the pharmaceutical composition together with the pharmaceutically acceptable carrier. Additionally, the pharmaceutical composition can include instructions for use of the composition for treating a patient, e.g., preferably for treating an inflammatory disease or condition or a neurodegenerative disease, to inhibit the opening of Cx43 hemichannels in astrocytes or osteocytes.

[0107] In one embodiment, the Cx43 ligand can be an anti-Cx43 antibody or an antigen-binding fragment thereof.

[0108] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and other excipients that are physiologically compatible. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, the active compound, e.g., a small molecule or a biological agent, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

[0109] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, as well as conventional excipients for preparing tablets, pills, capsules, etc. The use of such media and agents for the formulation of pharmaceutically active substances is known in the art. The use thereof in the pharmaceutical compositions provided herein is contemplated as long as any conventional media or agent is not incompatible with the active compound. Auxiliary active compounds can also be incorporated into the compositions.

[0110] The pharmaceutically acceptable carrier may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0111] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. If necessary, suitable fluidity can be maintained by, for example, the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. In many cases, it may be useful to include in the composition isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride. Sustained absorption of injectable compositions can be brought about by including in the composition agents that delay absorption such as monostearates and gelatin.

[0112] These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifying agents, and dispersing agents.

[0113] Therapeutic compositions typically must be sterile, non-phylogenic, and stable under manufacturing and storage conditions. The compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations.

[0114] A sterile injectable solution can be prepared by incorporating the required amount of the active compound in a suitable solvent with one or a combination of the ingredients enumerated above and then, if necessary, sterilizing, for example, by fine filtration. Generally, a dispersion is prepared by incorporating the active compound in a sterile medium that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder for preparing a sterile injectable solution, the method of preparation includes vacuum drying and freeze-drying (lyophilization) that yields a powder of the active ingredient plus any additional desired ingredients from its pre-filter sterilized solution. The active agent may be mixed under sterile conditions with an additional pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0115] Prevention of the presence of microorganisms can be ensured both by the sterilization procedures described above and by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenolsorbic acid, etc. Similarly, it may be desirable to include in the composition isotonic agents, for example, sugars, sodium chloride, etc. In addition, sustained absorption of injectable pharmaceutical dosage forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0116] A pharmaceutical composition containing a Cx43 ligand can be administered alone or in combination therapy. For example, the combination therapy can include a composition provided herein that contains a Cx43 ligand and at least one or more additional therapeutic agents, for example, an anti-inflammatory agent known in the art.

[0117] Adjust the dosing regimen to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the exigency of the therapeutic situation.

[0118] Exemplary dosage ranges for the administration of the antibody include: 10 - 1000 mg (antibody) / kg (patient body weight), 10 - 800 mg / kg, 10 - 600 mg / kg, 10 - 400 mg / kg, 10 - 200 mg / kg, 30 - 1000 mg / kg, 30 - 800 mg / kg, 30 - 600 mg / kg, 30 - 400 mg / kg, 30 - 200 mg / kg, 50 - 1000 mg / kg, 50 - 800 mg / kg, 50 - 600 mg / kg, 50 - 400 mg / kg, 50 - 200 mg / kg, 100 - 1000 mg / kg, 100 - 900 mg / kg, 100 - 800 mg / kg, 100 - 700 mg / kg, 100 - 600 mg / kg, 100 - 500 mg / kg, 100 - 400 mg / kg, 100 - 300 mg / kg, and 100 - 200 mg / kg. Exemplary dosing schedules include once every 3 days, once every 5 days, once every 7 days (i.e., once a week), once every 10 days, once every 14 days (i.e., once every 2 weeks), once every 21 days (i.e., once every 3 weeks), once every 28 days (i.e., once every 4 weeks), and once a month.

[0119] It may be advantageous to formulate the parenteral composition into unit dosage forms for ease of administration and uniformity of dosage. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for the patient to be treated, each unit containing a predetermined quantity of the active agent calculated to produce the desired therapeutic effect in association with any necessary pharmaceutical carrier. The specifications for the unit dosage form are directly dependent upon and are determined by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the art of mixing such active compounds for the treatment of sensitivity in individuals.

[0120] The actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein can vary in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. As used herein in the context of administration, "parenteral" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injections and infusions.

[0121] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral (i.e., via the digestive tract) and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injections and infusions. Intravenous injections and infusions are often (but not limited to) used for the administration of antibodies.

[0122] When the agents provided herein are administered to humans or animals as pharmaceuticals, they can be administered alone or in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition containing from 0.001% to 90% (e.g., from 0.005% to 70%, e.g., from 0.01% to 30%) of the active ingredient.

[0123] In one aspect, the improved efficacy of the combinations according to the present disclosure can be demonstrated by achieving a therapeutic synergistic effect.

[0124] The term "therapeutic synergy" is used when the combination of two products at a given dose is more effective than the best effect of each of the two products alone at the same dose. In one example, therapeutic synergy can be evaluated by comparing the combination to the best single agent using an estimate obtained from a two-way analysis of variance of repeated measurements (e.g., a time factor) on a desirable parameter.

[0125] The term "additive" refers to a combination of two or more products at a given dose being equally effective as the sum of the effectiveness obtained for each of the two or more products, while the term "supra-additive" refers to the combination being more effective than the sum of the effectiveness obtained for each of the two or more products.

[0126] Similarly, provided herein is a method of inhibiting the opening of Cx43 hemichannels in a cell, preferably for treating an inflammatory disease or condition, or a neurodegenerative disease, the method comprising administering to a subject in need thereof any one or more of the anti-Cx43 antibodies disclosed herein.

[0127] In various embodiments, the methods disclosed herein may comprise administering to a subject an effective amount of an anti-Cx43 antibody or an antigen-binding fragment thereof. Generally, the effective amount can be administered therapeutically and / or prophylactically.

[0128] Treatment can suitably be administered to a subject, particularly a human subject having, suffering from, susceptible to, or at risk of developing such a disease. The determination that such subjects are "at risk" can be made by a diagnostic test or any objective or subjective determination (e.g., genetic testing, enzyme or protein markers, family history, etc.) based on the opinion of the subject or a healthcare provider. Identifying subjects in need of such treatment can be at the discretion of the subject or a medical professional and can be subjective (e.g., an opinion) or objective (e.g., measurable by a test or diagnostic method). Administration of the formulation

[0129] The formulations of the present disclosure, including but not limited to the reconstituted formulations and liquid formulations, are administered to a mammal, preferably a human, in need thereof, together with an anti-Cx43 antibody, according to known methods, for example, as an intravenous bolus administration or by continuous infusion over a period of time, or by intramuscular, intracerebroventricular, intraperitoneal, intrathecal, subcutaneous, intra-articular, intrasynovial, intramedullary, oral, topical application, or inhalation routes.

[0130] In embodiments, the formulation is administered to a mammal by intravenous or subcutaneous (i.e., under the skin) administration. For such purposes, the formulation can be injected using a syringe. However, other devices for administration of the formulation are available, such as injection devices (e.g., INJECT-EASE™ and GENJECT™ devices); injection pens (e.g., GENPEN™); auto-injector devices, needle-free devices (e.g., MEDIJECTOR™ and BIOJECTOR™); and subcutaneous patch delivery systems.

[0131] In certain embodiments, the present disclosure is directed to a kit for a single-dose administration unit. Such a kit includes a container of an aqueous formulation of a therapeutic protein or antibody, including both single-chamber or multi-chamber pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0132] The appropriate dosage (the "therapeutically effective amount") of the protein depends, for example, on the condition being treated, the severity and course of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history, and response to the anti-Cx43 antibody, the format of the formulation used, and the discretion of the attending physician. The anti-Cx43 antibody is suitably administered to a patient in one or a series of treatments and can be administered to the patient at any time after diagnosis. The anti-Cx43 antibody can be administered as a single treatment or in combination with other drugs or treatments useful for treating the disease.

[0133] In the case of an anti-Cx43 antibody, the initial candidate dosage can range from about 0.1 to 100 or 1 to 20 mg / kg with respect to administration to a patient, and these can take the form of one or more individual administrations. However, other dosing regimens may be useful. The progression of such treatment is readily monitored by conventional techniques.

[0134] According to certain embodiments of the present disclosure, multiple doses of an anti-Cx43 antibody (or a pharmaceutical composition comprising a combination of an anti-Cx43 antibody and any of the additional therapeutic agents mentioned herein) may be administered to a subject over a period of time. The method according to this aspect of the present disclosure includes continuously administering multiple doses of the anti-Cx43 antibody of the present disclosure to a subject. As used herein, "administering continuously" means that each dose of the anti-Cx43 antibody is administered to the subject at different times, for example, on different days at a given interval (e.g., several hours, several days, several weeks, several months). The present disclosure includes methods that include continuously administering a single initial dose of an anti-Cx43 antibody to a patient, followed by administering one or more second doses of the anti-Cx43 antibody, and, if necessary, continuously administering one or more third doses of the anti-Cx43 antibody. The anti-Cx43 antibody can be administered at a dosage of 0.1 mg / kg to about 100 mg / kg.

[0135] The terms "first dose", "second dose", and "third dose" refer to the chronological order of administration of the anti-Cx43 antibodies of the present disclosure. Thus, the "first dose" is the dose administered first in the treatment regimen (also referred to as the "baseline dose"), the "second dose" is the dose administered after the first dose, and the "third dose" is the dose administered after the second dose. The first, second, and third doses all contain the same amount of anti-Cx43 antibody, but generally may differ from each other with respect to the dosing frequency. However, in certain embodiments, the amounts of anti-Cx43 antibody contained in the first, second, and / or third doses may differ from each other during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of the treatment regimen, and then subsequent doses are administered less frequently (e.g., "maintenance dose").

[0136] In certain exemplary embodiments of the present disclosure, each second and / or third dose is administered 1 to 26 weeks after the immediately preceding dose (e.g., 1 week later, 1 1 / 2 weeks later, 2 weeks later, 2 1 / 2 weeks later, 3 weeks later, 3 1 / 2 weeks later, 4 weeks later, 4 1 / 2 weeks later, 5 weeks later, 5 1 / 2 weeks later, 6 weeks later, 6 1 / 2 weeks later, 7 weeks later, 7 1 / 2 weeks later, 8 weeks later, 8 It is administered (after 1 / 2 week, 9 weeks, 9 1 / 2 weeks, 10 weeks, 10 1 / 2 weeks, 11 weeks, 11 1 / 2 weeks, 12 weeks, 12 1 / 2 weeks, 13 weeks, 13 1 / 2 weeks, 14 weeks, 14 1 / 2 weeks, 15 weeks, 15 1 / 2 weeks, 16 weeks, 16 1 / 2 weeks, 17 weeks, 17 1 / 2 weeks, 18 weeks, 18 1 / 2 weeks, 19 weeks, 19 1 / 2 weeks, 20 weeks, 20 1 / 2 weeks, 21 weeks, 21 1 / 2 weeks, 22 weeks, 22 1 / 2 weeks, 23 weeks, 23 1 / 2 weeks, 24 weeks, 24 1 / 2 weeks, 25 weeks, 25 1 / 2 weeks, 26 weeks, 26 1 / 2 weeks, or later). As used herein, the phrase "immediately preceding dose" means, in a series of multiple administrations, the dose of the anti-Cx43 antibody that is administered to the patient immediately prior to the administration of the very next dose in the order in which no dose intervenes therebetween.

[0137] The method according to this aspect of the disclosure may include administering to the patient any number of second and / or third doses of the anti-Cx43 antibody. For example, in certain embodiments, only one second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only one third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0138] In embodiments comprising a plurality of second doses, each second dose can be administered at the same frequency as the other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks or 1 to 2 months after the previous dose. Similarly, in embodiments comprising a plurality of third doses, each third dose can be administered at the same frequency as the other third doses. For example, each third dose can be administered to the patient 2 to 12 weeks after the previous dose. In certain embodiments of the present disclosure, the frequency of administering the second and / or third doses to the patient can vary over the course of the treatment regimen. The dosing frequency can also be adjusted by the physician during the course of treatment as needed for each individual patient following clinical examination.

[0139] The present disclosure includes a dosing regimen in which 1 to 10 or 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses administered to the patient at a less frequent rate. For example, in accordance with this aspect of the present disclosure, when the loading dose is administered, for example, once a month (e.g., administering 2, 3, 4, or more loading doses once a month), the maintenance dose can be administered to the patient once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks, etc.

Examples

[0140] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the present disclosure. (Example 1) Antibody binding affinity

[0141] The optimized antibody sequences were tested for binding affinity to Cx43 according to the following protocol.

[0142] The equilibrium dissociation constant (K DThe value) was determined using a biosensor (Octet HTX) assay based on a real-time biolayer interferometer. Anti-Cx43 antibody was captured using an Octet streptavidin (SA) biosensor coated with biotinylated antigen (0.01 ug / mL). The loaded sensors were immersed in serial two-fold dilutions of a purified control antibody starting from 100 nM. All binding tests were performed in assay buffer (PBS (pH 7.2) with 0.1% BSA, 0.02% Tween®-20) at 25 °C with the plate shaken at 1000 rpm. The association (K a ) and dissociation (K d ) rate constants were processed using Scrubber 2.0c curve fitting software and determined by fitting the data to a 1:1 binding model. The binding dissociation equilibrium constant (K D ) was calculated from the kinetic rate constants as follows: K D (M) = Kd / Ka

[0143] The results of the binding affinity (Table 1) generally show that the binding affinity was at least maintained and, in many cases, unexpectedly enhanced. Table 1. Binding Affinities of Various Antibodies

Table 1

[0144] (Example 2) Fc Receptor Binding Analysis

[0145] Fc effector functions are mediated by the binding of Fc to receptors. Receptors include FcRI, FcRIIa, FcRIIb, FcRIIIa, FcRIIIb, C1q, and FcRn. In general, it is desirable to reduce the binding affinity for most Fc receptors other than FcRn in order to minimize potential in-vivo toxicity while maintaining antibody half-life. The following surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) protocols were used to test different Fc receptor bindings to various antibodies. A. FcRI Binding

[0146] Experiment: Biacore 8K Chip: CM5

[0147] (1) Immobilization An activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor chip was activated with the mixture for 420 seconds. 30 μg / mL of THE (trademark) His-tag antibody in 10 mM NaAc (pH 4.5) was injected into channels 1-8 at a flow rate of 30 μL / min for 400 seconds. The chip was inactivated with 1 M ethanolamine HCl (GE).

[0148] (2) Ligand Capture and Analyte Running 2 μg / mL CD64 in running buffer (1×HBS-EP+) was injected into Fc2 of channels 1-4 at a flow rate of 10 μL / min for 30 seconds. Six concentrations (40, 20, 10, 5, 2.5, and 1.25 nM) of analytes Ab#C, Ab#G, and running buffer were sequentially injected into Fc1-Fc2 of channels 1-4 at a flow rate of 30 μL / min for 180 binding phases and then for 400 dissociation phases. The running of analytes according to ligand capture and analyte concentration was repeated in ascending order for 6 cycles. 10 mM glycine pH 1.5 as the regeneration buffer was injected after each dissociation phase. 2 μg / mL CD64 in running buffer (1×HBS-EP+) was injected into Fc2 of channels 1-6 at a flow rate of 10 μL / min for 30 seconds. Eight concentrations (10240, 5120, 2560, 1280, 640, 320, 160, and 80 nM) of analyte Abs #K, #O, #S, and running buffer were injected into Fc1-Fc2 of channels 1-6 in order at a flow rate of 30 μL / min between 60 binding phases and then between 90 dissociation phases. The capture of the ligand and the running of the analyte according to the analyte concentration were repeated 8 cycles in ascending order. 10 mM glycine pH 1.5 as the regeneration buffer was injected after each dissociation phase.

[0149] (3) Regeneration The chip was regenerated with 10 mM glycine pH 1.5.

[0150] (4) Data analysis The surface channel Fc1 without the capture ligand was used as a control surface for subtracting the reference. The final data for each interaction were estimated from the reference channel and buffer channel data. The experimental data of the binding of Abs #C, #G to CD64 were fitted in a 1:1 binding mode. To enable a better fit, the 10240 nM curves of analyte Abs #K, #O, #S were removed. The relative experimental data were fitted by steady-state affinity and are shown in Table 2 below. Table 2. FCRI binding

Table 2-1

Table 2-2

[0151] All antibodies showed low or no FCRI binding, which is advantageous. B. Binding to FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb Experiment: Biacore 8K Chip: CM5

[0152] (1) Immobilization The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor chip was activated with the mixture for 420 seconds. 30 μg / mL of THE (trademark) His-tag antibody in 10 mM NaAc (pH 4.5) was injected into channels 1 - 8 at a flow rate of 30 μL / min for 400 seconds. The chip was inactivated with 1 M ethanolamine HCl (GE).

[0153] (2) Ligand Capture and Analyte Running 1 μg / mL of FcγRIIa, FcγRIIb, FcγRIIIa, or FcγRIIIb in running buffer (1×HBS - EP+) was injected into Fc2 of channels 1 - 8 at a flow rate of 10 μL / min for 15 seconds. Analytes were injected into channels 1 - 8 respectively. A series of analyte concentrations (see Table 3 below) were monitored during a 60 - second binding phase at a flow rate of 30 μL / min, followed by a 90 - second dissociation phase. 10 mM glycine pH 1.5 as the regeneration buffer was injected after each dissociation phase. Table 3. Analyte Concentrations

Table 3

[0154] (3) Regeneration The chip was regenerated with 10 mM glycine pH 1.5.

[0155] (4) Data Analysis The surface channel Fc1 without the capture ligand was used as a control surface for subtracting the reference. The final data for each interaction was estimated from the reference channel and buffer channel data. The experimental data for antibody binding to FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb was fitted by the steady - state affinity mode and is shown in Table 4 below. Table 4. Binding of FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb

Table 4

[0156] All antibodies showed low or no binding to FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, which is advantageous. C. Binding to FcRn Experiment: Biacore 8K Chip: CM5

[0157] (1) Buffer exchange The buffer of human FcRn was exchanged to the running buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 150 mM NaCl, 0.05% Tween® 20, pH 6.0) using a desalting column according to the instruction manual. The concentration was determined by Nanodrop.

[0158] (2) Immobilization An activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection. The CM5 sensor chip was activated with the mixture at a flow rate of 10 μL / min for 420 s. Then, 5 μg / mL of the antibody in 10 mM NaAc (pH 5.5) was injected into Fc2 of channels 1 - 8 at a flow rate of 10 μL / min for 60 s each. The relative Fc1 was blocked. The chip was inactivated with 1 M ethanolamine HCl (GE) at a flow rate of 10 μL / min for 420 s.

[0159] (2) Running of the analyte Analyte FcRn was injected into channels 1 - 8 respectively. Eight concentrations of FcRn (0, 93.75, 187.5, 375, 750, 1500, 3000, and 6000 nM) were monitored during a 60 - s binding phase at a flow rate of 30 μL / min, followed by a 90 - s dissociation. After each cycle of interaction analysis, the sensor chip surface was regenerated with 1×PBS (pH 7.4) at a flow rate of 10 μL / min for 30 s.

[0160] (3) Regeneration The chip was regenerated with 1×PBS (pH 7.4).

[0161] (4) Data analysis The surface channel Fc1 without the immobilized antibody was used as a control surface for subtracting the reference. The final data for each interaction was estimated from the reference channel and buffer channel data. The experimental data was fitted by the steady-state affinity mode and is shown in Table 5 below. Table 5. FcRn binding

Table 5

[0162] All antibodies showed similar FcRn binding, which is desirable. D. Binding to C1q by ELISA

[0163] Plates (Nunc) were coated overnight at 4°C with 3 μg / mL antibody. After blocking and washing, C1q was titrated stepwise in half-log in blocking buffer (600, 189.75, 60.01, 18.98, 6.00, 1.90, 0.60, 0.19, 0.06, and 0.02 μg / mL) and incubated for 2 hours at room temperature. Next, the plates were washed and then incubated for 1 hour with the secondary antibody goat anti-human C1q Ab-HRP. After washing, the TMB substrate was added and the interaction was stopped with 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) and is shown in Table 6 below. Table 6. C1q binding

Table 6

[0164] All antibodies showed low or no C1q binding, which is advantageous. (Example 3) Epitope mapping

[0165] To reconstruct the epitope of the target molecule, a library of peptide-based epitope mimics was synthesized using solid-phase Fmoc synthesis. An amino-functionalized polypropylene support was obtained by grafting a proprietary hydrophilic polymer formulation and then reacting with Boc-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) together with N-hydroxybenzotriazole (HOBt), followed by cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on the amino-functionalized solid support by a custom-modified JANUS liquid handling station (Perkin Elmer) using standard Fmoc-peptide synthesis.

[0166] The synthesis of structural mimics was performed using the Chemically Linked Peptides on Scaffolds (CLIPS) technology. The CLIPS technology enables the structuring of peptides into single loops, double loops, triple loops, sheet folds, helical folds, and combinations thereof. The CLIPS template is coupled to cysteine residues. The side chains of multiple cysteines in the peptide are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (volume / volume)). This solution is added to the peptide array. The CLIPS template binds to the side chains of the two cysteines present in the solid-phase bound peptide of the peptide array (a 455-well plate with 3 μL wells). The peptide array is completely covered in the solution and gently shaken in the solution for 30 - 60 minutes. Finally, the peptide array is washed thoroughly with an excess of H2O and sonicated in a lysis buffer containing 1% SDS / 0.1% 2,2’-(ethylenedioxy)diethanethiol in PBS (pH 7.2) at 70 °C for 30 minutes and then sonicated in H2O for an additional 45 minutes. Peptides with T3 CLIPS were prepared similarly with three cysteines.

[0167] Different peptide sets were synthesized according to the following design. Note that in some cases, the actual order of the peptides on the mini - cards was randomized.

Table 7

[0168] The binding of antibodies to each of the synthesized peptides was tested by ELISA. The peptide array was incubated with the primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with a 1 / 1000 dilution of the appropriate antibody - peroxidase conjugate (SBA; goat anti - human HRP conjugate, Southern Biotech) for 1 hour at 25°C. After washing, the peroxidase substrate 2,2’ - azino - di - 3 - ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3 percent H2O2 were added. After 1 hour, the color development was measured. The color development was quantified by a charge - coupled device (CCD) camera and an image - processing system.

[0169] The values obtained from the CCD camera are in the range of 0 - 3000 mAU, similar to a standard 96 - well ELISA reader. The results are quantified and stored in the institute's database. Sometimes, the wells contain air bubbles, which can lead to false - positive values. So, the cards are manually inspected, and any values caused by air bubbles are scored 0.

[0170] To confirm the quality of the synthesized peptides, different sets of positive and negative control peptides were synthesized in parallel. These were screened with commercially available antibodies 3C9 and 57.9 (see Posthumus et al. (1990) J. Virol. 64:3304 - 3309).

[0171] A graphical overview of the complete data set is shown in Figure 1. Here, the box - and - whisker plots represent each data set, showing the mean ELISA signal, the distribution within each data set, and the outliers. Depending on the experimental conditions (amount of antibody, blocking strength, etc.), ELISA data with different distributions are obtained.

[0172] The antibodies were tested under high stringency conditions and binding to the peptides arrayed occurred (Figures 2 and 3). Each of the two peptide sets was analyzed individually.

[0173] From the analysis of the data recorded for substitution variants of the lead sequence FLSRPTEKTI, since most substitutions at positions R4, P5, E7, K8, and I10, except for R4K, reduced the signal intensity (Figure 2), it was suggested that these positions are important for antibody binding.

[0174] From the analysis of the data recorded for cleavage variants of the lead sequence FLSRPTEKTI, it was shown that the C-terminus of the sequence is essential for binding to occur (Figure 3), and the presence of at least 5 residues that do not tolerate substitution is essential.

[0175] In summary, antibodies were tested against a peptide array composed of two types of peptide variants derived from the lead sequence FLSRPTEKTI, namely single-residue mutants and cleavage variants. The antibodies produced detectable binding under high stringency conditions. Antibody binding was sensitive to substitutions at residues R4, P5, E7, K8, and I10 and showed preference for the C-terminal portion of the lead sequence. (Example 4) Stability of the antibody

[0176] The stability of the antibody is an important factor affecting development, efficacy, production cost, etc. After sequence optimization, important stability parameters were evaluated. The species distribution profiles of various antibodies under acidic and thermal conditions were tested. All the antibodies showed improved stability.

[0177] A. SE-UPLC (Size Exclusion Ultra Performance Liquid Chromatography) Formulation: PBS, pH 6.5 or 7.2 Concentration (mg / mL): 5.28, 5.13, 5.00, 5.17, 5.01, 5.26, 4.92, 5.04, 4.99, 5.12 (all approximately 5 mg / mL) Conditions: room temperature, after acid treatment, stored at 4°C for 1 week, or stored at 40°C for 1 week 2 μL of the sample was injected into an ACQUITY UPLC Protein BEH SEC 200, 1.7 μm, 4.6×150 mm column at a flow rate of 0.3 mL / min for 10 minutes. A mobile phase of 50 mM sodium phosphate, 500 mM NaCl, pH 6.2 was used. All antibodies showed the desired stability under various pH, heat, and storage conditions.

[0178] B.rCE - SDS (Reducing Capillary Electrophoresis - Sodium Dodecyl Sulfate) Formulation: PBS, pH 6.5, 7.2, 6.2 Concentration (mg / mL): 0.5 Conditions: room temperature, after acid treatment, stored at 4°C for 1 week or stored at 40°C for 1 week The sample was prepared in reducing labeling buffer and then submitted to a LabChip GXII system (PerkinElmer). All antibodies showed the desired stability under various pH, heat, and storage conditions. (Example 5) Assay for hemichannel opening A. In vitro assay

[0179] The antibodies disclosed herein can be tested in vitro for their effect on hemichannel opening or blockade, for example using a dye uptake assay. The dye can be a fluorescent tracer dye (e.g., ethidium bromide or lucifer yellow, or Alexa dyes).

[0180] In one example, a fluid flow loop apparatus (FFLA) (parallel plate flow chamber) or a modified version thereof can be used. The FFLA mimics the dynamic fluid microenvironment in bone to generate fluid flow shear stress (FFSS). Cells were cultured in a parallel plate flow chamber and exposed to a constant fluid laminar flow.

[0181] Osteocytes sense the mechanical stress generated by FFSS in the lacunae / canaliculi network of osteocytes. Fluid flow in bone is driven by extravascular pressure as well as the applied cyclic mechanical load on osteocytes, and the maximum physiological load is proposed to be 8 - 30 dyn / cm 2 It is proposed that it is. In certain embodiments, the FFSS levels were within the range of physiological values reported from previous studies measuring fluid flow in bone. The magnitude of the fluid shear stress can be varied by adjusting the column height of the flow loop.

[0182] Assays used to evaluate hemichannel functionality can use fluorescent tracer molecules small enough to pass through the pores of the hemichannel. When the hemichannel is closed, the molecules cannot pass through. When the hemichannel opens, the dye can pass through it and generate fluorescence in the cell, enabling quantification of the fluorescence. Ethidium bromide fluoresces when it binds to DNA. Lucifer yellow fluoresces when it is located inside the cell.

[0183] The dye movement method may involve exposing cells to an extracellular fluorescently permeable tracer. An extracellularly permeable tracer is a molecule that would otherwise remain outside the cell unless some condition increases the permeability of the cell membrane. In certain embodiments, the tracer has a mass of less than 1, 2, or 3 kDa. In other embodiments, the tracer has a net charge. Such permeable tracers include, but are not limited to, the anionic dye Lucifer Yellow (LY; net charge = -1) and the cationic probe ethidium bromide (EtBr; net charge = +1), propidium iodide (PI; net charge = +2). The fluorescence of EtBr is enhanced when bound to DNA, increasing the contrast and allowing for easier identification. In certain embodiments, the extracellular dye is removed after application of various periods or stimuli to open hemichannels and quantify the fluorescence intensity retained in each cell. In certain embodiments, the fluorescence intensity is quantified in a snapshot image.

[0184] Materials used in in vitro assays to test hemichannel opening / blocking include:

[0185] Hemichannel-expressing cells or cell lines. Cells or cell lines expressing various connexin hemichannels can be obtained, isolated, or manipulated using methods and / or expression vectors known in the art.

[0186] Osteocytes: Primary osteocytes isolated from animals (such as mice, rats, rabbits, chickens), or osteocyte cell lines including, but not limited to, MLO-Y4 cells and others.

[0187] Osteoblasts: MLO-A5 osteoblasts express connexin 43 and are used as a control since they do not appear to open upon stimulation with alendronate.

[0188] Tracer molecules include, but are not limited to, Lucifer Yellow, ethidium bromide, Evans blue, Alexa350, Alexa488, and Alexa594.

[0189] 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 opening of the hemichannel.

[0190] A method for determining whether an antibody opens or closes / blocks a hemichannel includes one or more of the following steps:

[0191] (a) Isolating, obtaining, or producing connexin-expressing cells or cell lines. For example, primary osteoblasts can be isolated from the calvarial crown. Other cell types can be isolated using other methods known in the art. In certain embodiments, calvarial bone cells are isolated from an animal (e.g., a 16-day-old chick embryo calvarial crown or a neonatal mouse). The animal is decapitated, the skull is dissected, and immediately immersed in 70% alcohol. Next, the skull is transferred to αMEM and washed multiple times with PBS. The cleaned bone is placed in fresh αMEM. The bone is minced and cut into pieces of 1.5 mm in size. The bone pieces are treated with collagenase to remove soft tissues and osteoid, and then decalcified using EDTA. Finally, the osteocytes are released from the bone chips by treatment with collagenase and vigorous agitation.

[0192] (b) Isolation of primary osteocytes from long bones. Osteocytes from long bones can be isolated from 2- to 3-week-old mice or rats. For example, an excessive amount of anesthesia is administered to the mouse, the neck is severed, the mouse is decapitated, and immersed in 70% ethanol. The femur and tibia with intact joint ends are isolated. The legs are quickly immersed in 70% alcohol and then placed in αMEM. The legs in αMEM are washed with PBS. Most of the muscle is removed and detached from the tendon / ligament. The cleaned bone is placed in fresh αMEM. After all the bones are cleaned, both ends of each bone are cut using a scalpel, and immediately thereafter, the bone marrow is flushed out using PBS. The bones are cut into lengths of 1.5 - 2 mm and treated with collagenase. In one example, the bone fragments are treated with collagenase continuously 9 times, and after removing all other tissues and osteoid, they are decalcified using EDTA.

[0193] (c) Culturing of cells or cell lines. For example, primary and / or osteocyte cell lines are cultured on collagen-coated plates and placed in a recording medium containing a permeable tracer (HCO3-free HEPES-buffered α-MEM medium).

[0194] (d) Administration of test antibodies. The cultured cells are contacted with the test antibody for a desired period.

[0195] (e) Determination of permeable tracer uptake. Permeable tracer uptake is determined by detecting the amount of tracer within the cells. In certain embodiments, time-lapse recording is used. Fluorescence can be recorded in the region of interest in different cells by an eclipse filter in a microscope based on the fluorescence wavelength of the tracer or other probe used. In certain embodiments, images are captured every 2 minutes by a chilled digital camera, and image processing is performed by ImageJ software. The collected data can be illustrated as the magnification of the difference between the initial fluorescence and the fluorescence at the desired time when compared to the basal fluorescence.

[0196] In the case of snapshot images, after exposing the cells to a permeable tracer for 5 - 10 minutes, they can be rinsed multiple times with PBS and fixed with formaldehyde. In certain embodiments, at least three micrographs of the fluorescence field are taken with a microscope. Image analysis is performed using ImageJ software. The average pixel density of random cells is measured.

[0197] Confirmation of the opening or blocking of connexin hemichannels can be obtained, for example, by treating osteocytes with a test antibody together with the shear stress of fluid flow and / or AD, which is known to open Cx43 hemichannels. When the test antibody blocks the Cx43 hemichannel, this channel block is reversed by the shear stress of fluid flow and / or AD. To control the blocking of Cx43 hemichannels, osteocytes are treated with a Cx43(E2) antibody, a polyclonal antibody that specifically inhibits the opening of Cx43 hemichannels.

[0198] In a specific example, MLO - Y4 osteocytes were treated with 1 μg / ml Cx43(E2) antibody or a test antibody for 30 minutes in the absence or presence of 20 μM AD. Ethidium bromide dye uptake was performed and quantified compared to the untreated basal level of uptake. The assay was performed in the presence of calcium. Low calcium conditions can be used as a control (to open hemichannels). The opening of osteocyte hemichannels induced by AD is blocked by the Cx43(E2) antibody or the test antibody.

[0199] Inhibition of the opening of Cx43 hemichannels in chondrocytes (e.g., by chemical reagents, etc.) can suppress the occurrence of inflammation and osteoarthritis. The opening of hemichannels in chondrocytes can be detected using the methods described herein. The release of pro - inflammatory factors (PGE2 and ATP) by Cx43 hemichannels is measured using an ELISA assay. An agent that blocks the opening of hemichannels can be used as a therapeutic agent for inflammatory disorders such as OA.

[0200] An in vitro cell model for evaluating Cx43 channel activity. Primary chondrocytes are isolated from the joints of the bones of the mouse leg. The effects of agents on the opening of Cx43 hemichannels and gap junction coupling in chondrocytes can be detected, and time- and dosage-dependent effects can be evaluated. For example, the opening of hemichannels is evaluated by a dye uptake assay using lucifer yellow or Alexa dyes. Downstream effects are measured by detecting the release of PGE2 and ATP using an ELISA assay.

[0201] A specific protocol is shown below.

[0202] Immunoblot. 3×10 5 MLO-Y4 cells were seeded in a 60 mm culture dish for 48 hours. Mouse heart tissue was collected in lysis buffer (5 mM Tris, 5 mM EDTA, 5 mM EGTA plus protease inhibitor, 20 μl / ml phenylmethylsulfonyl fluoride (PMSF), 20 μl / ml N-ethylmaleimide, 10 μl / ml NaVO4 and 10 μl / ml leupeptin), homogenized and centrifuged at 100,000×g for 30 minutes at 4°C, and resuspended in lysis buffer. The crude membrane proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, 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 secondary antibody, infrared IRDye® 800 anti-rabbit IgG (1:15000 dilution) (LI-COR, Lincoln, NE, USA), fluorescence was detected by an Odyssey infrared detection system (LI-COR, Lincoln, NE, USA).

[0203] Immunofluorescence. MLO-Y4 cells were cultured on collagen-coated cover glasses. The cells were rinsed twice with PBS and incubated with cold 70% ethanol at -20 °C for 20 min. The use of PFA is not recommended as it destroys lysine-rich epitopes. Next, to remove the ethanol, the cells were rinsed twice with PBS. The cells were then blocked overnight in blocking solution (2% goat serum, 2% fish skin gelatin, and 1% bovine serum albumin in PBS). The cells were then labeled with monoclonal antibodies at different concentrations in PBS, followed by labeling with FITC-conjugated goat anti-mouse antibody and WGA-alexa594 (Invitrogen) (1:400 dilution and 1:1500 dilution in blocking buffer, respectively). The cells were observed with an Olympus BH-2 fluorescence microscope and the images were processed offline with NIH Image J software.

[0204] Dye uptake related to hemichannel activity. Dye uptake measurements were evaluated using snapshot photographs. MLO-Y4 cells were placed in collagen-coated 35 mm culture dishes and incubated with recording medium, 10 mM HEPES salt composition, 154 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM glucose buffered with HCO3 - and free saline medium. 0.5 mM EGTA was added to the medium containing low concentrations of divalent cations (low [X 2 ), but CaCl2 and MgCl2 were not added. Low [X 2 recordings containing 50 μM EtBr for snapshot recording. The cells were exposed to 100 μM EtBr for 5 min, then rinsed three times with PBS and fixed with 2% formamide. At least three micrographs of the fluorescence field were taken with a 10x dry system objective lens in an inverted microscope (Carl Zeiss) equipped with a rhodamine filter. Image analysis was performed offline with software image J. The average pixel density of 30 random cells was measured.

[0205] Dye coupling assay of gap junctions. MLO-Y4 cells were seeded in a collagen-coated 35-mm culture dish and incubated with recording medium (HCO3 - -free αMEM medium) buffered with 10 mM HEPES. Cells were microinjected with alexafluor350 (Invitrogen, Eugene, Oregon, USA) (10 mM in PBS) at 37 °C using both an Eppendorf (Eppendorf) micromanipulator InjectMan NI 2 and Femtojet. The movement of the dye was measured 2 minutes after alexafluor350 injection. The index of dye coupling was scored by counting the number of cells in which the dye had moved. Dye coupling was observed under an inverted microscope equipped with a xenon arc lamp and a Nikon eclipse (Nikon, Japan) (excitation wavelength 330–380 nm; emission wavelength above 420 nm).

[0206] Cell parachute dye transfer assay of gap junctions. MLO-Y4 cells were grown to confluence in 12-well plates. 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 at gap junctions can be traced by simultaneously labeling the cells with calcein red-orange as a gap junction permeable tracer dye and Oregon Green 488 BAPTA-2 as a gap junction channel impermeable dye. Pre-loaded donor cells were removed from the plate by trypsinization. The pre-loaded cells were layered ("parachuted") on top of non-labeled recipient cells cultured at a ratio of 1:4 of donor to recipient. After the cells were allowed to bind for various periods of 1 hour, they were carefully washed 3 times, fixed in fresh 2% PFA at RT for 10 minutes, and rinsed 3 times again. The cells were examined by fluorescence microscopy. For calcein red-orange transfer, the threshold was adjusted to clearly distinguish the dye transfer boundary. The dye transfer positive criterion was to detect calcein red-orange / Oregon Green 488 BAPTA-2 by calcein red-orange positive and Oregon Green 488 BAPTA-2 negative contacting cells. The transfer of the dye was almost undetectable (<1%). Images were taken where the inventors found Oregon Green 488 BAPTA-2 green positive cells.

[0207] Shear stress of fluid flow to open hemichannels. Fluid flow was generated by a parallel plate flow chamber separated by a gasket of a given thickness by gravity-based fluid flow using a peristaltic pump. The thickness of the gasket determined the height of the channel, which was adjusted along the flow rate to generate a stress level of 16 dyn / cm 2 The circulating medium was α-MEM buffered with 10 mM HEPES. B. In vivo assay

[0208] In certain embodiments, Cx43 modulation in osteocytes is determined by injecting a candidate reagent into long bones and detecting in situ opening or blocking of hemichannels in osteocytes using a fluorescent tracer dye (e.g., calcein or Evans blue).

[0209] An example of an in vivo assay for analyzing hemichannels in osteocytes uses 3 - 4 - month - old mice or rats. The animal's body weight is measured. The test antibody is introduced into the animal via intraperitoneal (IP) injection. After 2 - 4 hours, a fluorescent tracer dye (i.e., Evans blue, Alexa594) is injected into the animal's lateral tail vein or by IP injection. Note that up to 1% of the animal's body weight per volume can be injected. In certain embodiments, the animal is warmed prior to tail vein injection to dilate the tail vein. After 2 - 4 hours, the animal is sacrificed and the tibia and femur without muscle tissue are dissected and washed several times with PBS. The bone is fixed in paraformaldehyde and decalcified in a 14% EDTA solution at 4°C for 2 weeks or at room temperature for 3 - 5 days with constant agitation. The bone is washed in PBS, immersed overnight in 30% sucrose in PBS, and embedded in OCT compound. The position of the bone is typically adjusted in a mold as needed. Frozen sections 5 μm thick are cut using a cryostat, the sections are rinsed in PBS, and mounted on a microscope using 50% glycerol in PBS. The bone sections are examined under a fluorescence microscope and the degree of osteocytes in the bone taking up the tracer dye is quantified using Image J.

[0210] Opening of Cx43 hemichannels in osteocytes can be confirmed by mechanical loading of the tibia that opens Cx43 hemichannels in osteocytes. This can serve as a negative control for hemichannel opening in osteocytes in vivo. For a positive control, mice with a deficiency of Cx43 in osteocytes are used. This mouse is generated by mating 10 - kb DMP - 1 Cre and Cx43 flox mice.

[0211] Cx43 conditional knockout (cKO) mice. Since homozygous Cx43 global knockout is lethal, osteocyte-specific Cx43 knockout mice were generated to examine the role of Cx43 expressed in osteocytes. To facilitate complete deletion of Cx43 in osteocytes, mice homozygous for the floxed Cx43 gene were mated with Cx43 global heterozygous mice. Next, 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 Cx43fl / -, DMP1 Cre+ or Cx43fl / -, DMP1 Cre- (a small percentage were Cx43fl / fl or Cx43- / -). Cx43-deficient osteocytes were confirmed by immunohistochemistry.

[0212] The study may include eight groups of mice: WT treated with Cx43 (E2), WT not treated with Cx43 (E2), cKO treated with Cx43 (E2), cKO not treated with Cx43 (E2), WT treated with the test antibody (TA), WT not treated with TA, cKO treated with TA, and cKO not treated with TA. Cx43 (E2) or TA was administered to the mice at 150 μg / kg body weight. It is expected that inflammation will increase in the KO compared to WT mice with Cx43 (E2) or TA treatment. Moreover, bone metastasis should be similar between WT and knockout mice without Cx43 (E2) or TA treatment.

[0213] The animal models of spinal cord injury that can be used to test the Cx43 antibodies disclosed herein are outlined, for example, by Sharif-Alhoseini et al., Spinal Cord. 2017 Aug;55(8):714-721, which is hereby incorporated by reference in its entirety. The animal models of inflammation (e.g., rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis) for use in testing the Cx43 antibodies disclosed herein are outlined by Webb, Biochem Pharmacol. 2014 Jan 1;87(1):121-30, which is hereby incorporated by reference in its entirety.

[0214] The various aspects of the present disclosure may be used alone, in combination, or in various arrangements not specifically contemplated in the embodiments described above, and thus, in their application, are not limited to the details and arrangements of the components described in the foregoing description or illustrated in the drawings. For example, the aspects described in the embodiments may be combined in any manner with the aspects described in other embodiments.

[0215] While particular embodiments of the present disclosure have been considered, the foregoing specification is illustrative and not restrictive. Many variations of the present disclosure will be apparent to those skilled in the art upon a review of this specification. The full scope of the present disclosure should be determined by reference to the claims, along with the full scope of equivalents, and by reference to this specification in light of such variations. Incorporation by reference

[0216] All publications, patents, and patent applications referenced herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The present invention provides, for example, the following items. (Item 1) The first, second, and third heavy chain complementarity determining region (CDR) sequences each having the amino acid sequences of SEQ ID NOs: 1, 2, and 3; and The first, second, and third light chain CDR sequences each having the amino acid sequences of SEQ ID NOs: 4, 5, and 6 An anti-Cx43 antibody or an antigen-binding fragment thereof comprising (Item 2) The antibody or fragment thereof according to item 1, comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 8. (Item 3) An anti-Cx43 antibody or an antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17 and a light chain having the amino acid sequence of SEQ ID NO: 18. (Item 4) An anti-Cx43 antibody or an antigen-binding fragment thereof that binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19) when binding to Cx43. (Item 5) The antibody or fragment thereof according to item 4, wherein the epitope comprises one or more amino acids selected from the group consisting of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. (Item 6) The antibody or fragment thereof according to item 4, wherein the epitope consists of R4, P5, E7, K8, and I10 of SEQ ID NO: 19. (Item 7) The antibody or fragment thereof according to item 4, wherein the epitope comprises all 10 amino acids of SEQ ID NO: 19. (Item 8) The antibody or fragment thereof according to item 4, wherein the epitope consists of all 10 amino acids of SEQ ID NO: 19. (Item 9) An isolated anti-Cx43 antibody or an antigen-binding fragment thereof that cross-competes with the antibody or fragment thereof according to any one of items 1-8 with respect to binding to Cx43. (Item 10) The antibody or fragment thereof cross-competes with the antibody or fragment thereof according to any one of items 1 to 3 with respect to binding to Cx43, preferably the antibody or fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19), more preferably the epitope contains one or more amino acids selected from the group consisting of R4, P5, E7, K8, and I10 of SEQ ID NO: 19, and even more preferably the epitope contains all 10 amino acids of SEQ ID NO: 19, an isolated anti-Cx43 antibody or antigen-binding fragment thereof. (Item 11) The antibody or fragment thereof according to any one of items 1 to 10 that inhibits the opening of Cx43 hemichannels in cells. (Item 12) A pharmaceutical composition for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating an inflammatory disease or condition or a neurodegenerative disease, comprising the antibody or fragment thereof according to any one of items 1 to 11 and a pharmaceutically acceptable carrier. (Item 13) The pharmaceutical composition according to item 12, wherein the neurodegenerative disease is spinal cord injury. (Item 14) Use of the antibody or fragment thereof according to any one of items 1 to 11 for the manufacture of a medicament for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury. (Item 15) A method for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, comprising the step of contacting a cell with an effective amount of the antibody or fragment thereof according to any one of items 1 to 11. (Item 16) A method for treating a disease or condition associated with the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof according to any one of items 1 to 11, preferably wherein the opening is an excessive opening or a long-term opening.

Claims

1. A nucleic acid comprising coding sequences for the heavy chain variable region and the light chain variable region of an anti-Cx43 antibody, wherein the anti-Cx43 antibody is first, second, and third heavy chain complementarity determining region (CDR) sequences having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and First, second, and third light chain CDR sequences having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. A nucleic acid comprising:

2. The nucleic acid described in claim 1, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 7 and the light chain variable region has the amino acid sequence of SEQ ID NO:

8.

3. A nucleic acid encoding a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, and 17, or a light chain having the amino acid sequence of SEQ ID NO:

18.

4. The nucleic acid described in claim 3, wherein the nucleic acid encodes the heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, 15, and 17, and the light chain having the amino acid sequence of SEQ ID NO:

18.

5. A nucleic acid described in any one of claims 1 to 4, further comprising a promoter operably linked to the nucleic acid.

6. An expression vector comprising the nucleic acid described in any one of claims 1 to 5.

7. The expression vector described in claim 6, wherein the vector is a viral vector.

8. A cell comprising a nucleic acid described in any one of claims 1 to 5 or an expression vector described in claim 6 or 7.

9. The cell described in claim 8, wherein the cell is a hybridoma cell line.

10. A method for producing an anti-Cx43 antibody, comprising the step of culturing a cell described in claim 8 or 9 under appropriate conditions, thereby producing the anti-Cx43 antibody.

11. The method described in claim 10, further comprising a step of purifying the anti-Cx43 antibody.

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