Connectin 43 antibodies and uses thereof

Abstract

The invention relates to connexin 43 antibodies and uses thereof. The present disclosure relates generally to compositions and methods for treating diseases or conditions associated with Cx43 semi-channel opening in stellate cells or bone cells, preferably for treating inflammatory diseases or conditions, or neurodegenerative diseases such as spinal cord injury.

Description

[0001] This application is a divisional application of Chinese patent application 202080002561.9 “Connexin 43 antibodies and uses thereof” filed on February 4, 2020.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 800,869, filed on February 4, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure generally relates to anti-connexin (Cx) 43 antibodies and their use in treating diseases or conditions associated with the opening of Cx43 hemichannels in cells, such as stellate cells and osteocytes. Background Art

[0005] It is estimated that approximately 400,000 patients suffer from spinal cord injury (SCI) in the United States alone, with more than 14,000 new cases added each year. Although the acute inflammatory response is a defense mechanism to preserve tissue integrity and differentiate between traumatic lesions, an overreaction will limit the possibility of successful recovery. Tissue swelling, especially under the tight confinement of the spinal canal, will reduce tissue perfusion and cause secondary ischemia. Delayed loss of tissue affects functional recovery in most patients, and there are currently no effective treatment options.

[0006] It was shown 50 years ago that injection of ATP in the absence of injury is sufficient to induce an acute inflammatory response. The key observation linking purinergic signaling to inflammatory mediators is that activation of the purinergic receptor P2RX7 triggers microglial maturation and secretion of IL-1β. Although adenosine nucleosides (i.e., ATP and its metabolites) are recognized as mediators of inflammation, little is known about the role of purinergic signaling in spinal cord injury. Spinal cord injury results in excessive and sustained ATP release in the area surrounding the injury, and P2RX7 inhibition reduces the inflammatory response and promotes functional recovery.

[0007] Previous studies have shown that astrocytes release ATP at least in part through the opening of connexin 43 (Cx43) hemichannels. Connexins are a class of proteins with dual channel functions. Their traditional role is to form gap junctions, which consist of two docked hemichannels connecting the cytoplasm of two adjacent cells. Gap junctions allow ions and small molecules, including Ca, to pass through the cytoplasm. 2+ cAMP, IP3, ATP, glutamine and glucose cell-to-cell transport. Unopposed hemichannels have been found to constitute channels for controlled release of gliotransmitters. Due to their relatively large pore diameter (approximately ), open hemichannels enable the efflux of small cytosolic compounds, many of which, including ATP and glutamine, act as transmitters once released. Hemichannel opening is usually tightly controlled, as prolonged opening of many hemichannels is incompatible with cell survival.

[0008] Cx43 has been found to be involved in other inflammatory diseases, such as sterile and infectious inflammation, for example infectious lung disease, osteoarthritis and spinal cord injury.

[0009] Therefore, there exists a need for effective methods and compositions for treating spinal cord injury, for example by inhibiting open Cx43 hemichannels. Summary of the invention

[0010] Provided herein are compositions and methods for treating a disease or condition associated with Cx43 hemichannel opening (eg, excessive or prolonged opening), such as an inflammatory disease or condition or a neurodegenerative disease, such as spinal cord injury, Alzheimer's disease, osteoarthritis, and the like.

[0011] In one aspect, provided herein is an anti-Cx43 antibody, or an antigen-binding fragment thereof, comprising:

[0012] A first, second and third heavy chain complementarity determining region (CDR) sequence having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and

[0013] The first, second and third light chain complementary determining region (CDR) sequences have the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0014] In some embodiments, the antibody or fragment thereof may have a heavy chain variable region having the amino acid sequence of SEQ ID NO:7, and a light chain variable region having the amino acid sequence of SEQ ID NO:8.

[0015] Another aspect relates to an anti-Cx43 antibody or an antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence selected from SEQ ID NOs: 9-17, and a light chain having an amino acid sequence of SEQ ID NO:18.

[0016] In another aspect, provided herein is an antibody that binds to an epitope located within the amino acid sequence FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may include one or more amino acids selected from: 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 some embodiments, the epitope may consist of all 10 amino acids of SEQ ID NO: 19.

[0017] Another aspect relates to an isolated anti-Cx43 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof cross-competes with any of the antibodies or fragments thereof disclosed herein for binding to Cx43. In some embodiments, the antibody or fragment thereof can inhibit or reduce or block the opening of a Cx43 hemichannel in a cell.

[0018] Another 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 having one or more of SEQ ID NOs: 1-18 disclosed herein for binding to Cx43, wherein preferably the antibody or fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19), wherein more preferably, the epitope comprises one or more amino acids selected from R4, P5, E7, K8 and I10 of SEQ ID NO: 19, wherein even more preferably, the epitope consists of all ten amino acids of SEQ ID NO: 19.

[0019] In some embodiments, the antibodies or fragments thereof disclosed herein can inhibit or reduce or prevent the opening of a Cx43 hemichannel in a cell. In some embodiments, the opening is excessive or prolonged.

[0020] Also disclosed herein is a pharmaceutical composition for inhibiting the opening of intracellular Cx43 hemichannels, preferably for treating inflammatory diseases or conditions or neurodegenerative diseases, such as spinal cord injury, comprising one or more antibodies or fragments thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the opening is excessive or prolonged opening.

[0021] Another aspect relates to the use of the antibodies or fragments thereof disclosed herein for the preparation of a medicament for inhibiting the opening of Cx43 hemichannels in a cell, preferably the medicament is used to treat an inflammatory disease or condition or a neurodegenerative disease, such as spinal cord injury. In some embodiments, the opening is excessive or prolonged opening.

[0022] Another aspect relates to a method of inhibiting the opening of a Cx43 hemichannel in a cell, preferably for treating an inflammatory disease or condition or a neurodegenerative disease, such as spinal cord injury, comprising contacting the cell with an effective amount of an antibody or fragment thereof disclosed herein. In some embodiments, the opening is excessive or prolonged.

[0023] Another aspect relates to a method for treating a disease or condition associated with the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury, 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 excessive or chronic.

[0024] The present invention specifically relates to the following solutions:

[0025] 1. An anti-Cx43 antibody or an antigen-binding fragment thereof, comprising:

[0026] A first, second and third heavy chain complementarity determining region (CDR) sequence having the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and

[0027] The first, second and third light chain complementary determining region (CDR) sequences have the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

[0028] 2. The antibody or fragment thereof according to Scheme 1, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7, and a light chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0029] 3. An anti-Cx43 antibody or an antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence selected from SEQ ID NOs: 9-17, and a light chain having an amino acid sequence of SEQ ID NO: 18.

[0030] 4. An anti-Cx43 antibody or antigen-binding fragment thereof, wherein when bound to Cx43, it binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19).

[0031] 5. The antibody or fragment thereof according to claim 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.

[0032] 6. The antibody or fragment thereof according to Scheme 4, wherein the epitope consists of R4, P5, E7, K8 and I10 of SEQ ID NO: 19.

[0033] 7. The antibody or fragment thereof according to claim 4, wherein the epitope comprises all ten amino acids of SEQ ID NO: 19.

[0034] 8. The antibody or fragment thereof according to claim 4, wherein the epitope consists of all ten amino acids of SEQ ID NO: 19.

[0035] 9. An isolated anti-Cx43 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof cross-competes with the antibody or fragment thereof of any one of Schemes 1 to 8 for binding to Cx43.

[0036] 10. An isolated anti-Cx43 antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof cross-competes with the antibody or fragment thereof of any one of Schemes 1-3 for binding to Cx43, wherein preferably the antibody or fragment thereof binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19), wherein more preferably, the epitope comprises one or more amino acids selected from R4, P5, E7, K8 and I10 of SEQID NO: 19, wherein even more preferably, the epitope comprises all ten amino acids of SEQID NO: 19.

[0037] 11. The antibody or fragment thereof according to any one of Schemes 1 to 10, which inhibits the opening of Cx43 hemichannels in cells.

[0038] 12. A pharmaceutical composition for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating inflammatory diseases or conditions or neurodegenerative diseases, comprising the antibody or fragment thereof described in any one of Schemes 1-11 and a pharmaceutically acceptable carrier.

[0039] 13. The pharmaceutical composition of Scheme 12, wherein the neurodegenerative disease is spinal cord injury.

[0040] 14. Use of any antibody or fragment thereof of schemes 1-11 for preparing a drug for inhibiting the opening of Cx43 hemichannels in cells, preferably for preparing a drug for treating inflammatory diseases or conditions or neurodegenerative diseases, such as spinal cord injury.

[0041] 15. A method for inhibiting the opening of Cx43 hemichannels in a cell, preferably for treating inflammatory diseases or conditions or neurodegenerative diseases, such as spinal cord injury, comprising contacting the cell with an effective amount of the antibody or fragment thereof according to any one of Schemes 1 to 11.

[0042] 16. A method for treating a disease or condition associated with the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating inflammatory diseases or conditions or neurodegenerative diseases, such as spinal cord injury, comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or fragment thereof as described in any one of Schemes 1-11, wherein the opening is preferably excessive or prolonged. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 .Box plot of raw data from antibody screening. Specifically, the bottom and top of the box are the 25th and 75th percentiles of the data. The bar near the middle of the box is the 50th percentile (median). The whiskers are at 1.5 of the inter-quantile range, indicating statistical anomalies in the data set (Mcgill et al., (1978) American Statistician, 32: 12-16).

[0044] Figure 2 . Letter plot of full substitution analysis of peptide FLSRPTEKTI probed with antibodies under high stringency conditions. The base sequence is listed below the plot, and the sense signal of the baseline sequence is at the red line. The substitution at a given position is plotted at the signal intensity recorded for that substitution.

[0045] Figure 3 . Antibodies probed on Group 2 peptides show increased binding to peptides containing the C-terminal residues of the target sequence. Lines are drawn from the start position to the end position in the peptide and the height is the height at which the signal is recorded. DETAILED DESCRIPTION

[0046] It is to be understood that both 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.

[0047] Disclosed herein are compositions and methods related to anti-Cx43 antibodies or their antigen-binding fragments. In some embodiments, the compositions disclosed herein unexpectedly show more remarkable activity, drugability (e.g., reduced toxicity), stability and / or developability (e.g., reduced production costs) relative to those disclosed in PCT Publication Nos. WO2015 / 027120 and WO2017 / 147561 (incorporated herein in their entirety for reference). In some embodiments, the advantages are unexpected.

[0048] definition

[0049] For convenience, specific terms used in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document belongs.

[0050] As used herein, the following terms and expressions shall have the following meanings:

[0051] The articles "a" and "an" are used herein to refer to one or more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0052] As used herein, the term "about" means an acceptable error within 20%, more preferably within 10%, and most preferably within 5% of the stated value.

[0053] An "anti-Cx43 antibody" is an antibody that immunospecifically binds to Cx43 (e.g., its extracellular domain). The antibody may be an isolated antibody. Such binding to Cx43 exhibits, for example, a K of no greater than 1 μM, no greater than 100 nM, or no greater than 50 nM. D K can be determined by any method known to those skilled in the art. D , such as surface plasmon resonance experiments or cell binding experiments. The anti-Cx43 antibody can be a monoclonal antibody or an antigen-binding fragment thereof.

[0054] "Antibody" as used herein is a protein comprising a binding domain that binds to a target epitope. The term antibody includes monoclonal antibodies, which include immunoglobulin heavy and light chain molecules, single heavy chain variable domain antibodies, and variants and derivatives thereof, including chimeric variants of monoclonal and single heavy chain variable domain antibodies. The binding domain is basically encoded by an immunoglobulin gene or an immunoglobulin gene fragment, wherein the protein immunospecifically binds to the antigen. The recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ or ε, which in turn determine the immunoglobulin class, which is IgG, IgM, IgA, IgD and IgE, respectively. For most vertebrates, including human and mouse species, the typical immunoglobulin structural unit includes a tetramer, which includes two pairs of identical polypeptide chains, each pair having a "light" (about 25kD) and a "heavy" chain (about 50-70kD). "VL" and "VH" refer to these light and heavy chains, respectively. L ” and “C H " refers to the constant region of the light chain and heavy chain. Each V L and V H The three β-strand loops on the Fab are responsible for antigen binding and are called the "complementarity determining regions" or "CDRs." The "Fab" (antigen binding fragment) region includes one constant region and one variable region, V, from each heavy and light chain of the antibody. L ,C L ,V H and C H 1.

[0055] Antibodies include intact immunoglobulins and antigen-binding fragments thereof. The term "antigen-binding fragment" refers to an antibody polypeptide fragment that binds to an antigen or competes with an intact antibody (i.e., an intact antibody from which it is derived) for 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 single-chain Fv antibodies (scFv), wherein V H and V L The chains are joined together (directly or through peptide linkers) to form a continuous polypeptide.

[0056] Antibodies also include variants, chimeric antibodies and humanized antibodies. The term "antibody variant" as used herein refers to an antibody having a single or multiple mutations in a heavy chain and / or light chain. In some embodiments, the mutation is present in the variable region. In some embodiments, the mutation is present in the constant region. "Chimeric antibody" refers to an antibody in which a portion of each of the heavy and light chain amino acid sequences is homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular family, but the remaining segments of the chain are homologous to the corresponding sequence of another species or family. Usually in these chimeric antibodies, the light and heavy chain variable regions simulate the variable regions of antibodies derived from mammalian species, while the constant regions are homologous to the sequences of antibodies derived from another species. A significant advantage of these chimeric forms is that, for example, readily available hybridomas or B cells from non-human host organisms can be used to conveniently derive variable regions from currently known sequences, while constant regions are derived from, for example, human cell preparations. Although the variable region has the advantage of being easy to prepare and its specificity is not affected by its source, the constant region from a person will be more difficult to induce an immune response when the antibody is injected into a human individual, compared to the corresponding antibody whose constant region is from a non-human source. However, the definition is not limited to this specific example. A "humanized" antibody refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, and the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may include a complete variable region fused to a constant region or a complementary determining region (CDR) that is only transplanted to a suitable framework region in the variable region. The antigen binding site may be wild type or modified with one or more amino acid substitutions, for example, modified to be more like a human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (e.g., humanized mouse antibodies, which contain all six CDRs from mouse antibodies). Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five or six), which are changed relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs.

[0057] As used herein, antibody amino acid residues may be numbered according to the universal numbering system of Kabat (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. NIH, Public Health Service, Bethesda, Maryland).

[0058] As used herein, the term "binding" in the context of binding between an antibody and an epitope of Cx43 as a target refers to the process of non-covalent interaction between molecules. Preferably, the binding is specific. The specificity of an antibody can be determined based on affinity. The binding affinity or dissociation constant K of a specific antibody to its epitope is D Less than 10 -7 M, preferably less than 10 -8 M.

[0059] The term "affinity" refers to the strength of the binding reaction between the binding domain of an antibody and an epitope. It 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 .

[0060] The term "antigen" refers to a molecule or portion of a molecule that can be bound to a selective binding agent such as an antibody and can be administered to an animal to produce antibodies that bind to an epitope of the antigen. An antigen can have one or more epitopes.

[0061] The term "epitope" includes any determinant region that can specifically bind to an immunoglobulin or T cell receptor, preferably a polypeptide determinant region. In certain embodiments, the epitope determinant includes a group of surface molecules (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl groups) that are chemically active, and in certain embodiments, the epitope determinant may have specific three-dimensional structural characteristics and / or specific charge characteristics. In one embodiment, the epitope is a region of an antigen bound by an antibody. In some embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is called specific binding to an antigen. Methods for epitope mapping are well known in the art, such as X-ray co-crystallization identification, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from continuous amino acids or discontinuous amino acids juxtaposed due to the folding of the tertiary structure of the protein. Epitopes formed by continuous amino acids are usually maintained after contact with a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. An epitope typically comprises at least 3, more usually at least 5, or about 8-10 amino acids in a unique spatial conformation.

[0062] The site on the antibody that binds to the epitope is called the "paratope," which generally includes the amino acid residues that are immediately adjacent to the epitope once bound to the epitope. See Sela-Culang et al., Front Immunol. 2013; 4:302.

[0063] "Immunohistochemistry" or "IHC" refers to a process of detection in cells or tissue sections that enables the binding of antibodies that immunospecifically recognize an antigen of interest in a biological tissue and its subsequent detection. An overview of IHC techniques can be found, for example, in Ramos-Vara et al., Veterinary Pathology, January 2014, Vol. 51, No. (1), 42-87, which is incorporated herein by reference in its entirety. In order to evaluate IHC results, different stereotypical and semiquantitative scoring systems have been developed. See, for example, Fedchenko et al., Diagnostic Pathology, 2014; 9:221, which is incorporated herein by reference in its entirety. An example is the H score, which is obtained by adding the percentage of cells with an ordinal value for staining intensity (a score ranging from 0 for "no signal" to 3 for "strong signal") multiplied by 300 possible values.

[0064] "Immunospecific" or "immunospecifically" (sometimes used interchangeably with "specifically") refers to an antibody that binds to one or more epitopes represented by an antigen through a domain encoded by an immunoglobulin gene or immunoglobulin gene fragment, but does not substantially recognize and bind to other molecules in a sample containing a mixed population of antigenic molecules. Typically, antibodies are detected using, for example, real-time label-free biolayer interferometry techniques, such as HTX biosensor, or using surface plasmon resonance such as BIACORE TM , or the K of the antibody immunospecifically binding to the cognate antigen measured by solution affinity ELISA D The value is no greater than 50 nM. The use of these techniques is well known in the art.

[0065] The term "surface plasmon resonance" refers to an optical phenomenon that can be used to analyze real-time biomolecular interactions by detecting changes in protein concentrations in a biosensor matrix, such as using BIACORE TM Systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscata, NJ).

[0066] Biolayer interferometry is a label-free technique for determining biomolecular interactions. It is an optical analysis technique that analyzes the interference pattern of two surface-reflected white light: a layer of immobilized protein and a layer of internal reference at the tip of the biosensor. Any change in the number of molecules bound to the tip of the biosensor results in a change in the interference pattern that can be measured in real time (Abdiche, YN, et al., Analytical Biochemistry, (2008), 377 (2), 209-217). In some embodiments, the binding characteristics of some anti-Cx43 antibodies disclosed herein are evaluated using "biosensors based on real-time biolayer interferometers (Octet HTX technology)".

[0067] The terms "cross-competition", "cross-competitiveness", "cross-blocking", "cross-blocking" and "cross-blocking" are used interchangeably herein and refer to the direct or indirect interference of an antibody or fragment thereof with the binding of a target Cx43 by allosteric modulation of an anti-Cx43 antibody herein. The extent to which an antibody or fragment thereof can interfere with the binding of another antibody or fragment thereof to a target and whether it can therefore be referred to as cross-blocking or cross-competing according to the present disclosure can be determined by competitive binding assays. A particularly suitable quantitative cross-competition assay uses FACS or an AlphaScreen-based method to determine the ability of a labeled (e.g., His-tagged, biotinylated or radiolabeled) antibody or fragment thereof to compete with another antibody or fragment thereof for binding to its target. Generally speaking, a cross-competing antibody or fragment thereof is thus an antibody that is capable of binding to a target in a cross-competition assay, such that during the assay or in the presence of a second antibody or fragment thereof, the immunoglobulin single variable domain or polypeptide according to the disclosure has a recorded displacement up to 100% (e.g. in a FACS-based competition assay) of the maximum theoretical displacement (e.g. by a cold (unlabeled) antibody or fragment thereof that is to be cross-blocked) by a given amount of a potential cross-blocking antibody or fragment thereof to be tested. Preferably, the cross-competing antibody or fragment thereof thus has a recorded displacement of between 10% and 100%, more preferably between 50% and 100%.

[0068] Cross-competitiveness between antibodies can also be determined using real-time label-free bio-layer interferometry experiments. Cross-competition between two antibodies can be expressed as binding of the second antibody below the signal due to self-binding (the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as % binding of the second antibody below baseline self-background binding (the first and second antibodies are the same antibody).

[0069] The terms "inhibit," "block," and "reduce" are used interchangeably herein and refer to any statistically significant reduction in biological activity (e.g., hemichannel opening). For example, "inhibit" or "block" can refer to a reduction in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0070] The term "subject" or "patient" includes a human or other mammal who is receiving prophylactic or therapeutic treatment.

[0071] As used herein, the terms "treatment", "treatment" and "treatment" refer to treatment or preventive measures such as those described herein. "Treatment" methods employ the administration of a Cx43 ligand provided herein to a patient (e.g., a patient suffering from an inflammatory disease or condition or a neurodegenerative disease) to prevent, cure, delay, reduce the severity of an inflammatory disease or condition or a neurodegenerative disease or to alleviate one or more symptoms thereof, or to extend the patient's survival beyond the survival expected in the absence of such treatment. "Treatment" methods also employ the administration of a Cx43 ligand (e.g., an antibody) provided herein to a patient to provide treatment in the patient beyond that expected in the absence of such treatment.

[0072] As used herein, the term "effective amount" refers to an amount of an agent (e.g., Cx43 ligand, such as an anti-Cx43 antibody) sufficient to achieve treatment, prognosis, or diagnosis of a disease when administered to a patient. The therapeutically effective amount can vary according to the patient and the disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the mode of administration, etc., which can be easily determined by a person of ordinary skill in the art. The dosage range can be, for example, 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 4,5 00mg, about 500ng to about 4,000mg, about 1μg to about 3,500mg, about 5μg to about 3,000mg, about 10μg to about 2,600mg, about 20μg to about 2,575mg, about 30μg to about 2,550mg, about 40μg to about 2,500mg, about 50μg to about 2,475mg, about 100μg to about 2,450mg, about 200μg to about 2,425mg, about 300μg 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 9 The present invention provides the antibody or its antigen-binding fragment provided herein.Dosing can be, for example, weekly, every two weeks, every three weeks, every 4 weeks, every 5 weeks or every 6 weeks.The dosage regimen can be adjusted to provide the best therapeutic response.The effective amount is also the amount that any toxicity or harmful effect (side effect) of the agent is minimized and / or offset by the beneficial effect.Dosing can be intravenous precise or about 6mg / kg or 12mg / kg intravenous administration every week, or with 12mg / kg or 24mg / kg intravenous administration every two weeks.Other dosage regimens are described below.

[0073] Those of ordinary skill in the appropriate field can generally understand the recombinant nucleic acid technology, microbiology, immunology, antibody engineering and other terms in the field of molecular and cell biology used herein. For example, conventional techniques (such as electroporation, transfection or lipofection) can be used to prepare recombinant DNA, perform oligonucleotide synthesis and implement tissue culture and transformation. Enzymatic reactions and purification techniques can be carried out according to the manufacturer's instructions or according to conventional operations in the art or as described herein. The above-mentioned techniques and methods can be generally carried out as described in the numerous comprehensive and monograph literatures as known in the art and also as cited and discussed in this specification. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference for all purposes. Unless otherwise specified, the nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. For chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients, standard techniques can be used.

[0074] As used herein, the terms "comprising" or "including" refer to compositions, methods and their respective components that are present in a given embodiment, but unspecified elements are not included.

[0075] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the disclosure.

[0076] The term "consisting of" refers to the compositions, methods, and respective components thereof described herein, excluding any elements not recited in the description of that embodiment.

[0077] In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless expressly stated otherwise. Thus, for example, a "method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and the like.

[0078] Various aspects and embodiments of the disclosure are described in further detail in the following subsections.

[0079] Cx43

[0080] Various cells are able to communicate with each other and with the extracellular environment through gap junctions formed by hemichannels and connexins. Connexins are ubiquitously expressed in the human body. Six connexins make up one hemichannel, and two hemichannels make up one gap junction channel. Gap junctions are a group of channels located in the plasma membrane between adjacent cells that mediate intercellular communication. Hemichannels are separate entities from gap junction channels. Hemichannels allow the exchange of molecules between intracellular compartments and the extracellular environment.

[0081] Many cells express hemichannels called connexin (Cx) 43 hemichannels. Connexin-43, also known as gap junction alpha-1 protein (GJA1), is a 4382 kDa protein consisting 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, retaining its C and N termini in contact with the cytoplasm. The C-terminal tail consists of 50 amino acids and includes sites for post-translational modifications as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. As a result, the C-terminal tail is essential for regulating functions such as pH gating and channel assembly. Notably, the DNA region of the GJA1 gene (NCBI Gene ID: 2697) encoding this tail is highly conserved, suggesting that it is either resistant to mutations or becomes lethal upon mutation. At the same time, the N-terminal domain is involved in channel gating and oligomerization, and thus controls the switching between the open and closed states of the channel. The transmembrane domain forms a gap junction channel, while the extracellular loops facilitate correct channel docking. In addition, the two extracellular loops form disulfide bonds that interact with two hexamers to form a complete gap junction channel.

[0082] Cx plays an important role in regulating many immune processes. Recent reports show that Cx43 hemichannels have pathological effects in sterile injury and infectious inflammatory diseases (Li et al., Scientific Reports (2018) 8: 166, doi: 10.1038 / s41598-017-18452-1). Sterile inflammation is a common event, which is triggered by physical, chemical or metabolic damage. Different damages lead to cell stress and subsequent stress responses. There are many types of stress responses (such as protein unfolding response, integrated stress response, oxidative stress), which are usually implicated with each other. Stress response triggers inflammation. When there is damage, inflammation cannot be resolved, resulting in a harmful cycle, which plays a key role in the pathophysiology of many human diseases, including cancer, metabolism and genetic diseases.

[0083] Acute conditions resulting from 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. Crystal deposition in joints leads to gouty arthritis, which causes the classic clinical symptoms of inflammation, including redness, pain, heat, swelling, and loss of function. Toxins such as acetaminophen or snake venom induce liver and muscle damage. Trauma, including crush injuries, triggers a sudden inflammatory response; endogenous and microbial triggers (due to exposure to bacteria) can also cause inflammation in this setting.

[0084] Chronic diseases that are triggered or caused by 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; certain cases of tumors; arthritis (such as osteoarthritis and rheumatoid arthritis (RA)) and autoimmune diseases.

[0085] Infectious inflammation can be caused by a variety of pathogens, such as bacteria and fungi in many tissues.

[0086] In various embodiments, regardless of the cause, inflammatory diseases can be treated by the anti-Cx43 antibodies disclosed herein. Inflammatory diseases as used herein refer to a large number of diseases 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. Autoimmune diseases refer to diseases in which the immune system attacks its own proteins, cells and tissues, or diseases in which immune effector T cells react to endogenous self-peptides and cause tissue destruction. Therefore, an immune response is generated against the subject's own antigens (called autoantigens). A complete list and review of autoimmune diseases can be found in Autoimmune Diseases (Rose and Mackay, 2014, Academic Press). Autoimmune diseases include, but are not limited to, rheumatoid arthritis, Crohn's disease, type 1 diabetes, alopecia, multiple sclerosis, lupus, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Cuban-Jackson syndrome, pemphigus (e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune infertility, glomerulonephritis (e.g., rapidly progressive glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjögren's syndrome, insulin resistance, and autoimmune diabetes.

[0087] Excessive or prolonged Cx43 hemichannel opening during chronic neurodegenerative diseases has also been shown to promote disease progression by interfering with metabolic gradients and excessive release of toxic molecules to induce cell death. See, for example, Orellana et al., (2011), Neurodegenerative Diseases - Progress, Prevention, Protection and Monitoring, Chapter 10, The 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, No. 1060, doi: 10.3389 / fphys.2017.01060; all are fully incorporated herein by reference. Thus, inhibition or reduction of Cx43 hemichannel opening using the compositions disclosed herein can be used to treat a variety of neurodegenerative diseases, such as Alzheimer's disease (AD), lysosomal storage diseases, bacterial meningitis, amyotrophic lateral sclerosis, hypoxia, ischemia, glaucoma, schizophrenia, major depression, bipolar disorder, epilepsy, brain trauma, 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, opiates, methamphetamine, phencyclidine, and cocaine).

[0088] Anti-Cx43 antibody

[0089] Inhibiting or reducing the opening of Cx43 hemichannels can reduce or inhibit the opening of Cx43 hemichannels in cells, thereby treating, for example, inflammatory diseases or disorders or neurodegenerative diseases. Thus, anti-Cx43 antibodies can be used as effective agents in anti-inflammatory and / or neuroprotective treatments.

[0090] 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, 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 on a human Cx43 protein (e.g., an extracellular loop or a portion thereof).

[0091] Exemplary anti-Cx43 antibodies may have one or more of the following CDR sequences:

[0092] Heavy Chain:

[0093] CDR1 (SEQ ID NO.: 1): GYTFTSYY

[0094] CDR2 (SEQ ID NO.: 2): INPSNAGT

[0095] CDR3(SEQ ID NO.:3):TREGNPYYTMNY

[0096] Light chain:

[0097] CDR1(SEQ ID NO.:4):QSLLNSGNQKTY

[0098] CDR2 (SEQ ID NO.: 5): GAS

[0099] CDR3 (SEQ ID NO.: 6): QNDHSYPFT

[0100] In some embodiments, it has been surprisingly found that antibodies with the above CDR sequences show excellent binding affinity and / or antibody stability compared to the antibodies disclosed in PCT Publication Nos. WO 2015 / 027120 and WO 2017 / 147561. Without wishing to be bound by theory, it is believed that the "NG" to "NA" mutation in the heavy chain CDR2 can reduce deamidation. Deamidation of antibodies, particularly in the CDR region, may lead to changes in binding affinity, antibody degradation, and charge variant changes, thereby affecting antibody function and increasing antibody production costs. In this way, the CDR disclosed herein provides improved binding affinity and antibody stability, resulting in advantageous technical effects superior to those disclosed in PCT Publication Nos. WO 2015 / 027120 and WO 2017 / 147561.

[0101] Monoclonal antibodies can be humanized and optimized using, for example, CDR grafting, germline modeling, and 3-D structural analysis to increase the drugability and / or developability of the antibody. In some embodiments, the anti-Cx43 antibody after humanization can have one or two of the following variable domains:

[0102] Heavy chain variable region (SEQ ID NO.: 7):

[0103] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGGINPSNAGTNFNEKFKNRATLTVDKSTSTAYMELSSLRSEDTAVYYCTREGNPYYTMNYWGQGTLVTVSS

[0104] Light chain variable region (SEQ ID NO.: 8):

[0105] DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKTYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPFTFGQGTKLEIK

[0106] In the embodiment of selection, anti-Cx43 antibody can have the variable domain of the constant region of fusion to human IgG1, IgG2 or IgG4, for example, which can optionally include one or more mutations. In some embodiments, mutations can be designed to reduce or minimize the cytotoxic effector function of the antibody while maintaining binding affinity and antibody stability. For example, anti-Cx43 antibody can have one or more following heavy chain sequences (wherein the bold portion corresponds to the variable region, and the non-bold portion corresponds to the constant region):

[0107] >Ab#C heavy chain (SEQ ID NO.: 9)

[0108]

[0109] >Ab#E heavy chain (SEQ ID NO.: 10)

[0110]

[0111] >Ab#G heavy chain (SEQ ID NO.: 11)

[0112]

[0113] >Ab#I heavy chain (SEQ ID NO.: 12)

[0114]

[0115]

[0116] >Ab#K heavy chain (SEQ ID NO.: 13)

[0117]

[0118] >Ab#M heavy chain (SEQ ID NO.: 14)

[0119]

[0120] >Ab#0 heavy chain (SEQ ID NO.: 15)

[0121]

[0122] >Ab#Q heavy chain (SEQ ID NO.: 16)

[0123]

[0124] >Ab#S heavy chain (SEQ ID NO.: 17)

[0125]

[0126] In some embodiments, the anti-Cx43 antibody may have one or more of the following light chain sequences (wherein the bold portion corresponds to the variable region and the non-bold portion corresponds to the constant region):

[0127] >Light chain of Ab#E, Ab#G, Ab#I, Ab#K, Ab#M, Ab#O, Ab#Q, Ab#S (SEQ ID NO.: 18)

[0128]

[0129] In yet another embodiment, the anti-Cx43 antibody may comprise a mixture or cocktail of two or more anti-Cx43 antibodies, each anti-Cx43 antibody binding to the same or a different epitope on Cx43.

[0130] In some embodiments, bispecific antibodies can be prepared in which at least one specificity is an anti-Cx43 antibody or antigen-binding fragment thereof disclosed herein. The other specificity can be directed to another target associated with the inflammatory disease or neurodegenerative disease being treated.

[0131] In one aspect, a use of a Cx43 ligand for preparing a medicament for treating an inflammatory disease or a neurodegenerative disease is provided. In another aspect, a method of inhibiting inflammation or neurodegeneration in a patient is provided, the method comprising administering an effective amount of a Cx43 ligand to the patient.

[0132] Preparation of anti-Cx43 antibody

[0133] Anti-Cx43 antibodies can be prepared using various methods generally known in the art. For example, phage display technology can be used to screen human antibody libraries to produce fully human monoclonal antibodies for treatment. High-affinity adhesives can be considered as candidates for neutralization studies. In addition, conventional monoclonal methods can be used, wherein human proteins can be used, identified and tested candidate binding substances, and humanized antibodies immunized mice or rabbits that are ultimately produced by transplanting the binding sites of heavy and light chains into human antibody coding sequences.

[0134] Antibodies typically comprise two pairs of identical polypeptide chains, 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 comprises a variable region of about 100-110 or more amino acids, which is typically responsible for antigen recognition. The carboxyl-terminal portion of each chain is defined as a constant region, which is typically responsible for effector function. The variable region of each of the heavy and light chains typically exhibits the same overall structure, including four relatively conserved framework regions (FRs) connected by three hypervariable regions (also called complementarity determining regions or CDRs). The CDRs of each pair of two chains are typically aligned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is generally consistent with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

[0135] With the development of monoclonal antibodies, antibodies have become useful and interesting drugs as drugs. Monoclonal antibodies can be produced using any method that produces antibody molecules through continuous cell lines 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 Technology and Applications, Marcel Dekker, Inc., New York, pp. 51-63).

[0136] Monoclonal antibodies can be modified for use as therapeutic agents. One example is a "chimeric" antibody, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Other examples are 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 a "CDR-grafted" antibody, in which the antibody comprises one or more complementary determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass.

[0137] Another development is "humanized" antibodies. 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 Cécile Vincke et al., J. Biol. Chem. 2009; 284: 3273-3284 for humanized lambda antibodies). Typically, humanized antibodies are produced by non-human animals, and then certain amino acid residues, typically from the non-antigen recognition portion of the antibody, are modified to be homologous to the residues in the corresponding isotype human antibody. Humanization can be performed, 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 replacing the corresponding region of a human antibody with at least a portion of a rodent variable region.

[0138] A recent development is human antibodies that are not exposed to human antigens ("fully human antibodies"). Transgenic animals (e.g., mice) that are capable of producing a human antibody repertoire without producing endogenous mouse immunoglobulins are used to produce such antibodies by immunization with an antigen (usually having at least 6 consecutive amino acids), which may optionally be conjugated to a carrier. 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 loci encoding mouse heavy and light chain immunoglobulin chains and inserting loci encoding human heavy and light chain proteins into their genomes. Animals with partial modifications that are less than all modifications are then hybridized to obtain animals with all desired immune system modifications. After administration of the immunogen, the antibodies produced by these transgenic animals are immunospecific for these antigens having human (rather than mouse) amino acid sequences (including variable regions). See PCT Publication Nos. WO96 / 33735 and WO94 / 02602, which are incorporated by reference. Other methods are described in U.S. Patent No. 5,545,807, PCT Publication No. WO91 / 10741, WO90 / 04036, and EP 546073B1 and EP546073A1, which are incorporated by reference. As described herein, human antibodies can also be produced by expressing recombinant DNA in host cells or expressing in hybridoma cells.

[0139] In some embodiments, phage display technology can be used to screen therapeutic antibodies. In phage display, antibody libraries can be displayed on the surface of filamentous phage, and the constructed library can be screened for phages combined with immunogens. Antibody phages are based on the genetic engineering of phages and repeated experiments of selection and phage breeding guided by antigens. This technology allows in vitro selection of Cx43 monoclonal antibodies. The phage display process begins with the preparation of the antibody library, and then variable heavy (VH) and variable light (VL) PCR products are connected to the phage display vector, and the clones of the monoclonal antibodies are finally analyzed. The VH and VL PCR products representing the antibody library are connected to the phage display vector (e.g., phagemid pComb3X), which can express VH and VL as scFv on the pIII minor capsid protein of the Escherichia coli filamentous phage originally derived from M13 phage through engineering. However, the phage display vector pComb3X does not have all the other genes necessary for encoding complete phages in Escherichia coli. For those genes, helper phage is added to the Escherichia coli transformed with the phage display vector library. The result is a phage library, each phage expresses Cx43 monoclonal antibody on its surface, and carries a carrier with a respective nucleotide sequence. Phage display can also be used to produce Cx43 monoclonal antibody itself (not connected to phage capsid protein) in some Escherichia coli strains. After VL and VH sequences, other cDNAs are engineered in phage display vectors to characterize and purify the mAb produced. Specifically, recombinant antibodies can have hemagglutinin (HA) epitope tags and polyhistidine to be easy to purify from solution.

[0140] The multi-antibody phage library consists of about 10 8 coli transformants. Using biopanning, libraries can be screened for phage binding to the above-mentioned immunogen sequences or fragments thereof through the surface of monoclonal antibody expression. Cyclic panning can extract antigen-binding clones that may be very rare and consists of multiple rounds of phage binding to antigen (immobilized on ELISA plates or in solution on the cell surface), washing, elution and re-amplification of phage binders in E. coli. In each round, specific binders are selected from the pool by washing away non-binders and selectively eluting bound phage clones. After three or four rounds, highly specific binding of phage clones by their surface Cx43 monoclonal antibodies is a feature of directed selection on immobilized immunogens.

[0141] Another approach is to add a C-terminal His tag suitable for purification by affinity chromatography to the immunogen sequence listed above. The purified protein can be inoculated into mice with a suitable adjuvant. The monoclonal antibodies produced in the hybridomas can be tested for binding to the immunogen, and positive binders can be screened as described in the assay herein.

[0142] Fully human antibodies can also be generated 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 immune selection by displaying antibody repertoires on the surface of filamentous phage and subsequently selecting by binding to a selected antigen. One such technique is described in PCT Publication No. WO 99 / 10494, which is incorporated herein by reference, which describes the use of this method to isolate high affinity and functional agonistic antibodies for MPL- and msk-receptor antibodies.

[0143] The nucleotide sequence encoding the above-mentioned antibody can be determined. Thereafter, chimeric, CDR-transferred, humanized and fully human antibodies can also be produced by recombinant methods. The nucleic acid encoding the antibody can be introduced into a host cell and expressed using materials and methods generally known in the art.

[0144] Antibodies to Cx43 are provided herein. Preferred antibodies bind to Cx43. In preferred embodiments, the present invention provides nucleotide sequences encoding heavy and light chain immunoglobulin molecules and amino acid sequences comprising them, particularly sequences corresponding to their variable regions. In preferred embodiments, sequences corresponding to CDRs, particularly from CDR1 to CDR3, are provided. In further embodiments, the present disclosure provides hybridoma cell lines expressing such immunoglobulin molecules and monoclonal antibodies produced therefrom, preferably purified human monoclonal antibodies to human Cx43.

[0145] The CDRs of the light chain and heavy chain variable regions of the anti-Cx43 antibodies of the present disclosure can be transferred to the framework region (FR) from the same or another species. In certain embodiments, the CDRs of the light chain and heavy chain variable regions of the anti-Cx43 antibodies can be transferred to the common human FR. In order to produce the common human FR, the FRs from several human heavy chain or light chain amino acid sequences are compared to identify the common amino acid sequence. The FRs of the anti-Cx43 antibody heavy chain or light chain can be replaced by the FRs from different heavy chains or light chains. The rare amino acids in the anti-Cx43 antibody heavy chain and light chain FRs are usually not replaced, while the remaining FR amino acids can be replaced. Rare amino acids are specific amino acids, which are located in the position that is not usually found in the FR. The variable regions transferred from the anti-Cx43 antibodies of the present disclosure can be used together with the constant regions that are different from the constant regions of the anti-Cx43 antibodies. Or, the variable regions transferred are part of single-chain Fv antibodies. CDR grafting is described in, e.g., U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference for any purpose.

[0146] In some embodiments, the antibodies of the present invention can be produced by hybridoma cell lines. In these embodiments, the antibodies of the present invention bind Cx43 with a dissociation constant (KD) between about 4pM and 1 μM. In certain embodiments of the present disclosure, the antibodies bind Cx43 with a KD less than about 100nM, less than about 50nM or less than about 10nM.

[0147] In an embodiment, the antibodies of the present disclosure are IgG1, IgG2, IgG3 or IgG4 isotypes, such as IgG1 isotypes. In certain embodiments, the antibodies comprise human kappa or lambda light chains and human IgG1, IgG2 or IgG4 heavy chains. In an embodiment, the variable region of the antibody is connected to a constant region of an IgG1, IgG2 or IgG4 isotype. In an embodiment, the variable region of the antibody is connected to a constant region other than a constant region of an IgG1, IgG2 or IgG4 isotype. In certain embodiments, the antibodies of the present disclosure have been cloned for expression in mammalian cells.

[0148] In alternative embodiments, the antibodies of the present invention can be expressed in cell lines other than hybridoma cell lines. In these embodiments, the sequence encoding a specific antibody can be used to transform a suitable host cell, such as a mammalian cell. According to these embodiments, any known method for introducing polynucleotides into a host cell can be used to achieve transformation, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with a virus (or vector) or by transfection methods known in the art. Such methods are, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (all of which are incorporated herein by reference for any purpose). Generally, the transformation method used may depend on the host to be transformed. The method of introducing heterologous polynucleotides into mammalian cells is well known in the art, including but not limited to dextran-mediated transfection, calcium phosphate precipitation, polyene-mediated transfection, protoplast fusion, electroporation, polynucleotides are encapsulated in liposomes, and DNA is directly microinjected into the nucleus.

[0149] According to certain embodiments of the disclosed method, 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 the disclosed Cx43 antibody is inserted into a suitable expression vector using standard connection techniques. In a preferred embodiment, the Cx43 antibody heavy chain or light chain constant region is attached to the C-terminus of the suitable variable region and connected to the expression vector. The vector is usually selected to work in the specific host cell used (i.e., the vector is compatible with the host cell mechanism, so that gene amplification and / or gene expression can occur). For a review of expression vectors, see Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press. New York.

[0150] Typically, the expression vector used in any host cell may contain sequences for plasmid maintenance and for cloning and expressing 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 comprising donor and acceptor splice sites, a sequence encoding a polypeptide secretion leader sequence, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. These sequences are well known in the art.

[0151] The expression vectors disclosed herein can be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all desired flanking sequences. When one or more of the flanking sequences are not already present in the vector, they can be obtained separately and connected to the vector. Methods for obtaining each flanking sequence are well known to those skilled in the art.

[0152] After constructing the vector and inserting the nucleic acid molecule encoding light chain or heavy chain or light chain and heavy chain of anti-Cx43 antibody into the appropriate site of the vector, the complete vector can be inserted into the cell for amplification and / or polypeptide expression in a suitable host. The expression vector of anti-Cx43 antibody can be transformed into the selected host cell by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection or other known techniques. The method of selection will depend in part on the type of host cell used. These methods and other suitable methods are well-known to technicians, and are, for example, proposed in Sambrook et al. above.

[0153] When the host cells are cultured under appropriate conditions, the anti-Cx43 antibody will be synthesized and can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells that produced it (if it is not secreted). The selection of an appropriate host cell will depend on a variety of factors, such as the desired expression level, the polypeptide modifications that are desirable or necessary for activity (e.g., glycosylation or phosphorylation), and the ease with which it folds into a biologically active molecule.

[0154] Mammalian cell lines that can be used as expression hosts are well known in the art, including but not limited to many immortalized cell lines that can be obtained from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cell, HeLa cell, hamster kidney (BHK) cell, human embryonic kidney cell (HEK), monkey kidney cell (COS), human hepatocellular carcinoma cell (such as Hep G2) and many other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high expression levels and produce antibodies with constitutive Cx43 binding properties. In another embodiment, cell lines (such as mouse myeloma cell lines NS0 and SP2 / 0) that do not produce their own antibodies but have the ability to produce and secrete heterologous antibodies can be selected from B cell lineage.

[0155] Epitope mapping and related technologies

[0156] The present disclosure provides anti-Cx43 antibodies that interact with one or more amino acids found in one or more domains of the Cx43 molecule, such as an extracellular loop. The epitope to which the antibody binds may include one or more continuous sequences of 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acids located within one or more extracellular loops. Alternatively or additionally, the epitope may include one or more non-continuous amino acids (or amino acid sequences) located within one or more extracellular loops (e.g., conformational epitopes).

[0157] Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids within a polypeptide or protein." Exemplary techniques include, for example, conventional cross-blocking experiments, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, New York). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and antigen chemical modification can be used (Tomer (2000) Prot. Sci. 9: 487-496).

[0158] Another method that can be used to identify amino acids in polypeptides that interact with antibodies is to detect hydrogen / deuterium exchange by mass spectrometry. In general, the hydrogen / deuterium exchange method involves deuterium labeling of the target protein, and then the antibody is combined with the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons in the amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons in the amino acids that do not belong to the interface. As a result, the amino acids that form part of the protein / antibody interface may retain deuterium, so their mass is relatively high compared to the amino acids that are not included in the interface. After the antibody dissociates, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues that correspond to specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

[0159] Modification Assisted Analysis (MAP), also known as Antigen Structure-Based Antibody Analysis (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen based on the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, incorporated herein by reference in its entirety). Each class can reflect a unique epitope that is significantly different from or partially overlaps with the epitope represented by another class. The technique can rapidly filter genetically identical antibodies, allowing characterization to focus on genetically different antibodies. When used for hybridoma screening, MAP may help identify rare hybridoma clones that produce mAbs with desired properties. MAP can be used to classify the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0160] The present disclosure provides anti-Cx43 antibodies that bind to the same epitope or a portion of an epitope. Likewise, the present disclosure also includes anti-Cx43 antibodies that compete with any specific exemplary antibody described herein for binding to Cx43 or its fragment. For example, the present disclosure includes anti-Cx43 antibodies that cross-compete with one or more antibodies obtained from those antibodies described herein for binding to Cx43.

[0161] By using conventional methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference anti-Cx43 antibody or competes to bind to it. For example, in order to determine whether a test antibody binds to the same epitope as a reference anti-Cx43 antibody of the present invention, the reference antibody can be made to bind to Cx43 or 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 with reference to the anti-Cx43 antibody, it can be concluded that the test antibody is different from the epitope bound to the reference anti-Cx43 antibody. On the other hand, if the test antibody cannot bind to Cx43 after saturation binding with reference to the anti-Cx43 antibody, the test antibody can bind to the same epitope as the reference anti-Cx43 antibody of the present disclosure.

[0162] In order to determine whether the antibody is combined with the reference anti-Cx43 antibody competition, the above-mentioned binding method can be performed in two directions: in the first direction, the reference antibody can be made to bind to Cx43 under saturation conditions, and then the combination of the test antibody and the Cx43 molecule is evaluated. In the second direction, the test antibody can be made to bind to Cx43 under saturation conditions, and then the combination of the reference antibody and the Cx43 molecule is evaluated. If only the first (saturated) antibody can bind to the Cx43 molecule in both directions, it can be concluded that the test antibody and the reference antibody compete for binding to Cx43. As will be understood by those of ordinary skill in the art, the antibody that competes for binding with the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may block the binding of the reference antibody in space by binding to overlapping or adjacent epitopes.

[0163] If two antibodies each competitively inhibit (block) the binding of the other antibody to the antigen, then the two antibodies will bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, a 1, 5, 10, 20 or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90% or even 99% (see, e.g., Junghans et al., Cancer Res. 1990 50: 1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the epitopes of the two antibodies overlap.

[0164] Other routine experiments (e.g., peptide mutations and binding analysis) can then 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 blocking (or other phenomena) is responsible for the lack of observed binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0165] In various embodiments, provided herein are antibodies that bind to an epitope located within the amino acid sequence FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may include one or more amino acids selected from: 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 some embodiments, the epitope may consist of all 10 amino acids of SEQ ID NO: 19.

[0166] Pharmaceutical composition and use thereof

[0167] In another aspect, there is provided a pharmaceutical composition useful in the methods disclosed herein, ie, for inhibiting the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating an inflammatory disease or condition or a neurodegenerative disease.

[0168] In some embodiments, the pharmaceutical composition comprises a Cx43 ligand and a pharmaceutically acceptable carrier. The Cx43 ligand can be formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition can include instructions for, for example, using the composition to treat a patient to inhibit the opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for the treatment of inflammatory diseases or conditions or neurodegenerative diseases.

[0169] In one embodiment, the Cx43 ligand can be an anti-Cx43 antibody or an antigen-binding fragment thereof.

[0170] As used herein, "pharmaceutically acceptable carriers" include 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., small molecule or biologic) may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0171] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, and conventional excipients for the preparation of tablets, pills, capsules, etc. The use of such media and agents for the preparation of pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, these media or agents should be considered for use in the pharmaceutical compositions provided herein. Supplementary active compounds may also be incorporated into the compositions.

[0172] The pharmaceutically acceptable carrier may include 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, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0173] Examples of suitable aqueous and non-aqueous vehicles 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, and injectable organic esters, such as ethyl oleate. When necessary, appropriate fluidity can be maintained by maintaining the desired particle size in the case of dispersions, and by using surfactants, for example, by using a coating material, such as lecithin. In many cases, isotonic agents are usefully included in the compositions, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Substances that delay absorption such as monostearate and gelatin in the compositions can prolong the absorption of injectable compositions.

[0174] These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifiers and dispersants.

[0175] The therapeutic compositions typically should be sterile, pyrogen-free, and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0176] The required amount of active compound and one of the above-mentioned components or a combination thereof can be mixed with a suitable solvent as required and then sterilized by, for example, filtration, to prepare a sterile injection. Typically, a dispersion can be prepared by adding an active activator to a sterile carrier containing an alkaline dispersion medium and the above-mentioned other required components. In the case of sterile powders for the preparation of sterile injections, the preparation method includes vacuum drying and freeze drying (freeze drying) to obtain a powder of the active ingredient and any other required components obtained by a solution previously aseptically filtered therefrom. Active agents can be mixed with other pharmaceutically acceptable carriers and any preservatives, buffers or propellants that may be required under aseptic conditions.

[0177] Prevention of the presence of microorganisms can be ensured by sterilization procedures (see above) and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0178] Pharmaceutical compositions comprising Cx43 ligands can be administered alone or in combination therapy. For example, combination therapy may include a composition provided herein comprising a Cx43 ligand and at least one or more other therapeutic agents, such as one or more additional therapeutic agents known in the art (e.g., anti-inflammatory drugs), which will be discussed in further detail below.

[0179] Dosage regimens may be adjusted to provide the optimum desired effect (eg, therapeutic effect), for example, by a single bolus, divided doses over time or by proportionally reduced or increased doses as indicated by the exigencies of the therapeutic situation.

[0180] Exemplary dosage ranges for administering 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 -800mg / kg, 50-600mg / kg, 50-400mg / kg, 50-200mg / kg, 100-1000mg / kg, 100-900mg / kg, 100-800mg / kg, 100-700mg / kg, 100-600mg / kg, 100-500mg / kg, 100-400mg / kg, 100-300mg / kg and 100-200mg / kg. Exemplary dosage schedules include once every three days, once every five days, once every seven days (i.e. once a week), once every ten days, once every fourteen days (i.e. once every two weeks), once every twenty-one days (i.e. once every three weeks), once every 28 days (i.e. once every four weeks) and once a month.

[0181] It is advantageous to formulate parenteral compositions in unit dosages for ease of administration and uniformity of dosage. As used herein, unit dosage refers to physically discrete units intended for individual dosages for the patient to be treated, each unit containing a predetermined amount of active agent and the required pharmaceutical carrier, the predetermined amount being calculated to produce any desired therapeutic effect. The specification for the unit dosage form of the present invention is dictated by and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of compounding such active compound for therapeutic sensitivity in an individual.

[0182] Actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. For administration, the term "parenteral administration" as used herein refers to forms of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion administration.

[0183] As used herein, the terms "parenteral administration" and "parenteral administration" refer to modes of administration other than enteral (i.e., through the digestive tract) and topical administration, usually by injection or infusion, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Intravenous injection and infusion are usually (but not limited to) used for antibody administration.

[0184] When the agents provided herein are administered to humans or animals as drugs, they can be administered alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (eg, 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0185] In one aspect, the improved efficacy of a combination according to the present disclosure may be demonstrated by achieving therapeutic synergy.

[0186] The term "therapeutic synergy" is used when the combination of two products at a given dose is better than the best effect of each of the two products at the same dose. In one example, therapeutic synergy can be assessed by comparing the combination to the best single agent using estimates obtained from a two-way analysis of variance for repeated measurements of the desired parameter (e.g., time factor).

[0187] The term “additive” means that the combination of two or more products at a given dose is equivalent to the sum of the effects achieved by the two or more products individually, while the term “superadditive” means that the combination is more effective than the sum of the effects achieved by the two or more products when used together.

[0188] Also provided herein is a method for inhibiting the opening of a Cx43 hemichannel in a cell, preferably for treating an inflammatory disease or condition or a neurodegenerative disease, comprising administering any one or more anti-Cx43 antibodies disclosed herein to a subject in need thereof.

[0189] In various embodiments, the methods disclosed herein can include administering to a subject an effective amount of an anti-Cx43 antibody or antigen-binding fragment thereof. Generally, an effective amount can be administered therapeutically and / or prophylactically.

[0190] Treatment may be appropriately administered to subjects, particularly humans, who are suffering from, have, are susceptible to, or are at risk of developing such diseases. Those subjects who are "at risk" may be arbitrarily determined objectively or subjectively by diagnostic tests or opinions of the subject or health care provider (e.g., genetic tests, enzyme or protein markers, family history, etc.). Identifying subjects in need of such treatment may rely on the judgment of the subject or health care professional, and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods).

[0191] Drug administration

[0192] The formulations of the present disclosure, including but not limited to reconstituted and liquid formulations, can be administered to a mammal, preferably a human, in need of treatment with an anti-Cx43 antibody according to known methods, for example, as a bolus intravenous push, or continuous infusion over a period of time, or by intramuscular, intraventricular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical or inhalation routes.

[0193] In embodiments, the formulation is administered to a mammal by intravenous or subcutaneous administration (i.e., under the skin). The formulation may be injected using a syringe for this purpose. However, other devices for administering the formulation may also be used, such as injection devices (e.g., INJECT-EASE™ and GENJECT TM device); sample injection pen (e.g., GENPEN TM ); auto-injector devices, needle-free devices (e.g. MEDIJECTOR TM and BIOJECTOR TM ); and subcutaneous patch delivery systems.

[0194] In a specific embodiment, the present disclosure relates to a kit for a single-dose dosing unit. Such a kit includes a container for an aqueous formulation of a therapeutic protein or antibody, including a single-chamber or multi-chamber prefilled syringe. Exemplary prefilled syringes are available from Vetter GmbH of Ravensburg, Germany.

[0195] The appropriate dose of the protein ("therapeutically effective amount") will depend on, for example, the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to anti-Cx43 antibodies, the form of the formulation used, and the discretion of the attending physician. The anti-Cx43 antibody may be appropriately administered to the patient at one time or over a series of treatments, and may be administered to the patient at any time from diagnosis. The anti-Cx43 antibody may be administered alone or in combination with other drugs or therapies useful for treating the disease.

[0196] For anti-Cx43 antibodies, the initial candidate dose for administration to the patient can be in the range of about 0.1-100 or 1-20 mg / kg, which can be taken in the form of one or more separate administrations. However, other dosage regimens may be useful. The progress of this treatment can be easily monitored by conventional techniques.

[0197] According to certain embodiments of the present disclosure, multiple doses of anti-Cx43 antibodies (or pharmaceutical compositions comprising a combination of anti-Cx43 antibodies and any other therapeutically active agents mentioned herein) can be administered to an object over a period of time. The method according to this aspect of the present disclosure includes sequentially administering multiple doses of anti-Cx43 antibodies of the present disclosure to an object. As used herein, "sequential administration" refers to the administration of each dose of anti-Cx43 antibodies to an object at different time points (e.g., on different days separated by predetermined intervals (e.g., hours, days, weeks or months). The present disclosure includes the following method, which includes: sequentially administering a single dose of an initial dose of anti-Cx43 antibodies to a patient, then sequentially administering one or more doses of anti-Cx43 antibodies, and optionally subsequently administering one or more third doses of anti-Cx43 antibodies. Anti-Cx43 antibodies can be administered at a dosage between 0.1 mg / kg and about 100 mg / kg.

[0198] The terms "initial dose", "second dose" and "third dose" refer to the time order of administration of the anti-Cx43 antibodies of the present invention. Therefore, the "initial dose" is the dose given at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "second dose" is the dose given after the initial dose; the "third dose" is the dose given after the second dose. The initial, second and third doses can all contain the same amount of anti-Cx43 antibodies, but may generally differ from each other in the frequency of administration. However, in certain embodiments, during the course of treatment, the amount of anti-Cx43 antibodies contained in the initial, second and / or third doses differs from each other (e.g., appropriately adjusted upward or downward). In certain embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered at the beginning of the treatment regimen with a "loading dose", followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency.

[0199] In certain exemplary embodiments of the present disclosure, each second and / or third dose is administered 1 to 26 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 1 / 2,2,2 1 / 2,3,3 1 / 2,4,4 1 / 2,5,5 1 / 2,6,6 1 / 2,7,7 1 / 2,8,8 1 / 2,9,9 1 / 2, 10, 10 1 / 2,11,11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2,14,14 1 / 2, 15, 15 1 / 2, 16, 161 / 2, 17, 17 1 / 2, 18, 18 1 / 2,19,19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "the immediately preceding dose" refers to a dose of an anti-Cx43 antibody in a sequence of multiple doses that is administered to a patient without intervening intervention prior to administration of the next dose in the sequence.

[0200] The method according to this aspect of the present disclosure may include applying any number of second doses and / or third doses of anti-Cx43 antibodies to the patient. For example, in certain embodiments, only a single second dose is applied to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are applied to the patient. For example, in certain embodiments, only a single third dose is applied to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are applied to the patient.

[0201] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1-2 weeks or 1-2 months after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered to the patient at the same frequency as other third doses. For example, each third dose can be administered to the patient 2-12 weeks after the previous dose. In certain embodiments of the present disclosure, the frequency of administering the second and / or third dose to the patient can change during the treatment regimen. The frequency of administration can also be adjusted during the doctor's treatment according to the needs of each patient after clinical examination.

[0202] The present disclosure includes an administration regimen in which 1-10 or 2-6 loading doses are administered to the 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), and then two or more maintenance doses are administered to the patient at a lower frequency. For example, according to this aspect of the present disclosure, if a loading dose is administered at a frequency of, for example, once a month (e.g., two, three, four or more loading doses once a month), the maintenance dose can be administered to the patient once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, etc.

[0203] Example

[0204] The following examples, including the experiments performed and the results obtained, are for illustrative purposes only and should not be construed as limiting the present disclosure.

[0205] Example 1: Antibody Binding Affinity

[0206] The optimized antibody sequences were tested for binding affinity to Cx43 according to the following protocol.

[0207] The equilibrium dissociation constant (K) of Cx43 binding to selected purified anti-Cx43 antibodies was determined using a real-time biolayer interferometer-based biosensor (Octet HTX) assay. D Octet streptavidin (SA) biosensors coated with biotinylated antigen (0.01 ug / mL) were used to capture anti-Cx43 antibodies. The loaded sensors were immersed in a two-fold dilution series of purified control antibodies (starting at 100 nM). All binding studies were performed in assay buffer (PBS (pH 7.2) containing 0.1% BSA, 0.02% Tween-20) at 25°C with the plate shaken at 1000 rpm. The kinetic association (K) was determined by processing the data and fitting it to a 1:1 binding model using Scrubber 2.0c curve fitting software. a ) and dissociation (K d ) rate constant. Binding and dissociation equilibrium constant (K D ) was calculated from the kinetic rate constant as:

[0208] K D (M) = Kd / Ka

[0209] The binding affinity results (Table 1) indicate that binding affinity was generally at least maintained and, in many cases, surprisingly improved.

[0210] Table 1. Binding affinity of various antibodies

[0211] Sample No. <![CDATA[K D (M)]]> Ka(1 / Ms) Kd(1 / s) Comparison 8.84E-09 1.87E+05 1.65E-03 Ab#E 2.15E-09 2.10E+05 4.51E-04 Ab#C 2.76E-09 2.54E+05 7.03E-04 Ab#G 2.31E-09 1.91E+05 4.42E-04 Ab#I 3.06E-09 2.00E+05 6.11E-04 Ab#M 2.98E-09 2.00E+05 5.95E-04 Ab#K 2.83E-09 2.22E+05 6.29E-04 Ab#O 3.36E-09 2.01E+05 6.77E-04 Ab#Q 2.53E-09 2.55E+05 6.47E-04 Ab#S 3.42E-09 1.96E+05 6.71E-04

[0212] Example 2. Fc receptor binding analysis

[0213] Fc effector functions are mediated by the binding of Fc to receptors. Receptors include FcRI, FcRIIa, FcRIIb, FCRIIIa, FCRIIIb, Clq and FcRn. It is generally desirable to reduce the binding affinity to most Fc receptors, which is expected to minimize the potential in vivo toxicity of FcRn while maintaining the antibody half-life. The following surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) protocols are used to test the binding of various antibodies to different Fc receptors.

[0214] A. FCRI binding

[0215] Experiment: Biacore 8K

[0216] Chip: CM5

[0217] (1) Immobilization

[0218] The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The mixture activated the CM5 sensor chip for 420 seconds. Then 30 μg / mL of THE in 10 mM NaAc (pH 4.5) was added. TM His tag antibody was injected into channels 1-8 at 30 μL / min for 400 seconds. The chip was inactivated with 1 M ethanolamine-HCl (GE).

[0219] (2) Capture ligand and run analyte

[0220] 2μg / mL CD64 in running buffer (1xHBS-EP+) was injected into Fc2 of channels 1-4 at 10μL / min for 30 seconds. Six concentrations (40, 20, 10, 5, 2.5 and 1.25nM) of analyte 20170905-Ab#C, AB#G and running buffer were injected into Fc1 to Fc2 of channels 1-4 at 30μL / min, with an association phase of 180 and then a dissociation phase of 400. The six cycles of capturing ligand and running analyte were repeated in ascending order according to the analyte concentration. 10mM glycine, pH 1.5, was injected as a regeneration buffer after each dissociation phase.

[0221] 2 μg / mL CD64 in running buffer (1xHBS-EP+) was injected into Fc2 of channels 1-6 at 10 μL / min for 30 seconds. Analytes Ab#K, Ab#O, Ab#S and running buffer at 8 concentrations (10240, 5120, 2560, 1280, 640, 320, 160 and 80 nM) were injected into Fc1 to Fc2 of channels 1-6 at 30 μL / min, with an association phase of 60 μL / min and then a dissociation phase of 90 μL / min. The 8 cycles of capturing ligand and running analyte were repeated in ascending order according to the analyte concentration. 10 mM glycine, pH 1.5, was injected as a regeneration buffer after each dissociation phase.

[0222] (3) Regeneration

[0223] The chip was regenerated with 10 mM glycine pH 1.5.

[0224] (4) Data analysis

[0225] The surface channel Fc1 without captured ligand was used as a control surface for reference subtraction. The final data for each interaction was subtracted from the reference channel and buffer channel data. The experimental data of AB#C, AB#G binding to CD64 were fitted by a 1:1 binding mode. The 10240nM curves of analytes Ab#K, Ab#O, and Ab#S were removed to obtain a better fit. The relative experimental data were fitted with steady-state affinity and are shown in Table 2 below.

[0226] Table 2. FCRI binding

[0227]

[0228] All antibodies showed low or no FCRI binding, which is advantageous.

[0229] B. Binding to FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb

[0230] Experiment: Biacore 8K

[0231] Chip: CM5

[0232] (1) Immobilization

[0233] The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The mixture activated the CM5 sensor chip for 420 seconds. Then 30 μg / mL of THE in 10 mM NaAc (pH 4.5) was added. TMHis tag antibody was injected into channels 1-8 at 30 μL / min for 400 seconds. The chip was inactivated with 1 M ethanolamine-HCl (GE).

[0234] (2) Capture ligand and run analyte

[0235] 1 μg / mL of FcγRIIa, FcγRIIb, FcγRIIIa or FcγRIIIb in running buffer (1xHBS-EP+) was injected into Fc2 of channels 1-8 at a flow rate of 10 μL / min for 15 seconds. The analytes were injected into channels 1-8 respectively. During the 60-second association phase, followed by a 90-second dissociation process, a series of analyte concentrations were monitored at a flow rate of 30 μL / min (see Table 3 below). 10 mM glycine, pH 1.5, was injected as a regeneration buffer after each dissociation phase.

[0236] Table 3. Analyte concentrations

[0237] Analytes Concentration tested (nM) Ab#C 0,160,320,640,1280,2560,5120,10240 other 0,320,640,1280,2560,5120,10240,20480,40960

[0238] (3) Regeneration

[0239] The chip was regenerated with 10 mM glycine pH 1.5.

[0240] (4) Data analysis

[0241] The surface channel Fc1 without captured ligand was used as a control surface for reference subtraction. The final data for each interaction was subtracted from the reference channel and buffer channel data. The experimental data for antibodies binding to FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb were fitted by steady-state affinity model and are shown in Table 4 below.

[0242] Table 4. FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb binding

[0243] Sample No. Binding to FcγRIIa Binding to FcγRIIb Binding to FcγRIIIa Binding to FcγRIIIb Ab#C 3.03E-06 9.72E-06 1.78E-06 4.31E-06 Ab#G 1.79E-05 1.32E-05 None / very weak binding None / very weak binding Ab#K None / very weak binding None / very weak binding 2.64E-05 None / very weak binding Ab#O 3.30E-05 1.78E-05 None / very weak binding None / very weak binding Ab#S None / very weak binding None / very weak binding None / very weak binding None / very weak binding

[0244] All antibodies showed low or no binding to FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, which is advantageous.

[0245] C. Binding to FcRn

[0246] Experiment: Biacore 8K

[0247] Chip: CM5

[0248] (1) Buffer exchange

[0249] According to the instruction manual, the buffer of human FcRn was exchanged into running buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 150 mM NaCl, 0.05% Tween 20, pH 6.0) using a desalting column, and the concentration was measured using Nanodrop.

[0250] (2) Immobilization

[0251] The activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection. The mixture was used to activate the CM5 sensor chip at a flow rate of 10 μL / min for 420 seconds. Then 5 μg / mL of 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 seconds. 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 seconds.

[0252] (2) Run Analyte

[0253] The analyte FcRn was injected into channels 1-8 respectively. During the 60-second association phase, followed by the 90-second dissociation process, 8 concentrations of FcRn (0, 93.75, 187.5, 375, 750, 1500, 3000 and 6000 nM) were monitored at a flow rate of 30 μL / min. After each round of interaction analysis cycle, the sensor chip surface was regenerated with 1xPBS (pH 7.4) at a flow rate of 10 μL / min for 30 seconds.

[0254] (3) Regeneration

[0255] The chip was regenerated with 1xPBS (pH 7.4).

[0256] (4) Data analysis

[0257] The surface channel Fc1 without immobilized antibody was used as a control surface for reference subtraction. The final data for each interaction were subtracted from the reference channel and buffer channel data. The relative experimental data were fitted with a steady state affinity model and are shown in Table 5 below.

[0258] Table 5. FcRn binding

[0259] Ligand <![CDATA[K D (M)]]> Ab#C 2.40E-06 Ab#G 2.39E-06 Ab#K 2.56E-06 Ab#O 2.57E-06 Ab#S 2.43E-06

[0260] All antibodies showed ideally similar FcRn binding.

[0261] D. Binding of C1q by ELISA

[0262] Plates (Nunc) were coated with 3 μg / mL of antibody at 4°C overnight. After blocking and washing, C1q was titrated 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 at room temperature for 2 hours. The plates were then washed and subsequently incubated with the secondary antibody sheep anti-human C1q Ab-HRP for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device) and is shown in Table 6 below.

[0263] Table 6. C1q binding

[0264] Antibody <![CDATA[K D ]]> Ab#C 61nM Ab#G No combination Ab#K Weak Binding Ab#O No combination Ab#S No combination

[0265] All antibodies showed low or no CIq binding, which is advantageous.

[0266] Example 3. Epitope mapping

[0267] To reconstruct the target molecular epitope, a peptide-based epitope mimetic library was synthesized using solid-phase Fmoc synthesis. Amino-functionalized polypropylene supports were obtained by grafting a proprietary hydrophilic polymer formulation followed by reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA), followed by cleavage of the Boc-group using trifluoroacetic acid (TFA). Peptides were synthesized on amino-functionalized solid supports using standard Fmoc-peptide synthesis methods using a custom-made modified JANUS liquid handling station (PerkinElmer).

[0268] The synthesis of structural mimetics was accomplished using the Chemically Ligated Peptides on Scaffolds (CLIPS) technique. CLIPS technology allows peptides to be structured as monocyclic, bicyclic, tricyclic, sheet-like folds, helical folds, and combinations thereof. CLIPS templates are coupled to cysteine ​​residues. The side chains of multiple cysteines in the peptide are coupled to one or two CLIPS templates. For example, 0.5 mM of P2 CLIPS (2,6-bis(bromomethyl)pyridine) solution is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (v / v)). This solution is added to the peptide array. The CLIPS template will bind to the side chains of two cysteines present in the solid-phase bound peptides of the peptide array (455-well plate with 3 μl wells). The peptide array is gently shaken in the solution for 30 to 60 minutes while it is completely covered in the solution. Finally, the peptide array was washed extensively with excess H2O and sonicated in a disruption buffer containing 1% SDS / 0.1% 2,2'-(ethylenedioxy)diethanethiol in PBS (pH 7.2) at 70°C for 30 min, followed by sonication in water for an additional 45 min. Peptides carrying T3 CLIPS were prepared in a similar manner, but now using three cysteines.

[0269] Different sets of peptides were synthesized according to the following designs. Please note that the actual order of the peptides on some of the microcards is randomized.

[0270]

[0271] The binding of the antibodies to each synthetic peptide was tested in an 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) at 25°C for 1 hour. After washing, the peroxidase substrate 2,2'-azido-di-3-ethylbenzothiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After one hour, the color development was measured. The color development was quantified using a charge coupled device (CCD)-camera and an image processing system.

[0272] The values ​​obtained from the CCD camera range from 0 to 3000mAU, similar to a standard 96-well plate ELISA reader. The results are quantified and stored in the laboratory database. Occasionally, wells will contain bubbles, resulting in false positive values, requiring manual inspection of the card and recording any values ​​caused by bubbles as 0.

[0273] To verify the quality of the synthesized peptides, an independent set of positive and negative control peptides were synthesized in parallel. Screening was performed using commercially available antibodies 3C9 and 57.9 (see Posthumus et al. (1990) J. Virol. 64: 3304-3309).

[0274] Figure 1 A graphical overview of the complete data set is given. The box plots here depict each data set and indicate the mean ELISA signal, distribution and outliers in each data set. Depending on the experimental conditions (antibody amount, blocking strength, etc.), different ELISA data distributions can be obtained.

[0275] The antibodies were tested under high stringency conditions and binding to the peptide array was obtained ( Figure 2 and Figure 3 ). The two peptide groups were analyzed separately.

[0276] The data recorded for the analysis of substitution variants with the leader sequence FLSRPTEKTI indicated that positions R4, P5, E7, K8, and I10 are critical for antibody binding and that many substitutions at these positions reduce signal intensity ( Figure 2 ), except for homologous substitutions such as R4K.

[0277] Analysis of the data recorded for truncated variants of the leader sequence FLSRPTEKTI indicated that the C-terminus of this sequence is necessary for binding to occur ( Figure 3 ), and the presence of at least 5 residues cannot tolerate substitution.

[0278] In summary, the antibody was tested on a peptide array containing two types of peptide variants derived from the leader sequence FLSRPTEKTI - a single residue mutant and a truncated variant. The antibody produced detectable binding under high stringency conditions. Antibody binding was sensitive to substitutions of residues R4, P5, E7, K8, and I10 and was biased toward the C-terminal portion of the leader sequence.

[0279] Example 4. Antibody stability

[0280] Antibody stability is an important factor affecting development, efficacy, production costs, etc. After sequence optimization, key stability parameters were evaluated. The species distribution profiles of various antibodies were tested under acidic and high temperature conditions. All antibodies showed improved stability.

[0281] SE-UPLC (Size Exclusion Ultra-Performance Liquid Chromatography)

[0282] Preparation: PBS, pH 6.5 or 7.2

[0283] Concentration (mg / mL): 5.28, 5.13, 5.00, 5.17, 5.01, 5.26, 4.92, 5.04, 4.99, 5.12 (all at about 5 mg / mL)

[0284] Conditions: Room temperature, acid treatment, then storage at 4°C for 1 week or storage at 40°C for 1 week

[0285] 2 μL of sample was injected into an ACQUITY UPLC Protein BEH SEC200, 1.7 μm, 4.6 x 150 mm column at a flow rate of 0.3 mL / min over 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, heating and storage conditions.

[0286] B. rCE-SDS (reduced capillary electrophoresis-sodium dodecyl sulfate)

[0287] Preparation: PBS, pH 6.5, 7.2, 6.2

[0288] Concentration (mg / mL): 0.5

[0289] Conditions: Room temperature, acid treatment, then storage at 4°C for 1 week or at 40°C for 1 week

[0290] Samples were prepared in reducing labeling buffer and submitted to the LabChip GXII system (PerkinElmer). All antibodies showed the desired stability under various pH, heating and storage conditions.

[0291] Example 5. Hemichannel opening experiment

[0292] A. In vitro experiments

[0293] The effects of the antibodies disclosed herein on hemichannel opening or blocking can be tested in vitro using, for example, a dye uptake assay. The dye can be a fluorescent tracer dye (eg, ethidium bromide or fluorescein yellow).

[0294] In one example, a fluid flow loop apparatus (FFLA) (parallel plate flow chamber) or a variation thereof can be used. The FFLA simulates the dynamic fluid microenvironment in bone to generate fluid flow shear stress (FFSS). Cells are cultured in a parallel plate flow chamber, exposing the cells to a steady laminar flow.

[0295] Osteocytes sense the mechanical stresses generated by FFSS in the osteocyte lumen / canalicular network. It has been proposed that osteolysis flow is driven by extravascular pressure as well as cyclic mechanical loads imposed by osteocytes, with a maximum physiological load of 8 to 30 dynes / cm 2 In some respects, FFSS levels were within the range of physiological values ​​reported in previous studies measuring intraosseous fluid flow. The magnitude of fluid shear stress can be altered by adjusting the column height of the flow circuit.

[0296] Experiments used to assess hemichannel function can use fluorescent tracer molecules that are small enough to pass through the hemichannel pore. If the hemichannel is closed, the molecule cannot pass through. If the hemichannel is open, the dye can pass through and cause the cell to fluoresce, allowing the fluorescence to be quantified. When ethidium bromide binds to DNA, it fluoresces. Once Lucifer Yellow is inside the cell, it fluoresces.

[0297] The dye transfer method may include exposing the cell to an extracellular fluorescent permeability tracer. The extracellular permeability tracer is a molecule retained outside the cell unless the cell membrane permeability increases under certain conditions. In some aspects, the mass of the tracer is less than 1, 2 or 3 kDa. In other aspects, the tracer will have a net charge. Such permeability tracers include but are not limited to anionic dye fluorescent yellow (LY; net charge = -1) and cationic probe ethidium bromide (Etd; net charge = +1), propidium iodide (PI; net charge = +2). After binding to DNA, the fluorescence of EtBr will be enhanced, thereby increasing the contrast and making it easier to identify. In some aspects, the extracellular dye is removed at different time periods or after applying a stimulus to open a hemichannel, and the fluorescence intensity retained in each cell is quantified. In some aspects, the fluorescence intensity is quantified in a snapshot image.

[0298] Substances used in in vitro assays to test hemichannel opening / blocking include:

[0299] Cells or Cell Lines Expressing Hemichannels Cells or cell lines expressing various connexin hemichannels can be obtained, isolated or engineered using methods and / or expression vectors known in the art.

[0300] Osteocytes: primary bone cells isolated from animals (including mice, rats, rabbits, chickens) or bone cell lines including but not limited to MLO-Y4 cells.

[0301] Osteoblasts: MLO-A5 osteoblasts were used as a control because they express connexin 43, but they do not appear to open upon alendronate stimulation.

[0302] Tracer molecules include, but are not limited to, fluorescein, ethidium bromide, Evans blue, Alexa350, Alexa488 and Alexa594.

[0303] Cx43(E2): The Cx43(E2) antibody is specific for Cx43 hemichannels. Cx43E2 binds to the second extracellular loop of the Cx43 hemichannel and prevents the hemichannel from opening.

[0304] Methods for determining whether an antibody opens or closes / blocks a hemichannel include one or more of the following steps:

[0305] (a) Isolate, obtain or produce cells or cell lines expressing connexins. For example, primary osteocytes can be isolated from bone ash. Other cell types can be isolated using other methods known in the art. In some aspects, skull osteocytes are isolated from animals (e.g., 16-day embryonic chicken skulls or newborn mice). The animal is decapitated, the skull is cut off and quickly immersed in 70% alcohol. The skull is then placed in αMEM and washed several times with PBS. The cleaned bones are placed in fresh αMEM. The bones are minced and cut into 1.5 mm area sizes. The bone blocks can be treated with collagenase to remove soft tissue and osteoid, and then decalcified using EDTA. Finally, osteocytes are released from the bone blocks by treatment with collagenase and vigorous stirring.

[0306] (b) Isolation of primary osteocytes from long bones. Long bone osteocytes can be isolated from mice or rats aged 2-3 weeks. For example, mice are given an overdose of anesthetics, then their necks are dislocated, their heads are decapitated and immersed in 70% ethanol. The femurs and tibiae, with the ends of the joints still intact, are separated. 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 separated from the tendons / ligaments. The cleaned bones are placed in fresh αMEM. After all bones are cleaned, both ends of each bone are cut with a scalpel before flushing the bone marrow with PBS. The bones are cut into lengths of 1.5 to 2 mm and treated with collagenase. In one example, the bone blocks are treated with collagenase 9 times in sequence to remove all other tissues and osteoid, and then decalcified using EDTA.

[0307] (c) Cultivating cells or cell lines: For example, primary and / or osteolytic cell lines are cultured on collagen-coated plates and immersed in recording medium (α-MEM medium without HCO3, buffered with HEPES) containing a permeable tracer.

[0308] (d) Administration of the test antibody. The cultured cells are contacted with the test antibody for a desired period of time.

[0309] (e) Determine the absorption of the permeability tracer. The uptake of the permeability tracer is determined by detecting the amount of tracer in the cell. In some aspects, time-lapse recording is used. The fluorescence of different cell regions of interest can be recorded on a microscope equipped with an eclipse filter based on the fluorescence wavelength of the tracer or other probe used. In some aspects, a fast-cooled digital camera captures images every 2 minutes and image processing is performed using ImageJ software. The collected data can be plotted as the multiple difference of the initial fluorescence and the fluorescence at the time of interest relative to the basal fluorescence.

[0310] For snapshot images, cells can be exposed to the permeabilizing tracer for 5-10 minutes, rinsed multiple times with PBS, and fixed with formaldehyde. In certain aspects, at least three micrographs of the fluorescent field are taken with a microscope. Image analysis is done with ImageJ software. The mean pixel density of random cells is measured.

[0311] The opening or blocking of connexin hemichannels can be confirmed by treating osteocytes with fluid flow shear stress and / or AD (all of which are known to open Cx43 hemichannels) and the test antibody, for example. If the test antibody blocks Cx43 hemichannels, the blocking of this channel will be reversed by fluid flow shear stress and / or AD. To control the blocking of Cx43 hemichannels, osteocytes are treated with Cx43 (E2) antibody (a polyclonal antibody that specifically inhibits the opening of Cx43 hemichannels).

[0312] In a specific example, MLO-Y4 osteolytic cells are treated with 1 μg / ml Cx43 (E2) antibody or test antibody for 30 minutes in the absence or presence of 20 μM AD. Ethidium bromide dye uptake is performed and quantified compared to untreated basal uptake levels. The assay is performed in the presence of calcium. Low calcium conditions can be used as a control (open hemichannels). Cx43 (E2) antibody or test antibody can block the opening of AD-induced osteocyte hemichannels.

[0313] Inhibiting the opening of Cx43 hemichannels in chondrocytes (e.g., by chemical agents, etc.) can inhibit the development of inflammation and osteoarthritis. The opening of hemichannels in chondrocytes can be detected using the methods described herein. Proinflammatory factors (PGE2 and ATP) released by Cx43 hemichannels are measured using ELISA analysis. Drugs that block the opening of hemichannels can be used to treat inflammatory diseases such as OA.

[0314] In vitro cell model for evaluating Cx43 channel activity. Primary chondrocytes were isolated from mouse leg joints. The effects of agents on Cx43 hemichannel opening and gap junction coupling in chondrocytes can be detected, and time and dose-dependent effects are evaluated. For example, hemichannel opening is evaluated by dye uptake assay using Lucifer yellow or Alexa dye. Downstream effects are measured by detecting the release of PGE2 and ATP using ELISA analysis.

[0315] The specific scenario is shown below.

[0316] Immunoblotting. MLO-Y4 cells were plated in 60 mm culture dishes at 3 × 10 548 hours after inoculation. Mouse heart tissue was collected in lysis buffer (5mMTris, 5mM EDTA, 5mM EGTA plus protease inhibitors, 20μl / ml phenylmethylsulfonyl fluoride (PMSF), 20μl / ml N-ethylmaleimide, 10μl / ml NaVO4 and 10μl / ml leupeptin), homogenized, and centrifuged at 100,000xg for 30 minutes at 4°C, and then resuspended in lysis buffer. Crude membrane proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blotted with anti-Cx43CT (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 was detected using the Odyssey infrared detection system (LI-COR, Lincoln, NE, USA). Fluorescence of 800 anti-rabbit IgG (1:15000).

[0317] Immunofluorescence analysis MLO-Y4 cells were cultured on collagen-coated glass coverslips. The cells were rinsed twice with PBS and incubated with cold 70% ethanol at -20 °C for 20 min. The use of PFA destroys lysine-rich epitopes and is therefore not recommended. The cells were then rinsed twice with PBS to remove the ethanol. Afterwards, the cells were blocked overnight with blocking solution (2% goat serum, 2% fish skin gelatin, and 1% bovine serum albumin in PBS). Next, the cells were labeled with different concentrations of monoclonal antibodies in PBS, followed by FITC-conjugated goat anti-mouse antibody and WGA-alexa594 (Invitrogen) (diluted 1:400 and 1:1500 in blocking solution, respectively). The cells were observed with an Olympus BH-2 fluorescence microscope, and the images were processed offline with NIHImage J software.

[0318] Analysis of Dye Uptake for Hemichannel Activity Dye uptake was assessed using snapshot photographs. MLO-Y4 cells were plated on collagen-coated 35 mm dishes and incubated with recording medium without HCO3. - The cells were incubated with saline medium (buffered with 10 mM HEPES) and a salt composition (containing 154 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, and 5 glucose in mM). 2 ]) 0.5 mM EGTA was added, but CaCl2 and MgCl2 were not added. Records containing 50 μM EtBr or low [X 2] medium was used for snapshot recording. Cells were exposed to 100 μM EtBr for 5 min, then rinsed 3 times with PBS and fixed with 2% formamide. At least 3 fluorescence field micrographs were taken with a 10x dry objective in an inverted microscope (Carl Zeiss) with a rhodamine filter. Image analysis was performed offline using the software Image J. The average value of the pixel density of 30 random cells was measured.

[0319] Dye coupling analysis of gap junctions. MLO-Y4 cells were seeded in 35 mm dishes coated with collagen and incubated with recording medium (αMEM medium without HCO3-, buffered with 10 mM HEPES). The cells were microinjected with alexafluor 350 (Invitrogen, Eugene, OR, USA) (10 mM in PBS) at 37 ° C using a micromanipulator InjectManNI 2 and Femtojet purchased from Eppendorf (Eppendorf). Dye transfer was measured 2 minutes after injection of alexafluor 350. The dye coupling index was counted to calculate the number of cells with dye transfer. Dye coupling was observed under an inverted microscope equipped with xenon arc illumination and Nikon Eclipse (Nikon, Japan) (excitation wavelength of 330-380 nm; emission wavelength of 420 nm or more).

[0320] Gap Junction Cell Parachute Dye Transfer Assay MLO-Y4 cells were grown to confluence in 12-well plates. Donor cells were incubated with 5 μM Calcein Orange-Red-AM (790 Da) and 5 μM Oregon Green 488 BAPTA-2-AM (1752 Da) at 37°C for 40 minutes. After gap junction intercellular communication, cells can be labeled simultaneously with Calcein Red-Orange, a gap junction permeable tracer dye, and Oregon Green 488 BAPTA-2, a gap junction channel impermeable dye. Preloaded donor cells were removed from the plate by trypsinization. Preloaded cells were layered on top of unlabeled acceptor cells ("parachute") and incubated at a donor to acceptor ratio of 1:4. Cells were allowed to adhere for any time, such as 1 hour, then carefully washed 3 times and fixed in fresh 2% PFA for 10 minutes at room temperature, then rinsed again 3 times. Cells were examined using a fluorescence microscope. For Calcein Red-Orange transfer, the threshold was adjusted to clearly distinguish the dye transfer boundaries. The positive standard for dye transfer is the detection of Calcein Red-Orange / Oregon Green 488BAPTA-2 touching cells, Calcein Red-Orange positive and Oregon Green 488BAPTA-2 negative. Dye transfer was barely detectable (<1%). Images were taken where we found Oregon Green 488BAPTA-2 green positive cells.

[0321] Open hemichannel fluid flow shear stress. Fluid flow was established using a peristaltic pump with gravity-driven fluid flow through the parallel plate flow chamber separated by a spacer of a certain thickness. The thickness of the spacer determines the channel height, which is adjusted together with the flow rate to produce 16 dynes / cm 2 The circulating medium was α-MEM buffered with 10 mM HEPES.

[0322] B. In vivo testing

[0323] In certain aspects, modulation of Cx43 in osteocytes is determined by injecting a candidate agent into long bones and using a fluorescent tracking dye (eg, calcein or Evans blue) to detect opening or blockage of hemichannels in osteocytes.

[0324] An example of an in vivo assay for analyzing hemichannels in bone cells uses 3-4 month old mice or rats. The animals are weighed. The test antibody is introduced into the animal by intraperitoneal (IP) injection. After 2-4 hours, a fluorescent tracer dye (i.e., Evans Blue, Alexa 594) is injected into the lateral tail vein of the animal or injected by IP. Note: Up to 1% of the animal's body weight (by volume) can be injected. In some aspects, the animal is heated to dilate the tail vein before injecting the tail vein. After 2-4 hours, the animal is killed, the tibia and femur without muscle tissue are cut, and washed with PBS several times. The bones are fixed in paraformaldehyde and decalcified in a 14% EDTA solution at 4°C for two weeks, or at room temperature for 3-5 days with constant stirring. The bones are washed in PBS and soaked in 30% sucrose in PBS overnight and then embedded in OCT compound. The position of the bones is usually adjusted in the mold as needed. 5 μm thick frozen sections are cut using a cryostat, the sections are rinsed with PBS, and then fixed with 50% glycerol in PBS. Bone sections can be examined under a fluorescent microscope and the extent of osteocytes in the bone that have taken up the tracer dye can be quantified using Image J.

[0325] The opening of Cx43 hemichannels in osteocytes can be confirmed by mechanical loading on the tibia, which opens Cx43 hemichannels in osteocytes. This can serve as a negative control for hemichannel opening in osteocytes in vivo. For a positive control, mice lacking Cx43 in osteocytes were used. This mouse was generated by crossing 10-kb DMP-1Cre and Cx43 flox mice.

[0326] Cx43 conditional knockout (cKO) mice. Since homozygous Cx43 knockout is lethal, in order to examine the role of Cx43 expressed in osteocytes, osteocyte-specific Cx43 knockout mice were generated. Homozygous mice of the floxed Cx43 gene were hybridized with mice heterozygous for Cx43 as a whole to promote the complete absence of Cx43 in osteocytes. Cx43f1 / - mice (50% of offspring) were then hybridized with mice expressing the Cre recombinase driven by the human DMP-1 promoter. The mice produced in this way are Cx43 fl / -, DMP1 Cre+ or Cx43 fl / -, DMP1 Cre- (a small proportion is Cx43fl / fl or Cx43- / -). Cx43-deficient osteocytes were confirmed by immunohistochemistry.

[0327] Studies can include 8 groups of mice: WT treated with Cx43(E2), WT without Cx43(E2), cKO treated with Cx43(E2), cKO without AD Cx43(E2), WT treated with test antibody (TA), WT without TA, cKO treated with TA, and cKO without TA. Mice are administered Cx43(E2) or TA at 150 μg / kg body weight. Increased inflammation in KO compared to WT mice can be expected with treatment with Cx43(E2) or TA. In the absence of Cx43(E2) or TA treatment, bone metastasis should be similar between WT and knockout mice.

[0328] Animal models of spinal cord injury that can be used to test the Cx43 antibodies disclosed herein are reviewed by, for example, Sharif-Alhoseini et al., Spinal Cord, 2017 Aug; 55(8): 714-721), which is incorporated herein by reference in its entirety. For example, Webb, Biochem Pharmacol., Biochem Pharmacol. 2014 Jan 1; 87(1): 121-30, which is incorporated herein by reference in its entirety) reviews animal models of inflammation (e.g., rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis) for testing the Cx43 antibodies disclosed herein.

[0329] The various aspects of the present disclosure may be used alone, in combination, or in various configurations not specifically discussed in the embodiments described above, and are therefore not limited in their application to the details and arrangements of the components set forth in the foregoing description or shown in the accompanying drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0330] Although the specific embodiments disclosed in the subject matter have been discussed, the above description is illustrative rather than restrictive. After reading this specification, many changes of the present disclosure will become apparent to those skilled in the art. The full scope of the present disclosure should be determined by reference to the full scope of the claims and their equivalents and the description and these changes.

[0331] Incorporation by Reference

[0332] All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference.

Claims

1. A pharmaceutical composition for inhibiting the opening of Cx43 hemichannels in cells, preferably for treating inflammatory diseases or conditions or neurodegenerative diseases, the pharmaceutical composition comprising an anti-Cx43 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, the anti-Cx43 antibody or an antigen-binding fragment thereof comprising: a first heavy chain complementarity determining region (CDR) sequence consisting of the amino acid sequence of SEQ ID NO: 1, a second heavy chain CDR sequence consisting of the amino acid sequence of SEQ ID NO: 2, and a third heavy chain CDR sequence consisting of the amino acid sequence of SEQ ID NO: 3; and A first light chain CDR sequence consisting of the amino acid sequence of SEQ ID NO:4, a second light chain CDR sequence consisting of the amino acid sequence of SEQ ID NO:5, and a third light chain CDR sequence consisting of the amino acid sequence of SEQ ID NO:

6.

2. The composition of claim 1, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

8.

3. The composition of claim 2, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 13, 15 and 17, and a light chain consisting of an amino acid sequence of SEQ ID NO:

18.

4. The pharmaceutical composition of any one of claims 1-3, wherein the inflammatory disease or condition or neurodegenerative disease is spinal cord injury.

5. An anti-Cx43 antibody or antigen-binding fragment thereof for use in the preparation of a medicament for inhibiting the opening of a Cx43 hemichannel in a cell, preferably for use in the preparation of a medicament for treating an inflammatory disease or condition or a neurodegenerative disease.

6. The use according to claim 5, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

8.

7. The use according to claim 6, wherein the anti-Cx43 antibody or antigen-binding fragment thereof comprises a heavy chain consisting of an amino acid sequence selected from SEQ ID NO: 11, 13, 15 and 17, and a light chain consisting of an amino acid sequence of SEQ ID NO:

18.

8. Use according to any one of claims 5 to 7, wherein the inflammatory disease or condition or neurodegenerative disease is spinal cord injury.

Images