Connexin 43 antibody and its use

Abstract

To provide compositions and methods for treating diseases or conditions related to opening deficiency of Cx43 hemichannels in osteocytes, preferably, for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia.SOLUTION: In one aspect, the disclosure provides an anti-Cx43 antibody or an antigen binding fragment thereof comprising: first, second and third heavy chain complementarity determining region (CDR) sequences comprising amino acid sequences of SEQ ID NOs: 1, 2, and 3 respectively; and first, second and third light chain complementarity determining region (CDR) sequences comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6 respectively.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 62 / 651,668, filed on April 2, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence List An ASCII text file named "172628_020301_sequence.txt", created on April 1, 2019, with a size of 43,787 bytes, submitted via EFS-Web, is incorporated herein by reference in its entirety.

[0003] field This disclosure generally relates to anti-connexin (Cx) 43 antibodies and their use in the treatment of diseases or conditions related to the opening of Cx 43 hemichannels, for example, in osteocytes. [Background technology]

[0004] background Despite multiple preventative and therapeutic approaches, cancer remains one of the leading causes of death worldwide. Between 2010 and 2020, the number of new cancer cases in the United States is projected to increase by approximately 24% to over 1 million cases per year in men and by approximately 21% to over 900,000 cases per year in women. The types of cancer expected to increase the most are melanoma in both men and women; prostate, kidney, liver, and bladder cancers in men; and lung, breast, uterine, and thyroid cancers in women. Cancer remains the second leading cause of death in the United States, accounting for nearly one in four deaths. Many cancers are difficult or impossible to treat with current approaches. Many cancers either evade current treatment regimens, become resistant to treatment, or recur after treatment.

[0005] Cancer metastasis occurs when cancer spreads from the body part of its origin (e.g., breast or prostate) to other body parts (e.g., liver or bone) and establishes secondary tumors. Bone is one of the most common sites of cancer metastasis. Cancers that metastasize to the bone include, but are not limited to, breast cancer, prostate cancer, lung cancer, and skin cancer (e.g., melanoma). Bone metastases can be identified in up to 75% of patients with advanced breast and prostate cancer. Bone metastases (mets) are associated with many significant clinical and quality-of-life consequences, such as, but are not limited to, intractable pain, pathological fractures, spinal cord and nerve compression, bone marrow infiltration, and motor impairment. Often, cancer present throughout the body can also make the cancer incurable.

[0006] Normal bone is composed of three main cell types: osteoblasts, resorbative osteoclasts, and osteocytes. Osteocytes make up about 95% of bone cells and maintain the bone regeneration process by regulating osteolytic and osteoblast activity. When cancer cells invade bone, much of normal bone function is affected. Cancer cells interact with the local microenvironment to promote cancer cell survival through bone destruction and angiogenesis.

[0007] Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels. These osteocyte hemichannels are normally closed and can open upon exposure to mechanical stimuli, releasing various factors into the bone microenvironment. Factors released by hemichannel opening may mediate other processes that can reduce tumor cell migration and bone metastases. Alendronate (AD), a commonly used and effective bisphosphonate drug, has been shown to open Cx43 hemichannels in osteocytes. Bisphosphonates are a class of drugs known for treating many bone disorders, including bone metastases. In patients with breast cancer, bisphosphonate administration has been shown to be associated with a reduced incidence of bone metastases and a reduction in mortality. AD is associated with reduced tumor growth as well as reduced bone destruction and pain. AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in osteocytes. However, AD administration is associated with several serious side effects.

[0008] Therefore, there is a need for effective methods and compositions for treating cancer metastasis, for example, by opening Cx43 hemichannels. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] Overview Compositions and methods for treating diseases or conditions associated with opening (e.g., under-opening or malfunction) of Cx43 hemichannels in osteocytes, such as cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, are provided herein.

[0010] In one aspect, an anti-Cx43 antibody or an antigen-binding fragment thereof, with the first, second, and third heavy-chain complementarity-determining region (CDR) sequences having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and the first, second, and third light-chain CDR sequences having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, is provided herein.

[0011] In some embodiments, the antibody or fragment thereof may have a heavy-chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light-chain variable domain having the amino acid sequence of SEQ ID NO: 8.

[0012] In another aspect, an anti-Cx43 antibody or an antigen-binding fragment thereof, comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 - 17 and a light chain having the amino acid sequence of SEQ ID NO: 18, is provided herein.

[0013] In another embodiment, antibodies are provided herein that bind to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may comprise one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In one embodiment, the epitope comprises F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In some embodiments, the epitope may comprise all 10 amino acids of SEQ ID NO: 19. In a particular embodiment, the epitope comprises all 10 amino acids of SEQ ID NO: 19.

[0014] In further embodiments, isolated anti-Cx43 antibodies or antigen-binding fragments thereof are provided herein, which cross-compete with any antibodies or fragments disclosed herein for binding to Cx43. In certain embodiments, the antibody or fragment promotes the opening of Cx43 hemichannels in osteocytes.

[0015] In another embodiment, pharmaceutical compositions for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising an antibody or fragment thereof disclosed herein and a pharmaceutically acceptable carrier, are provided herein.

[0016] The use of antibodies or fragments thereof disclosed herein for promoting the opening of Cx43 hemichannels in osteocytes, preferably for manufacturing pharmaceuticals for treating cancer, cancer metastasis, osteoporosis, or osteopenia, is also provided herein.

[0017] A method for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, is further provided herein, comprising contacting the osteocytes with an effective amount of an antibody or fragment thereof disclosed herein.

[0018] Methods for treating diseases or conditions associated with the opening (e.g., insufficient opening) of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising administering a therapeutically effective amount of an antibody or fragment thereof disclosed herein to a patient in need thereof. In certain embodiments, for example, the following are provided: (Item 1) An anti-Cx43 antibody or its antigen-binding fragment, The first, second, and third heavy chain complementarity-determining region (CDR) sequences having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; The first, second, and third light chains C, each having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. An anti-Cx43 antibody or its antigen-binding fragment containing a DR sequence. (Item 2) The antibody or fragment thereof according to item 1, comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain having the amino acid sequence of SEQ ID NO: 8. (Item 3) An anti-Cx43 antibody or its antigen-binding fragment, comprising a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs. 9 to 17, and a light chain having the amino acid sequence of SEQ ID NO. 18. (Item 4) An anti-Cx43 antibody or its antigen-binding fragment, wherein when bound to Cx43, it binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). (Item 5) The antibody or fragment thereof as described in item 4, wherein the epitope contains one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. (Item 6) The antibody or fragment thereof as described in item 4, wherein the epitope consists of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19. (Item 7) The antibody or fragment thereof as described in item 4, wherein the epitope contains all 10 amino acids of SEQ ID NO: 19. (Item 8) The antibody or fragment thereof as described in item 4, wherein the epitope consists of all 10 amino acids of sequence number 19. (Item 9) An isolated anti-Cx43 antibody or its antigen-binding fragment, which cross-competes with the antibody or fragment described in any one of items 1 to 8 for binding to Cx43. (Item 10) An isolated anti-Cx43 antibody or antigen-binding fragment thereof, which cross-competes with the antibody or fragment described in any one of items 1 to 3 for binding to Cx43, preferably binds to an epitope located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19), more preferably the epitope comprises one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9 and I10 of SEQ ID NO: 19, and even more preferably the epitope comprises all 10 amino acids of SEQ ID NO: 19. (Item 11) An antibody or fragment thereof, as described in any one of items 1 to 10, that promotes the opening of the Cx43 hemichannel in osteocytes. (Item 12) A pharmaceutical composition for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising an antibody or fragment thereof as described in any one of items 1 to 11 and a pharmaceutically acceptable carrier. (Item 13) Use of an antibody or fragment thereof as described in any one of items 1 to 11, preferably for the production of a medicament for treating cancer, cancer metastasis, osteoporosis or osteopenia, to promote the opening of Cx43 hemichannels in osteocytes. (Item 14) A method for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising contacting the osteocytes with an effective amount of an antibody or fragment thereof described in any one of items 1 to 11. (Item 15) A method for treating a disease or condition related to the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising administering a therapeutically effective amount of any one of items 1 to 11 of the antibody or fragment thereof to a patient in need thereof. [Brief explanation of the drawing]

[0019] [Figure 1] Box plot of raw antibody screening data.

[0020] [Figure 2] Letter plot graph of complete substitution analysis of peptide FLSRPTEKTI probed with antibody under high stringency conditions. The nucleotide sequence is shown below the graph, and the mean signal of the nucleotide sequence is the red line. Substitutions at a given position are plotted with the signal intensity recorded for that substitution.

[0021] [Figure 3] Letter plot graph of complete substitution analysis of peptide FLSRPTEKTI probed with antibody under high stringency conditions. The nucleotide sequence is shown below the graph, and the mean signal of the nucleotide sequence is the red line. Substitutions at a given position are plotted with the signal intensity recorded for that substitution. [Modes for carrying out the invention]

[0022] Detailed explanation It should be understood that both the general description above and the detailed description below are illustrative and descriptive, and do not limit the compositions and methods of this disclosure.

[0023] Compositions and methods relating to anti-Cx43 antibodies or their antigen-binding fragments are disclosed herein. In some embodiments, the compositions disclosed herein exhibit superior activity, pharmacokinetics (e.g., reduced toxicity), stability, and / or developability (e.g., reduced production costs) compared to those disclosed in International Publication No. 2015 / 027120 and International Publication No. 2017 / 147561 (both of which are incorporated herein by whole reference). In certain embodiments, the advantages are unexpected.

[0024] definition For convenience, specific terms used in this specification, the examples, and the appended claims are summarized herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0025] As used herein, the following terms and phrases are intended to have the following meanings:

[0026] The articles “a” and “an” are used herein to refer to one or more grammatical objects of the article. For example, “element” means one element or more than one element.

[0027] As used herein, the term “about” means an acceptable variation of 20%, more preferably 10%, and most preferably 5% of the stated value.

[0028] 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 is achieved by, for example, K with a K value of ≤1 μM, ≤100 nM, or ≤50 nM. D This indicates K Dcan be measured by any method known to those skilled in the art, such as surface plasmon resonance assay or cell binding assay. The anti-Cx43 antibody can be a monoclonal antibody or an antigen-binding fragment thereof.

[0029] As used herein, "antibody" is a protein that includes a binding domain that binds to a target epitope. The term antibody includes monoclonal antibodies, single-chain variable domain antibodies, including immunoglobulin heavy and light chain molecules, and their variants and derivatives, including chimeric variants of monoclonal and single-chain variable domain antibodies. The binding domain is substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene, and the protein binds immunospecifically to an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified into either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In most vertebrate organisms, including human and mouse species, a typical immunoglobulin structural unit includes a tetramer composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). "V L " and V H " refer to the variable domains of these light and heavy chains, respectively. "C L " and C H " refer to the constant domains of the light and heavy chains. V L and V H Each of the three β-strand loops above is involved in binding to an antigen and is referred to as a "complementary determining region" or "CDR". The "Fab" (fragment, antigen-binding) region includes one constant domain and one variable domain from each heavy and light chain of the antibody, i.e., V L , C L , V H and C H 1.

[0030] Antibodies include intact immunoglobulins and their antigen-binding fragments. The term "antigen-binding fragment" refers to a polypeptide fragment of an antibody that binds to an antigen or competes for antigen binding (i.e., specific binding) with an intact antibody (i.e., with the intact antibody from which it is derived). 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 V H , and V L Examples include single-chain Fv antibodies (scFv) in which chains are linked to each other (either directly or via peptide linkers) to form a continuous polypeptide.

[0031] Antibodies also include variants, chimeric antibodies, and humanized antibodies. As used herein, the term “antibody variant” refers to an antibody having one or more mutations in the heavy chain and / or light chain. In some embodiments, the mutations reside in the variable region. In some embodiments, the mutations reside in the constant region. A “chimeric antibody” refers to an antibody in which portions of each amino acid sequence in the heavy and light chains are homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, and the remaining segments of the chain are homologous to the corresponding sequence in another. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable region of an antibody derived from a certain mammal, and the constant portion is homologous to the sequence in an antibody derived from another. One clear advantage of such chimeric forms is that the variable region can be conveniently induced from currently known sources, for example, by combining it with a constant region derived from human cell preparations, using readily available hybridomas or B cells derived from non-human host organisms. Variable regions offer the advantage of ease of preparation, and their specificity is not affected by their source; however, human constant regions are less likely to induce an immune response in human subjects when the antibody is injected than constant regions derived from non-human sources. Nevertheless, the above definition is not limited to this particular example. “Humanized” antibodies refer to molecules having an antigen-binding site substantially derived from a non-human species immunoglobulin and the rest of the immunoglobulin molecular structure based on the structure and / or sequence of human immunoglobulin. The antigen-binding site may consist of either a complete variable domain fused to a constant domain, or only a complementarity-determining region (CDR) grafted onto an appropriate framework region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble human immunoglobulin. Some forms of humanized antibodies preserve all CDR sequences (e.g., humanized mouse antibodies containing all six CDRs derived from mouse antibodies).Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered from the original antibody, and these are also referred to as one or more CDRs "derived" from one or more CDRs.

[0032] Where applicable, the amino acid residues of antibodies may be numbered according to the general numbering system of Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, NIH, Bethesda, MD.

[0033] As used herein in the context of binding between an antibody and the epitope of Cx43 as a target, the term “binding” refers to the process of non-covalent interaction between molecules. Preferably, the binding is specific. The specificity of an antibody may be determined based on affinity. A specific antibody has an affinity of 10 to its epitope. -7 Less than M, preferably 10 -8 Binding affinity or dissociation constant K less than M D It may have.

[0034] The term "affinity" refers to the strength of the binding reaction between the antibody's binding domain and its epitope. It is the sum of the attractive and repulsive forces acting between the binding domain and the epitope. As used herein, the term affinity refers to the dissociation constant K D It refers to.

[0035] The term "antigen" refers to a molecule or part of a molecule that can be conjugated by a selective conjugate, such as an antibody, and can be used in an animal to produce an antibody that can further bind to the epitope of that antigen. An antigen may have one or more epitopes.

[0036] The term “cancer” broadly refers to the uncontrolled and abnormal growth of host cells, leading to the invasion of surrounding tissues and potentially distal tissues to the initial site of abnormal cell growth in the host. Major classes include carcinoma, which is cancer of epithelial tissue (e.g., skin, squamous epithelial cells); sarcoma, which is cancer of connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemia, which is cancer of hematopoietic tissue (e.g., bone marrow tissue); lymphoma and myeloma, which are cancers of immune cells; and central nervous system cancers, including cancers of brain and spinal cord tissue. The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably herein. As used herein, “cancer” refers to all types of cancer or neoplasms or malignant tumors, whether new or recurrent, including leukemia, carcinoma, and sarcoma. Specific examples of cancer include carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed tumors. Non-exclusive examples of cancer include new or recurrent cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovarian, prostate, sarcoma, stomach, uterus, and medulloblastoma.

[0037] The term “epitope” includes any determinant, preferably polypeptide determinant, that can specifically bind to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinant includes a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, phosphoryl, or sulfonyl, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. In one embodiment, the epitope is a region of an antigen to which an antibody binds. In certain embodiments, the antibody is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Methods for epitope mapping are well known in the art and include, for example, X-ray cocrystallography, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from both adjacent or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly five or eight to ten, amino acids in a unique spatial conformation.

[0038] The site on an antibody that binds to an epitope is called a "paratope," and typically contains amino acid residues that are adjacent to the epitope when bound. See Sela-Culang et al., Front Immunol. 2013;4:302.

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

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

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

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

[0043] The terms “cross-compete,” “cross-compete,” “cross-block,” “cross-blocked,” and “cross-blocking” are used interchangeably herein and mean the ability of an antibody or fragment to interfere with the binding of an antibody or fragment to target Cx43, either directly or indirectly through allosteric modulation of the anti-Cx43 antibody of this disclosure to that target. The extent to which an antibody or fragment can interfere with the binding of another to a target, and therefore whether it can be said to be cross-blocking or cross-competing according to this disclosure, can be determined using a competitive binding assay. One particularly suitable quantitative cross-competition assay uses a FACS or AlphaScreen-based approach to measure the competition between a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment and another antibody or fragment, in terms of their binding to a target. Generally, a cross-competitive antibody or fragment thereof can bind to a target in a cross-competitive assay, for example, in the presence of a secondary antibody or fragment thereof, such that the recorded substitution of the immunoglobulin monovariate domain or polypeptide of the present disclosure is up to 100% (in a FACS-based competitive assay) of the maximum theoretical substitution by the potential cross-blocking antibody or fragment thereof present in a given amount (e.g., substitution by a non-radioactive (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked). Preferably, the cross-competitive antibody or fragment thereof has recorded substitutions of 10% to 100%, more preferably 50% to 100%.

[0044] Antibody-antibody cross-competition can also be measured by real-time label-free biolayer interference assays. Cross-competition between two antibodies can be expressed as binding of the secondary antibody that is below the background signal due to self-binding (the primary and secondary antibodies are the same antibody). Cross-competition between two antibodies can also be expressed, for example, as the binding percentage of the secondary antibody that is below the baseline self-background binding (the primary and secondary antibodies are the same antibody).

[0045] Where used interchangeably in this specification, the terms “promote,” “enhance,” and “induce” refer to any statistically significant increase in biological activity (e.g., hemichannel opening). For example, “promote” could refer to an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.

[0046] The terms “subject” or “patient” include humans or other mammals receiving either prophylactic or therapeutic treatment.

[0047] As used herein, the terms “treat,” “treating,” and “treatment” refer to therapeutic or prophylactic measures, such as those described herein. A “treatment” method involves administering a Cx43 ligand provided herein to a patient, for example, a patient with cancer, to prevent, cure, delay, reduce or improve the severity of one or more symptoms of cancer or recurrent cancer, or to extend the patient’s survival beyond what would be expected in the absence of such treatment. A “treatment” method also involves administering a Cx43 ligand (e.g., an antibody) provided herein to a patient, to provide a cancer treatment in the patient beyond what would be expected in the absence of such treatment.

[0048] As used herein, the term “effective dose” refers to the amount of a drug, e.g., Cx43 ligand, e.g., anti-Cx43 antibody, sufficient to achieve the treatment, prognosis prediction, or diagnosis of cancer when administered to a patient. The therapeutic effective dose varies depending on the patient being treated and the symptoms of the disease, the patient’s weight and age, the severity of the symptoms of the disease, the method of administration, etc., and can be readily determined by those skilled in the art. Dosages for administration include, 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, and about 300 mg of the antibody or its antigen-binding moiety provided herein. ng~about 5,000mg, about 400ng~about 4,500mg, about 500ng~about 4,000mg, about 1μg~about 3,500mg, about 5μg~about 3,000mg, about 10μg~about 2,600mg, about 20 μg ~ approx. 2,575 mg, approx. 30 μg ~ approx. 2,550 mg, approx. 40 μg ~ approx. 2,500 mg, approx. 50 μg ~ approx. 2,475 mg, approx. 300μg to about 2,000, about 400μg to about 1,175mg, about 500μg to about 1,150mg, about 0.5mg to about 1,125mg, about 1mg to about 1,100mg, about 1.25mg to about 1,075m g, 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 m The dosage may range from approximately 4.5 mg to 900 mg, 5 mg to 875 mg, 10 mg to 850 mg, 20 mg to 825 mg, 30 mg to 800 mg, 40 mg to 775 mg, 50 mg to 750 mg, 100 mg to 725 mg, 200 mg to 700 mg, 300 mg to 675 mg, 400 mg to 650 mg, 500 mg, or 525 mg to 625 mg. Dosage may be, for example, weekly, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. The dosing regimen may be adjusted to provide the best possible therapeutic response.The effective dose is also the dose at which the toxicity or adverse effects (side effects) of the drug are minimized and / or outweighed by the beneficial effects. Administration may be intravenous, precisely or approximately 6 mg / kg or 12 mg / kg weekly, or 12 mg / kg or 24 mg / kg every other week. Further administration regimens are described below.

[0049] Where used herein, terms used in the fields of recombinant nucleic acid technology, microbiology, immunology, antibody manipulation, and molecular and cell biology will generally be understood by those skilled in the art. For example, prior art may be used to prepare recombinant DNA, carry out oligonucleotide synthesis, and perform tissue culture and transformation (e.g., electroporation, transfection, or lipofection). Enzymatic reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures may generally be carried out according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (which is incorporated herein by reference for any purpose). Unless otherwise specifically defined, the nomenclature, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are generally well-known in the art. Standard techniques may be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient care.

[0050] As used herein, the terms “comprising” or “comprises” are used with respect to compositions, methods, and their components that may include, but are not specified, elements present in a given embodiment.

[0051] As used herein, the term “essentially derived from” refers to elements necessary for a given embodiment. The term allows for the presence of further elements that do not substantially affect the basic and novel or functional features of that embodiment of the disclosure.

[0052] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not described in that description of the embodiments.

[0053] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise specifically indicated by the context. Thus, for example, a reference to “method” includes one or more types of methods and / or processes described herein and / or which would be apparent to a person skilled in the art who has read this disclosure, etc.

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

[0055] Cx43 Various cells can communicate with each other and with the extracellular environment via hemichannels and gap junctions formed by the protein connexin. Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins constitute one hemichannel, and two hemichannels constitute one gap junction channel. Gap junctions are clusters of channels located in the plasma membrane between adjacent cells and mediate intercellular communication. Hemichannels are distinct entities from gap junction channels. Hemichannels enable the exchange of molecules between intracellular compartments and the extracellular environment.

[0056] Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels. These osteocyte hemichannels are normally closed but can open upon exposure to mechanical stimuli, releasing various factors into the bone microenvironment. Factors released upon hemichannel opening may mediate other processes that could reduce tumor cell migration and bone metastasis.

[0057] Connexin-43, also known as gap junction alpha-1 protein (GJA1), is a 43.0 kDa protein composed of 382 amino acids (NCBI reference sequence: NP_000156.1). GJA1 contains a long C-terminal tail, an N-terminal domain, and multiple transmembrane domains. This protein passes through the phospholipid bilayer four times, with its C-terminus and N-terminus exposed to the cytoplasm. The C-terminal tail consists of 50 amino acids and contains post-translational modification sites, as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. Consequently, the C-terminal tail is central to functions such as pH gating and channel assembly regulation. In particular, the DNA region of the GJA1 gene (NCBI gene ID: 2697) encoding this tail is highly conserved, indicating that it is either mutation-resistant or would be lethal if mutated. The N-terminal domain, on the other hand, is involved in channel gating and oligomerization, and can therefore control the switching between open and closed states of channels. The transmembrane domain forms a gap junction channel, and the extracellular loop facilitates proper channel docking. Additionally, the two extracellular loops form a disulfide bond, interacting with the two hexamers to create a complete gap junction channel.

[0058] Anti-Cx43 antibody Promoting or enhancing Cx43 hemichannel opening can induce or promote Cx43 hemichannel opening in osteocytes, thereby potentially treating conditions such as cancer, cancer metastasis, osteosarcoma, osteoporosis, or osteopenia. Therefore, anti-Cx43 antibodies may be used as effective agents in cancer treatment.

[0059] In certain embodiments, the anti-Cx43 antibody may be a monoclonal antibody or its antigen-binding fragment. In certain embodiments, the anti-Cx43 antibody may be a modified antibody, for example, a chimeric or humanized antibody derived from a mouse anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody is an antibody or its antigen-binding fragment that binds to an epitope present on the human Cx43 protein, such as an extracellular loop or a portion thereof.

[0060] An exemplary anti-Cx43 antibody may have one or more of the following CDR sequences: Heavy chain: CDR1 (Sequence ID 1): GYTFTSYY CDR2 (Sequence ID 2): INPSNAGT CDR3 (Sequence ID 3): TREGNPYYTMNY Light chain: CDR1 (Sequence ID 4): QSLLESDGKTY CDR2 (Sequence ID 5): LVS CDR3 (Sequence ID 6): WQGTHFPWT

[0061] In some embodiments, surprisingly, antibodies having the above-described CDR sequence were found to exhibit superior binding affinity and / or antibody stability compared to those disclosed in International Publication No. 2015 / 027120 and International Publication No. 2017 / 147561. While not bound by theory, it is thought that the mutation from "NG" to "NA" in the heavy chain CDR2 may reduce deamidation. In particular, antibody deamidation in the CDR region can cause changes in binding affinity, antibody degradation, and changes in charge variants, affecting antibody function and increasing the cost of antibody production. Therefore, the CDRs disclosed herein offer improved binding affinity and antibody stability, resulting in more advantageous technical effects than those disclosed in International Publication No. 2015 / 027120 and International Publication No. 2017 / 147561.

[0062] For example, monoclonal antibodies can be humanized and optimized using CDR grafting, germline modeling, and 3D structural analysis to increase the drug efficacy and / or developability of the antibody. In some embodiments, after humanization, the anti-Cx43 antibody may have one or both of the following variable domains: Heavy chain variable domain (SEQ ID NO: 7): EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGLEWIGGINPSNAGTNFNEKFKNRATLTVDKSTSTAYMELSSLRSEDTAVYYCTREGNPYYTMNYWGQGTLVTVSS Light chain variable domain (SEQ ID NO: 8): DVVMTQSPLSLPVTIGQPASISCKSSQSLLESDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPWTFGGGTKVEIK

[0063] In selected embodiments, the anti-Cx43 antibody may have a variable domain fused to the constant region (the constant regain) of, for example, human IgG1 or IgG4, which may optionally contain one or more mutations. In some embodiments, the mutations may be designed to reduce or minimize the cytotoxic effector function of the antibody while maintaining binding affinity and antibody stability. For example, the anti-Cx43 antibody may have one or more of the following heavy chain sequences (bold portions correspond to the variable domain, and non-bold portions correspond to the constant region):

[0064] [ka] [ka] [ka]

[0065] In some embodiments, the anti-Cx43 antibody may have the following light chain sequence (the bolded portion corresponds to the variable domain, and the non-bolded portion corresponds to the constant region). [ka]

[0066] In yet another embodiment, the anti-Cx43 antibody may comprise a mixture or cocktail of two or more anti-Cx43 antibodies, each binding to the same or different epitopes on Cx43.

[0067] In some embodiments, a bispecific antibody can be produced in which at least one of the specificities is the anti-Cx43 antibody or its antigen-binding fragment disclosed herein. The other specificity may be against another target involved in the disease being treated.

[0068] In one embodiment, the use of Cx43 ligand for the manufacture of a pharmaceutical product is provided. In another embodiment, a method for inhibiting tumor growth and / or metastasis in a patient is provided, comprising administering an effective amount of Cx43 ligand to the patient.

[0069] Preparation of anti-Cx43 antibody Anti-Cx43 antibodies can be produced using a variety of methods commonly known in the art. For example, phage display technology can be used to screen human antibody libraries and produce fully human monoclonal antibodies for therapeutic purposes. High-affinity conjugates may be considered candidates for neutralization studies. Alternatively, a conventional monoclonal approach can be used, in which mice or rabbits are immunized with human proteins, candidate conjugates are identified and tested, and finally, humanized antibodies are produced by grafting heavy and light chain binding sites onto human antibody coding sequences.

[0070] Antibodies typically contain two identical pairs of polypeptide chains, each having one full-length "light" chain (typically with a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically with a molecular weight of about 50–70 kDa). The amino-terminal portion of each chain typically contains a variable region of about 100–110 or more amino acids, which are typically involved in antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region involved in effector function. The variable regions of the heavy and light chains typically exhibit the same general structure, containing four relatively conserved framework regions (FRs) connected by three hypervariable regions, also referred to as complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework region, and this alignment may enable binding to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is typically described in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes). The definition follows that of Health, Bethesda, Md., Chothia & Lesk, 1987, J.Mol.Biol.196:901-917 or Chothia et al., 1989, Nature 342:878-883).

[0071] The development of monoclonal antibodies has made antibodies useful and interesting as pharmaceuticals. Monoclonal antibodies are produced in serial cell lines in culture medium using any method for producing antibody molecules. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma method and the human B-cell hybridoma method by Kohler et al. (1975, Nature 256:495-497) (Kozbor, 1984, J.Immunol. 133:3001; and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63).

[0072] Monoclonal antibodies can be modified for therapeutic use. One example is a “chimeric” antibody in which part of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, and the rest of the chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass. Another example is fragments of such antibodies, insofar 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 graft” antibody, which contains one or more complementarity-determining regions (CDRs) originating from a particular species or belonging to a particular antibody class or subclass, and the rest of the antibody chain is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass.

[0073] Another development is "humanized" antibodies. Methods for humanizing non-human antibodies are well known in the art (see U.S. Patents 5,585,089 and 5,693,762; see also Cecile Vincke et al., J. Biol. Chem. 2009; 284: 3273-3284, for the humanization of llama antibodies). Generally, humanized antibodies are produced from non-human animals, and then, typically, specific amino acid residues derived from the non-antigen recognition portion of this antibody are modified to be homologous to those residues in the corresponding isotype of human antibody. Humanization can be carried out, for example, by replacing at least a portion of the rodent variable region with the corresponding region of a human antibody 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).

[0074] More recently, there has been the development of human antibodies ("fully human antibodies") that do not involve human exposure to antigens. Using transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies in the absence of endogenous mouse immunoglobulin production, such antibodies are produced by immunization with antigens (typically having at least six consecutive amino acids) conjugated to a carrier as needed. 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, the transgenic animal is produced by inactivating the endogenous mouse immunoglobulin loci that encode mouse heavy and light immunoglobulin chains and inserting loci that encode human heavy and light chain proteins into its genome. Next, partially modified animals having less than complete modifications are crossbred to obtain animals having all of the desired immune system modifications. Upon administration of immunogens, these transgenic animals produce antibodies that are immune-specific to these antigens and have a human amino acid sequence (not mouse) including a variable region. See International Publication No. 96 / 33735 and International Publication No. 94 / 02602 (these are incorporated by reference). Further methods are described in U.S. Patent No. 5,545,807, International Publication No. 91 / 10741, International Publication No. 90 / 04036, and European Patent No. 546073 and European Patent Application Publication No. 546073 (these are incorporated by reference). Human antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells, as described herein.

[0075] In some embodiments, phage display technology can be used to screen therapeutic antibodies. Phage display allows for the display of an antibody repertoire on the surface of a filamentous bacteriophage, enabling screening of a constructed library for phages that bind to an immunogen. Antibody phages are based on the genetic engineering of the bacteriophage, as well as repeated rounds of antigen-induced selection and phage proliferation. This technology enables in vitro selection of Cx43 monoclonal antibodies. The phage display process begins with antibody library preparation, followed by ligation of variable weight (VH) and variable light (VL) PCR products into a phage display vector, and then analysis of monoclonal antibody clones. VH and VL PCR products corresponding to the antibody repertoire are ligated into a phage display vector (e.g., phagemide pComb3X) engineered to express VH and VL as scFv fused to the pIII minor capsid protein of an E. coli filamentous bacteriophage originally derived from the M13 bacteriophage. However, the phage display vector pComb3X lacks all the other genes necessary to encode a complete bacteriophage in E. coli. For those genes, helper phages are added to E. coli transformed with the phage display vector library. The result is a library of phages, each expressing a Cx43 monoclonal antibody on its surface and containing a vector with each nucleotide sequence. Phage display can also be used to produce the Cx43 monoclonal antibody itself (not attached to the phage capsid protein) in specific strains of E. coli. Further cDNA is manipulated after the VL and VH sequences within the phage display vector to enable characterization and purification of the produced mAbs. Specifically, recombinant antibodies may have hemagglutinin (HA) epitope tags and polyhistidines to enable easy purification from solution.

[0076] A diverse antibody phage library contains approximately 10 helper phages that have been infected. 8It is produced from individual independent E. coli transformants. Using biopanning, the library can be screened for phages that bind to the immunogen sequence or fragments thereof via the expression surface of a monoclonal antibody. Periodic panning allows for the isolation of potentially very rare antigen-binding clones and consists of multiple rounds of phage binding to antigens (immobilized on ELISA plates or on the cell surface in solution), washing, elution, and re-amplification of phage conjugates in E. coli. During each round, specific conjugates are selected from the pool by washing away non-conjugated ones and selectively eluting conjugated phage clones. After 3 or 4 rounds, the highly specific binding of phage clones via the surface Cx43 monoclonal antibody is characteristic of direct selection in the immobilized immunogen.

[0077] Another method involves adding a C-terminal His tag suitable for purification by affinity chromatography to the immunogen sequence described above. The purified protein can be inoculated into mice with a suitable adjuvant. Monoclonal antibodies produced in hybridomas can be tested for binding to the immunogen, and positive conjugates can be screened as described in the assays herein.

[0078] Fully human antibodies can also be produced from phage display libraries (as disclosed in Hoogenboom et al., 1991, J.Mol. Biol. 227:381; and Marks et al., 1991, J.Mol. Biol. 222:581). These processes mimic immunoselection through the display of an antibody repertoire on the surface of a filamentous bacteriophage and subsequent phage selection by their binding to a selected antigen. One such technique is described in International Publication No. 99 / 10494 (which is incorporated by reference), which describes the isolation of high-affinity functional agonist antibodies against MPL and msk receptors using such an approach.

[0079] The nucleotide sequence encoding the above antibody can be determined. Subsequently, chimeric, CDR graft, humanized, and fully human antibodies can also be produced by recombinant methods. The nucleic acid encoding the antibody can be introduced into host cells and expressed using materials and procedures generally known in the art.

[0080] This disclosure provides antibodies against Cx43. Preferably, the antibodies bind to Cx43. In preferred embodiments, this disclosure provides heavy-chain and light-chain immunoglobulin molecules, in particular nucleotide sequences encoding sequences corresponding to their variable regions, and amino acid sequences containing them. In preferred embodiments, sequences corresponding to CDRs CDR1 to CDR3 are provided in particular. In further embodiments, this disclosure provides hybridoma cell lines expressing such immunoglobulin molecules and monoclonal antibodies produced therefrom, preferably purified human monoclonal antibodies against human Cx43.

[0081] The CDRs of the light and heavy chain variable regions of the anti-Cx43 antibody of this disclosure may be grafted onto a framework region (FR) of the same or a different species. In certain embodiments, the CDRs of the light and heavy chain variable regions of the anti-Cx43 antibody may be grafted onto a consensus human FR. To create a consensus human FR, several FRs derived from human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. The FR of the anti-Cx43 antibody heavy or light chain may be replaced with FRs derived from different heavy or light chains. Rare amino acids in the FRs of the heavy and light chains of the anti-Cx43 antibody are typically not replaced, but the rest of the FR amino acids may be replaced. Rare amino acids are specific amino acids that are not normally found in the FR. The graft variable region derived from the anti-Cx43 antibody of this disclosure may be used in conjunction with a constant region different from the constant region of the anti-Cx43 antibody. Alternatively, the graft variable region may be part of a single-chain Fv antibody. CDR grafting is described, for example, in U.S. Patents 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).

[0082] In some embodiments, the antibodies of the Disclosure may be produced by a hybridoma strain. In these embodiments, the antibodies of the Disclosure have a dissociation constant (K) of approximately 4 pM to 1 μM. D ) binds to Cx43. In certain embodiments of this disclosure, the antibody is K less than about 100 nM, less than about 50 nM, or less than about 10 nM. D Then connect it to Cx43.

[0083] In some embodiments, the antibody of this disclosure is an IgG1, IgG2, IgG3, or IgG4 isotype, for example, an IgG1 isotype. In certain embodiments, the antibody comprises a human kappa or lambda light chain and a human IgG1, IgG2, or IgG4 heavy chain. In some embodiments, the variable region of the antibody is ligated to the constant region of the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the variable region of the antibody is ligated to a constant region other than the constant region of the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the antibody of this disclosure is cloned for expression in mammalian cells.

[0084] In alternative embodiments, the antibodies of this disclosure may be expressed in cell lines other than hybridoma cell lines. In these embodiments, sequences encoding specific antibodies may be used for the transformation of suitable mammalian host cells. According to these embodiments, transformation may be achieved using any known method for introducing polynucleotides into host cells, for example, a method comprising packaging polynucleotides into a virus (or viral vector) and transfecting the host cell with the virus (or vector), or by transfection procedures known in the Art. Such procedures are exemplified in U.S. Patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (all incorporated herein by reference for any purpose). Generally, the transformation procedure used may depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and are not limited to, but include, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0085] According to certain embodiments of the methods of this disclosure, nucleic acid molecules encoding the amino acid sequences of the heavy chain constant region, heavy chain variable region, light chain constant region, or light chain variable region of a Cx43 antibody are inserted into a suitable expression vector using standard ligation techniques. In preferred embodiments, the Cx43 antibody heavy chain or light chain constant region is added to the C-terminus of a suitable variable region and ligated into the expression vector. The vector is typically selected to be functional in the specific host cell in which it is used (i.e., the vector is compatible with the host cell mechanisms 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. NY.

[0086] Typically, an expression vector used in any host cell may contain sequences for plasmid maintenance, as well as for the cloning and expression of exogenous nucleotide sequences. Such sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. These sequences are well known in the art.

[0087] The expression vectors disclosed herein can be constructed from starting vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired adjacent sequences. If one or more of the adjacent sequences described herein are not already present in the vector, they may be obtained individually and ligated into the vector. Methods used to obtain each adjacent sequence are well known to those skilled in the art.

[0088] After the vector is constructed and a nucleic acid molecule encoding a light chain or heavy chain or both containing an anti-Cx43 antibody is inserted into the appropriate site in the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of selected host cells with an anti-Cx43 antibody expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The selection method will, in part, depend on the type of host cell to be used. These methods and other suitable methods are well-known to those skilled in the art and are described, for example, by Sambrook et al., cited above.

[0089] Host cells, when cultured under appropriate conditions, synthesize anti-Cx43 antibodies, which can then be collected from the culture medium (if the host cells secrete it into the medium) or directly from the host cells producing it (if they do not secrete it). The selection of appropriate host cells will depend on various factors, including the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0090] Mammalian cell lines available as hosts for expression are well known in the art and are not limited to, but include many immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK), HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and many other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines have high expression levels and produce antibodies with constitutive Cx43 binding properties. In another embodiment, cell lines may be selected from B cell lines that do not produce antibodies themselves but have the ability to produce and secrete heterologous antibodies (e.g., mouse myeloma cell lines NS0 and SP2 / 0).

[0091] Epitope mapping and related technologies This disclosure provides an anti-Cx43 antibody that interacts with one or more domains of the Cx43 molecule, for example, one or more amino acids found within the extracellular loop. The epitope to which the antibody binds may include one or more adjacent sequences of two or more (e.g., two, three, four, five, six, seven, eight, nine or more) amino acids located within one or more extracellular loops. Alternatively, the epitope may include one or more non-adjacent amino acids (or amino acid sequences) located within one or more extracellular loops (e.g., structural epitopes).

[0092] Various techniques known to those skilled 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 routine cross-blocking assays, e.g., those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). 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 cleavage, epitope extraction, and chemical modification of antigens may be employed (Tomer (2000) Prot. Sci. 9:487-496).

[0093] Another method that can be used to identify amino acids within polypeptides that antibodies interact with is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterizing the protein of interest, followed by binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, where exchangeable protons within amino acids protected by the antibody complex undergo deuterium-hydrogen reverse exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore may exhibit a relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids that the antibody interacts with. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0094] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a number of monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see U.S. Patent Application Publication 2004 / 0101920, which is incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is distinctly different from or partially overlaps with the epitopes represented by another category. This technique enables rapid filtering of genetically identical antibodies, allowing for characterization to be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0095] This disclosure provides anti-Cx43 antibodies that bind to the same epitope or a portion of an epitope. Similarly, this disclosure also includes anti-Cx43 antibodies that compete for binding to Cx43 or a fragment thereof with any of the specific exemplary antibodies described herein. For example, this disclosure includes anti-Cx43 antibodies that cross-compete for binding to Cx43 with one or more antibodies derived from the antibodies described herein.

[0096] By using routine methods known in the art, it is possible to easily determine whether an antibody binds to the same epitope as the reference anti-Cx43 antibody, or whether it competes for binding. For example, to determine whether a test antibody binds to the same epitope as the reference anti-Cx43 antibody of the present invention, the reference antibody can be bound to Cx43 or a peptide under saturated conditions. The ability of the test antibody to bind to the Cx43 molecule is then evaluated. If the test antibody can bind to Cx43 after saturated binding with the reference anti-Cx43 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Cx43 antibody. On the other hand, if the test antibody cannot bind to Cx43 after saturated binding with the reference anti-Cx43 antibody, the test antibody may bind to the same epitope as the reference anti-Cx43 antibody of this disclosure.

[0097] To determine whether an antibody competes with a reference anti-Cx43 antibody for binding, the above binding methodology can be performed in two directions. In the first direction, the reference antibody can be bound to Cx43 under saturated conditions, and then the binding of the test antibody to the Cx43 molecule can be evaluated. In the second direction, the test antibody can be bound to the Cx43 molecule under saturated conditions, and then the binding of the reference antibody to the Cx43 molecule can be evaluated. In both directions, if only the first (saturated) antibody can bind to the Cx43 molecule, it can be concluded that the test antibody and the reference antibody compete for binding to Cx43. As will be recognized by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily have to bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0098] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, one, five, ten, twenty, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured by a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have the same epitope. If several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other, the two antibodies have overlapping epitopes.

[0099] Subsequently, further routine experiments (e.g., peptide mutation and binding analysis) may 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 blockade (or another phenomenon) is the cause of the observed lack of binding. These types of experiments may be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0100] In various embodiments, antibodies are provided herein that bind to an epitope located partially or entirely within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may comprise one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In one embodiment, the epitope comprises F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In some embodiments, the epitope may comprise all 10 amino acids of SEQ ID NO: 19. In a particular embodiment, the epitope comprises all 10 amino acids of SEQ ID NO: 19.

[0101] Pharmaceutical composition and its use In another embodiment, pharmaceutical compositions are provided that can be used in the manner disclosed herein, namely, pharmaceutical compositions for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis, or osteopenia.

[0102] In some embodiments, the pharmaceutical composition comprises a Cx43 ligand and a pharmaceutically acceptable carrier. The Cx43 ligand can be formulated into the pharmaceutical composition together with the pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may include instructions for use in the treatment of a patient, for example, to promote the opening of Cx43 hemichannels in osteocytes, preferably for the treatment of cancer, cancer metastasis, osteosarcoma, osteoporosis, or osteopenia.

[0103] In one embodiment, the Cx43 ligand may be an anti-Cx43 antibody or its antigen-binding fragment.

[0104] As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, buffers, and other excipients. 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 bioagent, may be coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

[0105] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, sterile powders for the immediate preparation of sterile injections or dispersions, and conventional excipients for the preparation of tablets, pills, capsules, and the like. The use of such media and agents for the formulation of pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions provided herein is intended. Auxiliary active compounds may also be incorporated into the compositions.

[0106] A pharmaceutically acceptable carrier may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0107] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, as well as injectable organic esters, such as ethyl oleate. Where necessary, appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be useful to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Sustained absorption of the injectable composition may be achieved by including absorption-delaying agents, such as monostearate salts and gelatin, in the composition.

[0108] These compositions may also contain functional excipients, such as preservatives, wetting agents, emulsifiers, and dispersants.

[0109] Therapeutic compositions are typically sterile, non-systematic, and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations.

[0110] Sterile injectable solutions may be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above, into a suitable solvent, and then sterilizing it, for example, by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, from which powders of the active ingredient and any further desired components are obtained from a solution that has been previously sterile-filtered. The active ingredient may be mixed under sterile conditions with additional pharmaceutically acceptable carriers and any preservatives, buffers, or sprays that may be required.

[0111] The presence of microorganisms can be ensured by the aforementioned sterilization procedures, as well as by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, sustained absorption of the injectable pharmaceutical form can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin.

[0112] Pharmaceutical compositions containing the Cx43 ligand may be administered alone or in combination therapy. For example, a combination therapy may include a composition provided herein comprising the Cx43 ligand and at least one or more further therapeutic agents, such as one or more chemotherapeutic agents known in the art, which are discussed in more detail below. Pharmaceutical compositions may also be administered in combination with radiotherapy and / or surgery.

[0113] The administration regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the emergency situation of the treatment.

[0114] Exemplary dosage ranges for antibody administration include 10-1000 mg (antibody) / kg (patient's 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, and 50 Examples of dosages include ~800 mg / kg, 50~600 mg / kg, 50~400 mg / kg, 50~200 mg / kg, 100~1000 mg / kg, 100~900 mg / kg, 100~800 mg / kg, 100~700 mg / kg, 100~600 mg / kg, 100~500 mg / kg, 100~400 mg / kg, 100~300 mg / kg, and 100~200 mg / kg. Exemplary dosing schedules include once every 3 days, once every 5 days, once every 7 days (i.e., once a week), once every 10 days, once every 14 days (i.e., once every 2 weeks), once every 21 days (i.e., once every 3 weeks), once every 28 days (i.e., once every 4 weeks), and once a month.

[0115] For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose for the patient to be treated; each unit contains a predetermined amount of the active agent calculated to produce the desired therapeutic effect in conjunction with any necessary pharmaceutical carrier. The specifications of a unit dosage form are determined and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the constraints inherent in the technique of formulating such an active compound for the treatment of susceptibility in an individual.

[0116] The actual dose levels of the active ingredients in the pharmaceutical compositions disclosed herein may vary to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. Where used herein in the context of administration, “parenteral” typically means modes of administration other than intestinal and topical administration by injection, and does not include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0117] As used herein, the terms “parenteral administration” and “administered parenterally” refer to, but are not limited to, modes of administration other than intra-intestinal (i.e., via the gastrointestinal tract) and local administration by injection or infusion, but include, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Intravenous injections and infusions are often used (but not exclusively) for antibody administration.

[0118] When the agents provided herein are administered to humans or animals as pharmaceuticals, they may be administered alone or in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition containing, for example, 0.001 to 90% (e.g., 0.005 to 70%, e.g., 0.01 to 30%) of the active ingredient.

[0119] In certain embodiments, methods and uses provided herein for treating cancer, cancer metastases, osteosarcoma, osteoporosis, or osteopenia may involve the administration of a Cx43 ligand and at least one additional anticancer agent that is not a Cx43 ligand, in order to promote the opening of Cx43 hemichannels in osteocytes.

[0120] In one embodiment, at least one further anticancer agent comprises at least one chemotherapeutic agent. Non-limiting examples of such chemotherapeutic agents include platinum-based chemotherapeutic agents (e.g., cisplatin, carboplatin), taxanes (e.g., paclitaxel (Taxol®), docetaxel (Taxotere®), EndoTAG-1® (a formulation of paclitaxel encapsulated in a positively charged lipid-based complex; MediGene), Abraxane® (a formulation of paclitaxel conjugated to albumin)), tyrosine kinase inhibitors (e.g., imatinib / Gleevec®, sunitinib / Sutent®, dasatinib / Sprycel®), and combinations thereof.

[0121] In another embodiment, at least one further anticancer agent includes an EGFR inhibitor, such as an anti-EGFR antibody or a small molecule inhibitor of EGFR signaling. An exemplary anti-EGFR antibody is cetuximab (Erbitux®), which is commercially available from ImClone Systems Incorporated. Other examples of anti-EGFR antibodies include matuzumab (EMD72000), panitumumab (Vectibix®; Amgen), nimotuzumab (TheraCIM®), and mAb806. An exemplary small molecule inhibitor of the EGFR signaling pathway is gefitinib (Iressa®), which is commercially available from AstraZeneca and Teva. Other examples of small molecule inhibitors of the EGFR signaling pathway include erlotinib HCl (OSI-774; Tarceva®, OSI Pharma); lapatinib (Tykerb®, GlaxoSmithKline); canertinib (canertinib dihydrochloride, Pfizer); peritinib (Pfizer); PKI-166 (Novartis); PD158780; and AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline).

[0122] In yet another embodiment, at least one further anticancer agent comprises a VEGF inhibitor. Exemplary VEGF inhibitors include anti-VEGF antibodies, such as bevacizumab (Avastatin®; Genentech).

[0123] In yet another embodiment, at least one further anticancer agent comprises an anti-ErbB2 antibody. Suitable anti-ErbB2 antibodies include trastuzumab and pertuzumab.

[0124] In one embodiment, the improved efficacy of the combinations of the present disclosure may be demonstrated by achieving therapeutic synergies.

[0125] The term "therapeutic synergy" is used when a combination of two products at a given dose is more effective than the best of each product alone at the same dose. For example, therapeutic synergy may be assessed by comparing the combination to the best monotherapy using estimates obtained from a two-way ANOVA with repeated measures of the parameter tumor volume (e.g., time factor).

[0126] The term "additive" refers to a combination of two or more products in a given dose that is as effective as the combined effectiveness of each of the two or more products, while the term "super-additive" refers to a combination that is more effective than the combined effectiveness of each of the two or more products.

[0127] Another way to quantify effectiveness (including combination effectiveness) is the following equation: log 10 Cell death = TC (number of days) / 3.32 × T d (In the formula, TC represents the delay in cell growth (this is the average time (in days) for the tumors in the treatment group (T) and the control group (C) to reach a predetermined value (e.g., 1 g or 10 mL)), T d The logarithm is determined according to the time (in days) required for the tumor volume to double in the control animal. 10This is done by calculating cell death. When applying this measurement, log 10 If cell death exceeds or is equal to 0.7, the product is considered active, log 10 If the cell death rate exceeds 2.8, the product is considered highly active.

[0128] Using this measurement, the combination used at its own maximum tolerated dose (each component is generally present at a dose below or equal to its maximum tolerated dose) is log 10 Cell death is the log of the best constituent components when administered alone. 10 A therapeutic synergistic effect is observed when the value exceeds the cell death threshold. In the example, the log of the combination 10 Cell death is the log of the best components in the combination. 10 The cell death value exceeds at least 1 log cell death.

[0129] Compositions and methods for providing cancer treatment are disclosed herein. The methods may include promoting the opening of Cx43 hemichannels in osteocytes of a subject requiring such treatment. Cx43 modulation (e.g., anti-Cx43 antibodies) may be used as a standalone cancer treatment or in combination with other cancer treatments.

[0130] Methods for promoting the opening of Cx43 hemichannels in osteocytes, preferably for treating cancer, cancer metastasis, osteosarcoma, osteoporosis or osteopenia, comprising administering one or more of the anti-Cx43 antibodies disclosed herein to a subject in need thereof.

[0131] In various embodiments, the methods disclosed herein may involve administering an effective amount of anti-Cx43 antibody or its antigen-binding fragment to a subject. Generally, the effective amount may be administered therapeutically and / or prophylactically.

[0132] The treatment may be appropriately administered to subjects, particularly humans, who have, possess, are susceptible to, or are at risk of developing such cancers. Determination of subjects "at risk" may be made by objective or subjective determination based on diagnostic tests or the opinion of the subject or healthcare provider (e.g., genetic testing, enzyme or protein markers, family history, etc.). Identification of subjects requiring such treatment may be within the discretion of the subject or healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods).

[0133] Administration of the preparation The formulations of this disclosure, including but not limited to reconstituted formulations and liquid formulations, are administered to mammals, preferably humans, that require treatment with anti-Cx43 antibodies by known methods, for example, by intravenous administration as a bolus, or by continuous infusion over a period of time via intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-sacral, intrathecal, oral, topical, or inhalation routes.

[0134] In embodiments, the formulation is administered to mammals intravenously or subcutaneously (i.e., under the skin). For such purposes, the formulation may be injected using a syringe. However, other devices for administering the formulation are available, such as injection devices (e.g., INJECT-EASE® and GENJECT® devices; injector pens (e.g., GENPEN); automated injection devices, needleless devices (e.g., MEDIJECTOR® and BIOJECTOR®)); and subcutaneous patch delivery systems.

[0135] In certain embodiments, this 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, which may include either one or more chambered pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0136] The appropriate dose of the protein ("therapeutic dose") will depend, for example, on the symptoms to be treated, the severity and course of the symptoms, whether the protein is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to anti-Cx43 antibodies, the form of formulation used, and the discretion of the attending physician. Anti-Cx43 antibodies may be administered to the patient at an appropriate time, in a single course, or over a series of treatments, or at any point after diagnosis. Anti-Cx43 antibodies may be administered as a monotherapy or in combination with other drugs or treatments useful in treating the symptoms in question.

[0137] For anti-Cx43 antibodies, the initial candidate dose may range from approximately 0.1 to 100 mg / kg or 1 to 20 mg / kg per patient, and may take the form of a single or more individual dose. However, other dosing regimens may be useful. The progression of such treatment can be easily monitored using conventional techniques.

[0138] According to certain embodiments of the Disclosure, multiple doses of anti-Cx43 antibody (or a pharmaceutical composition comprising a combination of anti-Cx43 antibody and any further therapeutic activators referred to herein) may be administered to a subject over a specified period of time. Methods of this aspect of the Disclosure include sequential administration of multiple doses of the anti-Cx43 antibody of the Disclosure to a subject. As used herein, “sequential administration” means that each dose of anti-Cx43 antibody is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The Disclosure includes methods of sequentially administering to a patient a single initial dose of anti-Cx43 antibody, followed by one or more secondary doses of anti-Cx43 antibody, and optionally followed by one or more tertiary doses of anti-Cx43 antibody. Anti-Cx43 antibodies may be administered in doses ranging from 0.1 mg / kg to approximately 100 mg / kg.

[0139] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to the chronological order of administration of the anti-Cx43 antibody in this disclosure. Thus, the “initial dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”); the “secondary dose” is the dose administered after the initial dose; and the “tertiary dose” is the dose administered after the secondary dose. While the initial, secondary, and tertiary doses may all contain the same amount of anti-Cx43 antibody, they may generally differ from one another in terms of administration frequency. However, in certain embodiments, the amounts of anti-Cx43 antibody contained in the initial, secondary, and / or tertiary doses may vary from one another during the course of treatment (e.g., adjusted up or down where appropriate). In certain embodiments, two or more doses (e.g., two, three, four, or five) may be administered as “loading doses” at the start of the treatment regimen, with subsequent doses administered on a lower frequency basis (e.g., “maintenance doses”).

[0140] In certain exemplary embodiments of this disclosure, each secondary and / or tertiary dose is 1 to 26 of the preceding dose (e.g., 1, 1) 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, 16 1 / 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 It is administered two weeks later (or more). As used herein, the term “previous dose” means the dose of anti-Cx43 antibody administered to the patient prior to the administration of the next dose in a multi-dose procedure.

[0141] Methods of this embodiment of the present disclosure may involve administering to a patient any number of secondary and / or tertiary doses of anti-Cx43 antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight or more) tertiary doses are administered to the patient.

[0142] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the previous dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the previous dose. In certain embodiments of this disclosure, the frequency at which secondary and / or tertiary doses are administered to the patient may vary throughout the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

[0143] This disclosure includes a dosing regimen in which a loading dose of -10 or 2-6 is administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by two or more maintenance doses administered to the patient at a lower frequency. For example, according to this aspect of the disclosure, if the loading dose is administered at a frequency of, for example, once a month (e.g., two, three, four or more loading doses are administered once a month), the maintenance dose may 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. [Examples]

[0144] The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the disclosure.

[0145] Example 1: Binding affinity The following protocol was used to test the optimal sequence for binding affinity to Cx43.

[0146] 1. Remove and reattach the Biacore T200. 2. Insert a new CM5 chip. 3. Prime three times with HBS-EP+ buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20 [tween20]). 4. Prerequisites: Start a new sensorgram at 100 ul / min. Using the regeneration command with a high viscosity solution (extra clean), inject 10 ul each of 2 × 100 mM HCl, 2 × 50 mM NaOH, and 2 × 0.5% SDS. This cleans and prepares the tip for coupling. Do this only for blank tips, not after protein has adhered to them. 5. Following the instructions included with the GE Human Antibody Capture Kit, separately amine-couple anti-human IgG Fc (GE:BR-1008-39) to each surface to be used (including the reference surface). Briefly, dilute Mab to 25 ug / ml with 10 mM sodium acetate pH 5. Start a new sensorgram at 5 ul / min. Immediately afterwards, activate 7' with NHS / EDC, inject Mab 7', and block with ethanolamine 7'. Typically, 10,000-12,000 RU / surface is obtained. After this, regenerate 10 times with 3M MgCl2 at 20 ul / min for 30 seconds each. (Note that the inventors conducted experiments using mouse IgG in a similar manner, except that they used the GE mouse antibody capture kit (GE:BR-1008-38). The only difference was that the anti-mouse IgG antibody was coupled at 30 ug / ml and regenerated at 10 mM glycine-HCl pH 1.7 at 20 ul / min for 3 minutes.) 6. After normalization, prime once with HBS-EP+ buffer. 7. Next, the experiment was performed immediately after amine coupling. If there was a considerable amount of time between experiments, the chips were removed and stored at 4°C. When returning the chips to the instrument, they were then primed three times with HBS-EP+, normalized, and primed once with HBS-EP+.

[0147] The program was created using the following parameters. overview: Buffer = HBS-EP+ Flow rate=100ul / min Data acquisition rate = 1Hz Sample compartment temperature = 15°C (This is the temperature at which the sample is held before injection) Assay run temperature = 25℃ Dual detection, Fc2-Fc1

[0148] For each cycle of the experiment: a. Mab Ab#K was trapped in FC2 by injecting 5 ul / min of 5 ug / ml Mab into FC2 for 180 seconds, followed by only additional washing after injection of 1% Tween20. b. 1M NaCl was injected into both FCs at a rate of 30 ul / min for 30 seconds, followed by additional buffer washing and stabilization for 180 seconds. The sample (peptide) was injected using a rapid injection method of 100 ul / min for 210 seconds, followed by dissociation for 300 seconds for both FCs, and then additional buffer washing and stabilization for 60 seconds. e. Both surfaces were regenerated with 3M MgCl2 at 20 ul / min for 30 seconds, a high viscosity solution was selected, followed by buffer washing and stabilization for 60 seconds.

[0149] I programmed the cycle as follows: 1. Ten startup injections of buffer to stabilize the instrument. 2. Concentration series of peptides (PEP1, PEP2, PEP3): 0, 4 nM, 12 nM, 37 nM, 111 nM, 333 nM, 1000 nM Cycles 1-10 Startup Cycles 11-17 PEP1(914) Cycles 18-24 PEP2(915) Cycles 25-31 PEP3(916) Cycles 32-38 PEP1(914) Cycles 39-45 PEP2 (915) Cycles 46-52 PEP3(916) Cycles 53-59 PEP1(914) Cycles 60-66 PEP2(915) Cycles 67-73 PEP3(916)

[0150] Data Analysis The data were analyzed using T200 evaluation software 2.0. Since equilibrium was reached under optimal conditions for the dynamics, the (Fc2-Fc1) data from each set of three replicates were globally fitted to either a 1:1 coupled dynamics model or a steady-state affinity model. The results obtained by both methods were similar.

[0151] In all experiments in which Mab was trapped on the surface of the CM5 chip, the above protocol was followed.

[0152] The CAP tip was also used according to the following protocol. 1. The CAP tip for the GE Healthcare Biotin Capture Kit (28920234) was prepared according to the manufacturer's instructions. Briefly, it was docked to the instrument (T200), primed three times with running buffer (HBS-EP+), and hydrated overnight in running buffer in standby mode. It was then conditioned by injecting regeneration solution (6M GuHCl, 250mM NaOH) at 30 ul / min for 3 × 60 seconds. After this, one normalization and one priming were performed. It was then ready for experimentation. The program was created using the following parameters.

[0153] overview: Buffer = HBS-EP+ Flow rate=100ul / min Data acquisition rate = 1Hz Sample compartment temperature = 15°C (This is the temperature at which the sample is held before injection) Assay run temperature = 25℃ Dual detection, Fc4-Fc3

[0154] For each cycle of the experiment: a. The biotin capture reagent from the kit is injected at a rate of 2 ul / min for 300 seconds to capture the biotin capture reagent in Fc3 and Fc4. b. The biotinylated peptide is captured by Fc4 by injecting 3 ug / ml peptide 2 at 5 ul / min for 120 seconds, followed by additional buffer washing and stabilization for 120 seconds. c. The sample (Mab) is injected using a rapid injection method of 100 ul / min for 210 seconds, and dissociation is performed on both FCs for 300 seconds. d. Regenerate both surfaces with 6M GuHCl and 250mM NaOH at 30 ul / min for 120 seconds (select a high viscosity solution), followed by buffer washing and stabilization for 120 seconds.

[0155] I programmed the cycle as follows: 1. Five startup injections of buffer to stabilize the instrument. 2. Each Mab(I or H) in the concentration series was run at 0, 6.2 nM, 18.5 nM, 55.6 nM, 166.7 nM, and 500 nM. Cycle 1-5 Startup Cycles 6-11 Mab I Cycles 12-17 Mab H Cycles 18-23 Mab I Cycle 24-29 Mab H

[0156] The binding affinity results (Table 1) generally show that binding affinity was maintained at least to some extent, and in many cases, surprisingly enhanced.

[0157] [Table 1]

[0158] Example 2. Fc receptor binding analysis Fc effector function is mediated by the binding of Fc to receptors. Receptors include FCRI, FCRIIa, FCRIIb, FCRIIIa, FCRIIIb, C1q, and FcRn. It is generally desirable to reduce binding affinity to most Fc receptors, except FcRn, while maintaining antibody half-life and minimizing potential in vivo toxicity. The binding of various antibodies to various Fc receptors was tested using the following surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) protocols.

[0159] A.FCRI combination Experiment: Biacore 8K Chip: CM5 (1) Immobilization An activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor tip was activated with the mixture for 420 seconds. Then, 30 μg / mL of THE(trademark)His-tagged antibody in 10 mM NaAc (pH 4.5) was injected into channels 1-8 at a flow rate of 30 μL / min for 400 seconds. The tip was deactivated with 1 M ethanolamine-HCl (GE).

[0160] (2) Ligand capture and analyte run 2 μg / mL CD64 in running buffer (1×HBS-EP+) was injected into Fc2 of channels 1-4 at a flow rate of 10 μL / min for 30 seconds. Analytes 20170905-Ab#C-02, 20170905-Ab#D-02, 20170908-Ab#G-02, 20170920-Ab#H-02 and running buffer at six concentrations (40, 20, 10, 5, 2.5, and 1.25 nM) were injected sequentially into Fc1-Fc2 of channels 1-4 at a flow rate of 30 μL / min, with 180 association steps followed by 400 dissociation steps. The ligand capture and analyte runs were repeated for 6 cycles in ascending order according to analyte concentration. After each dissociation step, 10 mM glycine pH 1.5 was injected as regeneration buffer.

[0161] 2 μg / mL CD64 in running buffer (1×HBS-EP+) was injected into the Fc2 of channels 1-6 at a flow rate of 10 μL / min for 30 seconds. Eight concentrations (10240, 5120, 2560, 1280, 640, 320, 160, and 80 nM) of analytes 20170907-Ab#K-02, 20170908-Ab#L-02, 20170915-Ab#O-02, 20170919-Ab#P-02, 20170919-Ab#S-02, and 20170920-Ab#T-02) and running buffer were injected sequentially into the Fc1-Fc2 of channels 1-6 at a flow rate of 30 μL / min with 60 association steps followed by 90 dissociation steps. The ligand capture and analyte runs were repeated for 8 cycles in ascending order according to the analyte concentration. After each dissociation step, 10 mM glycine pH 1.5 was injected as a regeneration buffer.

[0162] (3) Replay The chips were regenerated with 10 mM glycine at pH 1.5.

[0163] (4) Data analysis Surface channel Fc1 without ligand capture was used as the reference subtraction reference surface. Final data for each interaction were subtracted from the reference channel and buffer channel data. Experimental data for binding 20170905-Ab#C-02, 20170905-Ab#D-02, 20170908-Ab#G-02, and 20170920-Ab#H-02 to CD64 were fitted in 1:1 binding mode. The 10240nM curves for analytes 20170907-Ab#K-02, 20170908-Ab#L-02, 20170915-Ab#O-02, 20170919-Ab#P-02, 20170919-Ab#S-02, and 20170920-Ab#T-02 were removed to allow for a better fit. The relative experimental data were fitted using steady-state affinity and are shown in Table 2 below.

[0164] [Table 2]

[0165] All antibodies showed low or no FCRI binding, which is advantageous.

[0166] Binding experiments to B.FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb: Biacore 8K Chip: CM5 (1) Immobilization An activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor tip was activated with the mixture for 420 seconds. Then, 30 μg / mL of THE(trademark)His-tagged antibody in 10 mM NaAc (pH 4.5) was injected into channels 1-8 at a flow rate of 30 μL / min for 400 seconds. The tip was deactivated with 1 M ethanolamine-HCl (GE).

[0167] (2) Ligand capture and analyte run 1 μg / mL FcγRIIa, FcγRIIb, FcγRIIIa, or FcγRIIIb in running buffer (1×HBS-EP+) was injected into Fc2 of channels 1-8 at a flow rate of 10 μL / min for 15 seconds. The analytes were injected into channels 1-8, respectively. A series of analyte concentrations (see Table 3 below) were monitored at a flow rate of 30 μL / min for a 60-second association phase, followed by a 90-second dissociation phase. After each dissociation phase, 10 mM glycine pH 1.5 was injected as a regeneration buffer.

[0168] [Table 3]

[0169] (3) Replay The chips were regenerated with 10 mM glycine at pH 1.5.

[0170] (4) Data analysis Surface channel Fc1 without ligand capture was used as a reference subtraction control surface. Final interaction data were subtracted from the reference channel and buffer channel data. Experimental data for antibody binding to FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb were fitted using the steady-state affinity mode and are shown in Table 4 below.

[0171] [Table 4]

[0172] All antibodies showed either low or no FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb binding, which is advantageous.

[0173] Binding to C.FcRn Experiment: Biacore 8K Chip: CM5 (1) Buffer replacement Following the instructions, the human FcRn buffer was replaced with running buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 150 mM NaCl, 0.05% Tween20, pH 6.0) using a desalting column. The concentration was determined using Nanodrop.

[0174] (2) Immobilization An activator was prepared by mixing 400 mM EDC and 100 mM NHS (GE) immediately before injection. The CM5 sensor tip was activated with the mixture 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. Relative Fc1 was blocked. The tip was inactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds.

[0175] (2) The run of the analyte The analytes FcRn were injected into channels 1-8, respectively. Eight 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 for a 60-second association phase, followed by a 90-second dissociation phase. After each cycle of interaction analysis, the sensor tip surface was regenerated with 1 × PBS (pH 7.4) at a flow rate of 10 μL / min for 30 seconds.

[0176] (3) Replay The chips were regenerated in 1x PBS (pH 7.4).

[0177] (4) Data analysis Surface channel Fc1 without immobilized antibody was used as a reference subtraction control surface. Final interaction data were subtracted from the reference channel and buffer channel data. The experimental data were fitted using the steady-state affinity mode and are shown in Table 5 below.

[0178] [Table 5]

[0179] All antibodies showed similar FcRn binding, which is desirable.

[0180] D. Binding to C1q by ELISA Plates (Nunc) were coated overnight at 4°C with 3 μg / mL antibody. After blocking and washing, C1q was semi-logarithmically titrated with blocking buffers (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 incubated with the secondary antibody sheep anti-human C1qAb-HRP for 1 hour. After washing, TMB substrate was added and the interaction was stopped with 2M HCl. Absorbance at 450 nm was read using a microplate reader (Molecular Device) and is shown in Table 6 below.

[0181] [Table 6]

[0182] All antibodies showed either low C1q binding or no C1q binding at all, which is advantageous.

[0183] Example 3. Epitope Mapping To reconstruct the epitopes of target molecules, a library of peptide-based epitope mimics was synthesized using solid-phase Fmoc synthesis. These were grafted onto proprietary hydrophilic polymer formulations, subsequently reacted with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) together with N-hydroxybenzotriazole (HOBt), and then the Boc groups were cleaved using trifluoroacetic acid (TFA) to obtain amino-functionalized polypropylene supports. Peptides were synthesized on the amino-functionalized solid supports using standard Fmoc-peptide synthesis via a custom-modified JANUS liquid handling station (Perkin Elmer).

[0184] Structural mimics were synthesized using the Scaffold-Based Chemically Bound Peptide (CLIPS) technique. CLIPS technique allows for the structuring of peptides into single-loop, double-loop, triple-loop, sheet-like folds, helix-like folds, and combinations thereof. CLIPS templates are coupled to cysteine ​​residues. Multiple cysteine ​​side chains in the peptide are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (1:3 (v / v)). This solution is added to the peptide array. The CLIPS templates will bind to the two cysteine ​​side chains 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 solution for 30–60 minutes while completely covered in solution. Finally, the peptide array was thoroughly washed with excess H2O and sonicated at 70°C for 30 minutes in a disruption buffer (pH 7.2) containing 1% SDS / 0.1% 2,2'-(ethylenedioxy)diethanethiol in PBS, followed by further sonication in H2O for 45 minutes. T3 CLIPS containing peptides were then prepared using the same method, except that they contained three cysteine ​​groups.

[0185] Different sets of peptides were synthesized according to the following design. Note that the actual order of the peptides in some of the minicards was randomized. [Table 7]

[0186] Antibody binding to each synthetic peptide was tested by ELISA. The peptide array was incubated with the primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with a 1 / 1000 dilution of a suitable antibody peroxidase conjugate (SBA; goat anti-human HRP conjugate, Southern Biotech) at 25°C for 1 hour. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After 1 hour, the color development was measured. The color development was quantified using a charge-coupled device (CCD) camera and image processing system.

[0187] The values ​​obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA reader. Quantify the results and store them in the lab database. Occasionally, wells may contain air bubbles, leading to false positive values; therefore, manually inspect the cards and score any values ​​caused by air bubbles as 0.

[0188] To validate the quality of the synthetic peptides, separate sets of positive and negative control peptides were synthesized in parallel. These were screened using commercially available antibodies 3C9 and 57.9 (see Posthumus et al., (1990) J. Virol. 64:3304-3309).

[0189] A graphical overview of the complete dataset is shown in Figure 1. Here, the box plots represent each dataset, showing the mean ELISA signal, distribution, and outliers within each dataset. Different distributions of ELISA data can be obtained depending on experimental conditions (amount of antibody, blocking intensity, etc.). Specifically, the bottom and top of the box represent the 25th and 75th percentiles of the data. The band near the center of the box represents the 50th percentile (median). The whiskers represent the 1.5-interquartile range and indicate statistical outliers within the dataset (Mcgill et al., (1978) The American Statistician, 32:12-16).

[0190] Antibodies were tested at high concentrations under high stringency conditions. The recorded results are shown in Figures 2 and 3. Data for each of the two peptide sets were analyzed separately.

[0191] Analysis of data recorded with substitutional variants of the read sequence FLSRPTEKTI suggested that numerous substitutions of any residue in the sequence, to varying degrees, negatively affected antibody binding (Figure 2). The only exception was residue L2, which did not tolerate substitutions of L2P and L2Y, but remained insensitive to all other substitutions.

[0192] Analysis of data recorded with a shortened variant of the read sequence FLSRPTEKTI showed that the N-terminus of the sequence was preferred by the antibody (Figure 3). Many constructs derived from the central portion of FLSRPTEKTI were also well recognized.

[0193] In summary, antibodies were tested with a peptide array consisting of two types of peptide variants (single-residue mutants and truncated variants) derived from the read sequence FLSRPTEKTI. The antibodies exhibited detectable binding under high stringency conditions. Numerous substitutions across FLSRPTEKTI were undesirable for the antibodies. The antibodies were stronger binding truncated constructs derived from the N-terminal portion of the sequence.

[0194] Example 4. Antibody Stability Antibody stability is a crucial factor affecting development, efficacy, and production costs. After sequence optimization, key stability parameters were evaluated. The species distribution profiles of various antibodies were tested under acidic and thermal conditions. All antibodies exhibited improved stability.

[0195] A. SE-UPLC (Size Exclusion Ultrahigh-Speed ​​Liquid Chromatography) Formulation: PBS, pH 6.5 or 7.2 Concentration (mg / mL): 5.28, 5.13, 5.00, 5.17, 5.01, 5.26, 4.92, 5.04, 4.99, 5.12 (all approximately 5 mg / mL) Conditions: Room temperature, acid treatment, then storage at 4°C for 1 week or 40°C for 1 week. 2 μL of sample was injected into an ACQUITY UPLC Protein BEH SEC 200, 1.7 μm, 4.6 × 150 mm column at a flow rate of 0.3 mL / min for 10 minutes. A mobile phase of 50 mM sodium phosphate, 500 mM NaCl, pH 6.2 was used. All antibodies exhibited desirable stability under various pH, heat, and storage conditions.

[0196] B.rCE-SDS (Reduced capillary electrophoresis - sodium dodecyl sulfate) Formulation: PBS, pH 6.5, 7.2, 6.2 Concentration (mg / mL): 0.5 Conditions: Room temperature, acid treatment, then storage at 4°C for 1 week or 40°C for 1 week. Samples were prepared in reductive labeling buffer before being subjected to the LabChip GXII system (PerkinElmer). All antibodies exhibited the desired stability under various pH, heat, and storage conditions.

[0197] Example 5. Assay for hemichannel opening A. In vitro assay The antibodies disclosed herein may be tested in vitro for their effect on hemichannel opening using a dye uptake assay. The dye may be a fluorescent tracer dye (e.g., ethidium bromide or Lucifer yellow).

[0198] In one example, a Parallel Plate Flow Chamber (FFLA) or a modified version thereof may be used. The FFLA mimics the dynamic fluid microenvironment in bone to generate fluid-flow shear stress (FFSS). Cells are cultured in a parallel plate flow chamber, exposing them to steady laminar flow.

[0199] Osteocytes sense the mechanical strain induced by FFSS in their osteocyte lucate / tubule network. Bone fluid flow is driven by extravascular pressure and periodic mechanical load applied to osteocytes, with a peak physiological load of 8–30 dyn / cm². 2 It has been proposed that, in certain embodiments, the FFSS level was within the range of physiological values ​​reported from previous studies measuring intra-bone fluid flow. The magnitude of the fluid shear stress can be varied by adjusting the column height of the flow loop.

[0200] Assays used to evaluate the functionality of hemichannels may use fluorescent tracer molecules small enough to pass through the pores of the hemichannel. If the hemichannel is closed, the molecule cannot pass through. If the hemichannel is open, the dye can pass through and cause fluorescence emission in the cell, which may allow for the quantification of fluorescence. When ethidium bromide attaches to DNA, it begins to fluoresce. Lucifer yellow fluoresces when it is placed inside a cell.

[0201] Dye transfer methods may involve exposing cells to an extracellular fluorescence-permeable tracer. Unless certain conditions increase the permeability of the cell membrane, the extracellular fluorescence-permeable tracer is a molecule that remains outside the cell. In certain embodiments, the tracer has a mass of less than 1, 2, or 3 kDa. In other embodiments, the tracer will have a net charge. Examples of such permeable tracers, but not limited to, include the anionic dye Lucifer Yellow (LY; net charge = -1) and the cationic probes Ethidium bromide (Etd; net charge = +1) and Propidium iodide (PI; net charge = +2). Binding to DNA enhances the fluorescence of EtBr, increasing contrast and allowing for easier identification. In certain embodiments, the extracellular dye is removed at different time intervals or after the application of stimuli that open hemichannels, and the fluorescence intensity retained by each cell is quantified. In certain embodiments, the fluorescence intensity is quantified in snapshot images.

[0202] Materials used in in vitro assays to test hemichannel opening include hemichannel-expressing cells or cell lines. Cells or cell lines expressing various connexin hemichannels can be obtained, isolated, or manipulated using methods and / or expression vectors known in the art.

[0203] Osteocytes: Primary osteocytes isolated from animals (including mice, rats, rabbits, and chickens), or osteocyte lines, including but not limited to MLO-Y4 cells.

[0204] Cancer cells: Breast cancer cell lines: Includes ER, PR, HER, and TP53 positive / negative cells (e.g., MD-MBA-231, MCF7, T47D, or ZR751). MDA-MB-231 is a ductal carcinoma of the mammary gland. The Py8119 mammary tumor cell line was established from spontaneously occurring mammary tumors in C57Bl / 6 MMTV-PyMT female mice (mouse mammary tumor virus promoter-driven polyomamiddle T transgene). The expression of the oncogene (polyomamiddle T transgene) is driven by the mouse mammary tumor virus promoter.

[0205] Prostate cancer cell lines: including androgen receptor and 5α-reductase positive / negative and androgen-sensitive / insensitive cell lines (e.g., LNCaP-Rf, BM18, pRNA-1-1 / ras, RC58T / hTERT, PPC-1, etc.).

[0206] Osteoblasts: MLO-A5 osteoblasts are used as controls because they express connexin 43, but they do not seem to open when stimulated with alendronate.

[0207] Examples of tracer molecules include, but are not limited to, lucifer yellow, ethidium bromide, Evans blue, Alexa350, Alexa488, and Alexa594.

[0208] Cx43(E2): The Cx43(E2) antibody is specific for the Cx43 hemichannel. Cx43E2 binds to the second extracellular loop of the Cx43 hemichannel and prevents the opening of the hemichannel.

[0209] A method for determining whether an antibody opens a hemichannel includes one or more of the following steps: (a) Isolating, obtaining, or producing connexin-expressing cells or cell lines. For example, primary bone cells can be isolated from the calvaria. Other cell types can be isolated using other methods known in the art. In certain embodiments, calvarial bone cells are isolated from an animal (e.g., 16-day-old chick embryonic calvaria or neonatal mouse). The animal is decapitated, the calvarial bone is dissected, and quickly immersed in 70% alcohol. Then, the calvarial bone is placed in αMEM and washed multiple times with PBS. The cleaned bone is placed in fresh αMEM. The bone is minced and cut into 1.5 mm area sizes. The bone pieces can be treated with collagenase to remove soft tissue and osteoid, and subsequently decalcified using EDTA. Finally, the bone cells are released from the bone pieces by treatment with collagenase and vigorous agitation.

[0210] (b) Isolating primary bone cells from long bones. Long bone cells can be isolated from 2 - 3 week old mice or rats. For example, mice are given an overdose of anesthesia, cervical dislocation is performed, decapitated, and immersed in 70% ethanol. The femur and tibia with intact joint ends are isolated. The limb is quickly immersed in 70% alcohol and placed in αMEM. The limb in αMEM is washed with PBS. Most of the muscle is removed and separated from the tendon / ligament. The cleaned bone is placed in fresh αMEM. Once all the bones are cleaned, just before washing away the bone marrow using PBS, each bone's ends are cut using scissors. The bone is cut into lengths of 1.5 - 2 mm and treated with collagenase. In one example, the bone fragments are treated with collagenase nine times consecutively to remove all other tissues and osteoid, and then decalcified using EDTA.

[0211] (c) Culturing cells or cell lines. For example, primary and / or bone cell lines are cultured on collagen - coated plates and immersed in a recording medium containing a permeable tracer (α - MEM medium buffered with HEPES without HCO3).

[0212] (d) Administering a test antibody. The cultured cells are placed in contact with the test antibody for the desired time.

[0213] (e) Determining permeable tracer uptake. Permeable tracer uptake is determined by detecting the amount of tracer within the cells. In certain embodiments, time - lapse recording is used. Fluorescence can be recorded in the region of interest in different cells using an eclipse filter on a microscope based on the fluorescence wavelength of the tracer or other probe being used. In certain embodiments, images are captured every 2 minutes by a high - speed cooled digital camera and image processing is performed using ImageJ software. The collected data can be presented as the magnification difference of the initial fluorescence and the fluorescence at the desired time point compared to the basal fluorescence.

[0214] For snapshot images, cells may be exposed to a permeable tracer for 5–10 minutes, rinsed multiple times with PBS, and fixed with formaldehyde. In certain embodiments, at least three micrographs of the fluorescence field are taken using a microscope. Image analysis is performed using ImageJ software. The average pixel density of random cells is measured.

[0215] Confirmation of connexin hemichannel opening can be obtained, for example, by incubating osteocytes with a test antibody and a Cx43(E2) antibody (a polyclonal antibody that specifically inhibits the Cx43 hemichannel). If the test antibody opens the Cx43 hemichannel, this channel opening is blocked by the Cx43(E2) antibody. To control the opening of the Cx43 hemichannel, osteocytes are treated with fluid-flow shear stress and / or AD (both of which are known to open hemichannels in osteocytes).

[0216] In specific cases, MLO-Y4 osteocytes were treated with 20 μM AD or test antibody for 30 minutes in the absence or presence of 1 μg / ml Cx43(E2) antibody. Ethidium bromide dye uptake was performed and quantified compared to untreated basal uptake levels. The assay was performed in the presence of calcium. Low-calcium conditions can be used as a control (to open hemichannels). The opening of osteocyte hemichannels induced by AD or the test antibody is blocked by the Cx43(E2) antibody.

[0217] B. In vivo assay In certain embodiments, Cx43 modulation in osteocytes is determined by injecting a candidate reagent into long bone and detecting in situ hemichannel opening in osteocytes using a fluorescent tracer dye (e.g., calcein or Evans blue).

[0218] An example of an in vivo assay for analyzing hemichannels in osteocytes uses 3-4 month old mice or rats. Weigh the animals. Introduce the test antibody into the animals by intraperitoneal (IP) injection. 2-4 hours later, inject a fluorescent tracer dye (Evans Blue, Alexa 594) into the lateral tail vein of the animals or by IP injection. Note: Up to 1% of the animal's body weight may be injected. In certain embodiments, warm the animals to dilate the tail vein before tail vein injection. 2-4 hours later, dissect the animals, dissecting the tibia and femur without muscle tissue, and wash them multiple times with PBS. Fix the bones with paraformaldehyde and decalcify them in 14% EDTA solution at 4°C for 2 weeks, or at room temperature for 3-5 days with constant agitation. Wash the bones with PBS, immerse them overnight in 30% sucrose in PBS, and embed them in the OCT compound. Typically, adjust the position of the bones in a mold if necessary. Frozen sections with a thickness of 5 μm were cut using a cryostat, the sections were rinsed with PBS, and mounted using 50% glycerol in PBS. The bone sections could be examined under a fluorescence microscope, and the degree of osteocytes in the bone that had incorporated the tracer dye was quantified using Image J.

[0219] Opening of Cx43 hemichannels in osteocytes can be confirmed by mechanical loading onto the tibia, which opens Cx43 hemichannels in osteocytes. This can serve as a positive control for hemichannel opening in osteocytes in vivo. For a negative control, mice lacking Cx43 in osteocytes are used. These mice are generated by crossing 10kb DMP-1 Cre and Cx43 flox mice.

[0220] Example 6. Assay for cancer cell migration, viability, and metastasis. A. In vitro assay Cancer cell migration assay. Cx43 hemichannels in osteocytes are opened by administration of AD or FFSS. The opened hemichannels allow the release of various factors into the culture medium, resulting in conditioned medium (CM). Factors released in AD or FFSS-treated CM reduce cancer cell migration, as determined by soft agar and wound healing assays. Cancer cells treated with control CM exhibit normal migration. The soft agar assay is an assay for anchorage-independent growth, in contrast to anchorage-dependent growth. Only cancer cells can grow on soft agar, and their growth on this matrix indicates the degree of cancer cell proliferation.

[0221] In certain embodiments, cancer cells (e.g., breast or prostate) are incubated with CM to determine cancer cell proliferation, migration, and invasion.

[0222] Cancer cell growth and viability can be determined using the WST-1 (water-soluble tetrazolium salt) assay, live cell counting using trypan blue, BrdU DNA uptake, and cell proliferation assays. In the WST-1 assay, cell proliferation is measured at an emission wavelength of 450 nm using a Synergy HT multimode microplate reader (Biotek).

[0223] Cell migration assays are typically performed in 24-well tissue culture plates (BD Biosciences) using a Transwell membrane filter insert. The Transwell membrane filter insert may be, for example, a polycarbonate membrane with a diameter of 6.5 mm, a pore size of 8 μm, and a thickness of 10 nm.

[0224] The infiltration assay uses a BD Biocoat growth factor-reduced Matrigel infiltration chamber (BD This procedure is performed in Biosciences. Cancer cell lines are harvested and resuspended in osteocyte-derived CM regardless of the test antibody. The cancer cell suspension is added to the top of the insert. The cells are incubated at 37°C for various times. Non-migrating cells are removed through a filter, and migrating cells are fixed through the insert and then placed in a Hema 3 Stat Pack (Fisher). Stain with (Scientific). Count the number of migrating cells in 5 fields per insert under a light microscope.

[0225] When CM derived from osteocytes treated with AD, FFSS, or antibodies disclosed herein to stimulate Cx43 hemichannel opening, and incubated, breast cancer cell migration may be reduced. This inhibitory effect on cancer cell migration was attenuated when osteocyte Cx43 hemichannels were blocked with E2 antibodies. This reduction in cancer cell migration was not observed when incubated with CM collected from osteoblasts or when directly treated with AD. Cx43 hemichannel opening by antibodies disclosed herein is protective against breast cancer cell growth and migration.

[0226] B. In vivo assay The efficacy of test antibodies against bone metastases in vivo is determined using an intratibial injection bone metastasis model and / or an intracardiac injection cancer metastasis assay.

[0227] Intratibial injection bone metastasis model. The method involves anesthetizing 1-month-old normal or immunodeficient mice with isoflurane. Buprenorpine-HCl (0.3 mg / ml) is also administered to the mice as an analgesic. Intratibial injection is performed using cancer cells expressing a fluorescent or chemiluminescent marker (e.g., Py8119 cells expressing Luc-GFP for normal mice, or MD-MBA-231 expressing Luc-GFP for immunodeficient mice). Cancer cells are inoculated into the bone marrow region of the right tibia through a pre-made hole using a Hamilton syringe fitted with a 30-gauge needle. PBS is injected into the left tibia as a control. Test antibodies or saline are administered intraperitoneally twice weekly for 5 weeks. Intratibial tumor growth is monitored weekly starting 3 days after tumor cell inoculation using bioluminescence imaging or fluorescence. At the end of the study after sufficient bioluminescence imaging, X-ray images are taken to test bone quality, and labeled metastatic cancer cell colonies are observed and counted using a fluorescence microscope.

[0228] Intracardiac injection bone metastasis model. Normal or immunodeficient mice aged 2-3 months are anesthetized with isoflurane and given buprenorpine-HCl (0.3 mg / ml) as an analgesic. Cancer cells expressing a fluorescent or chemiluminescent marker (e.g., Py8119 cells expressing Luc-GFP for normal mice, or Luc-GFP-MD-MBA-231 for immunodeficient mice) are injected into the left ventricle of the mouse. The procedure includes: holding the needle tilted to the right toward the worker, insert it into the second intercostal space, approximately 3 mm to the left of the sternum. Advance the needle approximately 5 mm and gently rotate it until a pulsating flow of bright red arterial blood is observed entering the hub. Inject the cell suspension over 30 seconds. Withdraw the needle and apply pressure to the injection site for 30 seconds using an alcohol wipe. Place the mouse on a warmed surface until fully recovered from anesthesia. Bioluminescence or fluorescence imaging will be performed after intracardiac injection to verify the distribution of tumor cells weekly, starting 3 days after tumor cell inoculation. At the end of the study after sufficient bioluminescence imaging, X-ray images will be taken to assess bone quality, and labeled metastatic cancer cell colonies will be observed and counted using a fluorescence microscope.

[0229] Cx43 conditional knockout (cKO) mice. Since homozygous Cx43 global knockout is lethal and the inventors also wanted to investigate the role of Cx43 expressed in osteocytes, osteocyte-specific Cx43 knockout mice were generated. Mice homozygous for the floxed Cx43 gene were mated with Cx43 global heterozygous mice to promote complete deletion of Cx43 in osteocytes. Then, Cx43fl / - mice (50% of the progeny) were mated with mice expressing Cre recombinase driven by the human DMP-1 promoter. This resulted in the generation of Cx43 fl / -, DMP1 Cre+ or Cx43 fl / -, DMP1 Cre- mice (a very small portion were Cx43fl / fl or Cx43- / -). Cx43-deficient osteocytes were confirmed by immunohistochemistry.

[0230] The study may include eight groups of mice: WT treated with alendronate (AD), WT without AD, cKO treated with AD, cKO without AD, WT treated with a test antibody (TA), WT without TA, cKO treated with TA, and cKO without TA. AD or TA was administered to the mice at 150 μg / kg body weight. In the case of AD or TA treatment, in KO, bone metastasis is expected to increase compared to WT mice. Also, in the case of no AD or TA treatment, bone metastasis should be similar between WT mice and knockout mice.

[0231] Various aspects of the present disclosure can be used alone, in combination, or in various arrangements not specifically discussed in the above embodiments, so in its application, it is not limited to the details and arrangements of the components described in the foregoing description or shown in the drawings. For example, the aspects described in one embodiment can be combined with the aspects described in other embodiments in any way.

[0232] While specific embodiments of the subject matter disclosure have been discussed, this specification is illustrative and not limiting. A review of this specification will reveal many variations of this disclosure to those skilled in the art. The full scope of this disclosure should be determined by reference to the full scope of the claims and equivalents and to this specification and such variations.

[0233] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole for all purposes to the same extent as each individual publication, patent, or patent application is specifically indicated to be incorporated by reference.

Claims

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

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

8.

3. A nucleic acid encoding a heavy chain and a light chain of an anti-Cx43 antibody, wherein the heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 17, and the light chain has the amino acid sequence of SEQ ID NO:

18.

4. The nucleic acid according to any one of claims 1 to 3, further comprising a promoter operably coupled to the nucleic acid.

5. An expression vector comprising the nucleic acid according to any one of claims 1 to 4.

6. The expression vector according to claim 5, wherein the vector is a viral vector.

7. A cell comprising the nucleic acid according to any one of claims 1 to 4, or the expression vector according to claim 5 or 6.

8. The cell according to claim 7, wherein the cell is a hybridoma cell line.

9. A method for producing an anti-Cx43 antibody, wherein the method comprises the step of culturing the cells described in claim 7 or 8 under appropriate conditions, thereby producing the anti-Cx43 antibody.

10. The method according to claim 9, further comprising the step of purifying the anti-Cx43 antibody.

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