Compositions and Methods of Use for Treating Osteosarcoma

Abstract

Methods for treating or preventing osteosarcoma are provided. SOLUTION: A connexin 43 antibody or a fragment thereof is administered.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 811,938, filed Feb. 28, 2019. The entire content of the prior application is incorporated herein by reference.

[0002] Incorporation of Sequence Listing This application contains a Sequence Listing contained in a file named "21105_0070P1_SL.txt", which is 24,576 bytes in size, created on Feb. 25, 2020, and submitted via EFS - Web simultaneously with the filing of this application. The entire Sequence Listing is incorporated herein by reference.

Background Art

[0003] Connexin hemichannels play important roles in the functions of cells and tissues, and abnormal actions of connexin hemichannels can be involved in various pathological conditions as described herein. Thus, there remains a need for additional therapies for treating pathological conditions related to hemichannel activity, as well as methods for identifying such therapies.

[0004] Osteosarcoma occurs in patients including young adults and usually metastasizes to the lungs, making the disease incurable. Currently available treatments are related to symptom relief, but none of these treatments cure the disease. There is a need to treat osteosarcoma or lung metastases, bone metastases, brain metastases or metastases to other organs in osteosarcoma patients in need thereof.

Summary of the Invention

[0005] Disclosed herein is a method for treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti - connexin 43 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58.

[0006] Disclosed herein is a method for treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63.

[0007] Disclosed herein is a method for treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or fragment thereof, wherein the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58 and a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63.

[0008] Described herein is a method for treating or preventing osteosarcoma, lung metastasis, bone metastasis, brain metastasis, or metastasis to other organs in a subject having osteosarcoma, the method comprising administering to the subject an effective amount of an antibody that binds to the connexin 43 (Cx43) hemichannel and enhances channel opening or an expression vector encoding the antibody. In some embodiments, the method can comprise administering to the subject an effective amount of the antibody. In some embodiments, the method can comprise administering to the subject an effective amount of an expression vector encoding the antibody. In some embodiments, the antibody that binds to the connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap junction binding.

[0009] Described herein is a method of treating or preventing osteosarcoma in a subject, comprising administering to the subject, in an effective amount, an antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening, or an expression vector encoding the antibody. In some embodiments, the method comprises administering to the subject an effective amount of the antibody. In some embodiments, the method comprises administering to the subject an expression vector encoding an effective amount of the antibody. In some embodiments, the osteosarcoma has metastasized to the lungs of the subject. In some embodiments, the antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap junction binding.

[0010] Described herein is an expression vector encoding an antibody that can be administered in a pharmaceutically acceptable composition. In some embodiments, the antibody can be administered systemically. In some embodiments, the antibody can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

[0011] Disclosed herein is an antibody comprising a first VHCDR corresponding to SEQ ID NO: 19, a second VHCDR corresponding to SEQ ID NO: 20, a third VHCDR corresponding to SEQ ID NO: 21, a first VLCDR corresponding to SEQ ID NO: 49, a second VLCDR corresponding to SEQ ID NO: 50, and a third VLCDR corresponding to SEQ ID NO: 51. In some embodiments, the antibody can be a humanized antibody. In some embodiments, the antibody can be an IgG, IgM, IgA, IgD, IgE, or genetically engineered IgG class antibody comprising a first VHCDR corresponding to SEQ ID NO: 19, a second VHCDR corresponding to SEQ ID NO: 20, a third VHCDR corresponding to SEQ ID NO: 21, a first VLCDR corresponding to SEQ ID NO: 49, a second VLCDR corresponding to SEQ ID NO: 50, and a third VLCDR corresponding to SEQ ID NO: 51. In some embodiments, the antibody can be an antibody of the IgG class, wherein the IgG class antibody can be an antibody of the IgG1, IgG2, IgG3, or IgG4 class. In some embodiments, the antibody can comprise a VH amino acid sequence that is at least 90% identical to SEQ ID NO: 58 and / or a VL amino acid sequence that is at least 90% identical to SEQ ID NO: 63. In some embodiments, the antibody comprises a VH amino acid sequence according to SEQ ID NO: 58 and / or a VL amino acid sequence according to SEQ ID NO: 63.

[0012] In some embodiments, the disclosed method can further comprise administering at least a second anti-cancer therapy to the subject. In some embodiments, the second anti-cancer therapy can be surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0013] Disclosed herein are recombinant connexin 43 (Cx43) hemichannel-binding antibodies or fragments thereof. In some embodiments, the antibody comprises a first VHCDR corresponding to SEQ ID NO: 19 or a fragment thereof, a second VHCDR corresponding to SEQ ID NO: 20 or a fragment thereof, a third VHCDR corresponding to SEQ ID NO: 21 or a fragment thereof, a first VLCDR corresponding to SEQ ID NO: 49 or a fragment thereof, a second VLCDR corresponding to SEQ ID NO: 50 or a fragment thereof, and a third VLCDR corresponding to SEQ ID NO: 51 or a fragment thereof. In some embodiments, the antibody or fragment thereof can be a humanized antibody. In some embodiments, the antibody is an IgG, IgM, IgA, IgD, IgE, or genetically engineered IgG-class antibody comprising a first VHCDR corresponding to SEQ ID NO: 19, a second VHCDR corresponding to SEQ ID NO: 20, a third VHCDR corresponding to SEQ ID NO: 21, a first VLCDR corresponding to SEQ ID NO: 49, a second VLCDR corresponding to SEQ ID NO: 50, and a third VLCDR corresponding to SEQ ID NO: 51. In some embodiments, the antibody can be an IgG-class antibody, wherein the IgG-class antibody is an antibody of the IgG1, IgG2, IgG3, or IgG4 class. In some embodiments, the antibody comprises a VH amino acid sequence or fragment thereof that is at least 90% identical to SEQ ID NO: 58 and / or a VL amino acid sequence or fragment thereof that is at least 90% identical to SEQ ID NO: 63. In some embodiments, the antibody may comprise a VH amino acid sequence or fragment thereof according to SEQ ID NO: 58 and / or a VL amino acid sequence or fragment thereof according to SEQ ID NO: 63.

[0014] Disclosed herein is a method for treating osteosarcoma in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody according to the compositions described herein or an expression vector encoding an antibody according to the embodiments described herein. In some embodiments, the pharmaceutical composition comprises an expression vector encoding an antibody according to the embodiments described herein for the subject. In other embodiments, the pharmaceutical composition comprises an antibody according to the embodiments described herein for the subject. In some embodiments, the method may further be defined as a method for suppressing or preventing cancer lung metastasis in the subject. In some embodiments, the subject has osteosarcoma and / or has lung metastasis, bone metastasis, brain metastasis or metastasis to other organs. In some embodiments, the pharmaceutical composition may be administered systemically. In some embodiments, the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

[0015] In some embodiments, the pharmaceutical composition may comprise a first VH CDR identical to SEQ ID NO: 19, a second VH CDR identical to SEQ ID NO: 20, a third VH CDR identical to SEQ ID NO: 21, a first VL CDR identical to SEQ ID NO: 31, a second VL CDR identical to SEQ ID NO: 32, and a third VL CDR identical to SEQ ID NO: 65. In some embodiments, the method may further comprise administering to the subject at least a second anti-cancer therapy. In a further embodiment, the second anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0016] Disclosed herein are antibodies against hemichannel polypeptides and nucleic acid molecules encoding said antibodies. In some embodiments, the antibody can bind to an epitope having the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13), KRDPCPHQVD (SEQ ID NO: 14), or LSAVYTCKR (SEQ ID NO: 15). In some embodiments, the antibody can bind to an epitope having the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13).

[0017] In a further embodiment, the antibody for use according to the embodiment can be any of those described in International (PCT) Patent Publication No. WO2015-027120 or WO2017-147561, which are incorporated herein by reference for their teachings of the antibody, vector, and cell for making or expressing the antibody.

[0018] In some embodiments, the first heavy chain region can comprise an amino acid sequence having the amino acid sequence of residues 13 to 37 of SEQ ID NO: 2, the second heavy chain region has an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO: 2, and the third heavy chain region comprises an amino acid sequence having the amino acid sequence of residues 97 to 116 of SEQ ID NO: 2.

[0019] In some embodiments, the antibodies disclosed herein can include full-length antibodies, antibody fragments, single-chain antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies and antibody fusions, as well as fragments thereof.

[0020] Disclosed herein are pharmaceutical compositions comprising any of the antibodies or fragments thereof as described herein together with a pharmaceutically acceptable carrier. Also disclosed herein are antibodies or pharmaceutical compositions for use as a drug for treating osteosarcoma, or for suppressing or preventing cancer metastasis to the lung, or for use in a therapy.

[0021] Disclosed herein is a method for treating or preventing cancer metastasis. In some embodiments, the cancer can be bone metastasis, brain metastasis, or metastasis to other organs of lung cancer in an osteosarcoma patient. The method of treatment can include administering to a subject in need thereof an effective amount of an isolated antibody as described herein. Also disclosed herein is a method of using any of the antibodies described herein in the manufacture of a drug for the treatment or prevention of cancer metastasis (e.g., lung cancer).

[0022] Disclosed herein is an in vitro method of using any of the antibodies, compounds or reagents described herein to activate or enhance Cx43 hemichannels. In some embodiments, the methods described herein are used to (i) determine hemichannel opening by a dye uptake assay using Lucifer Yellow or Alexa dyes, (ii) evaluate the stimulatory effect on hemichannel opening by measuring ATP levels (e.g., an increase in ATP release from osteocytes through Cx43 hemichannels can indicate that the compound or antibody being tested can suppress tumors or arrest cell proliferation and / or colony formation), (iii) determine the effect on activation of Cx43 hemichannel opening in osteocytes by examining the stimulatory effect of a reagent on hemichannel opening by mechanical loading in the form of shear stress of fluid flow. In some embodiments, an antibody that binds to the connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap junction binding.

[0023] As used herein, the term "antigen" is a molecule to which an antibody or T cell receptor can bind. In some embodiments, binding moieties other than antibodies can be engineered to specifically bind to antigens such as aptamers, avimers, and the like.

[0024] The terms "antibody" or "immunoglobulin" are used to include intact antibodies and their binding fragments / segments. As used herein, the term "antibody" is intended to broadly refer to any immunological binding agent such as IgG, IgM, IgA, IgD, IgE and genetically engineered IgG, as well as polypeptides containing antibody CDR domains that retain antigen binding activity. Antibodies may be selected from the group consisting of chimeric antibodies, affinity matured antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, or antigen-binding antibody fragments or natural or synthetic ligands. Usually, fragments compete with the intact antibody from which they are derived for specific binding to an antigen. Fragments include separated heavy and light chains, Fab, Fab’, F(ab’)2, Fabc, and Fv. Fragments / segments may be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes one or more immunoglobulin chains that are chemically conjugated to or expressed as a fusion protein with another protein. The term "antibody" includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-21, 1990, Kostelny et al., J. Immunol. 148:1547-53, 1992.

[0025] The term "antibody" can include five different classes of human immunoglobulins, namely IgG, IgA, IgM, IgD, and IgE. In some embodiments, the disclosed antibodies can be antibodies of the IgG class that can be classified into four subclasses: IgG1, IgG2, IgG3, and IgG4. In some embodiments, the disclosed antibodies can be antibodies of the IgA class that can be classified into two subclasses: IgA1 and IgA2. The basic structure of an immunoglobulin is composed of two identical L chains (light chains) and two identical H chains (heavy chains). The class and subclass of an immunoglobulin are determined by the H chain. In some embodiments, the antibody(s) or its variant or fragment can be IgG4.

[0026] The antibody stability of IgG4 can be improved while maintaining the antigen-binding specificity. In some embodiments, the antibody can be improved, for example, by substituting arginine (R) of IgG4 with glutamic acid (E), phenylalanine (F), isoleucine (I), asparagine (N), glutamine (Q), serine (S), valine (V), tryptophan (W), tyrosine (Y), lysine (K), threonine (T), methionine (M), or leucine (L).

[0027] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). Further, an isolated compound refers to one that can be administered to a subject as an isolated compound. In other words, if a compound is attached to a column or embedded in an agarose gel, the compound may not simply be regarded as "isolated". Further, an "isolated nucleic acid fragment" or "isolated peptide" is a fragment of a nucleic acid or protein that does not exist naturally as a fragment and / or is not normally in a functional state.

[0028] Portions of the invention such as polypeptides, peptides, antigens, or immunogens may be conjugated, either covalently or non-covalently, to other portions such as adjuvants, proteins, peptides, supports, fluorescent moieties, or labels, or may be linked, either covalently or non-covalently. The terms "conjugate" or "immunoconjugate" are widely used to define an operative association of one portion with another agent, and are not intended to refer only to some kind of operative association, and are not particularly limited to chemically "conjugating".

[0029] The term "providing" is used according to its ordinary meaning of "supplying or giving for use". In some embodiments, the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid encoding the protein. In certain aspects, the invention contemplates compositions comprising various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0030] The expressions "specifically binds to" or "is specifically immunoreactive with" a target refer to a binding reaction that determines the presence of a molecule in the presence of a heterogeneous population of other biological agents. Thus, under the specified immunoassay conditions, the specified molecule binds preferentially to a particular target and does not bind in significant amounts to other biological agents present in the sample. Specific binding of an antibody to a target under such conditions requires that the antibody be selected for its specificity for the target. Various immunoassay formats may be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

[0031] Other aspects of the present invention are discussed throughout this application. Any aspect discussed with respect to one aspect of the present invention is similarly applicable to other aspects of the present invention, and vice versa. Each aspect described herein is understood to be an aspect of the present invention applicable to other aspects of the present invention. It is contemplated that any aspect discussed herein can be implemented with respect to any method or composition of the present invention, and vice versa. Further, the methods of the present invention can be achieved using the compositions and kits of the present invention.

[0032] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one", but is also consistent with the meanings of "one or more", "at least one", and "one or more than one".

[0033] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method used to determine that value.

[0034] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to only an alternative and unless the alternative is mutually exclusive, but the present disclosure supports definitions that refer to only the alternative and "and / or".

[0035] As used in this specification and the claims (if any), the word "comprising" (and any form such as, for example, "comprise" or "comprises"), "having" (and any form such as, for example, "have" or "has"), "includeing" (and any form such as, for example, "includes" or "include") or "containing" (and any form such as, for example, "contains" or "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0036] As used herein, the terms "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids or any unnatural analog that may be present in any of the disclosed antibodies, variants, or fragments. Thus, as used herein, "amino acid" means both naturally occurring amino acids and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the present invention. "Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chain may be in either the (R) configuration or the (S) configuration. In some embodiments, the amino acid is in the (S) configuration or the L-configuration. When non-naturally occurring side chains are used, non-amino acid substituents may be used, for example, to prevent or delay in vivo degradation.

[0037] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, so this detailed description and the specific examples are given by way of illustration only while showing specific embodiments of the present invention and are to be understood as such.

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

Brief Description of the Drawings

[0039]

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Modes for Carrying Out the Invention

[0040] The disclosed methods and compositions can be more readily understood by reference to the following detailed description of specific embodiments and the examples contained therein, as well as the figures and the description before and after them.

[0041] It should be understood that the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, unless otherwise specified, and thus may vary. It should also be understood that the terms used herein are for the purpose of merely describing specific embodiments and are not intended to be limiting.

[0042] Disclosed are materials, compositions, and components that can be used in the disclosed methods and compositions, can be used in conjunction with them, can be used in their preparation, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, specific mention of each of the various individual or collective combinations and permutations of these compounds may not be explicitly disclosed, but each is specifically contemplated and understood to be described herein. If classes of molecules A, B, and C and classes of molecules D, E, and F are disclosed and, as an example of a combination molecule, A-D is disclosed, each combination is individually and collectively contemplated even if not individually enumerated. Thus, in this example, from the disclosure of the examples of A, B, and C; D, E, and F; and the combination A-D, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F should be specifically contemplated and considered disclosed. Similarly, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, from the disclosure of the examples of A, B, and C; D, E, and F; and the combination A-D, the subgroups A-E, B-F, and C-E should be specifically contemplated and considered disclosed. This concept applies to all aspects of this application, including but not limited to the steps of methods for generating and using the compositions of the present disclosure. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the disclosed method and that each such combination is specifically contemplated and should be considered disclosed.

[0043] All publications mentioned in this specification are hereby incorporated by reference in their entirety for the purpose of disclosing and describing the publications and the methods and / or materials by which they are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure has no right to antedate such publications by virtue of prior disclosure. Further, the publication dates provided herein may be different from the actual publication dates and individual verification may be required.

[0044] Definitions The disclosed methods and compositions are not limited to the specific procedures, protocols, and reagents described, and it is understood that they may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0045] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. "Any" or "optionally" means that the event, situation, or substance described thereafter may or may not occur or exist, and the description includes both the case where the event, situation, or substance occurs or exists and the case where it does not occur or exist.

[0046] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0047] Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, it is to be specifically contemplated and considered disclosed, unless the context specifically indicates otherwise, as being the range extending from one particular value and / or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent “about”, then, unless the context specifically indicates otherwise, the particular value is to be considered as specifically contemplated to form another specifically contemplated embodiment that is disclosed. Further, unless the context specifically indicates otherwise, it will be understood that each endpoint of a range is effective both in relation to and independent of the other endpoint. Finally, it should be understood that all individual values and sub-ranges of values contained within the explicitly disclosed range are specifically contemplated and, unless the context specifically dictates otherwise, are to be considered disclosed. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0048] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, are meant to mean “including but not limited to”, and are not intended to exclude, for example, other additives, components, integers or steps. In particular, in a method described as including one or more steps, each step is specifically contemplated (unless the step includes a limiting term such as “consisting of”) as including what is recited, and is not intended to exclude, for example, other additives, components, integers or steps not recited in that step.

[0049] "Inhibit", "inhibiting", and "inhibition" mean weakening or reducing an activity, level, reaction, state, disease, or other biological parameter. This can include, but is not limited to, complete elimination of the activity, reaction, state, or disease. This can also include, for example, a 10% inhibition or decrease in the activity, reaction, state, or disease compared to a natural level or a control level. Thus, in some embodiments, the inhibition or decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of decrease therebetween compared to a natural level or a control level. In some embodiments, the inhibition or decrease is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% compared to a natural level or a control level. In some embodiments, the inhibition or decrease is 0-25, 25-50, 50-75, or 75-100% compared to a natural level or a control level.

[0050] As used herein, "modulate", "modulating", and "modulation" mean a change in an activity or function or number. The change can be an increase or decrease, improvement or inhibition of the activity, function, or number.

[0051] "Promote", "promotion", and "promoting" refer to an increase in activity, reaction, condition, disease, or other biological parameter. This can include, but is not limited to, the initiation of the activity, reaction, condition, or disease. This can also include, for example, a 10% increase in the activity, reaction, condition, or disease compared to a natural level or a control level. Thus, in some embodiments, the increase or promotion can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion therebetween, compared to a natural level or a control level. In some embodiments, the increase or promotion is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% compared to a natural level or a control level. In some embodiments, the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, compared to a natural level or a control level, such as 200, 300, 500, or 1000% more. In some embodiments, the increase or promotion can be greater than 100 percent compared to a natural level or a control level, such as 100, 150, 200, 250, 300, 350, 400, 450, 500%, or more, compared to the natural level or the control level.

[0052] "Treatment" and "treating" refer to the administration or application of a therapeutic agent (e.g., an anti-Cx43 antibody described herein) to a subject, or the performance of a procedure or treatment modality on a subject for the purpose of obtaining a therapeutic benefit in a disease or health-related condition. For example, treatment can include the administration of a pharmaceutically effective amount of an antibody that enhances or stimulates the opening of Cx43 hemichannels. In some embodiments, an antibody that binds to connexin 43 (Cx43) hemichannels and enhances channel opening can have no effect on gap junction binding.

[0053] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, reducing, delaying the onset of, suppressing or slowing the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment can be administered to a subject who does not exhibit symptoms of a disease, disorder, and / or condition and / or to a subject who exhibits only early symptoms of a disease, disorder, and / or condition for the purpose of reducing the risk of developing the pathology associated with the disease, disorder, and / or condition. For example, the disease, disorder, and / or condition can be osteosarcoma or cancer.

[0054] As used herein, the term "subject" refers to a target for administration, e.g., a human. Thus, the subject of the disclosed methods can be a vertebrate, e.g., a mammal, fish, bird, reptile, or amphibian. The term "subject" also includes companion animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, flies, etc.). In one aspect, the subject is a mammal. In another aspect, the subject is a human. The term does not denote a particular age or sex. Thus, both adult, pediatric, adolescent, and neonatal subjects, as well as fetuses, are intended to be encompassed regardless of sex.

[0055] As used herein, the term "patient" refers to a subject suffering from a disease or disorder. The term "patient" includes human and animal subjects. In some aspects of the disclosed methods, the "patient" is, for example, diagnosed as in need of treatment prior to the step of administration.

[0056] The term "fragment" can refer to a portion of a protein or nucleic acid molecule that is substantially identical to a reference protein or nucleic acid and retains the reference biological activity (e.g., at least 5, 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids, etc.). In some embodiments, the fragment or portion retains at least 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the biological activity of the reference protein or nucleic acid described herein. Further, a fragment of a reference peptide can be a continuous or adjacent portion of the reference polypeptide (e.g., a fragment of a 10 - amino - acid - long reference peptide can be any 2 - 9 adjacent residues within that reference peptide).

[0057] "Variant" can mean any difference from a reference sequence other than a mere deletion of N - terminal and / or C - terminal amino acid residue(s). When a variant contains an amino acid residue substitution, the substitution can be considered conservative or non - conservative. Conservative substitutions are substitutions within the following groups: Ser, Thr, and Cys; Leu, Ile, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gln, Asn, Glu, Asp, and His. A variant can contain at least one substitution and / or at least one addition, and there may be at least one deletion. A variant can also contain one or more non - naturally - occurring residues. For example, a variant may contain selenocysteine (e.g., seleno - L - cysteine) at any position, including the position of cysteine. Many other "non - natural" amino acid substitutes are known in the art and are available from commercial sources. Examples of non - naturally - occurring amino acids include D - amino acids, amino acid residues having an acetylaminomethyl group bonded to the sulfur atom of cysteine, PEGylated amino acids, and the formula NH2(CH2) nOmega amino acids of COOH (where n is from 2 to 6), neutral non-polar amino acids, such as sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine and norleucine. Phenylglycine can be an alternative to Trp, Tyr or Phe. Citrulline and methionine sulfoxide are neutral non-polar, cysteic acid is acidic, and ornithine is basic. Proline can be substituted with hydroxyproline and can retain the steric structure that brings about the properties of proline.

[0058] The term "single-chain variable fragment (scFv)" means a protein containing the variable regions of the heavy and light chains of an antibody. The scFv can be a fusion protein containing a variable heavy chain, a linker, and a variable light chain. In some embodiments, the linker can be a short, flexible fragment that can be about 8 to 20 amino acids in length. For example, (G4S) n can be used (n = 1, 2, 3 or 4).

[0059] The "fragment antigen-binding fragment (Fab)" is the region of an antibody that binds to an antigen. The Fab contains the constant and variable regions derived from both the heavy and light chains.

[0060] The "CDR" or complementarity-determining region is a region of hypervariability dispersed within a more conserved region called the "framework region" (FR).

[0061] The term "monoclonal antibody" (monoclonal antibody) should not be construed to require the production of an antibody by a specific method and refers to an antibody or a population of similar antibodies obtained from a substantially homogeneous population of antibodies, including but not limited to monoclonal antibodies produced by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 - 497, 1975) or by recombinant DNA methods.

[0062] The term "chimeric antibody" (or "chimeric immunoglobulin") refers to a molecule containing heavy and / or light chains that are identical or homologous to the corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but in which the remainder of the chain(s) is derived from a different species or belongs to a different antibody class or subclass, and that are identical or homologous to the corresponding sequences in fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et al. (1984), hereinafter Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851).

[0063] The term "humanized antibody" refers to a form of antibody that contains sequences derived from non-human (e.g., murine) antibodies as well as human antibodies. A humanized antibody can contain conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and / or biological activity. Such antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. A humanized antibody is mostly a human immunoglobulin (recipient antibody), and the residues of the recipient's complementarity-determining regions (CDRs) are replaced by the residues of the CDRs of a non-human species (donor antibody) such as mouse, rat, camel, goat, or rabbit that has the desired properties. Further, a humanized antibody can contain residues that are not found in the recipient antibody, nor in the transferred CDR sequences or framework sequences. These modifications are made to further improve and maximize antibody performance. Thus, generally, a humanized antibody can contain all or substantially all of at least one, and in one embodiment two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being the FRs of human immunoglobulin sequences. A humanized antibody can also optionally contain at least a portion of the immunoglobulin constant region (Fc), or that of a human immunoglobulin (see, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO86 / 01533; Neuberger, M.S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E.A. et al., European Patent Application No. 0,519,596 A1; Queen et al. (1989) Proc. Natl. Acad. Sci. USA, Vol 86:10029-10033).

[0064] As used herein, the term "M1H" refers to an antibody cloned from a hybridoma clone. "M1" refers to hybridoma monoclonal 1, and "H" refers to the variable heavy chain.

[0065] As used herein, the term "M1M7K" refers to the variable light chain identified from hybridoma clones M1 and M7.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the present invention, but the particularly useful methods, devices, and materials are as described. Publications cited herein and the materials to which they are cited are specifically incorporated herein by reference. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Reference thereto does not admit that it constitutes prior art. The discussion of references states what the authors assert, and the applicant reserves the right to challenge the accuracy and appropriateness of the cited documents. Although several publications are referred to herein, it will be clearly understood that none of these documents are admitted to form a part of the common general knowledge in the art.

[0067] Various cells can communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by the protein connexin. Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins form one hemichannel, and two hemichannels form one gap junction channel. Gap junctions are clusters of channels located in the plasma membranes between adjacent cells and mediate intercellular communication. Hemichannels are distinct entities from gap junction channels. Hemichannels allow for the exchange of molecules between the intracellular compartment and the extracellular environment.

[0068] Osteocytes express hemichannels known as connexin (Cx)43 hemichannels. These osteocyte hemichannels are normally closed and can open when exposed to mechanical stimuli, which leads to the release of various factors into the bone microenvironment. The factors released by hemichannel opening can intervene in other processes that can reduce the migration and bone metastasis of tumor cells.

[0069] Disclosed herein is a method of identifying a reagent that modulates the opening of connexin hemichannels. In some embodiments, modulation can mean stimulating or enhancing the opening of one or more connexin hemichannels. In some embodiments, the connexin hemichannel can be a Cx43 hemichannel. In some embodiments, the method can identify a compound or drug that positively modulates (i.e., stimulates or enhances) the opening of connexin hemichannels. Other embodiments are directed to methods of treating osteosarcoma by administering to a patient diagnosed with or having osteosarcoma a compound that opens or stimulates or enhances the opening of connexin 43 hemichannels. In some embodiments, the patient has a primary tumor. In some embodiments, a compound that opens or stimulates or enhances the opening of Cx43 hemichannels can be used to prevent, inhibit, or reduce metastasis to the lung. In some embodiments, a compound that opens or stimulates or enhances the opening of Cx43 channels can be used to treat osteosarcoma. In some embodiments, an antibody that binds to the connexin 43 (Cx43) hemichannel and enhances channel opening can be one that has no effect on gap junction binding.

[0070] Cancer metastasis occurs when cancer spreads from the part of the body where it originated (e.g., bone) to other parts of the body (e.g., lung) to establish secondary tumors. The lung is one of the most common sites of cancer metastasis in patients with osteosarcoma. Cancers that metastasize to the lung include, but are not limited to, breast cancer, prostate cancer, bone cancer, and skin cancer (e.g., melanoma). Lung metastases can be identified in patients with osteosarcoma. Lung metastases (mets) are associated with many serious clinical and quality of life outcomes, such as, but not limited to, intractable pain, nausea, headache, shortness of breath, hemoptysis, pleural effusion, and motor impairment. In many cases, the systemic presence of cancer can render the cancer incurable.

[0071] Normal bone is composed of three major cell types: osteoblastic osteoblasts, osteoclastic osteoclasts, and osteocytes. Osteocytes make up approximately 95% of the cells in bone and maintain the bone remodeling process by regulating osteolytic and osteogenic activities. When cancer cells invade bone, many of the normal bone functions are affected. Cancer cells interact with the local microenvironment to promote cancer cell survival through bone destruction and angiogenesis.

[0072] The opening of osteocyte connexin (Cx)43 hemichannels and their released factor(s) have an inhibitory role in cancer growth, migration, and metastasis, while inactivation of the hemichannels has the opposite effect. Cx43 hemichannels mediate the exchange of information between the intracellular and extracellular microenvironments. Cx43 hemichannels are abundant in the osteocytes of bone, and ATP released from osteocytes via Cx43 hemichannels plays a tumor-suppressive role against cancer bone metastasis. Therefore, enhancing the activation of Cx43 hemichannels in osteocytes can be an important strategy in protecting skeletal tissue against cancer cell growth and colony formation. In some embodiments, the antibodies described herein can reduce or inhibit the growth of tumor cells in bone, such as the growth of intratibial tumors.

[0073] The Cx43 hemichannels in osteocytes have been shown to be opened by treatment with alendronate (AD), an effective and commonly used bisphosphonate drug. Bisphosphonates are a class of drugs known for treating many bone disorders, including bone metastasis. Powles et al. have shown that administration of bisphosphonates is associated with a decrease in the incidence of bone metastasis and a decrease in mortality in breast cancer patients. AD has been associated with a decrease in tumor growth, as well as a decrease in bone destruction and pain. AD inhibits osteoclast activity and induces the opening of Cx43 hemichannels in osteocytes (Plotkin et al., 2002). However, AD administration is associated with multiple serious side effects.

[0074] antibody Disclosed herein are antibodies that can stimulate or enhance open hemichannels, particularly Cx43 hemichannels. In some embodiments, antibodies that bind to connexin 43 (Cx43) hemichannels and enhance channel opening can be those that have no effect on gap junction binding. Examples of identifying and isolating monoclonal antibodies are described below.

[0075] As used herein, the term "CDR" refers to the complementarity determining regions of the variable domains of an antibody. The systematic identification of residues included in CDRs has been developed by Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). The variable light chain (VL) CDRs are defined herein as including the residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3). The variable heavy chain (VH) CDRs are defined herein as including the residues at positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).

[0076] The CDRs disclosed herein may also include variants. Generally, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Thus, a "variant CDR" has a specific identity to the parent CDR or reference CDR of the invention and shares a biological function including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR. For example, a "variant CDR" can be a sequence containing 1, 2, 3 or 4 amino acid changes compared to the parent CDR or reference CDR of the invention and shares or improves the biological function, specificity and / or activity of the parent CDR.

[0077] In some embodiments, any of the CDR sequences disclosed herein can include a single amino acid change compared to a parental CDR or a reference CDR. In some embodiments, any of the CDR sequences disclosed herein can include at least two amino acid changes compared to a parental CDR or a reference CDR. In some embodiments, the amino acid change can be a change from a cysteine residue to another amino acid. In some embodiments, the amino acid change can be a change from a glycine residue to another amino acid. The amino acid identity between individual variant CDRs can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Thus, a "variant CDR" has an identity specified with respect to a parental CDR of the invention and shares a biological function including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parental CDR. For example, the parental CDR sequence can be one or more of SEQ ID NOs: 19, 20, 21, 49, 50 and / or 51. The variant CDR sequence can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 19, 20, 21, 49, 50 and / or 51. The variant CDR sequence can also share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parental CDR.

[0078] As discussed herein, minor changes in the amino acid sequence of any of the antibodies disclosed herein are contemplated to be encompassed by the present disclosure, provided that the change in the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity with the parental sequence. In some embodiments, conservative amino acid substitutions are contemplated. Conservative substitutions are substitutions that occur within a family of related amino acids in the side chain. Genetically encoded amino acids are generally divided into the following families: (1) acidic = aspartic acid, glutamic acid, (2) basic = lysine, arginine, histidine, (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are as follows. Serine and threonine are the aliphatic hydroxy family, asparagine and glutamine are the amide-containing family, alanine, valine, leucine, and isoleucine are the aliphatic family, and phenylalanine, tryptophan, and tyrosine are the aromatic family. For example, isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions with structurally related amino acids of the amino acid, especially when the substitution does not involve amino acids within the framework region, it is reasonable to expect that it will not have a major impact on the binding or properties of the resulting molecule. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are known to those of skill in the art.

[0079] In some embodiments, the amino acid substitutions can be ones that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter the binding affinity, and (4) confer or modify other physicochemical or functional properties of such analogs. In some embodiments, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the non-CDR sequences of the heavy chain, light chain, or both. In some embodiments, one or more amino acid substitutions can be made in one or more of the CDR sequences of the heavy chain, light chain, or both.

[0080] Many methods have been developed for the chemical labeling and enhancement of the properties of antibodies and their common fragments including Fab fragments and F(ab’)2 fragments. Somewhat selective reduction of some of the antibody disulfide bonds has been previously achieved, generating antibodies and antibody fragments that can be labeled at defined sites and enhancing their utility and properties. Selective reduction of the disulfide bonds of the two hinges present in F(ab’)2 fragments using mild reduction has been useful. In some embodiments, cysteine and methionine may be susceptible to rapid oxidation, which can adversely affect the cleavage of protecting groups during synthesis and subsequent peptide purification. In some cases, cysteine residues in peptides used for antibody production can affect the binding ability of the antibody. This is because free cysteine is rare in vivo and may not be recognized by the native peptide structure. In some embodiments, the disclosed antibodies and their fragments include sequences in which cysteine residues present outside the CDRs (e.g., non-CDR sequences of the heavy chain, light chain, or both) are substituted. In some embodiments, cysteine can be replaced with serine and methionine can be replaced with norleucine (Nle). Multiple cysteines in a peptide or one of the disclosed antibodies or its fragments may readily form disulfide bonds unless a reducing agent such as dithiothreitol (DTT) is added to the buffer or cysteine can be replaced with serine residues.

[0081] A site or region for introducing mutations in the amino acid sequence is predetermined, but the mutations themselves do not have to be predetermined. For example, in order to optimize the performance of mutations at a given site, random mutagenesis can be performed in the target codon or region, and the CDR variants of the expressed antigen-binding protein can be screened for the optimal combination of desired activities. Techniques for performing substitution mutations at a predetermined site of DNA having a known sequence are well known, and examples include M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using an assay of antigen-binding protein activity as described herein.

[0082] Amino acid substitutions are usually single residues. Insertions will usually be about 1 to about 20 amino acid residues, although much larger insertions are tolerated. Deletions range from about 1 to about 20 amino acid residues, although in some cases the deletion may be much larger.

[0083] Substitutions, deletions, insertions, or any combination thereof can be used to reach the final derivative or variant. Generally, these changes are made to a small number of amino acids in order to minimize changes in the molecule, particularly changes in the immunogenicity and specificity of the antigen-binding protein. However, in certain circumstances, larger changes may be tolerated.

[0084] As used herein, "Fab" or "Fab region" means a polypeptide comprising the immunoglobulin domains of VH, CH1, VL, and CL. Fab may refer to this region alone or in connection with a full-length antibody, antibody fragment or Fab fusion protein, or any other antibody embodiment as outlined herein.

[0085] As used herein, "Fv" or "Fv fragment" or "Fv region" means a polypeptide comprising the VL domain and the VH domain of a single antibody.

[0086] As used herein, the term "framework" means the region of the antibody variable domain excluding those regions defined as CDRs. The framework of each antibody variable domain can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by the CDRs.

[0087] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (e.g., a hemichannel). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL / VK, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fab' that can be a Fab with a part of the hinge (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed. 1993)); (iv) an Fd fragment consisting of the VH domain and the CH1 domain; (v) an Fv fragment consisting of the VL domain and the VH domain of a single arm of the antibody; (vi) a dAb fragment consisting of the VH domain (Ward et al., (1989), Nature, 341:544-546); (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, which is a heavy chain variable region containing a single variable domain and two constant domains.

[0088] The term "specifically binds" (or "immunologically specifically binds") is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody "specifically binds" if its affinity for its intended target is at least about 5-fold greater than its affinity for non-target molecules. Preferably, there is no significant cross-reactivity or cross-binding with undesirable substances. The affinity of the antibody will be, for example, at least about 5-fold, such as 10-fold, such as 25-fold, particularly 50-fold, and particularly 100-fold or more greater for the target molecule than for non-target molecules. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10 6 M -1 of binding affinity. An antibody can have, for example, an affinity between about 10 8 M -1 and about 10 9 M -1 , between about 10 9 M -1 and about 10 10 M -1 , or between about 10 10 M -1 and about 10 11 M -1 , such as at least about 10 7 M -1It can bind with an affinity. The antibody can bind with an EC50 of, for example, 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 μM or less. In some embodiments, the antibody can bind with an EC50 of about 60 μg / ml, 59 μg / ml, 58 μg / ml, 57 μg / ml, 56 μg / ml, 55 μg / ml, 54 μg / ml, 53 μg / ml, 52 μg / ml, 51 μg / ml, 50 μg / ml, or less. In some embodiments, the antibody can bind with an EC50 of about 50 μg / ml, 49 μg / ml, 48 μg / ml, 47 μg / ml, 46 μg / ml, 45 μg / ml, 44 μg / ml, 43 μg / ml, 42 μg / ml, 41 μg / ml, 40 μg / ml, or less. In some embodiments, the antibody can bind with an EC50 of about 40 μg / ml, 39 μg / ml, 38 μg / ml, 37 μg / ml, 36 μg / ml, 35 μg / ml, 34 μg / ml, 33 μg / ml, 32 μg / ml, 31 μg / ml, 30 μg / ml, or less.

[0089] In some embodiments, the antibodies described herein can specifically bind to their intended targets. In some embodiments, the antibodies described herein do not have off-target binding. For example, the antibodies described herein do not bind to or distribute in the heart, liver, or spinal cord.

[0090] The antibodies described herein can be variants that include fragments (e.g., Fab fragments or F(ab’)2 fragments of a tetrameric antibody), fragments of scFvs or diabodies, or tetrameric antibodies, scFvs, diabody variants, or fragments thereof that differ by the addition and / or substitution of one or more amino acid residues, but are not limited thereto. The antibody moiety can be further engineered, for example, as a bispecific diabody.

[0091] As is well known in the art, certain types of antibody fragments can be generated by enzymatic treatment of "full-length" antibodies. Digestion with papain produces two identical Fab fragments, each having a single antigen-binding site, and the remaining Fc fragment. The Fab fragment also contains the constant domain of the light chain and the Chi domain of the heavy chain. In contrast, digestion with pepsin yields F(ab’)2 fragments that have two antigen-binding sites and can still cross-link antigens.

[0092] The Fab’ fragment differs from the Fab fragment in that it has additional residues at the C-terminus of the Chi domain, including one or more cysteine residues from the antibody hinge region. The cysteine residues in the constant domain have free thiol groups. The F(ab’)2 antibody fragment is a pair of Fab’ fragments linked by the cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known in the art.

[0093] The Fv region is the smallest fragment containing a complete antigen recognition and binding site consisting of the variable domains of one heavy chain and one light chain. The three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. As is known in the art, a "single-chain" antibody or "scFv" fragment is a single-chain Fv variant formed when the VH and VL domains of an antibody are included in a single polypeptide chain that recognizes and binds an antigen. Typically, single-chain antibodies contain a polypeptide linker between the VH and VL domains such that the scFv can form the desired three-dimensional structure for antigen binding (see, for example, Pluckthun, In The Pharmacology of Monoclonal Antibodies, Rosenberg and Moore Eds., Springer-Verlag, New York, 113:269-315.1994).

[0094] In some embodiments, the antibody can be a diabody. A diabody is a small antibody fragment having two antigen-binding sites. Each fragment contains a VH domain linked to a VL domain. However, the linker between the domains is too short to allow them to pair with each other on the same chain, so the linked Vh-Vl domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites. Diabodies are further described, for example, in EP404,097; WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993.

[0095] In some embodiments, antibodies or fragments thereof are contemplated that bind to at least a portion of the Cx43 protein, stimulate, promote or enhance the opening of Cx43 hemichannels, stimulate or enhance signal transduction, and decrease cancer cell growth, proliferation and / or colony formation. In some embodiments, antibodies or fragments thereof are contemplated that bind to at least a portion of the Cx43 protein, stimulate, promote or enhance the opening of Cx43 hemichannels, stimulate or enhance signal transduction, decrease cancer cell growth, proliferation and / or colony formation, and have no effect on gap junction coupling. In some embodiments, the anti-Cx43 antibody can be a monoclonal antibody, a polyclonal antibody, or a humanized antibody. Thus, by known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs (including any of the engineered forms described above) specific for the Cx43 protein, to one or more of each of its epitopes, or to any of the conjugates described above, can be created, and such antigens or epitopes are isolated from a natural source or are either synthetic derivatives or variants of natural compounds.

[0096] Examples of suitable antibody fragments include, without limitation, (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains, (ii) "Fd" fragments consisting of the VII and Cm domains, (iii) "Fv" fragments consisting of the VL and VH domains of a single antibody, (iv) "dAb" fragments consisting of the VH domain, (v) isolated CDR regions, (vi) F(ab')2 fragments, which are bivalent fragments comprising two linked Fab fragments, (vii) single-chain Fv molecules ("scFv") in which the VII domain and the VL domain are linked by a peptide linker that joins the two domains to form a binding domain, (viii) bispecific single-chain Fv dimers (see U.S. Patent No. 5,091,513), and (ix) diabodies, which are multivalent or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication 20050214860). The Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulfide bridges that link the VH and VL domains. Minibodies comprising scFv linked to the CH3 domain may also be produced (Hu et al., 1996).

[0097] Antibody-like binding peptide mimics are also contemplated. Liu et al. (2003) described "antibody-like binding peptide mimics" (ABiP), which act as miniaturized antibodies and have certain advantages, such as a longer serum half-life and fewer cumbersome synthetic methods.

[0098] To produce an antibody specific for the Cx43 protein, an animal may be inoculated with an antigen such as the Cx43 extracellular domain protein. Frequently, to enhance the immune response, the antigen is bound or conjugated to another molecule. As used herein, a conjugate is a peptide, polypeptide, protein or non-proteinaceous substance that is bound to an antigen and used to elicit an immune response in an animal. Antibodies produced in an animal in response to inoculation with an antigen include a diverse population of non-identical molecules (polyclonal antibodies) produced from various individual antibody-producing B lymphocytes. Polyclonal antibodies are a mixed population of antibody species, each of which can recognize different epitopes on the same antigen. Given the appropriate conditions for the production of polyclonal antibodies in an animal, most of the antibodies in the animal's serum will recognize the collective epitopes on the antigen compound with which the animal was immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the antigen or epitope of interest.

[0099] Monoclonal antibodies are a single species of antibody and, since the antibody-producing cells are derived from a single B lymphocyte cell line, all antibody molecules recognize the same epitope. Methods for generating monoclonal antibodies (MAbs) generally begin in the same manner as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to generate monoclonal antibodies. In some embodiments, cells from rabbits, sheep, or frogs are used to generate monoclonal antibodies. The use of rats is well known and can offer certain advantages. Mice (e.g., BALB / c mice) are used routinely and generally provide a high percentage of stable fusions.

[0100] Hybridoma technology involves the fusion of immortalized myeloma cells (usually mouse myeloma) from a mouse pre-immunized with the Cx43 antigen with a single B lymphocyte. This technology provides a way to grow a single antibody-producing cell to an unlimited number of generations, such that an unlimited amount of structurally identical antibodies (monoclonal antibodies) with specificity for the same antigen or epitope can be produced.

[0101] Plasma B cells may be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for Cx43-binding cells. After enriching antibody-producing B cells, total RNA may be isolated and cDNA may be synthesized. The DNA sequences of the antibody variable regions from both the heavy and light chains may be amplified, constructed into a phage display Fab expression vector, and transformed into E. coli. Cx43-specific binding Fabs may be selected via multiple rounds of enrichment panning and sequenced. The selected Cx43-binding hits may be expressed as full-length IgG in rabbit and rabbit / human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and may be purified using a protein G resin equipped with a fast protein liquid chromatography (FPLC) separation unit.

[0102] In some embodiments, the antibody can be a chimeric antibody, e.g., an antibody that includes an antigen-binding sequence derived from a non-human donor transplanted into a heterologous non-human, human, or humanized sequence (e.g., a framework sequence and / or a constant domain sequence). Methods have been developed for replacing the light and heavy chain constant domains of a monoclonal antibody with similar domains of human origin while leaving the variable domains of the foreign antibody intact. Alternatively, “fully human” monoclonal antibodies can be produced in mice that have been genetically engineered with human immunoglobulin genes. Methods have also been developed for further converting the variable regions of monoclonal antibodies to a more human-like form by recombinantly constructing antibody variable domains that have both rodent, e.g., mouse, and human amino acid sequences. In “humanized” monoclonal antibodies, only the hypervariable CDRs are derived from the murine monoclonal antibody, and the framework and constant regions are derived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513 and 6,881,557). Replacing the amino acid sequences in antibodies characteristic of rodents with amino acid sequences found at corresponding positions in human antibodies is thought to reduce the likelihood of an adverse immune reaction during therapeutic use. Hybridomas or other antibody-producing cells may be subjected to genetic mutations or other changes, which may or may not alter the binding specificity of the antibodies produced by the hybridomas.

[0103] Methods for producing polyclonal antibodies in various animal species, as well as methods for producing various types of monoclonal antibodies, including humanized antibodies, chimeric antibodies, and fully human antibodies, are well known and readily predictable in the art. For example, the following U.S. patents and patent applications enable the description of such methods: U.S. Patent Application Nos. 2004 / 0126828 and 2002 / 0172677, as well as U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157; 6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; and 6,891,024. All patents, patent application publications, and other publications cited herein, as well as the patents, patent application publications, and other publications cited therein, are hereby incorporated by reference into this application.

[0104] Antibodies may be produced from any animal source, including birds and mammals. Preferably, the antibodies are antibodies from sheep, rodents (e.g., mice and rats), rabbits, goats, guinea pigs, camels, horses, or chickens. Additionally, new technologies enable the development and screening of human antibodies from human combinatorial antibody libraries. For example, bacteriophage antibody expression technology enables the production of specific antibodies without immunizing an animal, as described in U.S. Patent No. 6,946,546, which is incorporated herein by reference. These techniques are further described in Marks (1992); Stemmer (1994), Gram et al. (1992), Barbas et al. (1994); and Schier et al. (1996).

[0105] It is fully expected that antibodies to Cx43 will have the ability to neutralize or counteract the effects of Cx43, regardless of the animal species, monoclonal cell line, or other source of the antibody. Certain animal species may have a relatively low degree of preference for generating therapeutic antibodies because they are likely to cause allergic reactions due to the activation of the complement system through the "Fc" portion of the antibody. However, whole antibodies can be enzymatically digested into "Fc" (complement-binding) fragments and antibody fragments having binding domains or CDRs. Removal of the Fc portion reduces the potential of the antigen-antibody fragment to induce unwanted immunological responses, so that Fc-free antibodies may be preferred for prophylactic or therapeutic treatment. As described herein, antibodies may also be constructed to be chimeric or partially or fully humanized to reduce or eliminate the harmful immunological consequences resulting from administering to an animal antibodies produced in or having sequences from other species.

[0106] Substitution variants typically contain the replacement of one amino acid with another at one or more sites within a protein and may be designed to modulate one or more properties of the polypeptide, with or without loss of other functions or characteristics. The substitution may be conservative, i.e., one amino acid is replaced with one of similar shape or charge. Conservative substitutions are well known in the art and include, for example, the change of alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine to methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine. Alternatively, the substitution may be non-conservative such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with a chemically different one, for example, substituting a polar or charged amino acid with a non-polar or uncharged amino acid, and vice versa.

[0107] The protein may be recombinant or synthesized in vitro. Alternatively, non-recombinant or recombinant proteins may be isolated from bacteria. It is also contemplated that bacteria containing such variants may be applied in compositions and methods. As a result, the protein need not be isolated.

[0108] It is contemplated that the composition contains from about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. Thus, the protein concentration in the composition can be about, at least about, or up to about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therefrom). Of these, about, at least about, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% can be an antibody that binds Cx43.

[0109] The antibody or preferably the immunologically active portion of the antibody can be chemically conjugated to another protein or expressed as a fusion protein. For the purposes of this specification and the appended claims, all such fusion proteins are included within the definition of an antibody or immunologically active portion of an antibody.

[0110] Described herein are antibodies and antibody-like molecules against Cx43, polypeptides, and peptides that are linked to at least one agent to form an antibody conjugate or payload. To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, the antibody can be bound to, covalently attached to, or complexed with at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having a desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules attached to an antibody include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, and the like. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to an antibody include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin.

[0111] Several methods for attachment of an antibody to its conjugate moiety or for conjugation are known in the art. Some attachment methods involve, for example, the use of metal chelate complexes employing organic chelating agents attached to the antibody, such as diethylenetriaminepentaacetic anhydride (DTPA), diethylenetriamine tetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3,6-diphenylglycouril-3. Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanate.

[0112] In some embodiments, the anti-Cx43 antibodies described herein can include a heavy chain immunoglobulin variable region that includes a complementarity-determining region 1 (CDR1) that includes the sequence of SEQ ID NO: 19, a CDR2 that includes the sequence of SEQ ID NO: 20, and a CDR3 that includes the sequence of SEQ ID NO: 21. Table 2 shows examples of CDRs of the heavy chain.

[0113] In some embodiments, the anti-Cx43 antibodies described herein can include a light chain immunoglobulin variable region that includes a complementarity-determining region 1 (CDR1) that includes the sequence of SEQ ID NO: 49, a CDR2 that includes the sequence of SEQ ID NO: 50, and a CDR3 that includes the sequence of SEQ ID NO: 51. Table 2 shows examples of CDRs of the light chain.

[0114] In some embodiments, the anti-Cx43 antibodies described herein can include a heavy chain immunoglobulin variable region that includes a complementarity-determining region 1 (CDR1) that includes the sequence of SEQ ID NO: 19, a CDR2 that includes the sequence of SEQ ID NO: 20, and a CDR3 that includes the sequence of SEQ ID NO: 21, and a light chain immunoglobulin variable region that includes a complementarity-determining region 1 (CDR1) that includes the sequence of SEQ ID NO: 49, a CDR2 that includes the sequence of SEQ ID NO: 50, and a CDR3 that includes the sequence of SEQ ID NO: 51.

[0115] In some embodiments, the anti-Cx43 antibodies described herein can include a variable heavy chain that includes a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 19, 20, or 21 (see Table 2). In some embodiments, the anti-Cx43 antibodies described herein include a variable heavy chain that includes a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the sequence set forth in SEQ ID NO: 19, 20, or 21 (see Table 2).

[0116] In some embodiments, the anti-Cx43 antibodies described herein can include a variable light chain that includes a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 49, 50, or 51 (see Table 2). In some embodiments, the anti-Cx43 antibodies described herein include a variable light chain that includes a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the sequence set forth in SEQ ID NO: 49, 50, or 51.

[0117] Disclosed herein is a nucleic acid sequence encoding M1H that includes the sequence of SEQ ID NO: 52. Disclosed herein is a nucleic acid sequence encoding M1M7K that includes the sequence of SEQ ID NO: 57.

[0118] Disclosed herein is a nucleic acid sequence encoding M1H that includes a variable heavy chain that includes a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 52 (see Table 3). In some embodiments, M1H includes a variable heavy chain that includes a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the sequence set forth in SEQ ID NO: 52.

[0119] Disclosed herein is a nucleic acid sequence encoding M1M7K that includes a variable light chain that includes a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 57 (see Table 3). In some embodiments, M1M7K includes a variable light chain that includes a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the sequence set forth in SEQ ID NO: 57.

[0120] Disclosed herein is a nucleic acid sequence encoding an M1H region that includes a heavy chain immunoglobulin variable region that includes a CDR1 that includes the sequence of SEQ ID NO: 16, a CDR2 that includes the sequence of SEQ ID NO: 17, and a CDR3 that includes the sequence of SEQ ID NO: 18.

[0121] Disclosed herein is a nucleic acid sequence encoding an M1M7K region comprising a light chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 46, a CDR2 comprising the sequence of SEQ ID NO: 47, and a CDR3 comprising the sequence of SEQ ID NO: 48.

[0122] Disclosed herein is a nucleic acid sequence encoding an anti-Cx43 hemichannel antibody comprising a heavy chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 16, a CDR2 comprising the sequence of SEQ ID NO: 17, and a CDR3 comprising the sequence of SEQ ID NO: 18, and a light chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 46, a CDR2 comprising the sequence of SEQ ID NO: 47, and a CDR3 comprising the sequence of SEQ ID NO: 48.

[0123] Disclosed herein is an antibody or fragment thereof that binds to human Cx43. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to one of the variable heavy chain amino acid sequences provided in Table 2 or 4. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain comprising the sequence set forth in SEQ ID NO: 58.

[0124] Disclosed herein is an antibody or fragment thereof that binds to human Cx43. In some embodiments, the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to one of the variable light chain amino acid sequences provided in Table 2 or 4. In some embodiments, the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. In some embodiments, the antibody or fragment thereof comprises a variable light chain comprising the sequence set forth in SEQ ID NO: 63.

[0125] Disclosed herein are antibodies or fragments thereof that bind to human Cx43 hemichannels. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 58, and a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63. In some embodiments, the antibody comprises a variable heavy chain comprising the sequence shown in SEQ ID NO: 58 and a variable light chain comprising the sequence shown in SEQ ID NO: 63.

[0126] In some embodiments, the antibody or fragment thereof comprises an M1H region. In some embodiments, the M1H region comprises a heavy chain immunoglobulin variable region comprising CDR1 comprising the sequence of SEQ ID NO: 19, CDR2 comprising the sequence of SEQ ID NO: 20, and CDR3 comprising the sequence of SEQ ID NO: 21.

[0127] In some embodiments, the antibody or fragment thereof comprises an M1M7K region. In some embodiments, the M1M7K region comprises a light chain immunoglobulin variable region comprising CDR1 comprising the sequence of SEQ ID NO: 49, CDR2 comprising the sequence of SEQ ID NO: 50, and CDR3 comprising the sequence of SEQ ID NO: 51.

[0128] Optionally, the disclosed antibody or fragment thereof further comprises a tag sequence.

[0129] Disclosed herein are nucleic acid sequences encoding the disclosed antibodies or fragments thereof. For example, disclosed is a nucleic acid sequence comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 52. Disclosed herein are nucleic acid sequences encoding the disclosed antibodies or fragments thereof. For example, disclosed herein is a nucleic acid sequence comprising a variable heavy chain comprising the sequence shown in SEQ ID NO: 52. Also disclosed herein is a nucleic acid sequence comprising a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 57. Also disclosed is a nucleic acid sequence comprising a variable light chain comprising the sequence shown in SEQ ID NO: 57.

[0130] Disclosed herein is a nucleic acid sequence comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 52, and a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 57. Disclosed is a nucleic acid sequence comprising a variable heavy chain comprising the sequence shown in SEQ ID NO: 52 and a variable light chain comprising the sequence shown in SEQ ID NO: 57.

[0131] Disclosed herein is a nucleic acid sequence capable of encoding a single-chain variable fragment comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 52.

[0132] Disclosed is a nucleic acid sequence capable of encoding a single-chain variable fragment comprising a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 57.

[0133] Disclosed is a nucleic acid sequence capable of encoding a single-chain variable fragment comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 58, and a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63.

[0134] In some cases, the disclosed antibody or fragment thereof can be bispecific. For example, the antibody or fragment thereof can comprise a first Fab region comprising the heavy and light chains of SEQ ID NO: 58 and a second Fab region comprising the heavy and light chains of SEQ ID NO: 63, wherein the first Fab region and the second Fab region can be different.

[0135] In some cases, the bispecific antibody can be trifunctional.

[0136] In some cases, the disclosed antibody or fragment thereof can be murine, human, humanized, chimeric, or combinations thereof.

[0137] In some cases, the disclosed antibody or fragment thereof is monoclonal.

[0138] Method Disclosed herein is a method of treating or preventing osteosarcoma in a subject. The method can include administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or fragment thereof. In some embodiments, the antibody or fragment thereof can include a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58. In some embodiments, the antibody or fragment thereof can include a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. In some embodiments, the antibody or fragment thereof can include a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58 and a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. In some embodiments, the method can extend the lifespan of a subject with osteosarcoma. In some embodiments, the method can reduce, inhibit or prevent the growth of osteosarcoma cells, e.g., the growth of intratibial tumors. In some embodiments, the method can reduce, inhibit or prevent the growth of intratibial tumors. In some embodiments, the method can increase, enhance or promote the release of ATP in more cells, or in malignant tumors. In some embodiments, the method can reduce, inhibit or prevent the migration of osteosarcoma cells.

[0139] In some embodiments, the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising a first complementarity determining region 1 comprising a sequence having at least 60% identity to SEQ ID NO: 19, a second complementarity determining region 2 comprising a sequence having at least 60% identity to SEQ ID NO: 20, and a third complementarity determining region 3 comprising a sequence having at least 60% identity to SEQ ID NO: 21. In some embodiments, the antibody or fragment thereof can comprise a light chain immunoglobulin variable region comprising a first complementarity determining region 1 comprising a sequence having at least 60% identity to SEQ ID NO: 49, a second complementarity determining region 2 comprising a sequence having at least 60% identity to SEQ ID NO: 50, and a third complementarity determining region 3 comprising a sequence having at least 60% identity to SEQ ID NO: 51.

[0140] In some embodiments, the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising a first complementarity determining region 1 comprising a sequence having a single amino acid change compared to SEQ ID NO: 19, a second complementarity determining region 2 comprising a sequence having a single amino acid change compared to SEQ ID NO: 20, and a third complementarity determining region 3 comprising a sequence having a single amino acid change compared to SEQ ID NO: 21. In some embodiments, the antibody or fragment thereof can comprise a light chain immunoglobulin variable region comprising a first complementarity determining region 1 comprising a sequence having a single amino acid change compared to SEQ ID NO: 49, a second complementarity determining region 2 comprising a sequence having a single amino acid change compared to SEQ ID NO: 50, and a third complementarity determining region 3 comprising a sequence having a single amino acid change compared to SEQ ID NO: 51.

[0141] In some embodiments, the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising a) complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19 or a variant thereof, b) complementarity determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 20 or a variant thereof, and / or c) complementarity determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 21 or a variant thereof. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the heavy chain can comprise at least one amino acid substitution as compared to the parental CDR. In some embodiments, the at least one amino acid substitution can be to an amino acid other than a cysteine residue. In some embodiments, the at least one amino acid substitution can be to an amino acid other than a glycine residue.

[0142] In some embodiments, the antibody or fragment thereof can comprise a light chain immunoglobulin variable region comprising a) complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49 or a variant thereof, b) complementarity determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 50 or a variant thereof, and / or c) complementarity determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 51 or a variant thereof. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the light chain can comprise at least one amino acid substitution as compared to the parental CDR. In some embodiments, the at least one amino acid substitution can be to an amino acid other than a cysteine residue. In some embodiments, the at least one amino acid substitution can be to an amino acid other than a glycine residue.

[0143] In some embodiments, the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising a complementarity determining region 1 (CDR1) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 19, a complementarity determining region 2 (CDR2) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 20, and / or a complementarity determining region 3 (CDR3) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 21. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the light chain can comprise at least one amino acid substitution compared to the parental CDR. In some embodiments, at least one amino acid substitution can be another amino acid from a cysteine residue. In some embodiments, at least one amino acid substitution can be another amino acid from a glycine residue.

[0144] In some embodiments, the antibody or fragment thereof can comprise a light chain immunoglobulin variable region comprising a complementarity determining region 1 (CDR1) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 49, a complementarity determining region 2 (CDR2) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 50, and / or a complementarity determining region 3 (CDR3) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 51. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the light chain can comprise at least one amino acid substitution compared to the parental CDR. In some embodiments, at least one amino acid substitution can be another amino acid from a cysteine residue. In some embodiments, at least one amino acid substitution can be another amino acid from a glycine residue.

[0145] In some embodiments, the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region that comprises complementarity determining region 1 (CDR1) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 19, complementarity determining region 2 (CDR2) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 20, and / or complementarity determining region 3 (CDR3) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 21. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the light chain can comprise at least one amino acid substitution compared to the parental CDR. In some embodiments, the at least one amino acid substitution can be an amino acid other than a cysteine residue. In some embodiments, the at least one amino acid substitution can be an amino acid other than a glycine residue.

[0146] In some embodiments, the antibody or fragment thereof can comprise a light chain immunoglobulin variable region that comprises complementarity determining region 1 (CDR1) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 49, complementarity determining region 2 (CDR2) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 50, and / or complementarity determining region 3 (CDR3) comprising a sequence having a single amino acid change compared to the sequence set forth in SEQ ID NO: 51. In some embodiments, any one of the CDR1, CDR2, or CDR3 of the light chain can comprise at least one amino acid substitution compared to the parental CDR. In some embodiments, the at least one amino acid substitution can be an amino acid other than a cysteine residue. In some embodiments, the at least one amino acid substitution can be an amino acid other than a glycine residue.

[0147] In some embodiments, any of the methods disclosed herein can be further defined as a method for treating or preventing lung metastasis or brain metastasis in a subject having osteosarcoma. In some embodiments, the metastasis can be in the subject's breast, other bone, and / or other organ.

[0148] In some embodiments, any of the methods disclosed herein can include administering to a subject an effective amount of an expression vector encoding an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof can be administered in a pharmaceutically acceptable composition. In some embodiments, the pharmaceutical composition can be lyophilized. In some embodiments, the antibody or fragment thereof can be administered systemically. In some embodiments, the antibody or fragment thereof can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In some embodiments, the antibody or fragment thereof can be a humanized antibody or a humanized fragment thereof. In some embodiments, the antibody can be an IgG, IgM, IgA, IgD, IgE, or genetically engineered IgG class antibody comprising a first VH CDR corresponding to SEQ ID NO: 19, a second VH CDR corresponding to SEQ ID NO: 20, a third VH CDR corresponding to SEQ ID NO: 21, a first VL CDR corresponding to SEQ ID NO: 49, a second VL CDR corresponding to SEQ ID NO: 50, and a third VL CDR corresponding to SEQ ID NO: 51. In some embodiments, the antibody can be an antibody of the IgG class, and the antibody of the IgG class can be an antibody of the IgG1, IgG2, IgG3, or IgG4 class.

[0149] In some embodiments, any of the methods disclosed herein can further include subjecting the subject to at least a second anti-cancer therapy. In some embodiments, the second anti-cancer therapy can be surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0150] In some embodiments, in any of the methods disclosed herein, an antibody or a fragment thereof can bind to the Cx43 hemichannel. In some embodiments, in any of the methods disclosed herein, an antibody or a fragment thereof can stimulate the opening of the Cx43 hemichannel. In some embodiments, in any of the methods disclosed herein, an antibody or a fragment thereof can stimulate the opening of the Cx43 hemichannel and have no effect on gap junction coupling.

[0151] In some embodiments, the antibody or a fragment thereof can further comprise a tag sequence.

[0152] In some embodiments, the antibody or a fragment thereof can be a Fab fragment, a Fab’ fragment or an F(ab’)2 fragment.

[0153] Treatment of diseases Disclosed herein are antibodies and biological fragments thereof that can be used to treat osteosarcoma in an osteosarcoma subject or to prevent or reduce lung metastases and / or metastases to the liver, brain or breast. Enhancing Cx43 hemichannel signaling can be achieved by a suitable drug or therapeutic agent to prevent cancer cell proliferation, growth and / or colony formation. In some embodiments, the drug or therapeutic agent can be an anti-Cx43 antibody.

[0154] The compositions described herein can be administered to a subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of a clinical disease. Thus, in some embodiments, the patient can be a human patient. In therapeutic use, a composition can be administered to a subject (e.g., a human patient) already suffering from or diagnosed with osteosarcoma in an amount sufficient to at least partially improve the symptoms or signs, or to suppress (and preferably halt) the symptoms of the disease, its complications, and the progression of its course. The appropriate amount for achieving this is defined as a "therapeutically effective amount". The therapeutically effective amount of a composition (e.g., a pharmaceutical composition) can be an amount that achieves a cure, although the outcome is only one of several that can be achieved. As mentioned, the therapeutically effective amount includes an amount that provides a treatment that can delay, interfere with, or prevent the onset or progression of cancer, or improve or reduce the frequency of cancer or cancer symptoms. One or more of the symptoms can be less severe. Recovery can be promoted in the individual being treated. For example, the treatment of cancer can involve, for example, a reduction in tumor size, a decrease in tumor invasiveness, a decrease in the cancer growth rate, or the prevention of metastasis. The treatment of cancer can also refer to an increase in the survival period of a subject with cancer. In some embodiments, the antibodies described herein can extend the lifespan of a subject with cancer. In some embodiments, the antibodies described herein can reduce or inhibit the growth of bone tumor cells, e.g., the growth of intratibial tumors.

[0155] In some embodiments, the cancer can be a primary or secondary tumor. In some embodiments, the cancer can be a metastatic tumor. In other embodiments, the primary or secondary tumor is in the bone of the patient. In still other embodiments, the cancer has metastasized. In some embodiments, the cancer has its origin in the bone and can metastasize to one or more of the following sites: breast, lung, brain, liver, or other bone.

[0156] Disclosed herein is a method for treating cancer patients. The cancer can be osteosarcoma. In some embodiments, the cancer can be breast cancer, lung cancer, brain tumor, or liver cancer that has metastasized from bone cancer. In some embodiments, the subject has been diagnosed with cancer prior to the administration step. In some embodiments, the cancer can be osteosarcoma.

[0157] The compositions described herein can be formulated to contain a therapeutically effective amount of the compounds disclosed herein. In some embodiments, the antibodies disclosed herein can be included within a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation can be a unit dosage form formulation.

[0158] Any therapeutically effective amount or dosage of an antibody used in a method as disclosed herein for application to a mammal (e.g., a human) can be determined by one of ordinary skill in the art taking into account age, weight, gender, severity of the subject's symptoms, and the particular composition or route of administration selected, other drugs being administered, and individual variations in the judgment of the attending physician. Variations in the required dosage can be expected. Variations in dosage levels can be adjusted using standard empirical routes for optimization. The particular dosage of the pharmaceutical composition administered to a patient will depend on various considerations (e.g., the severity of the cancer symptoms), the age and physical characteristics of the subject, and other considerations known to one of ordinary skill in the art. The dosage can be established using clinical approaches known to one of ordinary skill in the art. A therapeutically effective dosage of an anti-hemichannel antibody can result in a decrease in the severity of one or more disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or the prevention of functional or physical impairment due to the pain of the disease. A therapeutically effective amount of a therapeutic compound or antibody can reduce tumor metastasis or otherwise improve symptoms in a subject.

[0159] The duration of treatment with any of the compositions provided herein can be for any length of time from a minimum of one day to the length of the host's life (e.g., many years). For example, the composition can be administered once a week (e.g., for 4 weeks to several months or years), once a month (e.g., for 3 - 12 months or years), or once a year for a period of 5 years, 10 years, or more. It should also be noted that the frequency of treatment can vary. For example, the present composition can be administered once a day, once a week, once a month, or once a year (or 2 times, 3 times, etc.).

[0160] The total effective amount of an antibody or composition as disclosed herein can be administered as a single dose to a subject either as a bolus or by infusion over a relatively short period of time, or using a divided treatment protocol with multiple doses administered over a longer period of time. Alternatively, continuous intravenous infusion sufficient to maintain a therapeutically effective concentration in the blood is also within the scope of the present disclosure.

[0161] The antibodies or compositions described herein can be administered to a subject in need of treatment in combination with other therapies. The compound can be administered before, simultaneously with, or after treatment with other agents or dosing regimens. For example, the antibodies disclosed herein can be administered alone or in combination with standard therapies used to treat cancer. In some embodiments, any of the antibodies or compositions described herein can be administered or used in conjunction with chemotherapy.

[0162] Pharmaceutical composition Disclosed herein is a composition, e.g., a pharmaceutical composition, comprising one monoclonal antibody or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, formulated with a pharmaceutically acceptable carrier. Such a composition may comprise one antibody or a combination of antibodies (e.g., two or more different) or an immunoconjugate as described herein. For example, the pharmaceutical composition of the invention can comprise a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0163] The pharmaceutical composition of the invention can also be administered as combination therapy, i.e., in combination with other agents. For example, combination therapy can comprise an anti-hemi-channel antibody combined with at least one other anti-cancer agent.

[0164] As used herein, the expression “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier can be suitable for intravenous, intramuscular, subcutaneous, or parenteral administration (e.g., by injection or inhalation). Depending on the route of administration, the active compound, i.e., the antibody or immunoconjugate, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0165] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical composition of the invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0166] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of injectable sterile solutions or dispersions. Such media and agents for pharmaceutical active substances are known in the art. Any conventional media or agents are contemplated for use in the pharmaceutical compositions of the present invention, except insofar as they are incompatible with the active compounds. Supplementary active compounds can also be incorporated into the compositions.

[0167] Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it will be preferable to include in the composition isotonic agents, for example, sugars such as polyhydric alcohols like mannitol, sorbitol, or sodium chloride. Sustained absorption of injectable compositions can be brought about by including in the composition agents that delay absorption, for example, monostearates and gelatin.

[0168] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, optionally with one or a combination of the ingredients listed herein, into a suitable solvent and then sterilizing and filtering precisely. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) whereby a powder is obtained that contains the active ingredient plus any additional desired ingredients from its pre-sterile filtered solution.

[0169] The amount of active ingredient that can be combined with a carrier material to create a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to create a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.01 percent to about 99 percent active ingredient, preferably from about 0.1 percent to about 70 percent, or most preferably from about 1 percent to about 30 percent active ingredient, in combination with a pharmaceutically acceptable carrier.

[0170] The dosage regimen is adjusted to provide the desired response (e.g., a therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dosage may be proportionally decreased or increased as dictated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, a unit dosage form refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of sensitivity in individuals.

[0171] For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg / kg per kg of the host body weight, and more usually from 0.01 to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 15 mg / kg, 15 mg / kg to 20 mg / kg or 20 mg / kg to 25 mg / kg. In some embodiments, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or in the range of 1-10 mg / kg. In some embodiments, the dosage can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 15 mg / kg body weight, 20 mg / kg body weight, 25 mg / kg body weight or 30 mg / kg body weight, or in the range of 1-30 mg / kg. In some embodiments, the dosage can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg / kg body weight. In some embodiments, the dosage can be 5 mg / kg body weight. In some embodiments, the dosage can be 15 mg / kg body weight. In some embodiments, the dosage can be 20 mg / kg body weight. In some embodiments, the dosage can be 25 mg / kg body weight. Example treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every 3-6 months. Preferred dosing regimens for the anti-hemichannel antibodies of the present invention include 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, and the antibody is administered using one of the following dosing schedules: (i) every four weeks for 6 doses, then every three months, (ii) every three weeks, (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight every three weeks.

[0172] In some methods, two or more monoclonal antibodies having different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the indicated range. Antibodies are usually administered on multiple occasions. The intervals between single doses can be, for example, weekly, monthly, every three months, or annually. The intervals can also be irregular when specified by measuring the blood level of the antibody against the target antigen in the patient. In some methods, the dosage is adjusted to achieve an antibody concentration in plasma of about 1 - 1000 μg / mL, and in some methods, an antibody concentration in plasma of about 25 - 300 μg / mL.

[0173] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will be influenced by the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds, and / or substances employed in combination with the particular composition employed, various pharmacokinetic factors including the age, sex, weight, condition, general health, and medical history of the patient being treated, as well as similar factors well known in the medical arts.

[0174] The compositions of the present invention can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be well understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Preferred routes of administration for the antibodies of the present invention include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous administration by injection or infusion, or other parenteral routes of administration. As used herein, the expression "parenteral administration" generally means a mode of administration other than enteral and topical administration by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular injections and infusions.

[0175] Combination therapy The compositions and methods described herein can include an antibody or an antibody fragment against Cx43 that stimulates the opening of Cx43 hemichannels to protect skeletal tissue against cancer cell proliferation and colony formation, for example, in combination with a second or additional therapy. Such therapies can be applied to the treatment of any disease associated with Cx43-mediated cell proliferation. For example, the disease may be osteosarcoma, lung metastasis, liver metastasis, brain metastasis, and / or breast metastasis.

[0176] Methods and compositions comprising combination therapies enhance the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer therapy or anti-proliferative disease therapy. The therapies and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect such as killing of cancer cells and / or suppression of cell overgrowth. This process may involve contacting the cells with both the antibody or antibody fragment and the second therapy. The tissue, tumor, or cell can be contacted with one or more compositions or pharmaceutical formulations comprising one or more of the agents (i.e., the antibody or antibody fragment or the anti-cancer agent), and it is also possible to contact the tissue, tumor, and / or cell with two or more separate compositions or formulations, where one composition provides 1) the antibody or antibody fragment, 2) the anti-cancer agent, or 3) both the antibody or antibody fragment and the anti-cancer agent. It is also contemplated that such combination therapies can be used in conjunction with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

[0177] The terms "contacting" and "exposing", when applied to cells, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to the target cells or placed in direct juxtaposition to the target cells. To achieve cell killing, for example, both agents are delivered to the cells in a combined amount effective to kill the cells or prevent the cells from dividing.

[0178] Antibodies and their biological fragments can be administered before, during, after, or in various combinations with respect to anti-cancer treatment. Administration may be carried out at intervals ranging from simultaneous to several minutes, days, or weeks. In the mode where the antibody or antibody fragment is provided to the patient separately from the anti-cancer agent, generally, it is ensured by a fairly long delivery that the expiration date does not expire between each delivery, so that the two compounds can more effectively exert the combined beneficial effect on the patient. In such an example, it is contemplated that antibody therapy and anti-cancer therapy may be provided to the patient within about 12 - 24 hours or within 72 hours, more specifically within about 6 - 12 hours, with respect to each other. In some situations, it may be desirable to considerably extend the treatment period with a state where several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration.

[0179] In some embodiments, the treatment process can continue for 1 - 90 days or more (such a range includes intervening days). One agent may be administered on any day from the 1st day to the 90th day (such a range includes intervening days), or in any combination of these days, or another agent may be administered on any day from the 1st day to the 90th day (such a range includes intervening days), or in any combination of these days. Within one day (a 24 - hour period), the patient may receive one or multiple administrations of the agent(s). Further, it is contemplated that there may be a period after the treatment process during which anti-cancer treatment is not performed. This period may continue for 1 - 7 days, and / or 1 - 5 weeks, and / or 1 - 12 months or more (such a range includes intervening days), depending on the patient's condition such as the patient's prognosis, endurance, health status, etc. The treatment cycle is expected to be repeated as necessary.

[0180] Various combinations may be employed. For example, hereinafter, antibody therapy is "A" and anti-cancer therapy is "B":

[0181] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A。

[0182] Administration of any compound or therapy disclosed herein to a patient, if any, will follow the general protocol for administration of such compounds, taking into account the toxicity of the agent. Thus, in some embodiments, there may be a step of monitoring for toxicities that may result from combination therapy.

[0183] Chemotherapy A wide variety of chemotherapeutic agents may be used. The term "chemotherapy" refers to the use of drugs to treat cancer. The term "chemotherapeutic agent" is used to encompass compounds or compositions that can be administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell, e.g., whether they affect the cell cycle and at which stage, or alternatively, the agent may be characterized based on its ability to induce chromosomal and mitotic abnormalities by directly crosslinking DNA, inserting into DNA, or affecting nucleic acid synthesis.

[0184] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bradykin and bradykinone); camptothecin (including the synthetic analogue topotecan); bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburevin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine); antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma1I and calicheamicin omegaII); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, actinomycins, actinomycin, aclacinomycins, azaserine, bleomycins, calicheamicin, carabicin, carminomycin, calycinophilins, chromomycins, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, for example, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, pteropterin, and trimethoprim; purine analogs, for example, fludarabine, 6-mercaptopurine, thiampurine, and thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, doxifluridine, enocitabine and floxuridine; androgens, for example, calusterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents, for example, mitotane, and trilostane; folic acid supplements, for example, folinic acid; aceglatone, aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; eptilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;Podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; schizophyllan; spirigermanium; tenuazonic acid; triazicone; 2,2’,2’’-trichloroethylamine; trichothecene (especially T2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"), cyclophosphamide; taxoids, for example, paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, for example, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, for example, retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl protein transferase inhibitor, transplatin, and any pharmaceutically acceptable salts, acids, or derivatives thereof described herein are included.;

[0185] Radiation therapy Other commonly used factors that cause DNA damage include those generally known as gamma rays, X-rays, and / or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are contemplated, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors are most likely to affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosomal assembly and maintenance. The X-ray dose ranges from a dose of 50 to 200 roentgens per day for long periods (3 to 4 times per week) to a single dose of 2,000 to 6,000 roentgens. The dose ranges of radioisotopes vary widely and depend on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumor cells.

[0186] Immunotherapy One of ordinary skill in the art will understand that additional immunotherapies may be used in combination with or in conjunction with the methods disclosed herein. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules that target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for a marker on the surface of a tumor cell. The antibody alone may function as an effector of the therapy, or it may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying surface molecules that interact directly or indirectly with the tumor cell target. Exemplary effector cells include cytotoxic T cells and NK cells.

[0187] In one aspect of immunotherapy, tumor cells must have a marker suitable for targeting, i.e., some marker that is not present on most other cells. There are many tumor markers, any of which could be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. Another aspect of immunotherapy is to combine the anti-cancer effect with an immune-stimulating effect. There are also immune-stimulating molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0188] Examples of immunotherapies currently under study or in use are immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169, Hui and Hashimoto, 1998, Christodoulides et al., 1998); cytokine therapy such as interferons a, (3, and y), IL-1, GM-CSF, and TNF (Bukowski et al., 1998, Davidson et al., 1998, Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998, Austin-Ward and Villaseca, 1998, U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012, Hanibuchi et al., 1998, U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed together with the antibody therapy described herein.

[0189] Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic, or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electro-surgery, and microscopic control surgery (Mohs surgery).

[0190] When part or all of a cancerous cell, tissue, or tumor is resected, a cavity may form in the body. Treatment may be achieved by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatment methods can also vary widely in dosage.

[0191] Other drugs It is contemplated that other agents may be used in combination with any of the methods or compositions disclosed herein to improve the therapeutic efficacy of treatment. These additional agents include, but are not limited to, agents that affect the upregulation of cell surface receptors and gap junctions, cell growth inhibitors and differentiating agents, inhibitors of cell adhesion, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intercellular signaling by increasing the number of gap junctions will enhance the antiproliferative effect on adjacent hyperproliferative cell populations. A cell growth inhibitor or differentiating agent can be used in combination with the compositions and methods disclosed herein to improve the antiproliferative effect of treatment. Inhibitors of cell adhesion are contemplated to improve the effectiveness of the compositions and methods disclosed herein. Examples of cell adhesion inhibitors include, but are not limited to, focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents, such as the antibody c225, that increase the sensitivity of hyperproliferative cells to apoptosis can be used in combination with the compositions and methods disclosed herein to improve the therapeutic effect.

[0192] Kits and diagnostic methods Disclosed herein is a kit comprising one or more therapeutic agents and / or other therapeutic and delivery agents. In some embodiments, the kit can be used to prepare and / or perform the treatments disclosed herein. The kit may include one or more sealed vials containing any of the pharmaceutical compositions disclosed herein. The kit may include, for example, at least one Cx43 antibody or fragment thereof, and reagents for preparing, formulating, and / or administering one or more components of the compositions disclosed herein or for performing one or more steps of the methods of the invention. In some embodiments, the kit may also include a suitable container that can be a container that does not react with the components of the kit, such as an Eppendorf tube, assay plate, syringe, bottle, or tube. The container may be made of a sterilizable material such as plastic or glass.

[0193] The kit may further include an instruction sheet that describes the general steps of the methods described herein and that will follow substantially the same procedures as those described herein or that are known to those of skill in the art. The instructional information may be on a computer-readable medium containing machine-readable instructions that, when executed using a computer, result in a display of the actual or virtual procedures for delivering a pharmaceutically effective amount of the therapeutic agent.

Examples

[0194] Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques found by the inventors to function well in the practice of the present invention and, accordingly, can be regarded as constituting a preferred mode for its practice. However, those skilled in the art will understand that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the invention, yet still obtain similar or analogous results.

[0195] Example 1 - Anti-Cx43 Monoclonal Antibody Anti-Cx43 monoclonal antibodies were generated and clones producing Cx43-binding monoclonal antibodies were identified. The DNA and amino acid CDR sequences for the antibody sequences are shown in the following table along with the correct pairing of each characterized antibody. The M1 antibody inhibits the opening of the Cx43 hemichannel. The M2 antibody activates, stimulates and / or enhances the opening of the Cx43 hemichannel.

[0196] Table 1: Pairing of heavy and light chains of two functional antibodies. TIFF0007710374000001.tif21138 Table 2: Sequences of antibody chains from hybridomas. TIFF0007710374000002.tif187146 The cloned variable domains are shown in the following chart.

[0197] Table 3: DNA sequence. Variable heavy chain (bold) and variable light chain (underlined). JPEG0007710374000003.jpg63164 JPEG0007710374000004.jpg63164 JPEG0007710374000005.jpg63164 JPEG0007710374000006.jpg62164 JPEG0007710374000007.jpg63164 JPEG0007710374000008.jpg62164

[0198] Table 4: Amino acid sequence. Variable heavy chain (bold) and variable light chain (underlined). JPEG0007710374000009.jpg33164 JPEG0007710374000010.jpg33164 JPEG0007710374000011.jpg33164 JPEG0007710374000012.jpg111164

[0199] Example 2 - Use in Diagnosis and Cancer Treatment The growth of osteolytic tumors was found to be enhanced in osteocyte-specific Cx43 knockout mice. Py8119-Luc cells were injected into the right tibiae of control and cKO female mice. PBS was injected into the left tibiae as a control. Tumor growth was recorded weekly for 4 weeks by bioluminescence imaging and quantified (Figures 1A - 1C).

[0200] MLO-Y4 osteocytes and primary mouse osteocytes were incubated with E2 (polyclonal), HMAb1 and HMAb2 antibodies, or carbenoxolone (CBX), a connexin channel blocker. An ethidium bromide (EtBr) dye uptake assay was performed (Figures 9A - 9B). The Cx43HMAb2 antibody was found to activate hemichannels.

[0201] Furthermore, it was found that the Cx43(M1) antibody was delivered to osteocytes in vivo and blocked the uptake of Evans blue induced by tibial loading. Evans blue dye was injected into the tail veins of WT and osteocyte-specific Cx43KO. Mouse IgG or Cx43(M1) mAb (25 mg / kg) was intraperitoneally injected 2 hours before dye injection. Thirty minutes after dye injection, the left tibia was mechanically loaded once every 10 minutes for 10 minutes. The mice were sacrificed and perfused with PBS. The tibiae were isolated and fixed tibial tissue sections were prepared. The results are shown in FIGS. 3A-3C.

[0202] Inhibition of osteolytic tumor growth by HMAb2 was also observed. Py8119-Luc cells were injected into the right tibia of female mice (FIG. 4A). PBS was injected into the left tibia as a control. HMAb2 at 25 mg / kg was intraperitoneally injected once or twice a week for 4 weeks. Control mice were injected with saline twice a week. Tumor growth was recorded and quantified weekly for 4 weeks by bioluminescence imaging (FIG. 4B).

[0203] Example 3 - Inhibition of osteoblast proliferation in osteosarcoma cells To determine the efficacy of the M2 antibody against the suppression of osteosarcoma in bone, two in vivo mouse models were used. The first model was the mouse osteosarcoma cell line, DLM8, which is syngeneic to the C3H mouse strain (Sottnik, J.L., et al., Clin. Exp. Metastasis, 2010. 27(3): p. 151-60). The second model was the OS17 human osteosarcoma cell line in immunodeficient (nude) mice (Kolb, E.A., et al. Pediatr Blood Cancer, 2010. 55(1): p. 67-75). Both osteosarcoma cells were transplanted into the bone by intratibial injection. The experiment is described in the legends of FIGS. 9 and 10A - B.

[0204] The results show that the M2 antibody significantly inhibited the growth of both mouse and human osteosarcoma tumors in WT mice and nude mice, respectively. This M2 antibody (e.g., mouse-house chimeric M2 antibody and optimized humanized M2 antibody) inhibits the growth of osteosarcoma in a dose-dependent manner.

[0205] All of the methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and steps described herein or to the order of the steps of the method without departing from the concept, spirit, or scope of the present invention. More specifically, it will be apparent that certain agents related to both chemistry and physiology may be replaced with the agents described herein, and yet the same or similar results will be achieved. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims. Aspects of the present invention include the following aspects. 1. A method of treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58. 2. A method of treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. 3. A method of treating or preventing osteosarcoma in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58 and a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. 4. The antibody or fragment thereof is a) a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19 or a variant thereof b) Complementary determining region 2 (CDR2) containing the sequence of SEQ ID NO: 20 or a variant thereof, and / or c) Complementary determining region 3 (CDR3) containing the sequence of SEQ ID NO: 21 or a variant thereof The method according to any one of items 1 or 3 above, comprising a heavy chain immunoglobulin variable region containing the same. 5. The antibody or a fragment thereof is a) Complementary determining region 1 (CDR1) containing the sequence of SEQ ID NO: 49 or a variant thereof, b) Complementary determining region 2 (CDR2) containing the sequence of SEQ ID NO: 50 or a variant thereof, and / or c) Complementary determining region 3 (CDR3) containing the sequence of SEQ ID NO: 51 or a variant thereof The method according to any one of items 1 or 2 above, comprising a light chain immunoglobulin variable region containing the same. 6. The method according to item 4 or 5 above, wherein any one of the heavy chain CDR1, CDR2 or CDR3 or the light chain CDR1, CDR2 or CDR3 contains at least one amino acid substitution compared to the parental CDR. 7. The method according to item 4 or 5 above, wherein any one of the variants of the heavy chain CDR1, CDR2 or CDR3 or the variants of the light chain CDR1, CDR2 or CDR3 contains at least one amino acid substitution compared to the parental CDR. 8. The method according to item 7 or 8 above, wherein the at least one amino acid substitution is a substitution from a cysteine residue to another amino acid or from glycine to another amino acid. 9. The antibody or a fragment thereof is (a) Complementary determining region 1 (CDR1) containing a sequence having at least 60% identity to the sequence shown in SEQ ID NO: 19, (b) Complementary determining region 2 (CDR2) containing a sequence having at least 60% identity to the sequence shown in SEQ ID NO: 20, and / or (c) Complementary determining region 3 (CDR3) containing a sequence having at least 60% identity to the sequence shown in SEQ ID NO: 21 The method according to any one of items 1 or 3 above, comprising a heavy chain immunoglobulin variable region containing the same. 10. The antibody or fragment thereof is (a) Complementary determining region 1 (CDR1) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 49, (b) Complementary determining region 2 (CDR2) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 50, and / or (c) Complementary determining region 3 (CDR3) comprising a sequence having at least 60% identity to the sequence set forth in SEQ ID NO: 51 and comprises a light chain immunoglobulin variable region, the method according to any one of 1 or 2 above. 11. Further defined as a method for treating or preventing metastasis to the lung, liver, brain or breast in a subject having osteosarcoma, the method according to any one of 1 to 5 above. The method according to 1 above, comprising administering to the subject an expression vector encoding an effective amount of said antibody. 12. The method according to any one of 1 to 5 above, wherein the antibody is administered in a pharmaceutically acceptable composition. 13. The method according to any one of 1 to 5 above, wherein the antibody is administered systemically. 14. The method according to 13 above, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. 15. The method according to 12 above, wherein the antibody is a humanized antibody. 16. Further comprising administering to the subject at least a second anti-cancer therapy, the method according to any one of 1 to 5 above. 17. The method according to 16 above, wherein the second anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. 18. The method according to any one of 1 to 5 above, wherein the antibody or fragment thereof binds to the Cx43 hemichannel. 19. The method according to any one of 1 to 5 above, wherein the antibody or fragment thereof further comprises a tag sequence. 20. The method according to any one of 1 to 5 above, wherein the antibody or fragment thereof stimulates the opening of the Cx43 hemichannel. 21. The method according to any one of 1 to 5 above, wherein the fragment is a Fab fragment, a Fab' fragment or an F(ab')2 fragment. 22. The method according to 12 above, wherein the pharmaceutical composition is lyophilized. 23. The body or a fragment thereof is (a) i) A first complementarity-determining region 1 comprising a sequence having at least 60% identity to SEQ ID NO: 19, ii) A second complementarity-determining region 2 comprising a sequence having at least 60% identity to SEQ ID NO: 20, and iii) A third complementarity-determining region 3 comprising a sequence having at least 60% identity to SEQ ID NO: 21 comprising a heavy-chain immunoglobulin variable region, and (b) i) A first complementarity-determining region 1 comprising a sequence having at least 60% identity to SEQ ID NO: 49, ii) A second complementarity-determining region 2 comprising a sequence having at least 60% identity to SEQ ID NO: 50, and iii) A third complementarity-determining region 3 comprising a sequence having at least 60% identity to SEQ ID NO: 51 comprising a light-chain immunoglobulin variable region The method according to 3 above, comprising the same. 24. The method according to 23 above, wherein the antibody is a humanized antibody. 25. The antibody or a fragment thereof is (a) A complementarity-determining region 1 (CDR1) comprising a sequence having a single amino acid change compared to the sequence shown in SEQ ID NO: 19, (b) A complementarity-determining region 2 (CDR2) comprising a sequence having a single amino acid change compared to the sequence shown in SEQ ID NO: 20, and / or (c) A method according to any one of 1 or 3 above, comprising a heavy-chain immunoglobulin variable region comprising a complementarity-determining region 3 (CDR3) comprising a sequence having a single amino acid change compared to the sequence shown in SEQ ID NO: 21. 26. The antibody or a fragment thereof is (a) A complementarity-determining region 1 (CDR1) comprising a sequence having a single amino acid change compared to the sequence shown in SEQ ID NO: 49, (b) Complementary determining region 2 (CDR2) comprising a sequence having a single amino acid change as compared to the sequence shown in SEQ ID NO: 50, and / or (c) Complementary determining region 3 (CDR3) comprising a sequence having a single amino acid change as compared to the sequence shown in SEQ ID NO: 51 The method according to any one of 1 or 3 above, comprising a light chain immunoglobulin variable region comprising the same. 27. The antibody or fragment thereof is (a) i) A first complementarity region 1 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 19, ii) A second complementarity region 2 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 20, and iii) A third complementarity region 3 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 21 Comprising a heavy chain immunoglobulin variable region, and (b) i) A first complementarity region 1 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 49, ii) A second complementarity region 2 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 50, and iii) A third complementarity region 3 comprising a sequence having a single amino acid change as compared to SEQ ID NO: 51 Comprising a light chain immunoglobulin variable region The method according to 3 above, comprising the same.

Claims

1. A pharmaceutical composition for inhibiting osteoblast proliferation of osteosarcoma cells in a subject, comprising an anti-connexin 43 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 58 and a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63, wherein the antibody or the antigen-binding fragment thereof, a) a complementarity-determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19, b) a complementarity-determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 20, and c) a complementarity-determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 21, and comprises a heavy-chain immunoglobulin variable region, and wherein the antibody or the antigen-binding fragment thereof, a) a complementarity-determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49, b) a complementarity-determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 50, and c) a complementarity-determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 51, and comprises a light-chain immunoglobulin variable region, characterized in that the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, for treating or preventing metastasis to the lung, liver, brain or breast in a subject having osteosarcoma.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody or the antigen-binding fragment thereof is contained in a pharmaceutically acceptable composition.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or the antigen-binding fragment thereof is for systemic administration.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or the antigen-binding fragment thereof is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody is a humanized antibody.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising a second anti-cancer therapy.

8. The pharmaceutical composition according to claim 7, wherein the second anti-cancer therapy is surgery, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody or the antigen-binding fragment thereof binds to the Cx43 hemichannel.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the antibody or the antigen-binding fragment thereof further comprises a tag sequence.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antibody or an antigen-binding fragment thereof stimulates the opening of Cx43 hemichannels.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the antigen-binding fragment thereof is a Fab fragment, a Fab' fragment or an F(ab')2 fragment.

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