Compositions and methods of use for treating osteosarcoma

Abstract

Disclosed herein are antibodies that bind to connexin 43 half-channels to activate channel opening. Also disclosed herein are methods of detecting and treating osteosarcoma using activated Cx43 semi-channel open antibodies.

Description

[0001] This application is a divisional application of the PCT international application PCT / US2020 / 020343 filed on February 28, 2020, which entered the Chinese national phase on August 27, 2021, and the Chinese patent application number is 202080017438.4, and the invention name is “Compositions and methods of use for treating osteosarcoma”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 811,938, filed on February 28, 2019. The contents of the prior-filed application are hereby incorporated by reference in their entirety.

[0004] Incorporation of Sequence Listing

[0005] This application includes a sequence listing submitted through EFS-Web at the same time as the filing of this application, containing a file named "21105_0070P1_SL.txt" created on February 25, 2020, with a size of 24,576 bytes, and the file is incorporated herein by reference in its entirety. Background Art

[0006] Connexin hemichannels play an important role in cell and tissue function, and malfunction of connexin hemichannels may be involved in various pathological conditions, such as those described herein. Therefore, there remains a need for additional therapies for treating pathological conditions associated with hemichannel activity, as well as methods for identifying such therapies.

[0007] Osteosarcoma occurs in patients, including young adults, and often metastasizes to the lungs, making the disease incurable. Currently available therapies are associated with relief of symptoms, but none of these therapies are curative for the disease. There is a need to treat osteosarcoma or metastases to the lungs, bones, brain, or other organs in patients with osteosarcoma who are in need. Summary of the invention

[0008] Disclosed herein are methods 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 fragment thereof, wherein 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.

[0009] Disclosed herein are methods 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 fragment thereof, wherein the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63.

[0010] 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 D 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.

[0011] Described herein is a method for treating or preventing osteosarcoma or lung, bone, brain metastasis or other organ metastasis in a subject with osteosarcoma, comprising administering to the subject an effective amount of an antibody or an expression vector encoding the antibody that is bound to connexin 43 (Cx43) hemichannels and enhances channel opening. In some aspects, the method may include administering to the subject an effective amount of an antibody. In some aspects, the method may include administering to the subject an effective amount of an expression vector encoding an antibody. In some aspects, the antibody that is bound to connexin 43 (Cx43) hemichannels and enhances channel opening has no effect on gap channel coupling.

[0012] Described herein is a method for treating or preventing osteosarcoma in a subject, comprising administering to the subject an effective amount of an antibody or an expression vector encoding the antibody that binds to connexin 43 (Cx43) hemichannels and enhances channel opening. In some aspects, the method comprises administering to the subject an effective amount of an antibody. In some aspects, the method comprises administering to the subject an effective amount of an expression vector encoding an antibody. In some aspects, osteosarcoma has metastasized to the lungs of the subject. In some aspects, the antibody that binds to connexin 43 (Cx43) hemichannels and enhances channel opening has no effect on gap channel coupling.

[0013] Expression vectors encoding antibodies that can be administered with pharmaceutically acceptable compositions are described herein. In some aspects, the antibodies can be administered systemically. In some aspects, the antibodies can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

[0014] Disclosed herein are antibodies 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 aspects, the antibody may be a humanized antibody. In some aspects, the antibody may be an IgG, IgM, IgA, IgD, IgE, or a genetically modified 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 aspects, the antibody may be an IgG class antibody, wherein the IgG class antibody is an IgG1, IgG2, IgG3, or IgG4 class antibody. In some aspects, the antibody can comprise a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 and / or a VL amino acid sequence at least 90% identical to SEQ ID NO: 63. In some aspects, 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.

[0015] In some aspects, the disclosed methods may further comprise administering to the subject at least a second anti-cancer therapy. In some aspects, the second anti-cancer therapy may be surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0016] Disclosed herein are recombinant connexin 43 (Cx43) hemichannel binding antibodies or fragments thereof. In some aspects, the antibody comprises a first VH CDR or fragment thereof corresponding to SEQ ID NO: 19, a second VH CDR or fragment thereof corresponding to SEQ ID NO: 20, a third VH CDR or fragment thereof corresponding to SEQ ID NO: 21, a first VL CDR or fragment thereof corresponding to SEQ ID NO: 49, a second VL CDR or fragment thereof corresponding to SEQ ID NO: 50, and a third VLCDR or fragment thereof corresponding to SEQ ID NO: 51. In some aspects, the antibody or fragment thereof may be a humanized antibody. In some aspects, the antibody may be an IgG, IgM, IgA, IgD, IgE, or a genetically modified IgG class antibody comprising a first VH CDR corresponding to SEQ I 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 VLCDR corresponding to SEQ ID NO: 51. In some aspects, the antibody can be an IgG class antibody, wherein the IgG class antibody is an IgG1, IgG2, IgG3 or IgG4 class antibody. In some aspects, the antibody comprises a VH amino acid sequence or a fragment thereof that is at least 90% identical to SEQ ID NO: 58 and / or a VL amino acid sequence or a fragment thereof that is at least 90% identical to SEQ ID NO: 63. In some aspects, the antibody can comprise a VH amino acid sequence or a fragment thereof according to SEQ ID NO: 58 and / or a VL amino acid sequence or a fragment thereof according to SEQ ID NO: 63.

[0017] 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 a composition described herein or an expression vector encoding an antibody according to aspects described herein. In some aspects, the pharmaceutical composition comprises an expression vector encoding an antibody according to aspects described herein for a subject. In other aspects, the pharmaceutical composition comprises an antibody according to aspects described herein for a subject. In some aspects, the method may be further defined as a method for inhibiting or preventing lung metastasis of cancer in a subject. In some aspects, the subject suffers from osteosarcoma and / or lung, bone, brain metastasis or other organ metastasis. In some aspects, the pharmaceutical composition may be administered systemically. In some aspects, the pharmaceutical composition is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously or topically.

[0018] In some aspects, 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 several aspects, the method may further comprise administering to the subject at least a second anti-cancer therapy. In additional aspects, the second anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0019] Disclosed herein are antibodies against hemichannel polypeptides and nucleic acid molecules encoding the antibodies. In some aspects, the antibodies can bind to an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13), KRDPCPHQVD (SEQ ID NO: 14), or LSAVYTCKR (SEQ ID NO: 15). In some aspects, the antibodies can bind to an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13).

[0020] In further embodiments, the antibody used in accordance with the embodiments may be any of those described in International (PCT) Patent Publication Nos. WO 2015-027120 or WO 2017-147561, which are incorporated herein by reference for their teachings of antibodies, vectors, and cells for making or expressing antibodies.

[0021] In some aspects, the first heavy chain region can comprise an amino acid sequence having an amino acid sequence of residues 13 to 37 of SEQ ID NO:2; a second heavy chain region having an amino acid sequence corresponding to residues 46 to 66 of SEQ ID NO:2; and a third heavy chain region comprising an amino acid sequence having an amino acid sequence of residues 97 to 116 of SEQ ID NO:2.

[0022] In some aspects, the antibodies disclosed herein may include full length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, and antibody fusions and fragments thereof.

[0023] Disclosed herein are pharmaceutical compositions comprising any of the antibodies or fragments thereof described herein and a pharmaceutically acceptable carrier. Also disclosed herein are antibodies or pharmaceutical compositions for use as medicaments or in therapies for treating osteosarcoma or inhibiting or preventing metastasis of lung cancer.

[0024] Disclosed herein are methods for treating or preventing cancer metastasis. In some aspects, the cancer may be a lung cancer bone, brain metastasis, or other organ metastasis in a patient with osteosarcoma. The method of treatment may include administering an effective amount of an antibody separated as described herein to a subject in need thereof. In addition, disclosed herein are methods for using any antibody described herein in a medicament for the preparation of a drug for treating or preventing cancer metastasis (e.g., lung cancer).

[0025] Disclosed herein is an in vitro method for activating or enhancing Cx43 hemichannels using any antibody, compound or reagent described herein. In some aspects, the methods described herein can be used to determine the impact of Cx43 hemichannel opening activation in bone cells by: (i) using Lucifer yellow or Alexa dye, determining hemichannel opening by dye uptake assay, (ii) assessing the stimulation of hemichannel opening by measuring ATP levels (e.g., the increase in ATP released from bone cells by Cx43 hemichannels can indicate that the tested compound or antibody can inhibit tumors or cancel cell growth and / or colonization, (iii) testing agents for the stimulation of hemichannel opening by mechanical loading in the form of fluid flow shear stress. In some aspects, antibodies that bind to connexin 43 (Cx43) hemichannels and enhance channel opening have no effect on gap channel coupling.

[0026] As used herein, the term "antigen" is a molecule that can be bound by an antibody or T cell receptor. In some aspects, binding moieties other than antibodies can be engineered to specifically bind to an antigen, such as aptamers, avimers, and the like.

[0027] The term "antibody" or "immunoglobulin" is used to include complete antibodies and their binding fragments / segments. As used herein, the term "antibody" is intended to refer to any immunobinder, such as IgG, IgM, IgA, IgD, IgE and genetically modified IgG and polypeptides containing antibody CDR domains that retain antigen binding activity. The antibody can 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. Generally, the fragment competes with the complete antibody from which it is derived for specific binding to the antigen. The fragment includes a single heavy chain, a light chain, Fab, Fab'F(ab')2, Fabc and Fv. The fragment / segment is produced by recombinant DNA technology, or by enzymatic or chemical separation of a complete immunoglobulin. The term "antibody" also includes one or more immunoglobulin chains that are chemically conjugated to or expressed as fusion proteins with other proteins. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with 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 connection 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.

[0028] The term "antibody" may include five different classes of human immunoglobulins, i.e., IgG, IgA, IgM, IgD, and IgE. In some aspects, the disclosed antibodies may be IgG class antibodies, which may be classified into four subclasses of IgG1, IgG2, IgG3, and IgG4. In some aspects, the disclosed antibodies may be IgA class antibodies, which may be classified into two subclasses of IgA1 and IgA2. The basic structure of an immunoglobulin is composed of two homologous L chains (light chains) and two homologous H chains (heavy chains). Immunoglobulin classes and subclasses are determined by the H chain. In some aspects, one or more antibodies or variants or fragments thereof may be IgG4.

[0029] While maintaining antigen binding specificity, the antibody stability of IgG4 can be improved. In some aspects, the antibody can be improved by, for example, replacing the 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).

[0030] The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of the cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) or chemical precursors or other chemicals (when chemically synthesized) from which it was derived. In addition, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, if the compound is adhered to a column or embedded in an agarose gel, it cannot simply be considered "isolated." In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not exist naturally as a fragment and / or is not normally in a functional state.

[0031] Moieties of the invention (such as polypeptides, peptides, antigens or immunogens) can be covalently or non-covalently conjugated or linked to other moieties, such as adjuvants, proteins, peptides, supports, fluorescent moieties or labels. The term "conjugate" or "immunoconjugate" is used broadly to define the effective combination of one moiety with another agent, and is not intended to refer to only any type of effective combination, and is particularly not limited to chemical "conjugation".

[0032] The term "providing" is used according to its ordinary meaning of "supplying or providing 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 present invention contemplates compositions comprising various combinations of nucleic acids, antigens, peptides and / or epitopes.

[0033] The phrase "specific binding" to a target or "specific immunoreactivity" to a target refers to a binding reaction that determines the presence of a molecule in the presence of a heterogeneous population of other biological agents. Thus, under specified immunoassay conditions, a particular molecule preferentially binds to a particular target and does not bind to other biological agents present in the sample in significant amounts. Specific binding of an antibody to a target under such conditions requires that the antibody be selected based on its specificity for the target. A variety of immunoassay formats are available for selecting antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with a protein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0034] Other aspects of the present invention are discussed throughout this application. Any aspect discussed about one aspect of the present invention is also 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 considered that any aspect discussed herein can be implemented about any method or composition of the present invention, and vice versa. In addition, the composition and kit of the present invention can be used to realize the method of the present invention.

[0035] As used herein, when used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" may mean "one", but it also conforms to the meaning of "one or more", "at least one" and "one or more than one".

[0036] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0037] Use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring to only alternatives and "and / or."

[0038] As used in this application and in the claims, the words "comprising" (and any forms of comprising, such as "includes" and "containing"), "having" (and any forms of having, such as "having" and "having"), "including" (and any forms of including, such as "covers" and "including"), or "containing" (and any forms of containing, such as "including" and "including") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0039] As used herein, the terms "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids or any non-natural analogs that may be in any disclosed antibody, variant or fragment. Thus, as used herein, "amino acid" refers to both naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline, and norleucine are considered amino acids. "Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chains may be in the (R) or (S) configuration. In some aspects, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example, to prevent or delay degradation in vivo.

[0040] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although specific embodiments of the present disclosure are indicated, the detailed description and specific examples are given by way of illustration only, because from this detailed description, various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

[0041] Description of the drawings

[0042] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the embodiments of the specification presented herein.

[0043] Figure 1A -C shows inhibition of breast cancer growth in bone by human-mouse chimeric anti-Cx43 antibody HMAb2 (this antibody contains the same murine variable domains and CDRs as the "M2" antibody). Py8119-Luc cells were injected into the right tibia of control and cKO female mice. The left tibia was injected with PBS as a control. Figure 1A Tumor growth was recorded weekly for 4 weeks and quantified by bioluminescent imaging. Data are presented as mean ± SEM. **, PO.01. n = 7 / group. Figure 1B Representative images of Cx43 cKO mice showing tumor spread to the lungs and brain indicated by white arrows. Figure 1C Representative X-rays showing tibiae injected with Py8119 cells, indicating the location of tumor cell injection and the location of osteolytic lesions (arrows). Left tibiae injected with PBS showed no osteolytic lesions.

[0044] Figure 2A -B shows that Cx43 hemichannels in MLO-Y4 osteocytes (A) or primary mouse osteocytes (B) are activated by HMAb2 but blocked by HMAb1. Cells were incubated with E2 (polyclonal), HMAb1, and HMAb2 antibodies or carbenoxolone (CBX), a connexin channel blocker. Ethidium bromide (EtBr) dye uptake assay was performed. Data are expressed as SEM. ***, P < 0.001 compared to basal control.

[0045] Figure 3A -B shows activation of hemichannels by MHAb2 in osteocytes in vivo. Evans blue dye was injected into the tail vein of WT mice, and 25 μg / ml MHAb2 was injected IP. Two hours after injection, mice were sacrificed and perfused with PBS. Tibiae were isolated and fixed tibial tissue sections were prepared. Figure 3AThe presence of antibodies detected with rhodamine-conjugated anti-human IgG is shown. Bars, 50 μm. Figure 3B Dye uptake in cortical and trabecular bone was measured and quantified by Evans blue (EB) fluorescence. *, P < 0.05; ***, P 0.001.

[0046] Figure 4A -C shows that HMAb2 inhibits the osteolytic growth of breast cancer cells and protects bones from fracture. Figure 4A Py8119-Luc breast cancer cells were shown injected into the tibia of female mice. Figure 4B HMAb2 was injected intraperitoneally once or twice a week at 25 mg / kg for four weeks. Control mice were injected with saline twice a week. Tumor growth was recorded weekly for 4 weeks and quantified by bioluminescent imaging (lower figure). Data are expressed as mean ± SEM. For HMAb2 and saline, n = 6. Figure 4C Shown are X-ray images of mice injected with MHAb2 or saline. *, PO.05.

[0047] Figure 5A -B shows that both HMAb2 and HAb2 antibodies recognize Cx43 and bind to Cx43 on the surface of osteocytes. Figure 5A Immunolabeling of parental HeLa or HeLa cells expressing Cx43 with HMAb2 (MHC2) or HAb2 (HC2) antibodies is shown. Figure 5B Immunofluorescence labeling of non-permeabilized osteocytic MLO-Y4 cells with anti-HMAb2 (MHC2) or HAb2 (HC2) antibodies is shown.

[0048] Figure 6 Dose-dependent inhibition of osteolytic breast cancer growth by MHAb2 is shown. Py8119-Luc breast cancer cells were injected into the tibia of female mice. HMAb2 was injected intraperitoneally once a week at 5, 15, and 25 mg / kg for four weeks. Control mice were injected with saline once a week. Tumor growth was recorded weekly for 4 weeks and quantified by bioluminescent imaging. Data are expressed as mean ± SEM. For HMAb2 and saline, n = 6. *, PO.05.

[0049] Fig. 7A -D shows that HMAb2 increases trabecular bone mass, volume, and thickness. Four-month-old mice were injected intraperitoneally with 25 mg / kg HMAb2 antibody or saline once a week for two weeks. Bone parameters (A) Bone volume; (B) Trabecular thickness; (C) Trabecular number; (D) Bone mineral density (BMD) were determined and quantified by micro-CT imaging. Data are expressed as mean ± SEM. n = 6; *, PO.05; **, PO.01.

[0050] Fig. 8A -B shows the inhibition of osteolytic human breast cancer growth by MHAb2. Fig. 8A MDA-MB231 human breast cancer cells were injected into the tibia of female immunocompromised nude mice. HMAb2 was injected intraperitoneally at 25 mg / kg once a week for 7 weeks. Saline or human IgG was injected once a week in control mice. Tumor growth was recorded weekly for 7 weeks and quantified by bioluminescent imaging. Data are expressed as mean ± SEM. n = 6. *, PO.05. Figure 8B Mice were sacrificed after 7 weeks as shown and tumors were isolated.

[0051] Fig. 9 Inhibition of osteoblast growth of murine osteosarcoma cancer cells by chimeric MHC2 antibody (corresponding to mouse-human chimeric M2 antibody) is shown. DML8-Luc murine osteosarcoma cells were implanted into the tibia of C3H mouse strain. MHC2 was injected intraperitoneally once a week at 25 mg / kg. Saline was injected once a week in control mice. Tumor growth was recorded weekly for 6 weeks and quantified by bioluminescent imaging. n=6 / group. Data are expressed as mean ± SEM. *, PO.05.

[0052] Fig. 10A -B shows that the optimized (single amino acid change in the heavy chain CDR) humanized M2 antibody inhibits osteoblast growth in human osteosarcoma cancer cells. Fig. 10A Dose-dependent inhibition of osteoblast growth of human osteosarcoma cancer cells by optimized humanized HC2 antibody (corresponding to optimized humanized M2 antibody) is shown. OS17-Luc osteosarcoma cells were implanted into the tibia of immunocompromised mice. HC2 was injected intraperitoneally once a week at 5, 15 and 25 mg / kg. Normal saline was injected once a week in control mice. Tumor growth was recorded weekly for 7 weeks and quantified by bioluminescent imaging. Fig. 10B Shows dose-dependent inhibition by HC2 antibody (corresponding to optimized humanized M1 antibody) 42 days after tumor implantation. n=5 / group. Data are expressed as mean±SEM. *, P<0.05; **, P<0.01. DETAILED DESCRIPTION

[0053] The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included therein, and by reference to the Figures and their previous and following descriptions.

[0054] It will be understood that unless otherwise stated, the disclosed methods and compositions are not limited to specific synthetic methods, specific synthetic techniques or specific reagents, and thus these may vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0055] The present invention discloses materials, compositions and components that can be used, can be combined for, can be used to prepare, or are products and compositions of the disclosed methods. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, although specific references to each different individual and various combinations and arrangements of these compounds may not be explicitly disclosed, each should be specifically considered and described herein. Thus, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed and an example of a combination molecule AD is disclosed, each can be considered individually and collectively even if each is not listed individually. Thus, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE and CF is specifically considered and should be considered to be disclosed from the disclosure A, B and C; D, E and F; and the exemplary combination AD. Likewise, any subset or combination of these is also specifically considered and disclosed. Thus, for example, the subgroups AE, BF and CE are specifically considered and should be considered to be disclosed from the disclosure A, B and C; D, E and F; and the exemplary combination AD. This concept applies to all aspects of this application, including but not limited to steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically contemplated and considered disclosed.

[0056] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the publications cited. The publications discussed herein are provided only for their disclosure before the filing date of the present application. Anything herein shall not be construed as admitting that the present disclosure is not entitled to be earlier than this publication by virtue of prior disclosure. In addition, the publication date provided herein may be different from the actual publication date, which may require separate confirmation.

[0057] definition

[0058] It should be understood that the disclosed methods and compositions are not limited to the specific methods, protocols and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0059] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes instances where the event, circumstance, or material occurs or exists and instances where it does not or does not exist.

[0060] As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0061] Ranges can be expressed herein as from "about" a specific value and / or to "about" another specific value. Unless the context clearly indicates otherwise, when expressed, it is also specifically considered and considered that the disclosed range is from a specific value and / or to another specific value. Similarly, when the value is expressed as an approximation by using the antecedent "about", it should be understood that unless the context clearly indicates otherwise, the specific value forms another embodiment that should be considered to be disclosed. It should be further understood that unless the context clearly indicates otherwise, the endpoints of each range are important relative to the other endpoint and independently of the other endpoint. Finally, it should be understood that unless the context clearly indicates otherwise, all single values ​​and sub-ranges of values ​​contained in the clearly disclosed range are also specifically considered and should be considered to be disclosed. Whether or not some or all of these embodiments are clearly disclosed in a particular case, the foregoing applies.

[0062] Throughout the description and claims of this specification, the word "comprise" and variations of the word such as "comprising / comprises" mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers or steps. Specifically, in a method recited as including one or more steps or operations, each step is specifically considered to include the listed items (unless the step includes a limiting term such as "consisting of"), which means that each step is not intended to exclude, for example, other additives, components, integers or steps not listed in the step.

[0063] "Inhibit," "suppress," and "inhibit" mean to reduce or lower an activity, level, response, condition, disease, or other biological parameter. This may include, but is not limited to, complete ablation of an activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction of an activity, response, condition, or disease compared to a natural or control level. Thus, in some aspects, the inhibition or reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction therebetween, compared to a natural or control level. In some aspects, the inhibition or reduction is 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%, compared to a natural or control level. In some aspects, the inhibition or reduction is 0%-25%, 25%-50%, 50%-75%, or 75%-100% compared to native or control levels.

[0064] As used herein, "modulation" refers to a change in activity, function or quantity. The change can be an increase or decrease, enhancement or inhibition of activity, function or quantity.

[0065] "Lifting" and "promoting" refer to an increase in activity, reaction, condition, disease or other biological parameters. This may include, but is not limited to, the onset of activity, reaction, condition or disease. This may also include, for example, an increase of 10% in activity, reaction, condition or disease compared to a natural or control level. Therefore, in some aspects, the increase or promotion may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, or any promotion amount in between, compared to a natural or control level. In some aspects, 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 or control level. In some aspects, compared to natural or control levels, the increase or improvement is 0%-25%, 25%-50%, 50%-75% or 75%-100% or more, such as 200%, 300%, 500% or 1000%. In some aspects, compared to natural or control levels, the increase or improvement can be greater than 100%, such as compared to natural or control levels, an increase or improvement of 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more.

[0066] "Treatment" refers to administering or applying a therapeutic agent (e.g., an anti-Cx43 antibody described herein) to a subject or performing a procedure or method on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment may include administering a pharmaceutically effective amount of an antibody that enhances or stimulates the opening of a Cx43 hemichannel. In some aspects, antibodies that bind to connexin 43 (Cx43) hemichannels and enhance channel opening have no effect on gap channel coupling.

[0067] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, relieving one or more symptoms or features of a particular disease, disorder, and / or condition, delaying its onset, inhibiting or slowing its progression, reducing its severity, and / or reducing its incidence. Treatment may be administered to subjects who do not show signs of a disease, disorder, and / or condition and / or to subjects who show only early signs of a disease, disorder, and / or condition, with the goal of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. For example, the disease, disorder, and / or condition may be osteosarcoma or cancer.

[0068] As used herein, the term "subject" refers to the target to be administered, such as humans. Therefore, the subject of the disclosed method can be a vertebrate, such as a mammal, fish, bird, reptile or amphibian. The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). In one aspect, the subject is a mammal. In another aspect, the subject is a human being. The term does not represent a specific age or gender. Therefore, it is intended to cover adults, children, adolescents and neonatal subjects, as well as fetuses, whether male or female.

[0069] As used herein, the term "patient" refers to a subject suffering from a disease or condition. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the "patient" has been diagnosed as needing treatment, such as, for example, prior to the administering step.

[0070] 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 biological activity of the reference (e.g., at least 5, 10, 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, or 500 (etc.) amino acids or nucleic acids). In some aspects, 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. In addition, a fragment of a reference peptide can be a continuous or contiguous portion of a reference polypeptide (e.g., a fragment of a ten amino acid long reference peptide can be any 2 to 9 contiguous residues within the peptide).

[0071] The term "variant" may mean a difference in some aspects from a reference sequence, rather than simply missing one or more N-terminal and / or C-terminal amino acid residues. When a variant includes a substitution of an amino acid residue, the substitution may be considered conservative or non-conservative. Conservative substitutions are those within the following groups: Ser, Thr and Cys; Leu, Ile and Val; Glu and Asp; Lys and Arg; Phe, Tyr and Trp; and Gin, Asn, Glu, Asp and His. A variant may include at least one substitution and / or at least one addition, and at least one deletion may also be present. A variant may also include one or more non-naturally occurring residues. For example, it may include selenocysteine ​​(e.g., seleno-L-cysteine) at any position (including at the position of cysteine). Many other "non-natural" amino acid substituents are known in the art and may be obtained from commercial sources. Examples of non-naturally occurring amino acids include D-amino acids, amino acid residues having an acetamidomethyl group attached to the sulfur atom of cysteine, pegylated amino acids, and ω amino acids of the formula NH2(CH2)nCOOH (wherein n is 2-6), neutral amino acids, non-polar amino acids such as sarcosine, tert-butylalanine, tert-butylglycine, N-methylisoleucine, and norleucine. Phenylglycine can replace Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral, non-polar, cysteine ​​is acidic, and ornithine is basic. Proline can be replaced by hydroxyproline and retain the conformation that confers the properties of proline.

[0072] "Single chain variable fragment (scFv)" means a protein comprising the variable regions of the heavy and light chains of an antibody. An scFv can be a fusion protein comprising a variable heavy chain, a linker, and a variable light chain. In some aspects, the linker can be a short, flexible fragment that can be about 8 to 20 amino acids in length. For example, (G4S) can be used n (n=1, 2, 3 or 4).

[0073] "Fragment antigen binding fragment (Fab)" is the region of an antibody that binds to an antigen. Fab contains the constant and variable regions from the heavy and light chains.

[0074] "CDRs" or complementarity determining regions are regions of hypervariability interspersed with more conserved regions called "framework regions" (FR).

[0075] The term "monoclonal antibody" (monoclonal antibody) refers to an antibody or similar antibody population obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method, including, but not limited to, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein (Nature, 256:495-497, 1975) or by recombinant DNA methods.

[0076] The term "chimeric antibody" (or "chimeric immunoglobulin") refers to a molecule that comprises heavy and / or light chains that are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly et al. (1984), see below; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851).

[0077] The term "humanized antibody" refers to an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Humanized antibodies may include conservative amino acid substitutions or non-natural residues from the same or different species, which do not significantly change their binding and / or biological activity. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), in which residues from the complementary determining regions (CDRs) of receptors are replaced by residues from the CDRs of non-human species (donor antibodies), such as mice, rats, camels, cattle, goats or rabbits with desired properties. In addition, humanized antibodies may include residues that are neither found in the receptor antibodies nor in the CDRs or framework sequences imported. These modifications are carried out to further improve and maximize antibody performance. Thus, in general, a humanized antibody may comprise all or substantially all of at least one variable domain (and in one aspect, two variable domains), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally can also comprise at least a portion of an immunoglobulin constant region (Fc), or at least a portion of the constant region of a human immunoglobulin (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 Bl; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Padlan, EA et al., European Patent Application No. 0,519,596 A1; Queen et al. (1989) Proc. Natl. Acad. Sci. USA, Vol. 86: 10029-10033).

[0078] As used herein, the term "MIH" refers to an antibody cloned from a hybridoma clone. "M1" refers to hybridoma monoclonal 1, and "FT" refers to variable heavy chain.

[0079] As used herein, the term "MIM7K" refers to the variable light chain identified from hybridoma clones M1 and M7.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the methods and compositions of the present invention, particularly useful methods, devices and materials are as described. The publications cited herein and the materials cited therein are hereby specifically incorporated by reference. Nothing herein shall be construed as admitting that the present invention is not entitled to precede this disclosure by virtue of prior invention. It is not admitted that any reference constitutes prior art. The discussion of the references states the viewpoints advocated by the author, and the applicant reserves the right to question the accuracy and relevance of the cited documents. It should be clearly understood that, although many publications are mentioned herein, such citation does not constitute an admission that any of these documents constitute part of the common general knowledge in the art.

[0081] Various cells are able to communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by the protein connexins. Connexins are ubiquitously expressed throughout the body. Six connexins make up one hemichannel, and two hemichannels make up one gap junction channel. Gap junctions are a group of channels located in the plasma membrane between adjacent cells that mediate intercellular communication. Hemichannels are entities independent of gap junction channels. Hemichannels allow for the exchange of molecules between intracellular compartments and the extracellular environment.

[0082] Osteocytes express hemichannels called connexin (Cx) 43 hemichannels. These osteocyte hemichannels are normally closed and can open when mechanically stimulated, which results in the release of various factors into the bone microenvironment. Factors released through hemichannel opening can mediate other processes that can reduce tumor cell migration and bone metastasis.

[0083] Disclosed herein is a method for identifying an agent for regulating the opening of a connexin hemichannel. In some aspects, regulation can mean stimulating or enhancing the opening of one or more connexin hemichannels. In some aspects, a connexin hemichannel can be a Cx 43 hemichannel. In some aspects, the method can identify a compound or drug that positively regulates (i.e., stimulates or enhances) the opening of a connexin hemichannel. Other embodiments relate to a method for treating osteosarcoma by administering an open connexin 43 hemichannel or stimulating or enhancing the opening of a connexin 43 hemichannel to a patient diagnosed with or having osteosarcoma. In some aspects, the patient suffers from a primary tumor. In some aspects, an open Cx43 hemichannel or a compound that stimulates or enhances the opening of a Cx43 hemichannel can be used to prevent, inhibit or reduce metastasis to the lung. In some aspects, an open Cx43 channel or a compound that stimulates or enhances the opening of a Cx43 hemichannel can be used to treat osteosarcoma. In some aspects, antibodies that bind to connexin 43 (Cx43) hemichannels and enhance channel opening have no effect on gap channel coupling.

[0084] Cancer metastasis occurs when cancer spreads from the body part (e.g., bone) where it originates to other parts of the body (e.g., lungs) and forms secondary tumors. Lungs are one of the most common sites of cancer metastasis in patients with osteosarcoma. Cancers that metastasize to the lungs include, but are not limited to, breast cancer, prostate cancer, bone cancer, and skin cancer (e.g., melanoma). Lung metastasis can be identified in patients with osteosarcoma. Lung metastasis (mets) is associated with many important clinical and quality of life consequences, such as, but not limited to, intractable pain, nausea, headache, shortness of breath, hemoptysis, pleural effusion, and impaired motor ability. In many cases, the systemic presence of cancer also makes cancer incurable.

[0085] Normal bone is composed of three major cell types: bone-forming osteoblasts, bone-resorbing osteoclasts, and osteocytes. Osteocytes account for approximately 95% of skeletal cells and maintain the bone remodeling process by coordinating osteolytic and osteogenic activities. When cancer cells invade the bone, many normal bone functions are affected. Cancer cells interact with the local microenvironment to promote cancer cell survival through bone destruction and vascularization.

[0086] The opening of bone cell connexin (Cx) 43 hemichannels and the factors released therefrom have an inhibitory effect in cancer growth, migration and metastasis, while the inactivation of hemichannels has the opposite effect. The Cx43 hemichannels mediate the communication between the intracellular and extracellular microenvironments. The Cx43 hemichannels are present in large quantities in skeletal osteocytes, and the ATP released by osteocytes through the Cx43 hemichannels exerts a tumor suppressive effect on cancer bone metastasis. Therefore, enhancing the activation of the Cx43 hemichannels in osteocytes may be an important strategy to protect bone tissue from cancer cell growth and colonization. In some aspects, the antibodies described herein can reduce or inhibit bone tumor cell growth, such as tumor growth in the tibia.

[0087] Cx43 hemichannels in osteocytes have been shown to be opened by treatment with alendronate (AD), which is an effective and commonly used bisphosphonate drug. Bisphosphonates are a class of drugs known to be used to treat many bone disorders including bone metastasis. Powles et al. have shown that the administration of bisphosphonates is associated with a reduction in the incidence of bone metastasis and a reduction in mortality in patients with breast cancer. AD is associated with reduced tumor growth and a reduction 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 accompanied by a variety of serious side effects.

[0088] Antibody

[0089] Disclosed herein are antibodies that can stimulate or enhance open hemichannels, and in particular Cx43 hemichannels. In some aspects, antibodies that bind to connexin 43 (Cx43) hemichannels and enhance channel opening have no effect on gap channel coupling. Examples of identifying and isolating monoclonal antibodies are described below.

[0090] As used herein, the term "CDR' refers to the complementarity determining region of an antibody variable domain. Rabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda) have developed a systematic identification of residues included in the CDR. The variable light chain (VL) CDR is defined herein as comprising residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3). The variable heavy chain (VH) CDR is defined herein as comprising residues at positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).

[0091] CDR disclosed herein may also include variants. Typically, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Therefore, "variant CDR" has specific identity with the parent of the present invention or reference CDR and shares biological function, including but not limited to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR. For example, "variant CDR" can be a sequence containing 1, 2, 3 or 4 amino acid changes compared with the parent of the present invention or reference CDR and sharing or improving the biological function, specificity and / or activity of the parent CDR.

[0092] In some aspects, any CDR sequence disclosed herein may include a single amino acid change compared to a parent or reference CDR. In some aspects, any CDR sequence disclosed herein may include at least two amino acid changes compared to a parent or reference CDR. In some aspects, the amino acid change may be to change from a cysteine ​​residue to another amino acid. In some aspects, the amino acid change may be to change from a glycine residue to another amino acid. The amino acid identity between individual variant CDRs may 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" may be a variant CDR that has a specific identity with a parent 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, the parent CDR sequence may be one or more of SEQ ID NOs: 19, 20, 21, 49, 50 and / or 51. The variant CDR sequence may be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any of SEQ ID NOs: 19, 20, 21, 49, 50 and / or 51. The variant CDR sequence may 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 parent CDR.

[0093] As discussed herein, minor changes in the amino acid sequence of any antibody disclosed herein are considered to be covered 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 parent sequence. In some aspects, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic hydroxyl families; asparagine and glutamine are amide-containing families; alanine, valine, leucine and isoleucine are aliphatic families; and phenylalanine, tryptophan, tyrosine are aromatic families. For example, it is reasonable to expect that replacement of leucine by isoleucine or valine, aspartic acid by glutamic acid, threonine by serine alone, or replacement of similar amino acids by structurally related amino acids will not have a significant effect on the binding or properties of the resulting molecule, particularly where the replacement does not involve amino acids within framework sites. By measuring the specific activity of the polypeptide derivatives, it can be easily determined whether an amino acid change will result in a functional peptide. The determination is known to those of ordinary skill in the art.

[0094] In some aspects, the amino acid substitutions may be those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (4) confer or modify other physicochemical or functional properties of such analogs. In some aspects, 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 aspects, one or more amino acid substitutions may be made in one or more CDR sequences of the heavy chain, light chain, or both.

[0095] Many methods for chemical labeling and enhancing the properties of antibodies and common fragments thereof (including Fab and F(ab')2 fragments) have been developed. Some selective reduction of certain antibody disulfide bonds has been previously achieved, producing antibodies and antibody fragments that can be labeled at specific sites, thereby enhancing their utility and properties. It is useful to selectively reduce the two hinge disulfide bonds present in the F(ab')2 fragment using mild reduction. In some aspects, cysteine ​​and methionine are susceptible to rapid oxidation, which may negatively affect the cracking of the protective group during synthesis and subsequent peptide purification. In some cases, the cysteine ​​residues in the peptides used for antibody production may affect the affinity of the antibody because free cysteine ​​is not common in vivo and therefore may not be recognized by the natural peptide structure. In some aspects, the disclosed antibodies and fragments thereof include sequences in which the cysteine ​​(e.g., in the non-CDR sequences of heavy chain, light chain, or both) located outside the CDR is replaced. In some aspects, cysteine ​​can be replaced by serine and methionine can be replaced by norleucine (Nle). Multiple cysteines on a peptide or in one of the disclosed antibodies or fragments thereof may be susceptible to disulfide bond formation unless a reducing agent such as dithiothreitol (DTT) is added to the buffer or the cysteines may be replaced with serine residues.

[0096] Although the site or region for introducing amino acid sequence variation is predetermined, the mutation itself does not need to be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region, and the antigen-binding protein CDR variants expressed can be screened for the best combination of desired activity. The technology of performing substitution mutations at predetermined sites in DNA with known sequences is well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed using the determination of antigen-binding protein activity as described herein.

[0097] Amino acid substitutions will generally be of single residues; insertions will generally be on the order of from about one (1) to about twenty (20) amino acid residues, although larger insertions may be tolerated. Deletions will range from about one (1) to about twenty (20) amino acid residues, although deletions may be larger in some cases.

[0098] Substitution, deletion, insertion or any combination thereof may be used to obtain the final derivative or variant. These changes are made on a few amino acids to minimize changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in some cases.

[0099] As used herein, "Fab" or "Fab region" means a polypeptide comprising VH, CHI, VL and CL immunoglobulin domains. Fab can refer to this region alone, or in the context of a full-length antibody, antibody fragment or Fab fusion protein or any other antibody embodiment outlined herein.

[0100] "Fv" or "Fv fragment" or "Fv region" as used herein means a polypeptide comprising the VL and VH domains of a single antibody.

[0101] As used herein, "framework" refers to the region of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0102] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more antibody fragments 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 encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL / VK, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which may be a Fab with a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul, ed., 3rd ed. 1993); (iv) a Fd fragment, which consists of the VH and CH1 domains; (v) a Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains.

[0103] The term "specific binding" (or "immunospecific binding") is not intended to indicate that an antibody binds exclusively to its intended target. Instead, an antibody "specifically binds" if its affinity for its intended target is about 5 times higher than its affinity for non-target molecules. Suitably, there is no significant cross-reaction or cross-binding with undesired substances. For example, the affinity of the antibody for the target molecule will be at least about 5 times, such as 10 times, such as 25 times, especially 50 times, and especially 100 times or more of its affinity for non-target molecules. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10 6 M-1 The antibody can, for example, have a binding affinity of at least about 10 7 M- 1 , such as 10 8 M- 1 to about 10 9 M- 1 , about 10 9 M- 1 to about 10 10 M- 1 Or about 10-^M- 1 to about 10 11 M- 1 The antibody can be combined with an affinity of 50nM or less, 10nM or less, 1nM or less, 100pM or less or more preferably 10pM or less EC50. In some aspects, the antibody can be combined with an EC50 of about 60μg / ml, 59μg / ml, 58μg / ml, 57μg / ml, 56μg / ml, 55μg / ml, 54μg / ml, 53μg / ml, 52μg / ml, 51μg / ml, 50μg / ml or less. In some aspects, the antibody can be combined with an EC50 of about 50μg / ml, 49μg / ml, 48μg / ml, 47μg / ml, 46μg / ml, 45μg / ml, 44μg / ml, 43μg / ml, 42μg / ml, 41μg / ml, 40μg / ml or less. In some aspects, the antibody may bind at 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.

[0104] In some aspects, the antibodies described herein can specifically bind to their intended targets. In some aspects, the antibodies described herein do not have off-site binding. For example, the antibodies described herein do not bind or are not distributed to the heart, liver, or spinal cord.

[0105] The antibodies described herein may be variants, including but not limited to fragments (e.g., Fab fragments or F(ab')2 fragments of tetrameric antibodies), fragments of scFv or diabodies, or variants of tetrameric antibodies, scFv, diabodies or fragments thereof that differ by addition and / or substitution of one or more amino acid residues. The antibody portion may be further engineered into, for example, a di-diabody.

[0106] As is well known in the art, certain types of antibody fragments can be produced by enzymatic treatment of "full length" antibodies. Two identical Fab fragments (each with a single antigen binding site) are produced with papain digestion, and a residual Fc fragment. The Fab fragment also contains the constant domain of the light chain and the C1 domain of the heavy chain. In contrast, pepsin digestion produces a F (ab ') 2 fragment with two antigen binding sites and is still capable of cross-linking antigens.

[0107] The difference between Fab' fragment and Fab fragment is that it includes additional residues at the C-terminal of Chi domain, including one or more cysteine ​​residues from antibody hinge region. The cysteine ​​residues of constant domain carry a free thiol group. F(ab')2 antibody fragment is a paired Fab' fragment connected by the cysteine ​​residues in hinge region. Other chemical couplings of antibody fragments are also known in the art.

[0108] The Fv region is the smallest fragment containing a complete antigen recognition and binding site consisting of a heavy chain and a light chain variable domain. The three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. In general, six CDRs confer antibody antigen binding specificity. As 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, a single-chain antibody includes a polypeptide linker between the VH and VL domains that allows scFv to form a desired three-dimensional structure for antigen binding (see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore, Springer-Verlag, New York, 113: 269-315.1994).

[0109] In some aspects, the antibody can be a diabody. Diabodies are small antibody fragments with two antigen binding sites. Each fragment comprises a VH domain connected to a VL domain. However, because the linker between the domains is too short to allow them to pair on the same chain, the connected Vh-Vl domains are forced to pair with the complementary domains of another chain, thereby generating two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993.

[0110] In some aspects, it is contemplated that at least a portion of the Cx43 protein is bonded to and stimulates, promotes or enhances the opening of Cx43 hemichannels and stimulates or enhances signal transduction and reduces cancer cell growth, proliferation and / or colonization antibodies or fragments thereof. In some aspects, it is contemplated that at least a portion of the Cx43 protein is bonded to and stimulates, promotes or enhances the opening of Cx43 hemichannels and stimulates or enhances signal transduction and reduces cancer cell growth, proliferation and / or colonization and that gap junction coupling is not affected by antibodies or fragments thereof. In some aspects, anti-Cx43 antibodies can be monoclonal antibodies, polyclonal antibodies or humanized antibodies. Therefore, by known means and as described herein, polyclonal or monoclonal antibodies, antibody fragments and binding domains and CDRs (including any aforementioned engineered forms) having specificity to Cx43 protein, one or more of its corresponding epitopes or any aforementioned conjugates can be produced, regardless of whether such antigens or epitopes are separated from natural sources or synthetic derivatives or variants of natural compounds.

[0111] Examples of suitable antibody fragments include, but are not limited to: (i) a Fab fragment, which consists of the VL, VH, CL and CH1 domains; (ii) a "Fd" fragment, which consists of the VII and Cm domains; (iii) a "Fv" fragment, which consists of the VL and VH domains of a single antibody; (iv) a "dAb" fragment, which consists of the VH domain; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule ("scFv"), in which the VII domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) a bispecific single-chain Fv dimer (see U.S. Pat. No. 5,091,513); (ix) diabodies, multivalents or multispecific fragments constructed by gene fusion (U.S. Patent Application Publication No. 20050214860). Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bonds linking the VH and VL domains. Minibodies comprising a scFv joined to a CH3 domain can also be prepared (Hu et al., 1996).

[0112] Antibody-like binding peptide mimetics are also contemplated. Liu et al. (2003) described "antibody-like binding peptide mimetics" (ABiPs), which are peptides that are stripped-down antibodies and have certain advantages of longer serum half-lives and simpler synthetic methods.

[0113] Animals can be inoculated with antigens, such as Cx43 extracellular domain proteins, to produce antibodies specific to Cx43 proteins. Typically, antigens are combined or conjugated with another molecule to enhance immune response. As used herein, conjugates are any peptides, polypeptides, proteins or non-protein substances combined with antigens for eliciting immune responses in animals. The antibodies produced in response to antigen inoculation in animals include various molecules (polyclonal antibodies) made from various individual antibodies that produce B lymphocytes. Polyclonal antibodies are mixed populations of antibody species, and each species can recognize different epitopes on the same antigen. In view of the correct conditions for the production of polyclonal antibodies in animals, most of the antibodies in animal serum will recognize the collective epitopes on the antigenic compounds that animals have been immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the antigen or epitope of interest.

[0114] Monoclonal antibody is a single species of antibody, wherein each antibody molecule recognizes the same epitope, because the cell producing the antibody is derived from a single B lymphocyte cell line. The method for producing a monoclonal antibody (MAb) is generally the same as the method for preparing a polyclonal antibody. In some aspects, rodents (such as mice and rats) are used to produce monoclonal antibodies. In some aspects, rabbits, sheep or frog cells are used to produce monoclonal antibodies. The use of rats is well-known and can provide certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally provide high percentage stable fusion.

[0115] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with the Cx43 antigen with an immortal myeloma cell (usually a mouse myeloma). This technology provides a means to propagate a single antibody-producing cell indefinitely, allowing the production of unlimited quantities of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0116] Plasma B cells can be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immune rabbits and further selected for Cx43 binding cells. After enrichment of B cells producing antibodies, total RNA can be isolated and cDNA synthesized. DNA sequences from the variable regions of antibodies of heavy and light chains can be amplified, constructed into phage display Fab expression vectors, and transformed into Escherichia coli. Cx43 specific binding Fab can be selected and sequenced by multiple rounds of enrichment panning. Selected Cx43 binding hits can be expressed as full-length IgG in human embryonic kidney (HEK293) cells (Invitrogen) using mammalian expression vector systems in rabbit and rabbit / human chimeric forms, and purified using protein G resin with fast protein liquid chromatography (FPLC) separation unit.

[0117] In some aspects, the antibody can be a chimeric antibody, such as an antibody comprising an antigen binding sequence from a non-human donor transplanted to a heterologous non-human, human or humanized sequence (such as a framework and / or constant domain sequence). A method for replacing the light chain and heavy chain constant domains of a monoclonal antibody with a similar domain of human origin has been developed, so that the variable region of the exogenous antibody remains intact. Alternatively, a "completely human" monoclonal antibody can be produced in a mouse transgenic for a human immunoglobulin gene. A method for converting the variable domains of a monoclonal antibody into more human forms has also been developed by recombinantly constructing an antibody variable domain with a rodent (such as a mouse) and a human amino acid sequence. In a "humanized" monoclonal antibody, only the hypervariable CDR is derived from a mouse monoclonal antibody, and the framework and constant region are derived from a human amino acid sequence (see U.S. Patent Nos. 5,091,513 and 6,881,557). It is believed that replacing the amino acid sequence in a rodent-specific antibody with an amino acid sequence found in the corresponding position of a human antibody will reduce the possibility of adverse immune reactions during treatment use. Hybridomas or other cells that produce antibodies may also undergo genetic mutations or other changes that may or may not alter the binding specificity of the antibody produced by the hybridoma.

[0118] Methods for producing polyclonal antibodies in various animal species, as well as various types of monoclonal antibodies (including humanized, chimeric and fully human) are well known in the art and are highly predictable. For example, the following U.S. patents and patent applications provide descriptions of these methods: U.S. Patent Application Nos. 2004 / 0126828 and 2002 / 0172677; and 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; 5,196,0 66; 5,223,409; 5,403,484; 5,420,2535,565,332; 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; 5,87 All patents, patent application publications, and other publications cited herein and therein are hereby incorporated by reference into this application.

[0119] Antibodies can be produced from any animal source, including birds and mammals. Preferably, the antibody is sheep, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse or chicken. In addition, newer technology allows the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows the production of specific antibodies in the absence of animal immunity, as described in U.S. Patent No. 6,946,546, which is incorporated herein by reference. These technologies are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).

[0120] It is fully expected that Cx43 antibodies will have the ability to neutralize or counteract the Cx43 effect, regardless of animal species, monoclonal cell lines or other antibody sources. Some animal species may not be suitable for producing therapeutic antibodies because they are more likely to cause allergic reactions due to the activation of the complement system by the "Fc" part of the antibody. However, complete antibodies can be enzymatically digested into "Fc" (complement binding) fragments, as well as antibody fragments with binding domains or CDRs. The removal of the Fc part reduces the possibility that the antigen-antibody fragment triggers an adverse immune response, and therefore, antibodies without Fc may be preferentially used for preventive or therapeutic treatments. As described herein, antibodies may also be constructed as chimeric or partially or completely humanized, so as to reduce or eliminate the adverse immunological consequences caused by administering antibodies that have been produced in other species or have sequences from other species to animals.

[0121] Substitution variants typically contain an exchange of one amino acid with another at one or more sites within a protein, and can be designed to modulate one or more properties of a polypeptide with or without loss of other functions or properties. Substitutions can be conservative, i.e., one amino acid is replaced by one with similar shape and charge. Conservative substitutions are well known in the art and include, for example, alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartate; 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 or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions can be non-conservative, such that the function or activity of a polypeptide is affected. Nonconservative changes typically involve replacing one residue with a residue that is chemically different, such as replacing a nonpolar or uncharged amino acid with a polar or charged amino acid, or vice versa.

[0122] The protein can be recombinant or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein can be isolated from bacteria. It is also contemplated that bacteria containing such variants can be implemented in compositions and methods. Thus, there is no need to isolate the protein.

[0123] It is contemplated that the composition contains between about 0.001 mg and about 10 mg of total polypeptide, peptide and / or protein per milliliter. Thus, the concentration of protein in the composition may be about 0.001 mg / ml, 0.010 mg / ml, 0.050 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10.0 mg / ml, 11.0 mg / ml, 12.0 mg / ml, 13.0 mg / ml, 14.0 mg / ml, 15.0 mg / ml, 16.0 mg / ml, 17.0 mg / ml, 18.0 mg / ml, 19.0 mg / ml, 20.0 mg / ml, 21.0 mg / ml, 22.0 mg / ml, 23.0 mg / ml, 24.0 mg / ml, 25.0 mg / ml, 26.0 mg / ml, 27.0 mg / ml, 28.0 mg / ml, 29.0 mg / ml 0 mg / ml, 10.0 mg / ml or more (or any range derivable therein). Wherein, about less than, at least about less than, or at most about less than can be an antibody that binds to Cx43: 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 1 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0124] The antibody or preferably the immunological portion of an antibody can be chemically conjugated to or expressed as a fusion protein with other proteins. For the purpose of this specification and the appended claims, all such fusion proteins are included in the definition of an antibody or the immunological portion of an antibody.

[0125] Described herein are antibodies and antibody-like molecules, polypeptides and peptides for Cx43, which are connected to at least one agent to form an antibody conjugate or payload. In order to increase the efficacy of the antibody molecule as a diagnostic or therapeutic agent, the antibody can be connected or covalently bound or complexed to at least one desired molecule or part. This molecule or part can be but is not limited to at least one effector molecule or reporter molecule. The effector molecule comprises a molecule with a desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules connected to the antibody include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. In contrast, reporter molecules are defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent markers, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0126] Several methods of linking or conjugating antibodies to their conjugate moieties are known in the art. Some methods of linking involve the use of metal chelate complexes, such as organic chelators, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenylglycoluril-3 linked to the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein labels are prepared in the presence of these coupling agents or by reaction with isothiocyanates.

[0127] In some aspects, an anti-Cx43 antibody described herein can comprise a heavy chain immunoglobulin variable region comprising: a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19; a CDR2 comprising the sequence of SEQ ID NO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21. Table 2 shows examples of heavy chain CDRs.

[0128] In some aspects, an anti-Cx43 antibody described herein can comprise a light chain immunoglobulin variable region comprising: a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49; a CDR2 comprising the sequence of SEQ ID NO: 50; and a CDR3 comprising the sequence of SEQ ID NO: 51. Table 2 shows examples of CDRs in a light chain.

[0129] In some aspects, the anti-Cx43 antibodies described herein may comprise a heavy chain immunoglobulin variable region comprising: a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19; a CDR2 comprising the sequence of SEQ ID NO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21, and a light chain immunoglobulin variable region comprising: a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49; a CDR2 comprising the sequence of SEQ ID NO: 50; and a CDR3 comprising the sequence of SEQ ID NO: 51.

[0130] In some aspects, the anti-Cx43 antibodies described herein can comprise a variable heavy chain comprising 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 aspects, the anti-Cx43 antibodies described herein comprise a variable heavy chain comprising 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.

[0131] In some aspects, the anti-CX43 antibodies described herein can comprise a variable light chain comprising 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 aspects, the anti-Cx43 antibodies described herein comprise a variable light chain comprising 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.

[0132] Disclosed herein is a nucleic acid sequence encoding M1H comprising the sequence of SEQ ID NO: 52. Disclosed herein is a nucleic acid sequence encoding M1M7K comprising the sequence of SEQ ID NO: 57.

[0133] Disclosed herein are nucleic acid sequences encoding M1H comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 52 (see, Table 3). In some aspects, M1H comprises a variable heavy chain comprising 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.

[0134] Disclosed herein are nucleic acid sequences encoding M1M7K comprising a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 57 (see, Table 3). In some aspects, M1M7K comprises a variable light chain comprising 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.

[0135] Disclosed herein is a nucleic acid sequence encoding an M1H region 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.

[0136] 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.

[0137] Disclosed herein is a nucleic acid sequence encoding an anti-Cx43 hemichannel antibody comprising a heavy chain immunoglobulin variable region and a light chain immunoglobulin variable region, wherein the heavy chain immunoglobulin variable region comprises: 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 the light chain immunoglobulin variable region comprises: 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.

[0138] Disclosed herein are antibodies or fragments thereof that bind to human Cx43. In some aspects, 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 aspects, 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 aspects, the antibody or fragment thereof comprises a variable heavy chain comprising a sequence set forth in SEQ ID NO: 58.

[0139] Disclosed herein are antibodies or fragments thereof that bind to human Cx43. In some aspects, 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 aspects, the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63. In some aspects, the antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 63.

[0140] Disclosed herein are antibodies or fragments thereof that bind to human Cx-43 hemichannels. In some aspects, 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. In some aspects, the antibody comprises: a variable heavy chain comprising the sequence set forth in SEQ ID NO: 58; and a variable light chain comprising the sequence set forth in SEQ ID NO: 63.

[0141] In some aspects, the antibody or fragment thereof comprises an M1H region. In some aspects, the M1H region comprises a heavy chain immunoglobulin variable region comprising: a CDR1 comprising the sequence of SEQ ID NO: 19; a CDR2 comprising the sequence of SEQ DNO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21.

[0142] In some aspects, the antibody or fragment thereof comprises an M1M7K region. In some aspects, the M1M7K region comprises a light chain immunoglobulin variable region comprising: a CDR1 comprising the sequence of SEQ ID NO: 49; a CDR2 comprising the sequence of 50; and a CDR3 comprising the sequence of SEQ ID NO: 51.

[0143] In some cases, the disclosed antibodies or fragments thereof further comprise a tag sequence.

[0144] Disclosed herein are nucleic acid sequences encoding the disclosed antibodies or fragments thereof. For example, disclosed herein are nucleic acid sequences comprising a variable heavy chain, the variable heavy chain comprising a sequence having at least 90% identity with 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 are nucleic acid sequences comprising a variable heavy chain, the variable heavy chain comprising the sequence shown in SEQ ID NO: 52. Also disclosed herein are nucleic acid sequences comprising a variable light chain, the variable light chain comprising a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 57. Also disclosed are nucleic acid sequences comprising a variable light chain, the variable light chain comprising the sequence shown in SEQ ID NO: 57.

[0145] Disclosed herein are nucleic acid sequences comprising: a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 52; and a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 57. Disclosed herein are nucleic acid sequences comprising: a variable heavy chain comprising the sequence set forth in SEQ ID NO: 52; and a variable light chain comprising the sequence set forth in SEQ ID NO: 57.

[0146] Disclosed herein are nucleic acid sequences capable of encoding a single chain variable fragment comprising a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:52.

[0147] 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.

[0148] Disclosed are nucleic acid sequences 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.

[0149] In some cases, the disclosed antibodies or fragments thereof can be bispecific. For example, the antibody or fragment thereof can comprise: a first Fab region comprising the heavy chain and light chain of SEQ ID NO: 58 and a second Fab region comprising the heavy chain and light chain of SEQ ID NO: 63, wherein the first and second Fab regions can be different.

[0150] In some cases, bispecific antibodies can be trifunctional.

[0151] In some cases, the disclosed antibodies or fragments thereof may be murine, human, humanized, chimeric, or a combination thereof.

[0152] In some cases, the disclosed antibodies or fragments thereof are monoclonal.

[0153] method

[0154] Disclosed herein are methods for treating or preventing osteosarcoma in a subject. The method may include administering to the subject a therapeutically effective amount of an anti-connexin 43 antibody or fragment thereof. In some aspects, the antibody or fragment thereof may comprise a variable heavy chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 58. In some aspects, the antibody or fragment thereof may comprise a variable light chain comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 63. In some aspects, the antibody or fragment thereof may comprise: 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 aspects, the method may extend the lifespan of a subject suffering from osteosarcoma. In some aspects, the method may reduce or inhibit or prevent the growth of bone tumor cells, such as tumor growth within the tibia. In some aspects, the method may reduce or inhibit or prevent tumor growth within the tibia. In some aspects, the method can increase, enhance or promote ATP release in or more cells or in malignant tumors. In some aspects, the method can reduce, inhibit or prevent osteosarcoma cell migration.

[0155] In some aspects, an antibody or fragment thereof may comprise: a heavy chain immunoglobulin variable region comprising: a first complementarity determining region 1 comprising a sequence having at least 60% identity with SEQ ID NO: 19; a second complementarity determining region 2 comprising a sequence having at least 60% identity with SEQ ID NO: 20; and a third complementarity determining region 3 comprising a sequence having at least 60% identity with SEQ ID NO: 21. In some aspects, an antibody or fragment thereof may comprise: a light chain immunoglobulin variable region comprising: a first complementarity determining region 1 comprising a sequence having at least 60% identity with SEQ ID NO: 49; a second complementarity determining region 2 comprising a sequence having at least 60% identity with SEQ ID NO: 50; and a third complementarity determining region 3 comprising a sequence having at least 60% identity with SEQ ID NO: 51.

[0156] In some aspects, an antibody or fragment thereof may 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 aspects, an antibody or fragment thereof may comprise: a light chain immunoglobulin variable determining 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.

[0157] In some aspects, the antibody or fragment thereof may comprise a heavy chain immunoglobulin variable region comprising: a) a complementarity determining region 1 (CDR1) comprising a sequence of SEQ ID NO: 19 or a variant thereof; b) a complementarity determining region 2 (CDR2) comprising a sequence of SEQ ID NO: 20 or a variant thereof; and / or c) a complementarity determining region (CDR3) comprising a sequence of SEQ ID NO: 21 or a variant thereof. In some aspects, any one of the heavy chain CDR1, CDR2 or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0158] In some aspects, the antibody or fragment thereof may comprise a light chain immunoglobulin variable region comprising: a) a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49 or a variant thereof; b) a complementarity determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 50 or a variant thereof; and / or c) a complementarity determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 51 or a variant thereof. In some aspects, any of the light chain CDR1, CDR2 or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0159] In some aspects, the antibody or fragment thereof may 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 aspects, any of the light chain CDR1, CDR2, or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0160] In some aspects, an antibody or fragment thereof may 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 aspects, any one of the light chain CDR1, CDR2, or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0161] In some aspects, an antibody or fragment thereof may comprise a heavy chain immunoglobulin variable region comprising: a 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; a 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 a 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 aspects, any of the light chain CDR1, CDR2, or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0162] In some aspects, an antibody or fragment thereof may comprise a light chain immunoglobulin variable region comprising: a 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; a 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 a 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 aspects, any one of the light chain CDR1, CDR2, or CDR3 may comprise at least one amino acid substitution compared to the parent CDR. In some aspects, at least one amino acid substitution may be a cysteine ​​residue to another amino acid. In some aspects, at least one amino acid substitution may be a glycine residue to another amino acid.

[0163] In some aspects, any method disclosed herein can be further defined as a method for treating or preventing lung or brain metastasis in a subject with osteosarcoma. In some aspects, the metastasis can be in the breast, other bones, and / or other organs of the subject.

[0164] In some aspects, any method disclosed herein may include administering an effective amount of an expression vector encoding an antibody or a fragment thereof to a subject. In some aspects, an antibody or a fragment thereof may be administered as a pharmaceutically acceptable composition. In some aspects, the pharmaceutical composition may be lyophilized. In some aspects, an antibody or a fragment thereof may be administered systemically. In some aspects, an antibody or a fragment thereof may be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously or topically. In some aspects, an antibody or a fragment thereof may be a humanized antibody or a humanized fragment thereof. In some aspects, an antibody may be an IgG, IgM, IgA, IgD, IgE or a genetically modified 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 VLCDR corresponding to SEQ ID NO: 51. In some aspects, the antibody can be an IgG class antibody, wherein the IgG class antibody is an IgG1, IgG2, IgG3, or IgG4 class antibody.

[0165] In some aspects, any method disclosed herein may further comprise administering to the subject at least a second anti-cancer therapy. In some aspects, the second anti-cancer therapy may be surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or cytokine therapy.

[0166] In some aspects, in any method disclosed herein, the antibody or its fragment can bind to a Cx43 hemichannel. In some aspects, in any method disclosed herein, the antibody or its fragment can stimulate the opening of a Cx43 hemichannel. In some aspects, in any method disclosed herein, the antibody or its fragment can stimulate the opening of a Cx43 hemichannel and have no effect on gap junction coupling.

[0167] In some aspects, the antibody or fragment thereof may further comprise a tag sequence.

[0168] In some aspects, the antibody or fragment thereof can be a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0169] Disease treatment

[0170] Disclosed herein are antibodies and biological fragments thereof that can be used to treat osteosarcoma or prevent or reduce lung metastasis and / or liver, brain or breast metastasis in subjects with osteosarcoma. Signaling of Cx43 hemichannels can be enhanced by any suitable drug or therapeutic agent to prevent cancer cell proliferation, growth and / or colonization. In some aspects, the drug or therapeutic agent can be an anti-Cx43 antibody.

[0171] The pharmaceutical compositions described herein can be applied to subjects (e.g., human patients) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease. Therefore, in some aspects, the patient can be a human patient. In therapeutic applications, the composition can be applied to subjects (e.g., human patients) who have suffered from or been diagnosed with osteosarcoma in an amount sufficient to at least partially improve signs or symptoms or inhibit (preferably prevent) the symptoms of the condition, its complications and consequences. The amount sufficient to achieve 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 cure, but the result is only one of several results that can be achieved. As noted, a therapeutically effective amount includes an amount for providing treatment, wherein the onset or progression of cancer is delayed, hindered, or prevented, or cancer or symptoms of cancer are improved or their frequency can be reduced. One or more of the symptoms may not be too severe. Recovery in the treated individual can be accelerated. For example, the treatment of cancer can involve, for example, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in cancer growth rate, or the prevention of metastasis. The treatment of cancer can also refer to extending the survival of subjects with cancer. In some aspects, the antibodies described herein can extend the lifespan of a subject with cancer.In some aspects, the antibodies described herein can reduce or inhibit bone tumor cell growth, such as tumor growth within the tibia.

[0172] In some aspects, the cancer may be a primary or secondary tumor. In some aspects, the cancer may be a metastatic tumor. In other aspects, the primary or secondary tumor is in the patient's bones. In other aspects, the cancer has metastasized. In some aspects, the cancer may originate in the bones and metastasize to one or more of the following sites: breast, lung, brain, liver or other bones.

[0173] Disclosed herein are methods for treating a patient with cancer. The cancer may be an osteosarcoma. In some aspects, the cancer may be a breast cancer, lung cancer, brain cancer, or liver cancer that has metastasized from a bone cancer. In some aspects, the subject has been diagnosed with cancer prior to the administering step. In some aspects, the cancer may be an osteosarcoma.

[0174] The compositions described herein can be formulated to include a therapeutically effective amount of an antibody disclosed herein. In some aspects, the antibodies disclosed herein can be included in a pharmaceutical formulation. In some aspects, the pharmaceutical formulation can be a unit dose formulation.

[0175] The therapeutically effective amount or dosage of any antibody used in the methods disclosed herein applied to a mammal (e.g., a human) can be determined by a person of ordinary skill in the art taking into account individual differences in age, weight, sex, severity of the subject's symptoms and the specific composition or route of administration selected, other drugs administered, and the judgment of the attending physician. Changes in the required dose can be expected. Changes in dose levels can be adjusted by optimizing using standard empirical routes. The specific dose of the pharmaceutical composition administered to the patient will depend on a variety of considerations (e.g., the severity of cancer symptoms), the age and physical characteristics of the subject, and other considerations known to a person of ordinary skill in the art. The dosage can be determined using clinical methods known to a person of ordinary skill in the art. The therapeutically effective dose of an anti-hemichannel antibody can cause a decrease in the severity of one or more disease symptoms, an increase in the frequency and duration of disease-free symptom periods, or the prevention of damage or disability caused by disease affliction. A therapeutically effective amount of a therapeutic compound or antibody can reduce tumor metastasis, or otherwise improve the symptoms of a subject.

[0176] The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the host life span (e.g., many years). For example, the composition can be administered once a week (e.g., for 4 weeks to many months or years); once a month (e.g., for three months to twelve months or for many years); or once a year (for 5 years, 10 years or more). It should also be noted that the frequency of treatment can be variable. For example, the composition of the present invention can be administered once a day, weekly, monthly, or annually (or twice, three times, etc.).

[0177] The total effective amount of the antibody or composition as disclosed herein can be administered to the subject as a single dose, or as a bolus or by infusion over a relatively short period of time, or can be administered using a graded treatment regimen in which multiple doses are 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.

[0178] The antibodies or compositions described herein can be administered to a subject in need of treatment in combination with other treatment modalities. The compounds of the invention can be administered before, simultaneously with, or after treatment with other agents or regimens. For example, the antibodies disclosed herein can be administered alone or in combination with standard therapies for treating cancer. In some aspects, any antibody or composition described herein can be administered or used together with chemotherapy.

[0179] Pharmaceutical composition

[0180] Disclosed herein are compositions, such as pharmaceutical compositions, comprising one or a combination of monoclonal antibodies or antigen-binding portions thereof formulated with a pharmaceutically acceptable carrier. These compositions may include one or a combination of (e.g., two or more different) antibodies or immunoconjugates described herein. For example, the pharmaceutical compositions of the invention may include a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0181] The pharmaceutical compositions of the invention may also be administered as combination therapy, ie, in combination with other agents. For example, the combination therapy may include an anti-hemichannel antibody in combination with at least one other anti-cancer agent.

[0182] As used herein, the phrase "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier can be suitable for intravenous, intramuscular, subcutaneous or parenteral administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody or immunoconjugate) can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0183] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size (in the case of dispersions), and by using surfactants.

[0184] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Unless any conventional media or medicaments are incompatible with the active compound, they are considered for use in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the composition.

[0185] The therapeutic composition must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the required particle size (in the case of a dispersion) and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as a sugar, a polyol (such as mannitol, sorbitol) or sodium chloride. The extended absorption of the injectable composition can be caused by including an agent (for example, monostearate and gelatin) that delays absorption in the composition.

[0186] Sterile injectable solutions can be prepared by incorporating the active compound in a desired amount with one or a combination of ingredients listed herein in a suitable solvent, followed by sterilization microfiltration as required. Typically, dispersions are prepared by incorporating the active compound into a dispersion containing a basic dispersion medium and other ingredients required from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze drying (lyophilization) of a powder that produces the active ingredient plus any additional required ingredients from its previously sterile filtered solution.

[0187] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Typically, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, most preferably about 1% to about 30% of the active ingredient and a pharmaceutically acceptable carrier.

[0188] The dosage regimen is adjusted to provide the desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased, as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as single doses for a subject to be treated; each unit contains a predetermined amount of active compound in combination with a desired pharmaceutical carrier calculated to produce the desired therapeutic effect. The specifications of the dosage unit forms of the present invention are determined by and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the formulation technology of such active compounds for treating individual sensitivities.

[0189] For the administration of antibodies, the dosage range is between 0.0001 to 100 mg / kg, more generally 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 host body weight. In some aspects, 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 aspects, 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 aspects, the dosage may be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg / kg body weight. In some aspects, the dosage may be 5 mg / kg body weight. In some aspects, the dosage may be 15 mg / kg body weight. In some aspects, the dosage may be 20 mg / kg body weight. In some aspects, the dosage may be 25 mg / kg body weight. Exemplary treatment regimens require 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 three to six months. The preferred administration regimen of the anti-hemichannel antibody of the present invention includes administration of 1 mg / kg body weight or 3 mg / kg body weight by intravenous administration, wherein the antibody is administered using one of the following administration regimens: (i) every four weeks for six doses, followed by every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight every three weeks.

[0190] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the indicated range. Antibodies are typically administered in a variety of situations. The intervals between single doses may be, for example, weekly, monthly, every three months or annually. Intervals may also be irregular, as indicated by blood levels of antibodies to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, about 25-300 pg / ml in some methods.

[0191] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed; the route of administration; the time of administration; the rate of excretion of the particular compound employed; the duration of treatment; other drugs, compounds and / or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health and previous medical history of the patient being treated; and similar factors well known in the medical field.

[0192] The compositions of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, routes of administration and / or modes will vary according to the desired results. Preferred routes of administration for antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular injection and infusion.

[0193] Combination therapy. The compositions and methods described herein may involve antibodies or antibody fragments thereof directed against Cx43 to stimulate the opening of Cx43 hemichannels, for example to protect bone tissue from cancer cell growth and colonization, and a second or additional therapy. Such therapy may be used to treat 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.

[0194] The method and composition comprising combined therapy enhances the treatment or protective effect of another anti-cancer or anti-hyperproliferative therapy, and / or increases its therapeutic effect. Treatment and prevention methods and compositions can be provided in a combined amount that effectively achieves the desired effect (e.g., killing cancer cells and / or inhibiting cell hyperproliferation). This process may involve contacting cells with antibodies or antibody fragments and a second therapy. Tissues, tumors or cells can be contacted with one or more compositions or pharmacological preparations comprising one or more agents (i.e., antibodies or antibody fragments or anticancer agents), or by contacting tissues, tumors, and / or cells with two or more different compositions or preparations, wherein a composition provides 1) antibodies or antibody fragments, 2) anticancer agents, or 3) both antibodies or antibody fragments and anticancer agents. In addition, it is contemplated that this combined therapy can be used in combination with chemotherapy, radiotherapy, surgical therapy or immunotherapy.

[0195] When applied to cells, the terms "contacting" and "exposing" are used herein to describe the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or in direct juxtaposition with a target cell. For example, to achieve cell killing, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0196] Antibodies and their biological fragments can be used before, during, after or in various combinations of anticancer treatment. The interval between administration can range from simultaneously to a few minutes to a few days to a few weeks. In the aspect that the antibody or antibody fragment is provided to the patient separately from the anticancer agent, it is usually ensured that there is a considerable period of time between each delivery time without expiration, so that the two compounds will still be able to produce a favorable joint effect to the patient. In this case, it is considered that antibody therapy and anticancer therapy can be provided to the patient within about 12 to 24 or 72 hours, more specifically, within about 6-12 hours. In some cases, it may be necessary to significantly extend the treatment period, wherein a few days (2, 3, 4, 5, 6 or 7 days) to a few weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) are spaced between each administration.

[0197] In some aspects, a course for the treatment of can last 1-90 days or longer (this scope includes the middle number of days). It is considered that one medicament can be given on any day of the 1st to the 90th day (this scope includes the middle number of days) or any combination thereof, and another medicament can be given on any day of the 1st to the 90th day (this scope includes the middle number of days) or any combination thereof. In one day (24 hour period), one or more medicament administrations can be given to the patient. In addition, after a course for the treatment of, it is considered that there may be a period in which anticancer therapy is not administered. This period may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or longer (this scope includes the middle number of days), depending on the patient's condition, such as its prognosis, strength, health, etc. It is expected that the treatment cycle will be repeated as needed.

[0198] Various combinations can be used. For the example below, the antibody therapy is "A" and the anti-cancer therapy is "B":

[0199] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A.

[0200] Administration of any compound or therapy disclosed herein to a patient will follow the general regimen for administration of these compounds, taking into account the toxicity, if any, of the agent. Thus, in some aspects, there may be a step of monitoring toxicity attributable to the combination therapy.

[0201] Chemotherapy. A variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition that can be administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether and at which stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0202] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bulatacin) and acetogenins. latacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); 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, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard mustard; nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma ll and calicheamicin omega ll); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid (such as mycophenolic acid), acid), nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and estolactone; antiadrenal substances such as mitotane and trilostane; folic acid supplements such as acetic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine compounds, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid); 2-ethylhydrazide; methylbenzylhydrazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine;trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, for example, paclitaxel and docetaxel gemcitabine; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinols such as retinoic acid (retinoic acid) acid); capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum, and any pharmaceutically acceptable salt, acid or derivative thereof. ;

[0203] Radiation therapy. Other factors that cause DNA damage and have been widely used include what are commonly referred to as gamma rays, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV irradiation are also considered. It is likely that all of these factors will affect extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dose range of X-rays ranges from a dose of 50 to 200 roentgens per day for a long period of time (3 to 4 times per week) to a single dose of 2000 to 6000 roentgens. The dose range of radioisotopes varies greatly, depending on the half-life of the isotope, the intensity and type of radiation, and the uptake of neoplastic cells.

[0204] Immunotherapy. Those skilled in the art will appreciate that additional immunotherapies may be used 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 to target and destroy cancer cells. Rituximab This is just one example.Immune effectors can be antibodies that are specific, for example, to certain markers on the surface of tumor cells.Single antibody can be used as an effector for treatment, or it can recruit other cells to actually affect cell killing.Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and used as targeting agents.Or, effectors can be lymphocytes that carry surface molecules that interact directly or indirectly with tumor cell targets.Various effector cells include cytotoxic T cells and NK cells.

[0205] In one aspect of immunotherapy, tumor cells must carry some markers suitable for targeting, i.e., not present on most other cells. There are many tumor markers and any of these markers may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. Another aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN; chemokines such as MIP-1, MCP-1, IL-8; and growth factors such as FLT3 ligands.

[0206] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., 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, e.g., interferon alpha (β, and γ), IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., T cells (Bukowski et al., 1998); NF, 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, e.g., 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 with the antibody therapies described herein.

[0207] Surgery. Approximately 60% of cancer patients 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 therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

[0208] After removing part or all of the cancer cells, tissues or tumors, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of additional anticancer therapy to the area. 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 treatments may also have different dosages.

[0209] Other agents. It is contemplated that other agents may be used in combination with any method or composition disclosed herein to improve the efficacy of treatment. These additional agents include, but are not limited to, agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP connections will increase the anti-overproliferation effect on neighboring overproliferation cell groups. Cell growth inhibitors or differentiation agents may be used in combination with the compositions and methods disclosed herein to improve the anti-overproliferation efficacy of treatment. It is contemplated that cell adhesion inhibitors are used to improve the efficacy 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 also contemplated that other agents (such as antibody c225) that increase the sensitivity of overproliferation cells to apoptosis may be used in combination with the compositions and methods disclosed herein to improve the efficacy of treatment.

[0210] Kits and Diagnostics

[0211] Disclosed herein is a kit comprising one or more therapeutic agents and / or other therapeutic agents and a delivery agent. In some aspects, the kit can be used to prepare and / or administer the therapy disclosed herein. The kit can include one or more sealed vials, and the vial contains any pharmaceutical composition disclosed herein. The kit can include, for example, at least one Cx43 antibody or its fragment and a reagent for preparing, preparing and / or administering one or more components of the compositions disclosed herein or performing one or more steps of the method of the present invention. In some aspects, the kit can also include a suitable container, which can be a container that will not react with the components of the kit, such as Eppendorf tubes, assay plates, syringes, bottles or tubes. The container can be made of sterilizable materials, such as plastic or glass.

[0212] The kit may also include an instruction sheet outlining the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or known to those of ordinary skill in the art. The instruction information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, cause a real or virtual procedure to be displayed for delivering a pharmaceutically effective amount of a therapeutic agent.

[0213] Example

[0214] It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present invention and therefore can be considered to constitute preferred modes for its practice. However, in light of this disclosure, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining similar or similar results.

[0215] Example 1 - Anti-Cx43 Monoclonal Antibody

[0216] Anti-Cx43 monoclonal antibodies were generated and clones that produced Cx43 binding monoclonal antibodies were identified. The DNA and amino acid CDR sequences of the antibody sequences are shown in the table below 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.

[0217] Table 1: Heavy and light chain pairings of two functional antibodies.

[0218] Antibody Name Heavy chain Light chain M1 (HmAb1) M1H M1K1 M2 (HmAb2) M1H M1M7K

[0219] Table 2: Antibody chain sequences from hybridomas.

[0220]

[0221]

[0222] The cloned variable domains are shown below.

[0223] Table 3. DNA sequences. Variable heavy chain (bold) and variable light chain (underlined).

[0224]

[0225]

[0226]

[0227] Table 4. Amino acid sequences. Variable heavy chain (bold) and variable light chain (underlined).

[0228]

[0229]

[0230] Example 2 - Diagnostic and cancer treatment uses

[0231] Osteolytic tumor growth was found to be increased in osteocyte-specific Cx43 knockout mice. Py8119-Luc cells were injected into the right tibia of control and cKO female mice. The left tibia was injected with PBS as a control. Tumor growth was recorded weekly for 4 weeks and quantified by bioluminescence imaging ( Figures 1A-1C ).

[0232] MLO-Y4 osteocytes and primary mouse osteocytes were incubated with E2 (polyclonal), HMAb1, and HMAb2 antibodies or carbenoxolone (CBX), a connexin channel blocker. Ethidium bromide (EtBr) dye uptake assays were performed ( Figure 2A-2B ). It was found that the Cx43 HMAb2 antibody activated the hemichannel.

[0233] In addition, Cx43 (M1) antibody is delivered to osteocytes in vivo, and it is found that blocking the Evans blue uptake induced by tibia load. Evans blue dye is injected into the tail vein of WT and osteocyte-specific Cx43 KO. Mouse IgG or Cx43 (M1) mAb (25 mg / kg) is injected intraperitoneally 2 hours before dye injection. 30min after dye injection, the left tibia is mechanically loaded once for 10 minutes. Mice are killed and perfused with PBS. Tibia is separated and fixed tibia tissue sections are prepared.

[0234] Inhibition of osteolytic tumor growth by HMAb2 was also observed. Py8119-Luc cells were injected into the right tibia of female mice ( Figure 4A ). The left tibia was injected with PBS as a control. HMAb2 was injected intraperitoneally at 25 mg / kg once or twice a week for four weeks. Control mice were injected with saline twice a week. Tumor growth was recorded weekly for 4 weeks and quantified by bioluminescence imaging ( Figure 4B ).

[0235] Example 3 - Inhibition of osteoblast growth in osteosarcoma cells

[0236] To determine the efficacy of the M2 antibody in inhibiting osteosarcoma in bone, two in vivo mouse models were used. The first model was the mouse osteosarcoma cell line DLM8, which is isogenic to the C3H mouse strain (Sottnik, JL et al., Clin Exp Metastasis, 2010. 27 (3): pp. 151-60). The second model was the OS17 human osteosarcoma cell line in immunocompromised (nude) mice (Kolb, EA et al. Pediatr Blood Cancer, 2010. 55 (1): pp. 67-75). Both osteosarcoma cells were implanted into the bone by intratibial injection.

[0237] The experiment is described in Fig. 9 and Fig. 10A -B's legend.

[0238] The results showed that the M2 antibody significantly inhibited the growth of murine and human osteosarcoma tumors in WT and nude mice, respectively. Such M2 antibodies (e.g., mouse-mouse chimeric M2 antibodies and optimized humanized M2 antibodies) inhibited osteosarcoma growth in a dose-dependent manner.

[0239] In view of the present disclosure, all methods disclosed and claimed herein can be carried out and performed without undue experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that the methods described herein and the steps or order of steps of the methods may be varied without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain chemical and physiologically related agents may be substituted for the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications apparent 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.

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Claims

1. 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 shown in SEQ ID NO:

58.

2. 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 light chain comprising a sequence having at least 90% identity to the sequence shown in SEQ ID NO:

63.

3. 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 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.

4. The method of any one of claims 1 or 3, wherein the antibody or fragment thereof comprises a heavy chain immunoglobulin variable region comprising: a) a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19 or a variant thereof; b) a complementarity determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 20 or a variant thereof; and / or c) a complementarity determining region (CDR3) comprising the sequence of SEQ ID NO: 21 or a variant thereof.

5. The method of any one of claims 1 or 2, wherein the antibody or fragment thereof comprises a light chain immunoglobulin variable region comprising: a) a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49 or a variant thereof; b) a complementarity determining region 2 (CDR2) comprising the sequence of SEQ ID NO: 50 or a variant thereof; and / or c) a complementarity determining region 3 (CDR3) comprising the sequence of SEQ ID NO: 51 or a variant thereof.

6. The method of claim 4 or 5, wherein any one of the heavy chain CDR1, CDR2 or CDR3 or the light chain CDR1, CDR2 or CDR3 comprises at least one amino acid substitution compared to a parent CDR.

7. The method of claim 4 or 5, wherein any of the heavy chain CDR1, CDR2 or CDR3 variant or the light chain CDR1, CDR2 or CDR variant comprises at least one amino acid substitution compared to the parent CDR.

8. The method of claim 7 or 8, wherein the at least one amino acid substitution is a cysteine ​​residue to another amino acid or a glycine to another amino acid.

9. The method of any one of claims 1 or 3, wherein the antibody or fragment thereof comprises a heavy chain immunoglobulin variable region comprising: (a) a complementarity determining region 1 (CDR1) comprising a sequence having at least 60% identity to the sequence shown in SEQ ID NO: 19; (b) a complementarity determining region 2 (CDR2) comprising a sequence that is at least 60% identical to the sequence shown in SEQ ID NO: 20; and / or (c) a complementarity determining region 3 (CDR3) comprising a sequence having at least 60% identity to the sequence shown in SEQ ID NO:

21.

10. The method of any one of claims 1 or 2, wherein the antibody or fragment thereof comprises a light chain immunoglobulin variable region comprising: (a) a complementarity determining region 1 (CDR1) comprising a sequence having at least 60% identity to the sequence shown in SEQ ID NO: 49; (b) a complementarity determining region 2 (CDR2) comprising a sequence that is at least 60% identical to the sequence shown in SEQ ID NO: 50; and / or (c) a complementarity determining region 3 (CDR3) comprising a sequence having at least 60% identity to the sequence shown in SEQ ID NO:51.

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