Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• Connexin hemichannels play important roles in the cell and tissue function, and abnormal function of connexin hemichannels may be involved various pathological conditions, such as those described herein. Thus, there remains a need for additional therapies for treating pathological conditions associated with hemichannels activity, as well as methods for identifying such therapies.
• Osteoporosis is a major prevailing skeletal disease that causes reduced bone strength and increases the risk of fragility fractures (Hemlund, E. et al. Arch Osteoporos. 8, 136 (2013)). Disused and aging are two major causes of osteoporosis and bone loss caused by reduced physical loading on the bones and reduced sensitivity of bone cells to mechanical stimulation, respectively. The examples of disuse include prolonged bed rest, physical inactivity, or space flight, leading to a decrease in bone mass, osteopenia and osteoporosis (Lang, T. et al. J Bone Miner Res. 19, 1006-1012 (2004); and Gabel, L prefer et al. J Bone Miner Res. 32, 1525-1536 (2017)).
• the antibodies for use according to the embodiments can be any of those described in international (PCT) patent publication no. WO 2015-027120 or WO 2017-147561, which is incorporated herein by reference for their teaching of antibodies, vectors and cells for making or expressing antibodies.
• SEQ ID NOs: 1-12 described in WO 2017-147561 are incorporated herein by reference.
• Disclosed herein are pharmaceutical compositions comprising any of antibodies or fragments thereof as described herein with a pharmaceutically acceptable carrier.
• antibodies or pharmaceutical compositions for use as a medicament or for use in therapy for treating or preventing osteoporosis, preventing or reducing bone loss, inhibiting osteocyte apoptosis, enhancing prostaglandin E2 release in osteocytes, suppressing sclerostin expression in osteocytes, increasing bone formation, treating or preventing osteopenia, and promoting or enhancing fracture healing.
• FIGS. 1A-F show that anti-Cx43 (M2) antibody enhanced HC opening in osteocytes under both tibia loading and unloading in vivo.
• M2 anti-Cx43
• FIG. 1 A shows representative fluorescence images of EB dye uptake in mid-diaphyseal cortical bone, for both control and HLU unloading tibias in the absence or presence of an anti-Cx43 (M2) antibody.
• FIG. IB shows the quantitation of EB fluorescence intensity in osteocytes.
• FIG. ID shows the representative fluorescence images of EB dye uptake in diaphyseal 37% cortical bone, for both loaded and contralateral tibias in the absence or presence of anti-Cx43 (M2) antibody.
• M2 anti-Cx43
• FIGS. 2A-D show the experimental set-up for hindlimb suspension to mimic mechanical unloading.
• FIG. 2A shows a schematic workflow of the experimental plan.
• FIG. 2B shows that a copper wise was fixed to the tail of anesthetized mouse for the hindlimb suspension (HLS) apparatus.
• FIG. 2C shows weekly monitoring of body weights in control and unloading mice of vehicle- and anti-Cx43 (M2) antibody-treated mice.
• FIGS. 2D, E show the change of soleus muscle mass (FIG. 2D) and gastrocnemius muscle (FIG. 2E) of control and unloading mice treated with vehicle and anti-Cx43 (M2) antibody.
• n 7 ⁇ 8 per group. *, PO.05; ****, po.OOOl ;
• Statistical analysis was performed using two-way ANOVA with Tukey test among different groups.
• FIGS. 3A-N show enhanced Cx43 HC activity by anti-Cx43 (M2) antibody treatment prevents trabecular and cortical bone loss caused by mechanical unloading.
• M2 anti-Cx43
• FIG. 3D shows representative 3D models of the metaphyseal trabecular bone of vehicle- and anti-Cx43 (M2) antibody-treated mice.
• FIGS. 3E- J show the results of micro-computed tomography (pCT) was used to assess structural parameters of mid-diaphyseal cortical bone: T.Ar (FIG. 3E), B.Ar (FIG. 3F), B.Ar/T.Ar (FIG. 3G), Ct. Th (FIG. 3H), M.Ar (FIG. 31), and TMD (FIG. 3 J).
• 3L-N show the results for the three-point bending assay performed for mid- diaphyseal femur bone of vehicle control and anti-Cx43 (M2) antibody-treated mice: elastic modulus (FIG. 3L), ultimate force (FIG. 3M), and ultimate stress (FIG. 3N).
• n 8 per group.
• Data are presented as mean ⁇ SD. *, P ⁇ 0.05; **, PO.Ol; ***, PO.OOI.
• Statistical analysis was performed using the two-way ANOVA with Tukey test for differences among groups.
• FIG. 4A-H shows that enhanced Cx43 HC activity by anti-Cx43 (M2) antibody treatment inhibited osteocyte apoptosis and osteoclastogenesis in 16-week-old male tibias induced by 28-day mechanical unloading.
• FIG. 4B shows the quantification of empty lacunae on mid- diaphyseal cortical bone.
• FIG. 4C shows representative images of TUNEL staining of apoptotic osteocytes in both control and unloading tibias of vehicle- and anti-Cx43 (M2) treated-treated mice.
• White arrowheads indicate the TUNEL-positive osteocytes.
• n 4 per group.
• Scale bar 50 pm.
• FIG. 4D shows representative RANKL immunohistostaining
• FIG. 4D shows representative RANKL immunohistostaining
• FIG. 4E shows the quantification of RANKL-positive osteocytes in mid-diaphyseal cortical bone for both control and unloading tibias of vehicle- and anti-Cx
• FIGS. 5A-F show anti-Cx43 (M2) antibody treatment sustains PGE2 levels and prevents increased SOST expression by mechanical unloading in osteocytes. After a four-week HLS on 16-week-old male mice, PGE2 levels and SOST were assessed.
• FIG. 5 A and FIG. 5B show ELISA analysis of PGE2 levels in bone marrow-flushed femur diaphysis (FIG. 5A) and hindlimb bone marrow (FIG. 5B) for both control and unloading tibias of vehicle- and anti- Cx43 (M2) antibody -treated mice.
• FIG. 5C shows the representative COX-2 immunohistostaining and FIG.
• FIG. 5E shows representative SOST immunohistostaining and
• the red and black arrowheads indicate the positive and negative osteocytes, respectively.
• Data are expressed as mean ⁇ SD. *, P ⁇ 0.05; **, PO.OL Statistical analysis was performed using the tw o-w ay ANOVA with Tukey test for difference among groups.
• FIGS. 6A-D show the effects of enhanced activity of Cx43 HCs by anti-Cx43 (M2) antibody treatment on osteogenesis in control and unloading mice.
• Bone formation rate/bone surface (BFR/BS) (FIG. 6 A), mineral apposition rate (MAR) (FIG. 6B), and surface/bone surface MS/BS (FIG. 6C) were assessed along cortical surfaces of control and unloading mice treated with vehicle an anti-Cx43 (M2) antibodies.
• FIGS. 7A-N show that anti-Cx43 (M2) antibody increased the anabolic effects of mechanical loading in aged mice. Bone structure and mechanical properties were analyzed in 22 -month-old male mice loaded 5 days/week for 2 weeks.
• FIG. 7A bone mineral density
• Tb.Th trabecular thickness
• Tb.N trabecular number
• FIGS. 7E-J shows the results of pCT that was used to assess structural parameters of cortical bone located 37% distal from the proximal end: total cross-sectional area (T.Ar) (FIG. 7E), bone area (B. Ar) (FIG. 7F), bone area fraction (B.Ar/T.Ar) (FIG. 7G), cortical thickness (Ct.Th) (FIG. 7H), bone marrow ⁇ area (M.Ar) (FIG. 71), and tissue mineral density (TMD) (FIG. 7J).
• T.Ar total cross-sectional area
• B. Ar bone area
• FIG. 7G. 7G bone area fraction
• Ct.Th cortical thickness
• FIG. 7H bone marrow ⁇ area
• M.Ar bone marrow ⁇ area
• TMD tissue mineral density
• FIGS. 8A-L show enhanced Cx43 hemi channel activity improved load-induced endosteal osteogenesis in aged mice.
• bone dynamic histomorphometric analyses were performed on the tibias within cortical bone located 37% distal from the proximal end of 22-month-old vehicle- and anti-Cx43 (M2) antibody-treated mice.
• Mineral apposition rate (MAR) (FIGS. 8A, 8D), mineralizing surface/bone surface (MS/BS) (FIGS. 8B, 8E), and bone formation rate (BFR/BS) (FIGS. 8C, 8F) were assessed along endosteal (FIGS. 8D-F) and periosteal (FIGS.
• FIG. 8G shows representative images of calcein (green) and alizarin (red) double labeling at the 37% diaphysis for the groups. Scale bar: 200 pm. Quantification of TRAP- positive osteoclast surface per bone perimeter (Oc.S/BS) and osteoclast number per bone perimeter (N.Oc/BS) on endosteal (FIGS. 8H, 81) and periosteal (FIGS. 8J. 8K) surfaces of 37% di
• 8L shows representative images of TRAP-positive osteoclasts (black arrow) on cortical bone located 37% distal from the proximal end, for both loaded and contralateral tibias of vehicle- and anti-Cx43 (M2) antibody-treated mice. Scale bar: 40 pm. Data are expressed as mean ⁇ SD. *, P ⁇ 0.05; **, PO.Ol. Statistical analysis was performed using the paired t test for loaded and contralateral tibias, two-way ANOVA with Tukey test for difference among groups.
• FIGS. 9A-H shows enhanced activity of Cx43 hemi channel improved load-induced PGE2 secretion and decreased SOST expression in osteocytes.
• FIG. 9C shows representative COX-2 immunohistostaining and
• FIG. 9D shows quantification of COX-2- positive osteocytes in diaphyseal 37% cortical bone. Scale bar, 40 pm.
• Statistical analysis was performed using the paired t-test for loaded and contralateral tibias, two-way ANOVA with Tukey test for difference among groups.
• FIGS. 10A-C show that the cartilage matrix is not altered after cyclic compressive loading of the knee joint.
• FIG. 10A show Safranin O-fast green staining of the medial articular cartilage in both vehicle control and anti-Cx43 (M2) antibody-treated mice. Scale bar, 50 pm.
• FIGS. 11 A-B show a model demonstrating the role of enhanced Cx43 hemichannels in regulating the response to mechanical loading and unloading (FIG. 11 A) and a model illustrating the impact of enhanced HCs via anti- Cx43 (M2) antibody treatment on osteocyte responses to mechanical loading in disused and aged bones (FIG. 1 IB).
• FIG. 11A shows enhanced Cx43 hemichannel activity will release PGE2 in an autocrine manner (Cherian et al. J Biol Chem. 278, 43146-43156 (2003) to reduce SOST expression in osteocytes during both mechanical loading and unloading (Galea, et al. FEBS Lett. 585, 2450-2454 (201 1).
• FIG. 1 IB shows that with aging, there is a decline in osteocytic Cx43 levels and a reduced responsiveness of Cx43 HCs to mechanical loading.
• FIGS. 12A-D show the experimental setup for tibia axial compressive loading.
• FIGS. 12A-B show schematic illustrations of the experimental plan and tibia loading setup.
• FIG. 12C shows a diagram of the left tibia positioned at the loading device and the direction of loading.
• FIG. 12D shows the weekly body weights of vehicle- and anti-Cx43 (M2) antibody -treated groups.
• M2 vehicle- and anti-Cx43
• FIGS. 13A-C show that anti-Cx43 (M2) antibody inhibits osteoclasts in tibial metaphyseal trabecular bone during mechanical loading.
• FIG. 13A shows representative images of TRAP -positive osteoclasts (red) in tibial metaphyseal trabecular bone. Scale bar. 80 pm.
• FIG. 13B shows the quantification of TRAP -positive osteoclast number per bone perimeter (N.Oc/BS) and
• FIG. 13C shows the osteoclast surface per bone perimeter (Oc.S/BS) in tibial metaphyseal trabecular bone.
• n 5/group.
• Data are expressed as mean ⁇ SD. *, P ⁇ 0.05.
• Data are expressed as mean ⁇ SD.
• Statistical analysis was performed using the paired t test for loaded and contralateral tibias, two-way ANOVA with Tukey test for difference among groups.
• each of the combinations A-E, A-F, B-D, B-E, B-F. C- D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
• any subset or combination of these is also specifically contemplated and disclosed.
• the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A. B, and C: D, E. and F; and the example combination A-D.
• This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
• steps in methods of making and using the disclosed compositions are if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
• Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range- from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
• the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.
• each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
• “Inhibit,” “inhibiting” and “inhibition” mean to diminish or decrease an activity', level, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% inhibition or reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the inhibition or reduction can be a 10, 20, 30, 40. 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
• the inhibition or reduction is 10-20, 20- 30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% as compared to native or control levels. In some aspects, the inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% as compared to native or control levels.
• Modulate means a change in activity or function or number.
• the change may be an increase or a decrease, an enhancement or an inhibition of the activity, function or number.
• “Promote,” “promotion,” and “promoting” refer to an increase in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the initiation of the activity, response, condition, or disease. This may also include, for example, a 10% increase in the activity, response, condition, or disease as compared to the native or control level. Thus, in some aspects, the increase or promotion can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or more, or any amount of promotion in between compared to native or control levels. 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% as compared to native or control levels.
• the increase or promotion is 0-25, 25-50, 50-75, or 75-100%, or more, such as 200, 300, 500, or 1000% more as compared to native or control levels. In some aspects, the increase or promotion can be greater than 100 percent as compared to native or control levels, such as 100. 150, 200, 250, 300, 350, 400, 450, 500% or more as compared to the native or control levels.
• Treatment and “treating” refer to administration or application of a therapeutic agent (e.g., an anti-Cx43 antibody described herein) to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health- related condition.
• a treatment may include administration of a pharmaceutically effective amount of an antibody that enhances or stimulates the opening of the Cx43 hemichannel.
• the antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap channel coupling.
• treating refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting or slowing progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
• Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology' associated with the disease, disorder, and/or condition.
• the disease, disorder, and/or condition can be osteoporosis or bone loss.
• the term “subject” refers to the target of administration, e.g., a human.
• the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
• the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
• a subject is a mammal.
• a subject is a human.
• the term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
• the term “patient” refers to a subject afflicted with a disease or disorder.
• the term “patient” includes human and veterinary subjects.
• the “patient” has been diagnosed with a need for treatment, such as, for example, prior to the administering step.
• fragment can refer to a portion (e.g., at least 5, 10, 25, 50, 100, 125. 150, 200, 250. 300, 350, 400 or 500, etc. amino acids or nucleic acids) 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. 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.
• a fragment of a referenced peptide can be a continuous or contiguous portion of the referenced polypeptide (e.g., a fragment of a peptide that is ten amino acids long can be any 2-9 contiguous residues within that peptide).
• a “variant” can mean a difference in some way from the reference sequence other than just a simple deletion of an N- and/or C-terminal amino acid residue or residues. Where the variant includes a substitution of an amino acid residue, the substitution can be considered conservative or non-conservative. Conservative substitutions are those within the following groups: Ser, Thr, and Cys; Leu, He, and Vai; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gin, Asn, Glu, Asp, and His. Variants can include at least one substitution and/or at least one addition, there may also be at least one deletion. Variants can also include one or more non-naturally occurring residues.
• selenocysteine e.g., seleno- L- cysteine
• cysteine e.g., seleno- L- cysteine
• Many other “unnatural” amino acid substitutes are known in the art and are available from commercial sources.
• non-naturally occurring amino acids include D-amino acids, amino acid residues having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, and omega amino acids of the formula NH2(CH2) n COOH wherein n is 2-6 neutral, nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine.
• Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic.
• Proline may be substituted with hydroxyproline and retain the conformation conferring properties of proline.
• MIH refers to an antibody that was cloned from hybridoma clones.
• M l refers to hybridoma monoclonal 1
• H refers to the variable heavy chain.
• MIM7K refers to the variable light chain identified from hybridoma clones Ml and M7.
• isolated can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized).
• an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel.
• an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state.
• Moieties of the invention such as polypeptides, peptides, antigens, or immunogens, may be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
• conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
• conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
• conjugates or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical “conjugation.”
• the term “providing” is used according to its ordinary meaning “to supply or furnish for use.”
• the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid that encodes the protein.
• the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and/or epitopes.
• the phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologies.
• a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologies present in the sample.
• Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity 7 to the target.
• a variety 7 of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
• solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g. , Harlow and Lane, Antibodies: A Laboratory 7 Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
• the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
• amino acid and “amino acid identity” refers to one of the 20 naturally occurring amino acids or any non-natural analogues that may be in any of the antibodies, variants, or fragments disclosed.
• amino acid as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention.
• Amino acid also includes amino acid residues such as proline and hydroxyproline.
• the side chain may be in either the (R) or the (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 retard in vivo degradation.
• Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins make up one hemichannel, and two hemichannels make up one gap junction channel.
• Gap junctions are a cluster of channels that are located in the plasma membrane between adjoining cells and they mediate intercellular communication.
• Hemichannels are a separate entity from gap junction channels. Hemichannels permit the exchange of molecules between the intracellular compartments and the extracellular environment.
• Osteocytes express hemichannels known as connexin (Cx) 43 hemichannels. These osteocyte hemichannels are normally closed and can be opened when exposed to mechanostimulation, which leads to the release of various factors into the bone microenvironment.
• Cx connexin
• modulate can mean stimulate or enhance the opening of one or more connexin hemichannels.
• the connexin hemichannel can be a Cx 43 hemichannel.
• the methods can identify compounds or drugs that positively modulate (i.e., stimulate or enhance) the opening of connexin hemi channels.
• Other embodiments are directed to methods of treating osteoporosis by administering a compound that open connexin 43 hemichannels or stimulate or enhance the opening of connexin 43 hemichannels to a patient diagnosed with or having or at risk for osteoporosis or bone loss.
• the patient can be over the age of 50.
• the compounds that open Cx43 hemichannels or stimulate or enhance the opening of Cx43 hemichannels can be used to prevent, inhibit osteocyte apoptosis or suppress sclerostin expression in osteocytes.
• compounds that open Cx43 channels or stimulate or enhance the opening of Cx43 hemichannels can be used to treat osteoporosis.
• the antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap channel coupling.
• Cx43 hemichannel activating antibodies that can be used in methods to increase the sensitivity of aged bones to mechanical stimulation and promote bone formation in subjects.
• Osteoporosis, osteopenia and bone loss are major health issues. Exercise associated with mechanical stimulation is beneficial to young bone, but not old bone. Combining Cx43 hemichannel activating antibodies with exercise can promote new bone formation and prevent bone loss and be useful for treating osteoporosis.
• Osteocytes comprise the majority of bone tissue cells and are thought to be a major mechanosensory cells in the adult skeleton (Burra, S. et al. Proc Natl Acad Sci U S A. 107, 13648-13653 (2010)). Osteocytes have been shown to primarily express the membrane protein connexin 43 (Cx43) (Civitelli, R. Arch Biochem Biophys. 473, 188-192 (2008)).
• Cx43 membrane protein connexin 43
• Cx43 formed hemichannels (HCs) in osteocytes that allow the passage of small anabolic molecules ( ⁇ 1.2 kDa) between the intracellular and the extracellular microenvironment (Loiselle. A. E., et al. Bone. 54, 205-212 (2012)).
• Cx43 HCs are highly responsive to mechanical stimulation in osteocytes leading to the release of small molecules such as prostaglandin E2 (PGE2) and ATP (Jiang, J. X. and Cherian, P. P. Cell Commun Adhes. 10, 259-264 (2003); and Cherian, P. P. et al. Mol Biol Cell. 16, 3100-3106 (2005)).
• PGE2 prostaglandin E2
• ATP acetet al. Mol Biol Cell. 16, 3100-3106 (2005).
• Cx43 HCs participate in the response to mechanical disuse. Changes in gravity during parabolic flight were found to decrease the expression of Cx43 in osteocytes (Di SM, et al. Adv. Space Res. 2011;48(6): 1161-1166).
• Cx43 deficiency provides protection against bone loss induced by unloading.
• Cx43 deficiency affects Cx43 HCs as well as Cx43 gap junctions and Cx43 protein retained in the cytoplasm of the osteocytes (Goodenough DA, Paul DL. Nat Rev Mol Cell Biol. 2003;4(4):285-294).
• FFSS fluid flow shear stress
• Cx43 HCs Given the important role of the Cx43 HCs in sensing and response to mechanical loading, it is thought that impaired Cx43 HCs in osteocytes attenuated the mechanosensitivity in aged bone and in disused bone. Previous in vitro studies showed that Cx43 HCs also participate in the response to mechanical disuse. To assess whether Cx43 HCs participate in the response to mechanical disuse a monoclonal anti-Cx43 antibody (e.g., M2 antibody) which specifically enhances Cx43 HC activity' was used. The results described herein show' that administration of Cx43 hemichannel activating antibodies disclosed herein (e.g.. the M2 antibody) is effective and enhanced Cx43 HC activity, resulting in the release of PGE2.
• M2 antibody monoclonal anti-Cx43 antibody
• an anti-Cx43 antibodies that stimulate or enhance the opening of Cx43 hemichannels can be referred to as “M2 antibodies’; An example of identify ing and isolating a monoclonal antibody is described below.
• CDR refers to a Complementarity' Determining Region of an antibody variable domain. Sy stematic identification of residues included in the CDRs have been developed by Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda).
• VL Variable light chain
• VH Variable heavy chain
• CDR1 residues at positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3).
• the CDRs disclosed herein may also include variants.
• the amino acid identity' between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
• a “variant CDR” is one with the specified identity' to the parent or reference CDR of the invention, 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.
• a “variant CDR” can be a sequence that contains 1, 2, 3 or 4 amino acid changes as compared to the parent or reference CDR of the invention, and shares or improves biological function, specificity and/or activity’ of the parent CDR.
• 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.
• the antibody can be a humanized antibody.
• the antibody can 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.
• the antibody can be an IgG class of antibody, wherein the IgG class antibody is an IgGl, IgG2, IgG3, or IgG4 class antibody.
• 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.
• the antibody comprises a first VH CDR corresponding to SEQ ID NO: 19 or a fragment thereof, a second VH CDR corresponding to SEQ ID NO: 20 or a fragment thereof, a third VH CDR corresponding to SEQ ID NO: 21 or a fragment thereof, a first VL CDR corresponding to SEQ ID NO: 49 or a fragment thereof, a second VL CDR corresponding to SEQ ID NO: 50 or a fragment thereof, and a third VL CDR corresponding to SEQ ID NO: 51 or a fragment thereof.
• the antibody or fragment thereof can be a humanized antibody.
• the antibody can 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.
• the antibody can be an IgG class of antibody, wherein the IgG class antibody is an IgGl, IgG2. IgG3, or IgG4 class antibody.
• the antibody comprises a VH amino acid sequence at least 90% identical to SEQ ID NO: 58 or a fragment thereof and/or a VL amino acid sequence at least 90% identical to SEQ ID NO: 63 or a fragment thereof.
• the antibody may comprise a VH amino acid sequence according to SEQ ID NO: 58 or a fragment thereof and/or a VL amino acid sequence according to SEQ ID NO: 63 or a fragment thereof.
• Disclosed herein are antibodies directed against hemichannel polypeptides, and nucleic acid molecules encoding said antibodies.
• the antibody can bind an epitope having an ammo acid sequence of FLSRPTEKTI (SEQ ID NO: 13), KRDPCPHQVD (SEQ ID NO: 14), or LSAVYTCKR (SEQ ID NO: 15). In some aspects, the antibody can bind an epitope having an amino acid sequence of FLSRPTEKTI (SEQ ID NO: 13).
• a 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.
• SEQ ID NO: 2 EVQLEQPGAELVKPGASVKLSCKASGYTFTSYYMYWVKQRPGQGLEWIGGINPSNG GTNFNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCTREGNPYYTMNYWGQ GTSVTVSSAKTTPPSVY.
• the antibodies disclosed herein can include full-length antibodies, antibody fragments, single chain antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies and antibody fusions, and fragments thereof.
• the term ‘'antigen” is a molecule capable of being bound by an antibody or T-cell receptor.
• binding moieties other than antibodies can be engineered to specifically bind to an antigen, e.g. , aptamers, avimers, and the like.
• antibody or “immunoglobulin” is used to include intact antibodies and binding fragments/segments thereof.
• the term “antibody” is intended to refer broadly to any immunologic binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG as well as polypeptides comprising antibody CDR domains that retain antigen binding activity.
• the antibody may be selected from the group consisting of a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment or a natural or synthetic ligand.
• fragments compete with the intact antibody from which they were derived for specific binding to an antigen.
• Fragments include separate heavy chains, light chains. Fab, Fab’ F(ab')2. Fabc, and Fv. Fragments/segments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.
• the term “antibody” also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins.
• antibody also includes bispecific antibodies.
• a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
• Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g, Songsivilai and Lachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol. 148: 1547-53, 1992.
• the term “antibody” can include five different classes of human immunoglobulins, namely IgG, IgA, IgM, IgD, and IgE.
• the disclosed antibodies can be an IgG class of antibody which can be classified into the 4 subclasses of IgGl, IgG2, IgG3, and IgG4.
• the disclosed antibodies can be an IgA class of antibody which, can be classified into the 2 subclasses of IgAl and IgA2.
• the basic structure of immunoglobulin is made up of 2 homologous L chains (light chains) and 2 homologous H chains (heavy chains). The immunoglobulin class and subclass are determined by H chains.
• the antibody or antibodies or variants or fragments thereof can be an IgG4.
• antibody stability of IgG4 can be improved.
• the antibody can be improved, for example, by substituting arginine (R) of IgG4 with glutamic acid (E), phenylalanine (F), isoleucine (I), asparagine (N), glutamine (Q), serine (S), valine (V), tryptophan (W), tyrosine (Y), lysine (K), threonine (T), methionine (M), or leucine (L).
• any of CDR sequences disclosed herein can include a single amino acid change as compared to the parent or reference CDR. In some aspects, any of the CDR sequences disclosed herein can include at least two amino acid changes as compared to the parent or reference CDR. In some aspects, the amino acid change can be a change from a cysteine residue to another amino acid. In some aspects, the amino acid change can be a change from a glycine residue to another amino acid. In some aspects, the at least one amino acid change or substitution can decrease deamidation.
• the amino acid identity between individual vanant CDRs can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%.
• a “variant CDR” can be one with the specified identity to the parent CDR of the invention, 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.
• the parent CDR sequence can be one or more of SEQ ID NOs: 19. 20. 21. 49, 50, and/or 51.
• the variant CDR sequence can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 19, 20, 21, 49, 50, and/or 51.
• the variant CDR sequence can also share at least 80%, 81%, 82%, 83%. 84%. 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. or 99% of the specificity and/or activity of the parent CDR.
• amino acid sequences of any of the antibodies disclosed herein are contemplated as being encompassed by the instant disclosure, providing that the variations in the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the parent sequence.
• conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
• serine and threonine are aliphatic-hydroxy family: asparagine and glutamine are an amide-containmg family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family.
• amino acid substitutions can be those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility' to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, (5) reduces or decreases deamidation; and (6) confer or modify other physiocochemical or functional properties of such analogs.
• single or multiple amino acid substitutions may be made in the non-CDR sequence of the heavy chain, the light chain or both.
• one or more amino acid substitutions can be made in one or more of the CDR sequences of the heavy chain, the light chain or both.
• the at least one amino acid change can be to substitute an NG motif (amino acid asparagine followed by a glycine).
• the glycine residue can be substituted or replaced with hydrophobic amino acid residue.
• the glycine residue can be substituted or replaced with alanine, aspartic acid, glutamic acid, or valine.
• the glycine residue can be substituted or replaced with arginine, lysine, or glutamine.
• cysteine and methionine can be susceptible to rapid oxidation, which can negatively influence the cleavage of protecting groups during synthesis and the subsequent peptide purification.
• cysteine residues in peptides used for antibody production can affect the avidity' of the antibody, because free cysteines are uncommon in vivo and therefore may not be recognized by the native peptide structure.
• the disclosed antibodies and fragments thereof comprise a sequence where a cysteine reside outside of the CDR (e.g., in the non-CDR sequence of the heavy chain, the light chain or both) is substituted.
• cysteine can be replaced with serine and methionine replaced with norleucine (Nle).
• Multiple cysteines on a peptide or in one of the disclosed antibodies or fragments thereof may be susceptible to forming disulfide linkages unless a reducing agent such as dithiothreitol (DTT) is added to the buffer or the cysteines can be replaced with serine residues.
• DTT dithiothreitol
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activity .
• Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, Ml 3 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of antigen binding protein activities as described herein.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about one (1) to about tw enty (20) amino acid residues, although considerably larger insertions may be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases deletions may be much larger.
• substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative or variant.
• these changes are done on a few- amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein.
• larger changes may be tolerated in certain circumstances.
• a “fragment antigen-binding fragment (Fab)” is a region of an antibody that binds to antigen.
• An Fab comprises constant and variable regions from both heavy and light chains.
• Fab or “Fab region” as used herein is meant the polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains.
• Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein, or any other antibody embodiments as outlined herein.
• Fv or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.
• CDR complementarity determining region
• FR framework regions
• antibody portion refers to one or more fragments of an antibody that retain the ability' to specifically bind to an antigen (e g., hemi channel). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL/VK, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab ? fragment, which can be an Fab with part of the hinge region (see. Fundamental Immunology (Paul ed., 3rd ed.
• the term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5 -fold greater when compared to its affinity for a non-target molecule. Suitably there is no significant cross-reaction or crossbinding with undesired substances.
• the affinity' of the antibody will, for example, be at least about 5-fold, such as 10-fold, such as 25-fold, especially 50-fold, and particularly 100-fold or more, greater for a target molecule than its affinity for a non-target molecule.
• specific binding between an antibody or other binding agent and an antigen means a binding affinity of at least 10 6 M- 1 .
• Antibodies may, for example, bind with affinities of at least about 10 7 M- 1 , such as between about 10 8 M- 1 to about 10 9 M- 1 . about 10 9 M- 1 to about IO 10 M- 1 , or about lO- ⁇ M- 1 to about 10 11 M- 1 .
• Antibodies may, for example, bind with an EC50 of 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 pM or less.
• the antibodies can bind with an EC50 of about 60 pg/ml, 59 pg/ml, 58 pg/ml, 57 pg/ml, 56 pg/ml, 55 pg/ml. 54 pg/ml, 53 pg/ml, 52 pg/ml, 51 pg/ml, 50 pg/ml or less. In some aspects, the antibodies can bind with an EC50 of about 50 pg/ml.
• the antibodies can bind with an EC50 of about 40 pg/ml, 39 pg/ml, 38 pg/ml, 37 pg/ml, 36 pg/ml, 35 pg/ml, 34 pg/ml, 33 pg/ml, 32 pg/ml, 31 pg/ml, 30 pg/ml or less.
• the antibodies described herein can be specifically bind to their intended target. In some aspects, the antibodies described herein have no off site binding. For example, the antibodies described herein do not bind or are not distributed to the heart, liver or spinal cord.
• the antibodies described herein can be variants including, without limitation, a fragment (e.g.. an Fab fragment or an F(ab’)2 fragment of, e.g.. a tetrameric antibody), a fragment of an scFv or diabody, or a variant of a tetrameric antibody, an scFv, a diabody, or fragments thereof that differ by virtue of the addition and/or substitution of one or more amino acid residues.
• the antibody moiety can be further engineered as, for example, a di-diabody.
• antibody fragments can be generated by enzymatic treatment of a “full-length” antibody. Digestion with papain produces two identical Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. The Fab fragment also contains the constant domain of the light chain and the Chi domain of the heavy 7 chain. In contrast, digestion with pepsin yields the F(ab')2 fragment that has two antigenbinding sites and is still capable of cross-linking antigen.
• Fab' fragments differ from Fab fragments in that they include additional residues at the C -terminus of the Chi domain, including one or more cysteine residues from the antibody hinge region.
• the cysteine residues of the constant domains bear a free thiol group.
• F(ab')2 antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known in the art.
• the Fv region is a minimal fragment that contains a complete antigen-recognition and binding site consisting of one heavy 7 chain and one light chain variable domain.
• the three CDRs of each variable domain interact to define an antigen-biding site on the surface of the VH-VL dimer.
• the six CDRs confer antigen-binding specificity to the antibody.
• a “single-chain variable fragment (scFv)” means a protein comprising the variable regions of the heavy and light chains of an antibody.
• a “singlechain”’ 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.
• single-chain antibodies include a polypeptide linker between the VH and VL domains that allows the scFv to form a desired three-dimensional structure for antigen binding (see. e.g., Pluckthun, In The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore Eds., Springer-Verlag, New York, 113:269-315. 1994).
• a scFv can be a fusion protein comprising a variable heavy chain, a linker, and a variable light chain.
• the linker can be a short, flexible fragment that can be about 8 to 20 amino acids in length.
• the antibody can be a diabody.
• Diabodies are small antibody fragments that have two antigen-binding sites. Each fragment contains a VH domain concatenated to a VL domain. However, since the linker between the domains is too short to allow pairing between them on the same chain, the linked Vh-Vl domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites. Diabodies are described more fully, for example, in EP 404,097; WO 93/1 1161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.
• the anti-Cx43 antibody can be a monoclonal antibody, polyclonal antibody or a humanized antibody.
• polyclonal or monoclonal antibodies, antibodyfragments, and binding domains and CDRs may be created that are specific to Cx43 protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.
• antibody fragments suitable include without limitation: (i) the Fab fragment, consisting of VL, VH, CL, and CHI domains; (ii) the “Fd” fragment consisting of the VII and Cm domains; (iii) the “Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the “dAb” fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (“scFv”), wherein a VII domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bi- specific single chain Fv dimers (see U.S.
• Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains.
• Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. , 1996).
• Antibody-like binding peptidomimetics are also contemplated. Liu et al. (2003) describe “antibody like binding peptidomimetics” (ABiPs). which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
• Animals may be inoculated with an antigen, such as a Cx43 extracellular domain protein, in order to produce antibodies specific for Cx43 protein. Frequently an antigen is bound or conjugated to another molecule to enhance the immune response.
• a conjugate is any peptide, polypeptide, protein, or non-proteinaceous substance bound to an antigen that is used to elicit an immune response in an animal.
• Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) made from a variety of individual antibody producing B lymphocytes.
• a polyclonal antibody is a mixed population of antibody species, each of which may recognize a different epitope on the same antigen.
• monoclonal antibody refers to an antibody, or population of like antibodies, obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method, including but not limited to, monoclonal antibodies can be made by the hybridoma method first described by Kohler and Milstein (Nature, 256: 495-497, 1975), or by recombinant DNA methods.
• a monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because the antibody producing cells are derived from a single B-lymphocyte cell line.
• the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
• rodents such as mice and rats are used in generating monoclonal antibodies.
• rabbit, sheep, or frog cells are used in generating monoclonal antibodies.
• the use of rats is well known and may provide certain advantages.
• Mice e.g., BALB/c mice
• BALB/c mice are routinely used and generally give a high percentage of stable fusions.
• Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a Cx43 antigen with an immortal myeloma cell (usually mouse myeloma).
• This technology provides a method to propagate a single antibody-producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced.
• Plasma B cells may be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for Cx43 binding cells. After enrichment of antibody producing B cells, total RNA may be isolated and cDNA synthesized. DNA sequences of antibody variable regions from both heavy chains and light chains may be amplified, constructed into a phage display Fab expression vector, and transformed into E. coli. Cx43 specific binding Fab may be selected out through multiple rounds enrichment panning and sequenced.
• Selected Cx43 binding hits may be expressed as full length IgG in rabbit and rabbit/human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and purified using a protein G resin with a fast protein liquid chromatography (FPLC) separation unit.
• HEK293 human embryonic kidney
• FPLC fast protein liquid chromatography
• the antibody can be a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous nonhuman. human, or humanized sequence (e.g. , framework and/or constant domain sequences).
• chimeric antibody refers to a molecule comprising a heavy and/or light chain which is identical with 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 chain(s) is identical with or homologous to corresponding sequences in antibodies denved 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), infra; Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851).
• a hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.
• Antibodies may be produced from any animal source, including birds and mammals.
• the antibodies are ovine, murine (e g. , mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken.
• newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries.
• bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by reference. These techniques are further described in: Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996).
• antibodies to Cx43 will have the ability to neutralize or counteract the effects of Cx43 regardless of the animal species, monoclonal cell line, or other source of the antibody.
• Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the “Fc” portion of the antibody.
• whole antibodies may be enzymatically digested into “Fc” (complement binding) fragment, and into antibodyfragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immunological response, and thus, antibodies without Fc may be preferential for prophylactic or therapeutic treatments.
• antibodies may also be constructed so as to be chimeric or partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.
• humanized antibody refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies.
• a humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity.
• Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties.
• CDR complementary-determining region
• humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance.
• a humanized antibody can comprise all or substantially all of at least one, 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 also can comprise at least a portion of an immunoglobulin constant region (Fc). or that of a human immunoglobulin (see. e.g., Cabilly et al., U.S. Pat. No.
• Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
• Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; 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.
• substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
• Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
• Proteins may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
• compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
• concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, 1.0, 1.5. 2.0, 2.5, 3.0. 3.5, 4.0, 4.5. 5.0, 5.5, 6.0. 6.5, 7.0, 7.5. 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
• about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69. 70. 71. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91. 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that binds Cx43.
• An antibody or preferably an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
• a fusion protein with other proteins.
• all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.
• Described herein are antibodies and antibody-like molecules against Cx43, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload.
• to the antibody can be linked or covalently bound or complexed to at least one desired molecule or moiety.
• a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
• Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
• Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like.
• reporter molecule is defined as any moiety that may be detected using an assay.
• reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
• Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody.
• an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody.
• Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
• Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an
• the anti-Cx43 antibody described herein can comprise a heavy chain immunoglobulin variable region comprising complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19; CDR2 comprising the sequence of SEQ ID NO: 20 or SEQ ID NO: 66; and a CDR3 comprising the sequence of SEQ ID NO: 21.
• CDR1 complementarity determining region 1
• CDR2 comprising the sequence of SEQ ID NO: 20 or SEQ ID NO: 66
• CDR3 comprising the sequence of SEQ ID NO: 21.
• Table 3 shows examples of CDRs of the heavy chain.
• the anti-Cx43 antibody described herein can comprise a light chain immunoglobulin variable region comprising complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49; CDR2 comprising the sequence of SEQ ID NO: 50; and a CDR3 comprising the sequence of SEQ ID NO: 51.
• CDR1 complementarity determining region 1
• CDR2 comprising the sequence of SEQ ID NO: 50
• CDR3 comprising the sequence of SEQ ID NO: 51.
• Table 3 shows examples of CDRs in the light chain.
• the anti-Cx43 antibody described herein can comprise a heavy chain immunoglobulin variable region comprising complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 19; CDR2 comprising the sequence of SEQ ID NO: 20 or SEQ ID NO: 66; and a CDR3 comprising the sequence of SEQ ID NO: 21; and a light chain immunoglobulin variable region comprising complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 49; CDR2 comprising the sequence of SEQ ID NO: 50: and a CDR3 comprising the sequence of SEQ ID NO: 51.
• CDR1 complementarity determining region 1
• CDR2 comprising the sequence of SEQ ID NO: 20 or SEQ ID NO: 66
• CDR3 comprising the sequence of SEQ ID NO: 21
• CDR1 complementarity determining region 1
• the anti-Cx43 antibody described herein can comprise a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NOs: 19, 20, or 21 (see, Table 3). In some aspects, the anti-Cx43 antibody described herein 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 a sequence set forth in SEQ ID NOs: 19, 20 (or SEQ ID NO: 66) or 21.
• the anti-Cx43 antibody described herein can comprise a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NOs: 49, 50 or 51 (see, Table 3). In some aspects, the anti-Cx43 antibody described herein 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 a sequence set forth in SEQ ID NOs: 49, 50 or 51.
• nucleic acid sequences that encode M1H comprising the sequence of SEQ ID NO: 52.
• nucleic acid sequences that encode M1M7K comprising the sequence of SEQ ID NO: 57.
• M1H comprises a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 52 (see, Table 4).
• 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 a sequence set forth in SEQ ID NO: 52.
• M1M7K comprising a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 57 (see, Table 4).
• 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 a sequence set forth in SEQ ID NO: 57.
• nucleic acid sequences that encode the M1H region comprising a heavy chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 16; a CDR2 comprising a comprising the sequence of SEQ ID NO: 17; a CDR3 comprising a comprising the sequence of SEQ ID NO: 18.
• nucleic acid sequences that encode the M1M7K region comprising a light chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 46; a CDR2 comprising a comprising the sequence of SEQ ID NO: 47; a CDR3 comprising a comprising the sequence of SEQ ID NO: 48.
• nucleic acid sequences that encode anti-Cx43 hemichannel antibody comprising a heavy chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 16; a CDR2 comprising a comprising the sequence of SEQ ID NO: 17; a CDR3 comprising a comprising the sequence of SEQ ID NO: 18; and a light chain immunoglobulin variable region comprising a CDR1 comprising the sequence of SEQ ID NO: 46; a CDR2 comprising a comprising the sequence of SEQ ID NO: 47; a CDR3 comprising a comprising the sequence of SEQ ID NO: 48.
• 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 Tables 2 or 4.
• the anti-Cx43 antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 58.
• the anti-Cx43 antibody or fragment thereof comprises a variable heavy 7 chain comprising a sequence set forth in SEQ ID NO: 58.
• the anti-Cx43 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 Tables 2 or 4. In some aspects, the anti-Cx43 antibody or fragment thereof comprises a variable light chain comprising a sequence having at least 90% identity' to a sequence set forth in SEQ ID NO: 63. In some aspects, the anti-Cx43 antibody or fragment thereof comprises a variable light chain comprising a sequence set forth in SEQ ID NOs: 63.
• the anti-Cx43 antibody or fragment thereof comprises a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 58, and a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 63.
• the anti-Cx43 antibody comprises a variable heavy chain comprising a sequence set forth in SEQ ID NO: 58 and a variable light chain comprising a sequence set forth in SEQ ID NO: 63.
• the anti-Cx43 antibody or fragment thereof comprises a M1H region.
• 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 ID NO: 20; and a CDR3 comprising the sequence of SEQ ID NO: 21.
• the anti-Cx43 antibody or fragment thereof comprises a M1M7K region.
• 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.
• the disclosed antibodies or fragments thereof further comprise a tag sequence.
• nucleic acid sequences that encode the disclosed antibodies or fragments thereof.
• nucleic acid sequences comprising a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 52.
• nucleic acid sequences that encode the disclosed antibodies or fragments thereof.
• nucleic acid sequences comprising a variable heavy chain comprising a sequence set forth in SEQ ID NO: 52.
• nucleic acid sequences comprising a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 57.
• nucleic acid sequences comprising a variable light chain comprising a sequence set forth in SEQ ID NO: 57 are also disclosed.
• nucleic acid sequences comprising a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 52; and a variable light chain comprising a sequence having at least 90% identity a sequence set forth in SEQ ID NO: 57.
• nucleic acid sequences comprising a variable heavy chain comprising a sequence set forth in SEQ ID NO: 52; and a variable light chain comprising a sequence set forth in SEQ ID NO: 57.
• nucleic acid sequences capable of encoding a single chain variable fragment comprising a variable heavy chain comprising a sequence having at least 90% identity a sequence set forth in SEQ ID NO: 52.
• nucleic acid sequences capable of encoding a single chain variable fragment comprising a variable light chain comprising a sequence having at least 90% identity a sequence set forth in SEQ ID NO: 57.
• the disclosed antibodies or fragments thereof can be bispecific.
• the anti-Cx43 antibody or fragment thereof can comprise a first Fab region comprising the heavy and light chain of SEQ ID NO: 58 and a second Fab region comprising the heavy and light chain of SEQ ID NO: 63, wherein the first and second Fab regions can be different.
• the bispecific antibodies can be trifunctional.
• the disclosed antibodies or fragments thereof can be mouse, human, humanized, chimeric, or a combination thereof.
• the disclosed antibodies or fragments thereof are monoclonal.
• methods of preventing or reducing bone loss in a subject with antibodies that activate Cx43 hemichannel opening are methods of inhibiting osteocyte apoptosis in a subject with antibodies that activate Cx43 hemichannel opening.
• Disclosed herein are methods of increasing bone formation with antibodies that activate Cx43 hemichannel opening. Disclosed herein are methods of treating or preventing osteopenia with antibodies that activate Cx43 hemichannel opening. Disclosed herein are methods of promoting or enhancing fracture healing with antibodies that activate Cx43 hemichannel opening.
• the methods disclosed herein can comprise administering to the subj ect a therapeutically effective amount of an anti-connexin 43 antibody or fragment thereof.
• the anti-Cx43 antibody or fragment thereof can comprise a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 58.
• the anti-Cx43 antibody or fragment thereof can comprise a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 63.
• the anti-Cx43 antibody or fragment thereof can comprise a variable heavy chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 58 and a variable light chain comprising a sequence having at least 90% identity to a sequence set forth in SEQ ID NO: 63.
• the anti-Cx43 antibody or fragment thereof can comprise: a heavy chain immunoglobulin variable region comprising: a first complementarity determining region 1 comprising a sequence having at least 60% identity 7 to SEQ ID NO: 19; a second complementarity determining region 2 comprising a sequence having at least 60% identity 7 to SEQ ID NO: 20; and a third complementarity determining region 3 comprising a sequence having at least 60% identity to SEQ ID NO: 21.
• the anti-Cx43 antibody or fragment thereof can comprise: a light chain immunoglobulin variable region comprising: a first complementarity 7 determining region 1 comprising a sequence having at least 60% identity to SEQ ID NO: 49; a second complementarity determining region 2 comprising a sequence having at least 60% identity' to SEQ ID NO: 50; and a third complementarity determining region 3 comprising a sequence having at least 60% identity 7 to SEQ ID NO: 51.
• the anti-Cx43 antibody or fragment thereof can comprise: a heavy chain immunoglobulin variable region comprising: a first complementarity determining region 1 comprising a sequence having a single amino acid change compared to SEQ ID NO: 19; a second complementarity 7 determining region 2 comprising a sequence a single amino acid change compared to SEQ ID NO: 20; and a third complementarity 7 determining region 3 comprising a sequence a single amino acid change compared to SEQ ID NO: 21.
• the anti-Cx43 antibody or fragment thereof can comprise: a light chain immunoglobulin variable determining region comprising: a first complementarity 7 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.
• the anti-Cx43 antibody or fragment thereof can comprise 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 (e.g., SEQ ID NO: 66); and/or c) a complementarity determining region (CDR3) comprising the sequence of SEQ ID NO: 21 or a variant thereof.
• any one of the heavy chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid. In some aspects, the at least one amino acid substitution can be a glycine residue to another amino acid. In some aspects, the at least one amino acid substitution can decrease or reduce deamidation.
• the anti-Cx43 antibody or fragment thereof can 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.
• any one of the light chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid.
• the at least one amino acid substitution can be a glycine residue to another amino acid.
• the other amino acid can be an alanine.
• the at least one amino acid substitution can decrease or reduce deamidation.
• the anti-Cx43 antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising: a complementarity determining region 1 (CDR1) comprising a sequence having at least 60% identity to a sequence set forth in SEQ ID NO: 19; a complementarity determining region 2 (CDR2) comprising a sequence having at least 60% identity to a 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 a sequence set forth in SEQ ID NO: 21.
• any one of the light chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid. In some aspects, the at least one amino acid substitution can be a glycine residue to another amino acid. In some aspects, the other amino acid can be an alanine. In some aspects, the at least one amino acid substitution can decrease or reduce deamidation. For example, the at least one amino acid substitution can be a glycine residue to an alanine residue.
• the antibody or fragment thereof can comprise a heavy chain immunoglobulin variable region comprising a CDR2 comprising a sequence set forth in SEQ ID NO: 66 (INPSNAGT).
• the anti-Cx43 antibody or fragment thereof can comprise a light chain immunoglobulin variable region comprising: a complementarity’ determining region 1 (CDR1) comprising a sequence having at least 60% identity to a sequence set forth in SEQ ID NO: 49; a complementarity determining region 2 (CDR2) comprising a sequence having at least 60% identity to a 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 a sequence set forth in SEQ ID NO: 51.
• any one of the light chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid. In some aspects, the at least one amino acid substitution can be a glycine residue to another amino acid. In some aspects, the other amino acid can be an alanine. In some aspects, the at least one amino acid substitution can decrease or reduce deamidation.
• the anti-Cx43 antibody or fragment thereof can 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 a 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 a 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 a sequence set forth in SEQ ID NO: 21.
• any one of the light chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid. In some aspects, the at least one amino acid substitution can be a glycine residue to another amino acid. In some aspects, the other amino acid can be an alanine. In some aspects, the at least one amino acid substitution can decrease or reduce deamidation. For example, the at least one amino acid substitution can be a glycine residue to an alanine residue.
• the antibody or fragment thereof can comprise a heavy 7 chain immunoglobulin variable region comprising a CDR2 comprising a sequence set forth in SEQ ID NO: 66 (INPSNAGT).
• the anti-Cx43 antibody or fragment thereof can 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 a 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 a 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 a sequence set forth in SEQ ID NO: 51.
• any one of the light chain CDR1, CDR2 or CDR3 can comprises at least one amino acid substitution as compared to the parent CDR.
• the at least one amino acid substitution can be a cysteine residue to another amino acid. In some aspects, the at least one amino acid substitution can be a glycine residue to another amino acid. In some aspects, the other amino acid can be an alanine. In some aspects, the at least one amino acid substitution can decrease or reduce deamidation.
• any of the methods disclosed herein can comprise administering to the subject an effective amount of an expression vector encoding the anti-Cx43 antibody or fragment thereof.
• the anti-Cx43 antibody or fragment thereof can be administered in a pharmaceutically acceptable composition.
• the pharmaceutical composition can be lyophilized.
• the anti-Cx43 antibody or fragment thereof can be administered systemically.
• the anti-Cx43 antibody or fragment thereof can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• the anti-Cx43 antibody or fragment thereof can be a humanized antibody or humanized fragment thereof.
• the anti-Cx43 antibody can 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 (or SEQ ID NO: 66), 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.
• the anti-Cx43 antibody can be an IgG class of antibody, wherein the IgG class antibody is an IgGl, IgG2, IgG3, or IgG4 class antibody.
• any of the methods disclosed herein can further comprise administering at least a second therapeutic agent, a second therapy, or a combination thereof to the subject.
• the second therapeutic agent can be a bisphosphonate, calcitonin (e.g., Miacalcin, Fortical), teriparatide (e.g., Forteo), denosumab (e.g., Prolia) or romosozumab.
• the bisphosphonate can be alendronate (e.g., Fosamax, Binosto), ibandronate (e.g...
• the second therapy can be exercise.
• the exercise can be moderate exercise.
• moderate exercise can include any exercises that increase heart rate by at least 50% than its rate at rest.
• the anti-Cx43 antibody or fragment thereof can bind to a Cx43 hemichannel. In some aspects, in any of the methods disclosed herein the antibody or fragment thereof can stimulate the opening of a Cx43 hemichannel. In some aspects, in any of the methods disclosed herein the antibody or fragment thereof can stimulate the opening of a Cx43 hemichannel and have no effect on gap junction coupling. In some aspects, the anti-Cx43 antibody or fragment thereof can further comprise a tag sequence.
• the anti-Cx43 antibody or fragment thereof can be a Fab fragment an Fab’ fragment or an F(ab’)2 fragment.
• the methods described herein can be used to determine the effect on activation of Cx43 hemichannel opening in osteocytes by (i) determining hemichannel opening by dye uptake assay, using Lucifer yellow or Alexa dyes, (ii) assessing stimulating effects on hemichannels opening by measuring ATP levels (e.g., an increase in ATP release from osteocytes via Cx43 hemichannels can indicate that the compound or antibody being tested can suppressive tumor or cancel cell growth and/or colonization, (iii) test stimulatory effects of the reagents on hemichannels opening by mechanical loading in the form of fluid flow shear stress.
• the antibody that binds to a connexin 43 (Cx43) hemichannel and enhances channel opening can have no effect on gap channel coupling.
• the anti-Cx43 antibody useful in any of the methods disclosed herein can be any of the anti-Cx43 antibodies comprising one or more of the sequences set forth in Table 1.
• anti-Cx43 antibodies e.g., the M2 antibody
• biological fragments thereof that can be used to treat osteoporosis, prevent or reduce bone loss, inhibit osteocyte apoptosis, enhance prostaglandin E2 release in osteocytes, suppress sclerostin expression in osteocytes, increase bone formation, treat or prevent osteopenia, or promote or enhance fracture healing in a subject in need thereof with antibodies that activate Cx43 hemichannel opening
• compositions described herein can be administered to the subject (e.g., a human patient) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease.
• the patient can be a human patient.
• compositions can be administered to a subject (e.g., a human patient) already with, diagnosed or at risk for osteoporosis, bone loss, osteopenia or one or more bone fractures in an amount sufficient to at least partially improve a sign or symptom or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences.
• An amount adequate to accomplish this is defined as a "‘therapeutically effective amount.”
• a therapeutically effective amount of a composition e.g...
• a pharmaceutical composition can be an amount that achieves a cure, but that outcome is only one among several that can be achieved.
• a therapeutically effective amount includes amounts that provide a treatment in which the onset or progression of the disease or condition is delayed, hindered, or prevented, or the disease or condition or a symptom of the disease or condition is ameliorated or its frequency can be reduced.
• One or more of the symptoms can be less severe.
• Recovery can be accelerated in an individual who has been treated. For example, treatment of osteoporosis, bone loss, osteopenia, or fracture healing may involve, for example, increasing bone density, or increasing bone strength.
• the antibodies described herein can improve the quality of life of a subject with or at risk for osteoporosis, bone loss, osteopenia, or bone fracture(s). In some aspects, the antibodies described herein can prevent osteoporosis, prevent or reduce bone loss, inhibit osteocyte apoptosis, enhance prostaglandin E2 release in osteocytes, suppress sclerostin expression in osteocytes. increase bone formation, prevent osteopenia, or promote or enhance fracture healing. In some aspects, the methods can comprise administering an effective amount of the anti-Cx43 antibody to the subject. In some aspects, the method can comprise administering an effective amount of an expression vector encoding the anti-Cx43 antibody to the subject. In some aspects, the anti-Cx43 antibody that binds to a connexin 43 (Cx43) hemi channel and enhances channel opening can have no effect on gap channel coupling.
• Cx43 connexin 43
• the subject has been diagnosed with osteoporosis, bone loss, osteopenia, or one or more bone fractures prior to the administering step.
• compositions described herein can be formulated to include a therapeutically effective amount of the anti-Cx43 antibodies disclosed herein.
• anti-Cx43 antibodies disclosed herein can be contained within a pharmaceutical formulation.
• the pharmaceutical formulation can be a unit dosage formulation.
• the therapeutically effective amount or dosage of any of the antibodies used in the methods as disclosed herein applied to mammals can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, sex, the severity of the subject's symptoms, and the particular composition or route of administration selected, other drugs administered and the judgment of the attending clinician. Variations in the needed dosage may be expected. Variations in dosage levels can be adjusted using standard empirical routes for optimization.
• the particular dosage of a pharmaceutical composition to be administered to the patient will depend on a variety of considerations (e.g., the seventy of the osteoporosis, bone loss, or osteopenia symptoms), the age and physical characteristics of the subject and other considerations known to those of ordinary skill in the art.
• a therapeutically effective dosage of an anti-hemichannel antibody can result in a decrease in severity of one or more disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
• a therapeutically effective amount of a therapeutic compound or anti-Cx43 antibody can decrease the severity of the osteoporosis, bone loss, or osteopenia symptoms, or otherwise ameliorate symptoms in a subject.
• 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 life span of the host (e.g., many years).
• the compositions can be administered once a w eek (for, for example, 4 weeks to many months or years); once a month (for, for example, three to tw elve months or for many years); or once a year for a period of 5 years, ten years, or longer.
• the frequency of treatment can be variable.
• the present compositions can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.
• the total effective amount of the anti-Cx43 antibodies or compositions as disclosed herein can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol in which multiple doses are administered over a more prolonged period of time.
• continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are also within the scope of the present disclosure.
• the anti-Cx43 antibodies or compositions described herein can be administered in conjunction with other therapeutic modalities to a subject in need of therapy.
• the present compounds can be given to prior to. simultaneously with or after treatment with other agents or regimes.
• the anti-Cx43 antibodies disclosed herein can be administered alone or in conjunction with standard therapies used to osteoporosis, bone loss, or osteopenia.
• any of the anti-Cx43 antibodies or compositions described herein can be administered or used together with a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab.
• the bisphosphonate can be alendronate, ibandronate, resendronate, or zoledronic acid.
• any of the anti-Cx43 antibodies or compositions described herein can be administered or used together with exercise.
• compositions e.g., pharmaceutical compositions, comprising one or a combination of the anti-Cx43 antibodies, or antigen-binding portion(s) thereof formulated with a pharmaceutically acceptable carrier.
• Such compositions may include one or a combination of (e.g., two or more different) anti-Cx43 antibodies, or immunoconjugates described herein.
• a pharmaceutical composition of the invention can comprise a combination of anti-Cx43 antibodies that bind to different epitopes on the target antigen or that have complementary activities.
• compositions of the invention also can be administered as combination therapy, i.e., combined with other agents.
• the combination therapy can include an anti-Cx43 hemichannel antibody combined with at least one other therapeutic agent or therapy.
• the second therapeutic agent can be a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab.
• the bisphosphonate can be alendronate, ibandronate, resendronate, or zoledronic acid.
• the second therapy can be exercise.
• the phrase “pharmaceutically acceptable carrier” includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier can be suitable for intravenous, intramuscular, subcutaneous, or parenteral administration (e.g., by injection or infusion).
• the active compound i.e., antibody, or immunoconjugate
• the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
• aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
• compositions typically 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 to high drug concentration.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by sterilization microfiltration.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein.
• the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0. 1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
• Dosage regimens are 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 exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary’ dosages for the subjects to be treated; each unit contains a predetermined quantity’ of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 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 of the host body weight.
• the dosages can be 0.3 mg/kg body weight, 1 mg/kg body yveight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body yveight or w ithin the range of 1-10 mg/kg.
• the dosages can be 0.3 mg/kg body weight, 1 mg/kg body w eight, 3 mg/kg body w eight, 5 mg/kg body weight, 10 mg/kg body weight. 15 mg/kg body weight, 20 mg/kg body yveight, 25 mg/kg body weight or 30 mg/kg body weight or yvithin the range of 1-30 mg/kg. In some aspects, the dosages can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg/kg body weight. In some aspects, the dosages can be 5 mg/kg body weight. In some aspects, the dosages can be 15 mg/kg body weight. In some aspects, the dosages can be 20 mg/kg body weight. In some aspects, the dosages can be 25 mg/kg body weight.
• An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every' four weeks, once a month, once every' 3 months or once every three to 6 months.
• Preferred dosage regimens for an anti-Cx43 hemichannel antibody’ of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every' four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg bodyweight every three weeks.
• two or more anti-Cx43 (e.g., monoclonal) antibodies with different binding specificities are administered simultaneously, in which case the dosage of each anti- Cx43 antibody administered falls within the ranges indicated.
• the anti-Cx43 antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of the anti-Cx43 antibody to the target antigen in the patient.
• dosage is adjusted to achieve a plasma anti-Cx43 antibody concentration of about 1-1000 pg/ml and in some methods about 25-300 pg/ml.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which 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 upon a variety’ of pharmacokinetic factors including the activity’ of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• a composition of the present invention can be administered via one or more routes of administration using one or more of a variety- of methods known in the art.
• routes and/or mode of administration w ill vary depending upon the desired results.
• Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or other parenteral routes of administration, for example by injection or infusion.
• parenteral administration' as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular injection and infusion.
• the anti-Cx43 antibody disclosed herein can be administered systemically. In some aspects, the anti-Cx43 antibody disclosed herein can be administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
• compositions and methods described herein can involve an anti-Cx43 antibody or an anti-Cx43 antibody fragment thereof against Cx43 to stimulate the opening of the Cx43 hemichannel to, for example, prevent osteoporosis, prevent or reduce bone loss, inhibit osteocyte apoptosis, enhance prostaglandin E2 release in osteocytes, suppress sclerostin expression in osteocytes, increase bone formation, prevent osteopenia, or promote or enhance fracture healing, in combination with a second therapeutic or additional therapy.
• compositions including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another therapeutic or therapy.
• Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect. This process may involve contacting the cells with both an anti-Cx43 antibody or an anti-Cx43 antibody fragment, and a second therapy.
• a tissue or cell can be contacted with one or more compositions or pharmacological fonnulation(s) comprising one or more of the agents (e.g., anti-Cx43 antibody or anti-Cx43 antibody fragment or a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab), or by contacting the tissue and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an anti-Cx43 antibody or anti-Cx43 antibody fragment, 2) a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab, or 3) both an anti-Cx43 antibody or anti-Cx43 antibody fragment and a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab. Also, it is contemplated that such a combination therapy can be used in conjunction with exercise.
• the agents e.g
• contacted and exposed when applied to a cell, are used herein to describe the process by which a therapeutic agent is delivered to a target cell or are placed in direct juxtaposition with the target cell.
• the anti-Cx43 antibodies and biological fragments thereof can be administered before, during, after, or in vanous combinations relative to any second treatment or therapy.
• the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
• the anti-Cx43 antibody or anti-Cx43 antibody fragment is provided to a patient separately from a bisphosphonate, calcitonin, teriparatide, denosumab or romosozumab, one would generally ensure that a significant period of time did not expire between the time of each delivery', such that the two compounds would still be able to exert an advantageously combined effect on the patient.
• a course of treatment can last between 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there can be a period of time at which no second treatment or therapy is administered.
• This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.
• an antibody therapy is “A” and a second therapy is “B”:
• Administration of any compound or therapy disclosed herein to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some aspects there can be a step of monitoring toxicity that can be attributable to combination therapy.
• kits comprising one or more therapeutic agents and/or other therapeutic and delivery agents.
• the kit can be used for preparing and/or administering a therapy disclosed herein.
• the kit may comprise one or more sealed vials containing any of the pharmaceutical compositions disclosed herein.
• the kit may include, for example, at least one anti-Cx43 antibody or fragment thereof as well as reagents to prepare, formulate, and/or administer the components one or more of the compositions disclosed herein or perform one or more steps of the inventive methods.
• the kit may also comprise a suitable container, which can be a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube.
• the container may be made from sterilizable materials such as plastic or glass.
• the kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill in the art.
• the instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.
• Anti-Cx43 monoclonal antibodies were generated and clones were identified that produced Cx43-binding monoclonal antibodies. CDR sequences of both DNA and amino acids for the antibody sequences are shown in the tables below along with the correct pairing for each of the characterized antibodies.
• the Ml antibody inhibits the opening of a Cx43 hemichannel.
• the M2 antibody activates, stimulates and/or enhances the opening of a Cx43 hemichannel.
• Table 2 Pairing of heavy chain and light chain for two functional antibodies.
• Table 3 Sequence of antibody chains from the hybridomas.
• GGCACCACTCTCATAGTCTCCTCA SEQ ID NO: 53
• AAC SEQ ID NO: 55
• DIVMTOSPASLAVSLGORATISYRASKSVSTSGYSYMHWNOQKPGOPPRLLIYLVSN LESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGTKLEIK (SEQ ID NO: 62)
• HCs Mechanosensitive connexin 43 hemichannels
• anti-Cx43 (M2) antibodies inhibited unloading-induced bone loss and osteocyte apoptosis in 16- week-old mice, and improved bone mass in response to tibial loading in 22-month old mice.
• the HC opening enhanced prostaglandin E2 (PGE2) release and sclerostin (SOST) suppression in osteocytes, leading to the suppression of increased bone resorption and decreased bone formation during unloading, and increased formation of both trabecular and cortical bone during loading.
• PGE2 prostaglandin E2
• SOST sclerostin
• anti-Cx43 (M2) antibody- induced Cx43 HC opening with PGE2 release rescued unloading-induced bone loss and reversed unresponsiveness of aged bones to anabolic effects of mechanical loading.
• the anti-connexin 43 antibodies disclosed herein can overcome the limitations of existing treatment regimens and be used to treat bone loss and osteoporosis associated with aging and disuse.
• Anti-Cx43 (M2) antibody enhanced the opening ofCx43 HCs in osteocytes during both mechanical unloading and loading in vivo.
• M2 monoclonal anti Cx43
• EB Evans blue
• anti-Cx43 (M2) antibody treatment improved the EB dye uptake under mechanical loading in the metaphyseal trabecular bone, but such increase was not seen in vehicle-treated mice.
• anti-Cx43 (M2) antibody enhances HC opening induced by mechanical loading in aged trabecular bone in vivo. EB dye uptake was observed in metaphyseal trabecular bone for both loaded and contralateral unloaded tibias of vehicle- and Cx43(M2)-treated mice.
• Anti-Cx43 (M2) antibody treatment prevents hone loss caused by mechanical unloading.
• the baseline body weight of mice was not different among the four experimental groups (FIG. 2C).
• anti-Cx43 (M2) antibody treatment caused a slight weight loss in control mice during the first seven days of HLS. the weight was gradually regained (FIG. 2C).
• FIG. 2C There was clear decrease in the body weight of mice subjected to unloading compared to control mice during the four weeks of HLS, but the unloading-induced decrease was not observed in anti-Cx43 (M2)-treated mice.
• unloading-induced loss of body weight was significantly less in anti-Cx43 (M2)-treated mice than in vehicle-treated mice.
• the micro-computed tomography (pCT) analysis on the tibial metaphyseal region showed that four-weeks of mechanical unloading via HLS resulted in a comprised quality and structure of trabecular bone in vehicle-treated mice, as shown by decreased bone mineral density (BMD) (FIG. 3A) and trabecular thickness (Tb.Th) (FIG. 3B) and bone volume fraction (BV/TV) (FIG. 3C), whereas this deleterious effect was reduced in anti-Cx43 (M2) antibody- treated mice. In anti-Cx43 (M2)-unloading tibias the Tb.Th was even greater than in vehicleunloading treated tibias (FIG. 3B).
• BMD bone mineral density
• Tb.Th trabecular thickness
• BV/TV bone volume fraction
• FIG. 3D Representative images of trabecular bone are shown in FIG. 3D.
• Mechanical unloading did not change the total cross-sectional area (T.Ar), but it caused a decrease in cortical parameters including cortical bone area (B.Ar). cortical thickness (Ct. Th), the bone area ratio (B.Ar/T.Ar), and tissue mineral density (TMD) as well as enlarged bone marrow area (M. Ar) in vehicle control mice (FIGS. 3E-J).
• these parameters remained unchanged between anti-Cx43 (M2)-control and Cx43 (M2)-unloading mice.
• Th and TMD were even greater than vehicle-unloading tibias (FIGS. 3F-H, J).
• Representative images of cortical bone FIG. 3K.
• Mechanical testing of femurs via three-point bending after four weeks of HLS revealed a significant decrease in elastic modulus, ultimate force, and ultimate stress in vehicle-control compared to vehicle-unloading mice, but such a decrease was not found in anti-Cx43 (M2) antibody mice (FIGS. 3L-N). Together, these results demonstrate that enhanced Cx43 HCs by anti-Cx43 (M2) antibody treatment prevented the bone loss and microstructure changes of trabecular and cortical bone during mechanical unloading.
• Enhanced Cx43 HC activity inhibited osteocyte apoptosis and osteoclast activity caused by mechanical unloading.
• Hematoxylin and eosin (H&E) staining of tibial cortical bone showed that mechanical unloading by HLS resulted in a greater number of empty' lacunae in vehicle-treated mice than vehicle-unloading mice, whereas there was no difference between anti-Cx43 (M2) antibody-control and anti-Cx43 (M2) antibody-unloading mice (FIGS. 4A, 4B).
• Apoptotic osteocytes were confirmed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (FIG. 4C).
• TUNEL-positive osteocytes (red) (indicated by solid white arrows) were increased in vehicle- treated groups during mechanical unloading, but such increase was inhibited in anti-Cx43 (M2) antibody-treated groups (FIG. 4C).
• Osteocyte apoptosis induced by HLS was reported to trigger osteocyte RANKL and osteoclast-mediated resorption (Aguirre, J. I. et al. J Bone Miner Res. 21, 605-615 (2006); AND Cabahug-Zuckerman, P. et al. J Bone Miner Res. 31, 1356- 1365 (2016)).
• Enhanced Cx43 HC activity increased PGE2 release and suppressed scler ostin (SOST) expression in osteocytes during unloading.
• PGE2 released through Cx43 HCs acts in an autocrine manner to reduce osteocyte apoptosis (Xia, X. et al. Mol Cell Biol. 30, 206-219 (2010); and Kitase, Y. et al. J Bone Miner Res. 25, 2657-2668 (2010)).
• FIGS. 11 A-B which are models demonstrating the role of enhanced Cx43 hemi channels in regulating the response to mechanical loading and unloading.
• osteocytic Cx43 HCs using the anti-Cx43 (M2) antibody elevates PGE2 levels and suppresses SOST expression, leading to increase endostea osteoblast activity and bone formation, and simultaneously decreases endosteal osteoclast activity in aged mice under mechanical loading conditions.
• apoptotic osteocytes release higher levels of RANKL. which induces osteoclast differentiation and recruitment.
• M2 anti-Cx43
• PGE2 level was determined in the femurs of both anti- Cx43 (M2) antibody-control and anti-Cx43 (M2) antibody-unloading mice. Although there was no significant increase in PGE2 level in femur bone between anti-Cx43 (M2) antibodycontrol and anti-Cx43 (M2) antibody-unloading mice, the anti-Cx43 (M2) antibody-unloading mice showed a greater PGE2 level than vehicle-unloading mice (FIG. 5A). There was no significant change in hindlimb bone marrow' PGE2 level (FIG. 5B).
• Enhancing osteocytic Cx43 HCs by anti-Cx43 (M2) antibody treatment improved load- induced increase of trabecular and cortical microstructure in aged mice.
• M2 anti-Cx43
• anti-Cx43 (M2) antibody treatment increased cortical B.Ar (FIG. 7F) and Ct. Th (FIG. 7H) via decreased M.Ar (Fig. 71), without changing the total cross-sectional area (T.Ar) (Fig. 7E) in loaded tibias leading to significant increases in B.Ar/T.Ar and (TMD) (FIGS. 7G, 7J) compared to the contralateral non-loaded control tibias.
• Representative images of cortical bone are shown in FIG. 7K.
• Mechanical property 7 analysis showed significantly increased elastic modulus over the contralateral controls in anti-Cx43 (M2) antibody-treated mice, but such an increase was not found in vehicle-treated mice (FIG. 7L).
• Cx43 (M2) antibody loaded tibias have significantly greater ultimate force (FIG. 7M) and ultimate stress (FIG. 7N) compared to vehicle control tibias.
• enhanced Cx43 HC activity increased anabolic response to the loading in trabecular and cortical bone.
• Enhancing Cx43 HCs in osteocytes increases endosteal anabolism in response to mechanical loading.
• Reduced M.Ar in Cx43 (M2) antibody -loaded tibias resulted in changes in the activities of osteoblasts and osteoclasts on the endosteal surface.
• histomorphometric analysis of diaphysis located 37% distal from the proximal end was performed. Dynamic histomorphometry indicated that mechanical loading had no effect on both endosteal and periosteal bone formation.
• Anti-Cx43 (M2) antibody treatment did not alter periosteal MAR, MS/BS and BFR/BS (FIGS. 8A-C).
• anti-Cx43 (M2) antibody treatment exhibited a significant increase of MAR, MS/BS, and BFR/BS (FIG. 8F) in endocortical bone in response to mechanical loading.
• the MAR, MS/BS and BFR/BS in anti-Cx43 (M2) antibody-treated tibias are greater compared to vehicle control tibias (FIGS. 8D-F). Representative images are shown in FIG. 8G.
• Mechanical loading showed a significant decrease of the osteoclast surface on the endosteal surface compared to contralateral unloaded controls in anti-Cx43 (M2) antibody-treated mice, but this decrease was not found in vehicle-treated mice (FIGS. 8H. 81).
• FIG. 8L Representative images of TRAP -positive osteoclasts on endosteal surface are shown in FIG. 8L.
• enhanced Cx43 HCs by anti-Cx43 (M2) antibody treatment increased bone formation and decreased osteoclast resorption on the endosteal surface caused by mechanical loading, resulting in decreased marrow area (FIG. 71).
• Anti-Cx43 (M2) antibody treatment enhanced loading-induced PGE release and SOST expression.
• a significant increase of the PGE2 level was observed in loaded tibias compared to contralateral unloaded control tibias in anti-Cx43 (M2) antibody-treated mice, and the PGE2 level in anti-Cx43 (M2) antibody-loaded tibias was also greater compared to vehicle-treated tibias (FIG. 9A).
• the serum PGE2 level was similar between vehicle- and anti-Cx43 (M2) antibody-treated mice (FIG. 9B).
• anti-Cx43 (M2) antibody treatment significantly increased the COX-2 expression in 37% diaphysis region in loaded tibias compared to contralateral unloaded controls, whereas the increased COX-2 expression was not detected in vehicle-treated mice (FIGS. 9C, 9D). It has been shown that mechanical loading downregulated Wnt pathw ay antagonist, SOST, in osteocytes of young WT mice (Zhao, D. et al. Bone Res. 10, 49 (2022); and Zhao, D. et al. eLife. 11 (2022)).
• Tibial loading upregulated the P-catenin-positive osteoblasts on the endosteal surface, compared to the unloaded contralateral controls in anti-Cx43 (M2) antibody-treated mice, but the increase was not evident in vehicle-treated mice (FIGS. 9G-9H).
• M2 anti-Cx43
• mice Male C57BL/6 mice were fed in the animal care facility with a 12-hour light/dark cycle and room temperature of 25 °C under pathogen-free conditions. Mice were housed in each cage less than 5 before experiments and provided standard rodent chow.
• Anti-Cx43 (M2) monoclonal antibody generation and treatment.
• the monoclonal antibody was generated by Abmart (Tulsa, OK). Briefly, mice were immunized with a Cx43 extracellular domain peptide intraperitoneally with 50 pg of the peptide in complete Freund’s adjuvant and then boosted repeatedly with the peptide antigen formulated in incomplete Freund’s adjuvant. Spleens were harvested and fusions performed (Javaheri, B. et al. Sci Rep. 8, 6636 (2016); and Spesael, M. et al. Endocrinology). 156, 1343-1353 (2015)).
• anti-Cx43 (M2) antibody clone is selected, and antibody was purified by affinity' chromatography using protein A resin (Razi, H. et al. Acta Biomater. 13, 301-310 (2015)).
• Micro-computed tomography The micro-computed tomography (pCT) scanner (1172, SkyScan, Briiker microCT, Kontich, Belgium) was used for bone structure scanning under the following parameters: 59 Kvp, 167 pA beam intensity, 0.5 mm aluminum filter, 800 ms exposure, 1024 x 1024-pixel matrix, and a 10 pm isotropic voxel dimension.
• the metaphyseal trabecular VOI was positioned 45 slices (0.45 mm) distal to the proximal growth plate with an extension of 100 slices (1 mm) from the distal side, which represented the secondary spongiosa.
• a grayscale threshold of 80-256 was used for the trabecular VOI for the analysis.
• the cortical VOI positioned 50 slices (0.5 mm) centered at diaphysis 37% distal from the end of the proximal side to analyze the response to mechanical loading.
• This VOI matches previously published studies examining dynamic loading of tibias (Zhao, D. et al. Bone Res. 10, 49 (2022); and Holguin, N., et al. J Bone Miner Res. 31, 2215-2226 (2016)).
• the cortical VOI was positioned 70 slices (0.7 mm) distal to the proximal growth plate with an extension of 50 slices (0.5 mm) from the distal side.
• the mid-diaphyseal VOI was reported to respond to mechanical unloading (Morse, A.
• Bone mechanical and material property testing Three-point bending tests were performed when the tibia thawed to room temperature. After removing the muscle and fibula, the tibia was subjected to a three-point bending test along the medial-lateral direction in a micromechanical testing system (Mach-1 V500CST, Biomomentum, Laval. Canada) (Zhao, D. et al. Bone Res. 10, 49 (2022); and Zhao. D. et al. eLife. 11 (2022)).
• the loading parameters are 8 mm span with a loading speed at 0.05 mm/sec, and 200-Hz.
• the cross-sectional areas determined from pCT were used to calculate mechanical properties (Jepsen, K. I. et al. J Bone Miner Res. 30, 951-966 (2015)).
• mice were IP injected with calcein (C0875, Sigma-Aldrich, St. Louis, MO, USA) one day before tibial loading, followed by alizarin red injection (A5533, Sigma-Aldrich, St. Louis, MO, USA) three days before euthanization (Zhao, D. et al. Bone Res. 10, 49 (2022); and Zhao. D. et al. eLife. 11 (2022)).
• mice were IP injected with calcein one day before the start and end of mechanical unloading separately.
• Tibias were embedded in methylmethacrylate and 80-pm-thick transversal sections of the mid-diaphyseal site were cut using a precision wafering saw (PICO 155. PACE Technologies, Arlington, AZ, USA). The sections were then sanded to a thickness of 80 pm using sandpaper of grit Pl 200 on the PHOENIX 4000 system (BUEHLER, Lake Bluff, IL, USA). Fluorescent labels were imaged by a fluorescence microscope (BZ-X710, KEYENCE, Itasca, IL, USA). The MS/BS, MAR, and BFR/BS were calculated using the NIH ImageJ (Zhao, D. et al. Bone Res. 10, 49 (2022); and Zhao, D. et al. eLife. 11 (2022)).
• mice After 10 min loading and 40 min rest, anesthetized mice were under heart perfusion to fix the osteocytes in tibias. Tibias were embedded in the sagittal orientation in optimum cutting temperature compound (OCT) to cut I2-pm frozen sagittal sections. The nuclei were stained with 4’,6-diami- dino-2-phenylindole (DAPI). EB fluorescence intensity in osteocytes at the mid-diaphyseal site was quantified by the NIH ImageJ software (NIH, USA).
• OCT optimum cutting temperature compound
• the PGE2 level in the serum, bone marrow from hindlimbs, femurs, and tibias were quantified using the PGE2 ELISA kit.
• Four hours after the completion of the five-day tibial loading or 4-week mechanical unloading, serum, bone marrow, and tibial diaphysis without bone marrow and soft tissues were collected and stored at -80°C.
• the PGE2 level in the samples was quantified using the PGE2 ELISA kit (#514010, Cayman Chemical, Ann Arbor. MI, USA) and calibrated to total protein concentration determined by a BCA assay. Histology.
• tibias were embedded sagittally in paraffin blocks to obtain 5-pm-thick longitudinal sections.
• Tartrate resistant acid phosphatase (TRAP) was performed to determine the osteoclast activity (Xu, H. et al. J Bone Miner Res. 30, 436-448 (2015)).
• the multinucleated (> 3 nuclei) TRAP -positive osteoclasts were quantified on the endosteal and metaphyseal trabecular surface.
• Hematoxylin and eosin (H&E) staining was used to quantify the numbers of empty lacunae.
• Cx43 conditional knockout in osteoblasts/osteocytes instead preserved trabecular and cortical bone loss and attenuated osteoclast activity on the endosteal surface during unloading (Grimston, S. K. et al. J Bone Miner Res. 26, 2151-2160 (2011); Lloyd, S. A., et al. J Bone Miner Res. 27, 2359-2372 (2012); and Lloyd, S. A., et al. Bone. 57, 76-83 (2013)).
• Cx43 deficiency affects Cx43 channels which may also affect other channel-independent functions.
• PGE2 acts in an autocrine manner through EP2/4 receptors to block glucocorticoid-induced osteocyte apoptosis by increasing 0- catenin level (Xia, X. et al. Mol Cell Biol. 30. 206-219 (2010)).
• M2 anti-Cx43
• Cx43 HCs rescued osteocyte apoptosis either directly or by suppressing the SOST expression during mechanical unloading.
• Osteocytes are major producers of osteoclastogenic cytokine RANKL (Boyle, W. J., et al. Nature. 423, 337-342 (2003); and Xiong, J. et al. Nat Med. 17, 1235-1241 (2011)). Osteocyte apoptosis caused by mechanical unloading was indicated to increase the expression of RANKL in neighboring non-apoptotic osteocytes (Cabahug-Zuckerman. P. et al. J Bone Miner Res. 31 , 1356-1365 (2016); and Kennedy, O. D et al. Bone.
• osteocytic Cx43 HCs by the combined treatment of anti-Cx43 (M2) antibodies and mechanical loading in aged bone promotes osteoblast recruitment and differentiation on the endosteal surface during axial compression loading.
• M2 anti-Cx43
• Extracellular PGE2 is an important modulator in promoting endosteal bone formation (Jee, W. S., et al. Calcif Tissue Int. 37, 148-157 (1985); and Tian, X. Y. et al. J Musculoskelet Neuronal Interact. 7, 372-381 (2007)). Extracellular PGE2 is an important modulator in promoting endosteal bone formation, whereas PGE2 deficiency attenuated endosteal bone anabolism during mechanical loading (Forwood, M. R. J Bone Miner Res. 11, 1688-1693 (1996)).
• activated Cx43 HCs by anti- Cx43 (M2) antibody treatment inhibits the osteocyte apoptosis and bone loss induced with a HLS mechanical discus model and aging bones, and it can render the unresponsiveness of Cx43 HCs to active states and release anabolic factors such as PGE2 in promoting bone formation and reducing bone resorption.
• anti-Cx43 (M2) antibody-activated HC decreased SOST expression in osteocytes and increased osteocyte survival and anabolic response during both mechanical unloading and loading in both aged and disuse bones.
• anti- Cx43 (M2) antibody treatment increases trabecular as well as cortical bone formation given the increased cortical bone formation is a major challenge for the existing therapies.
• Anti-Cx43 (M2) antibody treatment may directly activate the mechanical sensitivity of Cx43 HCs.
• anti-Cx43 (M2) antibody treatment can be combined with mechanical loading to activate HC opening in osteocytes in aged mice, which may be related to the more active osteocytes (Tiede-Lewis LM, et al. Aging (Albany NY). 2017;9(10):2190-2208) and higher Cx43 expression (Kar R, et al. J Bone Miner Res. 2013;28(7): 1611-1621) in younger mice compared to aged mice.
• osteoporosis drugs despite their efficacy, often come with adverse effects that can limit their long-term safety.
• antiresorptive agents such as bisphosphonates and the monoclonal antibody to RANKL (e g., denosumab) inhibit bone resorption but also reduce bone formation, increasing the risk of osteonecrosis of the jaw and atypical femoral fracture (11).
• the anabolic agents such as PTH (e.g., teriparatide) or the PTH-related peptide analog abaloparatide stimulates both bone formation and bone resorption due to PTH's ability to increase RANK production.

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