Methods of treating osteoarthritis

Abstract

The present disclosure provides compositions and methods for treating osteoarthritis, such as osteoarthritis in a subject. The method comprises blocking the opening of the Cx43 hemichannel in cartilage cells by, for example, using a composition comprising an anticonnexin 43 antibody.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 511,563, filed June 30, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0003] By referencing and incorporating into the sequence list

[0004] The sequence list titled 172628-201003_PCT_SL.xml, created on June 19, 2024 and of size 34,860 bytes, is hereby incorporated in its entirety by reference. Background Technology

[0005] Osteoarthritis (OA) is a common type of arthritis affecting approximately 20% of American adults. Osteoarthritis causes degeneration of the joint, including articular cartilage and subchondral bone, characterized by the loss of articular cartilage leading to narrowing of the joint space, increased joint friction, and potential structural remodeling. Current treatments include exercise, lifestyle modifications, and pain medication. If symptoms become severe, joint replacement surgery is often performed. However, most existing treatments fail to address the underlying cause of osteoarthritis (i.e., cartilage deterioration).

[0006] Chondrocytes express the connexin Cx43 hemichannels, and these channels mediate the passage of small molecules (less than 1.2 kDa) between the intracellular and extracellular spaces. The hemichannels are normally closed, but under certain conditions, such as mechanical stress and inflammation, they are activated and opened. Open Cx43 hemichannels in chondrocytes promote inflammatory responses by releasing pro-inflammatory factors such as prostaglandin E2 (PGE2) and ATP. Inhibiting the opening of Cx43 hemichannels in chondrocytes (e.g., by chemical agents, etc.) can suppress inflammation and the development of osteoarthritis.

[0007] Therefore, there is still a need for effective treatments for osteoarthritis and other pathological conditions associated with increased hemichannel activity. Summary of the Invention

[0008] In some aspects of this disclosure, methods for treating osteoarthritis in subjects with this need are provided, comprising administering at least one dose of an anti-connector protein 43 (Cx43) antibody to the subject. In some embodiments, the anti-Cx43 antibody comprises a heavy chain CDR sequence and a light chain CDR sequence as follows:

[0009] HCDR1: SEQ ID NO: 1;

[0010] HCDR2: SEQ ID NO: 2;

[0011] HCDR3: SEQ ID NO: 3;

[0012] LCDR1: SEQ ID NO: 4;

[0013] LCDR2: SEQ ID NO: 5; and

[0014] LCDR3: SEQ ID NO: 6.

[0015] In some embodiments, the method includes administering a first dose of anti-Cx43 antibody on day 1 and a second dose on day 8. In some embodiments, the method further includes administering a third dose of anti-Cx43 antibody on day 15. In some embodiments, the method further includes administering a fourth dose of anti-Cx43 antibody on day 22. In some embodiments, the method further includes administering follow-up doses weekly after the fourth dose.

[0016] In other aspects of this disclosure, a method for treating osteoarthritis in subjects with this need is provided, comprising administering to the subject an effective amount of an anti-connector protein 43 (Cx43) antibody, wherein the anti-Cx43 antibody is administered according to the following dosage regimen:

[0017] i) The first dose on day 1;

[0018] ii) The second dose on day 8;

[0019] iii) The third dose on day 15; and

[0020] iv) The fourth dose on day 21.

[0021] In some implementations, the method further includes administering subsequent doses of the anti-Cx43 antibody weekly after the fourth dose.

[0022] In some implementations, the anti-Cx43 antibody comprises the following heavy chain CDR sequence and light chain sequence:

[0023] HCDR1: SEQ ID NO: 1;

[0024] HCDR2: SEQ ID NO: 2;

[0025] HCDR3: SEQ ID NO: 3;

[0026] LCDR1: SEQ ID NO: 4;

[0027] LCDR2: SEQ ID NO: 5; and

[0028] LCDR3: SEQ ID NO: 6.

[0029] In some embodiments of any of the methods disclosed herein, the first, second, third, fourth, and / or subsequent doses are from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the first, second, third, fourth, and / or subsequent doses are about 15 mg / kg. In some embodiments, the first, second, third, fourth, and / or subsequent doses are about 25 mg / kg. In some embodiments, the first, second, third, fourth, and / or subsequent doses are about 50 mg / kg.

[0030] In some embodiments of any of the methods disclosed herein, the anti-Cx43 antibody is administered intravenously.

[0031] In some embodiments of the methods disclosed herein, at least one indicator of osteoarthritis severity is assessed at least one day to one week after administration of each dose. In some embodiments, at least one indicator of osteoarthritis severity includes: (a) pain threshold of arthritis, (b) gait score, or (c) weight-bearing difference. In some embodiments, at least one indicator of osteoarthritis severity is improved after administration of at least one dose of the anti-Cx43 antibody.

[0032] In some embodiments of the methods disclosed herein, the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO: 8. In some embodiments of the methods disclosed herein, the anti-Cx43 antibody comprises the heavy chain sequence of any one of SEQ ID NO: 9 and 11-18, and / or the light chain sequence of SEQ ID NO: 10. In some embodiments, the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10.

[0033] In some embodiments of any of the methods disclosed herein, the anti-Cx43 antibody blocks the opening of the Cx43 hemichannel in the subject.

[0034] In some embodiments of any of the methods disclosed herein, the subject is a human being. Attached Figure Description

[0035] Figure 1The effect of anti-Cx43 antibody treatment on gait scores (paw pressure scores) in a rat model of osteoarthritis was depicted. The rat model of osteoarthritis was established by partial medial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT). Anti-Cx43 antibody was administered intravenously once weekly for a total of four times, starting on days 15, 22, 29, and 36 post-model establishment (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody (15 mg / kg, 25 mg / kg, and 50 mg / kg) were tested. Duloxetine was used as a control and was administered orally daily at a dose of 5 mg / kg. Gait scores (paw pressure scores) were measured on days 16, 23, 30, and 37 post-model establishment. Each data point corresponds to the mean gait score (mean ± standard error [SEM], n=8). As shown in the figure: control, control rats; model, osteoarthritis rat model (mediated treatment); and AM1, anti-Cx43 antibody ALMB-0166.

[0036] Figure 2 The effect of anti-Cx43 antibody treatment on plantar pain threshold in a rat model of osteoarthritis was investigated. The rat model of osteoarthritis was established using pMMx+ACLT. Anti-Cx43 antibody was administered intravenously four times weekly, starting on days 15, 22, 29, and 36 post-model establishment (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody (15 mg / kg, 25 mg / kg, and 50 mg / kg) were tested. Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. Plantar pain threshold was measured on days 1, 15, 16, 23, 30, and 37 post-model establishment. Each data point corresponds to the mean gait score (±SEM, n=8). As shown in the figure: control, control rats; model, osteoarthritis rat model (mediator treatment); AM1, anti-Cx43 antibody ALMB-0166; and 50% MWT, which showed a 50% probability of positive reaction at the stimulation intensity.

[0037] Figures 3A-3D The effect of anti-Cx43 antibody treatment on the plantar pain threshold in a rat model of osteoarthritis was depicted. The rat model of osteoarthritis was established using pMMx+ACLT. Anti-Cx43 antibody was administered intravenously four times weekly, starting on days 15, 22, 29, and 36 after model establishment (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody (15 mg / kg, 25 mg / kg, and 50 mg / kg) were tested. Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. The figure shows the effect on the plantar pain threshold on day 16 after model establishment. Figure 3A Day 23 Figure 3B ), Day 30 Figure 3C ) and the 37th day ( Figure 3D The plantar pain threshold was determined. Each data point corresponds to the mean gait score (±SEM, n=8). As shown in the figure: Control, control rats; Model, osteoarthritis rat model (mediator treatment); AM1, anti-Cx43 antibody ALMB-0166; 50% MWT, 50% probability of positive response at the stimulus intensity; ##, p<0.01 compared to control rats; Compared with the osteoarthritis model rats, p<0.05; Compared with the osteoarthritis model rats, p<0.01.

[0038] Figure 4 The effect of anti-Cx43 antibody treatment on hindlimb weight-bearing differences in a rat model of osteoarthritis was depicted. The rat model of osteoarthritis was established using pMMx+ACLT. Anti-Cx43 antibody was administered intravenously four times weekly, starting on days 15, 22, 29, and 36 post-model establishment (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody (15 mg / kg, 25 mg / kg, and 50 mg / kg) were tested. Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. Hindlimb weight-bearing differences were measured on days 1, 15, 16, 23, 30, and 37 post-model establishment. Each data point corresponds to a mean gait score (±SEM, n=8). Figures used: Control, control rats; Model, osteoarthritis rat model (mediator treatment); and AM1, anti-Cx43 antibody ALMB-0166.

[0039] Figures 5A-5D The effect of anti-Cx43 antibody treatment on weight-bearing differences in a rat model of osteoarthritis was depicted. The rat model of osteoarthritis was established using pMMx+ACLT. Anti-Cx43 antibody was administered intravenously four times weekly, starting on days 15, 22, 29, and 36 after osteoarthritis model establishment (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody (15 mg / kg, 25 mg / kg, and 50 mg / kg) were tested. Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. The figure shows the effect on weight-bearing differences on day 16 after osteoarthritis model establishment. Figure 5A Day 23 Figure 5B ), Day 30 Figure 5C ) and the 37th day ( Figure 5DDifferences in hindlimb weight-bearing in rats. Each data point corresponds to the mean gait score (±SEM, n=8). As shown in the figure: Control, control rats; Model, osteoarthritis rat model (mediated treatment); AM1, anti-Cx43 antibody ALMB-0166; ##, p<0.01 compared to control rats; Compared with the osteoarthritis model rats, p<0.01. Detailed Implementation

[0040] This disclosure provides methods and compositions for treating osteoarthritis in a subject or patient with this need, comprising administering at least one dose of an anti-Cx43 antibody to the subject or patient. In some embodiments, the anti-Cx43 antibody blocks or inhibits the opening of the Cx43 hemichannel. In some embodiments, the anti-Cx43 antibody comprises a specific CDR amino acid sequence. In some embodiments, the anti-Cx43 antibody is administered according to a dosing regimen.

[0041] I. Definition

[0042] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0043] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, and therefore variations are possible. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0044] As used in this article, the articles “a,” “one,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “one element” means one element or more.

[0045] The use of substitutes (e.g., "or") should be understood to mean any one, both or any combination of substitutes.

[0046] The term “and / or” should be understood to mean either or both of the substitutes.

[0047] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, dimensions, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length.

[0048] As used herein, the terms "substantially" or "truly" refer to a quantity, level, value, number, frequency, percentage, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 3%, 99%, or higher than a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length. In one embodiment, the terms "substantially identical" or "substantially identical" refer to a range of quantities, levels, values, numbers, frequencies, percentages, sizes, volumes, weights, or lengths that are approximately the same as a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length.

[0049] Throughout this specification, unless the context otherwise requires, the word "comprising" is to be understood as implying the inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements. In certain embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0050] "Composed of" means including and limited to anything that follows the phrase "composed of". Therefore, the phrase "composed of" indicates that the listed elements are necessary or mandatory, and that other elements may not be present.

[0051] "Substantially composed of..." means any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the specific activity or function of the listed element in this disclosure. Thus, the phrase "substantially composed of..." indicates that the listed element is necessary or mandatory, but no other element is optional and may or may not be present depending on whether they affect the activity or function of the listed element.

[0052] The term "provide" is used in its general sense for supplying or providing for use. In some embodiments, the protein is provided directly by administration (e.g., an antibody), while in other embodiments, the protein is provided efficiently by administration of a nucleic acid encoding the protein (e.g., an antibody). In some aspects, the invention contemplates compositions comprising various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0053] Throughout this specification, references to "an embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, indicate that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, a particular feature, structure, or characteristic may be combined in any suitable manner.

[0054] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a molecule having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, the term refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as polypeptides or proteins. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0055] As used herein, the term "sequence identity percentage" or "sequence identity" refers to the degree of identity between any given query sequence and a subject sequence. The percentage of identity of any query nucleic acid or amino acid sequence relative to another subject nucleic acid or amino acid sequence can be determined using tools and techniques known in the art, such as NCBI BLAST.

[0056] The term "pharmaceutical formulation" refers to a formulation containing a therapeutic agent (e.g., an anti-Cx43 antibody). It is in a form that allows the antibody's biological activity to be effective and does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0057] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T-cell receptor. In some embodiments, the binding portion other than the antibody is engineered to specifically bind the antigen, for example, an aptamer, an avimer, etc.

[0058] As used herein, the term "specific binding" is not intended to imply that an antibody binds exclusively to its intended target. Rather, an antibody is considered to have "specifically bound" if its affinity for its intended target is approximately 5 times greater than its affinity for a non-target molecule. Appropriately, there is no significant cross-reactivity or cross-binding with undesirable substances. An antibody's affinity for a target molecule can be, for example, at least 5 times greater than its affinity for a non-target molecule, such as 10 times, 25 times, especially 50 times, and particularly 100 times or more. In some embodiments, specific binding between an antibody or other binding agent and an antigen means at least 10... 6 M -1 The binding affinity. Antibodies can, for example, bind with at least about 10... 7 M -1 For example, in about 10 8 M -1 To about 10 9 M -1 Approximately 10 9 M -1 To about 10 10 M -1 Or about 10 10 M -1 To about 10 11 M -1 The antibodies bind with affinity between the antigens. Antibodies can bind, for example, at 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. As is known in the art, various immunoassays can be used to select antibodies that specifically respond to antigen-specific immune responses. For example, solid-phase ELISA is routinely used to select monoclonal antibodies that specifically respond to protein-specific immune responses. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press, 1988, which describes immunoassays and conditions that can be used to determine specific immunoreactivity.

[0059] Affinity-matured antibodies possess one or more alterations in one or more hypervariable regions, which lead to improved affinity for the antigen compared to parental antibodies without those alterations. In one aspect, affinity-matured antibodies may have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by methods known in the art. Marks et al., Biotechnology, 10:779-783 (1992) describes affinity maturation via VH and VL domain shuffling. The following literature describes the random mutagenesis of CDR and / or framework residues: Barbas et al., Proc Nat. Acad. Sci, USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992).

[0060] “Arthritis” is used in this article to refer to any condition that affects the joints. Symptoms typically include one or more of the following: joint pain, stiffness, tenderness, redness, heat, swelling, and reduced range of motion in the affected joint. In some types of arthritis, other organs may also be affected. The onset of arthritis in a subject can be gradual or sudden (e.g., caused by injury or infection). Exemplary types of arthritis include, but are not limited to, osteoarthritis, rheumatoid arthritis, gout, septic arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Still's disease. Arthritis may be a secondary condition caused by another primary disease such as Lyme disease, systemic lupus erythematosus, or Ehlers-Danlos syndrome.

[0061] As used in this article, “osteoarthritis” refers to a type of arthritis involving wear and tear on the articular cartilage. Osteoarthritis is caused by the deterioration of articular cartilage, which is made up of specialized cells called chondrocytes. Chondrocytes produce a collagenous extracellular matrix containing collagen and proteoglycans. Loss or damage to cartilage increases friction between the bones in the joint, which can lead to the growth of bone spurs (osteophytes) or the release of cartilage and bone fragments into the joint space. Symptoms of osteoarthritis include joint pain, stiffness, reduced range of motion, joint swelling, and weakness or numbness in the arms and legs. Osteoarthritis can occur in any joint, but it most commonly affects the hands and fingers, wrists, knees, hips, feet, neck, and back. The development of osteoarthritis can be due to age (e.g., wear and tear on cartilage over many years), or it can be caused by joint injury or infection.

[0062] The term "affinity" refers to the strength of the binding reaction between the binding domain and the epitope of an antibody. It is the sum of the attractive and repulsive forces acting between the binding domain and the epitope. As used herein, the term affinity refers to the dissociation constant KD.

[0063] The term "epitope" includes any determinant capable of specifically binding to immunoglobulins or T-cell receptors, preferably peptide determinants. In some embodiments, the epitope determinant includes chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. In one embodiment, an epitope is a region of an antigen that an antibody binds to. In some embodiments, an antibody-specifically-binding antigen is referred to as an antibody preferentially recognizing its target antigen in a complex mixture of proteins and / or macromolecules. Methods for epitope mapping are well known in the art, such as X-ray cocrystallation, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from consecutive amino acids or from both non-consecutive amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically consist of at least three, more often at least five, or eight to ten amino acids in a unique spatial conformation.

[0064] As used interchangeably in this document, the terms “reduction,” “inhibition,” and “blockage” refer to any statistically significant decrease in biological activity (e.g., half-channel opening). For example, “reduction” can refer to a decrease in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0065] II. Antibodies

[0066] The term “antibody” is used in the broadest sense herein and specifically encompasses full-length antibodies, antibody peptides or immunoglobulins, monoclonal antibodies, chimeric antibodies, polyclonal antibodies, human antibodies, humanized antibodies, and antibodies derived from non-human species, including human antibodies derived from human immunoglobulin sequences transduced into non-human species such as mice, sheep, chickens, or goats; recombinant antigen-binding forms such as monoclonal and biclonal antibodies, multispecific antibodies (e.g., bispecific antibodies); and individual antigen-binding fragments of any of the above, such as antibodies or antibodies derived therefrom, including dAb, Fv, scFv, Fab, F(ab)'2, Fab'.

[0067] Antibody-like binding peptide mimics are also covered in the methods described herein. Liu et al. (2003) described “antibody-like binding peptide mimics” (ABiP), which are peptides that act as simplified versions of antibodies and have some advantages such as a longer serum half-life and a less cumbersome synthetic method.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies comprising that group are identical and / or bind to the same epitopes. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method.

[0069] The antigen-binding fragment of the antibody preferably contains at least the variable region of the heavy chain and / or light chain of the anti-Cx43 antibody. For example, the antigen-binding fragment of the anti-Cx43 antibody may contain the amino acid sequences of SEQ ID NO: 7 and 8. Examples of such antigen-binding fragments include Fab fragments, Fab' fragments, Fv fragments, scFv, and F(ab')2 fragments. The antigen-binding fragment of the antibody can be generated by enzymatic cleavage or by recombinant technology. For example, Fab or F(ab')2 fragments can be generated by cleavage with papain or pepsin, respectively. Antibodies can also be generated in various truncated forms using antibody genes with one or more stop codons introduced upstream of the natural termination site. For example, a recombinant construct encoding the heavy chain of the F(ab')2 fragment can be designed to include a DNA sequence encoding the CH1 domain and hinge region of the heavy chain. In one aspect, the antigen-binding fragment blocks or inhibits the opening of the Cx43 hemichannel in a subject and the effects associated with the development of the Cx43 hemichannel.

[0070] As used herein, a “multispecific antibody” refers to an artificial antibody having two or more distinct portions, each portion comprising an antigen-binding region targeting a different antigen or epitope. The portions of a multispecific antibody can be, for example, a full-length antibody or an antibody-binding fragment. A bispecific or bifunctional antibody is a multispecific antibody having two distinct portions (e.g., two different heavy chain and light chain pairs, or two different antibody-binding fragments) binding to two different antigens or epitopes. Multispecific antibodies can be produced by a variety of methods, including hybridoma fusion or linking of antibody-binding fragments. For example, Songsivilai and Lachmann, Clin Exp Immunol, 79: 315-21, 1990; Kostelny et al., J. Immunol., 148: 1547-53 (1992). In some cases, multispecific antibodies (e.g., bispecific antibodies) include at least one portion of an anti-Cx43 antibody that blocks or inhibits the opening of the Cx43 hemichannel in a subject and the effects associated with the opening of the Cx43 hemichannel.

[0071] "Therapeutic monoclonal antibodies" are antibodies used in therapies for human subjects. The therapeutic monoclonal antibodies disclosed herein include anti-Cx43 antibodies. Antibody "effector function" refers to those biological activities attributable to the Fc region of the antibody (the native sequence Fc region or the Fc region of an amino acid sequence variant). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. To assess the ADCC activity of the molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337.

[0072] The anti-Cx43 antibody disclosed herein can also be conjugated to at least one pharmaceutical agent or portion to form an antibody conjugate. The antibody can be covalently or non-covalently linked to the pharmaceutical agent. The pharmaceutical agent or portion can enhance the diagnostic or therapeutic potential of the antibody and includes, but is not limited to, effector molecules or reporter molecules. Effector molecules include molecules with desired activity (e.g., cytotoxic activity). Non-limiting examples of effector molecules that can be conjugated to the antibody include toxins, small molecule drugs, therapeutic enzymes, cytokines, antibiotics, radiolabeled nucleotides, etc. Reporter molecules are molecules detectable by assay and include, but are not limited to, enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photosynthetic molecules, colored particles, or ligands (e.g., biotin).

[0073] Full-length antibodies can be assigned to different “classes” based on the amino acid sequence of their heavy chain constant domain. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into “subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of the different classes of antibodies are well known. The “light chain” of antibodies from any vertebrate species can be assigned to one of two distinct types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain.

[0074] Parts of this invention, such as polypeptides, peptides, antigens, or immunogens, may be covalently or non-covalently conjugated or linked to other parts such as adjuvants, proteins, peptides, supports, fluorescent moieties, or labels. The terms "conjugate" or "immunoconjugate" are used broadly to define the effective association of one part with another agent and are not intended to refer only to any type of effective association, and are particularly not limited to chemical "conjugation".

[0075] When used in this article, the term "hypervariate region" refers to the amino acid residues in an antibody that are responsible for antigen binding. Hypervariable regions typically contain amino acid residues from the complementarity-determining region (CDR) or CDR (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues from the hypervariable ring (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and residues 26-32 (L1), 50-52 (L2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mal. Biol.). 196:901-917 (1987)). “Frame region” or “FR” residues are variable domain residues other than hypervariable region residues as defined herein. Hypervariable regions or their CDRs can be transferred from one antibody chain to another or to another protein to confer antigen-binding specificity to the resulting (complex) antibody or binding protein.

[0076] As will be understood by those skilled in the art, the CDRs disclosed herein may also include variants. Typically, the amino acid identity between the various variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant CDR” is a CDR that has a specific identity with the parental CDR of the present invention and shares biological functions, including but not limited to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parental CDR.

[0077] Amino acid substitutions are typically single-residue substitutions; insertions are usually in the range of about one to about twenty amino acid residues, although larger insertions can be tolerated. Deletions range from about one to about twenty amino acid residues, although in some cases deletions can be much larger.

[0078] Substitution, deletion, insertion, or any combination thereof can be used to obtain the final derivative or variant. Typically, these changes are made on a few amino acids to minimize alterations to the molecular structure, particularly the immunogenicity and specificity of antigen-binding proteins. However, in some cases, larger changes can be tolerated.

[0079] As used herein, the term “Fab” or “Fab region” refers to a polypeptide containing the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to the isolated region, or the region in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody implementation described herein.

[0080] As used herein, the terms “Fv,” “Fv fragment,” or “Fv region” refer to a polypeptide containing the VL and VH domains of a single antibody.

[0081] As used herein, the term "framework" refers to the region of an antibody variable domain that excludes those regions defined as CDRs. Each antibody variable domain frame can be further subdivided into consecutive regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0082] The “humanized” form of a non-human (e.g., rodent) antibody is a chimeric antibody containing a minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody) with the desired specificity, affinity, and ability, such as mouse, rat, rabbit, or non-human primate. In some cases, the frame region (FR) residues of the human antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the receptor or donor antibody. These modifications are made to further improve antibody performance. For further details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0083] "Isolated" antibodies are antibodies that have been identified and isolated and / or recovered from components of their native environment. In some embodiments, the antibody will be purified (1) to a protein content greater than 95% by weight, as determined by the Lowry method, and alternatively, greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a rotary cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Brilliant Blue or silver staining. Isolated antibodies include recombinant intracellular in situ antibodies, since at least one component of the antibody's native environment will be absent. However, typically, isolated antibodies will be prepared by at least one purification step.

[0084] Anti-Cx43 antibody

[0085] Certain aspects of this disclosure provide compositions and methods for treating osteoarthritis in subjects. The methods and compositions comprise an anti-Cx43 antibody that specifically binds to Cx43 hemichannels and blocks or inhibits the opening of Cx43 hemichannels in chondrocytes.

[0086] The anti-Cx43 antibody can be any antibody known in the art that specifically binds to Cx43. In various embodiments, the anti-Cx43 antibody used in this method comprises an HCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 1, an HCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 2, and an HCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 3; and / or an LCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 4, an LCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 5, and an LCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 6. In some specific embodiments, the anti-Cx43 antibody comprises the same HCDR1 amino acid sequence as SEQ ID NO: 1, the same HCDR2 amino acid sequence as SEQ ID NO: 2, and the same HCDR3 amino acid sequence as SEQ ID NO: 3; and / or the same LCDR1 amino acid sequence as SEQ ID NO: 4, the same LCDR1 amino acid sequence as SEQ ID NO: 5, and the same LCDR3 amino acid sequence as SEQ ID NO: 6.

[0087] In various embodiments, the anti-Cx43 antibody used in this method comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 9 and 11-18; and / or a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 9; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 11; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 12; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 13; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 14; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 15; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10.In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 16; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 17; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 18; and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10.

[0088] In some embodiments, the anti-Cx43 antibody comprises the same heavy chain as any one of SEQ ID NO: 9 and 11-18, and / or the same light chain as any one of SEQ ID NO: 10. In a preferred embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 9, and the same light chain as any one of SEQ ID NO: 10. In one embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 11, and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 12, and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 13, and the same light chain as any one of SEQ ID NO: 10. In yet another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 14, and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 15 and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 16 and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 17 and the same light chain as any one of SEQ ID NO: 10. In another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 18 and the same light chain as any one of SEQ ID NO: 10.

[0089] In several embodiments, this document provides antibodies that bind to epitopes located partially or entirely within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitope may comprise one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, or I10 of SEQ ID NO: 19. In one embodiment, the epitope consists of F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In some embodiments, the epitope may comprise all ten amino acids of SEQ ID NO: 19. In some embodiments, the epitope consists of all ten amino acids of SEQ ID NO: 19.

[0090] The anti-Cx43 antibody is substantially pure and, ideally, substantially homogeneous (i.e., free of contaminating proteins, etc.). "Substantially pure" antibody means a composition containing at least about 90%, at least about 95%, or 97% of the antibody by weight based on the total weight of the protein in the composition. "Substantially homogeneous" antibody means a composition containing protein in which at least about 99% of the protein by weight is a specific antibody, for example, an anti-Cx43 antibody.

[0091] In some embodiments, the anti-Cx43 antibody is a humanized antibody. In some embodiments, the anti-Cx43 antibody is a monoclonal antibody. In some embodiments, the anti-Cx43 antibody is a humanized monoclonal antibody.

[0092] Pharmaceutical formulations containing anti-Cx43 antibodies

[0093] One aspect of this disclosure provides a pharmaceutical formulation for treating osteoarthritis in a subject, comprising an anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody blocks or inhibits the opening of Cx43 hemichannels in bone cells. The anti-Cx43 antibody may be a complete antibody or an antigen-binding fragment thereof.

[0094] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, buffers, and other physiologically compatible excipients. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, active compounds, such as small molecules or biopharmaceuticals, may be coated in the material to protect the compound from acids and other natural conditions that can inactivate the compound.

[0095] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions, as well as conventional excipients for the preparation of tablets, pills, capsules, etc. The use of such media and agents in the formulation of active pharmaceutical ingredients is known in the art. Unless any conventional media or agent is incompatible with the active compound to some extent, its use in the pharmaceutical compositions provided herein should be considered. Additional active compounds may also be incorporated into the compositions.

[0096] Pharmaceutically acceptable carriers may include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0097] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. When needed, appropriate flowability can be maintained, for example by using a coating material such as lecithin, by maintaining the desired particle size in the case of a dispersion, or by using surfactants such as polysorbate, sodium lauryl sulfate, and nonionic surfactants. In many cases, including isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, in the composition can be useful. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as monostearate and gelatin, in the composition.

[0098] These compositions may also contain functional excipients, such as preservatives, wetting agents, emulsifiers, and dispersants.

[0099] Therapeutic compositions must generally be sterile, pyrogen-free, and stable under the conditions of preparation and storage. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above into a suitable solvent, followed by sterilization, for example, by microfiltration (if required). Dispersions are typically prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods include vacuum drying and lyophilization (freeze-drying), which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution. The active agent can be mixed under sterile conditions with other pharmaceutically acceptable carriers and any preservatives, buffers, or propellants that may be required.

[0100] The presence of microorganisms can be prevented by both the sterilization process described above and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It is also desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, prolonged absorption of injectable drugs can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0101] The pharmaceutical compositions described herein may also have various viscosities or osmotic pressures. Methods for measuring the viscosity of antibody formulations are known to those skilled in the art and may include, for example, a rheometer (e.g., an Anton Paar MCR301 rheometer with 50 mm, 40 mm, or 20 mm plate attachments). Methods for measuring the osmotic pressure of antibody formulations are known to those skilled in the art and may include, for example, an osmometer (e.g., an Advanced Instrument Inc 2020 freezing point depression osmometer).

[0102] The pharmaceutical compositions described herein may also have multiple pH levels. The pH of the pharmaceutical composition can be adjusted by any method known in the art, such as, for example, by adding a buffer.

[0103] In some embodiments, the pharmaceutical composition comprises the anti-Cx43 antibody described herein, a histidine / histidine hydrochloride buffer, polysorbate 80, and sucrose. In some embodiments, the pharmaceutical composition comprises about 40 mg / mL to about 60 mg / mL of the anti-Cx43 antibody described herein; about 10 mM to about 40 mM of histidine / histidine hydrochloride buffer; about 0.005% w / v to about 0.05% w / v of polysorbate 80; and about 1% w / v to about 20% w / v of sucrose. In some embodiments, the pharmaceutical composition comprises about 50 mg / mL of the anti-Cx43 antibody described herein; about 20 mM of histidine / aspartate buffer; about 0.02% w / v of polysorbate 80; and about 8% w / v of sucrose. In some embodiments, the pharmaceutical composition has a pH of about 5.4 to about 5.6. In some embodiments, the pharmaceutical composition has a pH of about 5.5.

[0104] Formulating parenteral compositions or unit dosage forms may be advantageous for ease of administration and dosage uniformity. As used herein, a unit dosage form refers to a physically discrete unit suitable as a unit dose for a patient to be treated: each unit contains a predetermined amount of active agent calculated to produce the desired therapeutic effect, along with any desired drug carrier. The specifications of a unit dosage form are determined by and directly depend on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the techniques used to formulate such active compounds for the sensitivity of the individual being treated.

[0105] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and administration mode without toxicity to the patient. As used herein, in the context of administration, “parenteral” means administration modes other than enteral and local administration, typically by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0106] As used herein, the phrases “parenteral administration” and “extragastric administration” refer to modes of administration other than intravenous (i.e., via the digestive tract) and local administration, typically by injection or infusion, and including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Intravenous injection and infusion are commonly (but not exclusively) used for antibody administration.

[0107] The formulation described herein is administered, preferably to human subjects requiring treatment with anti-Cx43 antibodies, according to known methods, such as intravenous administration via bolus injection or continuous infusion over a period of time, via intramuscular, intradermal, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, local, or inhalation routes.

[0108] In some implementations, the anti-Cx43 antibody is administered to the subject via intravenous or subcutaneous (i.e., under the skin). For this purpose, a syringe can be used to inject the formulation. However, other devices for administering the formulation are available, such as injection devices (e.g., INJECT-EASE™ and GENJECT™ devices); injection pens (e.g., GENPEN™); auto-injector devices, needle-free devices (e.g., MEDIJECTOR™ and BIOJECTOR™); and subcutaneous patch delivery systems.

[0109] In specific embodiments, this disclosure relates to kits for single-dose administration. Such kits comprise containers of aqueous formulations of therapeutic proteins or antibodies, including both single-compartment and multi-compartment pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0110] III. Methods for treating osteoarthritis

[0111] Certain aspects of this disclosure provide methods for treating osteoarthritis in subjects. These methods may include blocking the opening of Cx43 hemichannels in bone cells of the subject in need by using, for example, an anti-Cx43 antibody. Cx43 modulation may be a standalone therapy for osteoarthritis or in combination with other therapies. Osteoarthritis can affect any joint in the subject, such as the hands and fingers, wrists, knees, hips, feet, neck, and back.

[0112] Osteoarthritis is a common type of arthritis that leads to the degeneration of joints, including articular cartilage and subchondral bone. The pathological features of osteoarthritis include the loss of articular cartilage, resulting in narrowing of the joint space, increased joint friction, and potential structural remodeling. Current treatment options for osteoarthritis include exercise, lifestyle modifications, pain medication, and, in severe cases, joint replacement surgery. However, specific pharmacological interventions for treating osteoarthritis are lacking.

[0113] Various cells can communicate with each other and with the extracellular environment through hemichannels and gap junctions formed by connexins. Connexins are widely expressed throughout the body. Six connexins form a hemichannel, and two hemichannels form a gap junction channel. Gap junctions are clusters of channels located in the plasma membrane between adjacent cells that mediate intercellular communication. Hemichannels are separate entities from gap junction channels. Hemichannels allow molecular exchange between intracellular compartments and the extracellular environment.

[0114] Chondrocytes express a hemichannel called the connexin (Cx)43 hemichannel. Under normal conditions, the Cx43 hemichannel in chondrocytes remains closed. Inflammatory conditions and mechanical stimulation can induce the opening of the Cx43 hemichannel, leading to the release of pro-inflammatory factors that contribute to inflammation associated with osteoarthritis. Therefore, inhibiting the opening of the Cx43 hemichannel in chondrocytes (e.g., using an anti-Cx43 antibody) can suppress the development and progression of osteoarthritis in subjects.

[0115] Cx43, also known as gap junction α-1 protein (GJA1), is a 43.0 kDa protein consisting of 382 amino acids (NCBI reference sequence: NP 000156.1). GJA1 contains a long C-terminal tail, an N-terminal domain, and multiple transmembrane domains. The protein crosses the phospholipid bilayer four times, exposing its C-terminus and N-terminus to the cytoplasm. The C-terminal tail consists of 50 amino acids and includes post-translational modification sites, as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. Therefore, the C-terminal tail is crucial for regulating functions such as pH gating and channel assembly. Notably, the DNA region encoding this tail in the GJA1 gene (NCBI gene ID: 2697) is highly conserved, suggesting it is either resistant to mutation or becomes lethal upon mutation. Meanwhile, the N-terminal domain is involved in channel gating and oligomerization, thus controlling the switching between the open and closed states of channels. The transmembrane domains form gap junction channels, while the extracellular loop facilitates proper channel docking. In addition, the two extracellular rings form disulfide bonds, which interact with the two hexamers to form complete gap junction channels.

[0116] “Treatment” refers to therapeutic treatment. The population requiring treatment includes those who already have a disease. Therefore, the subjects to be treated in this document (e.g., people) may have been diagnosed with a disease such as osteoarthritis. A disease, such as osteoarthritis, is “inhibited” or “treated” if at least one symptom of the condition (as determined by responsiveness / non-responsiveness or indicators known in the art and described herein) is reduced, terminated, slowed, minimized, or prevented. The terms “patient” and “subject” are used interchangeably herein.

[0117] The terms "subject" or "patient" refer to a human or non-human being, such as a primate, mammal, or vertebrate. In a particular implementation, the subject is a human.

[0118] Treatment may be appropriately administered to subjects who have, possess, are susceptible to, or are at risk of developing osteoarthritis, particularly humans. Subjects “at risk” can be identified in any objective or subjective manner, by diagnostic testing or the opinion of the subject or healthcare provider. Identification of subjects requiring such treatment may be made by the judgment of the subject or healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by testing or diagnostic methods).

[0119] Anti-Cx43 antibodies used in any of the methods disclosed herein may be stored as lyophilized solids or aqueous formulations or any other form known in the art. When anti-Cx43 antibodies are stored as lyophilized solids, the antibody is reconstituted in a solution such as water (e.g., for injection) prior to administration. If prepared for infusion from a lyophilized or aqueous formulation, the final concentration of the anti-Cx43 antibody, for example, after diluting the reconstituted antibody (e.g., in a saline, Ringer's, or 5% glucose infusion system), may be from about 0.1 mg / ml to about 80 mg / ml for administration. Final concentrations may be from about 0.1 mg / ml to about 80 mg / ml, from about 0.5 mg / ml to about 70 mg / ml, from about 1 mg / ml to about 60 mg / ml, from about 5 mg / ml to about 50 mg / ml, from about 10 mg / ml to about 40 mg / ml, from about 15 mg / ml to about 30 mg / ml, or from about 20 mg / ml to about 25 mg / ml. In some implementations, the final dosage form concentration may be about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, or higher than 80 mg / ml.

[0120] As used herein, the term "effective amount" refers to the amount of a drug (e.g., an anti-Cx43 antibody) sufficient to achieve the treatment, prognosis, or diagnosis of osteoarthritis when administered to a patient or subject. Dosing regimens may be adjusted to provide an optimal therapeutic response. An effective amount is also the amount in which any toxic or harmful effects (side effects) of the drug are minimized and / or exceeded by beneficial effects. Therapeutic effective amounts can vary depending on the patient and disease condition being treated, the patient's weight and age, the severity of the disease condition, the route of administration, the course of the disease, the patient's clinical history, and the response to the anti-Cx43 antibody, which can be readily determined by one of ordinary skill in the art. For example, the effective amount or dose of an anti-Cx43 antibody ranges from about 0.01 mg / kg to about 1000 mg / kg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is about 0.01 mg / kg to about 900 mg / kg, about 0.1 mg / kg to about 800 mg / kg, about 0.5 mg / kg to about 700 mg / kg, about 1 mg / kg to about 600 mg / kg, about 1.5 mg / kg to about 500 mg / kg, about 2 mg / kg to about 400 mg / kg, about 5 mg / kg to about 300 mg / kg, about 10 mg / kg to about 200 mg / kg, about 15 mg / kg to about 100 mg / kg, about 20 mg / kg to about 50 mg / kg, about 25 mg / kg to about 45 mg / kg, or about 30 mg / kg to about 40 mg / kg. In some implementations, the effective amount or dose of the anti-Cx43 antibody is about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, or greater than 1000 mg / kg.

[0121] In specific embodiments, the effective amount or dose of the anti-Cx43 antibody is from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is 15 mg / kg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is 25 mg / kg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is 50 mg / kg.

[0122] In some embodiments, the effective amount or dose of the anti-Cx43 antibody ranges from about 1 mg to about 800 mg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is about 2 mg to 7000 mg, about 5 mg to 6000 mg, about 10 mg to 5000 mg, about 15 mg to 4000 mg, about 20 mg to 3000 mg, about 25 mg to 2000 mg, about 30 mg to 1000 mg, about 40 mg to 900 mg, about 50 mg to 800 mg, about 60 mg to 700 mg, about 70 mg to 600 mg, about 80 mg to 500 mg, about 90 mg to 400 mg, about 100 mg to 300 mg, or about 150 mg to 250 mg. In some embodiments, the effective amount or dose of the anti-Cx43 antibody is less than 1 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, about 2000 mg, about 2250 mg, about 2500 mg. mg, approximately 2750 mg, approximately 3000 mg, approximately 4000 mg, approximately 5000 mg, approximately 6000 mg, approximately 7000 mg, approximately 8000 mg or more.

[0123] In the methods described herein, an effective amount or at least one dose of the anti-Cx43 antibody is administered in the following manner: approximately once daily, approximately every 2 days, approximately every 3 days, approximately every 4 days, approximately every 5 days, approximately every 6 days, approximately once a week, approximately every 8 days, approximately every 9 days, approximately every 10 days, approximately every 11 days, approximately every 12 days, approximately every 13 days, approximately every 2 weeks, approximately every 15 days, approximately every 16 days, approximately every 17 days, approximately every 18 days, approximately every 19 days, approximately every 20 days, approximately every 3 weeks, approximately every 22 days, approximately every 23 days, approximately every 24 days, approximately every 25 days, approximately every 26 days, approximately every 27 days, approximately every 4 weeks, or approximately every 29 days. Approximately every 30 days, approximately every 31 days, approximately every 32 days, approximately every 33 days, approximately every 34 days, approximately every 5 weeks, approximately every 36 days, approximately every 37 days, approximately every 38 days, approximately every 39 days, approximately every 40 days, or approximately every 41 days, approximately every 6 weeks, approximately every 7 weeks, approximately every 8 weeks, approximately every 9 weeks, approximately every 10 weeks, approximately every 11 weeks, approximately every 12 weeks, approximately every 13 weeks, approximately every 15 weeks, approximately every 16 weeks, approximately every 17 weeks, approximately every 18 weeks, approximately every 19 weeks, approximately every 20 weeks, approximately every 21 weeks, approximately every 22 weeks, approximately every 23 weeks, approximately every 24 weeks or 6 months, or approximately every 24 weeks or 6 months.

[0124] In some specific implementations, an effective amount or dose of the anti-Cx43 antibody is administered approximately once a week. In one particular implementation, the dose of the anti-Cx43 antibody is administered once a week. In another particular implementation, the dose of the anti-Cx43 antibody is administered once a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 weeks.

[0125] According to certain embodiments of this disclosure, multiple doses of an anti-Cx43 antibody (or a pharmaceutical composition comprising an anti-Cx43 antibody and any of the other therapeutically active agents mentioned herein) can be administered to a subject over a defined time period. A method according to this aspect of the disclosure includes sequentially administering multiple doses of the disclosed anti-Cx43 antibody to a subject.

[0126] As used herein, “sequential administration” means administering each dose of anti-Cx43 antibody to a subject at different time points, for example, on different dates separated by predetermined intervals (e.g., hours, days, weeks, or months). This disclosure includes a method comprising sequentially administering to a patient a single initial dose of anti-Cx43 antibody, followed by a second dose of anti-Cx43 antibody, followed by a third dose of anti-Cx43 antibody, followed by a fourth dose of anti-Cx43 antibody, and optionally followed by one or more subsequent doses of anti-Cx43 antibody. The anti-Cx43 antibody may be administered at doses from 0.01 mg / kg to about 1000 mg / kg.

[0127] The terms "first dose," "second dose," "third dose," and "fourth dose" refer to the temporal sequence of administration of the anti-Cx43 antibody of this disclosure. Thus, "first dose" is the dose administered at the start of the treatment regimen (also known as the "baseline dose"); "second dose" is the dose administered after the first dose; "third dose" is the dose administered after the second dose; and "fourth dose" is the dose administered after the third dose. The first, second, third, and fourth doses may all contain the same amount of anti-Cx43 antibody, but may generally differ from each other in terms of administration frequency. However, in some embodiments, the amount of anti-Cx43 antibody contained in the first, second, third, fourth, and / or subsequent doses differs from each other during the treatment process (e.g., appropriately adjusted up or down).

[0128] In some embodiments of the method disclosed herein, the method includes administering a first dose and a second dose of anti-Cx43 antibody. In some embodiments, the first dose of anti-Cx43 antibody is administered on day 1 of treatment. In some embodiments, the second dose of anti-Cx43 antibody is administered on day 8 of treatment. In some embodiments, the method disclosed herein includes administering a third dose of anti-Cx43 antibody. In some embodiments, the third dose of anti-Cx43 antibody is administered on day 15 of treatment. In some embodiments, the method further includes administering a fourth dose of anti-Cx43 antibody. In some embodiments, the fourth dose of anti-Cx43 antibody is administered on day 22 of treatment. In some embodiments, the method further includes administering subsequent doses of anti-Cx43 antibody after the fourth dose. In some embodiments, subsequent doses of anti-Cx43 antibody are administered weekly after the fourth dose.

[0129] In some embodiments of the method disclosed herein, the method includes administering an effective amount of anti-Cx43 antibody according to the following dosing regimen: a first dose on day 1 of treatment, a second dose on day 8 of treatment, a third dose on day 15 of treatment, and a fourth dose on day 22 of treatment. In some embodiments, the method further includes administering subsequent doses of anti-Cx43 antibody weekly after the fourth dose.

[0130] An effective dose or at least one dose of anti-Cx43 antibody may be administered to the patient once or as part of a series of treatments, and may be administered at any time from the onset of diagnosis. Anti-Cx43 antibody may be used as a monotherapy or in combination with other medications or therapies available for the treatment of osteoarthritis.

[0131] In the methods described herein, an effective amount or dose of anti-Cx43 antibody may be administered to a patient within less than 5 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes. In some specific embodiments, an effective amount or dose of anti-Cx43 antibody may be administered to a patient within about 30 minutes. In one specific embodiment, an effective amount or dose of anti-Cx43 antibody may be administered intravenously to a patient within 30 minutes.

[0132] In the methods described herein, anti-Cx43 antibodies are administered to a patient. If the anti-Cx43 antibody is in a solid, such as a dry, formulation, the administration process may include a step of converting the formulation into a liquid state. In one aspect, the dry formulation may be used for injection, such as intravenous, intradermal, intramuscular, intraperitoneal, or subcutaneous injection, for example, by liquid reconstruction as described above. In another aspect, the solid or dry formulation may be applied topically, for example, in the form of a patch, cream, aerosol, or suppository.

[0133] In the methods described herein, the anti-Cx43 antibody may be administered to the subject alone or in combination with another therapy. The anti-Cx43 antibody may be administered before, simultaneously with, or after the administration of the additional therapy. In one embodiment, the dose of the co-administered therapy may be reduced over time or completely gradually reduced during treatment with the anti-Cx43 antibody. In some embodiments, the additional therapy includes one or more of physical therapy, adaptive support, steroids, surgery (e.g., joint replacement surgery), cell-based therapies, or small molecule drugs.

[0134] In one aspect, the improved effectiveness of the combination according to this disclosure can be demonstrated by achieving a therapeutic synergy. The term "therapeutic synergy" or "synergistic effect" is used when a combination of two products at a given dose is more effective than each of the two optimal individual products at the same dose. In one instance, the therapeutic synergy can be assessed by comparing the combination to the optimal single agent using estimates obtained from a two-way analysis of variance with repeated measures (e.g., time factor) of osteoarthritis severity parameters.

[0135] Before, during, or after treatment with anti-Cx43 antibodies, osteoarthritis-related parameters of patients or subjects should be examined using techniques and methods known in the art. Non-limiting examples of common medical techniques and methods used for examining and diagnosing osteoarthritis include: physician physical examination, physical fitness tests, blood tests, X-rays, magnetic resonance imaging (MRI) radiography, computed tomography (CT) scans, Western University and McMaster University Osteoarthritis Index (WOMAC), Knee Injury and Osteoarthritis Outcomes Score (KOOS) questionnaire, quality of life (QOL) assessments (e.g., using the SF-12 scale or the Quality of Life Assessment-6 Dimensions or the “AQoL-6D” method), Patient Global Impression Change (PGIC) questionnaire, maximal voluntary isometric force measurement (e.g., using a dynamometer), dynamic weight-bearing (DMB) testing, observational gait scoring (OGS), and digital rating scales for assessing pain intensity. The selection of techniques and methods, as well as the frequency of examinations, can be determined and / or adjusted by those skilled in the art based on the specific circumstances of the patient or subject.

[0136] In some implementations, improvement in the subject's osteoarthritis is assessed before, during, or after treatment with the anti-Cx43 antibody, as evaluated by at least one indicator of osteoarthritis severity. In some implementations, at least one indicator of osteoarthritis severity is assessed at least one day to one week after administration of an effective amount or dose of the anti-Cx43 antibody to the subject. In some implementations, at least one indicator of osteoarthritis severity is assessed one, two, three, four, five, six, or one week after administration of an effective amount or dose of the anti-Cx43 antibody to the subject. In some implementations, at least one indicator of osteoarthritis severity includes the pain threshold of arthritis, gait score, and / or weight-bearing variation.

[0137] In some implementations, treatment of osteoarthritis with anti-Cx43 antibodies improves disease symptoms, such as as assessed by differences in pain threshold, gait scores, and / or weight-bearing capacity of arthritis compared to a control group, which may include, but is not limited to, osteoarthritis populations who have received or have not received the same line of treatment as the subject but have not received anti-Cx43 antibodies for osteoarthritis.

[0138] In some implementations, subjects treated with anti-Cx43 antibodies for osteoarthritis have a response duration of at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 1 month, at least 2 months, at least 3 months, or longer.

[0139] In some implementations, subjects treated with anti-Cx43 antibodies for osteoarthritis had response times of less than 3 months, less than 2 months, less than 1 month, less than 25 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

[0140] As used herein, “pain threshold” refers to the minimum intensity at which a given stimulus is perceived as pain, and is relatively constant between individuals for a given stimulus. Injury or disease (e.g., osteoarthritis) may cause a subject’s pain threshold to be lowered at a specific location (e.g., a limb). Pain threshold can be a mechanical pain threshold or a pressure pain threshold, and can be measured locally (e.g., at the affected joint) or remotely (e.g., at adjacent muscles) by any suitable method known in the art.

[0141] In some embodiments, treatment of a subject with osteoarthritis with at least one dose of anti-Cx43 antibody improves the subject's pain threshold for arthritis. In some embodiments, treatment of a subject with osteoarthritis with at least one dose of anti-Cx43 antibody increases the pain threshold for arthritis by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more.

[0142] As used herein, “gait” refers to the manner in which a limb moves during movement. A subject’s gait may be affected by the presence of a disease or condition that influences movement (e.g., osteoarthritis). “Gait score” refers to any scale or scoring system known in the art for assessing and analyzing a subject’s gait. Depending on the gait scoring system used, an improvement in a subject’s gait may be reflected as an increase or decrease in the subject’s gait score.

[0143] In some embodiments, treatment of a subject with osteoarthritis with an anti-Cx43 antibody improves the subject's gait score. In some embodiments, treatment of a subject with osteoarthritis with at least one dose of an anti-Cx43 antibody improves the subject's gait score by at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.2, at least 1.5, at least 1.8, at least 2, at least 2.2, at least 2.5, at least 3, at least 3.5, or more. In some implementations, treatment of a subject with osteoarthritis with at least one dose of anti-Cx43 antibody resulted in a gait score improvement of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more.

[0144] As used herein, “load-bearing” refers to the amount of weight or force that a particular limb is able to maintain. Load-bearing can be determined by any method known in the art. A reduction in load-bearing can be observed in cases of injury or disease (e.g., osteoarthritis), and / or a reduction in load-bearing can be recommended by a physician to reduce the risk of injury or pain in the subject. As used herein, “load-bearing variance” refers to the difference in load-bearing between limbs during a state of injury or disease (e.g., osteoarthritis) compared to the load-bearing of the corresponding limb without injury or disease. A decrease in load-bearing variance indicates that the limb is bearing load closer to its full load.

[0145] In some embodiments, treatment of subjects with osteoarthritis with anti-Cx43 antibodies improves subject weight-bearing variability. In some embodiments, treatment of subjects with osteoarthritis with at least one dose of anti-Cx43 antibodies reduces subject weight-bearing variability by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% or more.

[0146] As used herein, a “response” or “having a response” to treatment means that a subject has an improvement in at least one parameter of disease progression. A subject may have a partial or complete response to treatment. The response to treatment can be determined based on methods known in the art. Those skilled in the art can determine appropriate methods based on the type of disease being assessed. Non-limiting examples of methods include pain thresholds for arthritis, gait scores, and differences in weight-bearing capacity, as well as other methods described herein.

[0147] Example

[0148] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention and can therefore be considered as constituting preferred modes for its practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the invention, and similar or analogous results can still be obtained.

[0149] Example 1: Effect of anti-Cx43 antibody treatment on a rat model of osteoarthritis

[0150] This example describes the evaluation of the efficacy of anti-Cx43 treatment in a rat model of osteoarthritis.

[0151] Research Objective

[0152] The aim of this study was to test the efficacy of anti-Cx43 antibody ALMB-0166 in treating osteoarthritis symptoms in a rat model of osteoarthritis induced by partial meniscectomy and anterior cruciate ligament resection (pMMx + ACLT), when administered via weekly intravenous injection for four weeks.

[0153] Regulatory compliance

[0154] This experiment follows the standard operating procedure (SOP).

[0155] Materials and methods

[0156] Test materials

[0157] The anti-Cx43 antibody used in this study was ALMB-0166 (AlaMab Therapeutics Inc.). Duloxetine hydrochloride (MedChemExpress) was used as a positive control. Other materials used in this study included isoflurane (RWD LifeScience, Inc.), phosphate-buffered saline (Thermo), and physiological saline (Anhui Shuanghe Pharmaceutical Co., Ltd.).

[0158] For animal administration, 0.5 mg / mL duloxetine hydrochloride was prepared by dissolving the drug in physiological saline, while two ALMB-0166 solutions were prepared by diluting the antibody stock solution with PBS, one at 3 mg / mL and the other at 5 mg / mL.

[0159] equipment

[0160] The equipment used in this study included a non-rebreathing anesthesia machine (Midmark Corporation, model VWR), a VonFrey filament (IITC, Life Science, USA), and a Linton incapacitance tester (UK).

[0161] Animals and breeding

[0162] Male Sprague Dawley (SD) rats (specific pathogen-free grade; Shanghai Silex Laboratory Animal Co., Ltd.) were obtained for this study. Each rat was weighed at the start of treatment, with an average weight range of 200–220 grams. Upon arrival at the testing facility, the animals were housed in cages of five and allowed to acclimatize for 5–7 days before the start of treatment.

[0163] Animals were kept in an environment maintained at a temperature of 23±2℃, humidity of 40-70%, and a 12-hour light / 12-hour dark cycle. The 12-hour dark cycle was temporarily interrupted to allow for adaptation to the study procedure. SPF rats were provided with free access to growth and reproduction feed (Beijing Keao Xieli Feed Co., Ltd.) throughout the study period. Reverse osmosis water was freely available. Animals had free access to food and water throughout the study.

[0164] The animals used in the study were selected based on their overall health and adaptation to cage rearing; a total of 48 rats were selected to participate in the study.

[0165] Research Design

[0166] Animal group and drug

[0167] For this study, the animal groups were formed as shown in Table 1 below. The appropriate treatment was initiated one week after partial medial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT) in rats.

[0168] Table 1: Treatment and administration information for the animal test group.

[0169]

[0170] Models and Methods

[0171] After animals acclimatized, their normal pain threshold was assessed using the Von Frey method or the up-and-down method (on day 1 of the study), and bipedal balance was assessed using a bipedal balance tester on the operated side (right hind limb). Rats were then anesthetized with isoflurane, and the knee joint was shaved and disinfected. Osteoarthritis was induced in animals via partial medial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT). Briefly, the skin and joint cavity near the medial aspect of the right knee were incised, the knee joint was placed in maximal flexion, the joint cavity was exposed, and the medial meniscus was dissected. The medial third of the meniscus and the anterior cruciate ligament were then transected. For the control group (without osteoarthritis induction), only the skin was incised to open the joint cavity without removing the ligaments or meniscus. The osteoarthritis model was established on day 8 of the study.

[0172] One week after model establishment (day 15 of the study), the baseline pain threshold of the surgical side plantar fossa was assessed in each animal before treatment administration. Rats with osteoarthritis were randomly assigned to groups of eight based on their baseline Von Frey response values ​​(2–5). The groups included the osteoarthritis model group (carrier treatment), the positive control drug (duloxetine) 5 mg / kg group, and ALMB-0166 dose groups at 15 mg / kg, 25 mg / kg, and 50 mg / kg. Treatment began on day 15 of the study after grouping. For each ALMB-0166 treatment test group, the treatment was administered intravenously once weekly for a total of four doses (on days 15, 22, 29, and 36 of the study, corresponding to days 1, 8, 15, and 22 of treatment). For the positive control group, duloxetine was administered orally daily until the end of the experimental period.

[0173] Animals in each test group were weighed on days 15, 22, 29, and 36 of the study. On days 16, 23, 30, and 37, each animal was scored using the Coderre scale, the over-and-under method, and the bipedal balance pain test, and gait, changes in plantar pain threshold caused by knee lesions on the operated side, and weight-bearing on both lower limbs were measured.

[0174] Coderre rating system

[0175] The Coderre gait method was used to measure the gait of each animal. Gait was measured according to the following scores: Standard (Grade 0: normal walking, 0 points); Grade 1 (slight lameness, slight flexion below the knee, 2 points); Grade 2 (lameness, the affected limb can touch the ground, 4 points); Grade 3 (severe lameness, the affected limb leaves the ground and walking on three legs, 6 points).

[0176] Mechanical pain threshold measurement

[0177] The plantar pain threshold was measured using the up-and-down method. Each rat was placed in a transparent enclosure with a sieve-like metal plate at the bottom and allowed to acclimatize for approximately 15 minutes until spontaneous exploration and grooming activities ceased. Then, the mid-sole of the hind foot of each rat was vertically stimulated with a Von Frey probe to slightly S-shape the foot for 5 seconds, and the claw retraction response was observed. A positive response was indicated by abrupt claw retraction or withdrawal upon removal. The interval between measurements was approximately 10 seconds. A negative response was indicated by no observed response after probe stimulation.

[0178] To score mechanical pain, rats were stimulated with a series of Von Frey probes (0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, and 15 g). Testing began with a 2 g probe; a positive response was recorded as “X”, and testing continued with a 1.4 g probe. If no positive response was observed with a 2 g probe, it was recorded as “O”, and testing continued with a 4 g probe. Testing continued in this manner, using probes sequentially without avoidance. Each response was recorded, and when “OX” or “XO” was first observed, the test was repeated four times to obtain a sequence such as “OXOXOX”. If the rat did not respond positively after using a 15 g probe, the 50% withdrawal threshold (50% MWT) was recorded as 15 g. If a positive response was induced even with a 0.4 g probe, the 50% MWT was recorded as 0.4 g. If 15 g and 0.4 g probes are not used, the following formula is used: 50%MWT(g) = (10[Xf + kδ]) / 10000, where “50% MWT” is defined as a 50% chance of a positive response at this stimulus intensity, “Xf” is defined as the value of the last Von Frey probe used (in logarithmic units), “K” is defined as a value that can be obtained by looking up a table based on the measurement sequence, and “δ” is defined as the average of the differences between a series of stimuli (in logarithmic units, defined as 0.224).

[0179] Bipedal balance measurement

[0180] The weight-bearing capacity of each animal's hind limbs was assessed using a bipedal balance pain tester. Each animal was placed inside the plexiglass cover of the bipedal balance pain tester. After the animal completed its exploration behavior, the rat's forelimbs lay on the ramp of the device, while its hind limbs stood on the left and right balance boards, respectively. Once the animal was inactive, the average weight-bearing capacity of the left and right hind limbs was measured within 5 seconds of inactivity. The measurement was repeated three times for each animal, with a 10-second interval between each repetition.

[0181] Statistical analysis

[0182] Gait scores, plantar pain thresholds, and weight-bearing differences are expressed as mean and standard error (mean ± SD). Repeated measures ANOVA was used to analyze data between the model group and the treatment group at multiple time points using SPSS Statistics 21 software, and the Tamhane method was used for subsequent analyses. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. The final data were plotted using GraphPad Prism 6 software.

[0183] result

[0184] General clinical symptoms in rats after administration

[0185] No obvious abnormalities were observed in any of the animals during the experiment.

[0186] Effect of anti-Cx43 antibody treatment on weight in rats with pMMx+ACLT-induced osteoarthritis

[0187] Following the administration of the appropriate treatment, the body weight of the animals in each test group increased over time (Table 2). No significant differences were observed between the groups.

[0188] Table 2. Weight of the animal test group during the study period (mean ± SEM, n=8).

[0189]

[0190] Effects of anti-Cx43 antibody treatment on gait in rats with pMMx+ACLT-induced osteoarthritis

[0191] The osteoarthritis model rats exhibited lameness on day 16 of the study period (1 week post-surgery), with a mean gait score of 3.8 between the test groups (Table 3 and 1 week post-surgery). Figure 1 Gait scores improved during treatment, and by day 37 of the study, the gait scores of the osteoarthritis model rats were comparable to those of the control rats (Table 3 and ). Figure 1 Significant differences in gait scores were observed in the osteoarthritis model rat test group compared with control (non-osteoarthritis) rats (multivariate test, p < 0.05, α = 0.05; Table 3). Although a decrease in gait scores over time was observed in the ALMB-0166 and duloxetine dosage groups, no significant difference was observed compared with the mediator-treated control group (multivariate test, p > 0.05, α = 0.05; Table 3).

[0192] Table 3. Gait scores of the animal test groups during the study period (mean ± SEM, n=8).

[0193]

[0194] Note: # = P<0.01 vs. control

[0195] Effects of anti-Cx43 antibody treatment on mechanical pain in rats with pMMx+ACLT-induced osteoarthritis

[0196] One week after osteoarthritis induction, a baseline level of approximately 3.5 g of plantar pain threshold (50% MWT) was observed in osteoarthritis model rats. An increase in the plantar pain threshold (50% MWT) over time was observed after the initiation of each treatment compared to day 15 of the study (day 1 of treatment) (Tables 4-5 and 5). Figure 2 Improvement in the plantar pain threshold was correlated with the number of doses of ALMB-0166 administered (Table 5 and ). Figure 2 ).

[0197] Table 4. Foot pain threshold in animal test groups during the study period (mean ± SEM, n=8).

[0198]

[0199] Note: # p<0.01 vs. control; ## p=0.52 vs. control; p<0.05, p<0.01 vs. model

[0200] Table 5. Growth rate of plantar pain threshold in animal test groups during the study period (mean ± SEM, n=8).

[0201]

[0202] Significant differences in plantar pain thresholds were observed in all osteoarthritis model test groups compared to model rats (multivariate test, p < 0.01, α = 0.05; Table 4). Furthermore, when comparing plantar pain thresholds over time, significant increases were observed in the ALMB-0166 25 mg / kg and 50 mg / kg dose groups, as well as the duloxetine 5 mg / kg test group, compared to the osteoarthritis model (mediation-treated) test groups (multivariate test, p < 0.05, α = 0.05; Table 4).

[0203] When analyzing the plantar pain threshold at each time point, significant increases in the plantar pain threshold were observed in the ALMB-0166 25 mg / kg and 50 mg / kg dose groups and the duloxetine dose group on days 23, 30, and 37 of the study (p<0.05, α=0.05; Table 4). An increase in the plantar pain threshold was also observed in the ALMB-0166 15 mg / kg dose group during treatment, but the difference was not statistically significant compared to osteoarthritis (carrier-treated) animals (p=0.52, α=0.05; Table 4). Furthermore, the increase in plantar pain threshold induced by ALMB-0166 treatment showed a dose-dependent effect. Figures 3A-3D ).

[0204] Effect of anti-Cx43 antibody on differential weight-bearing in the hind limbs of rats with pMMx+ACLT-induced osteoarthritis

[0205] For all osteoarthritis test groups, the difference in hindlimb weight-bearing decreased over time (Table 6 and...). Figure 4 When compared with the osteoarthritis model (mediation treatment) test group, the reduction in hindlimb weight-bearing differences in the three ALMB-0166 dose groups and the duloxetine 5 mg / kg test group was significant (multivariate test, p < 0.05, α = 0.05; Table 6). When analyzing hindlimb weight-bearing at each time point, significant reductions in weight-bearing differences were observed in all ALMB-0166 dose groups and the duloxetine test group on days 16, 23, 30, and 37 of the study (multivariate test, p < 0.01, α = 0.05; Table 6). Furthermore, the reduction in hindlimb weight differences resulting from ALMB-0166 treatment showed a dose-dependent effect. Figures 5A-5D ).

[0206] Table 6. Differences in hindlimb load-bearing capacity among animal test groups during the study period (mean ± SEM, n=8).

[0207]

[0208] Note: ## P<0.01 vs. control; P<0.05, P<0.01 vs. model

[0209] in conclusion

[0210] Treatment of a rat model of osteoarthritis (induced by pMMX+ACLT) with the anti-CX43 antibody ALMB-0166 at doses of 15 mg / kg, 25 mg / kg, and 50 mg / kg resulted in improvements in gait scores over time. Furthermore, treatment of the osteoarthritis model rats with the anti-CX43 antibody ALMB-0166 at doses of 15 mg / kg, 25 mg / kg, and 50 mg / kg also led to significant improvements in mechanical pain threshold and hindlimb weight-bearing differences. The analgesic effect observed in osteoarthritis rats treated with the anti-CX43 antibody ALMB-0166 was dose-dependent, and the analgesic effects produced by treatment with 25 mg / kg and 50 mg / kg ALMB-0166 were comparable to or better than those produced by treatment with 5 mg / kg duloxetine.

[0211] Table 7. Sequence List

[0212]

[0213]

[0214]

[0215]

Claims

1. A method for treating osteoarthritis in a subject with this need, comprising administering to the subject at least one dose of an anti-connector protein 43 (Cx43) antibody, wherein the anti-Cx43 antibody comprises a heavy chain CDR sequence and a light chain CDR sequence as follows: HCDR1: SEQ ID NO: 1; HCDR2: SEQ ID NO: 2; HCDR3: SEQ ID NO: 3; LCDR1: SEQ ID NO: 4; LCDR2: SEQ ID NO: 5; and LCDR3: SEQ ID NO:

6.

2. The method of claim 1, comprising administering a first dose of the anti-Cx43 antibody on day 1 and a second dose on day 8.

3. The method of claim 2, further comprising administering a third dose of the anti-Cx43 antibody on day 15.

4. The method of claim 3, further comprising administering a fourth dose of the anti-Cx43 antibody on day 22.

5. The method of claim 4, further comprising administering subsequent doses weekly after the fourth dose.

6. The method according to any one of claims 1-5, wherein the first, second, third, fourth and / or subsequent doses are from about 0.01 mg / kg to about 100 mg / kg.

7. The method according to any one of claims 1-6, wherein the first, second, third, fourth and / or subsequent doses are about 15 mg / kg.

8. The method according to any one of claims 1-6, wherein the first, second, third, fourth and / or subsequent doses are about 25 mg / kg.

9. The method according to any one of claims 1-6, wherein the first, second, third, fourth and / or subsequent doses are about 50 mg / kg.

10. The method according to any one of claims 1-9, wherein the anti-Cx43 antibody is administered intravenously.

11. The method according to any one of claims 1-10, wherein at least one indicator of osteoarthritis severity is assessed at least one day to one week after each dose is administered.

12. The method of claim 11, wherein the at least one indicator of osteoarthritis severity comprises: (a) The pain threshold of arthritis, (b) Gait score, or (c) Load difference.

13. The method according to any one of claims 1-12, wherein at least one indicator of osteoarthritis severity is improved after administration of at least one dose of the anti-Cx43 antibody.

14. The method according to any one of claims 1-13, wherein the subject is a human.

15. The method according to any one of claims 1-14, wherein the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO:

8.

16. The method according to any one of claims 1-15, wherein the anti-Cx43 antibody comprises the heavy chain sequence of any one of SEQ ID NO: 9 and 11-18, and / or the light chain variable sequence of SEQ ID NO:

10.

17. The method according to any one of claims 1-16, wherein the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain variable sequence of SEQ ID NO:

10.

18. A method for treating osteoarthritis in a subject with this need, comprising administering to the subject an effective amount of an anti-Cx43 antibody, wherein the anti-Cx43 antibody is administered according to the following dosage regimen: i) The first dose on day 1; ii) The second dose on day 8; iii) The third dose on day 15; and iv) The fourth dose on day 21.

19. The method of claim 18, further comprising administering subsequent doses of the anti-Cx43 antibody weekly after the fourth dose.

20. The method of claim 18 or claim 19, wherein the first, second, third, fourth and / or subsequent doses are from about 0.01 mg / kg to about 100 mg / kg.

21. The method according to any one of claims 18-20, wherein the first, second, third, fourth and / or subsequent doses of the anti-Cx43 antibody are about 15 mg / kg.

22. The method according to any one of claims 10-20, wherein the first, second, third, fourth and subsequent doses of the anti-Cx43 antibody are about 25 mg / kg.

23. The method according to any one of claims 18-20, wherein the first, second, third, fourth and subsequent doses of the anti-Cx43 antibody are about 50 mg / kg.

24. The method according to any one of claims 18-23, wherein the anti-Cx43 antibody is administered intravenously.

25. The method according to any one of claims 18-24, wherein at least one indicator of osteoarthritis severity is assessed at least one day to one week after each dose is administered.

26. The method of claim 25, wherein the at least one indicator of osteoarthritis severity comprises: (a) The pain threshold of arthritis, (b) Gait score, or (c) Load difference.

27. The method according to any one of claims 18-26, wherein at least one indicator of osteoarthritis severity is improved after administration of at least one dose of the anti-Cx43 antibody.

28. The method according to any one of claims 18-27, wherein the subject is a human.

29. The method according to any one of claims 18-28, wherein the anti-Cx43 antibody comprises a heavy chain CDR sequence and a light chain sequence as follows: HCDR1: SEQ ID NO: 1; HCDR2: SEQ ID NO: 2; HCDR3: SEQ ID NO: 3; and LCDR1: SEQ ID NO: 4; LCDR2: SEQ ID NO: 5; and LCDR3: SEQ ID NO:

6.

30. The method according to any one of claims 18-29, wherein the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO:

8.

31. The method according to any one of claims 18-30, wherein the anti-Cx43 antibody comprises the heavy chain sequence of any one of SEQ ID NO: 9 and 11-18, and / or the light chain variable sequence of SEQ ID NO:

10.

32. The method according to any one of claims 18-31, wherein the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain variable sequence of SEQ ID NO:

10.

33. The method according to any one of claims 1-32, wherein the anti-Cx43 antibody blocks the opening of the Cx43 hemichannel in the subject.

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