method

Abstract

Disclosed herein are methods and treatments for cartilage degradation that involve administration of a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

Description

[Technical field]

[0001] This application claims priority to SG 10202100687X, filed January 21, 2021, and SG 10202109307T, filed August 25, 2021, the contents and elements of which are incorporated herein by reference for all purposes. The present invention relates generally to the field of molecular biology. In particular, the present invention relates to methods for detecting and treating arthritis. [Background technology]

[0002] Arthritis is a leading cause of joint pain and disability, affecting more than one in four adults worldwide; the two most common types of arthritis are rheumatoid arthritis (RA) and osteoarthritis (OA).

[0003] Currently, there is no effective treatment for either osteoarthritis or rheumatoid arthritis patients, who receive a variety of anti-rheumatic or immunomodulatory drugs that primarily target the immune system (e.g., anti-TNFα and anti-IL-6).

[0004] The main problem is the immune deficiency induced by these treatments. In addition, the treatments usually fail to completely halt the progression and recurrence of the disease. Patients with severe end-stage osteoarthritis or rheumatoid arthritis who fail to respond to such drugs must undergo joint replacement surgery, which carries its own risk of serious adverse events.

[0005] Thus, there is an unmet need for methods of detecting arthritis and / or methods of treating arthritis in a subject. Summary of the Invention

[0006] In one embodiment, the disclosure refers to a method of treating arthritis in a subject, the method comprising administration of a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

[0007] In another aspect, the present disclosure refers to a method for detecting the presence or absence of arthritis in a subject, comprising the steps of obtaining a synovial fibroblast sample from a subject, detecting the level of Tn antigen / Tn glycan in the sample obtained in step (i), and comparing the level of Tn antigen / Tn glycan in step (ii) with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, wherein an increase in the level of Tn antigen / Tn glycan shown in the sample compared to the control group indicates the presence of arthritis, and the control group consists of subjects without arthritis.

[0008] In one embodiment, the present disclosure refers to the use of an anti-calnexin (anti-CNX) antibody for the manufacture of a medicament for the treatment or prevention of cartilage degradation in a subject.

[0009] In another aspect, the disclosure refers to an anti-calnexin (anti-CNX) antibody for use in the treatment or prevention of cartilage degradation in a subject.

[0010] In another aspect, the disclosure refers to a method of treating or preventing cartilage degradation, the method involving administration of an anti-calnexin (anti-CNX) antibody to a subject.

[0011] In some embodiments, the subject has osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), arthritis flare, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, cartilage damage, or polychondritis.

[0012] In some embodiments, the subject has arthritis.

[0013] In some embodiments, the subject has osteoarthritis or rheumatoid arthritis.

[0014] In some embodiments, the cartilage degradation is in one or more joints.

[0015] In some embodiments, cartilage degradation is characterized by increased O-glycosylation and / or GALNT activation (GALA) in articular tissue, cartilage tissue, and / or synovial fibroblasts.

[0016] In some embodiments, cartilage degradation is characterized by increased O-glycosylation of calnexin in articular tissue, cartilage tissue, and / or synovial fibroblasts.

[0017] In some embodiments, cartilage degradation is characterized by increased cell surface expression of calnexin in articular tissue, cartilage tissue, and / or synovial fibroblasts.

[0018] In some embodiments, cartilage degradation is characterized by degradation of the extracellular matrix (ECM).

[0019] In some embodiments, cartilage degradation is mediated by the activity of synovial fibroblasts.

[0020] In some embodiments, antibodies to Cnx can reduce: (a) ECM degradation, or (b) oxidoreductase activity, or (c) Disulfide bond reductase activity.

[0021] In some embodiments, the antibody is capable of reducing cartilage degradation.

[0022] In some embodiments, the antibody is capable of reducing ECM degradation.

[0023] In some embodiments, the antibody is capable of reducing ECM degrading activity.

[0024] In some embodiments, the antibody is capable of decreasing the ECM degrading activity of CNX:PDIA3 (also known as Cnx / ERp57).

[0025] In some embodiments, the antibody is capable of reducing the ECM degrading activity of CNX.

[0026] In some embodiments, the antibody is capable of reducing the ECM degrading activity of fibroblasts.

[0027] In some embodiments, the antibody is capable of reducing the ECM degrading activity of synovial fibroblasts.

[0028] In some embodiments, the antibody is capable of decreasing oxidoreductase activity.

[0029] In some embodiments, the antibody is capable of decreasing the oxidoreductase activity of CNX:PDIA3 (also known as Cnx / ERp57).

[0030] In some embodiments, the antibody is capable of decreasing the oxidoreductase activity of CNX.

[0031] In some embodiments, the antibody is capable of reducing disulfide bond reductase activity.

[0032] In some embodiments, the antibody is capable of decreasing the disulfide bond reductase activity of CNX:PDIA3 (also known as Cnx / ERp57).

[0033] In some embodiments, the antibody is capable of reducing the disulfide bond reductase activity of CNX.

[0034] In some embodiments, the antibody is capable of reducing O-glycosylation.

[0035] In some embodiments, the antibody is capable of reducing O-glycosylation of CNX.

[0036] In some embodiments, the antibody is capable of reducing glycosylation of CNX.

[0037] In some embodiments, the antibody is capable of reducing GALA-mediated O-glycosylation of CNX.

[0038] In some embodiments, the antibody is capable of reducing CNX activity.

[0039] In some embodiments, the antibody is capable of decreasing PDIA3 activity.

[0040] In some embodiments, the antibody is capable of decreasing PDIA4 activity.

[0041] In some embodiments, the antibody is capable of reducing the number or percentage of cells expressing CNX.

[0042] In some embodiments, the antibody is capable of reducing the number or percentage of cells that express PDIA3.

[0043] In some embodiments, the antibody is capable of reducing the number or percentage of cells that express PDIA4.

[0044] In some embodiments, the antibody is capable of reducing the number or percentage of cells that express CNX:PDIA3.

[0045] In some embodiments, the antibodies are antagonistic.

[0046] In some embodiments, the antibody is monoclonal.

[0047] In some embodiments, the therapeutic agent is to be administered separately or sequentially together with an additional therapeutic agent, wherein the additional therapeutic agent is an anti-rheumatic agent.

[0048] In some embodiments, the methods comprise intravenous, subcutaneous or intraperitoneal administration of an anti-cnx antibody.

[0049] In some embodiments, the treatment or prevention is in a human subject.

[0050] In some embodiments, a subject is determined to be suitable for treatment with an anti-Cnx antibody through the following steps: i. detecting levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample; ii. comparing the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in step (i) with the levels of Tn antigen / Tn glycan in a control synovial fibroblast sample; Here, an increase in the levels of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation exhibited in the sample compared to a control group indicates suitability for treatment.

[0051] In one embodiment, a method is provided for detecting the presence or absence of a disorder associated with cartilage degradation in a subject, the method comprising the steps of: i. detecting levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample; ii. comparing the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in step (i) with the levels of Tn antigen / Tn glycan in a control synovial fibroblast sample; Here, an increase in the levels of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation exhibited in the sample compared to a control group indicates suitability for treatment.

[0052] In some embodiments, the method is for detecting the presence or absence of arthritis in a subject. In some embodiments, the method is for detecting the presence or absence of osteoarthritis or rheumatoid arthritis in a subject.

[0053] Numbered Description 1. A method of treating arthritis in a subject, comprising administration of a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

[0054] 2. The method of statement 1, wherein the therapeutic agent inhibits any one or more of the cell surface markers involved in the GalNAc-T activation (GALA) pathway.

[0055] 3. The method of any one of the preceding claims, wherein the therapeutic agent inhibits one or more of the targets of the GalNAc-T activation (GALA) pathway selected from the group consisting of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), calnexin-PDIA3 complex, and O-glycosylated matrix metalloproteinase-14 (MMP14).

[0056] 4. The method of claim 3, wherein the target of the GALA pathway is calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3).

[0057] 5. The method of any one of the preceding claims, wherein said therapeutic agent is selected from the group consisting of an antibody, a gene, a fusion protein, and an expression vector.

[0058] 6. The method of statement 5, wherein the therapeutic agent is administered separately or sequentially together with an additional therapeutic agent, and the additional therapeutic agent is an anti-rheumatic agent.

[0059] 7. The method of statement 5, wherein the fusion protein is ER-2Lec.

[0060] 8. The method of statement 5, wherein the antibody is an anti-calnexin antibody.

[0061] 9. The method of claim 5, wherein the expression vector is an adeno-associated virus (AAV) vector.

[0062] 10. The method of statement 5 or 9, wherein the expression vector expresses the protein ER-2Lec.

[0063] 11. The method of any one of the preceding statements, wherein one or more of said therapeutic agents is administered as gene therapy.

[0064] 12. A method for detecting the presence or absence of arthritis in a subject, comprising the steps of: (i) obtaining a synovial fibroblast sample from a subject; (ii) detecting the level of Tn antigen / Tn glycan in the sample obtained in step (i); (iii) comparing the level of Tn antigen / Tn glycan in step (ii) with the level of Tn antigen / Tn glycan in a control synovial fibroblast sample; wherein an increase in the level of Tn antigen / Tn glycan exhibited in said sample compared to said control group is indicative of the presence of arthritis, said control group consisting of subjects without arthritis.

[0065] 13. The method of any one of the preceding statements, wherein said arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), and arthritis flare / flare-up.

[0066] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [Brief description of the drawings]

[0067] [Figure 1A]This shows that O-glycosylation is enhanced in human samples with rheumatoid arthritis and osteoarthritis, indicating that high levels of O-glycosylation correlate with disease status. Representative images of immunohistofluorescence staining of nuclei with Hoechst (top row) and O-GalNAc glycan (Tn glycan) staining with Vicia Villosa lectin (VVL, bottom row) are shown in human tissue microarrays (TMA) containing joint tissue from healthy subjects (normal) and patients with osteoarthritis (OA), rheumatoid arthritis (RA), or psoriatic arthritis (PSA). Scale bar, 5 μm. [Figure 1B] This shows that O-glycosylation is enhanced in human samples with rheumatoid arthritis and osteoarthritis, indicating that high levels of O-glycosylation correlate with disease status. Quantification graphs of Tn glycan levels in individual tissue cores are shown. Osteoarthritis patients exhibited higher O-GalNAc glycan levels than healthy subjects, and most rheumatoid arthritis patients and two psoriatic arthritis patients showed higher O-GalNAc glycan levels than healthy individuals. Data are mean ± SEM and were combined from two different tissue microarray (TMA) slides consisting of tissue sections from 21 osteoarthritis patients, 18 rheumatoid arthritis patients, 6 psoriatic arthritis patients, and 7 healthy subjects. Each data point represents the raw integrated density of Vicia Villosa lectin (VVL) staining normalized to that of nuclear staining in each subject. Box plots show all values, boxes extend from the 25th to the 75th percentile and error bars range from maximum to minimum values. *, p<0.05; ****, p<0.0001; NS: not significant (one-way ANOVA, Kruskal-Wallis test). [Figure 2A]We show that Tn glycan levels are elevated in arthritis-induced mice and correlate with disease severity, indicating that high Tn glycan levels correlate with lesion status. Hematoxylin and eosin (HE) histology of synovial tissues from control mice (day 0) or from collagen II antibody-injected arthritis-induced (CAIA) mice on days 7, 10, and 14. S: synovium, B: bone, P: pannus, (*): immune cell infiltration in synovial sublining, arrow: bone erosion. [Figure 2B] We show that Tn glycan levels are elevated in arthritis-induced mice and correlate with disease severity, indicating that high Tn glycan levels correlate with lesion status. Representative immunofluorescence staining images for Vicia Villosa lectin (VVL, top row) and nuclei (bottom row) are shown, revealing a time-dependent increase in VVL staining in pannus tissues from days 7 to 10 in CAIA mice. Scale bar, 50 μm. [Figure 2C] Figure 1 shows that Tn glycan levels are elevated in arthritic mice and correlate with disease severity, indicating that high Tn glycan levels correlate with lesion status. Clinical score assessment in CAIA mice from day 0 to day 14. Data are the mean arthritis scores of 4 mice per time point. [Figure 2D] Figure 1 shows that Tn glycan levels are elevated in arthritic mice and correlate with disease severity, indicating that high Tn glycan levels correlate with lesion status. Figure 1 shows quantification of total Tn levels in synovium in CAIA mice from day 0 to day 14. Individual data points represent the mean Tn levels in individual joints, calculated for two joints in the front paws of each animal. Box plots show all values, with boxes extending from the 25th to the 75th percentile and error bars extending from maximum to minimum values. *, p<0.05; **, p<0.01; NS, not significant (one-way ANOVA). [Figure 3A] Data are presented indicating that synovial cells from collagen type II antibody-induced arthritis (CAIA) mice exhibit signs of GALA pathway activation, indicating that the GALA pathway is activated in synovial cells in this disease model, making it a therapeutic target. Co-staining images of Vicia Villosa lectin (VVL) and endoplasmic reticulum (ER) resident protein calnexin (CNX) are shown, showing a marked increase in Tn-glycan levels in the synovium of arthritis-induced mice. Scale bar 50 μm. Enlarged images show that VVL staining co-localized with CNX in arthritic joints, indicating an activated state of GALA. Enlargement scale 4x, S: synovium, B: bone, arrows indicate either the Golgi pattern of VVL (in untreated mice) or the ER staining pattern (in CAIA mice). [Figure 3B] Data are presented indicating that synovial cells from collagen type II antibody-induced arthritis (CAIA) mice exhibit signs of GALA pathway activation, indicating that the GALA pathway is activated in synovial cells in this disease model, making it a therapeutic target. Co-staining images of GALNT2 with endoplasmic reticulum (ER) resident protein calnexin (CNX) are shown, demonstrating a marked increase in Tn-glycan levels in the synovium of arthritis-induced mice. Scale bar 50 μm. Enlarged images show that the GALNT2 enzyme co-localized with CNX in arthritic joints, indicating an activated state of GALA. Enlargement scale 4x, S: synovium, B: bone, arrows indicate either the Golgi pattern (in untreated mice) or the ER staining pattern (in CAIA mice) of GALNT2. [Figure 4]Images reveal that synovial fibroblasts (SFs) are the major cell type exhibiting GALA activation in collagen type II antibody-induced arthritis (CAIA) mice, indicating that the GALA pathway is activated in synovial cells in this disease model, thereby indicating that the GALA pathway is a therapeutic target. Co-staining with Vicia Villosa lectin (lower right panel) and the fibroblast marker vimentin (lower left panel), immune cell marker anti-CD45 (upper right panel), and nuclei (upper left panel) shows the relative distribution of immune cells (circles), fibroblasts (squares), and their Tn expression levels in the pannus tissue of CAIA mice. Scale bar, 50 μm. [Diagram 5] Images are shown demonstrating that synovial lining fibroblasts from both osteoarthritis and rheumatoid arthritis patients exhibit strong GALA activation, indicating that the GALA pathway is activated in synovial cells in disease models, thereby making it a therapeutic target. Panel A shows the results of hematoxylin and eosin (H&E) histology of synovial tissues from rheumatoid arthritis and osteoarthritis patients. "*" denotes immune cell infiltration in the RA synovial sublining, SL: synovial lining. Scale bar, 100 μm. Panel B shows representative immunofluorescence images of osteoarthritis synovium (upper panel) and rheumatoid arthritis synovium (lower panel), showing intense Tn glycan levels in synovial lining fibroblasts (SFs) identified by FAPα (arrowhead) and sparse Tn staining in immune cells identified by CD45 in the sublining (delimited by dashed line). Scale bar, 50 μm. [Figure 6]Data are presented indicating that primary synovial fibroblasts (SFs) from osteoarthritis and rheumatoid arthritis patients induce robust GALA activation in response to stimulation with arthritis-promoting cytokines and cartilage extracellular matrix (ECM), implicating the GALA pathway in arthritic symptoms and disease progression. Panel A shows fluorescence-activated cell sorting (FACS) dot plots demonstrating high purity (>90%) of primary synovial fibroblast cultures established from healthy subjects (HCSFs), osteoarthritis patients (OASFs), and rheumatoid arthritis patients (RASFs). Panel B shows representative images of Helix pomatia lectin (HPL) staining, demonstrating that Tn glycan levels are higher in both osteoarthritis and rheumatoid arthritis synovial fibroblasts compared to HCSF synovial fibroblasts under basal conditions. The height increases exponentially after priming the cells with pro-arthritic cytokines (CYTO) including IL1β and TNFα and cartilage extracellular matrix. Scale bar, 20 μm. Panel C shows the quantification of Helix pomatia lectin (HPL) levels in HCSFs, OASFs and RASFs under basal conditions and stimulation with CYTO alone (no coating) or in combination with cartilage extracellular matrix or collagen type I extracellular matrix. Data are means ± SEM of two independent experiments. *p<0.05, ***<0.001, ****p<0.0001, NS: not significant (two-way ANOVA). [Figure 7A] We present data indicating that GALA activation in synovial fibroblasts promotes cartilage extracellular matrix degradation, implicating the GALA pathway in arthritic symptoms and disease progression.We present a strategy to inhibit GALA in synovial fibroblasts by stably transfecting SW982 synovial fibroblasts with a construct expressing the two lectin domains of GALNT2 (ER-2Lec) in the ER in a doxycycline (DOX)-inducible manner. [Figure 7B]This figure shows data indicating that GALA activation in synovial fibroblasts promotes the degradation of cartilage extracellular matrix, indicating that the GALA pathway is responsible for arthritis symptoms and disease progression. Representative images of matrix degradation activity results of SW982 cells and ER-2Lec-expressing SW982 cells after stimulation with rheumatoid arthritis-related cytokines (CYTO, IL1β, and TNFα) are shown. Arrows indicate degraded matrix. [Figure 7C] Figure 1 shows data indicating that GALA activation in synovial fibroblasts promotes cartilage extracellular matrix degradation, indicating that the GALA pathway contributes to arthritic symptoms and disease progression. Figure 2 shows a quantification graph showing reduced matrix degradative activity in GALA-inhibited SW982 cells after stimulation with CYTO (IL1β and TNFα). Data represent the mean ± SEM and are representative of three independent experiments. Each data point represents the total degraded area (μm) per nucleus per well. ***, p<0.001, ****p<0.0001 (one-way ANOVA). [Figure 8] Figure 1 shows data indicating that ER-2Lec expression in synovial fibroblasts in vivo suppresses GALA activation, revealing the efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Representative images shown indicate that ER-2Lec is expressed primarily in synovial fibroblasts identified by EGFP-positive staining (arrowhead) in the synovium of Col6a1Cre ER-2Lec mice, and that such cells exhibit reduced VVL staining, indicating inhibition of GALA. In other words, panel B shows the results of VVL staining showing reduced O-GalNAc glycans in fibroblasts (EGFP-positive cells) of GALA-inhibited joints after induction of arthritis. B: bone, S: synovium. Scale bar, 50 μm. [Figure 9A]1 shows images illustrating the effect of GALA inhibition by ER-2Lec expression in reducing paw swelling in CAIA mice, demonstrating the effectiveness of ER-2Lec expression in treating arthritis or reducing symptoms associated with arthritis. Analysis of paw thickness revealed that Col6a1Cre ER-2Lec mice had reduced swelling levels in both front and hind paws 7 days after arthritis induction compared to Col6a1Cre control mice. [Figure 9B] 1 shows a graph illustrating the effect of GALA inhibition by ER-2Lec expression in reducing paw swelling in CAIA mice, indicating the efficacy of ER-2Lec expression in treating arthritis or reducing symptoms associated with arthritis. Paw thickness analysis revealed that Col6a1Cre ER-2Lec mice had reduced swelling levels in both front and hind paws 7 days after arthritis induction compared to Col6a1Cre control mice. Data are mean ± SEM of two independent experiments, n=5 mice per group. **, p<0.01 (one-way ANOVA). [Figure 10] Data indicate that GALA inhibition by ER-2Lec expression in synovial fibroblasts (SF) reduces the clinical score in CAIA mice, thus indicating that ER-2Lec expression is effective in treating arthritis on a clinical scale. Clinical score measurements show that Col6a1Cre ER-2Lec mice have reduced arthritis severity 7 days after arthritis induction compared to Col6a1Cre control mice. Data are means ± SEM from two independent experiments, n = 5 mice per group. P = 0.09 (non-parametric t-test, Mann-Whitney test). [Figure 11A]1 shows images indicating that inhibition of GALA in ER-2Lec expression in synovial fibroblasts protects CAIA mice from cartilage degradation, demonstrating the efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Representative images showing Alcian Blue (AB) staining in untreated Col6a1Cre or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7 are shown. [Figure 11B] 1 shows images indicating that inhibition of GALA in ER-2Lec expression in synovial fibroblasts protects CAIA mice from cartilage degradation, demonstrating the efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Representative images showing Safranin-O (SO) staining in untreated Col6a1Cre or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7 are shown. [Figure 11C] Figure 1 shows data indicating that inhibition of GALA on ER-2Lec expression in synovial fibroblasts protects CAIA mice from cartilage degradation, demonstrating the efficacy of ER-2Lec expression in treating arthritis or alleviating arthritis-related symptoms. Quantification of AB staining positive area (scale bar) is shown. The data show that arthritis-induced cartilage matrix degradation is rescued in GALA-inhibited mice (Col6aCre ER-2Lec). Each data point represents the mean value of positive staining area per mm2 of articular cartilage in three different metacarpophalangeal joints from one animal. Data presented as mean ± SEM. *, p<0.05; **, p<0.01; **, p<0.001 (one-way ANOVA test). [Figure 11D]Figure 1 shows data indicating that inhibition of GALA on ER-2Lec expression in synovial fibroblasts protects CAIA mice from cartilage degradation, demonstrating the efficacy of ER-2Lec expression in treating arthritis or alleviating arthritis-related symptoms. Quantification of SO staining positive area (scale bar) is shown. The data show that arthritis-induced cartilage matrix degradation is reversed in GALA-inhibited mice (Col6aCre ER-2Lec). Each data point represents the mean value of positive staining area per mm2 of articular cartilage in three different metacarpophalangeal joints from one animal. Data presented as mean ± SEM. *, p<0.05; **, p<0.01; **, p<0.001 (one-way ANOVA test). [Figure 12] Data indicate that GALA activates O-glycosylation of calnexin (CNX) in arthritis-induced synovial fibroblasts, demonstrating the effect of the GALA pathway in pathological conditions and identifying calnexin as a therapeutic target. Panels A and B show (A) blot results and (B) quantification graphs of co-immunoprecipitation with VVL lectin, demonstrating that the levels of O-GalNAc glycosylated calnexin (CNX) are increased in SW982 cells after stimulation with arthritis-associated cytokines (CYTO) and cartilage extracellular matrix (ECM), but are decreased in doxycycline (DOX)-inducible ER-2Lec-expressing SW982 cells. Actin was used as a loading control. In panel B, data are shown as mean ± SEM and are representative of three independent experiments. *, p<0.05; **, p<0.01 (one-way ANOVA test). [Figure 13A]Figure 1 shows data indicating that GALA induces cell surface exposure of calnexin (CNX) in arthritic synovial fibroblasts, demonstrating the effect of the GALA pathway in pathological conditions and identifying calnexin as a therapeutic target. Figure 2 shows flow cytometry histogram plots showing an increase in the percentage of synovial fibroblasts expressing surface calnexin (CNX) after stimulation with arthritic cytokines (CYTOs) and cartilage extracellular matrix (ECM). Induction of cell surface CNX is reduced in ER-2Lec expressing SF cells. [Figure 13B] Figure 1 shows data indicating that GALA induces cell surface exposure of calnexin (CNX) in arthritic synovial fibroblasts, demonstrating the effect of the GALA pathway in the pathological condition and identifying calnexin as a therapeutic target. Figure 2 shows quantification graphs showing an increase in the percentage of synovial fibroblasts expressing surface calnexin (CNX) after stimulation with arthritic cytokines (CYTO) and cartilage extracellular matrix (ECM). Induction of cell surface CNX is reduced in ER-2Lec expressing SF cells. Data are shown as mean ± SEM and are representative of two independent experiments. **, p<0.01; ***, p<0.001 (one-way ANOVA test). [Figure 14A] Figure 1 shows data indicating that surface exposure of calnexin (CNX) is increased in primary synovial fibroblasts from both osteoarthritis (OA) and rheumatoid arthritis (RA) patients, thus identifying calnexin as a therapeutic target in the treatment of arthritis. Figure 2 shows flow cytometry histogram plots revealing a higher percentage of surface CNX positive cells in osteoarthritis synovial fibroblasts (OASF) or rheumatoid arthritis synovial fibroblasts (RASF) compared to healthy control synovial fibroblasts (HCSF) under basal conditions or under stimulation with pro-arthritis cytokines (CYTOs) and cartilage extracellular matrix (ECM). [Figure 14B]Figure 1 shows data indicating that surface exposure of calnexin (CNX) is increased in primary synovial fibroblasts from both osteoarthritis (OA) and rheumatoid arthritis (RA) patients, thus identifying calnexin as a therapeutic target in the treatment of arthritis. Figure 2 shows quantification graphs revealing that under basal conditions or stimulation with pro-arthritis cytokines (CYTOs) and cartilage extracellular matrix (ECM), the percentage of surface CNX positive cells in osteoarthritis synovial fibroblasts (OASFs) or rheumatoid arthritis synovial fibroblasts (RASFs) is higher than that in healthy control synovial fibroblasts (HCSFs). Data are shown as mean ± SEM and are representative of two independent experiments. *, p<0.05; ***, p<0.001 (one-way ANOVA test). [Figure 15] Images showing that disulfide bonds are abundant in cartilage extracellular matrix (ECM). This identifies the CNX:PDIA3 complex (i.e., its reducing effect on disulfide bonds) as a therapeutic target. Representative immunofluorescence staining images show staining of collagen fibers containing collagen type III (Col III), collagen type I (Col I) and fibronectin in cartilage extracellular matrix (ECM). Collagen disulfide bonds are chemically reduced with TCEP or left untreated (UT), which can be detected by staining with OX133 antibody. Scale bar, 5 μm. [Figure 16]Figure 1 shows data indicating that blockade of calnexin (CNX) reduces cartilage degrading activity in primary synovial fibroblasts from osteoarthritis patients, thus identifying calnexin as a therapeutic target in the treatment of arthritis. Panels A and B show representative images (A) and quantification graphs (B) showing reduced matrix degrading activity in primary fibroblasts isolated from osteoarthritis patients after incubation with anti-CNX or isotype control antibodies. Data represent mean ± SEM and are representative of three independent experiments. Each data point represents the total degraded area (μm) per nucleus per well. ***, p<0.001 (one-way ANOVA). [Figure 17] Figure 1 shows data indicating that treatment with antibodies against calnexin (CNX) reduces paw swelling in CAIA mice, demonstrating the efficacy of using anti-calnexin antibodies in treating arthritis. Panel A shows a schematic of the antibody treatment scheme. Panel B shows representative photographs of CAIA mice treated with anti-CNX antibodies or left untreated at day 10. Panel C shows a line graph plotting paw thickness measurements, demonstrating the reduction in paw swelling in CAIA mice following anti-CNX antibody injection. Data represent mean ± SEM, n = 4-5 mice per group. *, p < 0.05 (2-way ANOVA test). [Figure 18] Figure 1 shows a graph indicating that treatment with an antibody against calnexin (CNX) reduces the severity of arthritis in CAIA mice, thus demonstrating the efficacy of using anti-calnexin antibodies in the treatment of arthritis. Clinical scores of CAIA mice 10 days after treatment with isotype control or anti-CNX antibodies. Data are means ± SEM, n = 4–5 mice per group. P values ​​were estimated by non-parametric t-test (Mann-Whitney test). [Figure 19A]1 shows data indicating that treatment with antibodies against calnexin (CNX) protects CAIA mice from cartilage degradation, thus demonstrating the efficacy of using anti-calnexin antibodies in treating arthritis or preventing the progression of arthritis or arthritic symptoms. Representative histology images showing Alcian Blue (AB) staining (arrow box) in CAIA mice treated with isotype control or anti-CNX antibody are shown. [Figure 19B] 1 shows data indicating that treatment with antibodies against calnexin (CNX) protects CAIA mice from cartilage degradation, thus demonstrating the efficacy of using anti-calnexin antibodies in treating arthritis or preventing the progression of arthritis or arthritic symptoms.Representative histology images showing Safranin-O (SO) staining (arrow box) in CAIA mice treated with isotype control or anti-CNX antibody are shown. [Figure 19C] Figure 1 shows data indicating that treatment with antibodies against calnexin (CNX) protects CAIA mice from cartilage degradation, thus demonstrating the efficacy of the use of anti-calnexin antibodies in treating arthritis or preventing the progression of arthritis or arthritic symptoms. Quantification graph showing the increase in AB staining area in CAIA mice treated with anti-CNX antibodies compared to those treated with an isotype control antibody. Individual data points represent the mean positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal, n=4-5 mice per group. Data presented as mean ± SEM. *, p<0.05; **, p<0.01 (Mann-Whitney test). [Figure 19D]Figure 1 shows data indicating that treatment with antibodies against calnexin (CNX) protects CAIA mice from cartilage degradation, thus demonstrating the efficacy of the use of anti-calnexin antibodies in treating arthritis or preventing the progression of arthritis or arthritic symptoms. Quantification graph showing increased SO staining area in CAIA mice treated with anti-CNX antibodies compared to those treated with an isotype control antibody. Individual data points represent the mean positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal, n=4-5 mice per group. Data presented as mean ± SEM. *, p<0.05; **, p<0.01 (Mann-Whitney test). [Figure 20] A: Images indicating that anti-calnexin (CNX) antibodies are accumulated in the synovium of CAIA mice. Representative immunofluorescence images show co-staining of VVL with anti-CNX antibodies (arrowheads) at day 10 in CAIA mice injected with anti-CNX or isotype control antibodies. This reveals the accumulation of anti-CNX antibodies indicating their ability to bind and target CAIA mouse synovium, in contrast to the lack of binding of the control isotype antibody. This indicates that calnexin is expressed by cells in the synovium of CAIA mice, and that synovial cells can be specifically targeted with anti-CNX antibodies for therapeutic purposes. B: bone, S: synovium. Scale bar, 50 μm. [Figure 21A] Figure 1 shows data indicating the results of GALA target screening and validation of its effect on primary arthritic synovial fibroblasts. Schematic diagram of high content screening to select GALA targets and their blocking modalities. Synovial fibroblasts (SF) (SW982) were cultured on quenched fluorescent cartilage matrix component (DQ collagen) in the presence of GALA target blockers (antibodies or siRNAs). GALA degradative activity in synovial fibroblasts increases the fluorescent signal (control panel), whereas the degradative activity could be inhibited in synovial fibroblasts treated with blockers. [Figure 21B]Data are presented indicating the results of the GALA targeting screen and validation of its effect on primary arthritic synovial fibroblasts. Results are shown stained with VVL showing high expression of Tn glycans outside the Golgi apparatus (identified with the Golgi marker Giantin), indicating GALA activation in primary synovial fibroblasts (OASFs) isolated from osteoarthritis patients. Scale bar, 5 μm. [Figure 21C] 1 shows data indicating the results of GALA target screening and validation of its effect on primary arthritic synovial fibroblasts. Quantification graphs are shown demonstrating reduced matrix degrading activity in primary synovial fibroblasts (OASFs) isolated from osteoarthritis patients treated with anti-calnexin and anti-MMP14 antibodies. Each data point represents the ratio of raw integrated density of degraded DQ collagen to nuclei per field. Data are mean ± SEM, n = 20 fields per group. *, p < 0.05; **, p < 0.01; NS, not significant (one-way ANOVA test). [Figure 22] Data are shown for mice injected with anti-CNX or isotype control antibody. The data show that the weight change is comparable between the two. A: Weight change of CAIA mice treated with isotype control or anti-CNX antibody. B and C: Representative histology (B) and quantification of Safranin-O (SO) stained area of ​​CAIA mice treated with isotype control or anti-CNX antibody. Individual data points represent the mean positive staining area per mm2 of articular cartilage from individual metacarpophalangeal joints of one animal, n=4–5 mice per group. Data are shown as mean ± SEM. *, p<0.05; **, p<0.01 (Mann-Whitney test). [Figure 23]Figure 1 shows tissue analysis of O-GalNAc(Tn) glycans in synovial tissues from both arthritic patients and arthritic-induced animals. A: Representative immunohistofluorescence staining of O-GalNAc glycans with VVL lectin in human tissue microarray (TMA) sections from osteoarthritis (OA), psoriatic arthritis (PSA), rheumatoid arthritis (RA) and healthy subjects (Normal). Magnification 10X, scale bar, 500 μm. B: HE histology (top) and immunohistochemical staining with VVL lectin (bottom) in mice with collagen type II antibody-induced arthritis (CAIA) at day 7 or left untreated (UNT). [Figure 24] OA synovial tissue analysis and purification criteria for primary synovial fibroblasts. A: (Left) H&E histology of synovial tissue from OA patients. Scale bar, 100 μm, SL: synovium. (Right) Representative immunofluorescence images of OA synovium stained with VVL / CD45 and FAPα. Synovial lining fibroblasts were identified by FAPα (arrowheads). Immune cells were identified by CD45. The borders of the sublining and lining layers are defined by dashed lines. Scale bar, 50 μm. B: FACS dot plots showing high purity (>90%) of primary SF cultures established from healthy subjects (HCSF), OA patients (OASF) and RA patients (RASF). [Diagram 25] Figure 2 shows that GALA activation causes damage to cartilage in CAIA mice. A: Representative images of Safranin-O (SO) (B) staining of untreated Col6a1Cre or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice at day 7. Scale bar, 100 μm. B and C: Quantification of SO staining thickness (B) and total positive staining area (C). Arthritis-induced cartilage matrix degradation is indicated by arrowheads. Each data point represents the mean value of the positive staining area per mm2 of articular cartilage in three different metacarpophalangeal joints from one animal. Data are presented as mean ± SEM. p<0.01, ****, p<0.0001 (one-way ANOVA test). [Figure 26]Binding data for monoclonal anti-calnexin antibody clone 2G9. Data were generated using an ELISA assay. The assay revealed that clone 2G9 and the commercial monoclonal antibody ab10286 were highly specific for Cnx (left bar) and showed little binding to BSA-coated wells (right bar). The negative control hIgG did not significantly bind calnexin. Data represent the mean ± SEM and are representative of three independent experiments. [Figure 27] 1 shows further binding data for monoclonal anti-calnexin antibody clone 2G9 based on ELISA assays performed with serial dilutions of antibodies 2G9 and ab10286. The data suggest a higher affinity and / or binding activity for 2GP compared to the commercially available monoclonal antibody ab10286. [Figure 28] FIG. 1 shows ECM degradation assay data demonstrating the ECM degradation ability of monoclonal anti-calnexin antibody clone 2G9. Panels A and B show representative images (A) and quantification graphs (B) showing that 2G9 was able to reduce ECM degradation by 90% compared to untreated controls. Data represent the mean ± SEM and are representative of three independent experiments. [Figure 29] Further ECM degradation assay data is shown. 2G9 in IgG4 format is also able to block ECM degradation. Data represent mean ± SEM and are representative of three independent experiments. ***, p<0.001 (One-way ANOVA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] GalNAc-T Activation (GALA) Inhibitors in the Treatment of Cartilage Degradation Extracellular matrix degradation is an important factor in cartilage degeneration, cartilage degradation, disorders associated with cartilage degradation, and joint disorders. Specifically, extracellular matrix degradation can lead to the onset, progression, or aggravation of disorders such as osteoarthritis, psoriatic arthritis, rheumatoid arthritis, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage damage, and polychondritis.

[0069] In most tissues, fibroblasts are the key cell type involved in the production of extracellular matrix. However, fibroblasts can also degrade matrix, allowing the turnover of this essential component of tissues. How fibroblasts control these two opposing activities remains unclear.

[0070] Synovial fibroblasts (SFs), also called synoviocytes, are typical biphasic cells. In healthy individuals, SFs contribute to the viscosity of synovial fluid by secreting proteins such as hyaluronic acid and lubricin (Jay et al., J. Rheumatol. 27, 594-600, 2000). In arthritic diseases, SFs attach to and degrade cartilage, specifically the extracellular matrix (ECM) of cartilage. Understanding the changes in this activity in arthritis has been a major focus of research in recent years (Ospelt. RMD Open. 3, e000471, 2017).

[0071] The GALNTs activation pathway (GALA) regulates ECM degradation in cancer cells through glycosylation of MMP14 and calnexin. Here, we demonstrate for the first time that cartilage degradation, disorders related to cartilage degradation, and joint disorders are also associated with increased levels of GALA and O-glycosylation.

[0072] GALA induces matrix degradation through at least two mechanisms. First, it stimulates glycosylation of MMP14, which is required for its proteolytic activity (Nguyen et al., Cancer Cell. 32, 639-653. e6, 2017). Second, GALA induces glycosylation of the ER-resident protein calnexin, which forms a complex with ERp57 (also known as PDIA3) (Ros et al., Nat. Cell Biol. 22, 1371-1381. 2020). Following GALA-glycosylation, a portion of the Cnx-PDIA3 complex translocates to the surface of cancer cells. The complex accumulates in invadosomes and reduces disulfide bonds in the ECM (Ros et al., Nat. Cell Biol. 22, 1371-1381. 2020). This reduction is essential for effective degradation of ECM (Ros et al., Nat. Cell Biol. 22, 1371-1381. 2020).

[0073] The present inventors also demonstrate the treatment of cartilage degradation with GALA inhibitors. Specifically, inhibition of GALA through the use of anti-cnx antibodies has been shown to reduce ECM degradation and arthritic symptoms in vivo.

[0074] Our results point to GALA O-glycosylation as a key switch in the pathological activation of fibroblasts to degrade cartilage.

[0075] cartilage degradation Cartilage degradation can occur through certain disorders (e.g., arthritis, chondrocalcinosis, and polychondritis) and their progression, as well as through mechanical damage (e.g., sports injuries). People with cartilage degradation typically experience joint pain, stiffness, and inflammation, which can affect their quality of life.

[0076] Certain disorders and diseases are associated with cartilage degradation, and cartilage degradation may be caused by the mechanisms of certain disorders / diseases (e.g., arthritis, chondrocalcinosis, and polychondritis).

[0077] The present inventors have demonstrated that cartilage degradation can be treated with GALA inhibitors, such as anti-cnx antibodies.

[0078] There are many different disorders associated with cartilage degradation, many of which are difficult to treat. Krishnan and Grodzinsky (2018) in their publication entitled "Cartilage Diseases" describe that most diseases involving cartilage result in dramatic changes to the ECM, which can (1) dominate the progression of the disease (e.g., osteoarthritis), (2) cause the presenting symptoms of the disease (e.g., dwarfism caused by genetically inherited mutations), or (3) occur as collateral damage to pathological processes occurring in other nearby tissues (e.g., osteochondritis dissecans and inflammatory arthropathy). Furthermore, it is described that challenges associated with cartilage diseases include a lack of understanding of the etiology and pathogenesis (Krishnan and Grodzinsky. Matrix Biol. 2018 Oct; 71-72: 51-69).

[0079] Cartilage is an avascular, aneural, alymphatic connective tissue found in synovial joints, the spine, ribs, the external ear, nose, and airways, and in the growth plates of children and adolescents. There are three main types of cartilage found in humans: hyaline, fibrous, and elastic (Wachsmuth et al., Histol Histopathol. 2006 May;21(5):477-85). Thus, degradation of the ECM in cartilage can lead to many different disorders.

[0080] Cartilage degradation can occur in hyaline cartilage, fibrocartilage or elastic cartilage. The disorder associated with cartilage degradation can be a disorder involving hyaline cartilage degradation, fibrocartilage degradation or elastic cartilage degradation.

[0081] Hyaline cartilage is the most widespread and common type of cartilage and is the type that composes the fetal skeleton. In adults, it persists as articular cartilage at the ends of bones in freely moving joints, at the ends of the ribs, nose, larynx, trachea, and bronchi.

[0082] Fibrocartilage is a tough, durable tissue found primarily in the intervertebral discs and at the insertions of ligaments and tendons; it resembles other fibrous tissues but contains a cartilage matrix and chondrocytes.

[0083] Elastic cartilage is more flexible than the other two types because it contains elastic fibers in addition to collagen. In humans, it makes up the outer ear, the Eustachian tube in the middle ear, and the epiglottis.

[0084] Subjects with cartilage degradation may have arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, and / or polychondritis.

[0085] A subject with cartilage degradation may have arthritis. Cartilage degradation may be caused by or otherwise associated with the subject's arthritis.

[0086] Subjects with cartilage degradation may have osteoarthritis or rheumatoid arthritis.

[0087] Arthritis is a group of diseases that affect the joints (Barbour et al., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056). Rheumatoid arthritis (RA) and osteoarthritis (OA) are two of the most common types (Murphy and Nagase. Nat. Clin. Pract. Rheumatol. 4, 128-135. 2008). RA is an immune-mediated inflammatory disease in which autoreactive B and T cells produce autoantibodies, resulting in the formation of immune complexes within the joints (van Delft, et al., J. Autoimmun. 110, 102392. 2020). This leads to inflammation and recruitment of neutrophils, macrophages, and other immune cells to the area (Hirota et al., Immunity. 48, 1220-1232.e5.2018; Kuo et al., Sci. Transl. Med. 11.2019, doi:10.1126 / scitranslmed.aau8587; Takemura et al., J. Immunol. 167, 4710-4718.2001; Smolen et al., Nature Reviews Disease Primers. 4.2018. doi:10.1038 / nrdp.2018.1). These immune cells secrete cytokines such as IL-1 beta and TNF alpha that activate synovial cells. These cytokines are now being targeted therapeutically, which provides significant relief for most patients. OA is the most frequent and poorly understood form of arthritis, a stepwise progressive disease with no prominent immune component (Martel-Pelletier et al., Nat Rev Dis Primers. 2, 16072. 2016; Kapoor et al., Nat. Rev. Rheumatol. 7, 33-42. 2011; Goldring and Otero. Curr. Opin. Rheumatol. 23, 471-478. 2011).The generally accepted hypothesis is that mechanical injury to cartilage leads to a low-grade inflammatory state that mediates progressive cartilage loss (Kapoor et al., Nat. Rev. Rheumatol. 7, 33-42. 2011; Pap and Korb-Pap. Rheumatol. 11, 606-615. 2015).

[0088] In healthy synovial joints, the synovium surrounds and sequesters the joint cavity, secreting extracellular matrix proteins in synovial fluid. The main interstitial cells in the synovium are synovial fibroblasts, with resident macrophages occupying the space between them (Barbour et al., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056). During the active phase of RA, SFs become activated, express fibroblast activation protein alpha, and proliferate. SF cells, like other interstitial cells, express innate immune receptors such as Toll-like receptors. They detect local pathogens and molecular damage and secrete cytokines that activate immune cells (Ospelt et al., Arthritis Rheum. 58, 3684-3692. 2008). During inflammation, SFs proliferate and, together with infiltrating immune cells, form a thickened synovium called pannus (Choy. Rheumatology .51 Suppl 5,v3-11.2012). The pannus invades the joint cavity and degrades cartilage (Pap and Korb-Pap. Rheumatol.11,606-615.2015). In particular, it has been shown that SFs in the synovial lining layer mediate cartilage degradation, while sublining SFs tend to mediate inflammation (Croft et al., Nature.570,246-251.2019). ECM degrading activity is due to increased production of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs) and cathepsins (Rengel and Ospelt. Arthritis Res. Ther.9,221 2007).Arthritic synovial fibroblasts express both secreted MMPs (Jay et al., J. Rheumatol. 27, 594-600, 2000; Barbour et al., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056; Smolen et al., Nature Reviews Disease Primers. 4. 2018. doi:10.1038 / nrdp.2018.1) and cell surface MMPs (Lange-Brokaar et al., Osteoarthritis Cartilage. 20, 1484-1499. 2012; Nygaard and Firestein. Nat. Rev. Rheumatol. 16, 316-333. 2020; Bauer et al., Arthritis Res. Ther. 8, R171; 2006). In particular, MMP14 (MT1-MMP) is essential for the invasive properties of SFs.

[0089] Acquisition of abnormal matrix degradation is also a characteristic of SFs in OA (Fuchs et al., Osteoarthritis Cartilage. 12, 409-418. 2004). OA synovium usually has fewer immune cells than RA, but OA synovium undergoes cartilage degradation similar to RA. What controls the switch to ECM degradative mode of SFs is not fully understood. Gene expression changes are clearly suspected, and similar transcriptional signatures have been detected in both diseases (Cai et al., J Immunol Res. 2019, 4080735. 2019). Epigenetic changes have been detected and proposed to drive the arthritic SF phenotype (Nakano et al., Ann. Rheum. Dis. 72, 110-117. 2013). Whether these changes are sufficient remains unclear.

[0090] The phenotype of SFs in arthritis has been compared to that of malignant cancer cells. Indeed, cancer growth requires extensive remodeling of the ECM in the tissue of origin, with subsequent degradation of the ECM in the tissue of origin (Hotary et al., Cell. 114, 33-45. 2003). MMPs and other matrix degrading enzymes are particularly active in malignant cells (Castro-Castro et al., Cell Dev. Biol. 32, 555-576. 2016).

[0091] Gout is an inflammatory form of arthritis, also known as gouty arthritis. Gout is the most common inflammatory arthritis, with a prevalence of 2.5% in the UK. Although it can be cured, its treatment remains suboptimal (Abhishek et al.,Clin Med(Lond).2017 Feb;17(1):54-59). Ultrasonographic findings of gout include the double contour sign (deposition of MSU crystals on the hyaline articular cartilage surface). Normal adult articular cartilage consists of abundant ECM, composed mainly of type II collagen fibers, with scattered collagen types IX and XI. Cartilage loss tends to be a later feature of gouty arthropathy and is focal rather than diffuse, similar to bone erosions. Cartilage damage is often associated with erosions and has been described as occurring in areas of biomechanical stress. Treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss should be beneficial for gout patients. In some cases, the gout to be treated is associated with ECM degradation and / or cartilage loss.

[0092] Chondrocalcinosis, or cartilage mineralization, is calcification (accumulation of calcium salts) in hyaline cartilage and / or fibrocartilage. Accumulation of calcium phosphate in the ankle joint is seen in about 50% of the general population and may be associated with osteoarthritis (Hubert et al., BMC Musculoskelet Disord. 2018;19:169). It is frequently found in weight-bearing joints such as the hip, ankle, and knee. The molecular structure of calcium pyrophosphate has the potential to induce an inflammatory response. The presence of chondrocalcinosis is associated with the degradation of cartilage, meniscus, and synovial tissue. It has been reported that the presence of calcium-containing crystals associated with chondrocalcinosis was associated with a higher prevalence of cartilage and meniscus damage (Gersing et al., Eur Radiol. 2017 Jun;27(6):2497-2506. doi:10.1007 / s00330-016-4608-8. Epub 2016 Oct 4). Treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss should benefit chondrocalcinosis patients. In some cases, the chondrocalcinosis to be treated is associated with ECM degradation and / or cartilage loss.

[0093] Fibromyalgia (FM) is a condition characterized by chronic widespread pain and enhanced pain response to compression. FM is commonly seen in rheumatoid arthritis, axial spondyloarthritis and psoriatic arthritis and may therefore impact the management of these rheumatic conditions. FM is also associated with costochondritis.

[0094] Costochondritis is inflammation of cartilage in the rib cage. The condition usually affects the cartilage where the top of the rib attaches to the sternum (the area known as the costochondritis junction or costochondritis joint). Costochondritis can be caused by mechanical stress and can lead to cartilage loss and / or ECM degradation. Therefore, treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss can be beneficial for costochondritis patients. In some cases, the costochondritis to be treated is associated with ECM degradation and / or cartilage loss.

[0095] Osteochondritis dissecans (OCD or OD) is a disorder in which cracks form in the articular cartilage and the underlying subchondral bone. OCD usually causes pain during and after sports. In the later stages of the disorder, the affected joint swells and catches during movement and becomes immobilized. In the early stages, physical examination may identify pain as a symptom, and in the later stages, there may be exudate, tenderness, and cracking sounds with joint movement. Treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss should be beneficial for osteochondritis dissecans patients. In some cases, the osteochondritis dissecans to be treated is associated with ECM degradation and / or cartilage loss.

[0096] Polychondritis, or relapsing polychondritis (RP), is an immune-mediated systemic disease characterized by recurrent inflammatory episodes of cartilage and proteoglycan-rich tissues, including elastic cartilage of the ear and nose, hyaline cartilage of peripheral joints, fibrocartilage of the axial region, and cartilage of the tracheobronchial tree, resulting in progressive anatomical deformation and functional impairment of the involved structures (Borgio et al.,Biomedicines.2018 Sep;6(3):84). Unilateral or, more frequently, bilateral auricular chondritis is the most common feature of RP, present in up to 90% of patients during the course of the disease and is the initial symptom in 20% of cases. Onset is sudden, with painful red to purple erythema and edema localized to the cartilaginous portion of the ear, typically sparing the cartilageless earlobe. Acute inflammatory episodes tend to resolve spontaneously within days or weeks, but recur at variable intervals. As a long-term consequence of repeated flares, the cartilage matrix becomes significantly damaged and is replaced by fibrous connective tissue (Borgio et al., Biomedicines. 2018 Sep;6(3):84). Thus, treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss may be beneficial to polychondritis patients. In some cases, the polychondritis being treated is associated with ECM degradation and / or cartilage loss.

[0097] Cartilage damage can occur through many processes, for example through specific disease mechanisms such as arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, and polychondritis. Cartilage damage can also result from mechanical damage, for example through collision or hyperextension while playing sports. In some cases, cartilage damage requires surgery to remove the damaged tissue. Isolated cartilage and osteochondral defects in joints (such as the knee) are difficult clinical challenges, and options such as partial or total knee replacement are rarely recommended, especially in young patients. Numerous surgical techniques have been developed to address localized cartilage defects. Cartilage treatment strategies are characterized as palliative (e.g., chondroplasty and debridement), repair (e.g., drilling and microfracture [MF]), or restorative (e.g., autologous chondrocyte implantation [ACI], osteochondral autotransplantation [OAT], and osteochondral allotransplantation [OCA]) (Richter et al., Sports Health. Mar-Apr 2016;8(2):153-60. doi:10.1177 / 1941738115611350. Epub 2015 Oct 12). In some cases, treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss may be an alternative to surgery for patients with cartilage damage. Treatments that prevent, reduce, or reverse ECM degradation and / or cartilage loss may be beneficial for patients with cartilage damage.

[0098] In some cases, cartilage damage can lead to arthritis (e.g., osteoarthritis). Post-traumatic arthritis (PTA) develops after acute direct trauma to joints. PTA occurs in about 12% of all osteoarthritis cases, and patients with chronic inflammatory arthritis may also have a history of physical trauma. Treatment with GALA inhibitors after cartilage damage can prevent the development of arthritis after physical trauma.

[0099] Cartilage Degradation Related Disorders The present inventors have demonstrated that disorders associated with cartilage degradation can be treated with GALA inhibitors, such as anti-cnx antibodies.

[0100] The disorder associated with cartilage degradation may be arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage injury, or polychondritis.

[0101] The disorder associated with cartilage degradation may be arthritis.

[0102] The disorder associated with cartilage degradation may be osteoarthritis or rheumatoid arthritis.

[0103] Joint disorders The inventors have demonstrated that joint disorders can be treated with GALA inhibitors, such as anti-cnx antibodies.

[0104] A joint is defined as a connection between two bones in the skeletal system. Joints may be classified by the type of tissue present (fibrous, cartilaginous, or synovial), or the degree of motion (immobile, hemiarthrosis, or mobile). A joint disorder is therefore defined as a condition that affects the connection between two bones in the skeletal system. Definitions of specific joints and relevant anatomical aspects can be found in Netter, FH (2006). Atlas of human anatomy. Philadelphia, PA: Saunders / Elsevier, which is incorporated by reference in its entirety.

[0105] Joint disorders can affect fibrous, cartilaginous or synovial joints.

[0106] Fibrous joints are connected by dense connective tissue, which is mainly made up of collagen. Because these joints do not move, they are also called fixed or immovable joints. Fibrous joints do not have a joint cavity and are connected through fibrous connective tissue. The bones of the skull are connected by fibrous joints called sutures.

[0107] A cartilaginous joint is a type of joint in which the bones are attached entirely by cartilage (either hyaline or fibrocartilage). These joints generally allow more movement than fibrous joints, but less movement than synovial joints.

[0108] A synovial joint is characterized by the presence of a fluid-filled joint cavity contained within a fibrous capsule. It is the most commonly found type of joint in the human body and contains several structures not found in fibrous or cartilaginous joints. The three main features of a synovial joint are (i) the joint capsule, (ii) the articular cartilage, and (iii) the synovial fluid. The joint capsule surrounds the joint and is continuous with the periosteum of the articular bones. The articular surfaces of a synovial joint (i.e., the surfaces that directly contact each other as the bones move) are covered with a thin layer of hyaline cartilage. The main role of articular cartilage is twofold: (i) to minimize friction during joint movement, and (ii) to absorb shock. Synovial fluid is located within the joint cavity of a synovial joint. It has three main functions. A synovial joint may contain accessory structures such as tendons, ligaments, bursae, and the vasculature.

[0109] There are many types of synovial joints. In some cases, the joint disorder is a disorder of a gliding joint, a hinge joint, a pivot joint, an ellipsoid joint, a saddle joint, or a ball-and-socket joint.

[0110] Gliding joints, also known as planar or planiform joints, are a common type of synovial joint formed between bones that meet with flat or nearly flat articular surfaces. Gliding joints allow the bones to glide over each other in any direction along the plane of the joint: up and down, side to side, and diagonally. Slight rotation can also occur at these joints, but is limited by the shape of the bones and the elasticity of the joint capsule that surrounds them.

[0111] Hinge joints are bone joints whose articular surfaces are formed with each other in such a way that they can only move in one plane. According to one classification system, they are said to be uniaxial (have one degree of freedom) (Platzer, Werner (2008) Color Atlas of Human Anatomy, Volume 1). The direction that the distal bone takes in this movement is rarely in the same plane as the axial direction of the proximal bone, and in flexion there is usually some deviation from a straight line. The articular surfaces of the bones are connected by strong collateral ligaments. The best examples of hinge joints are the interphalangeal joints of the hand and the interphalangeal joints of the foot, as well as the joint between the humerus and the ulna. The knee and ankle joints are not typical, but they allow a small degree of rotation or side-to-side movement in certain positions of the limb. The knee is the largest hinge joint in the human body.

[0112] The pivot joint (trochlear joint, revolute joint, or lateral ginglymus joint) is a type of synovial joint whose axis of motion is parallel to the long axis of the proximal bone and generally has a convex articular surface. According to one classification system, the pivot joint has one degree of freedom (Platzer, Werner (2008) Color Atlas of Human Anatomy, Volume 1).

[0113] An oval joint (also called a condylar joint) is an oval-shaped articular surface, or condyle, housed in an oval cavity. It moves in two planes, allowing flexion, extension, adduction, abduction, and rotation, as seen in the wrist joint.

[0114] A saddle joint is a type of synovial joint in which opposing surfaces meet in one another. It is found in the big toe, rib cage, middle ear, and heel.

[0115] A ball-and-socket joint (or spherical joint) is a type of synovial joint in which the ball-shaped surface of one spherical bone fits into a cup-shaped recess in another bone. The distal bones can move about an indefinite number of axes with one common center. This allows the joint to move in many directions. Joint disorders can affect immobile syndesmosis, hemijoints, or diarthrodial joints. The hip and shoulder are ball-and-socket joints.

[0116] An immobile joint is a type of joint that does not move under normal conditions. Suture and peg joints are both immobile joints.

[0117] A hemiarthrosis is a joint that has limited mobility. An example of this type of joint is the cartilaginous joint that unites adjacent vertebral bodies.

[0118] A diarthrodial joint is a joint that can move freely. Sometimes the terms diarthrodial joint and synovial joint are used interchangeably.

[0119] Joint disorders may affect any joint. In some cases, joint disorders may affect any mammalian joint. In some cases, joint disorders may affect any human joint.

[0120] In some cases, joint disorders affect the hips, knees, ankles, feet, toes, shoulders, elbows, wrists, hands, fingers, neck, spine, ribs, or sacroiliac joints.

[0121] The joint disorder may be osteoarthritis, psoriatic arthritis, rheumatoid arthritis, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, polychondritis, cartilage injury, tendon injury, or ligament injury.

[0122] Bursitis is inflammation of the bursa, a fluid-filled pouch that acts as a cushion between bone and muscle, skin, or tendons. The type of bursitis depends on where the affected bursa is located. This soft tissue condition commonly affects the shoulder, elbow, hip, buttock, knee, and calf. Athletes, the elderly, and people who perform repetitive movements, such as manual laborers and musicians, are more likely to develop bursitis. Bursitis is sometimes mistaken for arthritis because it can cause pain in the joints.

[0123] Tendon injury, or tendon disorder, can be caused by many reasons, such as overuse, aging, wear and tear, or mechanical damage. Tendon injury can be tendinitis or tendonosis. Tendonitis refers to inflammation of the tendon, and tendonosis refers to tearing of the tissue of the tendon and the tissue surrounding the tendon. Tendon injury can be tendon overuse, tendon strain, tendon tear, partial rupture of the tendon, or complete rupture of the tendon. Treatments that prevent, reduce, or reverse ECM degradation and / or tendon loss can be beneficial for patients with tendon injury. In some cases, the tendon injury to be treated is associated with ECM degradation and / or cartilage loss.

[0124] Ligament injury can be caused by many reasons, such as overuse, aging, wear and tear, or mechanical damage. Ligament injury can be ligament overuse, ligament strain, ligament tear, partial ligament rupture, or complete ligament rupture. Treatments that prevent, reduce, or reverse ECM degradation and / or ligament loss can be beneficial for ligament injury patients. In some cases, the tendon injury to be treated is associated with ECM degradation and / or cartilage loss.

[0125] Characteristics of the disorder The disorders described herein (e.g., disorders associated with cartilage degradation, and joint disorders) may be characterized by a number of features. In some cases, the disorder is characterized by ECM degradation. In some cases, the disorder is characterized by increased ECM degradation. In some cases, the disorder to be treated is characterized by increased expression of calnexin. In some cases, the disorder to be treated is characterized by increased glycosylation of calnexin. In some cases, the disorder is characterized by increased O-glycosylation of calnexin. In some cases, the disorder is characterized by increased O-glycosylation. In some cases, the disorder is characterized by GALNT activation (GALA). In some cases, the disorder is characterized by increased GALNT activation (GALA). In some cases, the disorder is characterized by inflammation.

[0126] The ECM is a highly dynamic structure, undergoing a constant remodeling process in which ECM components are deposited, degraded, or otherwise modified. Extracellular matrix (ECM) degradation is mediated by cell surface and secreted proteases, in particular matrix metalloproteases (MMPs), including but not limited to MMP14 and O-glycosylated MMP14.

[0127] Calnexin (CNX) is a 90 kDa calcium-binding protein in the ER membrane that forms a complex with PDIA3. Calnexin is a target of GALA. Following glycosylation, the CNX-PDIA3 complex has previously been shown to translocate to the surface of cancer cells, where it reduces disulfide bridges in the extracellular matrix (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). Reduction of disulfide bridges has been shown to be essential for the effective activity of matrix metalloproteinases (MMPs) and thus for the degradation of the extracellular matrix in cancer.

[0128] When reference is made to a CNX polypeptide, this should be taken as a reference to any member of the CNX polypeptide family. Of particular interest are CNX polypeptides derived from genes in the group consisting of: mouse gene ID: 12330, rat gene ID: 29144, dog gene ID: 403908, feline gene ID: 101085686, and horse gene ID: 100067402.

[0129] For example, the Cnx polypeptide may constitute the human CNX sequence having GenBank Accession Number NP_001350929.1, NP_001350926.1, NP_001350923.1, NP_001350922.1, NP_001350924.1, NP_001350928.1, NP_001350927.1 or NP_001019820.1.

[0130] PDIA4 is one of the largest PDI members, containing 645 amino acids and possessing three classical CGHC active sites. Together with ER Hsp70 (BiP), Grp94, PDI and ERp29, they form a multiprotein chaperone complex that is an ER network capable of binding unfolded protein substrates (Meunier et al., Mol. Biol. Cell, 13 (2002), pp. 4456-4469, 10.1091 / mbc.e02-05-0311).

[0131] When reference is made to a PDIA4 polypeptide, this should be taken as a reference to any member of the PDIA4 polypeptide family. Of particular interest are PDIA4 polypeptides derived from genes in the group consisting of: Homo sapiens gene ID: 9601, Mus musculus gene ID: 12304, Rattus norvegicus gene ID: 116598, Bos taurus gene ID: 415110, Equus caballus gene ID: 100060535, Felis catus gene ID: 101087568 and Canis lupus familiaris gene ID: 482715.

[0132] For example, the PDIA4 polypeptide may constitute the human PDIA4 sequence having GenBank Accession Number NP_001358173.1, NP_001358174.1, or NP_004902.1.

[0133] PDIA3, also known as ERp57, is an isomerase. This protein is localized to the endoplasmic reticulum (ER) and interacts with the lectin chaperones calreticulin and calnexin (CNX) to regulate the folding of newly synthesized glycoproteins. Lectin-protein complexes are thought to mediate protein folding by promoting disulfide bond formation in their glycoprotein substrates.

[0134] When reference is made to a PDIA3(ERp57) polypeptide, this should be taken as a reference to any member of the PDIA3(ERp57) polypeptide family. Of particular interest are PDIA3(ERp57) polypeptides derived from genes in the group consisting of: Homo sapiens gene ID: 2923, Mus musculus gene ID: 14827, Rattus norvegicus gene ID: 29468, Bos taurus gene ID: 281803, Equus caballus gene ID: 100056198, Felis catus gene ID: 101097245, and Canis lupus familiaris gene ID: 478279.

[0135] For example, the ERp57 polypeptide may constitute the human PDIA3 (ERp57) sequence having GenBank Accession Number NP_005304.3.

[0136] Glycosylation is an enzymatic process that attaches glycans to proteins or other biomolecules. O-glycosylation is the addition of O-linked glycans to the side chain hydroxyl groups of, for example, serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residues of proteins.

[0137] The N-acetylgalactosaminyltransferase (GALNTs) activation (GALA) pathway is regulated by translocation of the O-glycosylation initiation enzyme GALNTs from the Golgi apparatus to the ER. GALNTs catalyze the formation of Tn glycans consisting of a single GalNAc residue. Tn can be detected with lectins such as Vicia Villosa lectin (VVL). Translocation of GALNTs to the ER dramatically increases the overall cellular staining level of Tn, which appears in the cytoplasm rather than the nuclear periphery (Gill, DJ et al. Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness. Proc. Natl. Acad. Sci. USA 110, E3152-61 (2013).

[0138] Inflammation is part of the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, or irritants. The function of inflammation is to eliminate the initial cause of cellular injury, remove necrotic cells and tissue damaged by the original insult and inflammatory process, and initiate tissue repair. Clinically and epidemiologically, inflammation is usually assessed by measuring circulating substances released as a cause or consequence of the inflammatory response, with C-reactive protein (CRP) being the most widely used biomarker of inflammation (Sproston and Ashworth. Front Immunol. 2018;9:754).

[0139] In some cases, the disorder to be treated is characterized by increased expression of calnexin, increased expression of PDIA4, increased expression of PDIA3, increased glycosylation of calnexin, increased O-glycosylation of calnexin, increased O-glycosylation, GALA, increased GALA, inflammation, and / or increased inflammation.

[0140] When disorder is characterized by the increase in expression, activity, or another characteristic, the characteristic increases compared to the subject not affected by the disorder.The increase can be more than 1-fold, for example, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold compared to the subject not affected by the disorder.

[0141] Where a disorder associated with cartilage degradation is characterized by an increase in the trait, affected subjects will have a greater level of the trait when compared to corresponding subjects not affected by the trait.

[0142] Where the joint disorder is characterized by an increase in the trait, affected subjects will have greater levels of the trait in their joints compared to matched controls (e.g., subjects not afflicted with the joint disorder, or unaffected joints).

[0143] Where arthritis is characterized by an increase in a trait, affected subjects have greater levels of the trait in their joints compared to matched controls (e.g., matched subjects not afflicted with arthritis, or matched joints not afflicted with arthritis).

[0144] Where the disorder is characterized by increased expression of calnexin, increased expression of PDIA4, increased expression of PDIA3, increased glycosylation of calnexin, increased O-glycosylation of calnexin, increased O-glycosylation, increased GALA, and / or increased inflammation, the trait is increased compared to a corresponding control (e.g., a subject not afflicted with the disorder).

[0145] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased o-glycosylation, affected subjects have greater levels of o-glycosylation compared to matched controls (e.g., subjects not affected by the disorder). The level of o-glycosylation can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to a subject not affected by the disorder.

[0146] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased o-glycosylation in articular tissue, cartilage tissue, and / or synovial fibroblasts, affected subjects will have greater levels of o-glycosylation in articular tissue, cartilage tissue, and / or synovial fibroblasts when compared to corresponding controls (e.g., subjects not affected by the disorder). The level of O-glycosylation in articular tissue, cartilage tissue, and / or synovial fibroblasts can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to a subject not affected by the disorder.

[0147] When a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased cellular levels of Tn (T nouvelle), affected subjects have greater levels of Tn (T nouvelle) compared to corresponding controls (e.g., subjects not affected by the disorder). The level of Tn (T nouvelle) can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to a subject not afflicted with the disorder.

[0148] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased cellular levels of Tn (T nouvelle) in articular tissue, cartilage tissue, and / or synovial fibroblasts, affected subjects have greater levels of Tn (T nouvelle) in articular tissue and / or synovial fibroblasts when compared to corresponding controls (e.g., subjects not affected by the disorder). The level of Tn (T nouvelle) in joint tissue and / or synovial fibroblasts can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to a subject not afflicted with the disorder.

[0149] When a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased CNX glycosylation levels, affected subjects have greater CNX glycosylation levels compared to corresponding controls (e.g., subjects not affected by the disorder). The level of CNX glycosylation can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to a subject not affected by the disorder.

[0150] When a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of CNX glycosylation in synovial fibroblasts, affected subjects have greater levels of CNX glycosylation in synovial fibroblasts compared to corresponding controls (e.g., subjects not affected by the disorder). The level of CNX glycosylation in synovial fibroblasts can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, compared to subjects not affected by the disorder.

[0151] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of CNX cell surface expression, affected subjects have greater levels of CNX cell surface expression compared to corresponding controls (e.g., subjects not affected by the disorder). The CNX cell surface expression level may be greater than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, as compared to a subject not afflicted with the disorder.

[0152] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of CNX cell surface expression in synovial fibroblasts, affected subjects have greater levels of CNX cell surface expression in synovial fibroblasts compared to corresponding controls (e.g., subjects not affected by the disorder). The level of CNX cell surface expression in synovial fibroblasts may be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, as compared to subjects not affected by the disorder.

[0153] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of cartilage ECM degradation, affected subjects will have greater levels of ECM degradation compared to matched controls (e.g., subjects not affected by the disorder). The level of cartilage ECM degradation can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, ≧10-fold, ≧20-fold, ≧30-fold, ≧40-fold, ≧50-fold, ≧60-fold, ≧70-fold, ≧80-fold, ≧90-fold, or ≧100-fold, as compared to a subject not afflicted with the disorder.

[0154] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of ECM degrading activity, affected subjects have greater levels of ECM degrading activity when compared to matched controls (e.g., subjects not affected by the disorder). The level of ECM degradation activity can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, ≧10-fold, ≧20-fold, ≧30-fold, ≧40-fold, ≧50-fold, ≧60-fold, ≧70-fold, ≧80-fold, ≧90-fold, or ≧100-fold, relative to a subject not afflicted with the disorder.

[0155] Where a disorder (e.g., a disorder associated with cartilage degradation, a joint disorder, and / or arthritis) is characterized by increased levels of CNX, CNX / ERP57, and / or synovial fibroblast ECM degrading activity, affected subjects have greater levels of CNX, CNX / ERP57, and / or synovial fibroblast ECM degrading activity when compared to corresponding controls (e.g., subjects not affected by the disorder). The level of ECM degrading activity of CNX, CNX / ERP57, and / or synovial fibroblasts can be one of more than 1-fold, e.g., ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2.0-fold, ≧2.5-fold, ≧3.0-fold, ≧3.5-fold, ≧4.0-fold, ≧4.5-fold, ≧5.0-fold, ≧5.5-fold, ≧6.0-fold, ≧6.5-fold, ≧7.0-fold, ≧7.5-fold, ≧8.0-fold, ≧8.5-fold, ≧9.0-fold, ≧9.5-fold, or ≧10-fold, relative to a subject not afflicted with the disorder.

[0156] When comparing patients or subjects with a disorder with subjects without the disorder, an assay or test can be used to determine the level of the property of interest in both subjects and to determine whether the property of interest is increased in subjects with the disorder. Many assays are known in the art that can determine the relative levels of biological properties.

[0157] One of skill in the art would be able to determine the relative levels of properties such as ECM degradation, glycosylation, O-glycosylation, gene expression (e.g., calnexin, PDIA4, and / or PDIA3 expression), protein expression, GALA, and inflammation through methods well known in the art.

[0158] One of skill in the art may also use methods known in the art to determine whether a disorder exhibits characteristics, for example, one of skill in the art may use known methods to determine whether a disorder is associated with the presence of ECM degradation, glycosylation, O-glycosylation, expression of certain genes (e.g., calnexin expression), expression of certain proteins (e.g., calnexin), GALA, and inflammation through methods known in the art.

[0159] The presence and level of ECM degradation can be determined through many methods, such as ECM degradation assays and histological analysis. Those skilled in the art will recognize many methods that can measure ECM degrading activity, such as those shown in the examples herein. Such methods can easily determine whether a Cnx / ERp57 inhibitor can inhibit ECM degrading activity, the ECM degrading activity of Cnx / ERp57, and / or the ECM degrading activity of Cnx.

[0160] The following steps are one way one of skill in the art can assay for ECM degrading activity.

[0161] Commercially available gelatin solution (2%) can be labeled with 5-carboxy-X-rhodamine, succinimidyl ester. The labeled gelatin can then be transferred to a sterile cover slip to create a thin layer, which can then be stabilized by glutaraldehyde fixation. Finally, a rat tail collagen solution can be used to coat the cover slip to create a thin layer of collagen on top of the gelatin.

[0162] The coverslips can then be transferred to a culture vessel, seeded with degradatively active cells (eg, human hepatocellular carcinoma Huh7) and incubated for 48 hours to allow degradation to occur.

[0163] After fixation, the coverslips can be stained with Hoescht to allow the cells to be counted. The coverslips can then be imaged with a confocal microscope. The images can then be analyzed using ImageJ. A threshold can be manually defined to reveal the surface of degraded gelatin, and the total area per field was measured. In parallel, the number of nuclei can be calculated and the final results can be normalized to the cells in each field.

[0164] The presence and level of glycosylation can be determined by methods known in the art, such as those used in the Examples, and by commercially available assay kits. Methods for detecting and analyzing glycosylation and glycoproteins include glycan staining with lectins, glycan labeling, glycoprotein purification or enrichment, high performance liquid chromatography (HPLC) analysis, and mass spectrometry analysis. Those skilled in the art will be aware of such methods.

[0165] The presence and level of O-glycosylation can be determined through methods known in the art, such as those used in the Examples herein. A hallmark of O-glycosylation is an increase in the cellular levels of Tn (T nouvelle), an O-glycan formed by the addition of GalNac to Ser or Thr residues. Tn can be detected by Tn-binding proteins such as Vicia Villosa lectin (VVL) and Helix Pomatia lectin (HPL) (Gill, et al., Proc. Natl. Acad. Sci. USA110, E3152-61 2013).

[0166] The presence and level of gene expression (e.g., calnexin expression) can be determined through many different methods known to those of skill in the art. The level of RNA encoding a given gene can be determined by techniques such as RNAseq, RT-PCR, and RT-qPCR, among other well-known methods.

[0167] The presence and level of protein expression (e.g., calnexin expression) can be determined by means well known to those of skill in the art. The level of a given protein / its isoforms can be determined, for example, by antibody-based methods including Western blot, immunohisto / cytochemistry, flow cytometry, ELISA, etc.

[0168] The presence and levels of GALA can be determined through methods known in the art, such as those used in the Examples herein. A hallmark of GALA is increased cellular levels of Tn (T nouvelle), an O-glycan formed by the addition of GalNac to Ser or Thr residues. Tn can be detected by Tn-binding proteins such as Vicia Villosa lectin (VVL) and Helix Pomatia lectin (HPL) (Gill, et al., Proc. Natl. Acad. Sci. USA110, E3152-61 2013).

[0169] The presence and level of inflammation can be determined through methods known in the art, for example, through analysis of biomarkers such as cytokines, chemokines, and immune cells. Such analysis can be performed using techniques known in the art, such as mass spectrometry.

[0170] Damage mediators (e.g., fibroblasts, synovial fibroblasts) The disorders described herein may be mediated by fibroblast activity. Fibroblasts are cells that synthesize collagen and ECM. However, fibroblasts can also degrade ECM, allowing the turnover of this essential component of tissue. Fibroblasts contribute to joint and cartilage health. In healthy individuals, fibroblasts contribute to the viscosity of synovial fluid by secreting proteins such as hyaluronic acid and lubricin. In joint and cartilage disorders such as arthritis, fibroblasts attach to and degrade the ECM of cartilage.

[0171] In some cases, the disorders described herein may be mediated by synovial fibroblasts (SFs). SFs, also called synoviocytes, are the primary fibroblasts in synovial joints.

[0172] Those skilled in the art can determine whether a disease is mediated by fibroblast activity through methods known in the art. For example, those skilled in the art will be aware of suitable biochemical assays for determining the level of fibroblast activity. Fibroblast activity of a known disease can be assayed to determine whether the disease is associated with an increase in fibroblast activity compared to a suitable control (e.g., non-diseased subject), which indicates that the disease is mediated by fibroblast activity. Alternatively, animal models of a particular disease can be utilized to determine whether a disease is mediated by fibroblast activity. For example, a mouse model of the disease of interest can be modified to ablate fibroblast populations (e.g., synovial fibroblast populations). Analysis of the modified model compared to a non-modified model can be used to determine whether a disease is mediated by fibroblast activity. For example, a decrease in disease characteristics observed after ablation of fibroblast populations should indicate that the disease is mediated by fibroblast activity.

[0173] arthritis Rheumatoid arthritis (RA), an autoimmune disease, and osteoarthritis (OA), a degenerative disease, are both characterized by the degradation of articular cartilage.

[0174] Rheumatoid arthritis is understood as an autoimmune inflammatory condition. The affected joints of patients with rheumatoid arthritis are infiltrated with immune cells, including but not limited to macrophages, T cells, and B cells. These cells are concentrated in the enlarged synovium called pannus. Consistent with an autoimmune disease, autoantibodies are found in the serum of patients with rheumatoid arthritis. These autoantibodies are presumed to form immune complexes within the joints, thereby activating immune cells. However, not all patients with rheumatoid arthritis exhibit detectable autoantibodies, suggesting a complex pathogenesis.

[0175] In contrast, without being bound by theory, osteoarthritis is believed to occur largely independent of inflammation and immune cells, although inflammation may also be present. Osteoarthritis is further believed to result from mechanical damage to cartilage, which leads to progressive cartilage degradation and ultimately bone degradation.

[0176] Although rheumatoid arthritis and osteoarthritis are different diseases, it is understood that they share cellular and molecular characteristics. For example, damage to the cartilage extracellular matrix is ​​a pathological characteristic common to both osteoarthritis and rheumatoid arthritis and is a typical early symptom. In general, a key pathological characteristic of arthritis is the destruction of cartilage. This destruction begins with the degradation of the extracellular matrix (ECM) of cartilage and can eventually lead to the loss of chondrocytes, the cells that synthesize and maintain cartilage. In addition, as the disease progresses, it can also lead to bone erosion and loss. Thus, the methods disclosed herein are adapted for use in treatments performed during the development of the disease, up to the later stages of the diseases disclosed herein, to prevent cartilage damage.

[0177] This application presents evidence of GALA pathway activation in arthritic human samples, e.g., rheumatoid arthritis and osteoarthritis joint tissues. Synovial fibroblasts, responsible for cartilage degradation, rely on the GALA pathway to activate matrix degradation. This is believed to occur specifically through cell surface exposure of the calnexin (CNX) complex (i.e., the CNX-PDIA3 complex), which mediates the reduction of disulfide bonds. In other words, inhibition of the disulfide isomerase activity of the CNX-PDIA3 complex is believed to improve cartilage degradation. Genetic inhibition of the GALA pathway in synovial fibroblasts has been shown to block matrix degradation in vitro and reduce arthritic symptoms in vivo. Targeting calnexin (CNX), for example, with antibodies, produces similar results, pointing to therapeutic approaches. Overall, the results disclosed herein indicate that the GALA glycosylation pathway is a key determinant of arthritic pathology.

[0178] Treatment of disorders with GALA inhibitors Also disclosed herein is the treatment of cartilage disorders, joint disorders, and arthritic conditions, including but not limited to rheumatoid arthritis (RA) and osteoarthritis (OA). Currently, there is no cure for either rheumatoid arthritis (RA) or osteoarthritis (OA) patients. Rheumatoid arthritis patients receive disease-modifying anti-rheumatic drugs or immunomodulatory drugs, such as but not limited to anti-TNF alpha and anti-IL-6, which mainly target the immune system. The main problem is the immune dysfunction induced by these treatments. It has also been shown that these known treatments are unable to completely stop disease progression and prevent disease recurrence. For example, severe end-stage rheumatoid arthritis (RA) or osteoarthritis (OA) patients who have previously failed to respond to such anti-rheumatic drugs or immunomodulatory drugs must undergo total joint replacement surgery, which is associated with a high risk of serious adverse events. Therefore, alternative pathway-based therapies for patients with disorders related to cartilage degradation, joint damage, and arthritis represent a significant unmet medical need.

[0179] Arthritis-active synovial fibroblasts (SFs) were found to exhibit activation of the glycopathway known as GalNAc-T activation (GALA). GALA activation has been shown in some human rheumatoid arthritis, most human osteoarthritis samples, and is induced in mouse models of rheumatoid arthritis. In addition, inhibiting GalNAc-T activation (GALA) in synovial fibroblasts reduces cartilage degradation in vitro. For example, targeting calnexin, a target of GalNAc-T activation (GALA), has been shown to be similarly effective. In vivo, inhibiting GalNAc-T activation (GALA) in mouse models of rheumatoid arthritis using protein inhibitors, such as the protein inhibitor ER-2Lec, almost completely blocks cartilage degradation.

[0180] Thus, it has been shown that inhibition of GalNAc-T activation (GALA) in synovial fibroblasts has therapeutic applications.In one example, a method for treating arthritis in a subject is disclosed, comprising administering a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.In another example, the use of a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway in the manufacture of a medicament for treating arthritis is described.Also disclosed herein is one or more therapeutic agents that inhibit the GalNAc-T activation (GALA) pathway for use in treating arthritis.

[0181] In one example of therapeutic use, the nucleic acid sequence encoding protein ER-2Lec is transduced or transfected into synovial fibroblasts, for example, but not limited to, by using adeno-associated virus (AAV) or lentivirus transfection system, or any other gene delivery method.In another example, therapeutic use is to inhibit one or more targets selected from, but not limited to, calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), thioreductase activity of calnexin-PDIA3 complex, glycosylated MMP14 (e.g., O-glycosylated MMP14), or any cell surface-associated GALA-associated protein on the surface of synovial fibroblasts.In one example, such inhibition can be brought about or caused by using blocking antibodies, fragments thereof, or derivatives thereof.

[0182] Thus, in one example, in a method or use as described herein, the therapeutic agent inhibits any one or more of the cell surface markers involved in the GalNAc-T activation (GALA) pathway.

[0183] In another example, targets for treatment of disorders associated with cartilage degradation, joint damage, and arthritis as disclosed herein include targeting calnexin (CNX), PDIA4, PDIA3, and / or a complex of CNX and PDIA3, hi another example, the target is calnexin (CNX), PDIA4, and / or PDIA3 bound to or present on the cell surface.

[0184] In addition to targeting the immune system, it is also contemplated to control the synovium itself, for example by targeting cadherin-11.

[0185] The synovium is formed by hypertrophy of the synovial membrane. Normally, the synovium is confined to the periphery of the joint area, but during arthritis, this tissue infiltrates the joint area and attacks the cartilage. The synovium contains multiple cell types, but the key cells thought to be involved in the degradation of the extracellular matrix (ECM) are the synovial fibroblasts. Synovial fibroblasts are normally relatively few in number and quiescent, but in arthritic conditions, these cells proliferate, resulting in extracellular matrix degrading activity. What induces synovial fibroblasts to switch on their extracellular matrix degrading activity is not fully understood.

[0186] Extracellular matrix (ECM) degradation is mediated by cell surface and secreted proteases, particularly matrix metalloproteases (MMPs), including but not limited to MMP14 and O-glycosylated MMP14. These proteins are particularly active in cells of solid malignant tumors. Indeed, it has been shown that cancer growth requires extensive remodeling of the extracellular matrix in the original tissue, involving degradation and synthesis of new extracellular matrix.

[0187] The degradation of (extracellular) matrix in cancer cells has been shown to be controlled by a glycopathway known as GALA (for GALNT Activation). GALA controls the activity of GALNTs, enzymes that initiate O-glycosylation and add complex sugars to various proteins. The control of the GALA pathway (and thus the degradation of extracellular matrix in cancer cells) is mediated through the intracellular distribution of GALNTs, which involves translocation from the Golgi apparatus to the endoplasmic reticulum (ER). This translocation makes various substrates more readily O-glycosylated.

[0188] One exemplary target of GalNAc-T activation (GALA) is matrix metalloproteinase-14 (MMP14), whose O-glycosylation has been shown to be essential for the proteolytic activity of MMP14. Another target of GALA is the protein calnexin (CNX), located in the endoplasmic reticulum, which forms a complex with ERp57, also known as protein disulfide isomerase A3 (PDIA3). Following glycosylation, this complex translocates to the surface of cancer cells, where it reduces disulfide bridges in the extracellular matrix. Reduction of disulfide bridges has been shown to be essential for the effective activity of matrix metalloproteinases (MMPs), and thus for the degradation of the extracellular matrix. Thus, GalNAc-T activation (GALA) has been shown to coordinate or combine two enzymatic activities, one of which is proteolysis and the other of which is reduction of disulfide bonds.

[0189] Thus, in one example, a therapeutic agent disclosed herein inhibits one or more of the targets of the GalNAc-T Activation (GALA) pathway selected from the group consisting of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), calnexin-PDIA3 (CNX:PDIA3) complex, glycosylated matrix metalloproteinase-14 (MMP14), including O-glycosylated matrix metalloproteinase-14 (MMP14), and combinations thereof. In one example, the therapeutic agent inhibits calnexin (CNX). In another example, the therapeutic agent inhibits PDIA3. In a further example, the therapeutic agent inhibits the calnexin-PDIA3 complex. In yet another example, the therapeutic agent inhibits calnexin and PDIA3. In one example, the therapeutic agent inhibits glycosylated MMP14.

[0190] Experimental data described herein reveal that arthritis-active synovial fibroblasts display signs of significant GALA activation. The hallmark of GalNAc-T activation (GALA) is shown in some human rheumatoid arthritis and most human osteoarthritis samples. In mouse rheumatoid arthritis models, GALA is induced prior to the appearance of symptoms. In addition, inhibition of GALA in synovial fibroblasts using the protein inhibitor ER-2Lec has been shown to reduce cartilage degradation in vitro. In vivo, inhibition of GALA in mouse rheumatoid arthritis models using ER-2Lec expressed in synovial fibroblasts has been shown to specifically prevent disease progression, thereby also confirming the role of GALA in disease. Finally, in another example, targeting calnexin with an antibody has been shown to almost completely block cartilage degradation, demonstrating the need for the use of these concepts in therapy.

[0191] Osteoarthritis and rheumatoid arthritis patients share some common pathological features and show overlapping cellular and molecular characteristics. One of the pathological features of arthritis is the destruction of cartilage extracellular matrix (ECM) that occurs during the onset of disease, followed by irreversible bone loss. Synovial fibroblasts are reported to be a subset of synoviocytes and have been shown to cause damage to cartilage extracellular matrix through direct infiltration or through secretion of matrix remodeling enzymes (e.g., but not limited to, MMP14, ADAMTS).

[0192] Translocation of the N-acetylgalactosaminyltransferase (GalNAc-T) enzyme from the Golgi apparatus to the endoplasmic reticulum (ER), also known as the GalNAc-T activation (GALA) pathway, has been shown to be a key regulator of tumor extracellular matrix (ECM) remodeling. GalNAc-T catalyzes the addition of a single sugar to serine (Ser) and / or threonine (Thr) residues. The resulting glycans, called Tn antigens, are enriched in the endoplasmic reticulum (ER) of GALA-activated cells and serve as GALA activation markers. To test the GALA activation status in human samples from arthritic patients, we evaluated Tn glycan levels in synovial tissue from rheumatoid arthritis and osteoarthritis patients using a commercially available antibody. Rheumatoid arthritis patients showed a trend toward increased median Tn(O-GalNAc) glycan levels compared to healthy subjects (0.34 vs. 0.24, p=0.06, Figure 21). The median Tn glycan levels in osteoarthritis patients were significantly higher than both healthy subjects (0.59 vs. 0.24, p=0.002, FIG. 21) and rheumatoid arthritis patients (0.59 vs. 0.34, p=0.01). The majority of osteoarthritis patients (19 / 21, 90.4%) had higher median Tn glycan levels than healthy subjects (FIG. 21). These results revealed that arthritis patients showed higher GALA activation in synovial tissue compared to healthy individuals, and osteoarthritis patients exhibited GALA hyperactivation.

[0193] To understand the functional contribution of GALA activation in arthritis, we utilized an in vivo preclinical model of collagen antibody-induced arthritis. Consistent with the findings in clinical samples as outlined above, we found increased Tn levels in arthritis-induced joints (Figure 2B). This induction correlated with arthritis severity, reaching its peak at days 7–10 (Figures 2C and 2D). Furthermore, Tn glycan staining in control joints showed localization to the Golgi complex, whereas Tn glycan staining in arthritis revealed colocalization of Tn glycans with the endoplasmic reticulum resident protein calnexin (CNX). Taken together, these localizations may point to an activated state of GALA (Figure 3, enlarged panel). In addition, we identified synovial fibroblasts in the pannus as the major cell subset exhibiting GALA activation in arthritic joints (Figure 4).

[0194] To specifically inhibit the GalNAc-T activation (GALA) pathway in synovial fibroblasts, we engineered a genetically modified mouse line to express a GALA inhibitor (the two lectin domains of GalNAc-T2) only in the endoplasmic reticulum of synovial fibroblasts using the Cre / LoxP system (Figure 8A). We observed that inhibition of the GALA pathway using such an approach restored the integrity of the cartilage extracellular matrix and reduced the severity of arthritic disease (Figures 8B-8H). Taken together, these results indicate that the GALA pathway plays an important role in the degradation of cartilage extracellular matrix in arthritic disease.

[0195] The GalNAc-T activation (GALA) pathway controls the glycosylation of several cell surface and secreted proteins. For example, the GALA pathway has been shown to hyperglycosylate and activate the matrix metalloprotease MMP14, which is a driver of extracellular matrix degradation in both cancer and rheumatoid arthritis. Upon activation of the GalNAc-T (GALA) pathway, it has been shown that endoplasmic reticulum resident proteins (e.g., but not limited to, calnexin (CNX), PDIA3, PDIA4) are hyper-O-glycosylated. Furthermore, it has been shown that the calnexin (CNX) protein activated by GALA migrates to the cell surface, forms a complex with protein disulfide isomerase A3 (PDIA3), and causes the degradation of tumor extracellular matrix components via reduction of disulfide bonds in matrix constituent proteins. Without being bound by theory, calnexin (CNX) and PDIA3 have been shown to be targets of GALA pathway regulation / inhibition, and MMP14 and PDIA4 are also targets of GALA pathway regulation / inhibition.Therefore, glycosylated MMP14 is also a target of GALA pathway regulation / inhibition.PDIA4, a paralog of PDIA3, functions similarly to PDIA3 and is also a target of arthritis treatment similar to PDIA3.In one example, the arthritis is rheumatoid arthritis.

[0196] To exploit this concept, a high-throughput screening assay was developed to select the most suitable GALA-related targets for arthritis treatment. In this assay, synovial fibroblasts (cell line SW982) were cultured on quenched fluorescent cartilage matrix components (in this example, fluorogenic DQ collagen) in the presence of GALA target blocking agents (such as, but not limited to, antibodies or siRNAs). The degradative activity of synovial fibroblasts increased the fluorescent signal, which was quantitatively captured using a high-content imaging system (Figure 21A). Consistent with previous findings in liver tumors, this screen identified three GALA-related targets, calnexin, PDIA3, and MMP14, among the main hits that function as positive regulators of cartilage degradation in synovial fibroblasts (Figure 21C).

[0197] From the above screening, we identified GALA-targeted inhibitors that inhibit the degradation of cartilage extracellular matrix components in synovial fibroblasts. Among the identified inhibitors, a monoclonal antibody clone targeting calnexin (CNX) was selected for follow-up testing for therapeutic use.

[0198] Thus, in one example, the target of the GALA pathway is calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3).

[0199] In a further example, there is provided (a) a method for identifying a molecule that inhibits one or more targets of the GalNAc-T activation (GALA) pathway, the method comprising contacting the one or more targets with the molecule and determining whether the molecule inhibits the one or more targets; (b) a method for identifying an inhibitor of the GalNAc-T activation (GALA) pathway, the method comprising contacting a cell with the inhibitor and determining the level or activity of expression of one or more targets of the GalNAc-T activation (GALA) pathway in, on, or on the cell. (c) a method for identifying a molecule suitable for arthritis or a molecule suitable for the treatment, prevention or alleviation of arthritis, comprising determining whether the candidate molecule is an agonist or antagonist of calnexin (CNX), protein disulfide isomerase A3 (PDIA3), protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and protein disulfide isomerase A3 (PDIA3), preferably the candidate molecule is an agonist or antagonist of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and protein disulfide isomerase A3 (PDIA3), or (d) a method for identifying an agonist or antagonist of CNX, PDIA4, PDIA3, or CNX and PDIA3, comprising administering the candidate molecule to an animal and determining whether the animal is an agonist or antagonist of CNX, PDIA4, PDIA3, or CNX and PDIA3. The method includes determining whether the expression level or expression activity of DIA3, or CNX and PDIA3, is increased or decreased, wherein one or more targets of the GalNAc-T activation (GALA) pathway are selected from the group consisting of calnexin (CNX), protein disulfide isomerase A3 (PDIA3), protein disulfide isomerase A4 (PDIA4), CNX:PDIA3 complex, and O-glycosylated matrix metalloproteinase-14 (MMP14). In another example, the method disclosed herein optionally includes a step of isolating or synthesizing the molecule, modulator, agonist, or antagonist.

[0200] In one example, (a) a method for identifying a molecule that inhibits one or more targets of the GalNAc-T activating (GALA) pathway, comprising contacting the one or more targets with the molecule and determining whether the molecule inhibits the one or more targets; (b) a method for identifying an inhibitor of the GalNAc-T activating (GALA) pathway, comprising contacting a diseased cell with the inhibitor and detecting a decrease in the expression level or activity of one or more targets of the GalNAc-T activating (GALA) pathway in, on, or on the cell, wherein the disease is arthritis. (c) a method for identifying a molecule suitable for treating, preventing or alleviating arthritis, comprising determining whether a candidate molecule is an agonist or antagonist of calnexin (CNX), protein disulfide isomerase A3 (PDIA3), protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and protein disulfide isomerase A3 (PDIA3), preferably by subjecting the candidate molecule to an agonist or antagonist of CNX, PDIA4, or (d) a method for identifying an agonist or antagonist of CNX, PDIA4, PDIA3, or CNX and PDIA3, comprising exposing a candidate molecule to a cell expressing PDIA3, or CNX and PDIA3, or CNX, PDIA4, PDIA3, or CNX and PDIA3, and determining whether the animal model of arthritis exhibits an increase or decrease in the expression level or expression activity of CNX, PDIA4, PDIA3, or CNX and PDIA3. Disclosed is a method for treating osteoarthritis, comprising isolating or synthesizing a molecule, a modulator, an agonist, or an antagonist, comprising administering to said arthritis patient a therapeutically effective amount of at least one of the following:

[0201] In yet another example, the methods disclosed herein are methods for identifying or screening libraries of compounds or molecules capable of inhibiting one or more targets of the GalNAc-T activation (GALA) pathway. In another example, the compounds or molecules identified using the screening methods disclosed herein are administered to an animal or subject.

[0202] To model the complex physiological characteristics of synovial fibroblast cartilage degradation activity in the context of arthritis, primary synovial fibroblasts (also referred to as OASF cells) were isolated from osteoarthritis patients with active disease symptoms. Cultured OASF cells showed high GalNAc-T pathway activation (Figure 21B) and highly active dye quenching (DQ)-collagen matrix degrading activity, consistent with previous findings in synovial tissue. Selected antibody clones were found to inhibit the cartilage degrading activity of cultured OASF cells (p=0.001, Figure 21C).

[0203] Collectively, this data indicates that the GalNAc-T (GALA) activation pathway influences cartilage degradation in arthritis, and thus targeting the GALA activation pathway serves as a therapeutic target and approach for the treatment of arthritis.

[0204] Enhanced O-glycosylation in synovial membranes in rheumatoid arthritis and osteoarthritis Just as the GalNAc-T pathway is involved in the degradation of extracellular matrix in cancer cells, the same pathway is thought to be involved in cartilage degradation in arthritic episodes and arthritis. A marker of GalNAc-T pathway activation is increased levels of Tn (also referred to as Tn antigen), a single sugar O-glycan formed by the addition of GalNac to serine (Ser) or threonine (Thr) residues. Tn can be detected by lectins, such as, but not limited to, Vicia villosa lectin (VVL) and Helix pomatia lectin (HPL).

[0205] Tissue microarrays of joint tissues were analyzed by immunofluorescence using Vicia villosa lectin (VVL) as a counterstain for DNA. A clear increase in signal was detected in samples from patients with osteoarthritis and in some samples from rheumatoid arthritis patients (Figure 1A). The integrated fluorescence intensity of Vicia villosa lectin staining was then quantified, normalized against DNA signal intensity as a marker of cell density (Figure 1B). Healthy patient samples revealed little variability, whereas most, if not all, diseased patient samples showed increased Tn levels, with some rheumatoid arthritis and osteoarthritis samples showing a 5- to 7-fold increase in Vicia villosa lectin signal.

[0206] To further study this correlation, a murine rheumatoid arthritis model based on collagen antibody-induced arthritis (CAIA) was utilized. Briefly, mice were injected with antibodies against type II collagen, followed 3 days later by lipopolysaccharide (LPS). This treatment led to arthritic symptoms, including but not limited to paw swelling and lameness, on day 7, which persisted until day 14 and then gradually subsided (Figures 2A, 2C). Histologically, the formation of pannus that infiltrated the joint cavity was observed on day 7. The size of the pannus increased, and on day 10, the level of inflammation appeared to increase based on the influx of immune cells, and the pannus persisted until day 14 (Figure 2A). Using immunofluorescence labeling, an increase in Tn staining was observed on day 7, especially in the infiltrated pannus (Figure 2B). Tn staining persisted until day 10 and faded by day 14 (Figures 2B, 2D).

[0207] Arthritic synovium shows signs of GALA pathway activation Activation of the GalNAc-T pathway is well described as a mechanism leading to increased Tn levels. One of the hallmarks of GALA pathway activation is the redistribution of N-acetylgalactosaminyltransferases (GALNTs), particularly GALNT1 and GALNT2, from the Golgi apparatus to the endoplasmic reticulum. To verify that the GalNAc-T (GALA) pathway was indeed activated, we performed co-staining with Vicia villosa lectin (VVL) and calnexin, an endoplasmic reticulum marker (Figure 3A). In untreated samples, Vicia villosa lectin staining and calnexin staining were clearly separated, and Vicia villosa lectin was concentrated in a punctate pattern consistent with the Golgi complex (Figure 3A). In contrast, in collagen antibody-induced arthritis (CAIA) samples at day 7, Vicia villosa lectin staining and calnexin staining were clearly co-localized. We then performed a similar analysis with GALNT2 staining. Increased GALNT2 staining intensity was observed, indicating increased levels of the GALNT2 enzyme in disease states (Figure 3B). In addition, the distribution pattern of the enzyme followed a similar transition as Vicia villosa lectin, redistributing to an endoplasmic reticulum-like pattern and colocalizing with calnexin (Figure 3B).

[0208] Thus, in one example, the identification of GALNT1 and GALNT2 localized to the endoplasmic reticulum is used as a target for the treatment of rheumatoid arthritis and osteoarthritis. In another example, the target of the GALA pathway is calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3).

[0209] In one example, a method for detecting the presence or absence of a disorder associated with cartilage degradation in a subject is disclosed, comprising the steps of: (i) obtaining a synovial fibroblast sample from the subject; (ii) detecting levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in the sample obtained in step (i); and comparing the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (ii) with the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample of a control group, wherein an increase in the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates the presence of a disorder associated with cartilage degradation, and the control group consists of subjects without a disorder associated with cartilage degradation.

[0210] In another example, a method for detecting the presence or absence of arthritis, optionally osteoarthritis or rheumatoid arthritis, in a subject is disclosed, comprising the steps of: (i) obtaining a synovial fibroblast sample from the subject; (ii) detecting the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in the sample obtained in step (i); and comparing the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (ii) with the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample of a control group, wherein an increase in the levels of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates the presence of arthritis, and the control group consists of subjects without arthritis.

[0211] In one example, a method for detecting the presence or absence of a disorder associated with cartilage degradation in a subject is disclosed, comprising the steps of: (i) detecting the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample; (ii) comparing the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (i) with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, wherein an increase in the level of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates suitability for treatment. The control group may consist of subjects without a disorder associated with cartilage degradation.

[0212] In another example, a method for detecting the presence or absence of arthritis, optionally osteoarthritis or rheumatoid arthritis, in a subject is disclosed, comprising the steps of: (i) detecting the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample; (ii) comparing the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (i) with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, wherein an increase in the level of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates suitability for treatment. The control group may consist of subjects without arthritis, osteoarthritis, or rheumatoid arthritis.

[0213] In one example, there is a method for determining whether a subject is suitable for treatment with an anti-cnx antibody through the following steps: (i) detecting the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in a synovial fibroblast sample, (ii) comparing the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (i) with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, where an increase in the level of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates suitability for treatment. The control group can consist of subjects who do not have cartilage degradation, do not have a disorder related to cartilage degradation, do not have arthritis, do not have osteoarthritis, or do not have rheumatoid arthritis.

[0214] In another example, there is a method for determining whether a subject is suitable for treatment with an anti-cnx antibody by the following steps: (i) obtaining a synovial fibroblast sample from the subject, (ii) detecting the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation in the synovial fibroblast sample, and (iii) comparing the level of Tn antigen, Tn glycan, CNX cell surface expression, CNX glycosylation, and / or ECM degradation of step (ii) with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, where an increase in the level of Tn antigen, Tn glycan, CNX expression, CNX glycosylation, and / or ECM degradation shown in the sample compared to the control group indicates suitability for treatment. The control group can be composed of subjects who do not have cartilage degradation, do not have a disorder related to cartilage degradation, do not have arthritis, do not have osteoarthritis, or do not have rheumatoid arthritis.

[0215] In another example, a method for detecting the presence or absence of arthritis in a subject is disclosed, comprising the steps of: detecting the level of Tn antigen / Tn glycan in a sample obtained from the subject; and comparing the level of Tn antigen / Tn glycan detected in the preceding step with the level of Tn antigen / Tn glycan in a synovial fibroblast sample of a control group, wherein an increase in the level of Tn antigen / Tn glycan shown in the sample compared to the control group indicates the presence of arthritis, and the control group consists of subjects without arthritis.

[0216] Synovial fibroblasts are the predominant cell type expressing GALA The synovium of rheumatoid arthritis is a complex tissue that contains immune cells and synovial fibroblasts. To establish which cell types showed increased GalNAc-T levels, joint samples from collagen antibody-induced arthritis (CAIA) were co-stained with Vicia villosa lectin (VVL) and CD45, an immune cell marker, or vimentin, a fibroblast marker. Tn-positive cells in the pannus were predominantly vimentin-positive (vm + ), and CD45 negative (CD45 + ) (Figure 4). A set of CD45+ cells was also detected, but these were mostly located posterior to the anterior pannus. In human samples from patients with osteoarthritis and rheumatoid arthritis, fibroblast activation protein alpha (FAPα) was used as a marker for synovial lining fibroblasts (Figures 5A, 5B). Vicia villosa lectin (VVL) staining was shown to correlate with FAPα in both conditions. In rheumatoid arthritis samples, a CD45-rich compartment was prominent beneath the layer of FAPα-positive cells, but these immune cells were not significantly labeled by Vicia villosa lectin (Figures 5A, 5B).

[0217] Stimulation of synovial fibroblasts with cytokines and ECM induces higher GALA levels To understand how the GalNAc-T pathway is activated in vivo, primary human synovial fibroblasts from patients were used. Purity of the used cell preparations was established >90% by fluorescence-activated cell sorting (FACS) analysis using CD90 and CD45 as markers for synovial fibroblasts and immune cells, respectively (Figure 6A). Synovial fibroblasts were then stimulated with TNFα and IL1beta cytokines, which are thought to promote disease progression in rheumatoid arthritis. Although individual cytokines had limited effects on GALA activation, the combination of both cytokines (labeled CYTO) induced a two-fold increase in Vicia villosa lectin (VVL) cellular levels, especially in rheumatoid arthritis synovial fibroblasts (RASFs) (Figures 6B, 6C). The staining pattern of Vicia villosa lectin was clearly endoplasmic reticulum-like, indicating activation of GALA (Figure 6B). Healthy synovial fibroblasts showed little or no response, suggesting that the patient's cells were primed to the stimulus (Figures 6B, 6C).

[0218] It was suspected whether cartilage extracellular matrix proteins could contribute to the activation of synovial fibroblasts. Indeed, exposure of synovial fibroblasts to cartilage extracellular matrix activated the GalNAc-T pathway almost three-fold in both osteoarthritis and rheumatoid arthritis synovial fibroblasts. Again, in contrast, cells from healthy patients were unresponsive or only slightly responsive (Figure 6B, 6C). The combination of CYTO (TNFα and IL1 beta cytokines) and extracellular matrix had a slight additive effect (Figure 6C). The stimulation did not seem to be specific to cartilage extracellular matrix, since a similar activation was induced using type I collagen from rat tail (Figure 6C). Thus, combined stimulation with both cytokines and native cartilage extracellular matrix appears to elicit the greatest GALA response in synovial fibroblasts, with the latter being the strongest inducer, indicating that targeting GALA is appropriate for patients with early-onset rheumatoid arthritis who do not yet exhibit active inflammatory signs. Furthermore, this indicates that the treatments disclosed herein are also suitable for osteoarthritis patients who, despite having low levels of cytokine production, also exhibit GALA activation in fibroblasts.

[0219] Inhibition of GALA in synovial fibroblasts reduces cartilage extracellular matrix degradation Since activation of the GalNAc-T pathway leads to extracellular matrix degradation in cancer cells, we sought to test whether it is also involved in the degradation of cartilage extracellular matrix. This was done using a fluorescent gelatin sandwich assay as previously described. ER-2Lec is a chimeric protein composed of a fusion of two lectins, an endoplasmic reticulum (ER) targeting sequence and GALNT2. ER-2Lec inhibits the activity of GALA by interfering with ER-specific O-glycosylation (Figure 7A). Using SW982, a cell line derived from a synovial sarcoma patient, we generated stable ER-2Lec transfectants under the control of a doxycycline-inducible promoter system. SW982 cells were then stimulated with CYTO (TNFα and IL1 beta cytokines) to stimulate extracellular matrix degradation. An increase in extracellular matrix degradation was observed (Figure 7B). However, when expression of ER-2Lec was induced, extracellular matrix degradation was reduced more than two-fold (FIGS. 7B, 7C).

[0220] Expression of ER-2Lec in synovial fibroblasts attenuates arthritis in vivo To test whether ER-2Lec protein could alleviate arthritis in vivo, we generated ER-2Lec transgenic mice carrying a Lox cassette. These mice were then crossed with mice expressing Cre under the collagen VI alpha 1 (Col6a1) promoter (Figure 8A). Collagen VI is expressed by joint mesenchymal cells, of which fibroblasts are the most abundant cell type. As a result of this genetic cross, ER-2Lec is expressed primarily in synovial fibroblasts (Col6a1Cre ER-2Lec). Indeed, in the joint, we found that ER-2Lec was mainly expressed in synovial fibroblasts (Figure 8B).

[0221] We next induced collagen antibody-induced arthritis (CAIA) in Col6a1Cre ER-2Lec animals, and although these animals still formed pannus that invaded the joint cavity, articular cells showed reduced Tn levels, indicative of GALA inhibition (Fig. 8B).

[0222] Rheumatoid arthritis symptoms following collagen antibody-induced arthritis (CAIA) were monitored in animals expressing Cre alone or also ER-2Lec. Animals expressing ER-2Lec in synovial fibroblasts (Col6a1Cre ER-2Lec) showed reduced paw swelling (Figure 9A). Changes in paw thickness were measured from day 1 to day 7, with increases in control animals and some variation between paws (Figure 9B). In contrast, ER-2Lec animals showed little swelling. A blinded assessment using the internationally defined arthritis score showed consistent reductions in symptoms in Col6a1Cre ER-2Lec animals (Figure 10).

[0223] Histological analysis was then performed on day 7 using Alcian Blue (AB) and Safranin-O (SO) staining, which are commonly used to reveal the cartilage compartment of the joint. After inducing collagen antibody-induced arthritis (CAIA), pannus formation was observed in both groups of animals, with no significant difference in the size of the pannus (Figures 11A, 11B). Of note, in contrast, there were clear differences in the appearance of the cartilage. For example, the Alcian Blue area was severely reduced in CAIA Col6a1Cre animals compared to untreated animals, whereas it appeared to be largely preserved in Col6a1Cre ER-2Lec animals. Cartilage mm 2 Preservation of cartilage was observed in CAIA-treated Col6a1Cre ER-2Lec animals by measuring the percentage of Alcian blue-positive area per unit area (Figure 6C). Similar data were obtained after quantification of Safranin-O (SO) staining (Figure 6D).

[0224] Thus, in one example, expression of an ER-2Lec construct in synovial fibroblasts is illustrated to result in a GALA inhibitory effect.

[0225] GALA activates glycosylation and surface exposure of calnexin in synovial fibroblasts

[0226] Recently, calnexin (CNX) was described as a glycosylation target and effector of GALA. Once glycosylated, calnexin translocates to the cell surface and mediates disulfide bond cleavage in extracellular matrix proteins together with protein disulfide isomerase A3 (PDIA3). This reducing activity is essential for matrix degradation by cancer cells. In SW982 synovial fibroblasts, calnexin (CNX) was found to be overglycosylated by approximately six-fold after stimulation with cytokines and extracellular matrix (Figures 12A, 12B). Expression of ER-2Lec was able to reduce the glycosylation of calnexin, indicating that this glycosylation is GALA-dependent (Figures 12A, 12B).

[0227] In addition, using fluorescence-activated cell sorting (FACS), we found that surface expression of calnexin was increased after stimulating these cells with CYTO (TNFα and IL-1β cytokines) and extracellular matrix (Fig. 13A, 13B). Expression of ER-2Lec decreased calnexin expression, indicating that the cell surface expression of calnexin was closely correlated with GALA (Fig. 13A, 13B).

[0228] Thus, in one example, the calnexin inhibitor is ER-2Lec. In another example, ER-2Lec is used in gene therapy. In yet another example, an expression vector is used to express ER-2Lec. In other words, ER-2Lec is expressed using an expression vector. In another example, ER-2Lec can be transfected into cells using chemical transfection, viral transfection, lentiviral transduction or cloning. Viral vectors that can be used for such purposes include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses (AAV) and adenoviruses. In one example, the vector is an adeno-associated virus vector.

[0229] We next sought to confirm these results in primary cells obtained from patients. In healthy synovial fibroblasts, stimulation with cytokines (in this case, TNFα and IL-1β cytokines) and extracellular matrix induced an increase in the percentage of cell surface calnexin positive cells (Figure 14A). Synovial fibroblasts from patients with rheumatoid arthritis (RASF) or osteoarthritis (OASF) showed a three-fold increase in cell surface calnexin levels (Figure 14B). In contrast, synovial fibroblasts from healthy donors showed very limited induction of cell surface calnexin.

[0230] Overall, these results indicate that the glycosylation of calnexin and its exposure at the cell surface are dependent on GALA and indicate that calnexin is involved in the degradation of cartilage extracellular matrix.

[0231] Thus, described herein is the inhibition of calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) and / or protein disulfide isomerase A4 (PDIA4) with inhibitors of calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3, also known as ERp57) and / or protein disulfide isomerase A4 (PDIA4, also known as ERp72). It is further noted that PDIA4 is a paralog of PDIA3 and has similar functions as PDIA3.

[0232] As used herein, the term "inhibitors of calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) and / or protein disulfide isomerase A4 (PDIA4)" refers to any agent or compound capable of inhibiting the expression and / or function of CNX, or protein disulfide isomerase A3 (PDIA3) or protein disulfide isomerase A4 (PDIA4), or a complex containing calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3). Thus, agents or compounds capable of inhibiting PDIA4, or calnexin (CNX) and / or PDIA3, are referred to as "protein disulfide isomerase A4 (PDIA4) inhibitors" or "calnexin (CNX) / protein disulfide isomerase A3 (PDIA3) inhibitors," or "antagonists of protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3)."

[0233] In some examples, the protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) inhibitor is an inhibitor of calnexin (CNX). In some examples, the protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) inhibitor is an inhibitor of protein disulfide isomerase A3 (PDIA3). In some examples, the calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) inhibitor, or protein disulfide isomerase A4 (PDIA4) inhibitor is an inhibitor of a complex comprising calnexin (CNX) and protein disulfide isomerase A3 (PDIA3). In some instances, the protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) inhibitor is an inhibitor of protein disulfide isomerase A4 (PDIA4).

[0234] In some instances, an inhibitor of protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) comprises one or more of the following effects: decreasing the expression (e.g., gene and / or protein expression) of calnexin (CNX), protein disulfide isomerase A3 (PDIA3), protein disulfide isomerase A4 (PDIA4), and / or a complex comprising calnexin (CNX) and protein disulfide isomerase A3 (PDIA3). reducing the level of RNA encoding calnexin (CNX) and / or PDIA3 and / or PDIA4; reducing / inhibiting the transcription of nucleic acids encoding calnexin (CNX) and / or PDIA3 and / or PDIA4; increasing / promoting the degradation of RNA encoding calnexin (CNX) and / or PDIA3 and / or PDIA4; inhibiting post-transcriptional processing (e.g. splicing) of RNA encoding calnexin (CNX) and / or PDIA3 and / or PDIA4. (i) reducing / inhibiting the transcription, translation, and post-translational processing of calnexin (CNX) protein, PDIA4 protein, PDIA3 protein, and / or protein complexes containing calnexin (CNX) and PDIA3; (ii) reducing / inhibiting the transcription, translation, and post-translational processing of calnexin (CNX) protein, PDIA4 protein, PDIA3 protein, and / or protein complexes containing calnexin (CNX) and PDIA3; (iii) increasing / promoting the degradation of calnexin (CNX) protein, PDIA4 protein, PDIA3 protein, and / or protein complexes containing calnexin (CNX) and PDIA3; (ii) reducing / inhibiting the interaction between a complex comprising calnexin (CNX), PDIA4, PDIA3, and / or an interaction partner of a complex comprising calnexin (CNX) and PDIA3 (including, for example, reducing / inhibiting the interaction between calnexin (CNX) and PDIA3); reducing the functional levels of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3; calnexin (CNX), PDIA4, PDIA3,and / or reducing / inhibiting the degradation of extracellular matrix (ECM) by a complex comprising calnexin (CNX) and PDIA3; reducing / inhibiting the oxidoreductase activity of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3; and / or reducing / inhibiting the disulfide bond reductase activity of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3.

[0235] A given agent or compound can be evaluated for the properties detailed in the preceding paragraph using a suitable assay. The assays include, but are not limited to, in vitro assays, optionally cell-based or cell-free assays. When the assays are cell-based assays, they can include treating the cells to upregulate the expression and / or activity of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex that includes calnexin (CNX) and PDIA3, and treating the cells with a test agent or compound to determine whether the agent or compound exhibits one or more of the properties detailed.

[0236] Gene expression can be analyzed by means well known to those skilled in the art. The level of RNA encoding a given gene can be determined by techniques such as, but not limited to, RT-qPCR, qPCR, etc. Protein expression can also be determined by other means well known to those skilled in the art, such as chromatography and Western blot. The level of a given protein / its isoforms can be determined using antibody-based methods, such as, but not limited to, Western blot, immunohistology, immunostaining, immunohistochemistry, cytochemistry, flow cytometry, ELISA, etc.

[0237] Inhibition of gene or protein expression of a given gene can be less than 1-fold relative to expression levels observed in uninhibited conditions (e.g., in the absence of a Protein Disulfide Isomerase A4 (PDIA4), or Calnexin (CNX) and / or Protein Disulfide Isomerase A3 (PDIA3) inhibitor), specifically, 0.99-fold or less (≦0.99-fold), ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold. In some examples, a drug or compound capable of reducing / inhibiting the expression of a gene or protein inhibits said expression by more than 5%. In another example, gene expression is inhibited by 10% or more (≧10%), ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100% of the expression observed in the non-inhibited state. Such inhibition can be presented using the term "fold change", which is defined as the ratio between two quantities. That is, for quantities X and Y, the fold change of Y relative to X is Y / X. In other words, a change from 30 to 60 is defined as a fold change of 2. This is also referred to as a "2-fold increase." Similarly, a change from 30 to 15 is referred to as a "2-fold decrease."

[0238] Agents or compounds that can reduce / inhibit gene expression of protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3) (i.e., can reduce the level of RNA encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3), can reduce / inhibit the transcription of nucleic acid encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or PDIA3, and / or can increase / promote the degradation of RNA encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or PDIA3) can be identified using an assay that includes detecting the level of RNA encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or PDIA3, for example, by RT-qPCR. Such assays may involve treating cells / tissues with an agent and then comparing the levels of RNA encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or PDIA3 in the cells / tissues with the levels of RNA encoding protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or PDIA3 in the cells / tissues under appropriate control conditions (e.g., without limitation, untreated / vehicle-treated cells / tissues, mock-treated cells, or an appropriate negative control).

[0239] Agents or compounds capable of reducing / inhibiting protein expression of protein disulfide isomerase A4 (PDIA4), or calnexin (CNX) and / or protein disulfide isomerase A3 (PDIA3), as described herein, can be identified using assays that include detecting the levels of CNX protein, PDIA3 protein, PDIA4 protein, or protein complexes containing CNX and PDIA3, for example, using antibody / reporter-based methods (Western blot, ELISA, immunohistochemistry, immunohistology, cytochemistry, etc.). Such assays can include treating cells / tissues with an agent and then comparing the levels of protein / protein complexes in such cells / tissues to those in cells / tissues under appropriate control conditions.

[0240] Agents or compounds that can reduce / inhibit the interaction between (i) calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3, and (ii) an interaction partner of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3 (e.g., the interaction between calnexin (CNX) and PDIA3) can be identified, for example, using an assay that includes detecting the level of interaction (e.g., using an antibody / reporter-based method). The level of interaction between (i) calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex comprising calnexin (CNX) and PDIA3, and (ii) an interaction partner of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising CNX and PDIA3, may be analyzed using methods such as, but not limited to, resonance energy transfer techniques (e.g., FRET, BRET) or methods that analyze correlations of interactions. The assay may involve treating cells / tissues with an agent and then comparing the level of interaction between (i) calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3, and (ii) an interaction partner of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3 in such cells / tissues with the level of interaction observed in appropriate control cells / tissues (e.g., untreated / vehicle-treated cells / tissues). In addition, the level of interaction between (i) calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3, and (ii) an interaction partner of calnexin (CNX), PDIA4, PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3 (e.g., the interaction between calnexin (CNX) and PDIA3) may be analyzed using techniques such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, or biolayer interferometry.Assays can involve comparing the level of interaction in the presence of an agent with the level of interaction in suitable control conditions, for example, an untreated sample or a sample in the absence of agent.

[0241] (i) calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex containing calnexin (CNX) and protein disulfide isomerase A3 (PDIA3), and (ii) interaction proteins of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex containing calnexin (CNX) and PDIA3. The inhibition of the interaction between the toner (e.g., the interaction between calnexin (CNX) and PDIA3) is less than 1-fold, e.g., ≦0.99-fold, ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold, relative to the level of interaction observed in the uninhibited state. In some instances, the present invention reduces / inhibits the interaction between (i) PDIA4, calnexin (CNX), PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3, and (ii) an interaction partner of PDIA4, calnexin (CNX), PDIA3, and / or a complex comprising calnexin (CNX) and PDIA3 (e.g., the interaction between calnexin (CNX) and PDIA3). An agent or compound capable of inhibiting the interaction will inhibit more than 5% of the interaction observed under uninhibited conditions, e.g., one of ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0242] Agents or compounds capable of reducing / inhibiting the function of protein disulfide isomerase A4 (PDIA4), calnexin (CNX), protein disulfide isomerase A3 (PDIA3), and / or complexes comprising calnexin (CNX), PDIA3, may be identified using assays for the relevant function. Such assays may involve treating cells / tissues expressing PDIA4, CNX, PDIA3 and / or complexes comprising CNX and PDIA3 with an agent and then comparing the level of the relevant function to that found in appropriate control conditions, such as untreated / vehicle-treated cell / tissue samples, negative controls, or cell / tissue samples treated with an ineffective compound or a compound lacking the required effect.

[0243] Agents or compounds capable of reducing / inhibiting the function of protein disulfide isomerase A4 (PDIA4), calnexin (CNX), protein disulfide isomerase A3 (PDIA3), and / or a complex comprising calnexin (CNX) and PDIA3 may be identified using assays that include detecting levels of those that correlate with the function of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex comprising CNX and PDIA3, for example, the level of gene and / or protein expression and / or activity of one or more proteins whose expression is directly / indirectly upregulated or downregulated as a result of the function of CNX, PDIA4, PDIA3, and / or a complex comprising CNX and PDIA3. Such assays may involve treating cells / tissues expressing CNX, PDIA4, PDIA3, and / or complexes comprising CNX and PDIA3 with a drug or compound, and then comparing the levels of something that correlates with the function of CNX, PDIA4, PDIA3, and / or complexes comprising CNX and PDIA3 in such cells / tissues with the levels of something that correlates with the relevant function in appropriate control conditions as disclosed herein.

[0244] The function of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or a complex comprising CNX and PDIA3 can be, but is not limited to, extracellular matrix (ECM) degradation, oxidoreductase activity, or disulfide bond reductase activity. The correlate of the function of PDIA4, CNX, PDIA3, and / or a complex comprising CNX and PDIA3 can be, but is not limited to, a result of extracellular matrix (ECM) degradation, oxidoreductase activity, or disulfide bond reductase activity.

[0245] Agents capable of reducing / inhibiting extracellular matrix (ECM) degradation by calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or complexes comprising CNX and PDIA3 can be identified using assays that include detecting the level of extracellular matrix (ECM) or extracellular matrix (ECM) degrading activity (for example, but not limited to, using antibody / reporter-based methods). Extracellular matrix (ECM) degradation can be measured using methods known in the art that are available to those skilled in the art. The assays can include treating cells / tissues expressing CNX, PDIA4, PDIA3, and / or complexes comprising CNX and PDIA3 with an agent, and then comparing the level of extracellular matrix (ECM) degradation to the level of extracellular matrix (ECM) degradation observed in appropriate control conditions.

[0246] Agents or compounds capable of reducing / inhibiting the oxidoreductase activity and / or disulfide bond reductase activity of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or complexes comprising CNX and PDIA3 can be identified using an assay that includes detecting the level of the relevant activity. For example, the oxidoreductase activity and / or disulfide bond reductase activity can be measured, for example, using an insulin reduction assay known in the art. The assay can include comparing the level of the relevant activity exhibited by CNX, PDIA4, PDIA3, and / or complexes comprising CNX and PDIA3 in the presence of the agent with the level observed in the absence of the agent.

[0247] Inhibition of the function of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or complexes containing CNX and PDIA3 (e.g., but not limited to, extracellular matrix (ECM) degradation, oxidoreductase activity, disulfide bond reductase activity) reduces the level of activity observed in the uninhibited state. In contrast, it may be less than 1-fold, for example, 0.99-fold or less (≦0.99-fold), ≦0.95-fold, ≦0.9-fold, ≦0.85-fold, ≦0.8-fold, ≦0.75-fold, ≦0.7-fold, ≦0.65-fold, ≦0.6-fold, ≦0.55-fold, ≦0.5-fold, ≦0.45-fold, ≦0.4-fold, ≦0.35-fold, ≦0.3-fold, ≦0.25-fold, ≦0.2-fold, ≦0.15-fold, ≦0.1-fold, ≦0.05-fold, or ≦0.01-fold. In some instances, an agent or compound capable of reducing / inhibiting the function (e.g., extracellular matrix (ECM) degradation, oxidoreductase activity, disulfide bond reductase activity) of CNX, PDIA4, PDIA3, and / or a complex comprising CNX and PDIA3 inhibits the relevant activity observed in the uninhibited state by more than 5%, or by 10% or more (≧10%), ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0248] Protein disulfide isomerase A4 (PDIA4), or CNX and / or protein disulfide isomerase A3 (PDIA3) inhibitors according to the present disclosure can be any kind of drug or any type of compound with suitable inhibitory activity.

[0249] In some examples, the protein disulfide isomerase A4 (PDIA4), or CNX and / or protein disulfide isomerase A3 (PDIA3) inhibitor is selected from the following: a PDIA4 binding molecule, or a CNX binding molecule, a PDIA3 binding molecule, a CNX:PDIA3 complex binding molecule, a molecule capable of reducing levels of CNX, a molecule capable of reducing levels of PDIA3, a molecule capable of reducing levels of PDIA3, and a molecule capable of reducing levels of a CNX:PDIA3 complex.

[0250] As used herein, the term "CNX-binding molecule" refers to a molecule capable of binding to CNX. Similarly, a "PDIA3-binding molecule" or a "PDIA4-binding molecule" refers to a molecule capable of binding to protein disulfide isomerase A3 (PDIA3) or protein disulfide isomerase A4 (PDIA4). A "CNX:PDIA3 complex-binding molecule" refers to a molecule capable of binding to a complex comprising CNX and PDIA3.

[0251] Such binding molecules may be identified using any suitable assay for detecting binding of a molecule to an associated factor (which in the context of the present disclosure is CNX, PDIA4, PDIA3, or a complex comprising CNX and PDIA3). Such an assay may include detecting the formation of a complex between the associated factor and the molecule.

[0252] CNX-binding molecules, protein disulfide isomerase A4 (PDIA4)-binding molecules, protein disulfide isomerase A3 (PDIA3)-binding molecules, and CNX:PDIA3-binding molecules can be analyzed in suitable assays to identify antagonists of CNX or PDIA3. For example, molecules that specifically bind to CNX, PDIA4, PDIA3, and / or complexes containing CNX and PDIA3 can be evaluated for their ability to inhibit extracellular matrix (ECM) degradation, or for their ability to inhibit oxidoreductase and / or disulfide bond reductase activity.

[0253] CNX binding molecule, protein disulfide isomerase A4 (PDIA4) binding molecule, protein disulfide isomerase A3 (PDIA3) binding molecule or CNX:PDIA3 complex binding molecule can inhibit the ability of its target (i.e., CNX, PDIA3, CNX:PDIA3 complex) to interact with its interaction partner. In some examples, PDIA3 binding molecule, CNX binding molecule, PDIA3 binding molecule or CNX:PDIA3 complex binding molecule behaves as a competitive inhibitor of the interaction between its target and its interaction partner for that target. Binding molecule can occupy or otherwise reduce or block access to a region of the target that is necessary for binding with its interaction partner for that target. The ability of CNX binding molecule, PDIA4 binding molecule, PDIA3 binding molecule or CNX:PDIA3 complex binding molecule to inhibit the interaction between its target and its interaction partner for its target can be evaluated by, for example, but not limited to, interaction analysis in the presence of one or both of the interaction partners and the relevant binding molecule, or after incubation of one or both of the interaction partners with the relevant binding molecule.An example of a suitable assay for determining whether a given binding agent can inhibit the interaction between CNX, PDIA4, PDIA3 or CNX:PDIA3 complex and interaction partner is competitive enzyme-linked immunosorbent assay (ELISA).

[0254] Calnexin (CNX) binding molecules, protein disulfide isomerase A4 (PDIA4) binding molecules, protein disulfide isomerase A3 (PDIA3) binding molecules, and CNX:PDIA3 complex binding molecules include antigen binding molecules.

[0255] Antigen binding molecules capable of binding to calnexin (CNX), protein disulfide isomerase A4 (PDIA4) binding molecules, protein disulfide isomerase A3 (PDIA3), or complexes containing CNX and PDIA3 recognize regions of the relevant target protein / protein complex that are accessible to the antigen binding molecule when the protein / protein complex is expressed on the cell surface. This is the case, for example, when the protein / protein complex is exposed in or on the cell membrane of the cell expressing the protein / protein complex, and they are available for binding. In some examples, the antigen binding molecule binds to a region that is extracellular of CNX, PDIA4, PDIA3, or complexes containing CNX and PDIA3 when the protein / protein complex is expressed on the cell surface.

[0256] As used herein, the term "antigen-binding molecule" refers to a molecule capable of binding to a given target antigen, and includes, but is not limited to, antibodies (immunoglobulins), such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as, but not limited to, bispecific or trispecific antibodies), as well as fragments and derivatives thereof (such as, but not limited to, Fv, scFv, Fab, scFab, F(ab') 2 , Fab 2 , diabodies, triabodies, scFv-Fc, minibodies, and single domain antibodies (such as but not limited to VhH).

[0257] The antigen-binding molecule may specifically bind to a relevant target disclosed herein, which is, but is not limited to, calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), or a complex containing CNX and PDIA3. As used herein, the term "specific binding" refers to selective binding and can be distinguished from non-specific binding to non-target molecules. The antigen-binding molecule specifically binds to calnexin (CNX), PDIA4, PDIA3, or a complex containing CNX and PDIA3. Preferably, the antigen-binding molecule binds to a relevant target with higher affinity and / or longer duration than it binds to other non-target molecules.

[0258] The antigen-binding molecule may be or may comprise an antigen-binding peptide / polypeptide or an antigen-binding peptide / polypeptide complex. The antigen-binding molecule may be a non-covalent or covalent complex of more than one polypeptide (e.g., a complex containing 2, 3, 4, 6, or 8 polypeptides). In one example, such an antigen-binding molecule is an IgG-like antigen-binding molecule that contains two heavy chain polypeptides and two light chain polypeptides.

[0259] Generally, an antibody contains six complementarity determining regions (CDRs), three in the light chain variable region (VL), usually designated LC-CDR1, LC-CDR2, LC-CDR3, and three in the heavy chain variable region (VH), usually designated HC-CDR1, HC-CDR2, and HC-CDR3. The six CDRs together define the paratope of the antibody, the part of the antibody that binds to the target molecule. The VH and VL regions contain framework regions (FRs) on either side of each complementarity determining region (CDR), which provide the framework for the CDRs. By convention, from N-terminus to C-terminus, the heavy chain variable region (VH) comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0260] The present disclosure refers to antibodies or antibody antagonists of CNX, PDIA4, PDIA3, and / or complexes comprising CNX and PDIA3, including, but not limited to, antibody antagonists of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and / or complexes comprising CNX and PDIA3. In one example, an antigen-binding molecule according to the present disclosure comprises an antibody complementarity determining region (CDR) and / or a heavy chain variable region (VH) and a light chain variable region (VL) that specifically binds to a target antigen (e.g., CNX, PDIA4, PDIA3, or a complex comprising CNX and PDIA3).

[0261] Antibodies to a given target antigen can be made, derived or engineered using techniques known in the art. Such techniques include, but are not limited to, screening antibody gene phage display libraries for molecules capable of binding to the target antigen and raising antibodies to a given target antigen by animal immunization. Approaches to the production of monoclonal antibodies (monoclonal or otherwise) suitable for therapeutic use in humans or for use with the methods disclosed herein include, but are not limited to, the use of hybridoma technology, generation of xenogeneic antibodies followed by humanization, human antibody gene phage display, and production in transgenic mice bearing human antibody genes.

[0262] Phage display technology can also be utilized to identify antibodies capable of binding to a given target, target protein, or protein complex, and is well known to those of skill in the art.

[0263] Antibodies against calnexin (CNX) include, but are not limited to, monoclonal antibodies clone AF18 (Invitrogen Cat. No. MA3-027), clone AF8 (Merck Cat. No. MABF2067), clone TO-5 (Merck Cat. No. C7617), clone 3H4A7 (Invitrogen Cat. No. MA5-15389), clone ARC0648 (Invitrogen Cat. No. MA5-35588), clone GT1563 (GeneTex Cat. No. GTX629976), clone CANX / 1541 (GeneTex Cat. No. GTX34446), clone IE2.1C12 (Novus Biologicals Cat. No. NBP2-36571), clone 1C2.2D11 (Novus Biologicals Cat. No. NBP2-36572), and clone 2D11 (Novus Biologicals Cat. No. NBP2-36573). Examples of antibodies against Cnx include polyclonal antibodies ab10286 and ab22595 (Abcam), and the anti-Cnx antibodies disclosed in CN 101659702 A (e.g., the antibody produced by hybridoma CGMCC No. 3240). In one example, the antibody against calnexin (CNX) is ab22595 (Abcam), a rabbit polyclonal antibody raised against human CNX for an epitope beginning at amino acid 550 through the C-terminus.

[0264] Antibodies against protein disulfide isomerase A3 (PDIA3) include, but are not limited to, monoclonal antibody clone MaP.Erp57 (Enzo Life Sciences), clone CL2444 (Sigma-Aldrich Cat. No. AMAB90988), clone OTI3E1 (ThermoFisher Scientific Cat. No. TA504995), clone OTI3D2 (ThermoFisher Scientific Cat. No. TA504990), clone OTI4D7 (ThermoFisher Scientific Cat. No. TA505008) and polyclonal antibodies ab13506 and ab13507 (Abcam). In another example, the anti-PDIA3 antibody is ab13507 (Abcam).

[0265] Antibodies raised in non-human animals (such as, but not limited to, mice, rabbits, horses, dogs, donkeys, etc.) against a given target, target protein or protein complex can be engineered to improve their suitability for therapeutic use in humans, also known as humanization or humanized antibodies. In other words, humanized antibodies are antibodies whose protein sequence has been modified to increase their similarity to the antibody variants naturally produced in humans. For example, one or more amino acids of a monoclonal antibody raised by animal immunization can be replaced to arrive at an antibody sequence that is more similar to the human germline immunoglobulin sequence, thereby reducing the possibility of (anti-xeno) antibody immune responses in human subjects treated with the antibody. Modifications in antibody variable regions can focus on framework regions to preserve the antibody paratope. The requirement for humanization can be avoided, for example, by raising antibodies against a given target protein / protein complex in a transgenic model species expressing human immunoglobulin genes, and fully humanizing the antibodies raised in such animals.

[0266] Antigen-binding molecules also include peptide aptamers, thioredoxins, monobodies, anticalins, Kunitz domains, avimers, knottins, finomers, atrimers, DARPins, affibodies, nanobodies (including but not limited to single domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs) and obodies. Such antigen-binding peptides can be identified using methods known in the art, for example, by screening libraries of related peptides.

[0267] Antigen-binding peptides also include, but are not limited to, decoy interaction partners for related target antigens. For example, a decoy interaction partner for calnexin (CNX) may comprise or consist of a CNX-binding fragment of protein disulfide isomerase A3 (PDIA3) or a variant thereof, while conversely, a decoy interaction partner for PDIA3 may comprise or consist of a PDIA3-binding fragment of CNX or a variant thereof. A decoy interaction partner may be truncated or otherwise modified relative to its underlying peptide such that the decoy interaction partner associates with its target to form a non-functional complex and / or a complex that has a reduced level of functional properties of the complex formed by the target and the interaction partner on which the decoy interaction partner is based. Thus, in one example, the decoy interaction partner acts as a competitive inhibitor of the interaction between the target and the interaction partner of the target on which they are based.

[0268] Calnexin (CNX) binding molecules, protein disulfide isomerase A4 (PDIA4) binding molecules, protein disulfide isomerase A3 (PDIA3) binding molecules, and CNX:PDIA3 complex binding molecules include, but are not limited to, small molecules, for example, small molecules that can be identified by screening a small molecule compound library. As used herein, the term "small molecule" refers to a low molecular weight. Such low molecular weights are usually less than 1000 Daltons (<1000 Da). Typically, small molecules are between 300 and 700 Da and are organic compounds.

[0269] Examples of small molecule inhibitors include, but are not limited to, tenecteplase (eg, against calnexin (CNX)) and p-hydroxymercuribenzoic acid sodium salt (eg, against PDIA3).

[0270] Examples of calnexin (CNX) binding molecules, protein disulfide isomerase A3 (PDIA3) binding molecules, and CNX:PDIA3 complex binding molecules include aptamers. Nucleic acid aptamers can include DNA and / or RNA and can be single-stranded or double-stranded. In one example, they can include chemically modified nucleic acids, where the sugar and / or phosphate and / or base are chemically modified. Such modifications can improve the stability of the aptamer or make it more resistant to degradation. In one example, such modifications include, but are not limited to, modifications at the 2' position of the ribose. Nucleic acid aptamers can be chemically synthesized, for example, on a solid support. Solid-phase synthesis can include phosphoramidite chemistry. In one example, a solid-supported nucleotide is detritylated and then coupled with an appropriately activated nucleoside phosphoramidite to form a phosphite triester bond. Capping can then occur, followed by oxidation of the phosphite triester with an oxidizing agent, typically iodine. The cycle can then be repeated to construct the aptamer.

[0271] Molecules that can reduce the levels of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), protein disulfide isomerase A3 (PDIA3), and the CNX:PDIA3 complex include, but are not limited to, molecules that can reduce gene and / or protein expression of PDIA4, CNX, and / or PDIA3. In some embodiments, molecules that can reduce the levels of CNX, PDIA4, PDIA3, or the CNX:PDIA3 complex reduce or prevent expression of a polypeptide encoded by the genes PDIA4, CANX, or PDIA3, respectively.

[0272] Inhibition of gene or protein expression of a given target gene (e.g., but not limited to, genes CANX, PDIA4, or PDIA3) can include, but is not limited to, inhibiting transcription of the gene, inhibiting post-transcriptional processing (e.g., splicing) of RNA transcribed from the gene, reducing the stability of RNA transcribed from the gene, promoting the degradation of RNA transcribed from the gene, inhibiting translation of RNA transcribed from the gene into protein, inhibiting post-translational processing of a polypeptide encoded by the gene, reducing the stability of a polypeptide encoded by the gene, or promoting the degradation of a polypeptide encoded by the gene.

[0273] Inhibition of gene or protein expression can be achieved, for example, by modifying / disrupting the nucleotide sequence of the gene or by modifying / disrupting the nucleotide sequence required for expression of the gene (e.g., the regulatory sequence governing the expression of the gene). In some examples, inhibiting expression of a gene or protein includes modifying the nucleotide sequence. Such modification or alteration can be made, for example, by substituting, deleting or inserting one or more nucleotides. In certain examples, the present disclosure describes inhibiting expression of a gene or protein by deleting all or part of the nucleotide sequence of the relevant gene.

[0274] In some examples, altering / disrupting a nucleotide sequence may include altering or removing a control sequence for transcription of a gene (such as, but not limited to, a promoter or enhancer), introducing a premature stop codon into a sequence transcribed from a gene, altering a nucleotide sequence to encode a truncated and / or non-functional gene product, or altering a nucleotide sequence to encode a misfolded and / or degraded gene product. Nucleotide sequences may also be disrupted, for example, but not limited to, by homologous recombination or by targeted nucleic acid modification using site-specific nucleases (SSNs).

[0275] In some instances, altering / disrupting a nucleotide sequence to inhibit or prevent gene or protein expression from a gene can be referred to as "knocking out" the gene.

[0276] Modification by homologous recombination can involve the exchange of nucleic acid sequences through a crossover event induced by homologous sequences.

[0277] Gene editing is also a method that can be used to edit, change, or disrupt the expression or function of a nucleotide sequence. Enzymes that can generate site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence of interest. Double-strand breaks can be repaired by error-prone non-homologous end joining (NHEJ), in which the two cut ends are rejoined, often with the insertion or deletion of nucleotides. Alternatively, double-strand breaks can be repaired by highly homologous sequence-directed repair (HDR), in which a DNA template with ends homologous to the cut site is provided and introduced into the DSB site. Site-specific nucleases (SSNs) can be engineered to generate double-strand breaks specific to a target nucleic acid sequence, including, but not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats / CRISPR-associated protein 9 (CRISPR / Cas9) systems.

[0278] Zinc finger nuclease (ZFN) systems contain a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). The DNA binding domain can be identified by screening zinc finger arrays that can bind to target nucleic acid sequences. In another example, transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALENs contain a programmable DNA binding TALE domain and a DNA cleavage domain (e.g., a FokI endonuclease domain). TALEs contain a repeat domain consisting of between 33 and 39 amino acid repeats, where the repeats (also named repeat variable dipeptides (RVDs)) are identical except for two residues at positions 12 and 13 of each repeat. As known in the art, each RVD determines the binding of the repeat to a nucleotide in a target DNA sequence according to the following relationship: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G. Further examples of gene editing systems include, but are not limited to, CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3. These systems include an endonuclease (e.g., Cas9, Cpf1, etc.) and a single guide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to a nucleic acid sequence of interest.

[0279] Examples of CRISPR / Cas9 systems for targeted knockout of CNX expression are the systems encoded by the plasmids Calnexin CRISPR / Cas9 KO Plasmid (h) (Santa Cruz Biotechnology Cat. No. sc-400154) and Calnexin HDR Plasmid (h) (Santa Cruz Biotechnology Cat. No. sc-400154-HDR).

[0280] Destruction of a nucleotide sequence using a site-specific nuclease (SSN) can be achieved in an animal, for example, by administering to the animal a nucleic acid encoding a component of the relevant site-specific nuclease system. For example, one or more vectors can be administered to the animal, including a nucleic acid encoding a component of the site-specific nuclease system to target the relevant gene.

[0281] Inhibition of gene or protein expression can also be achieved, for example, by treatment with a drug or compound that can reduce gene or protein expression. For example, inhibition of gene or protein expression can be achieved using inhibitory nucleic acid, such as, but not limited to, antisense nucleic acid. Antisense nucleic acid can bind to target nucleic acid by complementary base pairing. When target nucleic acid is RNA (e.g., RNA transcribed from related gene), binding of antisense nucleic acid to target RNA can promote RNA degradation and / or inhibit translation of RNA. An example of inhibitory nucleic acid is antisense oligonucleotide (ASO).

[0282] The inhibitory nucleic acid can inhibit gene or protein expression by RNA interference (RNAi). RNAi involves the inhibition of gene expression and translation by targeting and disabling mRNA molecules. In some instances, the inhibitory nucleic acid is a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a microRNA (miRNA).

[0283] Small interfering RNA (siRNA) is derived by processing of long double-stranded RNA, and when found in nature, it is usually of exogenous origin. Microinterfering RNA (miRNA) is an endogenously encoded small non-coding RNA. Both siRNA and miRNA can inhibit the translation of mRNA with partially complementary target sequences and degrade mRNA with fully complementary sequences without cleaving the RNA. siRNA is typically double-stranded, and to optimize the effectiveness of RNA-mediated inhibition of target gene function, the length of the siRNA molecule is preferably selected to ensure that the siRNA is correctly recognized by the RNA-induced silencing complex (RISC), which mediates the recognition of the target mRNA by the siRNA. DNA sequences encoding miRNA include, but are not limited to, miRNA sequences and similar reverse complements. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse complement base-pair to form a partially double-stranded RNA segment.

[0284] Examples of siRNAs targeting the gene CANX include, but are not limited to, siRNAs based on or generated using the target sequence 5'-CAAGAGUGGUCCUAGGAGAUU-3' (SEQ ID NO: 2) as an exemplary target of calnexin.

[0285] Examples of siRNAs targeting PDIA3 include, but are not limited to, siRNAs based on or generated using any one of the following target sequences as exemplary targets of PDIA3: GGAAUAGUCCCAUUAGCAA (SEQ ID NO: 3), GGGCAAGGACUUACUUAUU (SEQ ID NO: 4), AGACCCAAAUAUCGUCAUA (SEQ ID NO: 5), AGGAGUUCUCGCGUGAUG (SEQ ID NO: 6), GAACGAGUAUGAUGAUAAU (SEQ ID NO: 7), GGACAAGACUGUGGCAUAU (SEQ ID NO: 8), GGCAAGGACUUACUUAUUG (SEQ ID NO: 9), UGAUAAAGAUGCCUCUAUA (SEQ ID NO: 10)), and combinations thereof. SEQ ID NOs: 3 to 6 refer to (L-003674-00-0005, ON-TARGETplus Human PDIA3(2923) siRNA·SMARTpool, 5 nmol), and SEQ ID NOs: 7 to 10 refer to M-003674-01-0005, siGENOME Human PDIA3(2923) siRNA·SMARTpool, 5 nmol.

[0286] Small hairpin RNA (shRNA) is considered to be more stable than synthetic siRNA. Small hairpin RNA (shRNA) consists of short inverted repeats separated by small loop sequences. One inverted repeat is complementary to the target gene. In cells, shRNA is processed into siRNA by endoribonuclease DICER, which degrades the target gene mRNA and suppresses its expression. Small hairpin RNA (shRNA) can be produced in cells, for example, by transcription from a vector.

[0287] Anti-calnexin antibody prevents arthritic symptoms in mice with collagen antibody-induced arthritis First, the presence of disulfide bonds in cartilage extracellular matrix was examined using the method previously described. Briefly, cartilage extracellular matrix was reduced with tris(2-carboxyethyl)phosphine (TCEP), then treated with N-ethylmaleimide (NEM), and then treated with anti-OX133 antibody. Similar to liver extracellular matrix, a large amount of signal was observed, indicating that cartilage extracellular matrix is ​​heavily cross-linked with disulfide bonds (Figure 15).

[0288] Next, the effect of anti-calnexin antibodies on extracellular matrix degradation was tested in vitro. Osteoarthritis synovial fibroblasts (OASFs) were seeded onto cartilage extracellular matrix overlying fluorescent gelatin, and their extracellular matrix degrading activity was measured as described above. The addition of polyclonal anti-calnexin antibodies was shown to block this degrading activity (Figure 16).

[0289] Collagen antibody-induced arthritis (CAIA) animals were treated with 25 micrograms (or the equivalent of 1.25 mg / kg based on an average body weight of 20 g per mouse model) of anti-calnexin (anti-CNX) antibody injections every 2 days from day 3 to day 7 after CAIA was induced (Figure 17A). Paw thickness was monitored at regular intervals, and animals were analyzed for arthritis score on day 10. Anti-calnexin treated animals showed only minimal paw swelling compared to isotype antibody treated control animals (Figures 17B, 17C). Although some redness and swelling of the digits still occurred and arthritis score was elevated, the average score of anti-calnexin (anti-CNX) antibody treated animals was half that of CAIA control animals (Figure 18).

[0290] Thus, in one example, a therapeutic agent disclosed herein is an antibody. In another example, the antibody is an anti-calnexin antibody. In yet another example, the antibody is an anti-PDIA3 antibody.

[0291] At the histological level, a reduction in Safranin-O (SO)-positive cartilage was evident in control animals at day 10, almost disappearing in some areas (Fig. 19A, 19B). In contrast, the cartilage architecture was generally preserved in animals treated with anti-calnexin antibody (Fig. 19A, 19B). Histological analysis revealed that the synovium of treated mice treated with anti-calnexin antibody was swollen and infiltrated into the joint, similar to that in CAIA animals, suggesting that an early part of the activation cascade had occurred (Fig. 19A, 19B). However, the pannus did not attack and deeply invade the cartilage as it did in control animals.

[0292] Finally, to verify that the antibody had indeed reached the synovium, it was detected using anti-rabbit IgG antibody. A signal was observed in synovial cells of animals treated with anti-calnexin antibody, but not in animals treated with control rabbit IgG, suggesting that anti-CNX antibody was specifically internalized in synovial fibroblasts (Figure 20).

[0293] Overall, these results indicate that inhibiting calnexin inhibits cartilage extracellular matrix degradation and may form the basis of an arthritis therapeutic. Thus, in one example, the therapeutic agent disclosed herein is an antibody. In another example, the antibody is an anti-calnexin antibody.

[0294] As shown herein, activation of the GALA-regulated pathway is described in synovial fibroblasts from both osteoarthritic and rheumatoid arthritis joints. Although these diseases are different, they share a key feature: the degradation of cartilage extracellular matrix. In both cases, the main driver of cartilage degradation is the synovial-derived fibroblasts.

[0295] It remains unclear how synovial fibroblasts switch from a healthy state to an aggressive matrix-degrading state. In the former, calnexin forms the intimal lining of the synovium and contributes to the composition of synovial fluid by secreting a variety of factors. In rheumatoid arthritis, calnexin proliferates and engages in collective migration to invade the joint cavity, degrade cartilage extracellular matrix, and ultimately invade cartilage itself. In osteoarthritis, proliferation is less evident, but synovial fibroblasts are still engaged in matrix degradation. The results disclosed herein point to GALA activation as a control switch in both diseases. Indeed, GALA has been shown to be specifically activated in synovial fibroblasts and is rarely found in immune cells found in inflamed synovium. Consistent with the important role of synovial fibroblasts in cartilage degradation, it has previously been reported that GALA controls the extracellular matrix degrading ability of cancer cells.

[0296] It is unclear how GALA is activated in these cells. The results described indicate that cytokines known to promote disease, including but not limited to IL-1β and TNFα, stimulate GALA in synovial fibroblasts in vitro. IL-1β has been reported to activate the tyrosine kinase Src. Src acts at the Golgi apparatus as a key regulator of GALA. In synovial fibroblasts, contact with the extracellular matrix also activates the GALA pathway, but the mechanism is currently unclear. It likely requires signaling through integrins as well as FAK and, again, Src kinases. Osteoarthritis synovial fibroblasts (OASF) and rheumatoid arthritis synovial fibroblasts (RASF) show comparable global methylation profiles, which are distinct from synovial fibroblasts from healthy subjects. Differences have been identified in genes involved in platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) signaling. Both growth factors have previously been shown to activate GALA, suggesting that rheumatoid arthritis synovial fibroblasts (RASFs) and osteoarthritis synovial fibroblasts (OASFs) may be primed by epigenetic imprinting to more readily activate the GALA pathway.

[0297] A feature of the GALA-regulated glycosylation enzymes, N-acetylgalactosaminyltransferases (GALNTs), is that in addition to the catalytic domain, they contain a lectin domain. This lectin domain has been shown to be essential for the GALA effect. This is further supported by data obtained using the ER-2Lec construct as a GALA-specific inhibitor, which resulted in the complete blocking of GALNT activity specifically in the endoplasmic reticulum. Expression of the ER-2Lec inhibitor using the Cre-Lox system under the collagen VI promoter allowed targeting to synovial fibroblasts. The alleviation of symptoms in transgenic animals expressing ER-2Lec clearly demonstrates the importance of the pathway to the disease.

[0298] Thus, in one example, the therapeutic agent disclosed herein is a fusion protein. In another example, the fusion protein is ER-2Lec. In yet another example, the fusion protein is ER-2Lec having the sequence of SEQ ID NO:1.

[0299] GALA regulates N-acetylgalactosaminyltransferases (GALNTs), glycosylation enzymes that act on thousands of proteins. One of the key targets of GALA is MMP14, whose glycosylation is critical for its proteolytic activity. In rheumatoid arthritis and osteoarthritis, MMP14 and other matrix metalloproteinases (MMPs) have been shown to be upregulated in synovial fibroblasts and play a critical role in collagen degradation.

[0300] In addition, GALA was recently shown to activate a complex composed of calnexin and protein disulfide isomerase A3 (PDIA3) on the surface of liver cancer cells. This complex mediates the cleavage of disulfide bonds in the extracellular matrix, an activity required for proteases to degrade the extracellular matrix in the liver. In this regard, it was shown that calnexin (CNX) is also required for the degradation of cartilage extracellular matrix by synovial fibroblasts. Indeed, both synovial fibroblast cell lines and cells derived from patients with osteoarthritis had reduced degradative capacity when incubated with anti-calnexin antibodies.

[0301] It should be noted that calnexin is a protein that is normally located in the endoplasmic reticulum within cells. Its transport towards the cell surface appears to be controlled by GALA-induced glycosylation. Therefore, anti-calnexin antibodies are expected to bind mostly or only to cells with high levels of GALA. However, in the case of healthy tissues, GALA levels are not high. This may have helped to concentrate anti-CNX antibodies in joint tissues. In other words, the high concentration of anti-CNX antibodies on the cell surface is caused by high levels of GALA, which leads to increased transport of CNX to the cell surface. This means that more CNX was detectable with anti-CNX antibodies. A complete characterization of the biodistribution is needed to confirm this point. It should also be noted that during these experiments with anti-calnexin antibodies, no adverse events were noted or detected, as shown, for example, in Figure 22, which shows that the weight of animals receiving CNX antibodies was not changed relative to the weight of animals receiving isotype control antibodies.

[0302] Overall, the results disclosed herein indicate that the GALA pathway is activated in both rheumatoid arthritis and osteoarthritis synovial fibroblasts and that the GALA pathway is essential for the pathology of rheumatoid arthritis in mice.

[0303] As used herein, the term "arthritis" refers to any disorder that affects joints, including but not limited to symptoms such as joint pain and stiffness. Other symptoms may include but are not limited to redness, warmth, swelling, and reduced range of motion of affected joints. In some types of arthritis, organs other than the target joints may also be affected. In the context of this disclosure, the methods disclosed herein will be applied to various forms of arthritis. This is indicated, for example, by the fact that GALA is activated in rheumatoid arthritis, psoriatic arthritis, and osteoarthritis. Without being bound by theory, it is believed that this common GALA activation indicates that said GALA activation is a relevant and targetable mechanism for treating any form of arthritis in which GALA is activated.

[0304] In the context of this disclosure, the term "rheumatoid arthritis" or "RA" refers to a chronic systemic autoimmune disease that primarily involves the joints. Rheumatoid arthritis causes damage mediated by cytokines, chemokines, and metalloproteases. Characteristically, peripheral joints (e.g., wrist, metacarpophalangeal joints) become symmetrically inflamed, leading to progressive destruction of articular structures and usually accompanied by systemic symptoms. Diagnosis is based on specific clinical, laboratory, and imaging features. Treatment involves drugs, physical procedures, and sometimes surgery. Treatments such as, but not limited to, disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, immunomodulatory drugs, cytotoxins, and immunosuppressants (e.g., azathioprine or cyclosporine) help manage symptoms and slow disease progression.

[0305] In the context of this disclosure, the term "osteoarthritis" or "OA" refers to a chronic disease of the joints (arthropathy) characterized by the destruction and potential loss of articular cartilage along with other joint changes including bone thickening (osteophyte formation). Symptoms include gradually progressive pain that is aggravated or precipitated by activity, stiffness on awakening and after inactivity lasting at least 30 minutes or more, and occasional joint swelling. Diagnosis is confirmed by x-ray, and treatment includes physical manipulation, rehabilitation, patient education, and drugs. Osteoarthritis can be classified as primary (idiopathic) or secondary, i.e., due to conditions that alter the cartilage microenvironment. Such conditions include, but are not limited to, major trauma, congenital joint abnormalities, metabolic defects (e.g., hemochromatosis, Wilson's disease), infections (leading to post-infectious arthritis), endocrine and neuropathic diseases, and disorders that alter the normal structure and function of hyaline cartilage (e.g., rheumatoid arthritis, gout, chondrocalcinosis).

[0306] As used herein, the term "arthritis flare", "arthritis flare-up", or "arthritis episode" is defined as an episode of increased disease activity or worsening symptoms. A flare is typically defined by a sudden intensity in joint pain accompanied by other characteristic symptoms such as, but not limited to, fever, fatigue, malaise, stiffness, or joint swelling. A flare may affect a single joint or multiple joints. For example, a person with osteoarthritis may have a single joint flare at the same time or recurrent flares in the same multiple joints. In contrast, those with autoimmune arthritis such as rheumatoid arthritis or psoriatic arthritis may often experience multiple joint flares simultaneously. In one example, the methods disclosed herein are used to alleviate the symptoms of an arthritis flare-up.

[0307] In one example, the arthritis is selected from the group including, but not limited to, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), osteoarthritis, and arthritis flare / flare-up. In another example, the arthritis is rheumatoid arthritis. In yet another example, the arthritis is osteoarthritis. In yet another example, the arthritis is psoriatic arthritis. In a further example, the condition to be treated is arthritis flare / flare-up.

[0308] In another example, the anti-rheumatic drug is, but is not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, azathioprine cyclosporine, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, rituximab, abatacept, anakinra, tumor necrosis factor (TNF) alpha antagonists (such as, but not limited to, adalimumab, etanercept, golimumab, certolizumab pegol, infliximab, and biosimilars thereof), sarilumab, tocilizumab, baricitinib, upadacitinib, filgotinib, and peficitinib, and combinations thereof.

[0309] As used herein, the term "treatment" refers to any and all uses of curing a disease state or condition, preventing the establishment of a disease, or otherwise preventing, hindering, slowing, or reversing the progression of a disease or other undesirable symptoms in any manner. The methods disclosed herein can also be used to alleviate any symptoms of a disease or condition disclosed herein.

[0310] Also disclosed herein is a kit or diagnostic kit for detecting arthritis in an individual or susceptibility to arthritis in an individual. The diagnostic kit may include a means for detecting the expression, amount or activity of CNX, protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3), or a complex of CNX and PDIA3 in an individual by any means as described herein. Thus, the diagnostic kit may include any one or more of the following: CNX, PDIA4, or PDIA3 polynucleotides or fragments thereof, complementary nucleotide sequences to CNX, PDIA4, or PDIA3 nucleic acids or fragments thereof, CNX, PDIA4, or PDIA3 polypeptides or fragments thereof, or antibodies to CNX, PDIA4, or PDIA3. The diagnostic kit may include instructions for use, or other instructions. The diagnostic kit may further include a means for treating or preventing arthritis, such as any of the compositions described herein, or any means for treating arthritis known in the art.

[0311] In one example, the diagnostic kit includes CNX, PDIA4, PDIA3, and / or CNX:PDIA3 inhibitors as described herein. In another example, the diagnostic kit includes CNX, PDIA4, PDIA3, and / or CNX:PDIA3 inhibitors obtained by screening. The term "CNX:PDIA3" refers to a complex containing CNX and PDIA3. The diagnostic kit may include a therapeutic agent such as, but not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, and combinations thereof, or other therapeutic agents as disclosed herein. The therapeutic agent may be, for example, an anti-CNX antibody, an anti-DPAI4 antibody, or an anti-PDIA3 antibody.

[0312] In yet another example, a kit for carrying out a method of detecting the presence or absence of arthritis in a subject according to the present disclosure is disclosed.

[0313] Materials required for screening compounds or agents to identify agonists, antagonists, ligands, receptors, substrates, enzymes of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3), for example, to identify polypeptides or compounds that reduce or enhance the production of CNX, PDIA4, or PDIA3, can be packaged into a screening kit. In one example, the screening kit includes any one or more or all of the following: CNX, PDIA4, or PDIA3 polypeptide, recombinant cells expressing CNX, PDIA4, or PDIA3 polypeptide, or antibodies against CNX, PDIA4, or PDIA3 polypeptide. The screening kit may further include a library. The screening kit may include any one or more of the components required for screening as described herein. The screening kit may optionally include instructions for use. Such screening kits may also be provided that can detect CNX, PDIA4, or PDIA3 expression at the nucleic acid level. Such kits may include a primer for amplification of CNX, PDAI4, or PDIA3, or a pair of primers for amplification. The primer(s) may be selected from any suitable sequence, for example, a portion of the CNX, PDIA4, or PDIA3 sequence. Methods for identifying primer sequences are well known in the art, and one of ordinary skill in the art would be able to easily design such primers. The kits may include a nucleic acid probe for CNX, PDIA4, or PDIA3 expression, as described herein. The kits may also optionally include instructions for use.

[0314] Also disclosed herein are polyclonal antibodies for use in the methods and treatments as described herein. If polyclonal antibodies are desired, a selected mammal (such as, but not limited to, mouse, rabbit, goat, and horse) is immunized with an immunogenic composition comprising a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptide or peptide. Depending on the host species, various adjuvants may be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as, but not limited to, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are human adjuvants utilized when purified amino acid sequences of the material are to be administered to immunologically compromised individuals with the aim of stimulating systemic defense.

[0315] The serum of the immunized animals is collected and processed according to known procedures to obtain antibodies. If the serum containing polyclonal antibodies against epitopes obtained from calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptide contains antibodies against other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. To generate such antibodies, the CNX, PDIA4, or PDIA3 amino acid sequence or a fragment thereof can be haptenized to another amino acid sequence for use as an immunogen in animals or humans.

[0316] Also included within the scope of the present disclosure are monoclonal antibodies directed against epitopes derived from calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptides or peptides. These monoclonal antibodies can be readily produced by those skilled in the art. The general methodology of producing monoclonal antibodies by hybridomas or hybridoma cell lines is well known in the art. Immortal antibody-producing cell lines can also be produced by cell fusion and other techniques such as, but not limited to, direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against surrounding epitopes can be screened for various properties, including, but not limited to, isotype and epitope affinity.

[0317] Monoclonal antibodies may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture, as known in the art.

[0318] Recombinant DNA technology can be used to improve antibodies as described herein. Thus, chimeric antibodies can be constructed to reduce their immunogenicity in diagnostic or therapeutic applications. Such techniques include, for example, splicing mouse antibody genes with human antibody genes to obtain molecules with appropriate antigen specificity and biological activity. Furthermore, immunogenicity can be minimized by humanizing antibodies, for example, by complementarity determining region (CDR) grafting and optional framework modifications, both of which are methods known in the art.

[0319] Both monoclonal and polyclonal antibodies directed against epitopes derived from calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptides or peptides are disclosed herein and are believed to be particularly useful in the methods disclosed herein, including diagnosis. In particular, monoclonal antibodies can be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins that bear against the "internal representation" of the substance and / or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies can also be useful in treatment methods and therapies as disclosed herein. In one example, the antibodies can be bound to a solid support and / or packaged into a kit in a suitable container along with suitable reagents, controls, instructions, etc.

[0320] Antibodies can also be produced by inducing in vivo production in lymphocyte populations or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents, as disclosed in the art.

[0321] Antibody fragments that contain specific binding sites for the target polypeptide or peptide can also be generated. For example, such fragments include, but are not limited to, F(ab') fragments that can be generated by pepsin digestion of the antibody molecule. 2 Fragments, and F(ab´) 2 and Fab fragments which can be generated by reducing the disulfide bridges of the fragment. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0322] Techniques known in the art for the production of single chain antibodies can also be adapted to produce single chain antibodies against calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptides. Transgenic mice, or other organisms, including other mammals, can also be used to express humanized antibodies.

[0323] The antibodies described herein may also be utilized to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography.

[0324] Recombinant DNA technology can be used to produce the antibodies in bacterial or mammalian cell culture following established procedures. The cell culture system selected can provide for secretion of the antibody product.

[0325] An exemplary process for producing an antibody includes culturing a host, such as, but not limited to, E. coli or a mammalian cell, that has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide, which is linked in proper reading frame to a second DNA sequence encoding the antibody protein, and isolating the protein.

[0326] In vitro production provides relatively pure antibody preparations and allows for scale-up to obtain large quantities of the desired antibody. Techniques for culturing bacterial, yeast or mammalian cells are known in the art and include, for example, homogenous suspension cultures in airlift reactors or continuous stirred reactors, or immobilized or entrapped immobilized cell cultures, for example, in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges.

[0327] Also provided are hybridoma cells secreting monoclonal antibodies, which are genetically stable, capable of secreting monoclonal antibodies of desired specificity, and can be activated from deep-freeze culture by thawing and recloning.

[0328] Also included herein is an exemplary process for preparing a hybridoma cell line secreting a monoclonal antibody directed against a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptide, comprising immunizing a suitable mammal, e.g., a Balb / c mouse, with one or more calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) polypeptides, or antigenic fragments thereof. Antibody-producing cells of the immunized mammal are fused with a suitable myeloma cell line cell, the hybrid cells obtained from the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells from Balb / c mice immunized with calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) are fused with myeloma cell line PAI or myeloma cell line Sp2 / 0-Ag14 cells, the resulting hybrid cells are screened for the desired antibody secretion, and positive hybrid cells are cloned.

[0329] Thus, the present invention provides a process for preparing a hybridoma cell line, comprising: 7 ~10 8The process is characterized by immunizing Balb / c mice by subcutaneous and / or intraperitoneal injection of between 100 and 1500 cells and a suitable adjuvant several times, e.g., 4-6 times, over a period of several months, e.g., 2-4 months, and harvesting the spleen cells of the immunized mice 2-4 days after the last injection and fusing them with myeloma cell line PAI cells in the presence of a fusion promoter such as polyethylene glycol. The myeloma cells may be fused with a 3-20 fold excess of the spleen cells of the immunized mice in a solution containing about 30% to about 50% polyethylene glycol with a molecular weight of about 4000. After fusion, the cells are grown in a suitable medium as described herein and supplemented with a selection medium, e.g., HAT medium, at regular intervals to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

[0330] Also disclosed is a recombinant DNA comprising an insert encoding the heavy and / or light chain variable domains of an antibody directed against calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) as disclosed herein. By definition, such DNA includes the encoding single-stranded DNA, or double-stranded DNA consisting of said encoding DNA and a complementary DNA thereto, or these complementary (single-stranded) DNAs themselves.

[0331] Furthermore, the DNA encoding the heavy and / or light chain variable domains of the antibody directed against calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) can be enzymatically or chemically synthesized DNA having an authentic DNA sequence encoding the heavy and / or light chain variable domain, or a variant thereof. A variant of the authentic DNA is a DNA encoding the heavy and / or light chain variable domain of said antibody, in which one or more amino acids are deleted or replaced with one or more other amino acids. The modification(s) can be, but are not limited to, in a region outside the complementarity determining region (CDR) of the heavy and / or light chain variable domain of the antibody. Such a variant DNA can also be a silent variant in which one or more nucleotides are replaced with other nucleotides, resulting in a new codon encoding the same amino acid(s). Such a variant sequence can also be a degenerate sequence. Degenerate sequences are degenerate in the sense of the genetic code in that an unlimited number of nucleotides may be substituted with other nucleotides without resulting in a change in the originally encoded amino acid sequence. Such degenerate sequences may be useful for obtaining optimal expression of the mouse heavy chain variable domain and / or the mouse light chain variable domain due to their various restriction sites and / or the frequency of certain codons preferred by certain hosts, particularly E. coli.

[0332] The term "mutants" also includes DNA mutants obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

[0333] For example, for the construction of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, recombinant DNA inserts encoding the heavy and light chain variable domains are fused to corresponding DNA encoding the heavy and light chain constant domains and then introduced into a suitable host cell, e.g., after incorporation into a hybrid vector.

[0334] Also disclosed herein is a recombinant DNA comprising an insert encoding a mouse heavy chain variable domain of an antibody directed against calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) fused to a human constant domain g, e.g., gamma 1, gamma 2, gamma 3, or gamma 4, e.g., gamma 1 or gamma 4. Also disclosed herein is a recombinant DNA comprising an insert encoding a mouse light chain variable domain of an antibody directed against calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) fused to a human constant domain kappa or lambda, e.g., kappa.

[0335] In another example, a recombinant DNA is disclosed that encodes a recombinant polypeptide in which a heavy chain variable domain and a light chain variable domain are linked via a spacer group, optionally including a signal sequence that facilitates processing of the antibody in a host cell and / or DNA encoding a peptide that facilitates purification of the antibody and / or a cleavage site and / or a peptide spacer and / or an effector molecule.

[0336] Anti-calnexin (CNX), anti-protein disulfide isomerase A4 (PDIA4), and anti-protein disulfide isomerase A3 (PDIA3) antibodies can be used in methods for detecting CNX, PDIA4, or protein disulfide isomerase A3 (PDIA3) polypeptides present in a biological sample. In one example, such a method includes: (a) providing an anti-CNX, anti-PDIA4, or anti-PDIA3 antibody, (b) incubating a biological sample with the antibody under conditions that allow for the formation of an antibody-antigen complex, and (c) determining whether an antibody-antigen complex comprising the antibody is formed.

[0337] As used herein, the term "biological sample" or "sample" includes, but is not limited to, any quantity of material from a living organism or formerly living thing. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such material includes, but is not limited to, blood, serum, plasma, urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, synovial cells, tissues (especially inflamed tissue), cartilage, and skin.

[0338] The antibodies disclosed herein, e.g., antibodies against CNX, PDIA4, or PDIA3 proteins, can be delivered intracellularly by techniques known in the art, e.g., by the use of liposomes, polymers, (e.g., but not limited to, polyethylene glycol (PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, polyamidoamine (PAMAM) dendrimers, HEMA, linear polyamidoamine polymers), and combinations thereof. Immunoglobulins and / or antibodies can also be delivered intracellularly, e.g., as protein fusions or conjugates with proteins capable of crossing the cell membrane and / or the nuclear membrane. For example, immunoglobulins and / or targets can be fused or conjugated to domains or sequences, such as proteins responsible for translocational activity. Translocational domains and sequences can include domains and sequences from HIV-1 transcriptional activator protein (Tat), Drosophila antennapedia homeodomain protein, and herpes simplex-1 virus VP22 protein.

[0339] While it is possible for a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) inhibitor to be administered alone, the active ingredient can be formulated as a pharmaceutical formulation, e.g., as a CNX, PDIA4, or protein disulfide isomerase A3 (PDIA3) nucleic acid, polypeptide, fragment, homolog, variant or derivative thereof, modulator, agonist or antagonist, structurally related compound, or acid salt of any.

[0340] Thus, also disclosed herein is a pharmaceutical composition comprising a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) inhibitor. Another example is a pharmaceutical composition comprising a calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) inhibitor. Such a pharmaceutical composition can be used to deliver a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) inhibitor, for example, in the form of a composition as described, to an individual for the treatment or alleviation of symptoms and / or diseases described herein. In one example, the pharmaceutical composition is a composition of matter comprising at least a calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3) inhibitor as an active ingredient.

[0341] A pharmaceutical formulation comprises an effective amount of a calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) inhibitor together with one or more pharma- ceutically acceptable carriers. As used herein, an "effective amount" refers to an amount sufficient to alleviate at least one symptom of a disease as described. This so-called effective amount will vary depending on the specific disease or syndrome to be treated or alleviated, as well as other factors, including the age and weight of the patient, how advanced the disease is, the general health of the patient, the severity of the symptoms, and whether the calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) inhibitor is administered alone or in conjunction with other therapies.

[0342] Suitable pharma- ceutically acceptable carriers are well known in the art and vary according to the desired form and mode of administration of pharmaceutical preparations. For example, such carriers include, but are not limited to, diluents or fillers, binders, wetting agents, disintegrants, surfactants, lubricants, and other excipients. Usually, carriers are solid, liquid, or vaporizable carriers, or combinations thereof. Each carrier should be "acceptable" in that it is compatible with other ingredients in the formulation and is not harmful to the patient. Thus, carriers should be biologically acceptable without inducing adverse reactions (e.g., immune or allergic responses) when administered to a host.

[0343] It is contemplated that the active ingredient(s) of the pharmaceutical composition exhibit therapeutic activity, for example, but not limited to, in alleviating arthritis, rheumatoid arthritis, osteoarthritis, arthritis flare-ups, and other related diseases or symptoms. Dosage regimens can be adjusted to obtain optimal therapeutic response. For example, doses can be administered daily in divided doses, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0344] The active compounds may be administered in any convenient manner, such as orally, intravenously (if water soluble), intramuscularly, subcutaneously, intranasally, intradermally or via suppository routes, or by implantation (e.g., using a sustained release molecule). Depending on the route of administration selected, the active ingredient may need to be coated with a material to protect the ingredient from the action of enzymes, acids and other natural conditions which may inactivate the ingredient.

[0345] The inhibitors disclosed herein may be administered alone or in combination with other therapeutic agents. Other therapeutic agents suitable for use herein are any compatible drugs that are effective for the intended purpose or drugs that complement the drug combination. The formulations utilized in combination therapy may be administered simultaneously or sequentially with other treatments so that a combined effect is achieved. Thus, in one example, the therapeutic agent is a group consisting of, but not limited to, antibodies, drugs, genes, fusion proteins, and expression vectors. In another example, the therapeutic agents will be administered together, separately, or sequentially. In a further example, the (first) therapeutic agent will be administered together with an additional therapeutic agent, and the additional therapeutic agent is an anti-rheumatic drug. In another example, such anti-rheumatic drugs are, but are not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, azathioprine cyclosporine, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, rituximab, abatacept, anakinra, tumor necrosis factor (TNF) alpha antagonists (such as, but not limited to, adalimumab, etanercept, golimumab, certolizumab pegol, infliximab, and biosimilars thereof), sarilumab, tocilizumab, baricitinib, upadacitinib, filgotinib, and peficitinib, and combinations thereof.

[0346] In some embodiments, the inhibitor of activity, expression or amount of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3), or the CNX:PDIA3 complex is provided as an oral composition and administered accordingly. The dosage of the inhibitor of activity, expression or amount of calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) can be between about 1 mg / day and about 10 mg / day.

[0347] The pharmaceutical composition may be administered in an oral formulation in the form of a tablet, capsule or solution. An effective amount of the oral formulation is administered to the patient 1 to 3 times daily until the symptoms of the disease are alleviated.

[0348] The effective amount of the drug depends on the age, weight and condition of the patient. In general, the daily oral dose of the drug is less than 1200 mg and more than 100 mg. The daily oral dose can be about 300 mg to about 600 mg. The oral formulation is conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical art. The composition can be formulated into any desired dosage form with a suitable pharma-ceutically acceptable carrier. Typical unit dosage forms include, but are not limited to, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, and suppositories. In general, the formulation is prepared by uniformly and intimately associating the pharmaceutical composition with liquid carriers or finely divided solid carriers or both, and shaping the product as necessary. The active ingredient can be incorporated into various base materials in the form of liquid, powder, tablet or capsule to obtain an effective amount of the active ingredient for the treatment of the disease.

[0349] The compositions may be suitably administered orally, for example, with an inert diluent or with an assimilable edible carrier, or the compositions may be enclosed in a hard or soft shell gelatin capsule, or compressed into tablets, or incorporated directly with the food of the diet. For oral therapeutic administration, the active compound.

[0350] In some embodiments, the inhibitor of calnexin (CNX), protein disulfide isomerase A4 (PDIA4), or protein disulfide isomerase A3 (PDIA3), or inhibitor of the CNX:PDIA3 complex, is provided as an injectable or intravenous composition and administered accordingly. The dosage of the calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) inhibitor can be between about 5 mg / kg / 2 weeks and about 10 mg / kg / 2 weeks. The calnexin (CNX) or protein disulfide isomerase A3 (PDIA3) inhibitor can be provided at a dosage between 10 mg / day and 300 mg / day, such as at least 30 mg / day, less than 200 mg / day, or between 30 mg / day and 200 mg / day.

[0351] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that the viscosity allows for syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, but not limited to, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0352] The compositions disclosed herein may be administered in adjuvants, co-administered with enzyme inhibitors, or in liposomes. The term "adjuvant" is used in its broadest sense and includes any immune stimulating compound, such as, but not limited to, interferon. Adjuvants contemplated herein include, but are not limited to, resorcinol, non-ionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include, but are not limited to, pancreatic trypsin. Liposomes include, but are not limited to, water-in-oil-in-water CGF emulsions, as well as conventional liposomes.

[0353] The term "control group", "negative control" or "control", when used in the context of sample analysis, refers to the use of samples obtained from disease-free or healthy subjects, so that these samples are then processed in the same manner as the other samples, with the difference that the control samples are, for example, processed with a buffer that does not contain the active compound or molecule of interest. Comparing the concentration of one or more targets (e.g., when comparing the absolute concentration or relative expression level of the target) or determining the presence or absence of one or more targets (e.g., one or more proteins, oligomers or oligonucleotides) as disclosed herein is determined based on a comparison of the levels determined in a sample obtained from a diseased subject and a sample obtained from a disease-free (or healthy) subject. In other words, the comparison of targets is based on a comparison of the level of one or more targets determined in a diseased subject with the level of the same one or more targets determined in a control group or control individual. In the present disclosure, the control sample is obtained from an individual without a disease. That is, the individual from which the control sample is obtained is free of the disease being tested. Usually, the term disease-free means that the subject is healthy.

[0354] As used herein, the term "differential expression" refers to measuring a target in comparison to a control or another sample, and thereby determining, for example, a difference in the concentration, presence or intensity of said target. The result of such a comparison can be given in absolute terms, that is, the target is present in the sample and absent in the control, or in relative terms, that is, the expression or concentration of the target is increased or decreased compared to the control. In this case, the terms "increased" and "decreased" can be used interchangeably with the terms "upregulated" and "downregulated".

[0355] As used herein, the term "pharmaceutically acceptable carrier and / or diluent" includes within its scope any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and combinations thereof. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0356] The present invention illustratively described herein may be suitably carried out without any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. In addition, the terms and expressions used herein are used as descriptive terms, not limiting terms, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein disclosed herein may be employed by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0357] Functional properties Agents, treatments, or other therapies described herein, such as antibodies and antigen-binding fragments, may be defined by their functional properties. For example, GALA inhibitors, calnexin (CNX) inhibitors, protein disulfide isomerase A4 (PDIA4) inhibitors, or protein disulfide isomerase A3 (PDIA3) inhibitors, or CNX:PDIA3 complex inhibitors may be defined by their functional properties. In addition, anti-calnexin (anti-CNX) antibodies, anti-protein disulfide isomerase A4 (anti-PDIA4) antibodies, or anti-protein disulfide isomerase A3 (anti-PDIA3) antibodies, or anti-CNX:PDIA3 complex antibodies may be defined by their functional properties.

[0358] In some cases, agents such as inhibitors, antibodies and antigen-binding fragments are capable of: -Reducing cartilage breakdown -Reducing ECM degradation -Reduce ECM degrading activity · Reducing the ECM degrading activity of CNX:PDIA3 (also known as Cnx / ERp57) - Suppressing the ECM degrading activity of CNX - Suppressing the ECM degrading activity of fibroblasts - Suppressing the ECM degrading activity of synovial fibroblasts - Suppressing oxidoreductase activity Decreasing the oxidoreductase activity of CNX:PDIA3 (also known as Cnx / ERp57) - Suppressing the oxidoreductase activity of CNX - Reducing disulfide bond reductase activity · Reducing the disulfide bond reductase activity of CNX:PDIA3 (also known as Cnx / ERp57) - Reducing the disulfide bond reductase activity of CNX Reduce O-glycosylation Reduce O-glycosylation of CNX Reduce glycosylation of CNX · Reducing GALA-mediated O-glycosylation of CNX - Reducing CNX activity - Reducing PDIA3 activity - Reducing PDIA4 activity Decreasing the number or percentage of cells expressing CNX Decreasing the number or percentage of cells expressing PDIA3 Decreasing the number or percentage of cells expressing PDIA4 Decreasing the number or percentage of cells expressing CNX:PDIA3

[0359] All of the functional properties highlighted above can be effectively assayed through methods known in the art, for example, through microscopy, qPCR, qrtPCR, Western blot, biochemical assays, enzymatic assays, and other techniques known to those of skill in the art.

[0360] The relative levels of cartilage degradation and cartilage degrading activity can be determined through many methods known in the art. For example, it can be determined through well-known methods such as microscopic analysis, conventional radiographs (X-rays), magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound imaging. In addition, those skilled in the art will be aware of biochemical and biomarker assays that can be used to determine the level of cartilage degradation. Serum and urinary biomarkers can be used to assay the level of cartilage degradation. Biomarkers include type II procollagen C propeptide (CPII), cartilage oligomeric matrix protein (COMP), collagenase-generated type II collagen carboxy-terminal neoepitope (sC2C), cartilage intermediate layer protein 2 (CILP-2), type II collagen C telopeptide (CTX-II), and collagenase-generated type II collagen peptide(s) (C2C-HUSA).

[0361] A person skilled in the art would be able to determine whether cartilage degradation or cartilage degrading activity is reduced / inhibited by comparison with a relevant control.

[0362] The relative levels of ECM degradation and ECM degrading activity can be determined through several methods. Those skilled in the art are aware of many methods that can measure ECM degradation and ECM degrading activity, such as those shown in the examples herein. Those skilled in the art can easily determine whether a Cnx / ERp57 inhibitor can inhibit ECM degrading activity, the ECM degrading activity of Cnx / ERp57, and / or the ECM degrading activity of Cnx.

[0363] The following steps are one way one skilled in the art can assay ECM degrading activity.

[0364] Commercially available gelatin solution (2%) can be labeled with 5-carboxy-X-rhodamine, succinimidyl ester. The labeled gelatin can then be transferred to a sterile cover slip to create a thin layer, which can then be stabilized by glutaraldehyde fixation. Finally, a rat tail collagen solution can be used to coat the cover slip to create a thin layer of collagen on top of the gelatin.

[0365] The coverslips can then be transferred to a culture vessel, seeded with degradatively active cells (eg, human hepatocellular carcinoma Huh7) and incubated for 48 hours to allow degradation to occur.

[0366] After fixation, the coverslips can be stained with Hoescht to allow the cells to be counted. The coverslips can then be imaged with a confocal microscope. The images can then be analyzed using ImageJ. A threshold can be manually defined to reveal the surface of degraded gelatin, and the total area per field was measured. In parallel, the number of nuclei can be calculated and the final results can be normalized to the cells in each field.

[0367] A person skilled in the art would be able to determine whether ECM degrading activity is reduced / inhibited by comparison to a relevant control.

[0368] The relative level of oxidoreductase activity can be determined by many methods. Those skilled in the art are aware of many methods that can measure oxidoreductase activity, such as those shown in the examples herein. Those skilled in the art can easily determine whether a Cnx / ERp57 inhibitor can inhibit oxidoreductase activity, the oxidoreductase activity of Cnx / ERp57, and / or the oxidoreductase activity of Cnx.

[0369] For example, use of an insulin reduction assay, as described in Hirano et al., Eur J Biochem. (1995) 234(1):336-42, is one way that one of skill in the art could test for oxidoreductase activity.

[0370] A person skilled in the art would be able to determine whether oxidoreductase activity is reduced / inhibited by comparison with relevant controls.

[0371] The relative level of disulfide bond reductase activity can be determined by many methods. Those skilled in the art are aware of many methods that can measure disulfide bond reductase activity, such as those shown in the examples herein. Those skilled in the art can easily determine whether a Cnx / ERp57 inhibitor can inhibit disulfide bond reductase activity, the disulfide bond reductase activity of Cnx / ERp57, and / or the disulfide bond reductase activity of Cnx.

[0372] The following steps are one way one of skill in the art can assay for disulfide bond reductase activity: (1) plating ECM (1 mg / ml collagen with 25 ug / ml fibronectin (polymerized at 37°C for 1 hr) or 0.5 mg / ml Matrigel (polymerized at RT for 2.5 hr)), (2) seeding cells at the appropriate density, (3) cell attachment (approximately 45 min), (4) addition of antibody at 10 ug / ml with 10-25 mM GM6001, (5) incubation for 72 hr, (6) addition of Hoechst to visualize nuclei, (7) decellularization with warm 20 mM ammonium hydroxide, 0.5% (v / v) Triton X-100 for 1-2 min, (8) washing with 1% BSA in PBS, (9) addition of 2.5 mM TCEP for control at RT for 30 min, (10) washing with 1% BSA in PBS, (11) incubation at RT for 30 min with 1% BSA in PBS, (12) incubation at RT for 30 min with 1% BSA in PBS, (13) incubation at RT for 30 min with 1% BSA in PBS, (14) incubation at RT for 30 min with 1% BSA in PBS, (15) incubation at RT for 30 min with 1% BSA in PBS, (16) incubation at RT for 30 min with 1% BSA in PBS, (17) incubation at RT for 30 min with 1% BSA in PBS, (18) incubation at RT for 30 min with 1% BSA in PBS, (19) incubation at RT for 30 min with 1% BSA in PBS, (20) incubation at RT for 30 min with 1% BSA in PBS, (21) incubation at RT for 30 min with 1 Addition of 5 mM NEM in BSA, (12) washing with 1% BSA in PBS, (13) fixation with 4% PFA, (14) washing with PBS, (15) primary antibody mouse anti-NEM OX133 (1:200) and IgG control overnight at 4°C, (16) washing with PBS, (17) secondary antibody anti-mouse 647 (1:400) and secondary control only at RT for 45 minutes, (18) washing with PBS, and (19) revisiting the same wells to measure cysteine ​​reduction signal and normalize to cell number.

[0373] A person skilled in the art would be able to determine whether disulfide bond reductase activity is reduced / inhibited by comparison with relevant controls.

[0374] The presence and relative levels of glycosylation can be determined by methods well known in the art, such as those used in the Examples, and by commercially available assay kits. Methods for detecting and analyzing glycosylation and glycoproteins include glycan staining or labeling, glycoprotein purification or enrichment, and analysis by mass spectrometry. Those of skill in the art will be aware of such methods.

[0375] The presence and relative levels of O-glycosylation can be determined through methods known in the art, such as those used in the Examples herein. A hallmark of O-glycosylation is increased cellular levels of Tn (T nouvelle), an O-glycan formed by the addition of GalNac to Ser or Thr residues. Tn can be detected by Tn-binding proteins such as Vicia Villosa lectin (VVL) and Helix Pomatia lectin (HPL) (Gill, et al., Proc. Natl. Acad. Sci. USA110, E3152-61 2013).

[0376] The relative levels of CNX, PDIA3 can be determined through methods known in the art. For example, the relative levels of gene expression (e.g., calnexin expression) can be determined through many different methods known to those skilled in the art. The level of RNA encoding a given gene can be determined by techniques such as, for example, RNAseq, RT-PCR, and RT-qPCR, among other well-known methods. In addition, the relative levels of protein expression (e.g., calnexin expression) can be determined by means well known to those skilled in the art. The level of a given protein / its isoforms can be determined by antibody-based methods including, for example, Western blot, immunohisto / cytochemistry, flow cytometry, ELISA, and the like. Furthermore, CNX, PDIA3, and / or PDIA4 activity can be determined by isolating the enzyme and performing an enzyme assay.

[0377] The number or percentage of cells expressing CNX, PDIA3, PDIA4, and / or CNX:PDIA3 can be determined by several methods, including single-cell transcriptome analysis, immunohistochemical microscopy, and flow cytometry.

[0378] As highlighted above, a therapeutic agent (e.g., a GALA inhibitor, a calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA4) inhibitor, or a protein disulfide isomerase A3 (PDIA3) inhibitor, a CNX:PDIA3 complex inhibitor, or an anti-calnexin antibody) can lead to a decrease in a certain property or activity (e.g., ECM degradation, oxidoreductase activity, disulfide bond reductase activity, glycosylation, protein activity, or the number or proportion of a particular cell type). In some cases, this reduction may be more than 5% of the level seen in a relevant control, for example one of: ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0379] A relevant control may be the uninhibited state, ie, the level of the trait before treatment.

[0380] In some embodiments, the therapeutic agent (e.g., a GALA inhibitor, a calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA4) inhibitor, or a protein disulfide isomerase A3 (PDIA3) inhibitor, a CNX:PDIA3 complex inhibitor, or an anti-calnexin antibody) leads to a decrease in the level of ECM degradation. In some cases, this reduction in ECM degradation can be more than 5% of the level observed in a relevant control (e.g., in an uninhibited state), for example, one of: ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0381] In some embodiments, the therapeutic agent (e.g., a GALA inhibitor, a calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA4) inhibitor, or a protein disulfide isomerase A3 (PDIA3) inhibitor, a CNX:PDIA3 complex inhibitor, or an anti-calnexin antibody) leads to a decrease in the level of ECM degrading activity (e.g., CNX ECM degrading activity, CNX / ERP57 ECM degrading activity, or synovial fibroblast ECM degrading activity). In some cases, this reduction in ECM degrading activity can be more than 5% of the level observed in a relevant control (e.g., in an uninhibited state), such as one of: ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0382] In some embodiments, the therapeutic agent (e.g., a GALA inhibitor, a calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA4) inhibitor, or a protein disulfide isomerase A3 (PDIA3) inhibitor, a CNX:PDIA3 complex inhibitor, or an anti-calnexin antibody) leads to a decrease in the level of oxidoreductase activity (e.g., CNX ECM degrading activity, CNX / ERP57 ECM degrading activity, or synovial fibroblast ECM degrading activity). In some cases, this reduction in oxidoreductase activity can be more than 5% of the level observed in a relevant control (e.g., in an uninhibited state), such as one of: ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0383] In some embodiments, the therapeutic agent (e.g., a GALA inhibitor, a calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA4) inhibitor, or a protein disulfide isomerase A3 (PDIA3) inhibitor, a CNX:PDIA3 complex inhibitor, or an anti-calnexin antibody) leads to a decrease in the level of disulfide bond reductase activity (e.g., CNX ECM degrading activity, CNX / ERP57 ECM degrading activity, or synovial fibroblast ECM degrading activity). In some cases, this reduction in disulfide bond reductase activity can be more than 5% of the level observed in a relevant control (e.g., in the uninhibited state), such as one of: ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧35%, ≧40%, ≧45%, ≧50%, ≧55%, ≧60%, ≧65%, ≧70%, ≧75%, ≧80%, ≧85%, ≧90%, ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99% or 100%.

[0384] General terminology As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations of genetic markers.

[0385] As used herein, in the context of formulation component concentrations, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0386] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, as well as each individual numerical value within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as each individual numerical value within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0387] Certain embodiments may also be broadly and generically described herein, and each of the more specific species and subgeneric groupings falling within the generic disclosure also form part of this disclosure, including the generic description of embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0388] The invention has been described broadly and generically herein, and each of the more specific species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a provisos or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0389] Other embodiments are within the scope of the claims and non-limiting examples. Additionally, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described thereby in terms of any individual element or subgroup of elements of the Markush group. EXAMPLES

[0390] Example 1 Patient samples, cell lines and mouse strains Patient samples: Synovial tissue specimens were obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) undergoing joint replacement surgery at Tan Tock Seng Hospital (Singapore). The procedure was approved by the Ethics Committee of The National Healthcare Group domain specific review board under protocol number 2018 / 00980. Written informed consent was obtained from all patients, who met the diagnostic criteria for rheumatoid arthritis or osteoarthritis.

[0391] Cell line: SW982 cells (ATCC HTB-93) are synovial fibroblasts derived from the synovium of a patient with synovial sarcoma. SW982 cells were maintained in Leibovitz´s L-15 Medium (Gibco, ThermoFisher Scientific) supplemented with 10% (v / v) fetal calf serum (FCS) and 1% (w / v) penicillin / streptomycin (Gibco, ThermoFisher Scientific) at 37°C with free gas exchange in air. SW982 cells were engineered to stably express a doxycycline-inducible gene encoding ER-2Lec using the Sleeping Beauty transposon system.

[0392] Mice: Col6a1Cre mice expressing collagen VI promoter driven on a C57BL / 6J background were provided by G. Bressan (University of Milano, Milano, Italy). It should be noted that comparable mouse models known in the art can be used as backgrounds to generate the required mice according to the present disclosure. ER-2Lec mice on the same background expressing endoplasmic reticulum (ER)-localized dual lectin domain (ER-2Lec) under the control of a loxP-flanked STOP cassette were custom-generated by us following the details provided by Ozgene. Col6a1Cre ER-2Lec mice were generated by crossing Col6a1Cre and ER-2Lec mice to obtain mice expressing ER-2Lec in the mesenchymal cell lineage. All animals were bred and maintained under specific pathogen-free conditions in microisolator cages with access to food and water at the Biological Resource Centre (ASTAR, Singapore). Experiments were performed using age- and sex-matched animals and followed guidelines approved by the Animal Ethics Committees at Biological Research Centre (ASTAR, Singapore) under IACUC protocol number 201548.

[0393] Isolation of primary synovial fibroblasts (SF) from human tissue Synovial tissue from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) was obtained at synovectomy or synovial biopsy. After removal, the tissue was immediately minced and digested with collagenase type IV (1 mg / ml, Gibco) in Dulbecco's modified Eagle's medium (DMEM) for 1.5 h at 37°C with gentle agitation. The mixture was passed through a 70 μm mesh cell strainer and a cell pellet was obtained after centrifugation at 250 g for 10 min. Human synovial fibroblasts (OASF and RASF, respectively) from patients with osteoarthritis and rheumatoid arthritis were used between passages 3 and 9 (Rosengren et al., 2007, Methods in Molecular Medicine, Vol. 135: Arthritis Research, Volume 1). The purity of the cultures was confirmed by staining with the fibroblast identification marker CD90 prior to carrying out the experiments. Normal human synovial fibroblasts were derived from synovial tissue (HCSF) of healthy human donors and obtained from Cell Applications, Inc. HCSF were cultured in DMEM supplemented with 10% (v / v) fetal calf serum (FCS) and 1% (w / v) penicillin / streptomycin (Gibco, ThermoFisher Scientific) at 37 °C under 5% CO 2 The incubation was completed at 37°C in a humidified atmosphere containing

[0394] reagent Antibodies: Anti-CNX (ab10286, ab22595), anti-vimentin (ab92547), anti-beta-actin (ab8226) antibodies were purchased from Abcam. Agarose-conjugated Vicia Villosa lectin (VVL, AL-1233) was purchased from Vector Laboratories. PE.C7-conjugated anti-CD45 and PE-conjugated anti-CD90 were purchased from Biolegend. Anti-FAPα was purchased from R&D systems. Anti-NEM OX133 antibody was purchased from Absolute Antibody. Anti-rabbit IgG-HRP antibody and anti-mouse IgG-horseradish peroxidase (HRP) antibody were purchased from GE Healthcare Life Sciences.

[0395] Plasmid: A plasmid expressing doxycycline-inducible ER-2Lec was constructed as previously described (Gill et al. 2013, PNAS, 2013, E3152-E3161). The resulting vector was used together with pPGK-SB13 expressing Sleeping Beauty 208 transposase to transfect SW982 cells.

[0396] Immunofluorescence staining Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated in xylene substitution buffer (Sub-X, Leica Biosystems). For mouse joint tissues and tissue microarrays (provitro AG, Berlin, Germany), antigen retrieval was performed by immersion in Epitope Retrieval Solution pH 6 (Leica Biosystems) and incubation in an oven at 60°C for 18 h. For human tissue specimens, antigen retrieval was performed in a pressure chamber (2100 Retriever, Akribis Scientific Limited, WA16 0JG, GB) using Epitope Retrieval Solution pH 6 (Leica Biosystems). Sections were washed twice with phosphate-buffered saline (PBS) and immersed in blocking buffer (5% horse serum, 1% Triton X100). After 1 h, sections were incubated overnight at 4°C with primary antibody mix containing primary antibodies including rabbit anti-CD45, rabbit anti-vimentin, rabbit anti-calnexin, rat anti-FAPα, or biotin-conjugated Vicia Villosa lectin (VVL). Samples were washed three times with blocking buffer and incubated with corresponding secondary antibodies including anti-rat Alexa Fluor647, anti-rabbit Alexa Fluor594 conjugate, or Alexa Fluor488 streptavidin (ThermoFisher Scientific, 1:400) for 2 h. After washing, cell nuclei were stained with Hoechst 33342 (ThermoFisher Scientific, 1:1000) for 5 min before mounting. All images were acquired using the same settings on an LSM-700 (Zeiss) confocal microscope. Raw integrated densities of VVL and nuclear signals were measured with a FIJI image calculator.

[0397] Collagen antibody-induced arthritis On day 0, mice were injected via intraperitoneal (IP) injection with an antibody cocktail mix (Chondrex Inc.) containing five different monoclonal antibody clones against type II collagen protein at a dose of 2 mg / mouse (200 μl). On day 3, animals were injected via IP injection with 50 μg / mouse LPS (100 μl). From day 3 onwards, arthritis severity was assessed daily by measuring paw thickness with a digital caliper. Assessments were also performed by blinded investigators using a qualitative clinical scoring system provided by Chondrex, Inc.

[0398] Histological analysis and cartilage extracellular matrix staining After removing the skin, both front and hind paws were formalin fixed and paraffin embedded. Tissues were deparaffinized and rehydrated as described above in the immunofluorescence staining protocol. Tissue sections were stained with hematoxylin and eosin (HE), safranin-O (SO), and Alcian Blue (AB) stains. Slides were scanned at 20x using a Leica SCN400 slide scanner (Leica Microsystems, Germany). Images were exported and viewed in Slidepath Digital Image Hub (Leica Microsystems, Germany). Selected areas were analyzed using the Measure Stained Area Assay in the software Slidepath Tissue Image Analysis 2.0 (Leica Microsystems, Germany). Quantitative analysis of cartilage extracellular matrix (ECM) stained area was performed using the FIJI image calculator.

[0399] Flow cytometry The skin of the paw was removed and the joint was amputated 3 mm above the heel. The marrow cavity of the tibia was thoroughly washed with Hank's Balanced Salt Solution (HBSS) to avoid contamination of the bone marrow. The joint was cut into small pieces and incubated in digestion buffer (1 mg / ml type IV collagenase and 1 mg / ml DNase I in HBSS) at 37°C for 60 min. Cells released during digestion were filtered through a 70 μm cell strainer and red blood cells were lysed using red blood cell lysis buffer (BD Biosciences). Cells were stained with live / dead Aqua (Invitrogen) viability dye, incubated with Fc Block (BD Biosciences) at 1:50, and stained with fluorochrome-conjugated antibodies including PE-conjugated anti-CD90 (Biolegend), PECy7-conjugated anti-CD45 (Biolegend) and FITC-conjugated Vicia Villosa lectin (VVL, Life Technologies) for 30 min. For intracellular staining, cells were fixed using BD Cytofix / Cytoperm solution (BD Biosciences). Fixed cells were permeabilized with 1x BD Perm / Wash buffer (BD Biosciences) prior to staining with FITC-conjugated Vicia Villosa lectin (VVL). Samples were acquired on a FACS BD LSRII and analyzed using the software Kaluza.

[0400] Western blot and VVL-coimmunoprecipitation (VVL-CoIP) The cells were plated at 2x10 5Cells were seeded at 2000 cells / ml and left overnight. After stimulation with 100 μg / ml TNFα (PeproTech) and 100 μg / ml IL-1β (PeproTech) for 24 h, cells were harvested and lysed in low stringency RIPA lysis buffer (50 mM Tris, 200 mM NaCl, 0.5% NP-40, Complete and PhoStop inhibitors [Roche Applied Science]) for 30 min at 4°C. Lysates were then clarified by centrifugation at 13000 g for 10 min at 4°C. The clarified tissue lysates were incubated with agarose-conjugated Vicia Villosa lectin (VVL) beads (Vector Laboratories) overnight at 4°C. Beads were washed three times with RIPA lysis buffer and precipitated proteins were eluted in 2x LDS sample buffer containing 50 mM DTT. Lysates were boiled at 95°C for 5 min and separated by SDS-PAGE electrophoresis using 4-12% Bis-Tris 80 NuPage gels (Invitrogen) at 180V for 70 min. Samples were then transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen) and blocked with 3% bovine serum albumin (BSA) in TBST (Tris-buffered saline (TBS) and polysorbate 20 (also known as Tween 20)-50 mM Tris, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. The nitrocellulose membranes were then incubated with primary antibodies (diluted 1 / 1000 in 3% BSA-TBST) overnight at 4°C. The next day, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The membrane was washed three more times with TBST before electrochemiluminescence (ECL) exposure.

[0401] Matrix degradation assay Red gelatin coverslips were prepared as previously described (Ros et al., Nat Cell Biol, 2020, vol 22, November 2020, 1371-1381). Coverslips were coated with 0.2 mg / ml cartilage extracellular matrix (ECM, Xylyx Bio) for 3 hours at 37°C. Synovial fibroblasts (including SW982 cells, osteoarthritis synovial fibroblasts (OASF), rheumatoid arthritis synovial fibroblasts (RASF) and healthy human donor synovial fibroblasts (HCSF)) were plated in 24-well plates at 5x10 4 Cells were seeded at 1000 cells / ml / well overnight. Cells were then stimulated with 100 μg / ml TNFα and 100 μg / ml IL-1β. After 24 h, cells were fixed with 4% paraformaldehyde (PFA) and nuclei were stained using Hoechst 33342 (Life Technologies). Stained coverslips were mounted on microscope slides and 10–30 images were acquired for each condition. The area of ​​matrix degradation normalized to cell number was measured using the software ImageJ as previously described (Martin et al., J Vis Expr, 2012, vol 66, e4119). Briefly, the degradation area was determined using thresholded fluorescent gelatin images, and the same threshold was applied to all images. The number of nuclei was counted using a cell counter and the area of ​​gelatin degradation per total number of cells was calculated. Experiments were performed in triplicate biological replicates.

[0402] statistical analysis GraphPad Prism (version 8.4.3, GraphPad Software, CA, USA) was used for statistical analysis and graphing. Data analysis was performed by one-way (Kruskal-Wallis test), two-way ANOVA (Tukey's multiple comparison test) or Mann-Whitney test, as indicated. Differences were considered statistically significant at p values ​​< 0.05.

[0403] Example 2 result Enhanced O-glycosylation in rheumatoid arthritis and osteoarthritis synovium A hallmark of GALA is the increase in cellular levels of Tn (T nouvelle), an O-glycan formed by the addition of GalNac to Ser or Thr residues, which can be detected by Tn-binding proteins such as Vicia Villosa lectin (VVL) and Helix Pomatia lectin (HPL) (Gill, et al., Proc. Natl. Acad. Sci. USA 110, E3152-61 2013).

[0404] We analyzed joint tissue microarrays by immunofluorescence with VVL, counterstaining DNA. We detected a clear increase in Tn samples in 18 / 21 samples from OA patients, 2 / 6 samples from psoriatic arthritis patients, and 9 / 18 samples from RA patients (Figure 1A and Figure 23). We quantified the integrated fluorescence intensity of VVL staining normalized by DNA signal intensity as a marker of cell density (Figure 1B). Healthy patient samples showed little change, while RA and OA samples showed most samples exhibited elevated Tn levels, with some areas showing up to a 7-fold increase in VVL signal.

[0405] To further characterize GALA in arthritis, we employed a murine RA model based on collagen antibody-induced arthritis (CAIA) (32). Briefly, animals were injected with antibodies against type II collagen, followed 3 days later by lipopolysaccharide (LPS), and symptoms were allowed to develop starting on day 5. Initial immunohistochemical analysis revealed that animals with arthritis had significantly increased Tn levels in and around the joints (Figure 23).

[0406] In the CAIA model, symptoms peaked at approximately day 7 and continued for 10 days before gradually relieving. We sampled animals at days 0, 7, 10, and 14 and quantified Tn levels using immunofluorescence staining. Histologically, pannus infiltrating the joint cavity was evident at day 7 and increased in size at day 10 with the influx of immune cells. By day 14, the amount of immune cells had decreased dramatically, but synovial tissue remained within the joint cavity (Figures 2A and B). High levels of Tn were observed in cells infiltrating the joint cavity at day 7 and persisted until day 10 (Figures 2C and D). By day 14, cellular Tn levels had subsided in the majority of animals. We observed some Tn staining in fibrous tissue devoid of cells, which did not decrease significantly even at day 14.

[0407] High Tn levels in arthritic synovium coincide with GALA activation High cellular Tn levels can be induced by the GALA pathway, which is characterized by abundant Tn signals in the ER. Because the ER is distributed throughout the cell body, GALA activation usually appears as an increase in a diffuse signal (Bard et al., Trends Cell Biol. 26, 379-388, 2016). We co-stained CAIA synovial samples for Tn and the ER marker calnexin (Figure 3A). In untreated samples, Tn and calnexin staining were clearly separated, and Tn was concentrated in a perinuclear pattern consistent with the Golgi apparatus. In contrast, in day 7 CAIA samples, Tn staining was elevated, filling the cellular space, and co-localized with the ER marker calnexin, strongly suggesting GALA induction. To corroborate GALA induction in the context of CAIA, we stained for GALNT2, a ubiquitously expressed GALNT transferase previously shown to relocalize to the ER in GALA-activated cancers. We observed a similar change in localization from the nuclear periphery in untreated samples to an ER pattern in CAIA samples where it colocalized with CNX (Figure 3B). Our results suggest that translocation of GALNT2 from the Golgi apparatus to the ER is the basis for increased Tn levels in the context of CAIA. These results are consistent with previously reported descriptions of the GALA phenotype in breast and liver cancer (Gill, et al., Proc. Natl. Acad. Sci. USA110, E3152-61, 2013; Nguyen, et al., Cancer Cell. 32, 639-653. e6, 2017).

[0408] Synovial fibroblasts are the predominant cell type expressing GALA The synovium in RA disease is a complex tissue that contains immune cells and synovial fibroblasts (Choy et al., Rheumatology .51 Suppl 5, v3-11, 2012). To establish which cell types showed increased GALA, 7d mouse CAIA joint samples were co-stained with VVL, the immune cell marker CD45, and the fibroblast marker vimentin. We found vimentin-positive cells at the very front of the invaded pannus (Figure 4). CD45-positive cells were typically clustered and located posterior to the invasion front of the pannus. Of note, Tn-positive cells within the pannus mostly co-stained with vimentin-positive areas but not with CD45-positive areas. This result suggests GALA activation in synovial fibroblasts (Figure 4). We verified this result in RA and OA human samples using fibroblast activation protein alpha (FAPα) as a marker of synovial lining fibroblasts (Bauer et al. Arthritis Res. Ther. 8, R171, 2006). FAPα positive cells formed a layer located at the edge of the pannus, with a larger layer of CD45 positive cells located posteriorly in RA samples (Figures 5A, 5B and Figure 24A). In OA samples, the number of CD45 cells was reduced compared to RA samples, but FAPα cells showed similar VVL staining (Figure 24). Strikingly, VVL staining exclusively colocalized with FAPα under both RA and OA conditions. Thus, in OA and RA synovium, synovial lining fibroblasts are the main cells exhibiting GALA activation.

[0409] Stimulation of SFs with cytokines and ECM induces higher GALA levels To understand how GALA is activated in vivo, we used primary human SFs from patients. We established the purity of the cell preparations we used >90% by FACS analysis using CD90 and CD45 as markers for SFs and immune cells, respectively (Figure 24B). We then performed high content imaging analysis of Tn levels in these patient-derived SF cells using HPL staining. Seeding the cells on plastic wells showed increased Tn levels in OA cells, and even more so in RA cells, compared to healthy SFs (Figure 6). We then stimulated SFs with TNFα and IL1β cytokines, which are thought to promote disease progression in RA (Kagari and Shimozato. J. Immunol. 169, 1459-1466, 2002). While each individual cytokine had a relatively limited effect on GALA activation, the combination of both cytokines (denoted CYTO) induced a two-fold increase in Tn cell levels. Interestingly, the effect was prominent in RASFs, more limited in OASFs, and nearly absent in healthy SFs (HCSFs).

[0410] Next, we wondered whether cartilage ECM proteins might contribute to the activation of SFs. Exposure of SFs to cartilage ECM activated GALA up to three-fold in both OASFs and RASFs. In contrast, HCSF cells showed little reaction. Combination of CYTO and ECM had an additive effect on Tn levels (Figure 6). The stimulation was not specific to cartilage ECM, since similar activation was induced using type I collagen from rat tail (Figure 6).

[0411] Under non-stimulated conditions, Tn staining was detected exclusively in the Golgi apparatus of HCSF, whereas OASF and RASF showed additional ER-like Tn staining (Figure 6). Upon stimulation with a combination of CYTO and cartilage ECM, ER-localized Tn staining was enhanced in both OASF and RASF, but not in HCSF (Figure 6).

[0412] In summary, this analysis revealed that SFs from OA and RA patients had elevated GALA in cell cultures compared to healthy SFs. While RA SFs further activated GALA in response to cytokines, both RA and OA SFs activated GALA immediately upon exposure to ECM. In contrast, healthy SFs showed a very limited GALA response, suggesting that the pathway was primed for activation in patient cells.

[0413] Inhibition of GALA in SFs reduces ECM degradation and arthritis in vivo Since GALA promotes ECM degradation in cancer cells, we sought to test whether it is also involved in cartilage ECM degradation by SFs. We found that human synovial sarcoma SW982 cells can degrade collagen (in a sandwich assay with fluorescent gelatin as described previously) (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). We previously described that the ER-2Lec chimeric protein is composed of a fusion of two lectins, an ER targeting sequence and GALNT2 (Gill, et al., Proc. Natl. Acad. Sci. USA110, E3152-61 2013). ER-2Lec specifically inhibits GALA activity by interfering with O-glycosylation in the ER.

[0414] We generated stable SW982 ER-2Lec transfectants under a doxycycline-inducible promoter system. Similar to RA SF, SW982 cells stimulated with CYTO mix were more active in ECM degradation (Figures 7B and 7C). However, when ER-2Lec expression was induced, degradation was significantly reduced by more than two-fold (Figures 7B and 7C).

[0415] To test whether ER-2Lec can alleviate arthritis in vivo, we generated ER-2Lec transgenic mouse lines carrying a Lox cassette. We crossed them with mice expressing Cre under the collagen VI alpha 1 (Col6a1) promoter (Figure 3C). Collagen VI is expressed by joint mesenchymal cells, especially SFs (Danks et al., Annals of the Rheumatic Diseases. 75, 2016, pp. 1187-1195; Armaka, et al., J. Exp. Med. 205, 331-337, 2008). Therefore, this genetic cross is expected to result in ER-2Lec being expressed primarily in SFs and to reduce Tn levels in arthritic mice. We induced CAIA in these mice and monitored ER-2Lec-GFP expression and Tn levels in the pannus 7 days later. Tn was significantly decreased, consistent with GALA inhibition (Fig. 8 ).

[0416] We monitored RA symptoms in Col6a1Cre ER-2Lec animals and in Cre-expressing controls. ER-2Lec-expressing animals showed significantly reduced paw swelling (Figure 9A and B). We measured changes in paw thickness over time and found a significant reduction in thickness in ER-2Lec animals compared to controls. We also found consistent symptom reduction in Col6a1Cre ER-2Lec animals using the internationally defined arthritis score in a blinded assessment (Figure 10).

[0417] We also performed histological analysis at day 7 using H&E and Alcian Blue (AB) and Safranin-O (SO) staining, which are commonly used to reveal the cartilage compartment of the joint. H&E staining of Col6a1Cre ER-2Lec joints showed a reduction in pannus size, consistent with reduced swelling (Figure 11). Interestingly, immune cell infiltration appeared to be significantly reduced in ER-2Lec expressing animals (Figure 11 and Figure 25A). AB-positive areas were also significantly preserved in CAIA Col6a1Cre ER-2Lec animals (Figure 11). Significant changes were also obtained after quantification of SO staining (Figure 25). Taken together, our results demonstrate that inhibition of GALA at ER-2Lec protein in SFs can limit arthritic disease progression.

[0418] GALA activates glycosylation and surface exposure of CNX in SFs We recently described calnexin (CNX) as a glycosylation target and effector of GALA (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). Once glycosylated, CNX immediately migrates to the cell surface and, together with PDIA3, mediates the cleavage of disulfide bonds in ECM proteins. This reducing activity is essential for matrix degradation by cancer cells (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). We found that CNX was approximately 6-fold overglycosylated in SW982 SF after stimulation with cytokines and ECM (Figures 12A and B). This glycosylation was dependent on GALA, since expression of ER-2Lec was able to significantly reduce the glycosylation of CNX.

[0419] In addition, we found that CNX surface expression was significantly increased by about 10% after stimulating SW982 SF cells with CYTO and ECM using FACS (Figures 13A and B). Remarkably, the increase in cell surface CNX signal was completely suppressed by expression of ER-2Lec (Figures 13A and B).

[0420] We sought to confirm this result in primary cells from patients. In healthy control SFs (HCSFs), the percentage of cell surface CNX positive cells was only about 7%, and stimulation with cytokines and ECM slightly increased the percentage (Figures 14A and B). In contrast, SF cells from patients with RA (RASFs) or SF cells from patients with OA (OASFs) showed a three-fold increase in the percentage of cells with cell surface CNX, a significantly increased level, and were more sensitive to stimulation (Figures 14A and B). Overall, these results indicate that glycosylation of CNX and its cell surface exposure are enhanced in arthritic SFs and are dependent on GALA.

[0421] Anti-CNX antibodies prevent arthritis symptoms in CAIA mice We previously showed that antibodies against calnexin can block ECM degradation by preventing the reduction of the necessary disulfide bonds (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). We hypothesized that blocking calnexin should similarly prevent cartilage ECM degradation. We first tested the presence of disulfide bonds in cartilage ECM using a previously described method (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). Briefly, cartilage ECM was reduced with TCEP, then exposed to N-ethylmaleimide (NEM), and then treated with anti-OX133 antibody. As previously described for liver ECM, we observed abundant OX133 signal colocalized with collagen 3 / collagen 1 and fibronectin / collagen 1 fibers, suggesting that cartilage ECM is highly cross-linked with disulfide bonds (Figure 15).

[0422] Next, we tested the effect of anti-CNX antibodies on ECM degradation. OASF cells seeded on cartilage ECM overlaid with fluorescent gelatin were allowed to degrade ECM overnight. Addition of polyclonal anti-CNX antibodies blocked this degradative activity (Figures 16 and B).

[0423] These results prompted us to aim to treat animals with anti-CNX antibodies. We first monitored the weight of animals that received three antibody injections over a 10-day period, and we did not detect any weight loss (Figure 22). We then treated CAIA animals with 25 micrograms of anti-CNX antibodies injected every 2 days from day 3 to day 7 after CAIA was induced (Figure 17A). We monitored paw thickness at regular intervals and measured arthritis scores on day 10. Remarkably, paw swelling was reduced in anti-CNX-treated animals compared to control animals treated with isotype antibodies (Figure 17B). Although some redness and swelling of the digits still occurred and arthritis scores were elevated, the average score of treated animals was half that of CAIA control animals (Figure 17C). This is reminiscent of the results obtained with ER-2Lec expression.

[0424] At the histological level, the loss of SO-positive cartilage was highly evident in control animals at day 10 (Figure 18). In addition, the synovium was attached to the underlying bone, possibly indicating the onset of bone remodeling. In contrast, in anti-CNX treated animals, the joint cavity was well preserved and cartilage remained abundant (Figure 18).

[0425] Our research hypothesis is that Cnx antibody binds to synovial lining fibroblasts and inhibits their decomposition activity. To test whether the antibody actually interacts with these cells, we stained the joints of treated animals with anti-rabbit IgG. A signal was clearly detected in synovial cells of animals treated with anti-CNX antibody, but no signal was observed in animals treated with control rabbit IgG (Figure 19C).

[0426] Overall, these results indicate that inhibition of CNX potently inhibits cartilage ECM degradation and may form the basis of a therapeutic agent for arthritis.

[0427] Experimental Example 3 Consideration In this study, we showed that GalNac O-glycosylation was significantly upregulated in arthritic synovial fibroblasts compared with healthy controls, and this increase was due to activation of the GALA pathway, which involves translocation of GALNTs from the Golgi apparatus to the ER.

[0428] In cancer cells, activation of EGF-R and especially Src kinases promotes GALA (Gill et al., J. Cell Biol. 189, 843-858, 2010; Chia et al., PLoS One. 14, e0214118, 2019). Other signaling molecules, such as ERK8 kinase, constitutively and dynamically inhibit the pathway (Chia et al., Elife. 3, e01828, 2014). In vitro, GALA levels are moderately elevated in fibroblasts from RA and OA patients compared to healthy human SFs. However, RA fibroblasts activate GALA in response to a mixture of IL-1 beta and TNF alpha cytokines. Interestingly, the response of RA SFs is more pronounced than that of normal SFs or OA SFs, suggesting that RA SFs are primed to respond to these cytokines. IL-1β has been reported to activate the tyrosine kinase Src (Mon et al., Oncol. Lett. 13, 955-960, 2017), suggesting a possible relationship between this cytokine and GALA.

[0429] Exposure to ECM activates GALA more readily and potently in both OA and RA fibroblasts than in healthy control cells. It has been previously proposed that adhesion of SFs to cartilage ECM elements plays a role in the development of arthritis (Pap et al., Arthritis Res. 2, 361-367, 2000). Injection of fibronectin into joints leads to degradation of cartilage proteoglycans (Homandberg, et al., J. Rheumatol. 20, 1378-1382, 1993). Integrins, the fibronectin receptors, are also activators of Src kinases (Shattil, Trends Cell Biol. 15, 399-403, 2005; Huveneers, and Danen, J. Cell Sci. 122, 1059-1069, 2009). Thus, a signaling cascade could link external ECM signals to integrins, Src, and then GALA, activating ECM degradation (Gill et al., J. Cell Biol. 189, 843-858, 2010). This hypothetical cascade should result in a pathological positive feedback loop. The reason for the much more limited GALA response to ECM in healthy SFs remains unclear. It has been proposed that SFs in arthritic joints are epigenetically primed for degradation (Nygaard et al., Nat. Rev. Rheumatol. 16, 316-333 (2020)). Indeed, OASFs and RASFs show comparable global methylation profiles, which are distinct from SFs in healthy subjects (Nakano et al., Ann. Rheum. Dis. 72, 110-117, 2013). Some differences were identified in genes involved in PDGF and EGF signaling, which are also regulators of GALA (Chia et al., PLoS One. 14, e0214118, 2019). Thus, epigenetic priming may include a stronger propensity to activate GALA. In addition, GALA glycosylation is probably synergistic with other regulatory mechanisms.For example, we found increased levels of CNX in the synovial tissue of arthritic mice, which is consistent with gene expression data reported in previous studies (Broeren et al., PLoS One. 11, e0167076, 2016; Nzeusseu Toukap, et al., Arthritis Rheum. 56, 1579-1588, 2007). In these studies, GALNT1, 3, and 5 were found to be upregulated, and we also found increased expression of GALNT1 and 2.

[0430] Regardless of whether activation is by immune signals or ECM proteins, GALA glycosylation may not be continuously activated in arthritis. Indeed, in CAIA mouse model, GALA levels are significantly decreased on day 10, after which animals fully recover (after day 14). In patient samples, GALA is detectable in OA, RA and psoriatic arthritis samples, but a significant proportion of samples show low GALA levels. This suggests that GALA is only fully activated during the active ECM degradation stage of the disease, which should correspond to a flare in patients. In contrast, there is less ECM degradation in the remission stage, and correspondingly low GALA levels.

[0431] Among the targets of GALA glycosylation is MMP14, a cell surface protease that degrades collagen fibers and activates other MMPs (Nguyen, et al., Cancer Cell. 32, 639-653.e6, 2017; Gialeli, et al., FEBS J. 278, 16-27, 2011). MMP14 is one of the MMPs involved in arthritis and activates MMP2 and 13 (Rose and Kooyman, Dis. Markers. 2016, 4895050, 2016). O-glycosylation of MMP14 is essential for its protease activity and occurs in clusters in low-complexity regions of the protein, i.e., six or more amino acids are modified with GalNAc or more complex O-glycans (Nguyen et al., Cancer Cell. 32, 639-653.e6 (2017)).

[0432] Calnexin also exhibits a clustered glycosylation pattern located in the N-terminal region (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). Clustered glycosylation is a frequent feature of GalNac glycosylation and is exemplified in mucin proteins. ER-2Lec chimeric proteins inhibit this clustered glycosylation (Gill et al., Proc. Natl. Acad. Sci. USA 110, E3152-61, 2013). As in cancer cells, ER-2Lec reduced Tn signal levels in SFs. Thus, ER-2Lec may at least partially inhibit glycosylation of MMP14 and Cnx in vitro and in vivo, inhibiting ECM degradation by SFs. Since GALNTs act on thousands of proteins and preliminary unpublished data indicate that GALA affects many proteins, additional glycoproteins may be involved in the pathological activity of SFs and influenced by ER-2Lec (Steentoft, et al., Nat. Methods. 8, 977-982, 2011).

[0433] ER-2Lec activated by Cre under collagen VI promoter prevented cartilage loss in CAIA-treated mice. ER-2Lec expression was mostly restricted to SFs, with no detectable expression in immune cells. Interestingly, expression of ER-2Lec reduced joint swelling and inflammation. Effective protection of cartilage ECM may reduce SF activation and prevent cytokine release, thus preventing inflammation. The fact that anti-Cnx antibody treatment also reduced inflammation supports this explanation.

[0434] The role of calnexin in ECM degradation has only recently been established. In complex with PDIA3, calnexin is involved in the reduction of disulfide bonds in ECM proteins in the liver (Ros et al., Nat. Cell Biol. 22, 1371-1381, 2020). Disulfide bonds, like other cross-links, prevent the action of proteases (Philp et al., Am. J. Respir. Cell Mol. Biol. 58, 594-603, 2018). Cartilage ECM is rich in disulfide bonds. Anti-calnexin antibodies blocked matrix degradation by SF in vitro and significantly protected the cartilage ECM in animals.

[0435] Other strategies to inhibit synovial cells have been developed, such as targeting the adhesion molecule cadherin 11 (Lee, et al., Science. 315, 1006-1010, 2007; Kiener, et al., Arthritis Rheum. 60, 1305-1310, 2009). More recently, it has been shown that targeting the tyrosine phosphatase PTPRS on the cell surface of SFs protects cartilage in RA mice (Svensson, et al., Sci Adv. 6, eaba 4353, 2020). In addition, targeting MMPs has also been studied for decades, with specific MMP inhibitors such as Trocade demonstrating protective effects against RA and OA in animal models (Lewis et al. Br. J. Pharmacol. 121, 540-546 (1997; Brewster, et al., Arthritis Rheum. 41, 1639-1644, 1998). Poor tolerability of these compounds led to failure of clinical trials (Close. Ann. Rheum. Dis. 60 Suppl 3, iii62-7 (2001)). Although some progress has been made to date, it remains relatively difficult to therapeutically inhibit MMPs (Fields. Cells. 8. 2019, doi:10.3390 / cells8090984). Targeting the calnexin-ERp57 complex with antibodies may represent a more attractive approach, as it is expected to be less toxic.

[0436] Overall, our data open perspectives for biomarker discovery and novel therapeutic approaches targeting Cnx with antibodies.More generally, our results suggest that activation of O-glycosylation through GALA is a critical control switch for ECM degradation in synovial fibroblasts, as in cancer cells, pointing to the broad pathological relevance of this pathway.

[0437] Example 4 Materials and Methods - Isolation and characterization of 2G9 monoclonal anti-calnexin antibody Identification and cloning of 2G9 by phage display:

[0438] To select antibodies against calnexin, we screened a human Fab domain library cloned into a phage display library. We isolated clones with affinity to calnexin. We selected clone 2G9 for further characterization.

[0439] Anti-Cnx antibodies were isolated from the HX02 human Fab phage display library (Humanyx Pte Ltd) via in vitro selection. We followed the procedures of biopanning, phage amplification, Fab expression and purification described by de Haard et al. (J Biol Chem 1999;274:18218 - 30;http: / / dx.doi.org / 10.1074 / jbc.274.26.18218;PMID:10373423). Briefly, biopanning was performed with biotinylated human calnexin. In the first two rounds of biopanning, calnexin was immobilized on M280 streptavidin-coated magnetic beads (Life Technologies), and in the third round, biotinylated calnexin was immobilized on neutravidin-coated microplates to avoid separation of streptavidin-magnetic bead binders. Approximately 1000 cfu of phage was used in 1 mL of casein-PBS blocking buffer in the first round, and 1000 cfu of phage was used in the second and third rounds. After three rounds of biopanning, selected Fab clones were expressed in E. coli TG1 cells (Stratagene) and screened for calnexin binders by ELISA.

[0440] Clone 2G9 was identified by DNA fingerprinting, confirmed by DNA sequencing, and converted to IgG1 and IgG4 formats for further analysis. Briefly, Fab was amplified by PCR and cloned in frame with the human Fc region of the respective IgG. Plasmids were then amplified and 2G9 was expressed by transient transfection into 293T cells.

[0441] ELISA Assay:

[0442] 100ng of recombinant CNX-His protein was diluted in 50mM sodium carbonate buffer, pH 9.6, and coated on a 96-well maxisorp plate (Nunc) overnight at 4°C. Similarly, 0.5ug / ml of bovine serum albumin (BSA) was coated on a maxisorp plate (Nunc) overnight at 4°C for negative control wells. The coated wells were then washed three times with 0.05% PBS-Tween (PBST) and PBS, respectively. The wells were blocked with 5% fetal bovine serum (FBS) for 1 hour at 37°C. After blocking, primary antibodies were added to the wells and incubated at 37°C for 2 hours. The wells were washed three times with PBST and PBS, respectively. Horseradish peroxidase (HRP)-conjugated secondary antibodies in 5% FBS-PBST were added to the wells and incubated at 37°C for 1 hour. The wells were washed three times with PBST and PBS, respectively. To detect HRP, TMB substrate was added to the wells and incubated for 5 min, after which the reaction was stopped by adding 1 M HCl and OD450 was measured on a Tecan microplate reader.

[0443] ECM Degradation Assay:

[0444] First, gelatin (G1393, Sigma) was conjugated to 5-carboxy-X-rhodamine succinimidyl ester (C-6125, ThermoFisher Scientific). Sterile coverslips were coated with gelatin for 20 min and then fixed with 0.5% glutaraldehyde (15960, Electron Microscopy Sciences) for 40 min. After washing, 0.5 mg ml -1 A layer of type I collagen (354236 rat tail, Corning) was coated onto these gelatin coverslips and incubated at 37°C for 4 h before seeding cells. Cells were incubated overnight before fixation and staining. A total of 10–30 images were acquired for each condition, and experiments were performed in three biological replicates. Resolved areas were quantified using ImageJ and normalized by the number of nuclei in each image.

[0445] Example 5 Results - Isolation and characterization of 2G9 monoclonal anti-calnexin antibody 2G9 has high binding affinity and specificity for calnexin:

[0446] An ELISA assay was used to test the binding affinity of 2G9. Purified His-tagged calnexin was adsorbed and coated onto plastic wells, and control wells were coated with BSA. We then incubated 2G9, control human IgG, or commercial monoclonal antibody ab10286 on the plate at 10 micrograms / ml. The assay revealed that both antibodies were highly specific for Cnx, with little binding to the BSA-coated wells (Figure 26). The negative control hIgG did not significantly bind to calnexin.

[0447] To quantitatively compare 2G9 and ab10286, we repeated the ELISA assay using serial dilutions of the antibodies (Figure 27). 2G9 showed saturation of binding at 0.1 micrograms / ml, with a calculated EC50 of approximately 3.10e-6 micrograms / ml. This concentration is lower than that of the commercially available monoclonal antibody ab10286, suggesting higher affinity and / or avidity.

[0448] 2G9 prevents ECM degradation by HUH7 liver cancer cells

[0449] The ability of 2G9 in IgG1 format to prevent ECM degradation was assessed using a degradation assay in which rat tail ECM was overlaid on a thin layer of fluorescently labeled gelatin. Upon activation of ECM degradation, it can be measured that the cells degrade the ECM and then the gelatin, forming dark spots in the gelatin layer (Figure 28A). HUH7 cells were seeded on top and incubated with 2G9 or control antibody for 48 hours. While the control IgG had no effect on or even seemed to stimulate matrix degradation, 2G9 was able to reduce ECM degradation by 90% compared to untreated controls (Figure 28B).

[0450] 2G9 in IgG4 format can block ECM degradation:

[0451] The 2G9 antibody was converted to an IgG4 format. We retested the ECM protection activity using a gelatin overlay assay. As found in the IgG1 format, we also found that 2G9 was able to protect against ECM degradation (Figure 29). This effect was most evident at 20ug / ml.

[0452] Sequence Listing [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

[0453] References 1. D.J.Gill,J.Chia,J.Senewiratne,F.Bard,Regulation of O-glycosylation through Golgi-to-ER relocation of initiation enzymes.J.Cell Biol.189,843-858(2010). 2. D.J.Gill,K.M.Tham,J.Chia,S.C.Wang,C.Steentoft,H.Clausen,E.A.Bard-Chapeau,F.A.Bard,Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasiveness.Proc.Natl.Acad.Sci.U.S.A.110,E3152-61(2013). 3. F.Bard,J.Chia,Cracking the Glycome Encoder:Signaling,Trafficking,and Glycosylation.Trends Cell Biol.26,379-388(2016). 4. A.T.Nguyen,J.Chia,M.Ros,K.M.Hui,F.Saltel,F.Bard,Organelle Specific O-Glycosylation Drives MMP14 Activation,Tumor Growth,and Metastasis.Cancer Cell.32,639-653.e6(2017). 5. M.Ros,A.T.Nguyen,J.Chia,S.Le Tran,X.Le Guezennec,R.McDowall,S.Vakhrushev,H.Clausen,M.J.Humphries,F.Saltel,F.A.Bard,ER-resident oxidoreductases are glycosylated and trafficked to the cell surface to promote matrix degradation by tumour cells.Nat.Cell Biol.22,1371-1381(2020). 6. J.Chia,F.Tay,F.Bard,The GalNAc-T Activation(GALA)Pathway:Drivers and markers.PLoS One.14,e0214118(2019). 7. J.Chia,K.M.Tham,D.J.Gill,E.A.Bard-Chapeau,F.A.Bard,ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration.Elife.3,e01828(2014).

Claims

1. 1. A pharmaceutical composition for the treatment or prevention of cartilage degradation in a subject, the pharmaceutical composition comprising an anti-calnexin (anti-CNX) antibody.

2. The pharmaceutical composition of claim 1 , wherein the subject has arthritis.

3. 3. The pharmaceutical composition of claim 2, wherein the arthritis is osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), or arthritis flare.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the cartilage degradation is in one or more joints.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the cartilage degradation is characterized by increased O-glycosylation and / or GALNT activation (GALA) in articular tissue, cartilage tissue and / or synovial fibroblasts.

6. The pharmaceutical composition of any one of claims 1-5, wherein the cartilage degradation is characterized by increased O-glycosylation of calnexin in articular tissue, cartilage tissue, and / or synovial fibroblasts.

7. The pharmaceutical composition of any one of claims 1-6, wherein the cartilage degradation is characterized by increased cell surface expression of calnexin in articular tissue, cartilage tissue, and / or synovial fibroblasts.

8. The pharmaceutical composition of any one of claims 1-7, wherein the cartilage degradation is characterized by degradation of the extracellular matrix (ECM).

9. The pharmaceutical composition of any one of claims 1-8, wherein said cartilage degradation is mediated by said activity of synovial fibroblasts.

10. The antibody against CNX is (a) ECM degradation, or (b) oxidoreductase activity, or (c) disulfide bond reductase activity; The pharmaceutical composition according to any one of claims 1 to 9, which is capable of reducing

11. The pharmaceutical composition of any one of claims 1-10, wherein the antibody is antagonistic.

12. The pharmaceutical composition of any one of claims 1-11, wherein the antibody is monoclonal.

13. The pharmaceutical composition of any one of claims 1-12, wherein the anti-CNX antibody is administered separately or sequentially together with a further therapeutic agent, the further therapeutic agent being an anti-rheumatic drug.

14. The pharmaceutical composition of any one of claims 1-13, wherein the method comprises intravenous, subcutaneous or intraperitoneal administration of the anti-CNX antibody.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment or prevention is in a human subject.

16. The subject is the following steps: i. detecting the level of Tn glycans in a synovial fibroblast sample; ii. comparing the level of Tn glycan in step (i) with the level of Tn glycan in a control synovial fibroblast sample; is determined to be suitable for treatment with the anti-CNX antibody, Wherein an increase in the level of Tn glycan exhibited in the sample compared to the control group indicates suitability for the treatment. A pharmaceutical composition according to any one of claims 1 to 15.

17. A method for treating a subject comprising the steps of: i. detecting the level of CNX cell surface expression in a synovial fibroblast sample; ii. comparing the level of CNX cell surface expression in step (i) with the level of CNX cell surface expression in a control synovial fibroblast sample; is determined to be suitable for treatment with the anti-CNX antibody, wherein an increase in the level of CNX cell surface expression in the sample compared to the control group indicates suitability for the treatment. A pharmaceutical composition according to any one of claims 1 to 16.

18. A method for treating a subject comprising the steps of: i. detecting the level of CNX glycosylation in a synovial fibroblast sample; ii. comparing the level of CNX glycosylation in step (i) with the level of CNX glycosylation in a control synovial fibroblast sample; is determined to be suitable for treatment with the anti-CNX antibody, Wherein an increase in the level of CNX glycosylation exhibited in the sample compared to the control group indicates suitability for the treatment. A pharmaceutical composition according to any one of claims 1 to 17.

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