CN117202931A

CN117202931A - Method

Info

Publication number: CN117202931A
Application number: CN202280011274.3A
Authority: CN
Inventors: F·巴德; L·S·特兰; K·P·里昂; J·Z·H·谢
Original assignee: Agency for Science Technology and Research Singapore; Tan Tock Seng Hospital
Current assignee: Agency for Science Technology and Research Singapore; Tan Tock Seng Hospital
Priority date: 2021-01-21
Filing date: 2022-01-21
Publication date: 2023-12-08

Abstract

Disclosed herein are methods and therapies for treating cartilage degradation, wherein the methods comprise administering a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

Description

Method

The present application claims priority from SG 10202100687X submitted at 21 st 2021 and SG 10202109307T submitted at 25 th 8 st 2021, the contents and elements of which are incorporated herein by reference for all purposes.

Technical Field

The present application relates generally to the field of molecular biology. In particular, the application relates to methods for detecting and treating arthritis.

Background

Arthritis is the leading cause of joint pain and disability, affecting more than one quarter of adults worldwide, with Rheumatoid Arthritis (RA) and Osteoarthritis (OA) being the two most common types of arthritis.

Currently, there is no effective treatment for patients with either osteoarthritis or rheumatoid arthritis. Rheumatoid arthritis patients receive various antirheumatic drugs or immunomodulators (e.g. anti-TNFa and anti-IL-6) directed mainly to the immune system.

One major problem is the immunodeficiency caused by these treatments. Furthermore, these treatments often do not completely prevent disease progression and recurrence. Patients with severe end-stage symptomatic osteoarthritis or rheumatoid arthritis who do not respond to these drugs have to undergo joint replacement surgery, which in itself carries the risk of serious adverse events.

Thus, there is an unmet need for methods of detecting arthritis in a subject and/or treating arthritis in a subject.

Summary of The Invention

In one aspect, the disclosure relates to a method of treating arthritis in a subject, wherein the method comprises administering a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

In another aspect, the present disclosure relates to a method of detecting the presence or absence of arthritis in a subject, wherein the method comprises the steps of: obtaining a synovial fibroblast sample from a subject; detecting the level of Tn antigen/Tn glycan (glycon) in the sample obtained in step (i); comparing the level of Tn antigen/Tn glycan in step (ii) with the level of Tn antigen/Tn glycan in a synovial fibroblast sample from the control group; wherein an increased level of Tn antigen/Tn glycan present in the sample as compared to the control group is indicative of the presence of arthritis; wherein the control group consisted of subjects without arthritis.

In one aspect, the disclosure relates to the use of an anti-calnexin (anti-CNX) antibody in the manufacture of a medicament for treating or preventing cartilage degradation in a subject.

In another aspect, the disclosure relates to an anti-calnexin (anti-CNX) antibody for treating or preventing cartilage degradation in a subject.

In another aspect, the disclosure relates to a method of treating or preventing cartilage degradation, wherein the method involves administering an anti-calnexin (anti-CNX) antibody to a subject.

In some aspects, the subject has osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), an arthritic episode, bursitis, gout, cartilage cancer, fibromyalgia, costal chondritis (costochondritis), cartilage damage, or multiple chondritis.

In some aspects, the subject has arthritis.

In some aspects, the subject has osteoarthritis or rheumatoid arthritis.

In some aspects, the cartilage degradation is in one or more joints.

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

In some aspects, the cartilage degradation is characterized by increased O-glycosylation of calnectin in articular tissue, cartilage tissue, and/or synovial fibroblasts.

In some aspects, the cartilage degradation is characterized by increased cell surface expression of calnectin in articular tissue, cartilage tissue, and/or synovial fibroblasts.

In some aspects, the cartilage degradation is characterized by extracellular matrix (ECM) degradation.

In some aspects, the cartilage degradation is mediated by the activity of synovial fibroblasts.

In some aspects, antibodies to Cnx can reduce:

(a) ECM degradation; or (b)

(b) Oxygen reductase (ox reducing) activity; or (b)

(c) Disulfide reductase activity.

In some aspects, the antibody is capable of reducing cartilage degradation.

In some aspects, the antibody is capable of reducing ECM degradation.

In some aspects, the antibody is capable of reducing ECM degradation activity.

In some aspects, the antibodies are capable of reducing the ECM-degrading activity of CNX: PDIA3 (also known as Cnx/ERp 57).

In some aspects, the antibody is capable of reducing ECM-degrading activity of CNX.

In some aspects, the antibody is capable of reducing ECM degrading activity of a fibroblast.

In some aspects, the antibody is capable of reducing ECM degrading activity of synovial fibroblasts.

In some aspects, the antibody is capable of reducing the activity of an oxidoreductase.

In some aspects, the antibodies are capable of reducing the oxygen reductase activity of CNX: PDIA3 (also known as Cnx/ERp 57).

In some aspects, the antibody is capable of reducing the oxidoreductase activity of CNX.

In some aspects, the antibody is capable of reducing disulfide reductase activity.

In some aspects, the antibodies are capable of reducing disulfide reductase activity of CNX: PDIA3 (also known as Cnx/ERp 57).

In some aspects, the antibody is capable of reducing disulfide reductase activity of CNX.

In some aspects, the antibody is capable of reducing O-glycosylation.

In some aspects, the antibody is capable of reducing O-glycosylation of CNX.

In some aspects, the antibody is capable of reducing glycosylation of CNX.

In some aspects, the antibodies are capable of reducing GALA-mediated O-glycosylation of CNX.

In some aspects, the antibody is capable of reducing CNX activity.

In some aspects, the antibody is capable of reducing PDIA3 activity.

In some aspects, the antibody is capable of reducing PDIA4 activity.

In some aspects, the antibodies are capable of reducing the number or proportion of cells expressing CNX.

In some aspects, the antibodies are capable of reducing the number or proportion of cells expressing PDIA 3.

In some aspects, the antibodies are capable of reducing the number or proportion of cells expressing PDIA 4.

In some aspects, the antibodies are capable of reducing the number or proportion of cells expressing CNX PDIA 3.

In some aspects, the antibody is antagonistic.

In some aspects, the antibody is monoclonal.

In some aspects, the therapeutic agent is administered together, separately or sequentially with a further therapeutic agent, wherein the further therapeutic agent is an antirheumatic.

In some aspects, the method comprises intravenous, subcutaneous, or intraperitoneal injection of an anti-cnx antibody.

In some aspects, the treatment or prevention is in a human subject.

In some aspects, a subject suitable for treatment with an anti-cnx antibody is determined by:

i. detecting levels of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in a synovial fibroblast sample;

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 the synovial fibroblast sample of the control group;

wherein an increase in the level of Tn antigen, tn glycan, CNX expression, CNX glycosylation, and/or ECM degradation present in the sample as compared to the control group is indicative of a suitable treatment.

In one aspect, a method for detecting the presence or absence of a cartilage degradation related disease in a subject is provided, wherein the method comprises the steps of:

i. Detecting levels of Tn antigen, tn glycans, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in a synovial fibroblast sample;

In some aspects, the methods are used to detect the presence or absence of arthritis in a subject.

In some aspects, the methods are used to detect the presence or absence of osteoarthritis or rheumatoid arthritis in a subject.

Numbered statements

1. A method of treating arthritis in a subject, wherein the method comprises administering a therapeutic agent that inhibits the GalNAc-T activation (GALA) pathway.

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

3. The method of any one of the preceding statements, wherein the therapeutic agent inhibits one or more of the following targets of a GalNAc-T activation (GALA) pathway selected from the group consisting of: calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), calnexin-PDIA 3 complex and O-glycosylated matrix metalloproteinase-14 (MMP 14).

4. The method of statement 3, wherein the target of the GALA pathway is Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3).

5. The method of any one of the preceding statements, wherein the therapeutic agent is selected from the group consisting of: antibodies, genes, fusion proteins and expression vectors.

6. The method of statement 5, wherein the therapeutic agent is administered together, separately or sequentially with a further therapeutic agent, wherein the further therapeutic agent is an antirheumatic drug.

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

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

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

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

11. The method of any one of the preceding statements, wherein the one or more therapeutic agents are administered as gene therapy.

12. A method of detecting the presence or absence of arthritis in a subject, wherein the method comprises the steps of:

(i) Obtaining a synovial fibroblast sample from a subject;

(ii) Detecting the level of Tn antigen/Tn glycans 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 the synovial fibroblast sample of the control group;

Wherein an increase in the level of Tn antigen/Tn glycan present in the sample as compared to the control group indicates the presence of arthritis; wherein the control group consisted of subjects without arthritis.

13. The method of any one of the preceding statements, wherein the arthritis is selected from the group consisting of: rheumatoid arthritis, osteoarthritis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA) and arthritis onset/recurrence.

Brief Description of Drawings

The invention will be better understood with reference to the detailed description in conjunction with the non-limiting examples and the accompanying drawings, in which:

FIG. 1 shows that O-glycosylation is enhanced in human samples of rheumatoid arthritis and osteoarthritis, indicating that high levels of O-glycosylation are associated with disease states. Panel A shows representative images of human Tissue Microarrays (TMA) immunohistochemical fluorescent staining of nuclei with Hoechst (upper panel) and immunohistochemical fluorescent staining of O-GalNAcs (Tn glycans) with pea lectin (Vicia Villosa lectin, VVL, lower panel), TMA comprising joint tissue of healthy subjects (normal) and Osteoarthritis (OA), rheumatoid Arthritis (RA) or psoriatic arthritis (PSA) patients. Scale bar, 5 μm. Panel B shows a quantitative plot of Tn glycan levels in a single tissue core. Osteoarthritis patients showed higher levels of O-GalNAc glycans than healthy people, while most rheumatoid arthritis patients and two psoriatic arthritis patients showed higher levels of O-GalNAc glycans than healthy people. The data are mean ± SEM, combined from two different Tissue Microarray (TMA) sections consisting of tissue sections of 21 osteoarthritis patients, 18 rheumatoid arthritis patients, 6 psoriatic arthritis patients and 7 healthy subjects. Single data points represent the raw integrated density of the virusha lectin (VVL) staining normalized to the raw integrated density of the nuclear staining of a single subject. Single data points represent raw integrated density of pea lectin (VVL) staining normalized to raw integrated density of nuclear staining of each subject. The block and whisker plots show all values, with boxes extending from the 25 th percentile to the 75 th percentile, error bars spanning from maximum to minimum, p <0.05, p <0.0001, ns: no significance (one-way ANOVA, kruskal-Wallis test).

Figure 2 shows that in arthritis-induced mice, increased Tn glycan levels correlated with disease severity. This suggests that high Tn glycan levels are associated with disease states. Panel a shows Hemoglobin and Eosin (HE) histological results of synovial tissue obtained from control mice (day 0) or collagen II antibody-injected induced arthritis (CAIA) mice on days 7, 10 and 14. S: a sliding film; b: bone; p: callus (): immersing immune cells into the lubricant film lining; arrow: bone erosion. Panel B shows representative immunofluorescent staining images of pea lectin (VVL; upper panel) and cell nuclei (lower panel), showing that VVL staining increases in callus tissue of the CAIA mice over time from day 7 to day 10. Scale bar, 50 μm. Panels C and D show the results of clinical scores (C) and quantitative determination of total Tn levels in synovium (D) for CAIA mice from day 0 to day 14. In panel C, the data are the average of 4 mice per time point for arthritis scores. In panel D, a single data point represents the average Tn level for a single joint, two joints for each animal's forepaw were calculated. The box plot shows all values, with boxes from the 25 th percentile to the 75 th percentile, and error bands from maximum to minimum. * P <0.05; * P <0.01; NS, not significant (one-way anova).

The data shown in fig. 3 demonstrate that synovial cells of collagen II antibody-induced arthritis (CAIA) mice show signs of activation of the GALA pathway. This suggests that the GALA pathway is active in synovial cells in disease models, making the GALA pathway a therapeutic target. Panels a and B show images of pea lectin (VVL; a) or GALNT2 (B) co-stained with Endoplasmic Reticulum (ER) resident protein, calnexin (CNX), showing a significant increase in Tn glycan levels in arthritis-induced mouse synovial membranes. The scale bar is 50 microns. The magnified image shows VVL staining or GALNT2 enzyme co-localized with CNX in arthritic joints, indicating GALA activation status. Scaling by a factor of 4; s: a sliding film; b: bone; arrows show golgi (untreated mice) or ER staining pattern (CAIA mice) of VVL or GALNT 2.

Fig. 4 shows that Synovial Fibroblasts (SF) are the major cell type exhibiting GALA activation in collagen II antibody-induced arthritis (CAIA) mice, indicating that the GALA pathway is active in synovial cells in the disease model, thereby making the GALA pathway a therapeutic target. The peas lectin (bottom right panel) co-developed with the fibroblast marker vimentin (bottom left panel), the immune cell marker anti-CD 45 (top right panel) and the nucleus (top left panel) showed the relative distribution of immune cells (circles), fibroblasts (rectangles) and their Tn expression levels in pannus tissues (pannus tissues) of CAIA mice. Scale bar, 50 μm.

The images shown in fig. 5 show that synovial lining fibroblasts from both osteoarthritis and rheumatoid arthritis patients show strong GALA activation, indicating that the GALA pathway is active in synovial cells in the disease model, thus making the GALA pathway a therapeutic target. Panel a shows hemoglobin and eosin (H & E) histology results of synovial tissue obtained from rheumatoid arthritis and osteoarthritis patients. ". Times." indicates that immune cells infiltrate the RA synovial lining; SL: and (5) lining the sliding film. Scale bar: 100 μm. Panel B shows representative immunofluorescence images of synovial membranes of osteoarthritis (upper panel) and rheumatoid arthritis (lower panel), showing higher levels of Tn glycans in Synovial Fibroblasts (SF) identified by FAP alpha (indicated by arrows) and lower Tn staining in immune cells identified by CD45 (divided by dashed lines). Scale bar, 50 μm.

The data shown in fig. 6 demonstrate that primary Synovial Fibroblasts (SF) from osteoarthritis and rheumatoid arthritis patients induce strong GALA activation in response to stimulation of arthritis driving cytokines and chondrocyte extracellular matrix (ECM). This suggests that the GALA pathway is responsible for the symptoms and disease progression of arthritis. Panel A shows Fluorescence Activated Cell Sorting (FACS) dot plots showing the high purity (> 90%) of primary synovial fibroblast cultures established from healthy subjects (HCSF), osteoarthritis (OASF) and Rheumatoid Arthritis (RASF) patients. Panel B shows representative images of snail (Helix pomatia) lectin (HPL) staining, showing that Tn glycan levels of osteoarthritis and rheumatoid arthritis synovial fibroblasts were higher than HCSF under basal conditions. The proliferation of arthritis-driven cytokines, including IL1 beta and TNF alpha (CYTO) and chondrocyte extracellular matrix, is accelerated after these cells are stimulated. Scale bar, 20 μm. Panel C shows quantification of snail lectin (HPL) levels in HCSF, OASF and RASF under basal conditions, either with CYTO stimulation alone (no coating) or in combination with either chondrocyte extracellular matrix or type I collagen extracellular matrix. Data are mean ± SEM of two independent experiments. * p <0.05, <0.001, < p <0.0001, ns: no significance (two-way analysis of variance).

Figure 7 shows data indicating that GALA activation in synovial fibroblasts drives degradation of the chondrocyte extracellular matrix, indicating that the GALA pathway is responsible for arthritic symptoms and disease progression. Panel A shows a strategy for inhibiting GALA in synovial fibroblasts, i.e., stable transfection of SW982 synovial fibroblasts with a construct expressing the 2 lectin domains of GALNT2 in Doxycycline (DOX) -induced ER (ER-2 Lec). Panel B shows representative images of results of matrix degrading activity of SW982 cells and SW982 cells expressing ER-2Lec after stimulation with rheumatoid arthritis-associated cytokines (CYTO; IL 1. Beta. And TNF. Alpha.). Arrows show the degraded matrix. Panel C shows a quantitative plot showing reduced matrix degrading activity in GALA-inhibited SW982 cells following CYTO (il1β and tnfα) stimulation. Data are mean ± SEM representing the results of three independent experiments. Each data point represents the total degradation area (μm) per core per well. * P <0.001, p <0.0001 (one-way anova).

FIG. 8 shows data indicating that ER-2Lec expression in synovial fibroblasts inhibits GALA activation, showing the efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Representative images showed that ER-2Lec was expressed predominantly in synovial fibroblasts identified by EGFP positive staining (arrow) in Col6a1Cre ER-2Lec mouse synovial membranes, and that such cells showed reduced VVL staining, indicating inhibition of GALA. In other words, panel B shows VVL staining results, showing that GALA inhibits the loss of O-GalNAcs in articular fibroblasts (EGFP-positive cells) after arthritis induction. B: bone, S: and (5) sliding films. Scale bar, 50 μm.

The images shown in FIG. 9 (panel A) and (panel B) illustrate the effect of inhibiting GALA in reducing the swelling of the paw of a CAIA mouse by ER-2Lec expression, showing the efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Paw thickness analysis showed a reduction in the extent of paw swelling in the Col6a1Cre ER-2Lec mice, both on day 7 post arthritis induction, compared to the Col6a1Cre control mice. Data are mean ± SEM of two independent experiments, n=5 mice per group. * P <0.01 (one-way analysis of variance).

The data shown in fig. 10 demonstrate that inhibition of GALA by ER-2Lec expression in Synovial Fibroblasts (SF) reduces clinical scores in CAIA mice, thereby demonstrating that ER-2Lec expression is clinically effective in treating arthritis. Clinical scores of Col6a1Cre ER-2Lec mice at day 7 post-arthritis induction showed reduced severity of arthritis compared to Col6a1Cre control mice. Data are mean ± SEM of two independent experiments, n=5 mice per group. P=0.09 (non-parametric t-test, mann-wheatstone test).

The images and data shown in fig. 11 demonstrate that inhibition of GALA by ER-2Lec expression in synovial fibroblasts protects CAIA mice from cartilage degradation, demonstrating efficacy of ER-2Lec expression in treating arthritis or alleviating symptoms associated with arthritis. Panels a and B show representative images of untreated Col6a1Cre mice or arthritis-induced Col6a1Cre and Col6a1Cre ER-2Lec mice stained with Alcian Blue (AB) (a) and Sha Fulin-O (SO) (B) at day 7. Panels C and D show the quantitative determination results (scale) of AB (C) and SO (D) positive staining areas. The data show that arthritis-induced cartilage matrix degradation was restored in the GALA-inhibited mice (Col 6aCre ER-2 Lec). Each data point represents the average of the areas of positive articular cartilage staining per square millimeter of three different metacarpophalangeal joints of one animal. Data are expressed as mean ± SEM. * P <0.05, < p <0.01; * P <0.001 (one-way analysis of variance).

The data shown in fig. 12 demonstrate that GALA-activated arthritis triggers O-glycosylation of Calnexin (CNX) in synovial fibroblasts. This shows the role of the GALA pathway in disease states and identifies calnexin as a therapeutic target. Panels a and B show the results of the blot (a) and quantification of the co-immunoprecipitation using VVL lectin (B), showing that O-GalNAc glycosylated Calnexin (CNX) levels were increased in SW982 cells but decreased in Doxycycline (DOX) -induced ER-2 Lec-expressed SW982 cells following stimulation with arthritis-associated Cytokines (CYTO) and chondrocyte extracellular matrix (ECM). Actin served as a load control. In panel B, the data are shown as mean ± SEM, representing 3 independent experiments. * P <0.05; * P <0.01 (one-way analysis of variance).

The data shown in fig. 13 demonstrate that GALA induces cell surface exposure of Calnexin (CNX) in arthritis-stimulated synovial fibroblasts. This shows the role of the GALA pathway in disease states and identifies calnexin as a therapeutic target. Panels a and B show flow cytometry histograms (a) and quantification (B) showing an increase in the proportion of synovial fibroblasts expressing surface Calnexin (CNX) following stimulation by arthritis-induced Cytokines (CYTO) and by chondrocyte extracellular matrix (ECM). In SF cells expressing ER-2Lec, the induction of cell surface CNX was reduced. In B, data are expressed as mean ± SEM representing 2 independent experiments. * P <0.01; * P <0.001 (one-way analysis of variance test).

The data shown in fig. 14 demonstrate that the surface exposure of Calnexin (CNX) in primary synovial fibroblasts from Osteoarthritis (OA) and Rheumatoid Arthritis (RA) patients is enhanced, confirming that calnexin is a therapeutic target for the treatment of arthritis. Panels a and B show flow cytometry histograms (a) and quantification (B) showing a higher proportion of surface CNX positive osteo-or rheumatoid arthritis synovial fibroblasts (OASF) compared to Healthy Control Synovial Fibroblasts (HCSF) under basal conditions or arthritis driving Cytokines (CYTO) and chondrocyte extracellular matrix (ECM) stimulation. In panel B, the data are shown as mean ± SEM, representing 2 independent experiments. * P <0.05; * P <0.001 (one-way analysis of variance test).

The image shown in fig. 15 indicates that disulfide bonds are present in large amounts in the chondrocyte extracellular matrix (ECM). This suggests that the CNX-PDIA 3 complex (i.e., complex reduction of disulfide bonds) is a therapeutic target. Representative immunofluorescent staining images show staining of collagen fibers containing type III collagen (Col III), type I collagen (Col I) and fibronectin in the chondrocyte extracellular matrix (ECM). Collagen disulfide bonds were chemically reduced with TCEP and detected by staining with OX133 antibodies or Untreated (UT). Scale bar, 5 μm.

The data shown in fig. 16 demonstrate that blocking Calnexin (CNX) can reduce cartilage degradation activity of primary synovial fibroblasts in osteoarthritis patients, thus determining that calnexin is a therapeutic target for the treatment of arthritis. Panels a and B are representative images (a) and quantitative images (B) showing reduced matrix degrading activity in primary fibroblasts isolated from osteoarthritis patients after incubation with anti-CNX antibodies or isotype control antibodies. Data are mean ± SEM of three independent experiments and representative data. Each data point represents the total degradation area (μm) per core per well. * P <0.001 (one-way anova).

Figure 17 shows data for anti-Calnexin (CNX) antibody treatment to reduce CAIA mouse paw swelling, showing the efficacy of anti-calnexin antibody treatment for arthritis. Panel A is a schematic of an antibody treatment regimen. Panel B is a representative photograph of CAIA mice treated or untreated with anti-CNX antibodies on day 10. Panel C shows a paw thickness measurement plot showing the reduction in paw swelling in the CAIA mice following injection of anti-CNX antibody. Data represent mean ± SEM, n=4-5 mice per group. * P <0.05 (one-way analysis of variance test).

Figure 18 shows a graph of the reduction of the severity of CAIA mouse arthritis for treatment with anti-Calnexin (CNX) antibodies, thus showing the efficacy of treatment of arthritis with anti-calnexin antibodies. (shown) clinical scores of CAIA mice on day 10 after treatment with isotype control antibodies or anti-CNX antibodies. Data are mean ± SEM, n=4-5 mice per group. The P-value is estimated by a non-parametric t-test (mann-wheatstone test).

Fig. 19 shows data for anti-Calnexin (CNX) antibody treatment to protect CAIA mice from cartilage degradation, thus showing the efficacy of anti-calnexin antibody treatment for arthritis or prevention of exacerbation of arthritis or symptoms of arthritis. Panels a and B show representative histological images showing Alcian Blue (AB) (a) and Sha Fulin-O (SO) (B) staining of CAIA mice treated with isotype control or anti-CNX antibodies (arrow bars). Panels C and D show quantitative plots showing increased AB and SO staining areas for CAIA mice treated with anti-CNX antibodies compared to CAIA mice treated with isotype control antibodies. Single data points represent the average of the area of positive articular cartilage staining per square millimeter of metacarpophalangeal joint of one animal, n=4-5 mice per group. Data are expressed as mean ± SEM. * P <0.05; * P <0.01 (mann-wheatstone test).

The images shown in fig. 20 demonstrate the accumulation of anti-Calnexin (CNX) antibodies in CAIA mouse synovial membranes. Representative immunofluorescence images show co-staining of VVL and anti-CNX antibodies (indicated by arrows) in CAIA mice injected with anti-CNX antibodies or isotype control antibodies on day 10. This shows that accumulation of anti-CNX antibodies suggests that they are able to bind and target CAIA mouse synovial membrane, whereas in contrast, control isotype antibodies did not bind. This suggests that calnexin is expressed in CAIA mouse synovial cells, and that synovial cells can be specifically targeted by anti-CNX antibodies for treatment. B: bone, S: and (5) sliding films. Scale bar: 50 μm.

The data shown in figure 21 demonstrate the results of GALA target screening and validation of primary arthritic synovial fibroblasts effect. Panel A shows a schematic of high content screening of GALA targets and their blocking patterns. Synovial Fibroblasts (SF) (SW 982) were cultured on quenched fluorescent cartilage matrix components (DQ-collagen) in the presence of a GALA target blocker (antibody or siRNA). The degradation activity of GALA in synovial fibroblasts resulted in an increase in fluorescence signal (control group), whereas synovial fibroblasts treated with blocking agent can inhibit the degradation activity. Panel B shows VVL staining results, showing high expression of Tn glycans outside the golgi apparatus (recognized by the golgi marker megalin (Giantin)), indicating ALA activation in primary synovial fibroblasts isolated from osteoarthritis patients (OASF). Scale bar, 5 μm. Panel C shows a quantitative assay graph showing reduced matrix degrading activity in primary synovial fibroblasts isolated from osteoarthritis patients (OASF) treated with anti-calnexin and anti-MMP 14 antibodies. Each data point represents the ratio of the original integrated density of degraded DQ-collagen to nuclei in each field. Data are mean ± SEM, n=20 fields per group. * P <0.05, < p <0.01; NS, no significance (one-way analysis of variance test).

Figure 22 shows data from mice injected with anti-CNX antibodies or isotype control antibodies. This data shows that the weight changes of both are comparable. A: changes in body weight of CAIA mice treated with isotype control antibodies or anti-CNX antibodies. B and C: representative histological images and quantitative determination results of safranin-O (SO) stained area (B) of CAIA mice treated with isotype control antibodies or anti-CNX antibodies. Single data points represent the average of the area of positive articular cartilage staining per square millimeter of metacarpophalangeal joint of one animal, n=4-5 mice per group. Data are expressed as mean ± SEM. * P <0.05; * P <0.01 (mann-wheatstone test).

FIG. 23 shows O-GalNAc (Tn) glycan tissue analysis in synovial tissue of arthritis patients and arthritis-induced animals. A: representative immunofluorescent staining of O-GalNAc glycans with VVL lectin was performed on human Tissue Microarray (TMA) sections of Osteoarthritis (OA), psoriasis (PSA), rheumatoid Arthritis (RA) and healthy subjects (normal). Magnification 10X, scale bar 500 μm. B: HE histology (top panel) and VVL lectin immunohistochemical staining of collagen II-induced arthritis (CAIA) mice or untreated mice (UNT) at day 7 (bottom panel).

FIG. 24 shows OA synovial tissue analysis and primary synovial fibroblast purification index. A: (left panel) H & E histological properties of synovial tissue obtained from OA patients. Scale bar, 100 μm; SL: a slip film liner. (right panel) representative immunofluorescence image of OA synovium stained with VVL/CD45 and fapα. Lining synovial fibroblasts are recognized by fapα (arrows). Immune cells were identified by CD 45. The boundaries of the backing layer and the lining layer are defined by dash lines. Scale bar, 50 μm. B: FACS plots show high purity (> 90%) of primary SF cultures established from healthy subjects (HCSF), OA (OASF) and RA (RASF) patients.

Figure 25 shows that GALA activation results in d cartilage injury in CAIA mice. A: representative images of safranin-O (SO) (B) staining in untreated Col6a1Cre mice or arthritis induced Col6a1Cre and Col6a1Cre ER-2Lec mice on day 7. Scale bar, 100 μm. B and C: quantitative determination of SO staining thickness (B) and total positive staining area (C). Arrows indicate arthritis-induced degradation of cartilage matrix. Each data point represents the average of the positive areas of articular cartilage staining per square millimeter of three different metacarpophalangeal joints of one animal. P <0.01; * P <0.0001 (one-way anova test).

FIG. 26 shows binding data for monoclonal anti-calnexin antibody clone 2G 9. Data were generated using ELISA assays. This assay showed a high specificity of clone 2G9 and commercial monoclonal antibody ab10286 for Cnx (left bar) with little binding to BSA coated wells (right bar). Negative control hIgG did not bind significantly to calnexin. Data are mean ± SEM of three independent experiments and representative data.

Figure 27 shows further binding data for monoclonal anti-calnexin antibody clone 2G9 based on ELISA experiments performed with serial dilutions of antibodies 2G9 and ab 10286. The data indicate that 2G9 has higher affinity and/or avidity for 2GP than the commercial monoclonal antibody ab 10286.

FIG. 28 shows ECM degradation assay data indicating the ECM degradation capacity of monoclonal anti-calnexin antibody clone 2G 9. Panels a and B show representative images (a) and quantitative plot (B) with 2G9 being able to reduce ECM degradation by 90% compared to untreated control. The data correspond to mean ± SEM of three independent experiments and representative data.

Fig. 29 shows further ECM degradation detection data. The IgG4 form of 2G9 is also capable of blocking ECM degradation. The data correspond to mean ± SEM of three independent experiments and representative data. * P <0.001 (one-way anova).

Detailed description of the invention

GalNAc-T activation (GALA) inhibitors for the treatment of cartilage degradation

Extracellular matrix degradation is a key factor in cartilage degeneration, cartilage degeneration related diseases and joint diseases. Specifically, extracellular matrix degradation can lead to the occurrence, development, or exacerbation of diseases such as osteoarthritis, psoriatic arthritis, rheumatoid arthritis, bursitis, gout, chondrocalcification, fibromyalgia, costal chondritis, osteochondritis, cartilage damage, and polychondritis.

In most tissues, fibroblasts are the key cell types involved in the production of extracellular matrix. However, fibroblasts can also degrade the matrix, reversing this important component of the tissue. How fibroblasts regulate these two opposite activities is not clear.

Synovial Fibroblasts (SF), also known as synovial cells, are typically "double-sided" cells. SF increases the viscosity of synovial fluid by secretion of proteins such as hyaluronic acid and lubricin in healthy humans (Jay et al, J. Rheumatoid.27, 594-600,2000). In arthritic diseases, SF adheres to and degrades cartilage, particularly the extracellular matrix (ECM) of cartilage. Understanding this change in activity during arthritis is an important part of recent research (ospelt. Rmd open.3, e000471,2017).

The galns activation pathway (GALA) regulates ECM degradation in cancer cells through glycosylation of MMP14 and calnexin. The inventors herein demonstrate for the first time that cartilage degradation, cartilage degradation related diseases and joint diseases are also associated with elevated levels of GALA and O-glycosylation.

GALA induces matrix degradation by 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 ER-resident protein, glycosylation of calnexin, which forms a complex with ERp57 (alias PDIA 3) (Ros et al, nat.cell biol.22, 1371-1381.2020). After GALA-glycosylation, a portion of the Cnx-PDIA3 complex is transported to the surface of cancer cells. The complex aggregates and reduces disulfide bonds in the ECM in the invar (Ros et al, nat.cell biol.22, 1371-1381.2020). This reduction is critical for efficient degradation of the ECM (Ros et al, nat. Cell biol.22, 1371-1381.2020).

The inventors have also demonstrated treatment of cartilage degradation with GALA inhibitors. In particular, inhibition of GALA by use of anti-cnx antibodies has been shown to lead to in vivo ECM degradation and alleviation of arthritic symptoms.

The results of the inventors' studies indicate that GALA O-glycosylation is a key switch for pathological cartilage degradation activation of fibroblasts.

Cartilage degeneration

Cartilage degradation can occur through the occurrence and progression of certain diseases (such as arthritis, cartilage calcification and polychondritis) and mechanical injury (such as sports injury). People with cartilage degradation often experience joint pain, stiffness and inflammation, affecting quality of life.

Certain disorders and diseases are associated with cartilage degradation. In addition, cartilage degradation can be caused by the mechanisms of certain disorders/diseases (such as arthritis, chondrocalcification and polychondritis).

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

There are many different diseases associated with cartilage degradation, many of which are difficult to treat. Krishnan and grodzisky (2018) indicate in their publications entitled "cartilage disorders", most cartilage-related disorders result in severe changes in the ECM that can either (1) govern the progression of the disease (e.g., osteoarthritis), (2) lead to the main symptoms of the disease (e.g., dwarfism due to genetic mutations) or (3) act as collateral lesions to pathological processes occurring in other nearby tissues (e.g., osteochondritis and inflammatory joint diseases). It is further pointed out that challenges associated with cartilage disease include a poor understanding of etiology and pathogenesis (Krishnan and Grodzinsky. Matrix biol.2018, month 10; 71-72:51-69).

Cartilage is a avascular, nerve-free, lymph-free connective tissue found in synovial joints, the spine, ribs, outer ear, nose, respiratory tract, and growth plates of children and adolescents. There are three main types of cartilage found in humans: hyaline cartilage, fibrocartilage and elastic cartilage (Wachsmuth et al, histol histopathol.2006, month 5; 21 (5): 477-85). Thus, ECM degradation in cartilage can lead to a number of different diseases.

Cartilage degradation can occur in hyaline cartilage, fibrocartilage, or elastic cartilage. The cartilage degradation-related disease may be a disease involving hyaline cartilage degradation, fibrocartilage degradation or elastic cartilage degradation.

Hyaline cartilage is the most common cartilage type, and also the cartilage type that makes up embryonic bones. In adults, it exists as articular cartilage in the bone ends, rib ends, and nose, throat, trachea, and bronchi of freely movable joints.

Fibrocartilage is a tough tissue that is primarily found at the insertion site of intervertebral discs and ligaments and tendons; it is similar to other fibrous tissues but contains cartilage matrix and chondrocytes.

Elastic cartilage is more flexible than the other two forms of cartilage because it contains elastic fibers in addition to collagen. In the human body, it constitutes the auditory canal and epiglottis of the outer ear, middle ear.

Subjects with cartilage degradation may have arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, gout, chondrocalcification, fibromyalgia, costal chondritis, osteochondritis, cartilage injury, and/or polychondritis.

Subjects with cartilage degradation may suffer from arthritis. Cartilage degradation may be caused by or associated with arthritis in a subject.

Subjects with cartilage degradation may suffer from osteoarthritis or rheumatoid arthritis.

Arthritis is a group of diseases affecting the joints (Barbaur et al, report on morbidity and mortality Morbidity and Mortality Weekly report.65.2016, pages 1052-1056). Rheumatoid Arthritis (RA) and Osteoarthritis (OA) are the two most common types (Murphy and Nagase. Nat. Clin. Practice. Rheumatoid. 4, 128-135.2008). RA is an immune-mediated inflammatory disease in which autoantibodies are produced from reactive B cells and T cells, leading to the formation of intra-articular immune complexes (van Delft et al, j.autoimmunimun.). This drives the recruitment of inflammatory and neutrophil granulocytes, macrophages and other immune cells to this region (Hirota et al, immunity 48,1220-1232.E5.2018; kuo et al, sci. 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-1Beta and TNFalpha that activate synoviocytes. These cytokines have now become therapeutic targets, providing significant relief in most patients. OA is the most common, but less well known for arthritis; it is a gradually evolving disease whose immune components are not prominent (Martel-Pelletier et al, nat Rev Dis primers.2,16072.2016; kapore et al, nat. Rev. Rheumatoid.7, 33-42.2011; goldring and Otere. Curr. Opin. Rheumatoid.23, 471-478.2011). The widely accepted hypothesis is that mechanical injury to cartilage results in low-grade inflammation that mediates progressive loss of cartilage (Kapore et al, nat. Rev. Rheumatol.7,33-42.2011; pap and Korb-Pap. Rheumatol.11, 606-615.2015).

In a healthy synovial joint, the synovium surrounds and isolates the joint cavity, secreting extracellular matrix proteins into the synovial fluid. Synovial fibroblasts are the primary stromal cells of the synovium, interspersed with normal macrophages (Barbur et al, issue. 2016, pages 1052-1056.) SF proliferate during inflammation, forming together with infiltrated immune cells an enlarged synovium called "pannus" (Chuy. Rheumatology.51 journal 5, v 3-11.2012.) pannus activates the articular cavity and proliferates the cartilage degradation (Pap and Pap. Rheumatoid. 11, 606-615.2015), which in particular is proved to be a membrane-mediated layer that mediates the inflammatory response of the local pathogens and molecules, and a small-scale factor (Arthritis Rheumatoid. 58, 3684-3692.2008) in the inflammatory process of the synovium, SF proliferates, together with infiltrated immune cells, forming an enlarged synovium called "pannus" (Chuy. Rheumatology.51 journal 5, v 3-11.2012), pannus (Pap and Pap. Rheumatoid. 11, 606-615.2015), whereas the membrane-lining has been demonstrated to mediate the inflammatory response of the endothelial growth of the endothelial cells (Barbur. 58, 3684-3692.2008), and the endothelial growth of the endothelial cells (Flupulous tissue-supporting enzyme (Fluer. 96, fluer. 96. 35, metrop. 96. 35), the endothelial growth factor (Fluer. 96. 35, metrop. 96. 35) and the inflammatory response of the endothelial growth of the endothelial cells; smolen et al, nature Reviews Disease primers.4.2018.doi: 10.1038/nrdp.2018.1) and cell surface MMP (Lange-Brokar et al, osteoarthris cartilage.20,1484-1499.2012; nygaard and Firestein. Nat. Rev. Rheumatoid.16, 316-333.2020; bauer et al, arthris Res. Ther.8, r171; 2006). MMP14 (MT 1-MMP) is particularly critical for the invasive nature of SFs.

Abnormal matrix degradation is also characteristic of SF in OA (Fuchs et al, osteoarthritis cartilage.12, 409-418.2004). Although immune cells in OA synovium are usually less than RA, they drive cartilage degradation as RA. It is not yet clear what controls the conversion of SF to ECM degradation modes. Variations in gene expression are clearly suspicious, 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 are believed to be a driver of the arthritic SF phenotype (Nakano et al, ann. Rheum. Dis.72, 110-117.2013). Whether these changes are sufficient is still unclear.

The phenotype of SF during arthritis was compared to that of malignant cells. In fact, the growth of cancer cells requires deep remodeling of the ECM in the primary tissue and degradation of the ECM of the primary tissue (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).

Gout is an inflammatory arthritis, also known as gouty arthritis. Gout is the most common inflammatory arthritis with a incidence of 2.5% in the uk. Although it has a cure potential, its therapeutic effect is still not ideal (Abhishaek et al, clin Med (Lond.) february 2017; 17 (1): 54-59). Ultrasonic examination results for gout included double profiling (MSU crystals deposited on hyaline articular cartilage surface). Normal adult articular cartilage consists of a rich ECM, consisting mainly of type II collagen fibers intercalated with type IX, XI collagen. Cartilage defects are often late features of gouty arthropathy, similar to bone erosion, and are localized rather than diffuse. Cartilage damage is generally associated with erosion and is described as occurring in areas of biological mechanical stress. Treatments that prevent, reduce or reverse ECM degradation and/or cartilage loss may benefit gout patients. In some cases, gout in need of treatment is associated with ECM degradation and/or cartilage loss.

Cartilage calcification or cartilage calcification refers to calcification (calcium salt accumulation) in hyaline cartilage and/or fibrocartilage. The accumulation of calcium phosphate in the ankle joint has been found in about 50% of the general population and may be associated with osteoarthritis (Hubert et al BMC Musculoskelet Disord.2018; 19:169). It is commonly found in weight bearing joints such as the hip, ankle and knee joints. The molecular structure of calcium pyrophosphate may trigger an inflammatory reaction. The presence of cartilage calcification is associated with the degradation of cartilage meniscus and synovial tissue. The presence of calcium-containing crystals associated with cartilage calcification has been reported to be associated with a high prevalence of cartilage and meniscus damage (gersting et al, eur radio. Doi:10.1007/s00330-016-4608-8. Electronic publication 2016, 10, 4). Treatment to prevent, reduce or reverse ECM degradation and/or cartilage loss would benefit a chondrocalcified patient. In some cases, the cartilage calcification in need of treatment is associated with ECM degradation and/or cartilage loss.

Fibromyalgia (FM) is a condition characterized by chronic widespread pain and an enhanced pain response to stress. FM is common in rheumatoid arthritis, axial spinal arthritis and psoriatic arthritis and thus may affect the treatment of these rheumatism. FM is also associated with costal chondritis.

Costal chondritis is an inflammation of costal cartilage. The disease usually affects the cartilage at the junction of the upper rib and the sternum or sternum, an area known as the chest rib joint or chest rib junction. Costal chondritis can be caused by mechanical stress, possibly leading to cartilage loss and/or ECM degradation. Thus, a treatment that prevents, reduces or reverses ECM degradation and/or cartilage loss may benefit patients with costal chondritis. In some cases, costal chondritis in need of treatment is associated with ECM degradation and/or cartilage loss.

Exfoliative osteochondritis (OCD or OD) is a disease that forms cracks in articular cartilage and subchondral bone. OCDs generally cause pain during and after exercise. In the late stages of the disease, the affected joints may swell, seize and lock during exercise. Early physical examination may reveal symptoms of pain and late stage may have exudation, tenderness and cracking during joint movement. Treatment to prevent, reduce or reverse ECM degradation and/or cartilage loss would be beneficial to patients with osteoarthritis. In some cases, exfoliative osteochondritis in need of treatment is associated with ECM degradation and/or cartilage loss.

Polychondritis, or Recurrent Polychondritis (RP), is an immune-mediated systemic disease characterized by repeated attacks of cartilage and proteoglycan-rich tissue, including the elastic cartilage of the ear and nose, the hyaline cartilage of the peripheral joints, the fibrocartilage of the axial region and the inflammation of the tracheobronchial cartilage, resulting in progressive anatomical deformation and dysfunction of the affected structure (Borgio et al, biomedicines.2018, month 9; 6 (3): 84). Unilateral or more common bilateral otochondritis is the most common feature of RP that is observed in up to 90% of patients during the course of the disease, with otochondritis being the first symptom in 20% of cases. The onset of the flash, painful red to severe erythema and edema is localized to the cartilage region of the ear, usually without the involvement of the cartilage-free earlobe. The onset of acute inflammation often self-subsides within days or weeks, with varying intervals of recurrence. The long-term consequence of recurrent attacks is that the cartilage matrix is severely damaged and replaced by fibrous connective tissue (Borgio et al, biomedicines.2018, month 9; 6 (3): 84). Thus, treatment to prevent, reduce or reverse ECM degradation and/or cartilage loss may benefit a patient with subchondral inflammation. In some cases, the polychondritis in need of treatment is associated with ECM degradation and/or cartilage loss.

Cartilage damage can occur through a variety of processes, for example, through mechanisms of specific diseases such as arthritis, gout, cartilage calcification, fibromyalgia, costal chondritis, osteochondritis, and polychondritis. Cartilage damage can also be caused by mechanical injury, such as being impacted or overstretched during exercise. In some cases, cartilage damage requires surgical removal of damaged tissue. Isolated cartilage and osteochondral defects of joints such as knee joints are a troublesome clinical problem, especially for young patients, and partial or total knee replacement is rarely recommended. A number of surgical techniques have been developed to address focal cartilage defects. Cartilage treatment strategies are characterized by relief (e.g., chondroplasty and debridement), repair (e.g., drilling and microfracture [ MF ]), or restoration (e.g., autologous chondrocyte implantation [ ACI ], osteochondral autograft [ OAT ], and osteochondral allograft [ OCA ]) (Richter et al, sports health.doi:10.1177/1941738115611350. Electronic publication 2015, 10 month 12). In some cases, treatment to prevent, reduce or reverse ECM degradation and/or cartilage loss may replace surgical treatment of cartilage damaged patients. Treatment to prevent, reduce or reverse ECM degradation and/or cartilage loss may benefit cartilage damage patients.

In some cases, cartilage damage can lead to arthritis (e.g., osteoarthritis). Post-traumatic arthritis (PTA) occurs after an acute direct trauma to the joint. In all cases of osteoarthritis, PTA accounts for about 12%, and chronic inflammatory arthritis patients may also have a history of physical trauma. Treatment with a GALA inhibitor following cartilage injury can prevent the occurrence of arthritis following physical trauma.

Cartilage degradation related diseases

The inventors have demonstrated that inhibitors of GALA, such as anti-cnx antibodies, can be used to treat cartilage degradation related diseases.

The cartilage degradation related disease may be arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis, gout, cartilage calcification, fibromyalgia, costal chondritis, osteochondritis, cartilage injury, or polychondritis.

The cartilage degradation related disease may be arthritis.

The cartilage degradation related disease may be osteoarthritis or rheumatoid arthritis.

Joint diseases

The inventors have demonstrated that GALA inhibitors, such as anti-cnx antibodies, can be used to treat joint diseases.

A joint is defined as the connection between two bones in the skeletal system. Joints may be classified according to the type of tissue present (fibrous, cartilage or synovial) or the degree of movement allowed (synovial arthropathy, binrthrosis or diarthrosis). Joint diseases are thus defined as disorders affecting the connection between two bones in the skeletal system. The definition of a particular joint and related anatomical aspects can be found in "Netter, f.h. (2006). Human anatomical atlas. Philadelphia, pennsylvania: saunders/Elsevier ", which is incorporated by reference in its entirety.

Joint diseases may affect fibro-joints, cartilage joints, or synovial joints.

The fibrous joints are connected by dense connective tissue consisting mainly of collagen. These joints are also referred to as fixed joints or immobilized joints because they do not move. The fibrous joint has no joint cavity and is connected by fibrous connective tissue. The skull is articulated by fibers called sutures.

Cartilage joints are joints where bones are completely connected by cartilage (hyaline cartilage or fibrocartilage). These joints typically allow more movement than fibrous joints, but less movement than synovial joints.

Synovial joints are characterized by a fluid-filled joint cavity within the fibrous capsule. It is the most common type of joint in the human body and comprises several structures that are not found in fibrous or cartilage joints. Three main features of synovial joints are: (i) joint capsule, (ii) articular cartilage, (iii) synovial fluid. The joint capsule surrounds the joint and is continuous with the periosteum of the joint bone. The articular surfaces of the synovial joint (i.e., the surfaces that directly contact each other as the bone moves) are covered by a thin layer of hyaline cartilage. Articular cartilage has two main roles: (i) Minimizing friction during articulation, and (ii) absorbing shock. The synovial fluid is positioned in the joint cavity of the synovial joint. It has three main functions. Synovial joints may include accessory structures such as tendons, ligaments, bursa, and blood vessels.

There are many types of synovial joints. In some cases, the joint disorder is a glide joint, a hinge joint, a pivot joint, an oval joint, a saddle joint, or a ball and socket joint.

A sliding joint, also known as a planar joint or flat joint, is a common synovial joint formed between bones on a flat or nearly flat articular surface. The glide joint allows the bone to slide in any direction along the joint plane, including up and down, side-to-side, and diagonal directions. These joints may also rotate slightly, but are limited by the bone shape and elasticity of the joint capsule around the joint.

A hinge joint is a bone joint whose articular surfaces are shaped relative to each other in a manner that allows movement in only one plane. According to a sorting system, they are considered uniaxial (with one degree of freedom) (Platzer, werner (2008) Color Atlas of Human Anatomy, volume 1). The direction of the distal bone in this motion is rarely in the same plane as the axis of the proximal bone; there is typically some straight line deviation during buckling. The articular surfaces of the bones are connected by a strong collateral ligament. The interphalangeal joints of the hand, the interphalangeal joints of the foot, and the joints between the humerus and the ulna are the best examples of the sheath-like joints. Knee joints and ankle joints are less typical because they allow for slight rotation or lateral movement of the limb at certain locations. The knee joint is the largest hinge joint of the human body.

The pivot joint (the sleeve, rotary or lateral sheath (lateral ginglymus)) is a synovial joint with its axis of motion parallel to the long axis of the proximal bone, typically with a convex articular surface. According to a sorting system, the pivot joint has one degree of freedom (Platzer, werner (2008) Color Atlas of Human Anatomy, volume 1).

An ellipsoidal joint (also known as a condyloid joint) is an oval articular surface, or a condyloid articular surface received into an oval cavity. This allows movement in two planes, allowing flexion, extension, adduction, abduction and circumferential movements, as seen in the wrist.

The saddle joint is a synovial joint, and the opposite faces of the saddle joint are concave-convex mutually. It is present in the thumb, chest, middle ear and heel.

The ball-and-socket joint (or ball joint) is a synovial joint in which the spherical surface of one rounded bone conforms to the cupped recess of the other bone. The distal bone can move about an indefinite number of axes, which have a common center. This allows the joint to move in multiple directions. Joint diseases may affect the articular process, both joints, or both joints. The hip and shoulder joints are ball and socket joints.

A stationary joint is a joint that normally does not allow movement. Both the suture and the iris belong to the motionless joints.

Two joint diseases are joints with limited movement. Examples of such joints are cartilage joints connecting adjacent vertebral bodies.

A two-joint disease joint is a freely movable joint. Sometimes, the two terms articulation and synovial articulation are used interchangeably.

Joint diseases can affect any joint. In some cases, joint disease may affect any mammalian joint. In some cases, joint disease may affect any human joint.

In some cases, the joint disorder affects the hip joint, knee joint, ankle joint, foot, toe, shoulder, elbow, wrist, hand, finger, neck, spine, rib or sacroiliac joint.

The joint disease may be osteoarthritis, psoriatic arthritis, rheumatoid arthritis, bursitis, gout, cartilage calcification, fibromyalgia, costal chondritis, osteochondritis, polychondritis, cartilage injury, tendon injury or ligament injury.

Bursitis is inflammation of the bursa, which is a fluid-filled small sac that serves as a buffer between the bone and muscle, skin or tendon. The type of bursitis depends on the location of the affected bursa. Such soft tissue disorders are common in the shoulders, elbows, buttocks, knees and lower legs. Athletes, elderly people, and people engaged in repetitive motion, such as physical workers and music families, are more prone to bursitis. Bursitis is sometimes mistaken for arthritis because pain may occur at the joint site.

Tendon injury or tendinopathy can be caused in a variety of ways, such as overuse, aging, wear, or mechanical injury. Tendon injury may be tendinitis or tendinopathy. Tendinitis refers to inflammation of the tendon, and tendinosis is associated with tearing of the internal and surrounding tissues of the tendon. Tendon injury may be tendon strain, tendon sprain, tendon tear, partial tendon rupture, or complete tendon rupture. Treatment to prevent, reduce or reverse ECM degradation and/or tendon loss may benefit tendon injury patients. In some cases, tendon injury in need of treatment is associated with ECM degradation and/or cartilage loss.

Ligament injury can be caused by a variety of means, such as overuse, aging, abrasion, or mechanical injury. Ligament injury may be a ligament strain, ligament sprain, ligament tear, partial fracture of a ligament, or complete fracture of a ligament. Treatment to prevent, reduce or reverse ECM degradation and/or ligament loss may benefit ligament injury patients. In some cases, tendon injury in need of treatment is associated with ECM degradation and/or cartilage loss.

Characterization of disease

Diseases described herein (e.g., cartilage degradation related diseases and joint diseases) can be characterized by a number of features. In some cases, the disease is characterized by ECM degradation. In some cases, the disease is characterized by increased ECM degradation. In some cases, the disease to be treated is characterized by increased expression of calnexin. In some cases, the disease to be treated is characterized by increased calponin glycosylation. In some cases, the disease is characterized by increased O-glycosylation of calnexin. In some cases, the disease is characterized by increased O-glycosylation. In some cases, the disease is characterized by GALNT activation (GALA). In some cases, the disease is characterized by increased GALNT activation (GALA). In some cases, the disease is characterized by inflammation.

ECM is a highly dynamic structure that is continually subjected to a remodeling process in which ECM components deposit, degrade, or otherwise modify. Extracellular matrix (ECM) degradation is mediated by cell surface and secreted proteases, particularly Matrix Metalloproteinases (MMPs), such as, but not limited to, MMP14 and O-glycosylated MMP14.

Calnexin (CNX) is an ER membrane calbindin of 90kDa, forming a complex with PDIA 3. Calnexin is a target of GALA. After glycosylation, the CNX-PDIA3 complex has been shown to be transported to the surface of cancer cells, where it can reduce disulfide bonds in the extracellular matrix (Ros et al, nat.cell biol.22,1371-1381,2020). Reduction of disulfide bonds has been shown to be critical for the effective activity of Matrix Metalloproteinases (MMPs) and thus for the degradation of cancer extracellular matrix.

Where a CNX polypeptide is mentioned, it should be considered as any member of the CNX polypeptide family. Of particular note are CNX polypeptides derived from the following groups of genes: mouse gene ID:12330, rat gene ID:29144, dog Gene ID:403908, cat gene ID:101085686 and horse gene ID:100067402.

for example, the Cnx polypeptide can include a human CNX sequence with the following GenBank accession numbers: 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.

PDIA4 is one of the largest PDI members, consisting of 645 amino acids, with three classical CGHC active sites. Together with ER Hsp70 (BiP), grp94, PDI and ERp29, they form a multiprotein partner complex, which is an ER network, that can bind to unfolded protein substrates (Meunier et al mol. Biol. Cell,13 (2002), pages 4456-4469, 10.1091/mbc. E02-05-0311).

Reference to a PDIA4 polypeptide should be taken to refer to any member of the PDIA4 polypeptide family. Of particular interest are PDIA4 polypeptides derived from the following groups of genes: homo sapiens (Homo sapiens) gene sequence number: 9601; mouse (Mus musculus) gene sequence number: 12304; rat (Rattus norvegicus) gene sequence number: 116598; bovine (Bos taurus) gene sequence number: 415110; horse (Equus bacillus) gene sequence number: 100060535; cat (Felis catus) gene sequence number: 101087568 and canine (Canis lupus familiaris) gene sequence numbers: 482715.

for example, the PDIA4 polypeptide may include a human PDIA4 sequence having the following GenBank accession numbers: np_001358173.1, np_001358174.1 or np_004902.1.

PDIA3, also known as ERp57, is an isomerase. The protein localizes to the Endoplasmic Reticulum (ER) and interacts with the lectin partner calreticulin (calreticulin) and Calnexin (CNX) to regulate folding of newly synthesized glycoproteins. It is believed that the complex of lectin and this protein mediates protein folding by promoting disulfide bond formation in its glycoprotein substrate.

When referring to PDIA3 (ERp 57) polypeptides, it should be taken to refer to any member of the PDIA3 (ERp 57) polypeptide family. Of particular interest are PDIA3 (ERp 57) polypeptides derived from the following group of genes: homo sapiens (Homo sapiens) gene sequence number: 2923, mouse (Mus musculus) gene sequence number: 14827, rat (Rattus norvegicus) gene sequence number: 29468 bovine (Bos taurus) gene sequence number: 281803 horse (Equus calilus) gene sequence number: 100056198 cat (Felis catus) gene sequence number: 101097245 and canine (Canis lupus familiaris) gene sequence numbers: 478279.

for example, an ERp57 polypeptide may include a human PDIA3 (ERp 57) sequence with the following GenBank accession numbers: np_005304.3.

Glycosylation is an enzymatic process that links glycans to proteins or other biological molecules. O-glycosylation is the addition of O-linked glycans to the hydroxyl groups of the side chains of proteins, such as serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residues.

The N-acetylgalactosyltransferase (GALNT) activation (GALA) pathway is regulated by migration of the O-glycosylation initiating enzyme GALNT from the Golgi apparatus to the ER. GALNT catalyzes the formation of Tn glycans, which consist of a single GalNac residue. Tn can be detected by lectin (pea lectin (VVL)). GALNT ER migration significantly increases the total cellular staining level of Tn, with a cytoplasmic rather than perinuclear appearance (Gill, d.j. Et al. Initiation of GalNAc-type O-glycosylation in the endogenous endoplasmic reticulum promotes cancer cell invasiveness. Proc. Natl.110, E3152-61 (2013).

Inflammation is a part of the complex biological response of body tissue to a harmful stimulus (such as a pathogen, damaged cell or stimulus). The function of inflammation is to eliminate the primary cause of cellular injury, remove necrotic cells and damaged tissue due to the primary injury and inflammatory process, and initiate tissue repair. Clinically and epidemiologically, inflammation is often estimated by measuring circulating substances released as a cause or result of an inflammatory response, the most widely used biomarker of inflammation being C-reactive protein (CRP) (Sproston and ashworth. Front immunol.2018; 9:754).

In certain instances, the disease to be treated is characterized by increased expression of calponin, increased expression of PDIA4, increased expression of PDIA3, increased glycosylation of calponin, increased O-glycosylation, GALA, increased GALA, inflammation and/or increased inflammation.

If the disease is characterized by an increase in expression, activity, or another characteristic, the characteristic is increased as compared to a subject not affected by the disease. The increase 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, compared to a subject not affected by the disease.

If the characteristic of the cartilage degradation related disease is an increase in a characteristic, the affected subject has a higher level of the characteristic than a corresponding subject not affected by the characteristic.

If a joint disease is characterized by an increase in a characteristic, the level of the characteristic in the joint of the affected subject is higher than in a corresponding control (e.g., a subject not affected by the joint disease or an unaffected joint).

If an arthritis is characterized by an increase in a characteristic, the level of the characteristic in the joint of the affected subject is higher than a corresponding control (e.g., with a corresponding subject not affected by arthritis or a corresponding joint not affected by arthritis).

If the disease is characterized by increased calponin expression, increased PDIA4 expression, increased PDIA3 expression, increased calponin glycosylation, increased calponin O-glycosylation, increased GALA and/or increased inflammation, the characteristic is increased compared to a corresponding control (e.g., a subject not affected by the disease).

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by increased o-glycosylation, the affected subject has a higher level of o-glycosylation than a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the o-glycosylation 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.

If the disease (e.g., cartilage degradation related disease, joint disease and/or arthritis) is characterized by increased o-glycosylation in joint tissue, cartilage tissue and/or synovial fibroblasts, the level of o-glycosylation in joint tissue, cartilage tissue and/or synovial fibroblasts in the affected subject is higher than in a corresponding control (e.g., a subject not affected by the disease). Or compared to a subject not affected by the disease, the o-glycosylation level in the articular tissue, cartilage tissue and/or synovial fibroblasts 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, 10-fold.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated level of Tn (tenofovir) cells in the cells, the level of Tn (tenofovir) in the affected subject is greater than in a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the Tn (T knoop Weller) 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated cellular level of Tn (tenofovir) in joint tissue, cartilage tissue, and/or synovial fibroblasts, then the affected subject has a higher level of Tn (tenofovir) in joint tissue and/or synovial fibroblasts as compared to a corresponding control (e.g., a subject not affected by the disease). Compared to a subject not affected by the disease, the Tn (T Knowler) level in the joint tissue and/or synovial fibroblasts 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated level of CNX glycosylation, the affected subject has a higher level of CNX glycosylation than the corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the CNX glycosylation 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an increased level of CNX glycosylation in synovial fibroblasts, the level of CNX glycosylation in synovial fibroblasts in the affected subject is higher than in a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the level of CNX glycosylation in synovial fibroblasts 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated level of CNX cell surface expression, the affected subject has a higher level of CNX cell surface expression than a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an increased level of CNX cell surface expression in synovial fibroblasts, the level of CNX cell surface expression in synovial fibroblasts in the affected subject is higher compared to a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the level of CNX cell surface expression in synovial fibroblasts 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.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an increased level of cartilage ECM degradation, the level of ECM degradation in the affected subject is greater than in a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the cartilage ECM degradation 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, 10-fold, 20-fold, 40-fold, 50-fold, 60-fold, 80-fold or 100-fold.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated level of ECM degrading activity, the affected subject has a higher level of ECM degrading activity than a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by the disease, the ECM degradation activity 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, 10 fold, 20 fold, 40 fold, 50 fold, 60 fold, 80 fold, or 100 fold.

If a disease (e.g., cartilage degradation related disease, joint disease, and/or arthritis) is characterized by an elevated level of ECM degrading activity of CNX, CNX/ERP57 and/or synovial fibroblasts, the affected subject has a higher level of ECM degrading activity of CNX, CNX/ERP57 and/or synovial fibroblasts than a corresponding control (e.g., a subject not affected by the disease). Compared to subjects not affected by disease, the ECM degradation activity level of CNX, CNX/ERP57 and/or synovial fibroblasts 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.

When comparing a patient or subject with a disease to a subject not having the disease, an assay or test can be used to determine the level of a relevant feature in both subjects and to determine whether the relevant feature is increased in the subject with the disease. Many detection methods are known in the art to determine the relative level of a biological feature.

The skilled artisan can determine the relative levels of ECM degradation, glycosylation, O-glycosylation, gene expression (e.g., calnexin, PDIA4 and/or PDIA3 expression), protein expression, GALA, and inflammation, among other features, by methods known in the art.

The skilled artisan can also determine whether a feature is present in a disease using methods known in the art. For example, the skilled artisan can determine whether a disease is associated with ECM degradation, glycosylation, O-glycosylation, expression of a particular gene (e.g., expression of calnexin), expression of a particular protein (e.g., calnexin), the presence of GALA, and inflammation using methods known in the art.

The presence and level of ECM degradation can be determined by a variety of methods, such as ECM degradation assays and histological analysis. The skilled artisan is aware of a number of methods for measuring ECM degradation activity, such as those shown in the examples herein. These methods can readily determine whether the Cnx/ERp57 inhibitor is capable of inhibiting ECM degrading activity, cnx/ERp57 ECM degrading activity and/or Cnx ECM degrading activity.

The following procedure is one way for the skilled artisan to detect ECM degradation activity.

Commercial solutions of gelatin (2%) were labeled with 5-carboxy-X-rhodamine succinimidyl ester. The labeled gelatin was then transferred to a sterile coverslip to form a thin layer and fixed with glutaraldehyde. Finally, the rat tail collagen solution can be used to coat a cover slip to form a thin layer of collagen on the gelatin.

The coverslip may then be transferred to a culture vessel and cells with degradation activity (e.g., human hepatocellular carcinoma Huh 7) inoculated and cultured for 48 hours for degradation.

After fixation, the coverslips may be stained with Hoescht for cell counting. The coverslip was then imaged using a confocal microscope. The images were then analyzed using ImageJ. A threshold may be manually defined to show the surface of degraded gelatin and the total area of each field of view measured. At the same time, the number of nuclei can be counted and the final results normalized to the cells in each field of view.

The presence and level of glycosylation can be determined by methods known in the art, such as those used in the examples and commercial detection kits. Methods for detecting and analyzing glycosylated and glycoprotein include: lectin was used for glycan staining, glycan labeling, glycoprotein purification or enrichment, high Performance Liquid Chromatography (HPLC) analysis and mass spectrometry analysis. The skilled person will be aware of these methods.

The presence and level of O-glycosylation can be determined by methods known in the art, such as those used in the examples herein. The marker of O-glycosylation is an increase in the level of Tn (T knooviler) in the cell, tn being the O-sugar formed by the addition of GalNac to Ser or Thr residues. Tn can be detected by Tn binding proteins such as pea lectin (VVL) and snail lectin (HPL) (Gill et al, proc.Natl. Acad. Sci.U.S.A.110, E3152-61 2013).

The presence and level of gene expression (e.g., calnexin expression) can be determined by a variety of different methods known to the skilled artisan. The level of RNA encoding a given gene can be determined by techniques such as RNAseq, RT-PCR and RT-qPCR.

The presence and level of protein expression (e.g., calnexin expression) can be determined by methods well known to the skilled artisan. The level of a given protein/isoform may be determined by antibody-based methods such as Western blotting, immunohistochemistry/cytochemistry, flow cytometry, ELISA, and the like.

The presence and level of GALA can be determined by methods known in the art, such as those employed in the examples herein. One feature of GALA is an increase in the level of Tn (tenofovir) in the cell, which is an O-glycan formed by GalNac added to Ser or Thr residues. Tn can be detected by Tn binding proteins such as pea lectin (VVL) and snail lectin (HPL) (Gill et al, proc.Natl. Acad. Sci.U.S.A.110, E3152-61 2013).

The presence and level of inflammation may be determined by methods known in the art. For example, by analysis of biomarkers, such as cytokines, chemokines, and immune cells. Such analysis may be performed using techniques known in the art, such as mass spectrometry.

Disease medium (e.g. fibroblast, synovial fibroblast)

The diseases described herein may be mediated by the activity of fibroblasts. Fibroblasts are cells that synthesize collagen and ECM. However, fibroblasts can also degrade ECM, reversing this essential component of tissue. Fibroblasts contribute to the health of joints and cartilage. In healthy humans, fibroblasts increase the viscosity of synovial fluid by secreting proteins such as hyaluronic acid and lubricin. In joint and cartilage diseases (such as arthritis), fibroblasts adhere to and degrade the ECM of cartilage.

In certain instances, the diseases described herein may be mediated by Synovial Fibroblasts (SF). SF, also known as synovial cells, is the primary fibroblast in the synovial joint.

The skilled artisan can determine whether the disease is mediated by fibroblast activity by methods known in the art. For example, the skilled artisan is aware of biochemical assays suitable for determining the level of fibroblast activity. The fibroblast activity of a known disease can be tested to determine if the disease is associated with increased fibroblast activity as compared to a suitable control (e.g., unaffected subject), thereby indicating that the disease is mediated by fibroblast activity. Alternatively, animal models of certain diseases may be used to determine whether the disease is mediated by fibroblast activity. For example, a mouse model of a disease of interest may be engineered to deplete a population of fibroblasts (e.g., a synovial fibroblast population). Analysis of the modified model can be used to determine whether the disease is mediated by fibroblast activity, as compared to the unmodified model. For example, a decrease in the disease profile observed after depletion of the fibroblast population would indicate that the disease is mediated by fibroblast activity.

Arthritis treatment

Rheumatoid Arthritis (RA) is an autoimmune disease, while Osteoarthritis (OA) is a degenerative disease, both characterized by degeneration of articular cartilage.

Rheumatoid arthritis is an autoimmune inflammation. The affected joints of rheumatoid arthritis patients are infiltrated with immune cells such as, but not limited to, macrophages, T cells, and B cells. These cells are enriched in the enlarged synovium, known as "pannus". Autoantibodies are found in the serum of rheumatoid arthritis patients, which is consistent with autoimmune diseases. These autoantibodies are thought to form immune complexes within the joint, resulting in activation of immune cells. However, autoantibodies are not detectable in all rheumatoid arthritis patients, suggesting a complex etiology.

Without being bound by theory, in contrast, osteoarthritis is thought to occur independently of inflammation and immune cells, although inflammation may also be present. Osteoarthritis is also thought to result from mechanical damage to cartilage, leading to progressive degeneration of cartilage and ultimately bone degeneration.

Although rheumatoid arthritis and osteoarthritis are different diseases, they share common cellular and molecular features. For example, cartilage extracellular matrix damage is a common pathological feature and typical early manifestation of osteoarthritis and rheumatoid arthritis. The main pathological feature of arthritis is usually destruction of cartilage. This disruption begins with degradation of the extracellular matrix (ECM) of cartilage, which is a cell that synthesizes and maintains cartilage, and eventually leads to the loss of chondrocytes. In addition, bone erosion and bone loss can also result as the condition progresses. Thus, the methods disclosed herein can be used in the treatment of disease onset to prevent cartilage damage until the advanced stage of the disease disclosed herein.

In the present application, evidence of activation of the GALA pathway in human arthritic samples, such as rheumatoid arthritis and osteoarthritis joint tissues, is shown. Synovial fibroblasts, which lead to cartilage degradation, rely on the GALA pathway to activate matrix degradation. This is thought to occur through the cell surface exposed Calnexin (CNX) complex (i.e., CNX-PDIA3 complex), which mediates disulfide bond reduction. In other words, inhibiting disulfide isomerase activity of the CNX-PDIA3 complex can improve cartilage degradation. Genetic inhibition of the GALA pathway in synovial fibroblasts has been shown to prevent matrix degradation in vitro and to alleviate arthritic symptoms in vivo. For example, targeting Calnexin (CNX) using antibodies also produced similar results, indicating a therapeutic approach. Taken together, the results disclosed herein demonstrate that the GALA glycosylation pathway is a key determinant of arthritic pathology.

Treatment of disease with GALA inhibitors

Also disclosed herein are treatments for cartilage disorders, joint disorders, and arthritic conditions such as, but not limited to, rheumatoid Arthritis (RA) and Osteoarthritis (OA). At present, patients with Rheumatoid Arthritis (RA) and Osteoarthritis (OA) are incurable. Patients with rheumatoid arthritis are required to take antirheumatic drugs or immunomodulators (e.g., without limitation, anti-tumor necrosis factor alpha and anti-IL-6) that are directed primarily to the immune system for improving the condition. The main problem is that these treatments induce immunodeficiency. Moreover, these known treatments do not completely prevent disease progression and prevent disease recurrence. For example, if such antirheumatic drugs or immunomodulators were previously ineffective, severe end-stage symptomatic Rheumatoid Arthritis (RA) or Osteoarthritis (OA) patients had to undergo total joint replacement surgery, which would present a high risk of serious adverse events. Thus, alternative pathway-based therapies are an unmet significant medical need for patients with cartilage degeneration, joint disorders, and arthritis-related diseases.

Arthritis-active Synovial Fibroblasts (SF) have been found to exhibit an activation of sugar pathways known as GalNAc-T activation (GALA). GALA activation is present in several human rheumatoid arthritis, most human osteoarthritis samples, and is induced in a mouse model of rheumatoid arthritis. Furthermore, inhibition of GalNAc-T activation (GALA) in synovial fibroblasts may reduce cartilage degradation in vitro. For example, target calponins targeting GalNAc-T activation (GALA) are also effective. In vivo, inhibition of GalNAc-T activation (GALA) in a mouse model of rheumatoid arthritis using a protein inhibitor such as the protein inhibitor ER-2Lec may result in almost complete resistance to cartilage degradation.

Thus, inhibition of GalNAc-T activation (GALA) in synovial fibroblasts has therapeutic uses. In one example, a method of treating arthritis in a subject is disclosed, wherein the method comprises 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 the treatment of arthritis is described. Also disclosed herein are one or more therapeutic agents that inhibit the GalNAc-T activation (GALA) pathway for use in the treatment of arthritis.

In one example of a therapeutic application, a nucleic acid sequence encoding the protein ER-2Lec is transduced or transfected into synovial fibroblasts using, for example, but not limited to, an adeno-associated virus (AAV) or lentiviral transfection system or any other genetic delivery method. In another example, the therapeutic application is inhibition of one or more targets selected from (but not limited to) the group consisting of: calponin (CNX), protein disulfide-isomerase A4 (PDIA 4), protein disulfide-isomerase A3 (PDIA 3), the sulfur reductase activity of the calponin-PDIA 3 complex, MMP14 inhibiting glycosylation (e.g., O-glycosylated MMP 14), or any cell surface-bound GALA-associated protein on the surface of synovial fibroblasts. In one example, such inhibition may be achieved or caused by the use of blocking antibodies, fragments or derivatives thereof.

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

In another example, as disclosed herein, therapeutic targets for cartilage degradation related diseases, joint diseases, and arthritis include targeted Calnexin (CNX), PDIA4, PDIA3, and/or complexes of CNX and PDIA3. In another example, the target is cell surface bound or presented Calnexin (CNX), PDIA4, and/or PDIA3.

In addition to targeting the immune system, it is also contemplated to modulate the synovium itself by, for example, targeting cadherin 11.

The synovium is formed by synovial hyperplasia. Synovium is generally localized to the periphery of the joint area, and during arthritis, this tissue invades the joint area and attacks cartilage. Synovium contains multiple cell types, but key cells involved in extracellular matrix (ECM) degradation are thought to be synovial fibroblasts. While synovial fibroblasts are generally fewer and in a quiescent state, in the case of arthritis, these cells proliferate and lead to extracellular matrix degradation events. It is not clear what induces synovial fibroblasts to initiate extracellular matrix degradation activity.

Extracellular matrix (ECM) degradation is mediated by cell surface and secreted proteases, particularly Matrix Metalloproteinases (MMPs), such as, but not limited to, MMP14 and O-glycosylated MMP14. These proteins are particularly active in solid malignant cells. In fact, studies have shown that the growth of cancer requires deep remodeling of the extracellular matrix of the primary tissue with concomitant degradation and synthesis of new extracellular matrix.

Degradation of the (extracellular) matrix in cancer cells has been shown to be controlled by a sugar pathway known as GALA (GALNT activation). GALA modulates the activity of GALNT, an enzyme that initiates initiation of O-glycosylation and addition of complex carbohydrates on various proteins. Modulation of the GALA pathway (and thus the degradation of extracellular matrix in cancer cells) is mediated by intracellular distribution, with GALNT translocation from the golgi apparatus to the Endoplasmic Reticulum (ER). This migration results in a greater tendency for the various substrates to be O-glycosylated.

One of the exemplary targets for GalNAc-T activation (GALA) is matrix metalloproteinase-14 (MMP 14), whose O-glycosylation has been shown to be critical for its proteolytic activity. Another target of GALA is the endoplasmic reticulum localization protein Calnexin (CNX), which forms a complex with ERp57 (also known as protein disulfide isomerase A3 (PDIA 3)). After glycosylation, the complex is transported to the surface of the cancer cell, where disulfide bonds in the extracellular matrix are reduced. The reduction of disulfide bonds is critical to the effective activity of Matrix Metalloproteinases (MMPs) and the degradation of the extracellular matrix. Thus, galNAc-T activation (GALA) has been shown to coordinate or bind to two enzymatic activities, one proteolytic and one disulfide bond reducing.

Thus, in one example, a therapeutic agent disclosed herein inhibits one or more targets selected from the group consisting of: calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), calnexin-PDIA 3 (CNX. PDIA3) complexes, glycosylated matrix metalloproteinase-14 (MMP 14), including O-glycosylated matrix metalloproteinase-14 (MMP 14), and combinations thereof. In one example, the therapeutic agent inhibits Calnexin (CNX). In another example, the therapeutic agent inhibits PDIA3. In another example, the therapeutic agent inhibits the calnexin-PDIA 3 complex. In another example, the therapeutic agent inhibits calnexin and PDIA3. In one example, the therapeutic agent inhibits glycosylated MMP14.

The experimental data described herein show that arthritis-active synovial fibroblasts show clear signs of GALA activation. GalNAc-T activation (GALA) markers are present in several human rheumatoid arthritis and most human osteoarthritis samples. In a mouse model of rheumatoid arthritis, GALA is induced before symptoms appear. Furthermore, inhibition of GALA in synovial fibroblasts using the protein inhibitor ER-2Lec has been shown to reduce cartilage degradation in vitro. Inhibition of GALA in a mouse model of rheumatoid arthritis using ER-2Lec expressed in synovial fibroblasts has been demonstrated to specifically prevent disease progression in vivo, and thus the role of GALA in disease has also been demonstrated. Finally, in another example, targeting calnectin using antibodies has been demonstrated to result in near complete blockage of cartilage degradation, suggesting that these concepts may be useful in therapy.

Osteoarthritis and rheumatoid arthritis patients share some common pathological features and exhibit overlapping cellular and molecular features. One of the pathological features of arthritis is that the cartilage extracellular matrix (ECM) is destroyed at the onset, followed by irreversible bone loss. Synovial fibroblasts are reported to be a subset of synovial cells that have been demonstrated to disrupt cartilage extracellular matrix by direct invasion or secretion of matrix remodelling enzymes (e.g., without limitation, MMP14, ADAMTS).

The migration of N-acetylgalactosyltransferase (GalNAc-T) from the Golgi apparatus to the Endoplasmic Reticulum (ER) is also known as the GalNAc-T activation (GALA) pathway, which is shown to be a major regulator of tumor extracellular matrix (ECM) remodeling. GalNAc-Ts catalyzes the addition of monosaccharides 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 markers of GALA activation. To detect GALA activation status in human samples of arthritic patients, commercially available rheumatoid arthritis and osteoarthritis patients were evaluated for Tn glycan levels in synovial tissue. Studies showed that rheumatoid arthritis patients showed a trend of increasing median levels of Tn (O-GalNac) glycans compared to healthy subjects (0.34 versus 0.24, p=0.06, fig. 21). The median Tn glycans levels were significantly higher in the osteoarthritis patients (0.59 versus 0.24, p=0.002, fig. 21) and in the rheumatoid arthritis patients (0.59 versus 0.34, p=0.01). Notably, the median levels of Tn glycans were higher in most osteoarthritis patients than in healthy subjects (19/21, 90.4%) (fig. 21). These results indicate that the extent of GALA activation in synovial tissue is high in arthritic patients compared to healthy persons, and that osteoarthritis patients exhibit GALA overactivation.

In vivo preclinical models of collagen antibody-induced arthritis were used to understand the functional contribution of GALA activation in arthritis. Consistent with the findings of the clinical samples described above, elevated Tn levels were observed in arthritis-induced joints (fig. 2B). This induction correlated with the severity of arthritis, peaking at days 7-10 (figures 2C and 2D). It was further shown that Tn glycan staining in the control joint showed localization to golgi complex, while Tn glycan staining in arthritis showed co-localization of Tn glycans with endoplasmic reticulum resident protein Calnexin (CNX). Taken together, these locations are considered to be indicative of the activation state of the GALA (fig. 3, enlarged view). Furthermore, synovial fibroblasts in pannus were identified as the major cell subset in arthritic joints that showed GALA activation (fig. 4).

In order to specifically inhibit GalNAc-T activation (GALA) pathways in synovial fibroblasts, genetically modified mouse strains were engineered with the Cre/LoxP system (fig. 8A) to specifically express GALA inhibitors (two lectin domains of GalNAc-T2) in the endoplasmic reticulum of synovial fibroblasts. Inhibition of the GALA pathway using this approach was observed to restore the integrity of the chondrocyte extracellular matrix and reduce the severity of arthritic disease (fig. 8B-8H). Taken together, these results indicate that the GALA pathway plays an important role in the degradation of the chondrocyte extracellular matrix of arthritic diseases.

The GalNAc-T activation (GALA) pathway regulates glycosylation of several cell surface and secreted proteins. For example, the GALA pathway has been demonstrated to overslycosylate and activate the matrix metalloproteinase MMP14, MMP14 being a driving factor for extracellular matrix degradation in both cancer and rheumatoid arthritis. Upon activation of the GalNAc-T (GALA) pathway, endoplasmic reticulum resident proteins (e.g., but not limited to Calnexin (CNX), PDIA3, PDIA 4) are over-O-glycosylated. In addition, GALA-activated Calnexin (CNX) has been demonstrated to migrate to the cell surface and form complexes with the protein disulfide isomerase A3 (PDIA 3), driving degradation of tumor extracellular matrix components by reducing disulfide bonds of matrix component proteins. Without being bound by theory, MMP14 and PDIA4 are targets for GALA pathway modulation/inhibition, just as Calnexin (CNX) and PDIA3 have been demonstrated to be targets for GALA pathway modulation/inhibition. Therefore, glycosylated MMP14 is also a target for GALA pathway modulation/inhibition. PDIA4 is a PDIA3 paralog that functions similarly to PDIA3, and is also a target for arthritis treatment, similar to PDIA 3. In one example, the arthritis is rheumatoid arthritis.

To take advantage of this concept, a high throughput screening assay was developed to select the most appropriate GALA-associated target for arthritis treatment. In this assay, synovial fibroblasts (cell line SW 982) are cultured with quenched fluorescent cartilage matrix components (fluorescent DQ-collagen in this case) in the presence of a GALA target blocker (such as, but not limited to, antibodies or siRNA). The degradation activity of synovial fibroblasts resulted in an increase in fluorescence signal, which was captured quantitatively using a high resolution imaging system (fig. 21A). Consistent with previous findings in liver tumors, this screen found three GALA-related targets, of which calnexin, PDIA3 and MMP14 were hot targets as positive regulators of cartilage degradation in synovial fibroblasts (fig. 21C).

Through the above screening, GALA targeted inhibitors were identified that inhibited the degradation activity of the chondrocyte extracellular matrix components in synovial fibroblasts. Among the identified inhibitors, a monoclonal antibody clone targeting Calnexin (CNX) was selected for subsequent validation of therapeutic applications.

Thus, in one example, the target of the GALA pathway is Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3).

In a further example, a method of identifying: (a) A molecule that inhibits one or more targets of a GalNAc-T activation (GALA) pathway, the method comprising contacting one or more targets with the molecule and determining whether the molecule binds to and inhibits the one or more targets; (b) An inhibitor of the GalNAc-T activation (GALA) pathway, the method comprising contacting a cell with the inhibitor and detecting a decrease in expression, content or activity of one or more targets of the GalNAc-T activation (GALA) pathway in, on or in the cell; (c) A molecule suitable for or in the treatment, prevention or alleviation of arthritis, the method comprising determining whether the candidate molecule is an agonist or antagonist of Calnexin (CNX), protein disulfide isomerase A3 (PDIA 3), protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and protein disulfide isomerase A3 (PDIA 3), preferably exposing the candidate molecule to CNX, PDIA4, PDIA3 or CNX and PDIA3 or cells expressing CNX, PDIA4, PDIA3 or CNX and PDIA3, to determine whether the candidate molecule is an agonist or antagonist thereof; or (d) an agonist or antagonist of CNX, PDIA4, PDIA3, or CNX and PDIA3, the method comprising administering a candidate molecule to the animal and determining whether the animal exhibits an increase or decrease in expression, content or activity of CNX, PDIA4, PDIA3, or CNX and PDIA 3; 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 (PDIA 3), protein disulfide isomerase A4 (PDIA 4), CNX: PDIA3 complex and O-glycosylated matrix metalloproteinase-14 (MMP 14). In another example, the methods disclosed herein optionally include the step of isolating or synthesizing a molecule, modulator, agonist or antagonist.

In one example, a method of identifying: (a) A molecule that inhibits one or more targets of a GalNAc-T activation (GALA) pathway, the method comprising contacting one or more targets with the molecule and determining whether the molecule binds to and inhibits the one or more targets; (b) An inhibitor of the GalNAc-T activation (GALA) pathway, the method comprising contacting a diseased cell with the inhibitor and detecting a decrease in expression, content or activity of one or more targets of the GalNAc-T activation (GALA) pathway in, on or by the cell, wherein the disease is arthritis; (c) A molecule suitable for use in or for the treatment, prevention or alleviation of arthritis, the method comprising determining whether the candidate molecule is an agonist or antagonist of Calnexin (CNX), protein disulfide isomerase A3 (PDIA 3), protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and protein disulfide isomerase A3 (PDIA 3), preferably exposing the candidate molecule to CNX, PDIA4, PDIA3 or CNX and PDIA3 or cells expressing CNX, PDIA4, PDIA3 or CNX and PDIA3, to determine whether the candidate molecule is an agonist or antagonist thereof; or (d) an agonist or antagonist of CNX, PDIA4, PDIA3, or CNX and PDIA3, the method comprising administering a candidate molecule to an animal model of arthritis and determining whether the animal model of arthritis exhibits an increase or decrease in expression, content or activity of CNX, PDIA4, PDIA3, or CNX and PDIA 3; wherein one or more targets of the GalNAc-T activation (GALA) pathway are selected from the group consisting of: calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), calnexin-PDIA 3 complex and O-glycosylated matrix metalloproteinase-14 (MMP 14); wherein the arthritis is osteoarthritis, psoriatic arthritis or rheumatoid arthritis; wherein the method optionally comprises isolating or synthesizing a molecule, modulator, agonist or antagonist.

In yet another example, the methods disclosed herein are methods of identifying or screening a library of compounds or molecules capable of inhibiting one or more targets of the GalNAc-T activation (GALA) pathway. In another example, a compound or molecule identified using the screening methods disclosed herein will be administered to an animal or subject.

To model the complex physiological features of synovial fibroblast cartilage degradation activity in the context of arthritis, primary synovial fibroblasts (also called OASF cells) were isolated from osteoarthritis patients with symptoms of activated disease. Consistent with previous observations in synovial tissue, cultured OASF cells showed high GalNAc-T pathway activation (fig. 21B) and exhibited highly active dye-quenching (DQ) -collagen matrix degrading activity. As a result, the selected antibody clone was found to inhibit cartilage degradation activity in cultured OASF cells (p=0.001, fig. 21C).

Taken together, these data indicate that the GalNAc-T (GALA) activation pathway affects cartilage degradation in arthritis. Thus, targeting the GALNAc-T (GALA) activation pathway may be a therapeutic target and method for the treatment of arthritis.

Enhancement of O-glycosylation in rheumatoid arthritis and osteoarthritis synovium

Since the GalNAc-T pathway is involved in degradation of cancer cell extracellular matrix, it is thought that this pathway is also involved in the onset of arthritis and cartilage degradation during arthritis. One marker of GalNAc-T pathway activation is an increase in the level of Tn (also known as Tn antigen), which is a monosaccharide O-glycan formed by the addition of GalNAc to serine (Ser) or threonine (Thr) residues. Tn can be detected by, for example, lectins, such as, but not limited to, pea lectin (VVL) and snail lectin (HPL).

Joint tissue microarrays were analyzed by immunofluorescence using pea lectin (VVL) as an anti-stain for DNA. Significant signal enhancement was detected in the samples of osteoarthritis patients and some of the samples of rheumatoid arthritis patients (fig. 1A). Subsequently, integrated fluorescence intensities of pea lectin staining normalized to DNA signal intensity as a cell density marker were quantified (fig. 1B). Although healthy patient samples do not vary much, samples from most, if not all, disease patients show elevated levels of Tn, with some rheumatoid arthritis and osteoarthritis samples showing 5 to 7 fold increases in pea lectin signaling.

To further investigate this correlation, a collagen antibody-induced arthritis (CAIA) -based mouse model of rheumatoid arthritis was used. Briefly, mice were injected with anti-type II collagen antibodies, and Lipopolysaccharide (LPS) three days later. This treatment resulted in the appearance of arthritic symptoms such as, but not limited to, paw swelling and lameness on day 7, continuing until gradually remitting after day 14 (fig. 2A, 2C). Histologically, pannus formation into the joint cavity was observed on day 7. On day 10, pannus volume increased according to immune cell influx, the degree of inflammation was manifested as exacerbation, pannus continued until day 14 (fig. 2A). With immunofluorescent labeling, an increase in Tn staining was observed on day 7, especially in invasive pannus (fig. 2B). Tn staining continued on day 10 and resolved on day 14 (fig. 2b,2 d).

Arthritic synovium showed signs of activation of the GALA pathway

GalNAc-T pathway activation is a well known mechanism leading to elevated Tn levels. One of the markers of GALNAc-T pathway activation is N-acetylgalactosyltransferase (GALNT), particularly GALNT1 and GALNT2, redistribution from the golgi to the endoplasmic reticulum. To verify that the GalNAc-T (GALA) pathway was indeed activated, co-staining of lectin (VVL) with the endoplasmic reticulum marker calnexin was performed (fig. 3A). In the untreated samples, pea lectin staining and calnexin staining were significantly separated, pea lectin was concentrated in a punctate pattern consistent with golgi complex (fig. 3A). In contrast, pea lectin staining and calnectin staining were significantly co-localized in the collagen antibody-induced arthritis (CAIA) samples at day 7. Subsequently, a similar analysis was performed for GALNT2 staining. An increase in GALNT2 staining intensity was observed, indicating an increase in the level of GALNT2 enzyme under disease conditions (fig. 3B). In addition, the distribution pattern of the enzyme was similar to pea lectin, redistributed into an endoplasmic reticulum-like pattern and co-localized with calnexin (fig. 3B).

Thus, in one example, GALNT1 and GALNT2 identifying endoplasmic reticulum localization are employed as targets for rheumatoid arthritis and osteoarthritis treatment. In another example, the target of the GALA pathway is Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3).

In one example, a method of detecting the presence or absence of a cartilage degradation related disease in a subject is disclosed, the method comprising the steps of (i) obtaining a synovial fibroblast sample from the subject; detecting the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in the sample obtained in step (i); comparing the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in step (ii) with the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in the sample as compared to the control group is indicative of the presence of a cartilage degradation related disease; wherein the control group consisted of subjects without cartilage degradation related diseases.

In another example, a method of detecting the presence or absence of arthritis, optionally osteoarthritis or rheumatoid arthritis, in a subject is disclosed, wherein the method comprises the steps of: (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 sample obtained in step (i); comparing the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in step (ii) with the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in the sample as compared to the control group is indicative of the presence of arthritis; wherein the control group consisted of subjects without arthritis.

In one example, a method of detecting the presence or absence of a cartilage degradation related disorder in a subject is disclosed, wherein the method comprises 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 level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in step (i) with the level of Tn antigen/Tn glycan in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen, tn glycan, CNX expression, CNX glycosylation, and/or ECM degradation in the sample as compared to the control group is indicative of suitability for treatment. The control group may consist of subjects without cartilage degradation related diseases.

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

In one example, a method of determining whether a subject is suitable for anti-CNX antibody treatment is (provided) by: (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 level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in step (i) with the Tn antigen/Tn glycan level in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen, tn glycan, CNX expression, CNX glycosylation, and/or ECM degradation in the sample as compared to the control group is indicative of suitability for treatment. The control group may consist of subjects without cartilage degradation, without cartilage degradation related diseases, without arthritis, without osteoarthritis or without rheumatoid arthritis.

In another example, a method of determining whether a subject is suitable for anti-CNX antibody treatment is (provided) by: (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 a synovial fibroblast sample; (iii) Comparing the level of Tn antigen, tn glycan, CNX cell surface expression, CNX glycosylation, and/or ECM degradation in step (ii) with the level of Tn antigen/Tn glycan in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen, tn glycan, CNX expression, CNX glycosylation, and/or ECM degradation in the sample as compared to the control group is indicative of suitability for treatment. The control group may consist of subjects without cartilage degradation, without cartilage degradation related diseases, without arthritis, without osteoarthritis or without rheumatoid arthritis.

In another example, a method of detecting the presence or absence of arthritis in a subject is disclosed, wherein the method comprises the steps of: detecting the level of Tn antigen/Tn glycan in a sample obtained from a subject; comparing the Tn antigen/Tn glycan level detected in the previous step with the Tn antigen/Tn glycan level in the control group synovial fibroblast sample; wherein an increase in the level of Tn antigen/Tn glycan in the sample as compared to the control group indicates the presence of arthritis; wherein the control group consisted of subjects without arthritis.

Synovial fibroblasts are the major cell type exhibiting GALA

The synovium of rheumatoid arthritis is a complex tissue composed of immune cells and synovial fibroblasts. To determine which cell types showed elevated GalNAc-T levels, joint samples of Collagen Antibody Induced Arthritis (CAIA) were combined with pea lectin (VVL) and CD45 (marker of immune cells) or vimentin (adultMarkers of fibroblasts). The Tn positive cells in pannus are mostly vimentin positive (vm ⁺ ) And CD45 negative (CD 45) ⁺ ) (FIG. 4). A set of cd45+ cells was also detected, but these cells were typically located posterior to the anterior part of the pannus. In human samples from osteoarthritis and rheumatoid arthritis patients, fibroblast activation protein alpha (fapα) was used as a marker for synovial lining fibroblasts (fig. 5a,5 b). In both cases, pea lectin (VVL) staining was associated with fapα. In rheumatoid arthritis samples, there was a CD 45-rich compartment below the fapα positive cell layer, but these immune cells were not significantly labeled with pea lectin (fig. 5A, 5B).

Cytokine and ECM stimulation of synovial fibroblasts induces higher GALA levels

To understand how GalNAc-T pathway is activated in vivo, primary human synovial fibroblasts from patients were used. Fluorescence Activated Cell Sorting (FACS) analysis using CD90 and CD45 as synovial fibroblasts and immune cell markers, respectively, determined that the purity of the cell preparation used was greater than 90% (fig. 6A). Synovial fibroblasts were then stimulated with tnfα and IL1beta cytokines, both of which are thought to be driving factors in the progression of rheumatoid arthritis disease. The effect of individual cytokines on GALA activation was limited, but the combination of these two cytokines (labeled CYTO) induced a 2-fold increase in pea lectin (VVL) cell levels, especially in Rheumatoid Arthritis Synovial Fibroblasts (RASF) (fig. 6B, 6C). The staining pattern of pea lectin was clearly similar to that of endoplasmic reticulum, indicating that GALA was activated (fig. 6B). Healthy synovial fibroblasts responded little or no, indicating that the patient's cells had been activated to receive the stimulus (fig. 6B, 6C).

It is questionable whether the extracellular matrix protein of cartilage contributes to the activation of synovial fibroblasts. Indeed, exposure of synovial fibroblasts to chondrocyte extracellular matrix activates GalNAc-T pathways of osteoarthritis and rheumatoid arthritis synovial fibroblasts approximately 3-fold. Also, in contrast, cells of healthy patients did not respond or responded slightly (fig. 6B, 6C). The combination of CYTO (tnfα and IL1beta cytokine) and extracellular matrix had a slight additive effect (fig. 6C). This stimulation appears to be not specific for the chondrocyte extracellular matrix, as similar activation was induced using rat tail extracted collagen I (fig. 6C). That is, the combined stimulation of cytokines and the native chondrocyte extracellular matrix is believed to elicit the greatest GALA response of synovial fibroblasts, which is the strongest inducer, suggesting that treatment with GALA is applicable to early rheumatoid arthritis patients who have not yet developed active inflammatory symptoms. Furthermore, this suggests that the methods of treatment disclosed herein are also applicable to osteoarthritis patients, whose fibroblasts also show GALA activation, despite lower cytokine production levels.

Inhibition of GALA in synovial fibroblasts reduces cartilage extracellular matrix degradation

Since activation of the GalNAc-T pathway drives degradation of extracellular matrix in cancer cells, it was tested whether it was also associated with degradation of chondrocyte extracellular matrix. As previously described, a fluorescent gelatin sandwich assay was used. ER-2Lec is a chimeric protein fused by two lectins of the Endoplasmic Reticulum (ER) targeting sequence and GALNT 2. ER-2Lec inhibited GALA activity by interfering with endoplasmic reticulum specific O-glycosylation (FIG. 7A). A stable ER-2Lec transfectant (transfected cell line) was generated under the control of a doxycycline-inducible promoter system using the SW982 cell line from synovial sarcoma patient. SW982 cells were then stimulated with CYTO (tnfα and IL1beta cytokine) to stimulate extracellular matrix degradation. An increase in extracellular matrix degradation was observed (fig. 7B). However, when ER-2Lec expression was induced, extracellular matrix degradation was reduced 2-fold more (fig. 7B, 7C).

Expression of ER-2Lec in synovial fibroblasts in vivo reduces the incidence of arthritis

To test whether ER-2Lec proteins can alleviate arthritis in vivo, ER-2Lec transgenic mice with Lox cassettes were generated. Subsequently, these mice were crossed with mice expressing Cre under the collagen VI alpha1 (Col 6a 1) promoter (FIG. 8A). Collagen VI is expressed by joint mesenchymal cells, wherein fibroblasts are the cell type with the greatest expression level. This gene hybridization resulted in the expression of ER-2Lec mainly in synovial fibroblasts (Col 6a1Cre ER-2 Lec). Indeed, in the joints, ER-2Lec was expressed mainly in synovial fibroblasts (FIG. 8B).

Subsequently, col6a1Cre ER-2Lec animals were stimulated with Collagen Antibody Induced Arthritis (CAIA). In these animals, although pannus formation into the joint cavity still occurred, the joint cells showed reduced Tn levels, indicating that GALA was inhibited (fig. 8B).

Following collagen antibody-induced arthritis (CAIA), animals expressing Cre alone or simultaneously expressing ER-2Lec were monitored for symptoms of rheumatoid arthritis. Animals expressing ER-2Lec in synovial fibroblasts (Col 6a1Cre ER-2 Lec) showed reduced paw swelling (FIG. 9A). The change in paw thickness between day one and day seven was measured and an increase in paw thickness was observed in the animals of the control group, with some differences between paws (fig. 9B). In contrast, ER-2Lec animals had little swelling. Using the internationally defined arthritis scores in the blind evaluation, a sustained reduction in symptoms was observed in the Col6a1Cre ER-2Lec animals (fig. 10).

Subsequently, histological analysis was performed on day 7 using Alcian Blue (AB) and safranin-O (SO) staining, which are commonly used to reveal articular cartilage portions. Following collagen antibody-induced arthritis (CAIA) induction, pannus formation was observed in both groups of animals with no significant difference in pannus size (fig. 11A, 11B). Notably, there was a significant difference in the appearance of cartilage in comparison. For example, the alcian blue region of the CAIA Col6a1Cre animals was significantly reduced compared to untreated animals, while the alcian blue region of the Col6a1Cre ER-2Lec animals was largely preserved. The ratio of alxin blue positive areas per square millimeter of cartilage was measured and cartilage retention was observed in CAIA treated Col6a1Cre ER-2Lec animals (fig. 1C). Similar data were also obtained after quantification of safranin-O (SO) staining (fig. 11D).

Thus, in one example, expression of the ER-2Lec construct in synovial fibroblasts is described, resulting in GALA inhibition.

GALA activates calnexin glycosylation and surface exposure in synovial fibroblasts

Recently, calnexin (CNX) has been described as a glycosylation target and effector of GALA. Upon glycosylation, calnexin migrates to the cell surface and, together with protein disulfide isomerase A3 (PDIA 3), mediates cleavage of disulfide bonds in extracellular matrix proteins. This reducing activity is critical for the degradation of the matrix by cancer cells. In SW982 synovial fibroblasts, it was found that, following stimulation of cytokines and extracellular matrix, calnexin (CNX) was hyperglycosylated approximately 6-fold (fig. 12A, 12B). This glycosylation proved to be GALA-dependent, as expression of ER-2Lec was able to reduce the glycosylation of calnexin (FIGS. 12A, 12B).

Furthermore, using Fluorescence Activated Cell Sorting (FACS), it was found that the surface expression of calnexin increased after stimulation of these cells with cytoo (tnfα and IL-1 β cytokines) and extracellular matrix (fig. 13A, 13B). Cell surface expression of calnexin is closely related to GALA, since expression of ER-2Lec reduces expression of calnexin (FIGS. 13A, 13B).

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

Subsequently, attempts were made to confirm these results in primary cells obtained from the patient. In healthy synovial fibroblasts, stimulation of cytokines (tnfα and IL-1 β cytokines in this case) and extracellular matrix induced an increase in the proportion of cell surface calnexin-positive cells (fig. 14A). Cell surface calnexin levels of synovial fibroblasts were increased 3-fold in Rheumatoid Arthritis (RASF) or Osteoarthritis (OASF) patients (fig. 14B). In contrast, synovial fibroblasts derived from healthy donors exhibit very limited induction of cell surface calnexin.

Overall, these results indicate that calnexin glycosylation and its cell surface exposure are dependent on GALA, suggesting that calnexin is involved in cartilage extracellular matrix degradation.

Thus, described herein is the inhibition of Calnexin (CNX) and/or protein disulfide-isomerase A3 (PDIA 3) and/or protein disulfide-isomerase A4 (PDIA 4; also known as ERp 72) by inhibitors of Calnexin (CNX) and/or protein disulfide-isomerase A3 (PDIA 3) and/or protein disulfide-isomerase A4 (PDIA 4). It is further noted that PDIA4 is a paralog of PDIA3 and functions similarly to PDIA 3.

The term "inhibitor of Cadherin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) and/or protein disulfide isomerase A4 (PDIA 4)" as used herein refers to any agent or compound capable of inhibiting the expression and/or function of CNX, or protein disulfide isomerase A3 (PDIA 3) or protein disulfide isomerase A4 (PDIA 4), or a complex comprising Cadherin (CNX) and/or protein disulfide isomerase A3 (PDIA 3). Thus, an agent or compound capable of inhibiting PDIA4 or Calnexin (CNX) and/or PDIA3 is referred to as a "protein disulfide isomerase A4 (PDIA 4) inhibitor" or "Calnexin (CNX)/protein disulfide isomerase A3 (PDIA 3) inhibitor" or "protein disulfide isomerase A4 (PDIA 4) or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) antagonist.

In some examples, the protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX), and/or the protein disulfide isomerase A3 (PDIA 3) inhibitor is an inhibitor of Calnexin (CNX). In some examples, the protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX), and/or protein disulfide isomerase A3 (PDIA 3) inhibitor is an inhibitor of protein disulfide isomerase A3 (PDIA 3). In some examples, the Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) inhibitor or protein disulfide isomerase A4 (PDIA 4) inhibitor is an inhibitor of a complex comprising Calnexin (CNX) and protein disulfide isomerase A3 (PDIA 3). In some examples, the protein disulfide isomerase A4 (PDIA 4) or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) inhibitor is an inhibitor of protein disulfide isomerase A4 (PDIA 4).

In some examples, inhibitors of protein disulfide isomerase A4 (PDIA 4) or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) include one or more of the following effects: lowering the expression level (e.g., gene and/or protein expression level) of Calnexin (CNX), protein disulfide isomerase A3 (PDIA 3), protein disulfide isomerase A4 (PDIA 4), and/or complexes comprising Calnexin (CNX) and protein disulfide isomerase A3 (PDIA 3); lowering the level of RNA encoding Calponin (CNX) and/or PDIA3 and/or PDIA 4; reducing/inhibiting transcription of nucleic acids encoding Calnexin (CNX) and/or PDIA3 and/or PDIA 4; increase/promote degradation of RNAs encoding Calponin (CNX) and/or PDIA3 and/or PDIA 4; reducing/inhibiting post-transcriptional processing (e.g., splicing, translation, post-translational processing) of an RNA encoding Cadherin (CNX) and/or PDIA3 and/or PDIA 4; lowering the level of Calponin (CNX), PDIA4 protein, PDIA3 protein and/or protein complexes comprising Calponin (CNX) and PDIA 3; increase/promote degradation of Calnexin (CNX) protein, PDIA4 protein, PDIA3 protein and/or protein complexes comprising Calnexin (CNX) and PDIA 3; reducing/inhibiting interactions between (i) Calnectin (CNX), PDIA4, PDIA3, and/or complexes comprising Calnectin (CNX) and PDIA3, and (ii) Calnectin (CNX), PDIA4, PDIA3, and/or interaction partners comprising complexes of Calnectin (CNX) and PDIA3 (e.g., comprising reducing/inhibiting interactions between Calnectin (CNX) and PDIA 3); lowering the functional level of Calnexin (CNX), PDIA4, PDIA3 and/or complexes comprising Calnexin (CNX) and PDIA 3; reducing/inhibiting extracellular matrix (ECM) degradation caused by Calnexin (CNX), PDIA4, PDIA3 and/or complexes comprising Calnexin (CNX) and PDIA 3; reducing/inhibiting the oxidoreductase activity of Calnectin (CNX), PDIA4, PDIA3 and/or complexes comprising Calnectin (CNX) and PDIA 3; and/or reducing/inhibiting disulfide reductase activity of Calnexin (CNX), PDIA4, PDIA3, and/or complexes comprising Calnexin (CNX) and PDIA 3.

Suitable detection methods may be employed to evaluate the properties described in the preceding paragraph for a given reagent or compound. Detection methods include, but are not limited to, in vitro detection, optional cell-based detection, or cell-free detection. If the assay is a cell-based assay, it may comprise treating the cells to up-regulate the expression and/or activity of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising 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 indicated properties.

Gene expression can be analyzed by methods well known to the skilled artisan. The level of RNA encoding a given gene can be determined by techniques such as RT-qPCR, etc. Expression of the protein may also be determined by other methods well known to the skilled artisan, such as chromatography and Western blotting. For example, the level of a given protein/isoform thereof may be determined using, but not limited to, antibody-based methods including, but not limited to, western blot, immunohistochemistry, immunostaining, immunohistochemistry, cytochemistry, flow cytometry, ELISA, and the like.

The inhibition of expression of a given gene or protein may be less than 1-fold, specifically, less than or equal to 0.99-fold (.ltoreq.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 of the expression level observed in the absence of a protein disulfide isomerase A4 (PDIA 4) or Calponin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) inhibitor. In certain examples, an agent or compound capable of reducing/inhibiting gene or protein expression inhibits said expression by greater than 5%. In another example, gene expression is inhibited to greater than or equal to 10% (. Gtoreq.10%),. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100% of the observed in the uninhibited state. Such inhibition may also be provided using the term "fold change", which is defined as the ratio between two amounts; for the quantities X and Y, then the fold change of Y with respect to X is Y/X. In other words, a change from 30 to 60 is defined as a 2-fold change. This is also referred to as a "2-fold increase". Also, the change from 30 to 15 is referred to as a "2-fold drop".

An assay comprising detecting the level of RNA encoding protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and/or PDIA3 may be employed, for example by RT-qPCR to identify agents or compounds capable of reducing/inhibiting the transcription of a nucleic acid encoding protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) (i.e., capable of reducing the level of RNA encoding protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3)), reducing/inhibiting the transcription of a nucleic acid encoding protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and/or protein disulfide isomerase A3 (PDIA 3), and/or increasing/promoting the degradation of RNA encoding protein disulfide isomerase A4 (PDIA 4), or Calnexin (CNX) and/or PDIA 3. Such assays may include treating cells/tissues with an agent, and subsequently comparing the RNA level encoding protein disulfide isomerase A4 (PDIA 4) or Calnexin (CNX) and/or PDIA3 in the cells/tissues to the RNA level encoding protein disulfide isomerase A4 (PDIA 4) or Calnexin (CNX) and/or PDIA3 in cells/tissues under appropriate control conditions (e.g., without limitation, untreated/vehicle treated cells/tissues).

Assays involving detection of levels of CNX protein, PDIA3 protein, PDIA4 protein, or protein complexes comprising CNX and PDIA3 may be employed, for example, employing antibody/reporter-based methods (western blotting, ELISA, immunohistochemistry, cytochemistry, etc.) to identify agents or compounds described herein that are capable of reducing/inhibiting protein disulfide isomerase A4 (PDIA 4) or Calponin (CNX) and/or protein disulfide isomerase A3 (PDIA 3) protein expression. Such assays may include treating cells/tissues with reagents and then comparing the levels of protein/protein complexes in such cells/tissues to the levels in cells/tissues under appropriate control conditions.

Agents or compounds capable of reducing/inhibiting the interaction between (i) Calnectin (CNX), PDIA4, PDIA3 and/or a complex comprising Calnectin (CNX) and PDIA3 and (ii) Calnectin (CNX), PDIA4, PDIA3 and/or a complex comprising Calnectin (CNX) and PDIA3, such as the interaction between Calnectin (CNX) and PDIA3, may be identified, for example, using assays comprising detecting the level of interaction, such as using antibody/reporter-based methods. The level of interaction between (i) Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising Calnexin (CNX) and PDIA3 and (ii) Calnexin (CNX), PDIA4, PDIA3 and/or complexes comprising CNX and PDIA3 may be analyzed using methods such as, but not limited to, resonance energy transfer techniques (e.g., FRET, BRET) or analysis of interaction relatives. The assay may comprise treating cells/tissues with an agent and then comparing the level of interaction between (i) Calnexin (CNX), PDIA4, PDIA3 and/or complexes comprising Calnexin (CNX) and PDIA3 and (ii) Calnexin (CNX), PDIA4, PDIA3 and/or complexes comprising Calnexin (CNX) and PDIA3 in these cells/tissues with the level of interaction observed in cells/tissues of the appropriate control conditions (e.g., untreated/vehicle treated cells/tissues). Techniques such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, or biological layer interferometry analysis may also be employed to analyze the level of interaction between (i) Calnexin (CNX), PDIA4, PDIA3, and/or complexes comprising Calnexin (CNX) and PDIA3, and (ii) Calnexin (CNX), PDIA4, PDIA3, and/or interaction partners of complexes comprising Calnexin (CNX) and PDIA3 (e.g., interaction between Calnexin (CNX) and PDIA 3). The assay may comprise comparing the level of interaction in the presence of the agent to the level of interaction under appropriate control conditions, e.g. an untreated sample or a sample in the absence of the agent.

The interaction between (i) Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or a complex comprising Calnexin (CNX) and protein disulfide isomerase A3 (PDIA 3) and (ii) Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or a complex comprising Calnexin (CNX) and PDIA3 (e.g., the interaction between Calnexin (CNX) and PDIA 3) may be less than 1-fold, e.g., 0.99-fold, 0.9-fold, 0.85-fold, 0.8-fold, 0.75-fold, 0.7-fold, 0.6-fold, 0.55-fold, 0.5-fold, 0.45-fold, 0.15-fold, 0.5-fold, 0.35-fold, 0.5-fold, 0.7-fold, 0.65-fold, and/or-fold the level of the interaction observed in the non-inhibited state. In some examples, an agent or compound capable of reducing/inhibiting the interaction between (i) PDIA4, calnexin (CNX), PDIA3, and/or a complex comprising Calnexin (CNX) and PDIA3 and (ii) PDIA4, calnexin (CNX), PDIA3, and/or an interaction partner comprising a complex of Calnexin (CNX) and PDIA3, such as the interaction between Calnexin (CNX) and PDIA3, inhibits more than 5% of the interaction observed in the uninhibited state, for example 10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100%.

Agents or compounds capable of reducing/inhibiting the function of protein disulfide isomerase A4 (PDIA 4), calponin (CNX), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising Calponin (CNX), PDIA3 can be identified by assays of related functions. Such assays may include treating cells/tissues expressing PDIA4, CNX, PDIA3 and/or complexes comprising CNX and PDIA3 with reagents and then comparing the level of relevant function to that observed under appropriate control conditions, e.g., untreated/vehicle treated cell/tissue samples, negative controls, or cell/tissue samples treated with inactive compounds or compounds that do not have the desired effect.

Assays involving detection of the levels of functional relatives of Calponin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising CNX and PDIA3 may be employed to identify agents or compounds capable of reducing/inhibiting the function of protein disulfide isomerase A4 (PDIA 4), calponin (CNX), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising Calponin (CNX) and PDIA3, such as the expression and/or activity of genes and/or proteins of one or more proteins whose expression is directly/indirectly up-regulated or down-regulated by the function of CNX, PDIA4, PDIA3 and/or complexes comprising CNX and PDIA 3. Such assays may include treating cells/tissues expressing CNX, PDIA4, PDIA3 and/or complexes comprising CNX and PDIA3 with an agent or compound, and then comparing the level of a functional relator of CNX, PDIA4, PDIA3 and/or complexes comprising CNX and PDIA3 in such cells/tissues to the level of a relator of a related function under the appropriate control conditions disclosed herein.

The function of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), and/or complexes comprising CNX and PDIA3 may be, but is not limited to, extracellular matrix (ECM) degradation, oxidoreductase activity, or disulfide reductase activity. The functional relatives of PDIA4, CNX, PDIA3 and/or complexes comprising CNX and PDIA3 may be, but are not limited to, extracellular matrix (ECM) degradation products, oxidoreductase activity, or disulfide reductase activity.

Assays involving detection of extracellular matrix (ECM) levels or extracellular matrix (ECM) degradation activity may be employed to identify agents capable of reducing/inhibiting degradation of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), and/or extracellular matrix (ECM) comprising complexes of CNX and PDIA3, for example, but not limited to, using antibody/reporter-based methods. Extracellular matrix (ECM) degradation may be measured using methods known to those skilled in the art. The assay may comprise treating cells/tissues expressing CNX, PDIA4, PDIA3 and/or complexes comprising CNX and PDIA3 with an agent and then comparing the extracellular matrix (ECM) degradation level to that observed under appropriate control conditions.

Assays involving detection of relevant activity levels may be employed to identify agents or compounds capable of reducing/inhibiting the oxidoreductase activity and/or disulfide reductase activity of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and/or complexes comprising CNX and PDIA 3. For example, the oxidoreductase activity and/or disulfide reductase activity can be determined using insulin reduction assays known in the art. Assays may include comparing the level of activity associated with that exhibited by CNX, PDIA4, PDIA3 and/or complexes comprising CNX and PDIA3 in the presence of an agent to that observed in the absence of an agent.

Inhibition of the function of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), and/or complexes comprising CNX and PDIA3 (e.g., without limitation, extracellular matrix (ECM) degradation, oxidoreductase activity, disulfide reductase activity) may be less than 1-fold, such as less than or equal to 0.99-fold (.ltoreq.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, 0.01-fold or 0.01-fold of the level of activity observed in the uninhibited state. In some examples, agents or compounds capable of reducing/inhibiting CNX, PDIA4, PDIA3, and/or complexes containing CNX and PDIA3 (e.g., extracellular matrix (ECM) degradation, oxidoreductase activity, disulfide reductase activity) inhibit the observed relative activity in the uninhibited state by greater than 5%, or less than or equal to 10% (. Gtoreq.10%),. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.96%. Gtoreq.97%. Gtoregtoregtoregtoregtto.97% or 100%).

The protein disulfide isomerase A4 (PDIA 4), or CNX and/or protein disulfide isomerase A3 (PDIA 3) inhibitors of the present disclosure may be any kind of agent or compound having suitable inhibitory activity.

In some examples, the protein disulfide isomerase A4 (PDIA 4) or CNX and/or protein disulfide isomerase A3 (PDIA 3) inhibitor is selected from: a PDIA4 binding molecule, or a CNX binding molecule, a PDIA3 binding molecule, a CNX: PDIA3 complex binding molecule, a molecule capable of reducing the level of CNX, a molecule capable of reducing the level of PDIA3, a molecule capable of reducing the level of CNX: PDIA3 complex.

The term "CNX binding molecule" as used herein refers to a molecule capable of binding to CNX. Similarly, a "PDIA3 binding molecule" or "PDIA4 binding molecule" refers to a molecule capable of binding to protein disulfide isomerase A3 (PDIA 3) or protein disulfide isomerase A4 (PDIA 4). "CNX-PDIA 3 complex binding molecule" refers to a molecule capable of binding to a complex comprising CNX and PDIA 3.

Any suitable assay for detecting binding of a molecule to a factor of interest (CNX, PDIA4, PDIA3 or a complex comprising CNX and PDIA3 in the present disclosure) may be used to identify such binding molecules. Such assays may include detecting the formation of complexes between the relevant factors and the molecule.

The CNX binding molecules, protein disulfide isomerase A4 (PDIA 4) binding molecules, protein disulfide isomerase A3 (PDIA 3) binding molecules, and CNX: PDIA3 binding molecules can be analyzed in appropriate assays to identify antagonists of CNX or PDIA 3. For example, molecules that specifically bind to CNX, PDIA4, PDIA3, and/or complexes comprising CNX and PDIA3 may be evaluated for their ability to inhibit extracellular matrix (ECM) degradation, or to inhibit oxidoreductase and/or disulfide reductase activity.

Molecules that bind to CNX, protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), or the CNX: PDIA3 complex can inhibit the ability of their targets (i.e., CNX, PDIA3, CNX: PDIA3 complex) to interact with an interaction partner. In some examples, the molecule that binds to PDIA3, CNX, PDIA3, or CNX: PDIA3 complex is a competitive inhibitor of interaction between its target and its interaction partner of the target. Binding molecules may occupy or otherwise reduce or block the area required for their target to bind to its interacting partner of the target. The ability of a molecule that binds to CNX, PDIA4, PDIA3, or CNX: PDIA3 complex to inhibit the interaction between its target and its interaction partner of the target can be assessed, for example, but not limited to, by an interaction assay in the presence of one or both interaction partners or after incubation of one or both interaction partners with the relevant binding molecule. An example of a suitable assay for determining whether a given binding agent is capable of inhibiting CNX, PDIA4, PDIA3 or CNX, the interaction between the PDIA3 complex and the interaction partner is a competitive enzyme-linked immunosorbent assay (ELISA).

Calnexin (CNX) -binding molecules, protein disulfide isomerase A4 (PDIA 4) -binding molecules, protein disulfide isomerase A3 (PDIA 3) -binding molecules and CNX-PDIA 3 complex-binding molecules include antigen binding molecules.

An antigen binding molecule capable of binding to Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) binding molecules, protein disulfide isomerase A3 (PDIA 3), or complexes comprising CNX and PDIA3 recognizes a region of the relevant target protein/protein complex that is 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 a cell expressing the protein/protein complex and allowed to bind thereto. In some examples, when the protein/protein complex is expressed on the cell surface, the antigen binding molecule binds to the extracellular region of CNX, PDIA4, PDIA3, or a complex comprising CNX and PDIA 3.

The term "antigen binding molecule" as used herein refers to a molecule capable of binding a given target antigen, a range of which includes antibodies (immunoglobulins) such as, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., but not limited to, bispecific antibodies or trispecific antibodies), and fragments and derivatives thereof (e.g., 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)).

The antigen binding molecules can specifically bind to related targets disclosed herein, including, but not limited to, calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), or complexes comprising CNX and PDIA 3. The term "specific binding" as used herein refers to binding that is selective and distinguishable from non-specific binding of a non-target molecule. The antigen binding molecule is capable of specifically binding to Calnexin (CNX), PDIA4, PDIA3 or a complex comprising CNX and PDIA 3. The antigen binding molecules preferably bind to the relevant target with an affinity and/or duration that is longer than the binding times of other non-target molecules.

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 comprising 2, 3, 4, 6, or 8 polypeptides). In one example, such antigen binding molecules are IgG-like antigen binding molecules comprising two heavy chain polypeptides and two light chain polypeptides.

Antibodies typically comprise 6 Complementarity Determining Regions (CDRs); 3 in the light chain variable region (VL), commonly denoted as LC-CDR1, LC-CDR2 and LC-CDR3, and 3 in the heavy chain variable region (VH), commonly denoted as HC-CDR1, HC-CDR2 and HC-CDR3. These 6 CDRs together define the secondary epitope (paratope) of the antibody, i.e., the portion of the antibody that binds to the target molecule. The VH and VL regions include Framework Regions (FR) on either side of each CDR, providing a scaffold for the Complementarity Determining Regions (CDRs). Conventionally, from the N-terminal to the C-terminal, the heavy chain variable region (VH) comprises the following structure: an N-terminal- [ HC-FR1] - [ HC-CDR1] - [ HC-FR2] - [ HC-CDR2] - [ HC-FR3] - [ HC-CDR3] - [ HC-FR4] -C-terminal; the VL region comprises the following structure: n-terminal- [ LC-FR1] - [ LC-CDR1] - [ LC-FR2] - [ LC-CDR2] - [ LC-FR3] - [ LC-CDR3] - [ LC-FR4] -C-terminal.

The present disclosure relates to antibodies or antibody antagonists to Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3), and/or complexes comprising CNX and PDIA3, which are but not limited to, antibody antagonists to CNX, PDIA4, PDIA3, and/or complexes comprising CNX and PDIA 3. In one example, the antigen binding molecules of the present disclosure include Complementarity Determining Regions (CDRs) and/or heavy chain variable regions (VH) and light chain variable regions (VL) of antibodies that specifically bind to an antigen of interest (e.g., CNX, PDIA4, PDIA3, or a complex comprising CNX and PDIA 3).

Antibodies to a given target antigen may 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 a target antigen, and raising antibodies to a given target antigen by animal immunization. Methods of producing monoclonal antibodies (monoclonal or otherwise) suitable for human therapeutic use or use in the methods disclosed herein include, but are not limited to, employing hybridoma technology, producing xenogeneic antibodies and subsequent humanization, phage display of human antibody genes, and production in transgenic mice with human antibody genes.

Phage display techniques can also be used to identify antibodies that are capable of binding to a given target, target protein or protein complex, as is well known to the skilled artisan.

Antibodies to Calnexin (CNX) include, but are not limited to, monoclonal antibody clone AF18 (Enjejun company, cat# C7617), clone 3H4A7 (Enjejun company, cat# MA 5-15389), clone ARC0648 (Enjejun company, cat# MA 5-35588), clone GT1563 (GeneTex, cat# GTX 629976), clone CANX/1541 (GeneTex, cat# GTX 34446), clone IE2.1C12 (Novus Biologicals, cat# NBP 2-36571), clone 1C2.2D11 (Novus Biologicals, cat# NBP2-36570 SS), clone 2A2C6 (protein technologies, cat# 66903-1-Ig), clone C5C9 (cell signaling technologies, cat# 2679), clone E-10 (SC biotechnology, SC-46669), polyclonal antibodies 32ab 10286 and ab22595 (Ai Bokang), and anti-Cnx antibodies disclosed in CN 101659702A (e.g., antibodies produced by hybridoma CC numbers CC 40). In one example, the antibody to Calnexin (CNX) is ab22595 (Ai Bokang), which is a rabbit polyclonal antibody to human CNX, an epitope starting at the C-terminal 550 amino acids.

Antibodies to protein disulfide isomerase A3 (PDIA 3) include, but are not limited to, monoclonal antibody clone map.erp57 (enzolife sciences), clone CL2444 (sigma-aldrich company, AMAB 90988), clone OTI3E1 (sameizerland technologies, TA 504995), clone OTI3D2 (sameizerland technologies, TA 504990), clone OTI4D7 (sameizerland technologies, TA 505008), and polyclonal antibodies ab13506 and ab13507 (Ai Bokang). In another example, the anti-PDIA 3 antibody is ab13507 (Ai Bokang).

Antibodies raised in non-human animals (e.g., without limitation, mice, rabbits, horses, dogs, donkeys, etc.) against a given target, target protein, or protein complex may be engineered to increase their applicability in human therapy, also known as humanized or humanized antibodies. In other words, a humanized antibody is an antibody whose protein sequence is modified to increase its similarity to naturally occurring antibody variants in humans. For example, one or more amino acids of a monoclonal antibody prepared by animal immunization may be replaced to obtain an antibody sequence more similar to a human germline immunoglobulin sequence, thereby reducing the likelihood of an immune response to an (anti-xenogeneic) antibody in a human subject treated with the antibody. Modifications in the antibody variable domain may be concentrated in the framework regions to retain the antibody epitope pair. For example, the humanization requirement can be circumvented by generating antibodies to specific target proteins/protein complexes in transgenic pattern species expressing human immunoglobulin genes, such that the antibodies generated in such animals are fully human antibodies.

Antigen binding molecules also include, but are not limited to, peptide aptamers, thioredoxins, monomers, anti-calproteins, kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies (e.g., but not limited to, single domain antibodies (sdAbs)), affilins, armRPs, and OBodies. These antigen binding peptides can be identified using methods known in the art, for example, by screening a library of related peptides.

Antigen binding peptides also include, but are not limited to decoy interaction partners of the relevant target antigen. For example, a decoy interaction partner of Calnexin (CNX) may comprise or consist of a CNX binding fragment of protein disulfide isomerase A3 (PDIA 3) or a variant thereof, whereas a decoy interaction partner of PDIA3 may comprise or consist of a PDIA3 binding fragment of CNX or a variant thereof. The decoy interaction partner may be truncated or otherwise modified relative to the polypeptide upon which it is based such that the decoy interaction partner binds to its target to form a nonfunctional complex and/or a complex with a reduced level of functional properties possessed by the complex formed by the target and the interaction partner upon which the decoy interaction partner is based. Thus, in one example, the interaction of the bait interaction partner is to competitively inhibit the interaction between the target and the interaction partner upon which it is based.

Calnexin (CNX) -binding molecules, protein disulfide isomerase A4 (PDIA 4) -binding molecules, protein disulfide isomerase A3 (PDIA 3) -binding molecules, and CNX: PDIA3 complex-binding molecules include, but are not limited to, small molecules that can be identified by screening libraries of small molecules, etc. The term "small molecule" as used herein refers to low molecular weight. Such low molecular weights are typically less than 1000 daltons (< 1000 daltons (Da)). Typically, small molecules have a molecular weight between 300 and 700Da and are an organic compound.

Examples of small molecule inhibitors include, but are not limited to, tenectepeptidase (e.g., for Calnexin (CNX)) and sodium p-hydroxy sulfhydryl benzoate (e.g., for PDIA 3).

Examples of Calnexin (CNX) -binding molecules, protein disulfide isomerase A3 (PDIA 3) -binding molecules, and CNX: PDIA3 complex-binding molecules include aptamers. The nucleic acid aptamer may comprise DNA and/or RNA, and may be single-stranded or double-stranded. In one example, they may include chemically modified nucleic acids in which sugar and/or phosphate and/or base are chemically modified. Such modifications may increase the stability of the aptamer, or make the aptamer more resistant to degradation. In one example, such modifications include, but are not limited to, modifications at the 2' position of ribose. The nucleic acid aptamer may be synthesized chemically, for example, on a solid support. Solid phase synthesis may include phosphoramidite chemistry. In one example, the solid supported nucleotide is debenzylated and then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. The capping is then carried out followed by oxidation of the phosphite triester with an oxidizing agent, typically iodine. The cycle is then repeated to assemble the aptamer.

Molecules capable of reducing the levels of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), protein disulfide isomerase A3 (PDIA 3) and the CNX: PDIA3 complex include, but are not limited to, molecules capable of reducing the expression of genes and/or proteins of PDIA4, CNX and/or PDIA 3. In some embodiments, molecules capable of reducing the level of CNX, PDIA4, PDIA3 or CNX: PDIA3 complex can reduce or prevent the expression of a polypeptide encoded by gene PDIA4, CANX or PDIA3, respectively.

Inhibiting gene or protein expression of a given target gene (e.g., without limitation, gene CANX, PDIA4, or PDIA 3) may include, without limitation, inhibiting gene transcription, inhibiting post-transcriptional processing (e.g., splicing) of RNA transcribed from a gene, reducing stability of RNA transcribed from a gene, promoting degradation of RNA transcribed from a gene, inhibiting translation of RNA transcribed from a gene into a protein, inhibiting post-translational processing of a polypeptide encoded by a gene, reducing stability of a polypeptide encoded by a gene, or promoting degradation of a polypeptide encoded by a gene.

For example, expression of a gene or protein may be inhibited by altering/disrupting the nucleotide sequence of the gene, or by altering/disrupting the nucleotide sequence required for gene expression (e.g., regulatory sequences controlling gene expression). In some examples, inhibiting gene or protein expression comprises altering the nucleotide sequence. Such changes or modifications may be effected, for example, by substitution, deletion or insertion of one or more nucleotides. In particular examples, the disclosure describes inhibiting expression of a gene or protein by deleting all or part of the nucleotide sequence of the associated gene.

In some examples, altering/disrupting the nucleotide sequence may include altering or removing regulatory sequences (e.g., without limitation, promoters or enhancers) of gene transcription, introducing premature stop codons into the sequence of gene transcription, altering the nucleotide sequence to encode truncated and/or nonfunctional gene products, or altering the nucleotide sequence to encode misfolded and/or degraded gene products. The nucleotide sequence may also be disrupted by homologous recombination or modification of the target nucleic acid with a site-specific nuclease (SSN).

In some examples, altering/disrupting a nucleotide sequence to inhibit or prevent gene or protein expression may be referred to as "knocking out" a gene.

Modification by homologous recombination may involve nucleic acid sequence exchange by homologous sequence directed crossover events.

Gene editing is also a method that can be used to edit, alter or disrupt nucleotide sequence expression or function. Enzymes capable of generating site-specific Double Strand Breaks (DSBs) can be engineered to introduce DSBs into target nucleic acid sequences of interest. Double strand breaks can be repaired by error-prone non-homologous end joining (NHEJ), i.e., the two ends of the break are religated, typically with an insertion or deletion of a nucleotide. Alternatively, double strand breaks can also be repaired by highly Homology Directed Repair (HDR), in which a DNA template homologous to the ends of the break site is provided and introduced into the DSB site. Site-specific nucleases (SSNs) can be engineered to produce target nucleic acid sequence-specific double-strand breaks including, but not limited to, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas 9) systems.

The Zinc Finger Nuclease (ZFN) system includes a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a fokl endonuclease domain). The DNA-binding domain can be determined by screening zinc finger arrays that bind to the target nucleic acid sequence. In another example, a transcription activator-like effector nuclease (TALEN) is a restriction enzyme that can be engineered to cleave a specific DNA sequence. TALENs include a programmable DNA binding TALE domain and a DNA cleavage domain (e.g., fokl endonuclease domain). TALE comprises a repeat domain consisting of repeats of 33 to 39 amino acids, which are identical except for the two residues at positions 12 and 13 of each repeat, also known as Repeat Variable Diradicals (RVDs). As known in the art, each RVD determines the binding of a repeat to a nucleotide in a target DNA sequence according to the following relationship: "HD" is bound to C, "NI" is bound to A, "NG" is bound to T, "NN" or "NK" is bound to G. Other examples of gene editing systems include, but are not limited to, CRISPR/Cas9 and related systems such as CRISPR/Cpf1, CRISPR/C2, and CRISPR/C2C3. These systems include endonucleases (e.g., cas9, cpf1, etc.) and single guide RNA (sgRNA) molecules. The sgrnas can be engineered to target endonuclease activity to a nucleic acid sequence of interest.

Examples of CRISPR/Cas9 systems for targeted knockdown of CNX expression are those encoded by the plasmids calnexin CRISPR/Cas9 KO plasmid (h) (SC biotechnology company cat# SC-400154) and calnexin HDR plasmid (h) (SC biotechnology company cat# SC-400154-HDR).

Disruption of the nucleotide sequence with a site-specific nuclease (SSN) can be accomplished in an animal, for example, by injecting the animal with a nucleic acid encoding a component of the relevant site-specific nuclease system. For example, the animal may be injected with one or more vectors comprising nucleic acids encoding components of a site-specific nuclease system in order to target the gene of interest.

Inhibition of gene or protein expression may also be achieved by the use of agents or compounds that reduce gene or protein expression. For example, expression of a gene or protein may be inhibited using an inhibitory nucleic acid, such as, but not limited to, an antisense nucleic acid. Antisense nucleic acids can bind to target nucleic acids by base-complementary pairing. If the target nucleic acid is RNA (e.g., RNA transcribed from a gene of interest), binding of the antisense nucleic acid to the target RNA can promote degradation of the RNA and/or inhibit translation of the RNA. An example of an inhibitory nucleic acid is an antisense oligonucleotide (ASO).

Inhibitory nucleic acids can inhibit the expression of a gene or protein by RNA interference (RNAi). RNAi involves the inhibition of gene expression and translation by targeted neutralization of mRNA molecules. In some examples, the inhibitory nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microrna (miRNA).

Small interfering RNAs (sirnas) are processed from long double-stranded RNAs, and are typically exogenous when found in nature. Micro-interfering RNAs (mirnas) are endogenously encoded small non-coding RNAs. Both siRNA and miRNA inhibit translation of mRNA with a partially complementary target sequence without causing RNA fragmentation and degrade mRNA with a fully complementary sequence. The siRNA is typically double-stranded, and in order to optimize the effect of RNA-mediated inhibition of target gene function, the length of the siRNA molecule is preferably selected to ensure that the RNA-induced silencing complex (RISC) correctly recognizes the siRNA, which RISC mediates the recognition of the mRNA target by the siRNA. DNA sequences encoding mirnas include, but are not limited to, miRNA sequences and near-reverse complement sequences. When the DNA sequence is transcribed into single-stranded RNA molecules, the miRNA sequence and its reverse complementary base pairing to form a partially double-stranded RNA segment.

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

Examples of sirnas targeting PDIA3 include, but are not limited to, sirnas generated based on or using any one of the following target sequences: GGAAUAGUCCCAUUAGCAA (SEQ ID NO: 3), GGGCAAGGACUUACUU AU (SEQ ID NO: 4), AGACCCAAAUAUCGUCAUA (SEQ ID NO: 5), AGGAGUUC UCGCGUGAUG (SEQ ID NO: 6), GAACGAGUAUGAUGAUAAU (SEQ ID NO: 7), GGACAAGACUGUGCAUAU (SEQ ID NO: 8), GGCAAGGACUUACUAUUG (SEQ ID NO: 9), UGAUAAAGAUGCCUCUAUA (SEQ ID NO: 10), and combinations thereof, are exemplary targets for PDIA 3. SEQ ID Nos 3 to 6 refer to L-003674-00-0005, ON-TARGET+human PDIA3 (2923) siRNA-SMART library, 5nmol, and SEQ ID Nos 7 to 10 refer to M-003674-01-0005, siGENOME human PDIA3 (2923) siRNA-SMART library, 5nmol.

Short hairpin RNAs (shrnas) are considered to be more stable than synthetic sirnas. Short hairpin RNAs (shrnas) consist of short inverted repeats separated by a small circular sequence. One of the inverted repeats is complementary to the gene target. In cells, shRNA is treated with endonuclease DICER to form siRNA, thereby degrading target gene mRNA and inhibiting its expression. Short hairpin ribonucleic acids (shRNA) can be produced intracellularly by transcription from vectors, and the like.

Anti-calnexin antibodies to prevent arthritic symptoms in collagen antibody-induced arthritic mice

First, the presence or absence of disulfide bonds in the chondrocyte extracellular matrix is detected using the methods previously described. Briefly, chondrocyte extracellular matrix was reduced with tris (2-carboxyethyl) phosphine (TCEP), then treated with N-ethylmaleimide (NEM), and then treated with anti-OX 133 antibody. As with the liver extracellular matrix, a large number of signals were observed, indicating the presence of a large number of disulfide crosslinks in the chondrocyte matrix (fig. 15).

Next, the effect of anti-calnexin antibodies on extracellular matrix degradation was tested in vitro. Osteoarthritis synovial fibroblasts (OASF) were seeded on chondrocyte matrix coated with fluorescent gelatin, and their extracellular matrix degrading activity was measured as described above. The results indicate that addition of polyclonal anti-calnexin antibodies blocked this degradation activity (FIG. 16).

Collagen antibody-induced arthritis (CAIA) animals were treated with anti-calnexin (anti-CNX) antibodies, and 25 μg (or equivalent to 1.25 mg/kg, calculated as 20 g average body weight per mouse) was injected every two days from day 3 to day 7 after CAIA onset (fig. 17A). The paw thickness of the animals was monitored at intervals and the animals were analyzed for arthritis scores on day 10. Paw swelling was small in animals treated with anti-calnexin antibody compared to control animals treated with isotype antibody (fig. 17B, 17C). While there was some redness of the finger, resulting in an increase in the arthritis score, the average score of the anti-calnexin (anti-CNX) antibody treated animals was half that of the CAIA control animals (fig. 18).

Thus, in one example, the therapeutic agents disclosed herein are antibodies. In another example, the antibody is an anti-calnexin antibody. In another example, the antibody is an anti-PDIA 3 antibody.

At the histological level, the control animals showed a significant decrease in safranin-O (SO) positive cartilage at day 10, with almost vanishing in some areas (fig. 19A, 19B). In contrast, the cartilage structure of anti-calnexin antibody-treated animals was overall preserved (fig. 19A, 19B). Histological analysis showed that, in the same manner as the CAIA animals, the anti-calnexin antibody treated mice, the synovium swelled and invaded the joints, indicating that the initial part of the activation cascade did occur (fig. 19A, 19B). Pannus, however, does not invade cartilage and invade deep cartilage as in animals.

Finally, to verify that the antibody did reach the synovium, it was detected using anti-rabbit IgG antibodies. Signals were observed in synovial cells of animals treated with anti-calnexin antibodies, whereas no signals were seen in animals treated with control rabbit IgG, indicating that anti-CNX antibodies were specifically internalized in synovial fibroblasts (fig. 20).

Overall, these results indicate that inhibition of calnexin inhibits degradation of the chondrocyte extracellular matrix and can lay a foundation for arthritis treatment. Thus, in one example, the therapeutic agents disclosed herein are antibodies. In another example, the antibody is an anti-calnexin antibody.

As shown herein, the activation of the GALA regulatory pathway in arthritic and rheumatoid arthritis synovial fibroblasts is described. Although these diseases are different, they all have a major characteristic of degradation of the extracellular matrix of cartilage. In both cases, the primary driving force for cartilage degradation is fibroblasts from the synovium.

It is not yet clear how synovial fibroblasts transition from a healthy state to an invasive matrix degradation state. In the former, calnexin forms the lining of the synovium and promotes the composition of synovial fluid by secreting various factors. In rheumatoid arthritis, calnexin proliferates and undergoes collective migration, invades the joint cavity, degrades the extracellular matrix of cartilage, and finally invades the cartilage itself. In osteoarthritis, proliferation is not apparent, but synovial fibroblasts remain involved in matrix degradation. The results disclosed herein indicate that activation of GALA is a control switch for both diseases. In fact, GALA shows specific activation in synovial fibroblasts, but not significant in immune cells in inflammatory synovium. Consistent with the critical role of synovial fibroblasts in cartilage degradation, it has been previously reported that GALA controls the ability of cancer cells to degrade extracellular matrix.

How GALA is activated in these cells is not known at present. The results indicate that cytokines driving disease, such as but not limited to IL-1 beta and tnfα, are known to stimulate GALA in synovial fibroblasts in vitro. GALA in fibroblasts in vitro. IL-1. Beta. Has been reported to activate the tyrosine kinase Src. Src is a key regulator of GALA in the golgi. Contact with the extracellular matrix also activates the GALA pathway of synovial fibroblasts, although its mechanism is currently unknown. It may involve integrins and signals emitted by FAK and Src kinases. Osteoarthritis synovial fibroblasts (OASF) and Rheumatoid Arthritis Synovial Fibroblasts (RASF) show similar global methylation patterns, which differ from synovial fibroblasts in healthy subjects. Differences were found 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 (RASF) and osteoarthritis synovial fibroblasts (OASF) may be stimulated to more easily activate the GALA pathway by epigenetic imprinting.

N-acetylgalactosyltransferase (GALNT) is a GALA controlled glycosylase characterized by a lectin domain in addition to the catalytic domain. The lectin domain is shown to be critical for the action of GALA. This was further confirmed by data obtained using ER-2Lec constructs with specificity that resulted in GALNT not possessing sufficient activity in the endoplasmic reticulum as GALA specific inhibitors. Expressing ER-2Lec inhibitors under the collagen VI promoter using the Cre-Lox system can target synovial fibroblasts. Alleviation of symptoms in transgenic animals expressing ER-2Lec clearly demonstrates the importance of this pathway to disease.

Thus, in one example, the therapeutic agents disclosed herein are fusion proteins. In another example, the fusion protein is ER-2Lec. In another example, the fusion protein is ER-2Lec having the sequence set forth in SEQ ID NO. 1.

GALA controls N-acetylgalactosyltransferase (GALNT), a glycosylase that acts on thousands of proteins. One of the important targets of GALA is MMP14, whose glycosylation is critical for proteolytic activity. In rheumatoid arthritis and osteoarthritis, MMP14 and other Matrix Metalloproteinases (MMPs) have been shown to be up-regulated in synovial fibroblasts and play a key role in collagen degradation.

In addition, GALA has recently been shown to activate a complex of calnexin and protein disulfide isomerase A3 (PDIA 3) on the surface of liver cancer cells. The complex is capable of mediating cleavage of disulfide bonds in the extracellular matrix, which is required for proteolytic degradation of the extracellular matrix of the liver. Here, synovial fibroblasts also show the need for Calnexin (CNX) to degrade the chondrocyte extracellular matrix. Indeed, both synovial fibroblast lines and cells derived from osteoarthritis patients have reduced degradability when incubated with anti-calnexin antibodies.

Notably, calnexin is typically an intracellular, endoplasmic reticulum-located protein. Its transport at the cell surface appears to be controlled by GALA-induced glycosylation. Thus, anti-calnexin antibodies are expected to bind predominantly or exclusively to cells of high GALA. However, high GALA levels are not present in healthy tissue. This is believed to help concentrate the anti-CNX antibodies in joint tissue. In other words, high concentrations of anti-CNX antibodies on the cell surface are caused by high levels of GALA, which in turn lead to increased migration of CNX to the cell surface. This means that more CNX can be detected using anti-CNX antibodies. To confirm this, a comprehensive characterization of the biodistribution is required. It is also notable that in these experiments with anti-calnexin antibodies, no adverse effects were shown or detected, e.g., figure 22 shows that the body weight of animals receiving CNX antibodies was unchanged from isotype control antibodies.

Overall, the results disclosed herein demonstrate that in rheumatoid arthritis and osteoarthritis, the GALA pathway is activated in synovial fibroblasts and is critical for the pathological changes of rheumatoid arthritis in mice.

The term "arthritis" as used herein refers to any disease affecting a joint, and generally includes symptoms such as joint pain and stiffness, but is not limited thereto. Other symptoms include, but are not limited to, redness, heat, swelling, and reduced range of motion of the affected joint. In some types of arthritis, organs other than the joints of the subject may also be affected. For the purposes of this disclosure, the methods disclosed herein are applicable 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 activation of GALA suggests that the GALA activation is a related and targetable mechanism for the treatment of any form of arthritis in which GALA is activated.

In the present disclosure, the term "rheumatoid arthritis" or "RA" refers to chronic systemic autoimmune diseases that involve mainly joints. Rheumatoid arthritis causes cytokine, chemokine and metalloprotease mediated damage. It is characterized by symmetrical inflammation of peripheral joints (such as wrist and metacarpophalangeal joints) leading to progressive destruction of joint structures, often accompanied by systemic symptoms. Diagnosis is based on specific clinical, laboratory and imaging features. Treatment includes pharmaceutical, physical, and sometimes surgical procedures. Therapeutic approaches such as, but not limited to, disease modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, immunomodulators, cytotoxic drugs and immunosuppressants (e.g., azathioprine or cyclosporine) help control symptoms and slow disease progression.

In the present disclosure, the term "osteoarthritis" or "OA" refers to chronic joint disease (arthropathy) characterized by the destruction and potential loss of articular cartilage and other joint changes, including hyperosteogeny (hyperosteogeny formation). Symptoms include progressive pain, exacerbation or induction of pain during activity, stiffness after getting up and after activity for at least 30 minutes or more, occasional joint swelling. Diagnosis requires confirmation by X-ray film, and treatment includes physical measures, rehabilitation, patient education, and medicines. Osteoarthritis can be divided into primary (idiopathic) and secondary, i.e. caused by conditions that alter the cartilage microenvironment. These disorders include, but are not limited to: major trauma, congenital joint abnormalities, metabolic defects (e.g., hemochromatosis, wilson's disease), infections (causing post-infection arthritis), endocrine and neurological disorders, and disorders that alter the normal structure and function of hyaline cartilage (e.g., rheumatoid arthritis, gout, cartilage calcification).

The terms "onset of arthritis", "exacerbation of arthritis" or "onset of arthritis" as used herein are defined as the onset of increased disease activity or worsening symptoms. Attacks are generally sudden exacerbations of joint pain with other characteristic symptoms such as, but not limited to, fever, fatigue, weakness, stiffness, or joint swelling. Attacks may involve a single joint or multiple joints. For example, an osteoarthritis patient is either afflicted with a single joint or repeatedly attacks the same joint, multiple joints. In contrast, autoimmune arthritis patients such as rheumatoid arthritis or psoriatic arthritis often experience multiple joint attacks simultaneously. In one example, the methods disclosed herein can be used to alleviate symptoms of an arthritic episode.

In one example, the arthritis is selected from, but not limited to, rheumatoid arthritis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), osteoarthritis, and arthritic episodes/exacerbations. In another example, the arthritis is rheumatoid arthritis. In another example, the arthritis is osteoarthritis. In another example, the arthritis is psoriatic arthritis. In another example, the symptom in need of treatment is an onset/exacerbation of arthritis.

In another example, the antirheumatic drug is, but is not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, azathioprine cyclosporin, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, rituximab, abatacipratropium, ananaproxen, tumor Necrosis Factor (TNF) -alpha antagonists (e.g., without limitation, adalimumab, etanercept, golimumab, cetuximab (certolizumab pegol), infliximab (infliximab), sha Lishan anti (sarilumab), toxizumab (tocilizumab), balitutinib (baritinib), dapatinib (upadapalitinib), phenanthrenetinib (gofilipib) and fipeib, and combinations thereof.

The term "treatment" as used herein refers to any and all uses that remedy a disease state or condition in any way, prevent the occurrence of a disease, or otherwise prevent, hinder, delay, or reverse the progression of a disease or other undesirable condition. The methods disclosed herein may also be used to alleviate any symptom of the diseases or conditions disclosed herein.

Also disclosed herein are kits or diagnostic kits for detecting arthritis or susceptibility to arthritis in an individual. The diagnostic kit may include a device for detecting the expression, amount or activity of CNX, protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3), or a complex of CNX and PDIA3 in an individual by any means described herein. Thus, the diagnostic kit may comprise any one or more of the following: CNX, PDIA4 or PDIA3 polynucleotides or fragments thereof; a complementary nucleotide sequence of a CNX, PDIA4 or PDIA3 nucleic acid or fragment thereof; CNX, PDIA4 or PDIA3 polypeptides or fragments thereof; or antibodies to CNX, PDIA4 or PDIA 3. The diagnostic kit may include instructions or other indicia. The diagnostic kit may also include means for treating or preventing arthritis, any of the compositions described herein, or any means for treating arthritis known in the art.

In one example, the diagnostic kit includes a CNX, PDIA4, PDIA3 and/or CNX: PDIA3 inhibitor as described herein. In another example, the diagnostic kit comprises CNX, PDIA4, PDIA3 and/or CNX: PDIA3 inhibitor obtained by screening. The term "CNX: PDIA3" means a complex comprising CNX and PDIA 3. The diagnostic kit may comprise a therapeutic agent such as, but not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, and combinations thereof, or other therapeutic agents disclosed herein. For example, the therapeutic agent may be an anti-CNX, anti-DPAI 4 or anti-PDIA 3 antibody.

In another example, a kit for performing the method of detecting whether a subject has arthritis of the present disclosure is disclosed.

Screening compounds or reagents may be packaged into screening kits for materials required to identify agonists, antagonists, ligands, receptors, substrates, enzymes of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3), e.g., to identify polypeptides or compounds that reduce or enhance production of CNX, PDIA4, or PDIA 3. In one example, the screening kit includes any one or more or all of the following: CNX, PDIA4 or PDIA3 polypeptides; recombinant cells expressing a CNX, PDIA4 or PDIA3 polypeptide; or antibodies to CNX, PDIA4 or PDIA3 polypeptides. The screening kit may also include a library. As described herein, a screening kit may include any one or more components required for screening. The screening kit also optionally includes instructions for use. Screening kits capable of detecting CNX, PDIA4 or PDIA3 expression at the nucleic acid level may also be provided. Such a kit may include primers for amplifying CNX, PDAI4 or PDIA3, or a pair of primers for amplification. The primer may be selected from any suitable sequence, for example a part of the CNX, PDIA4 or PDIA3 sequence. Methods for identifying primer sequences are well known in the art and the skilled artisan can readily design such primers. As described herein, the kit may include nucleic acid probes for CNX, PDIA4, or PDIA3 expression. The kit also optionally includes instructions for use.

Also disclosed herein are polyclonal antibodies for use in the methods and treatments described herein. If polyclonal antibodies are desired, selected mammals (e.g., without limitation, mice, rabbits, goats, and horses) are immunized with an immunogenic composition comprising a Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) polypeptide or peptide. Depending on the host species, various adjuvants may be utilized to enhance the immune response. Such adjuvants include, but are not limited to, freund's adjuvant, mineral gels (such as aluminum hydroxide) and surface active substances (such as, but not limited to, lysolecithin, polyaldehyde polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). BCG (Bacillli Calmette-Guerin) and Corynebacterium parvum (Corynebacterium parvum) are human adjuvants which can be used if the purified substance amino acid sequence is to be administered to immunocompromised individuals to stimulate systemic defenses.

Serum from immunized animals is collected and processed according to known procedures for obtaining antibodies. If the serum containing polyclonal antibodies against epitopes obtainable from Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) polypeptides 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. For the production of such antibodies, the CNX, PDIA4 or PDIA3 amino acid sequence or fragment thereof can be made hapten (haptenied) with another amino acid sequence for use as an animal or human immunogen.

The scope of the present disclosure also includes monoclonal antibodies directed against epitopes obtainable from Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) polypeptides or peptides. These monoclonal antibodies are readily produced by those skilled in the art. Conventional methods for producing monoclonal antibodies from hybridomas or hybridoma cell lines are well known in the art. Immortalized, antibody-producing cell lines may be produced by cell fusion, but may also be produced by other techniques such as, but not limited to, direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. The set of monoclonal antibodies produced against the orbital epitope (orbiter) can be screened for various characteristics such as, but not limited to, isotype and epitope affinity.

Monoclonal antibodies can be prepared using any technique known in the art that can produce antibody molecules by continuous culture of cell lines.

Recombinant DNA technology can be used to improve antibodies described herein. Thus, chimeric antibodies can be constructed to reduce their immunogenicity in diagnostic or therapeutic applications. For example, such techniques include splicing mouse antibody genes to human antibody genes to obtain molecules with the appropriate antigen specificity and biological activity. In addition, antibodies can be humanized by methods known in the art such as Complementarity Determining Region (CDR) grafting and framework modification to minimize immunogenicity.

Disclosed herein are antibodies, including monoclonal and polyclonal antibodies, directed against epitopes obtainable from Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) polypeptides or polypeptides, which are believed to be particularly useful in the methods disclosed herein, including diagnostics. In particular, monoclonal antibodies can be used to generate anti-idiotype antibodies. An anti-idiotype antibody is an immunoglobulin with an "internal image" of the substance and/or agent to which it is to be protected. Techniques for preparing anti-idiotype antibodies are well known in the art. These anti-idiotype antibodies can also be used in the methods of treatment described herein. In one example, the antibodies can be bound to a solid support and/or packaged in a suitable container with suitable reagents, controls, instructions, and the like.

Antibodies can also be generated by in vivo induction in lymphocyte populations, or by screening recombinant immunoglobulin libraries or high specificity binding reagent sets disclosed in the art.

Antibody fragments may also be generated that contain specific binding sites for the target polypeptide or peptide. For example, such fragments include, but are not limited to, F (ab') produced after pepsin digestion of an antibody molecule ₂ Fragments and reduction F (ab') ₂ Fab fragments generated after disulfide bridging of the fragments. Alternatively, a Fab expression library can be constructed to quickly and easily identify monoclonal Fab fragments with the desired specificity.

Techniques known in the art for producing single chain antibodies are also suitable for producing single chain antibodies directed against Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) polypeptides. In addition, transgenic mice or other organisms (including other mammals) can also be used to express humanized antibodies.

The antibodies described herein can also be used to isolate or identify clones expressing the polypeptide, or to purify the polypeptide by affinity chromatography.

Recombinant DNA technology can be used to produce antibodies in bacterial or mammalian cell culture according to a pre-determined procedure. The cell culture system of choice may secrete antibody products.

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

In vitro production provides relatively pure antibody preparations and can be scaled up to obtain large amounts of the desired antibody. Techniques for culturing bacterial cells, yeast cells or mammalian cells are known in the art and include homogeneous suspension culture, such as in an air-float reactor or a continuous stirred reactor, or fixed or entrained cell culture, such as on hollow fibers, microcapsules, agarose microbeads or ceramic cartridges.

Hybridoma cells that secrete monoclonal antibodies are also provided. Hybridoma cells are genetically stable, secrete monoclonal antibodies of the desired specificity, and can be activated from deep frozen cultures by thawing and recloning.

Also included herein are exemplary processes for preparing a hybridoma cell line that secretes a monoclonal antibody directed against a Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) polypeptide, characterized by immunizing a suitable mammal, such as a Balb/c mouse, with one or more Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) polypeptides, or antigenic fragments thereof; the antibody-producing cells of the immunized mammal are cell-fused with a suitable myeloma cell line, the fused hybrid cells are cloned, and cell clones secreting the desired antibody are selected. For example, spleen cells of Balb/c mice immunized with Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) are fused with cells of myeloma cell line PAI or myeloma cell line Sp2/0-Ag14, the resulting hybrid cells are selected for secretion of the desired antibodies, and positive hybridoma cells are cloned.

Thus, described herein is a method for the preparation of a hybridoma cell line characterized in that the hybridoma cell line is prepared by subcutaneous and/or intraperitoneal injection for between 10 and 10 months, e.g. 2-4 months ⁷ And 10 ⁸ The Balb/c mice are immunized, e.g., 4-6 times, with cells expressing Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) and appropriate adjuvants, and spleen cells from the immunized mice are removed 2 to 4 days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, such as polyethylene glycol. Myeloma cells can be fused with a3 to 20-fold excess of immunized mouse spleen cells in a solution containing about 30% to about 50% polyethylene glycol having a molecular weight of about 4000. After fusion, the cells are expanded in a suitable medium as described herein and periodically supplemented with a selection medium, such as HAT medium, to prevent growth of normal myeloma cells beyond the desired hybridoma cells.

Also disclosed are recombinant DNA comprising an insert encoding the heavy chain variable domain and/or the light chain variable domain of an antibody directed against Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) described herein. Such DNA by definition includes double-stranded DNA encoding or consisting of the encoding DNA and its complement, or these complementary (single-stranded) DNA itself.

Furthermore, the DNA encoding the heavy chain variable domain and/or the light chain variable domain of an antibody against Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) may be enzymatically or chemically synthesized DNA having a true DNA sequence encoding the heavy chain variable domain and/or the light chain variable domain or a mutant thereof. A mutant of true DNA is DNA encoding the heavy chain variable domain and/or the light chain variable domain of the above-described antibodies, wherein one or more amino acids are deleted or exchanged with one or more other amino acids. The modification may be, but is not limited to, a region outside the Complementarity Determining Regions (CDRs) of the heavy chain variable domain and/or the light chain variable domain of the antibody. Such mutant DNA may also be a silent mutant, in which one or more nucleotides are replaced with other nucleotides, thereby generating a new codon encoding the same amino acid. Such mutant sequences may also be degenerate sequences. Degenerate sequences are degeneracy in the sense of the genetic code, i.e., an unlimited number of nucleotides replaced by other nucleotides, without resulting in a change in the originally encoded amino acid sequence. Such degenerate sequences are useful because of the frequency of their different restriction sites and/or specific codons that are favored by a particular host (particularly E.coli) to obtain optimal expression of the heavy chain mouse variable domain and/or the light chain mouse variable domain.

The term "mutant" also includes DNA mutants obtained by in vitro mutagenesis of true DNA according to methods known in the art.

For assembly, e.g., intact tetrameric immunoglobulin molecules and 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 transferred into an appropriate host cell, e.g., after integration into a hybrid vector.

Also disclosed herein is a recombinant DNA comprising an insert encoding a heavy chain murine variable domain of an antibody against Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) fused to human constant domain g, e.g., γ1, γ2, γ3 or γ4, such as γ1 or γ4. Furthermore, recombinant DNA comprising an insert encoding the light chain murine variable domain of an antibody against Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) fused to a human constant domain, for example kappa or lambda, is disclosed.

In another example, recombinant DNA encoding a recombinant polypeptide is disclosed, wherein the heavy chain variable domain and the light chain variable domain are linked by a spacer group, optionally comprising a signal sequence that facilitates antibody processing in a host cell and/or a DNA and/or cleavage site and/or polypeptide spacer group and/or effector molecule encoding a polypeptide that facilitates antibody purification.

anti-Calnexin (CNX) antibodies, anti-protein disulfide isomerase A4 (PDIA 4) antibodies, and anti-protein disulfide isomerase A3 (PDIA 3) antibodies are useful in methods of detecting CNX, PDIA4, or protein disulfide isomerase A3 (PDIA 3) polypeptides present in a biological sample. In one example, the method includes: (a) providing an anti-CNX, anti-PDIA 4 or anti-PDIA 3 antibody; (b) Incubating the biological sample with the antibody under conditions that allow formation of an antibody-antigen complex; (c) Determining whether an antibody-antigen complex comprising the antibody is formed.

The term "biological sample" or "specimen" as used herein includes, but is not limited to, any number of substances from living or previously living organisms. Such living organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, plasma, urine, cells, organs, tissues, bones, bone marrow, lymph nodes, synovial tissue, synovial cells, tissues (especially inflamed tissues), cartilage and skin.

Antibodies disclosed herein, such as antibodies directed against CNX, PDIA4, or PDIA3 proteins, can be delivered into cells by techniques known in the art, such as by using liposomes, polymers (such as, but not limited to, polyethylene glycol (PEG), N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer, polyaminoamine (PAMAM) dendrimer, HEMA, linear polyaminoamine polymers), and combinations thereof. For example, immunoglobulins and/or antibodies may also be delivered to cells in fusion or conjugation with proteins capable of crossing the plasma and/or nuclear membranes. For example, immunoglobulins and/or targets may be fused or conjugated to domains or sequences in such proteins responsible for translocation activity. Translocation domains and sequences may include domains and sequences from HIV-1-transactivator (Tat), drosophila antenna homology domain proteins and herpes simplex-1 virus VP22 proteins.

While the Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) inhibitors may be administered alone, e.g., as CNX, PDIA4 or protein disulfide isomerase A3 (PDIA 3) nucleic acids, polypeptides, fragments, homologs, variants or derivatives thereof, modulators, agonists or antagonists, structurally related compounds or acidic salts of either, the active ingredients may be formulated into pharmaceutical formulations.

Accordingly, also disclosed herein are pharmaceutical compositions comprising an inhibitor of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3). In another example, the pharmaceutical composition comprises a Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) inhibitor. Such pharmaceutical compositions may be used to deliver Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) inhibitors, e.g., to an individual in the form of the compositions, to treat or ameliorate the symptoms and or diseases described herein. In one example, the pharmaceutical composition is a composition of matter comprising at least one Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4) or protein disulfide isomerase A3 (PDIA 3) inhibitor as an active ingredient.

The pharmaceutical formulation comprises an effective amount of an inhibitor of Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) and one or more pharmaceutically acceptable carriers. As used herein, an "effective amount" refers to an amount sufficient to alleviate at least one symptom of the disease. Such a so-called effective amount will vary depending on the particular disease or syndrome to be treated or alleviated, and other factors, including the age and weight of the patient, the advanced degree of disease, the general health of the patient, the severity of the symptoms, and whether the Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) inhibitor is administered alone or in combination with other therapies.

Suitable pharmaceutically acceptable carriers are well known in the art and will vary with the desired form and mode of administration of the pharmaceutical formulation. For example, such carriers may include, but are not limited to, diluents or excipients such as fillers, binders, wetting agents, disintegrants, surfactants, lubricants, and the like. The carrier is typically a solid, liquid, or vaporizable carrier, or a combination thereof. Each carrier should be "acceptable", i.e., compatible with the other ingredients of the formulation, and not injurious to the patient. Thus, the carrier should be biologically acceptable and not cause adverse reactions (e.g., immune or allergic reactions) when administered to a host.

The active ingredients of the pharmaceutical compositions exhibit therapeutic activity, for example, in alleviating arthritis, rheumatoid arthritis, osteoarthritis, arthritic episodes and other related diseases or symptoms. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, the dosage may be administered several times a day, or the dosage may be proportionally reduced as needed for the treatment situation.

The active compounds can be administered in a convenient manner, such as orally, intravenously (water-soluble), intramuscularly, subcutaneously, intranasally, intradermally or by suppository route or by implantation (e.g., using slow-release molecules). Depending on the route of administration selected, the active ingredient may need to be encapsulated in a material to protect the ingredient from enzymes, acids and other natural conditions that may inactivate the ingredient.

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 are complementary to the formulation recipe. The formulations used in the combination therapy may be administered simultaneously or sequentially with other therapies to achieve the effect of the combination therapy. Thus, in one example, therapeutic agents include, but are not limited to, antibodies, drugs, genes, fusion proteins, and expression vectors. In another example, the therapeutic agents may be administered together, separately or sequentially. In a further example, the (first) therapeutic agent will be administered with another therapeutic agent, wherein the other therapeutic agent is an antirheumatic. In another example, such antirheumatic drugs are, but are not limited to, celecoxib, ibuprofen, nabumetone, naproxen sodium, naproxen, piroxicam, azathioprine cyclosporin, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, rituximab, apatacide, anaolaquin, tumor Necrosis Factor (TNF) -alpha antagonists (e.g., without limitation, adalimumab, etanercept, golimumab, cetuximab (certolizumab pegol), infliximab (infliximab) and biopharmaceuticals thereof), sha Lishan anti (sarilumab), toxizumab (tocilizumab), basitinib (baritinib), dapatinib (upadapalitib), filtinib (glitinib) and fipeib (fintib) and combinations thereof.

In some embodiments, inhibitors of the activity, expression or amount of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) or CNX: PDIA3 complex are provided in the form of oral compositions and administered accordingly. The dosage of the inhibitor of the activity, expression or amount of Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) may be between about 1 mg/day and about 10 mg/day.

The pharmaceutical composition may be administered orally in the form of a tablet, capsule or solution. The patient is administered an effective amount of the oral formulation one to three times per day until the symptoms of the disease are relieved.

The effective amount of the agent depends on the age, weight and condition of the patient. Generally, the daily oral dosage of the medicament is less than 1200mg, more than 100mg. The daily oral dosage may be from about 300mg to about 600mg. The oral formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art of pharmacy. The compositions may be formulated with a suitable pharmaceutically acceptable carrier into any desired dosage form. Typical unit dosage forms include, but are not limited to, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories. Formulations are typically prepared by uniformly and intimately bringing into association the pharmaceutical composition with liquid carriers or finely divided solid carriers or both, and then shaping the product as necessary. The active ingredient may be added to the various base materials in the form of a liquid, powder, tablet or capsule to obtain an effective amount of the active ingredient to treat the disease.

The composition may suitably be administered orally, for example with an inert diluent or with an assimilable edible carrier, or by filling the composition into hard or soft shell gelatin capsules, or compressed into tablets, or directly with the food in the diet. For oral therapeutic administration, the active compounds

In some embodiments, an inhibitor of Calnexin (CNX), protein disulfide isomerase A4 (PDIA 4), or protein disulfide isomerase A3 (PDIA 3) or CNX: PDIA3 complex is provided in the form of an injectable or intravenous composition and administered accordingly. The dose of the inhibitor of Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) may be between about 5mg/kg/2 weeks to about 10mg/kg/2 weeks. The amount of the inhibitor of Calnexin (CNX) or protein disulfide isomerase A3 (PDIA 3) may be between 10 mg/day and 300 mg/day, for example, at least 30 mg/day, less than 200 mg/day or between 30 mg/day and 200 mg/day.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In any event, the medicament must be sterile and must be fluid to the extent that a syringe can be used. The pharmaceutical agent must remain stable under the conditions of manufacture and storage and be resistant to the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, for example containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained, for example, by the use of a coating such as, but not limited to, lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

The compositions disclosed herein may be administered in an adjuvant, co-administered with an enzyme inhibitor or in a liposome. The term "adjuvant" is used in its broadest sense and includes, but is not limited to, any immunostimulatory compound, such as an interferon. Adjuvants contemplated herein include, but are not limited to, resorcinol, nonionic surfactants such as polyoxyethylene oleyl ether and n-cetyl polyvinyl ether. Enzyme inhibitors include, but are not limited to, trypsin. Liposomes include, but are not limited to, water-in-oil-in-water CGF emulsions and conventional liposomes.

The term "control group", "negative control" or "control" as used in the analysis of a sample refers to the use of a sample obtained from a disease-free or healthy subject and then treating these samples in the same manner as other samples, except that the control sample is treated with, for example, a buffer that does not contain the relevant active compound or molecule. Comparing the concentration of one or more targets (e.g., comparing the absolute concentration or relative expression levels of the targets), or determining whether one or more targets disclosed herein (e.g., one or more proteins, oligomers, or oligonucleotides) are present, is based on comparing the levels determined in a sample obtained from a diseased subject and a sample obtained from a non-diseased (or healthy) subject. In other words, the comparison of targets is based on comparing the level of one or more targets determined in the diseased subject to the level of the same one or more targets determined in the control group or control individual. In the present disclosure, control samples are taken from individuals who are ill. In other words, the individuals from which the control samples were obtained were not examined for disease. In general, the term "disease-free" means that the subject is healthy.

The term "differential expression" as used herein refers to measuring a target as compared to a control or another sample, thereby determining a difference in, for example, concentration, presence, or intensity of the target. The result of such a comparison may be absolute, i.e., the target is present in the sample and not in the control; it may also be relative, i.e., the expression or concentration of the target is increased or decreased as compared to the control. "increase" and "decrease" are used interchangeably herein with "up" and "down".

The term "pharmaceutically acceptable carrier and/or diluent" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and combinations thereof. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional medium or formulation in a therapeutic composition is contemplated unless such medium or formulation is incompatible with the active ingredient. Supplementary active ingredients may also be added to the composition.

The invention illustratively described herein suitably may be practiced in the absence of any element, limitation or limitations which is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and are not limited thereto. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding 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, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Functional characteristics

The formulations, methods of treatment, or other therapeutic agents described herein, such as antibodies and antigen binding fragments, may be defined in terms of their functional properties. For example, GALA inhibitors, calnexin (CNX) inhibitors, protein disulfide isomerase A4 (PDIA 4) inhibitors, or protein disulfide isomerase A3 (PDIA 3) inhibitors, or CNX: PDIA3 complex inhibitors, may be defined by functional properties. In addition, anti-calnexin (anti-CNX) antibodies, anti-protein disulfide isomerase A4 (anti-PDIA 4) antibodies, anti-protein disulfide isomerase A3 (anti-PDIA 3) antibodies or anti-CNX: PDIA3 complex antibodies may be defined according to functional characteristics.

In some cases, agents such as inhibitors, antibodies, and antigen binding fragments are capable of:

-reducing cartilage degradation

-reducing ECM degradation

-reduction of ECM degradation activity

-decreasing ECM degrading activity of CNX PDIA3 (also known as Cnx/ERp 57)

-decreasing ECM degradation activity of CNX

-decreasing ECM degrading activity of fibroblasts

-decreasing ECM degrading activity of synovial fibroblasts

-decreasing the oxygen reductase (ox reducing) activity

-decreasing the oxygen reductase activity of CNX PDIA3 (also known as Cnx/ERp 57)

-decreasing the oxygen reductase activity of CNX

-reduction of disulfide reductase activity

Reduction of disulfide reductase Activity of CNX PDIA3 (also known as Cnx/ERp 57)

-reducing disulfide reductase activity of CNX

-reducing O-glycosylation

-reducing O-glycosylation of CNX

-reducing glycosylation of CNX

-reducing GALA-mediated O-glycosylation of CNX

-decreasing CNX activity

-reduction of PDIA3 activity

-reduction of PDIA4 activity

-decreasing the number or proportion of cells expressing CNX

-decreasing the number or proportion of cells expressing PDIA3

-decreasing the number or proportion of cells expressing PDIA4

-decreasing the number or proportion of cells expressing CNX PDIA3

The functional properties highlighted above can all be effectively detected by methods known in the art, for example by microscopy, qPCR, qrtPCR, western blotting, biochemical detection, enzymatic detection and other techniques known to the skilled person.

The cartilage degradation and the relative level of cartilage degradation activity can be determined by a variety of methods known in the art. For example, by well known methods such as microscopic analysis, conventional radiography (X-ray), magnetic Resonance Imaging (MRI), computed Tomography (CT), and ultrasound imaging. Furthermore, the skilled artisan is aware of biochemical and biomarker assays that can be used to determine the extent of cartilage degradation. Serum biomarkers and urine biomarkers can be used to detect the extent of cartilage degradation. Biomarkers include the carboxy-terminal propeptide of type II procollagen (CPI), cartilage Oligomeric Matrix Protein (COMP), the carboxy-terminal neoepitope of collagenase-generated type II collagen (sC 2C), cartilage intermediate layer protein 2 (CILP-2), the carboxy-terminal peptide of type II collagen (C-telopeptide, CTX-II), and the peptide of collagenase-generated type II collagen (C2C-HUSA).

The skilled artisan can determine whether cartilage degradation or cartilage degradation activity is reduced/inhibited by comparison to a relevant control.

The relative levels of ECM degradation and ECM degradation activity can be determined by a variety of methods. The skilled artisan is aware of many methods by which ECM degradation and ECM degradation activity can be measured, such as those shown in the examples herein. The skilled artisan will readily determine whether a Cnx/ERp57 inhibitor is capable of inhibiting ECM degradation activity, cnx/ERp57 ECM degradation activity and/or Cnx ECM degradation activity.

The following procedure is one method for the skilled artisan to detect ECM degradation activity.

Commercial solutions of gelatin (2%) were labeled with 5-carboxy-X-rhodamine succinimidyl ester. The labeled gelatin was then transferred to a sterile coverslip to form a thin layer and fixed with glutaraldehyde. Finally, a cover slip is coated with a rat tail collagen solution to form a thin layer of collagen on the gelatin.

The coverslip may then be transferred to a culture vessel, seeded with cells having degradation activity (e.g., human hepatocellular carcinoma Huh 7) and cultured for 48 hours for degradation.

After fixing, the coverslips can be stained with Hoescht for cell counting. The coverslip was then imaged using a confocal microscope. The images were then analyzed using ImageJ. A threshold may be manually defined to show the surface of degraded gelatin and the total area of each field of view measured. At the same time, the number of nuclei can be counted and the final results normalized to the cells in each field of view.

The skilled artisan can determine whether ECM degradation activity is reduced/inhibited by comparison to a relevant control.

The relative level of oxidoreductase activity can be determined by a variety of methods. The skilled artisan is aware of many methods by which the activity of an oxidoreductase can be measured, such as those shown in the examples of the present specification. The skilled artisan will readily determine whether a Cnx/ERp57 inhibitor is capable of inhibiting oxygen reductase activity, cnx/ERp57 oxygen reductase activity, and/or Cnx oxygen reductase activity.

The use of insulin reduction assays (as described in Hirano et al, eur J biochem. (1995) 234 (1): 336-42) is one method for detecting oxidoreductase activity by the skilled artisan.

The skilled artisan can determine whether the oxidoreductase activity is reduced/inhibited by comparison to a relevant control.

The relative level of disulfide reductase activity can be determined by a variety of methods. The skilled artisan is aware of a number of methods for measuring disulfide reductase activity, such as those shown in the examples of the present specification. The skilled artisan will readily determine whether a Cnx/ERp57 inhibitor is capable of inhibiting disulfide reductase activity, cnx/ERp57 disulfide reductase activity, and/or Cnx disulfide reductase activity.

The following procedure is one method for the skilled artisan to detect disulfide reductase activity: (1) coating ECM (1 mg/ml collagen with 25ug/ml fibronectin (1 hour polymerization at 37 ℃) or 0.5mg/ml Matrigel (2.5 hours polymerization at room temperature)), (2) seeding cells at appropriate density, (3) allowing cells to adhere (about 45 minutes), (4) adding 10ug/ml antibody and 10-25mM GM6001, (5) incubating for 72 hours, (6) adding hoechst to observe nuclei, (7) decellularizing for 1-2 minutes using warm 20mM ammonium hydroxide, 0.5% (v/v) triton X-100, (8) rinsing with 1% BSA in PBS, (9) adding 2.5mM TCEP as control, 30 minutes at room temperature; (10) rinsing with 1% BSA in PBS; (11) 5mM NEM in 1% BSA was added for 30 min at room temperature; (12) rinsing with 1% BSA in PBS; (13) immobilization with 4% PFA; (14) rinsing with PBS; (15) One anti-mouse anti-NEM OX133 (1:200) and IgG control were incubated overnight at 4 ℃, (16) washed with PBS, (17) secondary anti-mouse 647 (1:400) and secondary only control were incubated for 45 min at room temperature, (18) washed with PBS, and (19) identical wells were again visualized visually to determine cysteine reduction signal and normalized to cell number.

The skilled artisan can determine whether disulfide reductase activity is reduced/inhibited by comparison to a relevant control.

The presence and relative level of glycosylation can be determined by methods well known in the art, such as those employed in the examples and commercial detection kits. Methods for detecting and analyzing glycosylated and glycoprotein include: glycan staining or labeling, glycoprotein purification or enrichment, and mass spectrometry. The skilled person is aware of these methods.

The presence and relative level of O-glycosylation can be determined by methods known in the art, such as those employed in the examples herein. The marker of O-glycosylation is an increase in the level of Tn (T knooviler), an O-glycan formed by adding GalNac to Ser or Thr residues in the cell. Tn can be detected by Tn binding proteins such as pea lectin (VVL) and snail lectin (HPL) (Gill et al, proc.Natl. Acad. Sci. U.S.A.110, E3152-61 2013).

The relative levels of CNX, PDIA3 may be determined by methods known in the art. For example, the relative level of gene expression (e.g., calnexin expression) can be determined by a variety of different methods known to the skilled artisan. For example, the RNA level encoding a particular gene may be determined by techniques such as RNAseq, RT-PCR, and RT-qPCR, among other known methods. In addition, the relative levels of protein expression (e.g., calnexin expression) may also be determined by methods well known to the skilled artisan. The level of a given protein/isoform thereof can be determined, for example, by antibody-based methods, including western blotting, immunohistochemistry, flow cytometry, ELISA, and the like. Furthermore, the activity of CNX, PDIA3 and/or PDIA4 can be determined by isolating the enzyme and performing an enzymatic assay.

The number or proportion of cells expressing CNX, PDIA3, PDIA4 and/or CNX: PDIA3 can be determined by a variety of methods including single cell transcriptome analysis, microscopy and immunohistochemical methods and flow cytometry.

As highlighted above, a therapeutic agent, such as a GALA inhibitor, a Calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA 4) inhibitor, or a protein disulfide isomerase A3 (PDIA 3) inhibitor, a CNX: PDIA3 complex inhibitor, or an anti-calnexin antibody, may result in a decrease in a property or activity, such as ECM degradation, oxygen reductase activity, disulfide reductase activity, glycosylation, protein activity, or the number or proportion of a particular cell type. In some cases, this decrease may be more than 5% of the level observed in the relevant control, e.g., one of 10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.98%,. Gtoreq.99% or 100%.

The relevant control may be an uninhibited state, i.e. a characteristic level prior to treatment.

In some embodiments, a therapeutic agent (e.g., a GALA inhibitor, a Calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA 4) inhibitor, or a protein disulfide isomerase A3 (PDIA 3) inhibitor, a CNX: PDIA3 complex inhibitor, or an anti-calnexin antibody) results in a reduced level of ECM degradation. In some cases, the decrease in ECM degradation may be greater than 5% of the level observed in the relevant control (e.g., in the non-inhibited 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%, power99% or 100%.

In some embodiments, a therapeutic agent (e.g., a GALA inhibitor, a Calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA 4) inhibitor, or a protein disulfide isomerase A3 (PDIA 3) inhibitor, a CNX: PDIA3 complex inhibitor, or an anti-calnexin antibody) results in a decrease in the level of ECM degrading activity (e.g., ECM degrading activity of CNX/ERP57, or ECM degrading activity of synovial fibroblasts). In some cases, this decrease in ECM degradation activity may be greater than 5% of the level observed in the relevant control (e.g., in the non-inhibited state), such as one of ≡10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100%.

In some embodiments, a therapeutic agent (e.g., a GALA inhibitor, a Calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA 4) inhibitor, or a protein disulfide isomerase A3 (PDIA 3) inhibitor, a CNX: PDIA3 complex inhibitor, or an anti-calnexin antibody) results in a decrease in the level of oxidoreductase activity (e.g., ECM degrading activity of CNX/ERP57, or ECM degrading activity of synovial fibroblasts). In some cases, this decrease in oxygen reductase activity may be greater than 5% of the level observed in the relevant control (e.g., in the absence of inhibition), such as one of 10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100%.

In some embodiments, the therapeutic agent (e.g., a GALA inhibitor, a Calnexin (CNX) inhibitor, a protein disulfide isomerase A4 (PDIA 4) inhibitor, or a protein disulfide isomerase A3 (PDIA 3) inhibitor, a CNX: PDIA3 complex inhibitor, or an anti-calnexin antibody) results in a reduced level of disulfide reductase activity (e.g., ECM degrading activity of CNX/ERP57, or ECM degrading activity of synovial fibroblasts). In some cases, this decrease in disulfide reductase activity may be greater than 5% of the level observed in the relevant control (e.g., in the absence of inhibition), such as one of 10%,. Gtoreq.15%,. Gtoreq.20%,. Gtoreq.25%,. Gtoreq.30%,. Gtoreq.35%,. Gtoreq.40%,. Gtoreq.45%,. Gtoreq.50%,. Gtoreq.55%,. Gtoreq.60%,. Gtoreq.65%,. Gtoreq.70%,. Gtoreq.75%,. Gtoreq.80%,. Gtoreq.85%,. Gtoreq.90%,. Gtoreq.91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99% or 100%.

General terms

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

The term "about" as used herein in the context of formulation ingredient concentrations generally refers to +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, more typically +/-1% of the value, more typically +/-0.5% of the value.

In the present 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 interpreted as a limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual values within the range. For example, describing a range (e.g., 1 through 6) should be taken to have specifically disclosed sub-ranges (e.g., 1 through 3, 1 through 4, 1 through 5, 2 through 4, 2 through 6, 3 through 6, etc.) as well as individual values (e.g., 1, 2, 3, 4, 5, and 6) within the range. This applies regardless of the extent.

Certain embodiments may also be described broadly and generically herein. The narrower species and subgroups falling within the generic disclosure also form part of the disclosure. This includes the generic description of embodiments with a proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically recited herein.

The invention is described broadly and generally herein. Narrower species and subgroups falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limiting examples. Furthermore, while 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 thereby described in terms of any individual member or subgroup of members of the Markush group.

Experimental part

Example 1

Patient samples, cell lines and mouse lines

Patient samples: synovial tissue samples were taken from Rheumatoid Arthritis (RA) or Osteoarthritis (OA) patients undergoing joint replacement surgery at the Chen Dusheng hospital (singapore). The procedure was approved by the national healthcare group domain specific review board ethics committee according to protocol 2018/00980. All patients received written consent and met the diagnostic criteria for rheumatoid arthritis or osteoarthritis.

Cell line: SW982 cells (ATCC HTB-93) are synovial fibroblasts derived from the synovium of a synovial sarcoma patient. SW982 cells were maintained in Laibuweiz L-15 medium (Gibco, sieimer's technique) supplemented with 10% (v/v) Fetal Calf Serum (FCS) and 1% (w/v) penicillin/streptomycin (Gibco; sieimer's technique (ThermoFisher Scientific)) at 37℃in free gas exchange with the atmosphere. SW982 cells were engineered using the sleeping beauty transposon system to stably express the doxycycline-inducing gene encoding ER-2Lec.

Mice: col6a1Cre mice expressing the type VI collagen promoter in a C57BL/6J background were supplied by G.Bressan (university of Milan, italy). Notably, similar mouse models known in the art can be used as background to generate the mice required for the present invention. According to the details provided by the inventors of Ozgene, ER-2Lec mice were generated that expressed Endoplasmic Reticulum (ER) -localized double lectin domain (ER-2 Lec) under the control of a STOP-cassette flanked by loxP (loxP-expanded) under the same background. The Col6a1Cre ER-2Lec mice were generated by crossing the Col6a1Cre mice with ER-2Lec mice, and were allowed to express ER-2Lec in the mesenchymal cell line. All animals were bred and raised under specific pathogen-free conditions with food and water available in biological resource center (asar, singapore) mini-isolation cages. Experiments were performed using age and sex matched animals and following guidelines approved by the biological research center animal ethics committee (asar, singapore) according to IACUC protocol No. 201548.

Isolation of primary Synovial Fibroblasts (SF) from human tissue

Synovial tissue of Osteoarthritis (OA) and Rheumatoid Arthritis (RA) patients is obtained at the time of synoviotomy or synovial biopsy. Immediately after excision, the tissue was minced and digested with gentle agitation at 37℃for 1.5 hours in Dulbecco's Modified Eagle's Medium (DMEM) collagenase IV (1 mg/ml, gibco). The mixture was passed through a 70 μm mesh cell filter, and after centrifugation at 250g for 10 minutes, a cell pellet was obtained. Human synovial fibroblasts from between passage 3 and passage 9 of osteoarthritis and rheumatoid arthritis patients (OASF and RASF, respectively) were used (Rosengren et al, 2007, molecular medical methods, volume 135: arthritis study, volume 1). The purity of the cultures was confirmed by staining with the fibroblast identification marker CD90 prior to conducting the experiments. Normal human synovial fibroblasts were derived from synovial tissue of a healthy human donor (HCSF) obtained from cell applications company (Cell Applications, inc.). HCSF was completely cultured in DMEM supplemented with 10% (v/v) Fetal Calf Serum (FCS) and 1% (w/v) penicillin/streptomycin (Gibco; sesameimer's technology) at 37℃in humidified air with 5% CO 2.

Reagent(s)

Antibody: anti-CNX (ab 10286, ab 22595), anti-vimentin (ab 92547), anti- β -actin (ab 8226) antibodies were purchased from Ai Bokang (Abcam). Agar-bound fava lectin (VVL, AL-1233) was purchased from vector laboratories (Vector Laboratories). The PE.C7 conjugated anti-CD 45 and PE conjugated anti-CD 90 were purchased from Baida (Biolegend). Anti-fapα is available from R & D systems (R & D systems). anti-NEM OX133 antibodies were purchased from Absolute antibody company. Anti-rabbit IgG HRP antibodies and anti-mouse IgG horseradish peroxidase (HRP) antibodies were purchased from GE medical life sciences (GE Healthcare Life Sciences).

Plasmid: plasmids expressing doxycycline-inducible ER-2Lec were generated as described previously (Gill et al, 2013, PNAS,2013, E3152-E3161). The resulting vector was used to transfect SW982 cells with pPGK-SB13 expressing sleeping beauty 208 transposase.

Immunofluorescent staining

Formalin-fixed paraffin-embedded tissue sections were dewaxed in xylene replacement buffer (Sub-X, laica Biosystems) and rehydrated. For mouse joint tissues and tissue microarrays (provitro AG, berlin germany), antigen retrieval was performed by immersion in an epitope retrieval solution pH6 (the clada biosystem) and incubation in an oven at 60 ℃ for 18 hours. For human tissue specimens, antigen retrieval WAs performed in a pressure chamber (2100 retriever, jacobian technologies, inc. (Akribis Scientific Limited), WA16 0jg, gb) using epitope retrieval solution pH6 (lica biosystems). The sections were washed twice with Phosphate Buffered Saline (PBS) and immersed in blocking buffer (5% horse serum, 1% triton x 100). After 1 hour, sections were incubated overnight at 4 ℃ with primary antibody mixtures containing primary antibodies including rabbit anti-CD 45, rabbit anti-vimentin, rabbit anti-calnexin, rat anti-FAP alpha, or biotin-conjugated pea lectin (VVL). The samples were washed three times with blocking buffer and incubated with the corresponding secondary antibodies, including anti-rat Alexa Fluor 647, anti-rabbit Alexa Fluor 594 coupling or Alexa Fluor 488 streptavidin (Sieimer Feier technology, 1:400), for 2 hours. After washing, nuclei were stained with Herste 33342 (Siemens technology; 1:1000) for 5 minutes before loading. All images were captured on an LSM-700 (Zeiss) confocal microscope using the same setup. The raw integrated densities of the VVL and the nuclear signal were measured with a FIJI image calculator.

Collagen antibody induced arthritis

On day 0, mice were injected with an antibody mixture (Chondrex corporation) containing five different anti-type II collagen monoclonal antibody clones at 2 mg/mouse (200 μl) by intraperitoneal Injection (IP). On day 3, animals were injected with LPS at 50 μg/mouse (100 μl) by IP injection. From day 3, the severity of arthritis was assessed daily by measuring paw thickness using a digital caliper. The evaluation was also performed by an unknowing researcher using the qualitative clinical scoring system provided by Chondrex corporation.

Histological analysis and staining of chondrocyte extracellular matrix

After removal of the skin, the anterior and posterior paws were formalin fixed and embedded in paraffin. Tissues were deparaffinized and rehydrated as described in the immunofluorescent staining protocol above. Tissue sections were stained with Hematoxylin and Eosin (HE), safranin-O (SO) and Alcian Blue (AB). Slides were scanned 20 times using a Leika CN400 slide scanner (Leika microscopy system (Leica Microsystems), germany). The image was exported to the slide path digital image center (Leka microscopy, germany) for review. Selected areas were analyzed using a slide path tissue image analysis 2.0 software (Leika microscopy systems, germany) for measurement staining area analysis. The extracellular matrix (ECM) stained areas of cartilage were quantitatively analyzed using a FIJI image calculator.

Flow cytometry

The skin of the foot was removed and the joint was cut 3mm above the heel. To avoid bone marrow contamination, the bone marrow cavity of the tibia was thoroughly flushed with Hank's Balanced Salt Solution (HBSS). The joints were sectioned into small pieces and incubated in digestion buffer (1 mg/ml collagenase IV and 1mg/ml DNase I in HBSS) for 60 minutes at 37 ℃. Filtering the cells released during digestion through a 70 μm cell filter; erythrocytes were lysed using erythrocyte lysis buffer (BD biosciences). Cells were stained with live/dead Aqua (Invitrogen) reactive dye, incubated with Fc Block at 1:50 (BD biosciences), and stained with fluorochrome-conjugated antibodies including PE-conjugated anti-CD 90 (hundred), PECy 7-conjugated anti-CD 45 (hundred) and FITC-conjugated pea lectin (VVL; life technologies (Life Technologies))) for 30 minutes. For intracellular staining, cells were fixed by using BD Cytofix/Cytoperm solution (BD biosciences). The fixed cells were permeabilized in 1 XBD Perm/Wash buffer (BD Biotechnology) and then stained with FITC-conjugated pea lectin (VVL). Samples were collected on FACS BD LSRII and analyzed using Kaluza software.

Western blot and VVL-co-immunoprecipitation (VVL-CoIP)

The cells were treated at 2X10 ⁵ Cells/ml were seeded in 10cm dishes pre-coated with 2mg/ml chondrocyte extracellular matrix (ECM; xylyx organism) and allowed to stand overnight. In the case of 100. Mu.g/ml TNF. Alpha. (PeproTech) and 100. Mu.g/ml TNF. Alpha., peproTechAfter 24 hours of g/ml IL-1β (Pai Prateck) stimulation, cells were harvested and lysed in low stringency RIPA lysis buffer (50 mM Tris, 200mM NaCl, 0.5% NP-40, complete and PhoStop inhibitor (Roche applied sciences)) at 4℃for 30 minutes. The lysate is then clarified by centrifugation at 13000g for 10 minutes at 4 ℃. Clarified tissue lysates were incubated with agarose-bound pea lectin (VVL) beads (vector laboratory) overnight at 4 ℃. The beads were washed three times with RIPA lysis buffer and the precipitated proteins were eluted in 2xLDS sample buffer containing 50mM DTT. Lysates were boiled at 95℃for 5 min and separated by SDS-PAGE electrophoresis at 180V for 70 min using 4-12% Bis-Tris 80NuPage gel (Inje). The samples were then transferred onto nitrocellulose membranes using an iBlot transfer system (invitrogen) and blocked for 1 hour at room temperature using 3% Bovine Serum Albumin (BSA) dissolved in TBST (tris buffered saline (TBS) and polysorbate 20 (also known as tween 20) -50mM tris, 150mM NaCl and 0.1% tween 20). Nitrocellulose membranes were then incubated with primary antibody (1/1000 dilution in 3% BSA-TBST) overnight at 4 ℃. The next day, membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies for 2 hours at room temperature. The membranes were rewashed three times with TBST prior to Electrochemiluminescence (ECL).

Matrix degradation assay

Red gelatin coverslips were prepared as described previously (Ros et al, nat Cell Biol,2020, volume 22, month 11 2020, 1371-1381). The coverslips were coated with 0.2mg/ml chondrocyte extracellular matrix (ECM; xylyx organism) for 3 hours at 37 ℃. Synovial fibroblasts (including SW982 cells, osteoarthritis synovial fibroblasts (OASF), rheumatoid Arthritis Synovial Fibroblasts (RASF) and healthy human donor synovial fibroblasts (HCSF)) at 5×10 ⁴ Cells/ml/well were seeded overnight in 24-well plates. Cells were then stimulated with 100. Mu.g/ml TNF. Alpha. And 100. Mu.g/ml IL-1. Beta. After 24 hours, cells were fixed with 4% Paraformaldehyde (PFA) and nuclei stained using Hoechst 33342 (life technology). The stained coverslips were mounted on glass microscope slides and 10 to 30 images were acquired for each case. As previously described, image j software was used to measure relative to cell numberNormalized area of matrix degradation. (Martin et al, J Vis Expr,2012, volume 66, e 4119) briefly, the degradation area of the thresholded fluorescence gelatin image was used and the same threshold was applied to all images. The number of nuclei was counted using a cell counting tool and the area of gelatin degradation per total number of cells was calculated. Experiments were performed in three biological replicates.

Statistical analysis

Statistical analysis and graphic preparation were performed using GraphPad Prism (version 8.4.3, graphPad software, california, usa). Data analysis was performed using either single factor (Kruskal Wallis test), two-factor analysis of variance (Tukey multiple comparison test) or Mann-Whitney test. Differences with p-values <0.05 are considered statistically significant.

Example 2

Results

Enhanced O-glycosylation in rheumatoid arthritis and osteoarthritis synovium

One marker of GALA is an increase in the cellular level of Tn (T nouvelle), tn is an O-glycan formed by the addition of GalNac to Ser or Thr residues. Tn can be detected by Tn-binding proteins such as pea lectin (VVL) and snail lectin (HPL) (Gill et al, proc.Natl. Acad. Sci. U.S.A.110, E3152-61 2013).

We analyzed microarrays of joint tissue by immunofluorescence using VVL and counterstained DNA. We detected a significant 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 (fig. 1A and 23). We quantified the integrated fluorescence intensity of VVL staining normalized to the DNA signal intensity as a cell density marker (fig. 1B). The variation in healthy patient samples was small, but the Tn levels were increased in most RA and OA samples, with a 7-fold increase in VVL signal in certain areas.

To further characterize GALA in arthritis, we used a RA mouse model based on Collagen Antibody Induced Arthritis (CAIA) (32). Briefly, animals were injected with antibodies against type II collagen, followed by Lipopolysaccharide (LPS) after 3 days, and symptoms were developed from day 5. Preliminary immunohistochemical analysis showed a significant increase in Tn levels in and around the joints of arthritic animals (fig. 23).

In the CAIA model, symptoms peak approximately 7 days and last for 10 days, then slowly abate. We sampled animals on days 0, 7, 10 and 14 and quantified Tn levels using immunofluorescent staining. Histologically, there was a significant pannus invasion into the joint cavity on day 7 and increased with immune cell influx on day 10. By day 14, the number of immune cells decreased dramatically, but synovial tissue remained in the joint cavity (fig. 2A and B). High levels of Tn were observed in cells invading the joint cavity on day 7 and continued until day 10 (fig. 2C and D). By day 14, the cell Tn levels have decreased in most animals. We observed some Tn staining in the cell-free fibrous material and this staining was not significantly reduced at day 14.

High Tn levels in arthritic synovial membranes are consistent with GALA activation

High cellular Tn levels can be induced by the GALA pathway, characterized by an abundant Tn signal in the endoplasmic reticulum. Because the endoplasmic reticulum is distributed throughout the cell body, GALA activation is generally manifested as an increased spreading signal (Bard et al, trends cell biol.26,379-388,2016). We performed co-staining of the CAIA synovial samples for Tn and calnexin (an ER marker) (fig. 3A). In untreated samples, tn and calnexin staining were significantly separated, tn accumulated in a perinuclear pattern consistent with golgi. In contrast, in the CAIA sample on day 7, tn staining was elevated, filling the cell space and co-localized with ER-tagged calnexin, strongly suggesting GALA induction. To demonstrate the induction of GALA in the CAIA environment, we stained with GALNT2, a ubiquitously expressed GALNT transferase, which was previously shown to be relocated to the ER in GALA-activated cancers. Similar changes were observed from perinuclear localization in untreated samples to ER pattern localization in CAIA samples, accompanied by co-localization of CNX (fig. 3B). Our results indicate that transfer of GALNT2 from golgi to ER is the basis for increased Tn levels in the case of CAIA. These results are consistent with the description of the GALA phenotype in breast and liver cancers previously reported (Gill et al, proc. Natl. Acad. Sci. U.S. A.110, E3152-61, 2013; nguyen et al, cancer Cell 32, 639-653.e6, 2017).

Synovial fibroblasts are the predominant cell type exhibiting GALA

Synovial membrane in RA disease is a complex tissue that includes immune cells and synovial fibroblasts (Choy et al, rheumatology 51 journal 5, volumes 3-11, 2012). To determine which cell types showed increased GALA, we co-stained day 7 mouse CAIA joint samples with VVL, CD45 (immune cell marker) and vimentin (marker of fibroblasts). We observed vimentin-positive cells at the front of the invading pannus (fig. 4). CD45 positive cells typically accumulate and are located behind the front end of pannus invasion. Notably, tn positive cells in pannus were co-stained primarily with vimentin positive regions, rather than CD45 positive regions. This result indicated GALA activation in synovial fibroblasts (fig. 4). We validated this result in RA and OA human samples using fibroblast activation protein alpha (FAP alpha) as a marker for synovial lining fibroblasts (Bauer et al Arthritis Res. Ther.8, R171,2006). In RA samples, fapα positive cells formed a layer at the pannus edge, and a larger CD45 positive cell layer on the back side (fig. 5A, 5B and 24A). In OA samples, the number of CD45 cells was reduced compared to RA samples, but fapα cells showed similar VVL staining (fig. 24). Remarkably, in both RA and OA cases, VVL staining was co-localized only with fapα. Thus, synovial fibroblasts are the primary cells that exhibit GALA activation in OA and RA synovium.

Stimulation of SF by cytokines and ECM induces higher GALA levels

To understand how GALA is activated in vivo we used primary human SF from the patient. FACS analysis using CD90 and CD45 as markers for SF and immune cells, respectively, determined that the cell preparation we used was >90% pure (fig. 24B). We then performed high content imaging analysis of Tn levels in cells derived from these SF patients using HPL staining. Seed cells on plastic wells showed increased Tn levels in OA compared to healthy SF, even more in RA cells (fig. 6). We then stimulated SF with TNF alpha and IL1 beta cytokines, which are thought to drive disease progression in RA (Kagari and Shimiozato. J. Immunol.169,1459-1466,2002). The effect of a single cytokine on GALA activation was relatively limited, however the combination of the two cytokines (labeled CYTO) induced a 2-fold increase in Tn cell levels. Interestingly, this effect is pronounced in RASF, more limited in OASF, and almost absent in healthy SF (HCSF).

Next we want to know if the cartilage ECM proteins contribute to activation of SF. In OASF and RASF, exposure of SF to cartilage ECM can activate GALA up to three times. In contrast, HCSF cells hardly respond. The combination of CYTO and ECM had a superimposed effect on Tn levels (fig. 6). This stimulation was not specific to cartilage ECM, as similar activation was induced using rat tail-derived collagen I (fig. 6).

In the unstimulated condition, tn staining was only detected in the golgi of HCSF, while OASF and RASF showed additional ER-like Tn staining (fig. 6). After stimulation with a combination of CYTO and cartilage ECM, ER-localized Tn staining was enhanced in both OASF and RASF, but not in HCSF (fig. 6).

In summary, this analysis shows that OA and RA patients have elevated GALA in SF compared to healthy SF in cell culture. RA SF further activates GALA in response to cytokines, whereas both RA and OA SF activate GALA when exposed to ECM. In contrast, the GALA response of healthy SF is very limited, suggesting that this pathway is prepared for activation of patient cells.

Inhibition of GALA in SF reduces ECM degradation and arthritis in vivo

Since GALA drives ECM degradation in cancer cells, we tried to test whether it is also related to SF degradation of cartilage ECM. We (in the fluorescent gelatin sandwich assay described previously) found that human synovial sarcoma SW982 cells were able to degrade collagen (Ros et al, nat.cell biol.22,1371-1381,2020). We have previously described ER-2Lec chimeric proteins consisting of one ER targeting sequence and a fusion of two lectins of GALNT2 (Gill et al, proc. Natl. Acad. Sci. U.S.A.110, E3152-61 2013). ER-2Lec specifically inhibits GALA activity by interfering with the O-glycosylation of ER.

We generated ER-2Lec stable SW982 transfectants under the doxycycline inducible promoter system. Like RA SF, SW982 cells stimulated with CYTO mixtures were more active in degrading ECM (fig. 7B and 7C). However, when ER-2Lec expression was induced, degradation was significantly reduced by more than 2-fold (fig. 7B and 7C).

To test whether ER-2Lec can reduce arthritis in vivo, we generated transgenic mouse lines containing ER-2Lec to be Lox-boxed. We hybridized them with mice expressing Cre under the collagen VI α1 (Col 6a 1) promoter (fig. 3C). Collagen VI is expressed by joint mesenchymal cells, in particular SF (Danks et al, annual rheumatism (Annals of the Rheumatic Diseases) 75,2016, pages 1187-1195; armaka et al, J.Exp. Med.205,331-337,2008). Thus, this gene hybridization is expected to allow ER-2Lec to be expressed mainly in SF and to reduce Tn levels in arthritic mice. We induced CAIA in these mice and monitored expression of ER-2Lec-GFP and Tn levels in pannus after 7 days. Tn was significantly reduced, consistent with GALA inhibition (fig. 8).

We monitored the symptoms of RA in Col6a1Cre ER-2Lec animals and Cre expressing control groups. Paw swelling was significantly reduced in animals expressing ER-2Lec (FIGS. 9A and B). We measured the change in paw thickness over time and observed a significant decrease in paw thickness in ER-2Lec animals compared to the control group. We also used the internationally defined arthritis scores in the blind evaluation and observed a sustained reduction in symptoms in the Col6a1Cre ER-2Lec animals (fig. 10).

We also performed histological analysis on day 7 using H & E and Alcian Blue (AB) and safranin-O (SO) staining, which are commonly used to reveal the cartilage portion of the joint. H & E staining of the Col6a1Cre ER-2Lec joint showed a decrease in pannus volume consistent with a decrease in swelling (fig. 11). Interestingly, infiltration of immune cells appeared to be greatly reduced in animals expressing ER-2Lec (fig. 11 and 25A). The AB positive region was also significantly preserved in the CAIA Col6a1Cre ER-2Lec animals (fig. 11). A significant change was also obtained after quantification of SO staining (fig. 25). Taken together, our findings indicate that inhibition of GALA in SF with ER-2Lec protein can limit the progression of arthritic disease.

CNX glycosylation and surface exposure in GALA activated SF

We have recently described Calnexin (CNX) as a glycosylation target and effector of GALA (Ros et al, nat.cell biol.22,1371-1381,2020). After glycosylation, CNX translocates on the cell surface and, together with PDIA3, mediates cleavage of disulfide bonds in ECM proteins. This reducing activity is critical for the degradation of the cancer cell matrix (Ros et al, nat.cell biol.22,1371-1381,2020). In SW982 SF, we found that CNX was highly glycosylated approximately 6-fold after stimulation with cytokines and ECM (fig. 12A and B). This glycosylation is GALA dependent and expression of ER-2Lec can significantly reduce CNX glycosylation.

Furthermore, we found that CNX surface expression increased significantly by about 10% after stimulation of SW982 SF cells with CYTO and ECM using FACS (fig. 13A and B). Remarkably, ER-2Lec expression completely inhibited the increase in cell surface CNX signaling (FIGS. 13A and B).

We attempted to confirm this result in primary cells obtained from the patient. In SF from Healthy Control (HCSF), the proportion of cell surface CNX positive cells was only about 7%, which was slightly increased by stimulation with cytokines and ECM (fig. 14A and B). In contrast, patients with RA (RASF) or OA (OASF) showed significantly increased levels of SF cells and were more sensitive to stimulation, with a three-fold increase in the percentage of cells with cell surface CNX (fig. 14A and B). Taken together, these results indicate that CNX glycosylation and its cell surface exposure are enhanced in arthritis SF and dependent on GALA.

anti-CNX antibodies in CAIA mice for the prevention of arthritic symptoms

We have previously shown that antibodies to calnexin can block ECM degradation by preventing the necessary disulfide bond reduction (Ros et al, nat.cell biol.22,1371-1381,2020). We hypothesize that blocking calnexin can approximately prevent cartilage ECM degradation. We first tested for the presence of disulfide bonds in the cartilage ECM using the method described previously (Ros et al, nat.cell biol.22,1371-1381,2020). Briefly, cartilage ECM was reduced with TCEP, then exposed to N-ethylmaleimide (NEM), followed by treatment with anti-OX 133 antibodies. As previously described, we observed a large number of OX133 signals co-localized with collagen 3/collagen 1 and fibronectin/collagen 1 fibers, indicating that the cartilage ECM was heavily crosslinked by disulfide bonds (fig. 15).

We next tested the effect of anti-CNX antibodies on ECM degradation. OASF cells seeded on cartilage ECM covered with fluorescent gelatin were allowed to degrade ECM overnight. The addition of polyclonal anti-CNX antibodies blocked this degradation activity (fig. 16 and B).

Inspired by these results, we aimed at treating animals with anti-CNX antibodies. We first monitored the body weight of animals receiving three antibody injections over 10 days; we did not find any weight loss (figure 22). Next, we treated CAIA animals with anti-CNX antibodies, injected 25 micrograms every two days from day 3 to day 7 after CAIA onset (fig. 17A). We periodically monitored paw thickness and measured the arthritis score on day 10. Remarkably, paw swelling was reduced in animals treated with anti-CNX compared to control animals treated with isotype antibodies (fig. 17B). While the toes still appeared somewhat red and swollen, increasing the arthritis score, the average score for the treated animals was half that of the CAIA control animals (fig. 17C). This is similar to the results obtained with ER-2Lec expression.

At the histological level, a very pronounced decrease in SO-positive cartilage was seen in the control animals on day 10 (fig. 18). Furthermore, the synovium has adhered to the underlying bone, which may indicate the onset of bone remodeling. In contrast, anti-CNX treated animals had preserved joint cavities with a large amount of cartilage remaining (fig. 18).

Our working hypothesis was that the Cnx antibody bound to and inhibited the degradation activity of inner synovial fibroblasts. To test whether the antibodies actually interacted with these cells, we stained joints of the treated animals with anti-rabbit IgG. The signal was clearly detectable in synovial cells of animals treated with anti-CNX antibodies, whereas no signal was present in animals treated with control rabbit IgG (fig. 19C).

Taken together, these results indicate that inhibition of CNX is effective in inhibiting cartilage ECM degradation and may lay a foundation for arthritis treatment.

Example 3

Discussion of the invention

In this study we demonstrate that GalNac O-glycosylation is significantly up-regulated in arthritic synovial fibroblasts compared to healthy fibroblasts. This increase is due to activation of the GALA pathway, which transfers GALNT from the golgi to the endoplasmic reticulum.

In cancer cells, EGF-R activation, in particular Src kinase driven GALA (Gill et al, J.cell biol.189,843-858,2010; chia et al, PLoS one.14, e0214118,2019). Other signaling molecules (e.g., ERK8 kinase) constitutively and dynamically inhibit this pathway (Chia et al, elife.3, e01828, 2014). In vitro, RA and OA patient-derived fibroblasts have moderately elevated GALA levels compared to healthy human SF. However, RA fibroblasts activate GALA in response to IL-1 beta and TNF-alpha cytokine mixtures. Interestingly, the response to RA SF was more pronounced than normal or OA SF, indicating that RA SF is ready 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), indicating a possible link between cytokines and GALA.

Exposure to ECM activated GALA in OA and RA fibroblasts more strongly than healthy control cells. It has been previously proposed that the adhesion of SF to cartilage ECM elements is involved in the development of Arthritis (Pap et al, arthritis Res.2,361-367,2000). Injection of fibronectin in the joint results in degradation of the cartilage proteoglycans (Homandberg et al J. Rheumatol.20,1378-1382,1993). Integrins, the fibronectin receptor, are also activators of Src kinase (Shattil, trends Cell biol.15,399-403,2005; hufins and Danen, J.cell Sci.122,1059-1069,2009). Thus, the signaling cascade can link external ECM signals to integrin, src, and then to GALA, activating ECM degradation (Gill et al, j. Cell biol.189,843-858,2010). This hypothetical cascade would form a pathological positive feedback loop. It is currently unclear why the response of GALA to ECM in healthy SF is much more limited. SF from arthritic joints has been considered to be epigenetic in preparation for degradation (Nygaard et al, nat. Rev. Rheumatol.16,316-333 (2020)). In fact, OASF and RASF show similar overall methylation characteristics, unlike SF in healthy subjects (Nakano et al, ann.rheum. Dis.72,110-117,2013). Some differences were found in genes involved in PDGF and EGF signaling, as well as in the regulator of GALA (Chia et al, PLoS one.14, e0214118,2019). Thus, epigenetic priming may include a higher propensity to activate GALA. Furthermore, GALA glycosylation may have a synergistic effect with other regulatory mechanisms. For example, we found increased CNX levels in arthritic mouse synovial tissue, consistent with gene expression data reported in previous studies (Broeren et al, PLoS one.11, e0167076,2016; nzeusseuse Toukap et al, arthritis Rheum.56,1579-1588,2007). Up-regulation of GALNT1, 3 and 5 was found in these studies, and we also observed increased expression of GALNT1 and 2.

Regardless of whether activated by immune signals or ECM proteins, GALA glycosylation may not be activated continuously during arthritis. Indeed, in the CAIA mouse model, GALA levels were significantly reduced at day 10, prior to complete recovery of the animals (day 14 or later). In patient samples, GALA could be detected in OA, RA and psoriatic arthritis samples, but a significant fraction of the samples had lower levels of GALA. This suggests that GALA is fully activated only during the active ECM degradation phase of the disease, which corresponds to the onset of the patient. In contrast, during remission, ECM degradation was less, with corresponding lower GALA levels.

One of the targets of GALA glycosylation is MMP14, a cell surface protease that degrades collagen fibers and activates other MMPs (Nguyen et al, cancer cells 32, 639-653.e6, 2017, gialli et al, FEBS j.278,16-27,2011). MMP14 is one of the MMPs involved in arthritis and activates MMP-2 and 13 (Rose and Kooyman, dis. Markers.2016,4895050, 2016). MMP 14O-glycosylation is critical to its protease activity, which occurs in aggregated form in low complexity regions of the protein: six or more amino acids are modified with GalNAc or more complex O-glycans (Nguyen et al, cancer cells 32, 639-653.e6 (2017).

Calnexin also shows an aggregate glycosylation pattern, located in the N-terminal region (Ros et al, nat.cell biol.22,1371-1381,2020). Aggregation glycosylation is a common feature of GalNac glycosylation, for example in mucins. ER-2Lec chimeric proteins inhibit this aggregation glycosylation (Gill et al, proc. Natl. Acad. Sci. U.S. A.110, E3152-61, 2013). As in cancer cells, ER-2Lec reduced the level of Tn signaling in SF. Thus, it may inhibit MMP14 and Cnx glycosylation at least in part in vitro and in vivo, inhibiting SF degradation of ECM. Since GALNT acts on thousands of proteins, and preliminary, unpublished data suggests that GALA affects many proteins, additional glycoproteins may be involved in SF pathological activity and affected by ER-2Lec (Steentoft et al, methods 8,977-982,2011).

Cre-activated ER-2Lec under collagen type VI promoter resulted in CAIA treated mice protected from cartilage loss. Expression of ER-2Lec was primarily limited to SF, with no expression detected in immune cells. Interestingly, expression of ER-2Lec reduced swelling and inflammation of the joints. Effective protection of the cartilage ECM may reduce activation of SF, preventing release of cytokines and thus inflammation. This explanation is supported by the fact that anti-Cnx antibody treatment also reduces inflammation.

The role of calnexin in ECM degradation has only recently been determined. Calnexin, together with PDIA3, is involved in the reduction of disulfide bonds in liver ECM proteins (Ros et al, nat.cell biol.22,1371-1381,2020). Disulfide bonds, like other crosslinks, prevent protease activity (Philp et al, am. J. Respir. Cell mol. Biol.58,594-603,2018). Cartilage ECM contains abundant disulfide bonds. Anti-calnexin antibodies block SF degradation of matrix in vitro and provide significant protection to animal cartilage ECM.

Other strategies have been developed to inhibit synovial cells, 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). Recently, targeting SF cell surface tyrosine phosphatase PTPRS has also been shown to protect cartilage in RA mice (Svensson et al Sci adv.6, eaba4353,2020). Furthermore, the targeting of MMPs has been studied for decades, and specific MMP inhibitors such as Tarchium (Trocade) have demonstrated protection 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 tolerance of these compounds resulted in failure of the clinical trial (close. Ann. Rheum. Dis.60 journal 3, iii62-7 (2001)). To date, while some progress has been made, inhibition of MMP remains relatively challenging therapeutically (fields. Cells.8.2019, doi:10.3390/cells 8090984). Targeting the calnexin-ERp 57 complex with antibodies may be a more attractive approach because less toxicity is expected.

Overall, our data opens up the prospect of biomarker discovery and new therapeutic approaches targeting Cnx with antibodies. More broadly, our results indicate that activation of O-glycosylation by GALA is a key control switch for ECM degradation in synovial fibroblasts and cancer cells, suggesting that this pathway has a broad pathological relevance.

Example 4

Materials and methods isolation and characterization of 2G9 monoclonal anti-calnexin antibodies

Identification and cloning by phage display 2G9:

to select antibodies against calnexin, we screened a library of human Fab domains cloned in a phage display library. We isolated clones with affinity for calnexin. We selected clone 2G9 for further characterization.

anti-Cnx antibodies were isolated from HX02 human Fab phage display library (Humanyx private Co., ltd.) by in vitro screening. We followed the procedures described by de Haard et al for bioscreening, phage amplification, fab expression and purification (J Biol Chem 1999;274:18218-30; http:// dx. Doi. Org/10.1074/jbc.274.26.18218; PMID: 10373423). Briefly, biological screening was performed using biotinylated human calnexin. In the first two rounds of biological screening, calnexin was immobilized on M280 streptavidin-coated magnetic beads (life technologies); third, biotinylated calnexin was immobilized on neutravidin coated microwell plates to avoid separation of streptavidin magnetic bead conjugates. The first round used about one thousand cfu of phage in 1mL casein PBS blocking buffer, and the second and third rounds used one thousand cfu of phage. After three rounds of biological screening, the Fab of the selected clones were expressed in e.coli TG1 cells (Stratagene) to screen for calnexin binding agents by ELISA.

Clone 2G9 was identified by DNA fingerprinting, confirmed by DNA sequencing, and converted to IgG1 and IgG4 forms for further analysis. Briefly, fab was amplified by PCR and cloned in frame with the human Fc region of the corresponding IgG. The plasmid was then amplified and 2G9 was expressed in 293T cells by transient transfection.

ELISA assay:

100ng of recombinant CNX-His protein was diluted in 50mM sodium carbonate, pH9.6 buffer coated with 96-well maxisorp plate (Nunc) overnight at 4 ℃. Negative control wells were also coated with maxisorp plate (Nunc) overnight at 4 ℃ with 0.5ug/ml Bovine Serum Albumin (BSA). The coated wells were then washed 3 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 ℃. After blocking, the primary antibody was added to the wells and incubated at 37℃for 2 hours. Wells were washed 3 times with PBST and PBS, respectively. Horseradish peroxidase (HRP) -conjugated secondary antibody was added to 5% FBS-PBST and incubated for 1 hour at 37 ℃. Wells were washed 3 times with PBST and PBS, respectively. To detect HRP, TMB substrate was added to the wells and incubated for 5 minutes, followed by 1M hydrochloric acid to stop the reaction. OD450 was measured on a Tecan microplate reader.

ECM degradation assay:

gelatin (G1393, sigma) was first coupled to 5-carboxy-X-rhodamine succinimide ester (C-6125, siemens technology). Sterile coverslips were coated with gelatin for 20 minutes and then fixed with 0.5% glutaraldehyde (15960, electron microscopy (Electron Microscopy Sciences)) for 40 minutes. After washing, a layer of 0.5mg ml ^-1 Type I collagen (354236 rat tail, corning) was coated on these gelatin coverslips and incubated at 37 ℃ for 4 hours before cell seeding. Cells were incubated overnight prior to fixation and staining. Each of whichIn each case 10-30 images were taken and experiments were performed in three biological replicates. Degradation area was quantified using ImageJ and normalized to the number of nuclei in each image.

Example 5

Results isolation and identification of monoclonal anti-calnexin antibodies to 2G9

2G9 has high binding affinity and specificity for calnexin:

the 2G9 binding affinity was tested using an ELISA assay. Adsorbing the purified His-tagged calnexin and coating on plastic wells; control wells were coated with BSA. Then, we incubated 2G9, control human IgG or commercial monoclonal antibody ab10286 at a concentration of 10 μg/ml on the plates. This assay showed that both antibodies had very high specificity for Cnx with little binding to BSA coated wells (fig. 26). Negative control hIgG did not bind significantly to calnexin.

To quantitatively compare 2G9 and ab10286, we repeated the ELISA assay using serial dilutions of the antibodies (fig. 27). 2G9 exhibits saturated binding at 0.1. Mu.g/ml, with a calculated EC50 of about 3.10 e-6. Mu.g/ml. This concentration was lower than the commercial monoclonal antibody ab10286, indicating higher affinity and/or mobility.

2G9 prevents HUH7 liver cancer cell from degrading ECM

The rat tail ECM was overlaid on a thin layer of fluorescently labeled gelatin by assessing the ability of the IgG1 form of 2G9 to prevent ECM degradation using a degradation assay. As the active ECM degraded, the cells degraded the ECM, and then degraded the gelatin, creating a dark spot in the gelatin layer that could be measured (fig. 28A). HUH7 cells were seeded on top and incubated with 2G9 or control antibodies for 48 hours. Control IgG had no effect on matrix degradation, or even seemed to stimulate matrix degradation, but 2G9 was able to reduce ECM degradation by 90% compared to untreated control (fig. 28B).

The IgG4 form of 2G9 is capable of blocking ECM degradation:

the 2G9 antibody was converted to IgG4 form. We retest the protective activity of ECM using gelatin overlay. As found in IgG1 format, we also found that 2G9 was able to protect ECM degradation (fig. 29). This effect is most pronounced at 20 μg/ml.

Sequence listing

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Reference to the literature

O-glycosylation is regulated by Golgi apparatus relocation of the initiating enzyme to the endoplasmic reticulum (Regulation of O-glycosylation through Golgi-to-ER relocation of initiation enzymes) J.cell biol.189,843-858 (2010).

Initiation of GalNAc-type O-glycosylation in the endoplasmic reticulum promotes cancer cell invasion (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).

F.bard, j.chia, break sugar group coding: signaling, trafficking, and Glycosylation, trends Cell biol.26,379-388 (2016).

Organ-specific O-glycosylation drives MMP14 Activation, tumor Growth and Metastasis (Organelle Specific O-Glycosylation Drives MMP Activation, tumor Growth, and metatasis) in cancer cells. 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 the retained oxidoreductase is glycosylated and transported to the cell surface to promote degradation of the matrix by tumor cells (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).

J. Chia, F.Tay, F.Bard, galNAc-T activation (GALA) pathway: driver and marker (The GalNAc-T activity (GALA) Pathway: drivers and markers). PLoS one.14, e0214118 (2019).

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 (ERK 8 is a negative regulator of O-GalNAc glycosylation and cell migration). Elife.3, e01828 (2014).

Sequence listing

<110> Singapore scientific research bureau

Chen Dusheng Hospital

F.Bard

L.S. Telan

K.P.Freon

<120> method

<130> 008160186

<150> SG10202100687X

<151> 2021-01-21

<150> SG10202109307T

<151> 2021-08-25

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 9522

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ER-2Lec

<400> 1

aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60

tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120

tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180

gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg 240

gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300

tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360

taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420

aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800

aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 1920

tatcgataag cttgggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 1980

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 2040

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 2100

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 2160

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 2220

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 2280

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 2340

ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 2400

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg 2460

aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 2520

gaccgatcca gcctccggac tctagaggat ccctaccggt gatatcctcg agaccatggc 2580

tacaggctcc cggacgtccc tgctcctggc ttttggcctg ctctgcctgc cctggcttca 2640

agagggcagt gccttcccaa ccattccctt atccgtgagc aagggcgagg agctgttcac 2700

cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 2760

gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 2820

caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca 2880

gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 2940

cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 3000

cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 3060

cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 3120

ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca 3180

caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 3240

cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 3300

agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 3360

cactctcggc atggacgagc tgtacaaggg ctcgagccca tcaacaagtt tgtacaaaaa 3420

agcaggctcc gataacggga agcctatccc taaccctctc ctcggtctcg attctacgga 3480

gttaagggtt ccagaccatc aggatatagc ttttggggcc ttgcagcagg gaactaactg 3540

cctcgacact ttgggacact ttgctgatgg tgtggttgga gtttatgaat gtcacaatgc 3600

tgggggaaac caggaatggg ccttgacgaa ggagaagtcg gtgaagcaca tggatttgtg 3660

ccttactgtg gtggaccggg caccgggctc tcttataaag ctgcagggct gccgagaaaa 3720

tgacagcaga cagaaatggg aacagatcga gggcaactcc aagctgaggc acgtgggcag 3780

caacctgtgc ctggacagtc gcacggccaa gagcgggggc ctaagcgtgg aggtgtgtgg 3840

cccggccctt tcgcagcagt ggaagttcac gctcaacctg cagcaggcac agggagccca 3900

agggagatcc gataacggga agcctatccc taaccctctc ctcggtctcg attctacgga 3960

gttaagggtt ccagaccatc aggatatagc ttttggggcc ttgcagcagg gaactaactg 4020

cctcgacact ttgggacact ttgctgatgg tgtggttgga gtttatgaat gtcacaatgc 4080

tgggggaaac caggaatggg ccttgacgaa ggagaagtcg gtgaagcaca tggatttgtg 4140

ccttactgtg gtggaccggg caccgggctc tcttataaag ctgcagggct gccgagaaaa 4200

tgacagcaga cagaaatggg aacagatcga gggcaactcc aagctgaggc acgtgggcag 4260

caacctgtgc ctggacagtc gcacggccaa gagcgggggc ctaagcgtgg aggtgtgtgg 4320

cccggccctt tcgcagcagt ggaagttcac gctcaacctg cagcagaaag acgagctgta 4380

ggacccagct ttcttgtaca aagtggttga tatccagcac agtggcggcc gctcgagtct 4440

agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc tcgattctac 4500

gcgtaccggt tagtaatgat cgacaatcaa cctctggatt acaaaatttg tgaaagattg 4560

actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 4620

ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 4680

ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 4740

gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 4800

gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 4860

cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag 4920

ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 4980

ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 5040

gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 5100

gccgcctccc cgcctggcga tggtaccggt gtggaaagtc cccaggctcc ccagcaggca 5160

gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 5220

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 5280

ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 5340

gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc 5400

agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt 5460

gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat 5520

cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagcctt tgtctcaaga 5580

agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga 5640

ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt 5700

atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc 5760

ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag 5820

cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt 5880

gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta 5940

tgtgtgggag ggctaagcac aattcgagct cggtaccttt aagaccaatg acttacaagg 6000

cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 6060

cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 6120

gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 6180

cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 6240

tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta 6300

ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg 6360

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 6420

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc 6480

tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 6540

ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 6600

ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat 6660

tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac 6720

tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 6780

tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 6840

ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 6900

agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 6960

tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 7020

ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 7080

cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 7140

tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 7200

tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 7260

caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc 7320

ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 7380

cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 7440

gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 7500

ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 7560

tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 7620

aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 7680

ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 7740

agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 7800

gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 7860

gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 7920

gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 7980

tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 8040

ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 8100

cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 8160

gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 8220

cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 8280

tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 8340

aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 8400

aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 8460

gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 8520

cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 8580

ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 8640

accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 8700

tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 8760

cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 8820

gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 8880

ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 8940

cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 9000

tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 9060

ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 9120

ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 9180

ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 9240

gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 9300

acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 9360

ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 9420

tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa 9480

ttaaccctca ctaaagggaa caaaagctgg agctgcaagc tt 9522

<210> 2

<211> 21

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of Calnexin (CNX)

<400> 2

caagaguggu ccuaggagau u 21

<210> 3

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 3

ggaauagucc cauuagcaa 19

<210> 4

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 4

gggcaaggac uuacuuauu 19

<210> 5

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 5

agacccaaau aucgucaua 19

<210> 6

<211> 18

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 6

aggaguucuc gcgugaug 18

<210> 7

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 7

gaacgaguau gaugauaau 19

<210> 8

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 8

ggacaagacu guggcauau 19

<210> 9

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 9

ggcaaggacu uacuuauug 19

<210> 10

<211> 19

<212> RNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> siRNA target sequence of PDIA3

<400> 10

ugauaaagau gccucuaua 19

<210> 11

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> 2 (DOX) -inducible lectin domains of GALNT2

<400> 11

Thr Pro Thr Thr Val Gly Pro Thr Thr Val Gly Ser Thr Thr Val Gly

1 5 10 15

Claims

1. Use of an anti-calnexin (anti-CNX) antibody in the manufacture of a medicament for treating or preventing cartilage degradation in a subject.

2. An anti-calnexin (anti-CNX) antibody for use in the treatment or prevention of cartilage degradation in a subject.

3. A method of treating or preventing cartilage degradation, wherein the method comprises administering to a subject an anti-calnexin (anti-CNX) antibody.

4. The use of claim 1, the antibody for use of claim 2, or the method of treatment of claim 3, wherein the subject has osteoarthritis, rheumatoid arthritis, psoriatic arthritis, juvenile Idiopathic Arthritis (JIA), an arthritic episode, bursitis, gout, chondrocalcification, fibromyalgia, costal chondritis, cartilage injury, or polychondritis.

5. The use of claim 1, the antibody for use of claim 2, or the method of claim 3, wherein the subject has arthritis.

6. The use, antibody for use, or method of claim 5, wherein the arthritis is osteoarthritis or rheumatoid arthritis.

7. The use, antibody for use, or method of any preceding claim, wherein the cartilage degradation is in one or more joints.

8. The use, antibody for use, or method according to any preceding claim, wherein the cartilage degradation is characterized by increased O-glycosylation and/or GALNT activation (GALA) in articular tissue, cartilage tissue and/or synovial fibroblasts.

9. The use, antibody for use, or method according to any preceding claim, wherein the cartilage degradation is characterized by increased O-glycosylation of calnexin in joint tissue, cartilage tissue and/or synovial fibroblasts.

10. The use, antibody for use, or method according to any preceding claim, wherein the cartilage degradation is characterized by increased cell surface expression of calnexin in joint tissue, cartilage tissue and/or synovial fibroblasts.

11. The use, antibody for use, or method according to any preceding claim, wherein the cartilage degradation is characterized by extracellular matrix (ECM) degradation.

12. The use, antibody for use, or method according to any preceding claim, wherein the cartilage degradation is mediated by synovial fibroblast activity.

13. The use, antibody for use, or method of any preceding claim, wherein the antibody against Cnx is capable of reducing:

(a) ECM degradation; or (b)

(b) Oxygen reductase activity; or (b)

(c) Disulfide reductase activity.

14. The use, antibody for use, or method of any preceding claim, wherein the antibody is antagonistic.

15. The use, antibody for use, or method of any preceding claim, wherein the antibody is monoclonal.

16. The use, antibody for use, or method according to any preceding claim, wherein the therapeutic agent is to be administered together, separately or sequentially with another therapeutic agent, wherein the other therapeutic agent is an antirheumatic.

17. The use, antibody for use, or method of any preceding claim, wherein the method comprises intravenous, subcutaneous or intraperitoneal administration of an anti-cnx antibody.

18. The use, antibody for use, or method of any preceding claim, wherein the treatment or prophylaxis is in a human subject.

19. The use, antibody for use, or method of any preceding claim, wherein a subject suitable for treatment with an anti-cnx antibody is determined by:

20. A method for detecting the presence or absence of a cartilage degradation related disease in a subject, wherein the method comprises the steps of:

21. The method of claim 20, wherein the method is used to detect the presence or absence of arthritis in a subject.

22. The method of claim 20 or claim 21, wherein the method is for detecting the presence or absence of osteoarthritis or rheumatoid arthritis in a subject.