Method for detecting disease-associated calnexin

Abstract

The present disclosure provides methods of assessing whether a sample comprises disease-associated CNX comprising contacting the sample with antigen-binding molecules and performing a proximity assay. Also disclosed are methods of treating or preventing a disease or condition which comprise assessing a sample with said method.

Description

[0001] Method

[0002] This application claims priority from US 63 / 697,004 filed 20 September 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the field of molecular biology, more specifically assay technology. The present disclosure also relates to methods of determining whether a protein is present on the surface of a cell.

[0005] Background

[0006] Calnexin (CNX) is an endoplasmic reticulum (ER)-resident lectin chaperone protein, which binds to N- glycoproteins bearing monoglucosylated glycans, and recruits various other chaperones that mediate protein disulfide formation, proline isomerisation, and protein folding.

[0007] Recent studies have implicated cell-surface CNX, glycosylated CNX and CNX-containing complexes (e.g. CNX:ERp57 complexes) in the pathology of diseases / conditions including cancers, particularly through their ECM degrading activity (see Ros et al. Nat. Cell Biol. (2020) 22(11):1371-1381).

[0008] It is presently difficult to determine the level of disease-associated CNX in a way that is accurate, efficient, and suitable for use on different clinical samples. There remains a need to develop new methods of assessing whether a sample comprises disease-associated CNX.

[0009] Summary

[0010] In a first aspect, the present disclosure provides a method of assessing whether a sample comprises disease-associated CNX.

[0011] The present disclosure also provides a method of determining the level of disease-associated CNX in a sample.

[0012] The present disclosure also provides a method of selecting or stratifying a subject for treatment with a CNX-targeted agent.

[0013] The present disclosure also provides a method of selecting or stratifying a subject which would benefit from treatment with a CNX-targeted agent.

[0014] A method of assessing whether a sample comprises disease-associated CNX may alternatively be described as a method of detecting disease-associated CNX in a sample. In some embodiments, the method is a method of predicting whether disease-associated CNX is present in a sample. In some embodiments, the method is a method of predicting the amount of disease-associated CNX present in a sample. In some embodiments, the method is a method of predicting the relative amount of disease- associated CNX present in a sample. In some embodiments, the method comprises contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule.

[0015] In some embodiments, the method comprises performing a proximity assay. In some embodiments, the proximity assay comprises assessing proximity of the first antigen-binding molecule and the second antigen-binding molecule.

[0016] In some embodiments, a positive output from the proximity assay indicates that the sample comprises disease-associated CNX.

[0017] In some embodiments, the method comprises, i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, and ii. performing a proximity assay, wherein the proximity assay comprises assessing proximity of the first antigen-binding molecule and the second antigen-binding molecule, wherein a positive output from the proximity assay indicates that the sample comprises disease- associated CNX.

[0018] In some embodiments, the method comprises, i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to Tn, and ii. performing a proximity assay, wherein the proximity assay comprises assessing proximity of the first antigen-binding molecule and the second antigen-binding molecule, wherein a positive output from the proximity assay indicates that the sample comprises disease- associated CNX.

[0019] In some embodiments, the disease-associated CNX is cell-surface CNX and / or glycosylated CNX. In some embodiments, the disease-associated CNX is cell-surface CNX, In some embodiments, the disease-associated CNX is a CNX:ERp57 complex. In some embodiments, the disease-associated CNX is glycosylated CNX.

[0020] In some embodiments, the CNX-interacting molecule is Tn or ERp57. In some embodiments, the CNX- interacting molecule is Tn

[0021] In some embodiments, the proximity assay comprises the use of fluorescent probes.

[0022] In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a resonance energy transfer assay, a protein-fragment complementation assay, or a protein dimerization assay.

[0023] In some embodiments, the resonance energy transfer assay is a BRET assay or a FRET assay. In some embodiments, the protein-fragment complementation assay is a bimolecular fluorescence complementation (BiFC), bimolecular luciferase complementation (BiLC), yeast-two hybrid, or a split ubiquitin assay.

[0024] In some embodiments, the protein dimerization assay is a dimerization-dependent fluorescent protein (ddFP) assay.

[0025] In some embodiments, the positive output is determined through microscopy, PCR, nucleotide sequencing, and / or flow cytometry analysis.

[0026] In some embodiments, the antigen-binding molecule which binds to CNX is an antibody or an aptamer. In some embodiments, the antigen-binding molecule which binds to CNX is an antibody.

[0027] In some embodiments, the antigen-binding molecule which binds to a CNX-interacting molecule is a lectin, an antibody, or an aptamer. In some embodiments, the antigen-binding molecule which binds to a CNX-interacting molecule is a lectin. In some embodiments, the antigen-binding molecule which binds to a CNX-interacting molecule is an antibody.

[0028] In some embodiments, the first antigen-binding molecule which binds to CNX, and / or the second antigenbinding molecule which binds to a CNX-interacting molecule comprises a detectable tag.

[0029] In some embodiments, the first antigen-binding molecule which binds to CNX, and / or the second antigenbinding molecule which binds to a CNX-interacting molecule is bound by a further antigen-binding molecule, or a series of further antigen-binding molecules, comprising a detectable tag.

[0030] In some embodiments, the detectable tag comprises an oligonucleotide, a fluorophore, a fluorescent protein, and / or a fluorescent protein fragment.

[0031] In some embodiments, the sample is a tissue sample (e.g., a biopsy), a blood sample, or a serum sample. In some embodiments, the sample is a fixed tissue section. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a fixed tissue section.

[0032] In some embodiments, the method comprises quantitatively comparing the positive output with an output of a control sample.

[0033] In some embodiments, the subject has, is suspected of having, or is at risk of developing, a disease or disorder characterised by ECM degradation. In some embodiments, the subject has, is suspected of having, or is at risk of developing, a cancer. In some embodiments, the subject has, is suspected of having, or is at risk of developing, arthritis. In some embodiments, the subject is suspected of having, is at risk of developing, or has, cancer or arthritis.

[0034] In some embodiments, the method further comprises administering a therapeutically- or prophylactically- effective amount of a CNX-targeted agent to a subject.

[0035] In some embodiments, the CNX-targeted agent is an antigen-binding molecule which binds CNX.

[0036] The present disclosure also provides a method of treating or preventing a disease or condition.

[0037] The present disclosure also provides an antigen-binding molecule which binds to CNX for use in a in a method of treating or preventing a disease or condition.

[0038] The present disclosure also provides uses of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of a disease or condition.

[0039] In some embodiments, the method of treating or preventing a disease or condition comprises assessing whether a sample comprises disease-associated CNX according to a method of the present disclosure.

[0040] In some embodiments, the method of treating or preventing a disease or condition comprises determining the level of disease-associated CNX in a sample according to a method of the present disclosure.

[0041] In some embodiments, the method of treating or preventing a disease or condition comprises selecting or stratifying a subject which would benefit from treatment with a CNX-targeted agent according to a method of the present disclosure.

[0042] In some embodiments, the method of treating or preventing a disease or condition comprises assessing whether a sample comprises disease-associated CNX according to a method of the present disclosure, determining the level of disease-associated CNX in a sample according to a method of the present disclosure, and / or selecting or stratifying a subject which would benefit from treatment with a CNX- targeted agent according to a method of the present disclosure.

[0043] Description

[0044] Methods of identifying disease-associated CNX, and other related methods, are disclosed herein.

[0045] The inventors found that previously known methods of identifying disease-associated CNX (e.g., cellsurface CNX and glycosylated CNX) were not best suited to clinical use. For example, conventional pulldown approaches are cumbersome, time consuming, and require a substantial amount of fresh cells or tissue extracts. These requirements can be particularly limiting when working with patient samples.

[0046] Inventors have developed new methods which can identify disease-associated CNX in a more clinically appropriate way. Methods described herein may be used on samples comprising fresh cells (e.g., a fresh tissue biopsy sample) as well as fixed samples (e.g., FFPE tissue sections). Methods described herein may also be used on samples comprising fewer cells than other methods. Therefore, methods according to the present disclosure are especially advantageous for analysing clinical samples and archived specimens. The excellent results obtained for the disclosed methods could not have been predicted by the skilled person.

[0047] Calnexin (CNX)

[0048] Human CNX (also known as CNX, CANX or IP90) is the protein identified by UniProt P27824. Alternative splicing of mRNA encoded by the human CANX gene yields three main CNX isoforms: isoform 1 (SEQ ID NO:1), isoform 2 (SEQ ID NO:2) and isoform 3 (SEQ ID NO:3). Isoform 2 differs from isoform 1 by insertion of a 35 amino acid sequence after position 1 of SEQ ID NO:1. Positions 1 to 108 of SEQ ID NO:1 are absent from isoform 3.

[0049] Human CNX isoform 1 comprises an N-terminal signal peptide (SEQ ID NO:4), followed by a calcium- binding lumenal domain (SEQ ID NO:5), a single-pass transmembrane domain (SEQ ID NO:6) and an acidic cytoplasmic domain (SEQ ID NO:7) at the C-terminus. The lumenal domain comprises a globular lectin domain (SEQ ID NO:8), followed by an arm-like, proline-rich P-domain (SEQ ID NO:9) and a second lectin domain (SEQ ID NO:10). The mature form of human CNX isoform 1 is shown in SEQ ID NO:11.

[0050] In this specification ‘CNX’ refers to CNX from any species, and includes isoforms, fragments, variants or homologues from any species. In some embodiments CNX is CNX from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, or primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the CNX is human CNX or mouse CNX.

[0051] As used herein, a ‘fragment’, ‘variant’, ‘isoform’ or ‘homologue’ of a given protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform).

[0052] A ‘fragment’ generally refers to a fraction of the reference protein. A ‘variant’ generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An ‘isoform’ generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein (e.g. human CNX isoform 1 , isoform 2 and isoform 3 are all isoforms of one another). A ‘homologue’ generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. For example, human CNX isoform 1 (UniProt: P27824-1 , v2; SEQ ID NO:1) and mouse CNX (UniProt: P35564-1 , v1 ; SEQ ID NO:12) are homologues of one another. Homologues include orthologues. Isoforms, fragments, variants or homologues of CNX according to the present disclosure may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CNX isoform from a given species, e.g. human.

[0053] Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property / activity of the reference CNX (e.g. human CNX isoform 1), as determined by analysis by a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of CNX may display binding to a monoglucosylated glycan- bearing N-glycoprotein, and / or association with ERp57, cyclophilin B and / or ERp29.

[0054] In some embodiments, the CNX comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1 , 2, 3 or 12.

[0055] In some embodiments, the CNX comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:11 or 20.

[0056] A ‘fragment’ of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0057] A fragment of CNX may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 amino acids.

[0058] In some embodiments, a fragment of CNX comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:11 , 5, 6, 7, 8, 9 or 10.

[0059] In some embodiments, a fragment of CNX comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NQ:20, 14, 15, 16, 17, 18 or 19.

[0060] The structure and function of CNX is reviewed e.g. in Kozlov and Gehring, FEBS J. (2020) 287(20):4322- 4340, which is hereby incorporated by reference in its entirety.

[0061] CNX is an endoplasmic reticulum (ER)-resident lectin chaperone protein. CNX binds N-glycoproteins bearing monoglucosylated glycans, and recruits various other chaperones which mediate protein disulfide formation, proline isomerisation, and protein folding. CNX is able to associate with the protein folding enzyme ERp57 to catalyse glycoprotein-specific disulfide bond formation. CNX:ERp57 complexes have also been shown to translocate to the surface of cancer cells, where they reduce disulfide bridges in the extracellular matrix (Ros et al., Nat. Cell Biol. 22, 1371-1381 , 2020). The reduction of disulfide bridges has been shown to be essential for the effective activity of matrix metalloproteinases (MMPs), and thus for the degradation of the extracellular matrix in cancer. CNX also associates with the peptidyl-prolyl cistrans isomerase cyclophilin B (CypB), for the proline isomerisation of peptide bonds. CNX has also been reported to associate with ERp29 to form CNX:ERp29 complexes, which have a general chaperone function. CNX also functions as a chaperone for the folding of MHC class I a-chain in the membrane of the ER.

[0062] Processing by glucosidase II removes the glucose residue of the monoglucosylated N-glycan required for interaction ofthe glycoprotein with CNX, resulting in liberation of the mature, processed glycoprotein from CNX. For proteins that have not yet folded properly, UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by re-adding a glucose residue back onto the N-glycan, reconstituting the interaction site for CNX. In this way, misfolded proteins re-associate with CNX for additional rounds of chaperone-mediated refolding, and their exit from the ER and progression to the Golgi is prevented. If multiple folding cycles are unsuccessful, terminally misfolded proteins are transported to the cytoplasm for degradation via the ER-associated protein degradation (ERAD) pathway.

[0063] Disease-associated CNX

[0064] The present disclosure relates to methods of identifying cells comprising disease-associated CNX.

[0065] Disease-associated CNX may be CNX (e.g., CNX comprising a post-translational modification, CNX present in a specific location, a CNX variant, or a CNX-containing complex) which is positively associated with the onset, development or progression of a disease, disorder, or condition. In some embodiments, the disease-associated CNX is CNX (e.g., CNX comprising a post-translational modification, CNX present in a specific location, a CNX variant, or a CNX-containing complex) which is pathologically-implicated in the onset, development or progression of a disease, disorder, or condition. In some embodiments, the disease-associated CNX is CNX (e.g., CNX comprising a post-translational modification, CNX present in a specific location, a CNX variant, or a CNX-containing complex) which is associated with ECM degradation. In some embodiments, the disease-associated CNX is cell-surface CNX and / or glycosylated CNX. Therefore, the present disclosure also relates to methods of identifying cells comprising cell-surface CNX, and methods of identifying glycosylated CNX.

[0066] Disease-associated CNX may be a modified CNX or a CNX complex. The modification of CNX and / or the formation of a CNX complex may comprise the interaction of CNX with a CNX-interacting molecule.

[0067] The CNX-interacting molecule may be a molecule which is a constituent of a disease-associated CNX molecule (e.g., a constituent of CNX comprising a post-translational modification, CNX present in a specific location, a CNX variant, or a CNX-containing complex). In some embodiments, the CNX- interacting molecule is a molecule which binds (or is bound) to CNX. In some embodiments, the CNX- interacting molecule is a molecule which conjugates (or is conjugated) to CNX. In some embodiments, the CNX-interacting molecule is Tn or ERp57. In some embodiments, the CNX-interacting molecule is Tn. In some embodiments, the CNX-interacting molecule is ERp57.

[0068] Cell-surface CNX has been shown to be associated with diseases such as cancer (Ros et al, Nat Cell Biol. 2020 Nov;22(11):1371-1381), and arthritis (WO2022157281 A1).

[0069] The majority of CNX proteins are typically found in the endoplasmic reticulum (ER). ER-localised CNX is not glycosylated, because ER-localised CNX is not exposed to the enzymes required to glycosylate the CNX. Conversely, cell-surface CNX is glycosylated.

[0070] The amount of Tn glycosylation ( / .e., glycoproteins comprising Tn) is notably elevated in many cancers and is associated with increased metastatic potential and poorer patient prognosis (He et al. Signal Transduct Target Ther. 2024 Aug 5;9(1 ) : 194). In contrast, normal tissues generally exhibit much lower levels of Tn glycosylation.

[0071] Tn (or Tn antigen) is an abnormal mucin-type O-glycan. It has a relatively simple structure composed of N-acetyl-d-galactosamine with a glycosidic a linkage to serine / threonine residues in glycoproteins (GalNAca1-O-Ser / Thr) and was one of the first glycoconjugates to be chemically synthesized (Ju et al. Angew Chem Int Ed Engl. 2011 Feb 18; 50(8): 1770-1791).

[0072] Tn glycosylation is mediated by a group of enzymes known as UDP-N-acetyl-a-D-galactosamine N- acetylgalactosaminyltransferases (GalNAc-Ts), which are primarily located in the Golgi apparatus. These enzymes initiate O-glycosylation by adding N-acetylgalactosamine (GalNAc) to polypeptides (Bennett et al. Glycobiology. 2012 Jun;22(6):736-56).

[0073] In healthy cells, CNX is typically an endoplasmic reticulum chaperone protein, and is not normally exposed to GalNAc-Ts (Figure 1). However, in disease contexts involving overexpression of invasionpromoting kinases such as the EGF receptor (EGFR), platelet-derived growth factor receptor (PDGF-R), Src oncogene or active GalNAc-T activation (GALA) pathway, there is increased trafficking of GalNAc-Ts between the Golgi and the endoplasmic reticulum (Gill et al. Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):E3152-61). This altered trafficking results in the exposure of CNX to GalNAc-Ts, leading to Tn glycosylation of CNX and expression of CNX at the cell-surface (Ros et al, Nat Cell Biol. 2020 Nov;22(11):1371 -1381). Elevated levels of glycosylated CNX and cell-surface CNX are associated with higher levels of extracellular matrix (ECM) degradation (e.g., cartilage ECM degradation), and the progression of diseases such as cancer and arthritis.

[0074] As used herein, the term “disease-associated CNX” refers to CNX which is capable of contributing to the development / progression of a disease or disorder, or CNX which is associated with the development / progression of a disease or disorder.

[0075] CNX which is associated with the development / progression of a disease or disorder may be CNX (e.g., a modified CNX, a CNX complex, or CNX in a specific location) which is expressed or present at a higher level in a disease setting (e.g., in a subject with a disease, in diseased tissue, and / or in cells associated with a disease), compared to a relevant control (e.g., a non-disease setting, such as in a subject without the disease, in non-diseased tissue, and / or in cells not associated with the disease). For example, glycosylated CNX and cell-surface CNX are associated with the development / progression of a disease (e.g., cancer).

[0076] In some embodiments, the disease-associated CNX is CNX comprising a post-translational modification. In some embodiments, the disease-associated CNX is glycosylated CNX. In some embodiments, the disease-associated CNX is O-glycosylated CNX. In some embodiments, the disease-associated CNX is Tn-glycosylated CNX.

[0077] In some embodiments, the disease-associated CNX is CNX localized to a region of the cell where it is capable of contributing to the development / progression of a disease or disorder, or where it is associated with the development / progression of a disease or disorder. In some embodiments, the disease- associated CNX is cell-surface CNX.

[0078] In some embodiments, the disease-associated CNX is a protein complex comprising CNX. In some embodiments, the disease-associated CNX is a protein complex comprising CNX which is capable of contributing to the development / progression of a disease or disorder, or a protein complex comprising CNX which is associated with the development / progression of a disease or disorder. In some embodiments, the disease-associated CNX is a CNX / ERp57 complex.

[0079] Glycosylated CNX has been shown to form a complex with ERp57. Following GALA and CNX glycosylation, a CNX:ERp57 complex may form, and the CNX:ERp57 complex can be translocated to the surface of cancer cells (Ros et al., Nat. Cell Biol. 22, 1371-1381 . 2020). In some embodiments, the disease-associated CNX is a glycosylated CNX / ERp57 complex. In some embodiments, the disease- associated CNX is a cell-surface CNX / ERp57 complex.

[0080] In some embodiments, the disease is cancer. In some embodiments, the disease is arthritis. In some embodiments, the disease is associated with ECM degradation. In some embodiments, the disease is associated with the expression of glycosylated CNX. In some embodiments, the disease is associated with cell-surface CNX.

[0081] As used herein, the term “cell-surface CNX” refers to CNX at the surface of a cell.

[0082] In some embodiments, cell-surface CNX is attached to the cell membrane. In some embodiments, the cell-surface CNX is exposed to the extracellular environment. In some embodiments, the cell-surface CNX comprises an antigen which is accessible to an extracellular antibody.

[0083] In some embodiments, cell-surface CNX is present within 10 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 9 nm of the cell membrane. In some embodiments, cellsurface CNX is present within 8 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 7 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 6 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 5 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 4 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 3 nm of the cell membrane. In some embodiments, cell-surface CNX is present within 2 nm of the cell membrane. In some embodiments, cellsurface CNX is present within 1 nm of the cell membrane.

[0084] In some embodiments, the CNX is localised to an invadosome. In some embodiments, the cell-surface CNX is localised to an invadosome. In some embodiments, the cell-surface CNX is present at the surface of an invadosome. In some embodiments, the cell-surface CNX is attached to the cell membrane of an invadosome. In some embodiments, the cell-surface CNX is embedded in the cell membrane of an invadosome.

[0085] In some embodiments, the cell-surface CNX is removed from the surface of a cell during sample preparation. In some embodiments, the cell-surface CNX was present at the surface of a cell in vivo, but is removed from the surface of a cell during in vitro sample preparation. In some embodiments, cellsurface CNX remains on the surface of a cell in a biological sample (e.g., an in vitro sample).

[0086] As used herein, the term “glycosylated CNX” refers to CNX conjugated to an oligosaccharide.

[0087] Glycosylation is an enzymatic process that attaches glycans to proteins, or other biological molecules. O- glycosylation is the conjugation of O-linked glycan to the hydroxyl group of the side chain of e.g. a serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residue of a protein.

[0088] In some embodiments, the glycan is an O-linked glycan. In some embodiments, the O-linked glycan is Tn.

[0089] In some embodiments, the glycosylated CNX is O-glycosylated CNX. As used herein, the term “O- glycosylated CNX” refers to CNX conjugated to O-linked glycan.

[0090] In some embodiments, the glycosylated CNX is Tn glycosylated CNX. As used herein, the term “Tn glycosylated CNX” refers to CNX conjugated to a Tn glycan. A Tn glycosylated CNX comprises at least one Tn glycan.

[0091] Tn antigen

[0092] Mucin-type O-glycans are a class of glycans characterised by an N-acetylgalactosamine (GalNAc) linked by a glycosidic a linkage primarily to a Ser / Thr residue within a glycoprotein, and often extended or branched by sugars or saccharides. Many secretory and membrane-bound proteins receive O-glycan modification, which is important in regulating many biological processes.

[0093] Tn (also referred to as CD175, Tn antigen, or Tn glycan) is a CNX-interacting molecule. The Tn initials stand for Thomsen-nouveau. Further details on Tn are widely available in the published literature, for example Ju etal. (Angew Chem Int Ed Engl. 2011 Feb 18; 50(8): 1770-1791) review the structure and biology of Tn.

[0094] Tn is a small mucin-type O-glycan with a simple structure: GalNAc with a glycosidic a linkage to a serine / threonine residue of a glycoprotein (GalNAca1-O-Ser / Thr). GalNAc is an amino sugar derivative of galactose. The chemical formula of GalNAc is CaHisNOeand it has a molar mass of 221.21 g / mol. It is typically the first monosaccharide that connects serine or threonine in particular forms of protein O- glycosylation. Further information on GalNAc is widely available on public databases, such as ChEBI (CHEBI:40356) and CAS (1811-31-0).

[0095] Antigen binding molecules

[0096] The present disclosure provides antigen-binding molecules capable of binding to a given target antigen (e.g., CNX). An antigen-binding molecule that is capable of binding to a given target antigen may also be described as an antigen-binding molecule that binds to the given target antigen.

[0097] An ‘antigen-binding molecule’ refers to a molecule that binds to a given target antigen. In some embodiments, the antigen-binding molecule is an antibody, a lectin, or an aptamer. In some embodiments, the antigen-binding molecule is derived from an antibody, a lectin, or an aptamer.

[0098] Antigen-binding molecules include antibodies ( / .e. immunoglobulins (Igs)) and antigen-binding fragments thereof. As used herein, ‘antibodies’ include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, or single domain antibodies (e.g. VhH), etc.). Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments. In some embodiments, an antigen-binding molecule may be an antibody or an antigen-binding fragment thereof.

[0099] Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, e.g. molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules may comprise an antigen-binding region / domain that comprises, or consists of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody, or the whole antibody. For example, antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) comprising a (cytotoxic) drug moiety (e.g. as described hereinbelow). Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules such as immune cell engager molecules comprising a domain for recruiting (effector) immune cells (reviewed e.g. in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: 418-434 and Ellerman, Methods (2019) 154:102-117, both of which are hereby incorporated by reference in their entirety), including BiTEs, BiKEs and TriKEs. Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors providing both antigen-binding and T cell activating functions (CAR structure, function and engineering is reviewed e.g. in Dotti et al., Immunol Rev (2014) 257(1), which is hereby incorporated by reference in its entirety).

[0100] The antigen-binding molecule of the present disclosure comprises a moiety or moieties capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.

[0101] In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202).

[0102] In some embodiments, the moiety capable of binding to a target antigen comprises, or consists of, an antigen-binding peptide / polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody ( / .e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0103] As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0104] The antibodies of the present disclosure generally comprise an antigen-binding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen. The antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv region.

[0105] An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain. The polypeptides may associate covalently or non- covalently. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).

[0106] An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0107] The antigen-binding molecules of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to a target antigen. Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used / provided. An ‘antigen-binding region’ is any fragment of an antibody that binds to the target for which the given antibody is specific.

[0108] Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0109] The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.

[0110] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et a!., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDRs and FRs of antigenbinding molecules referred to herein are defined according to the IMGT information system.

[0111] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the FRs of an antigenbinding molecule that binds to CNX. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule that binds to CNX. That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule that binds to CNX.

[0112] In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and / or the VH and / or VL regions of an antibody described in the literature.

[0113] In some embodiments, the antigen-binding molecule which binds to CNX comprises the CDRs, FRs and / or the VH and / or VL regions of an antibody which binds to CNX, described in the literature.

[0114] In some embodiments, the antigen-binding molecule which binds to CNX comprises the CDRs, FRs and / or the VH and / or VL regions of a CNX-binding antibody clone described in W02024008960A1 , or CDRs, FRs and / or VH and / or VL regions which are derived from those of a CNX-binding antibody clone described in W02024008960A1 . In some embodiments, a CNX-binding antibody clone is selected from: 1 E1 , 1 D3, 1 D6, 1 E6, 2C6, 2H6, 3D1 , 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001 , C008, C010, C023, C025, C040, C046 and C117 described in W02024008960A1 . In some embodiments, the antigenbinding molecule which binds to CNX is 1 E1 described in W02024008960A1 .

[0115] In some embodiments, the antigen-binding molecule which binds to CNX comprises the CDRs, FRs and / or the VH and / or VL regions of 1 E1 , 1 D3, 1 D6, 1 E6, 2C6, 2H6, 3D1 , 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001 , C008, C010, C023, C025, C040, C046 or C117 (described in WG2024008960A1), AF18 (Invitrogen Cat. No. MA3-027), clone AF8 (Merck Cat. No. MABF2067), clone TO-5 (Merck Cat. No. C7617), clone 3H4A7 (Invitrogen Cat. No. MA5-15389), clone ARC0648 (Invitrogen Cat. No. MAS- 35588), clone GT1563 (GeneTex Cat. No. GTX629976), clone CANX / 1541 (GeneTex Cat. No. GTX34446), clone IE2.1C12 (Novus Biologicals Cat No. NBP2-36571), clone 1C2.2D11 (Novus Biologicals Cat No. NBP2-36570SS), clone 2A2C6 (Proteintech Cat. No. 66903-1 -Ig) clone C5C9 (Cell Signaling Technology, Inc Cat. No. 2679), clone E-10 (Santa Cruz Biotechnology Cat No. sc-46669), ab10286 (Abeam), ab22595 (Abeam), and anti-Cnx antibodies disclosed in CN 101659702 A (e.g., the antibody produced by hybridoma CGMCC No. 3240).

[0116] In some embodiments, the antigen-binding molecule which binds to CNX is 1 E1 , 1 D3, 1 D6, 1 E6, 2C6, 2H6, 3D1 , 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001 , C008, C010, C023, C025, C040, C046 or C117 (described in WC2024008960A1), AF18 (Invitrogen Cat. No. MA3-027), clone AF8 (Merck Cat. No. MABF2067), clone TO-5 (Merck Cat. No. C7617), clone 3H4A7 (Invitrogen Cat. No. MA5-15389), clone ARC0648 (Invitrogen Cat. No. MA5-35588), clone GT1563 (GeneTex Cat. No. GTX629976), clone CANX / 1541 (GeneTex Cat. No. GTX34446), clone IE2.1C12 (Novus Biologicals Cat No. NBP2-36571), clone 1C2.2D11 (Novus Biologicals Cat No. NBP2-36570SS), clone 2A2C6 (Proteintech Cat. No. 66903- 1 -Ig) clone C5C9 (Cell Signaling Technology, Inc Cat. No. 2679), clone E-10 (Santa Cruz Biotechnology Cat No. sc-46669), ab10286 (Abeam), ab22595 (Abeam), and anti-Cnx antibodies disclosed in CN 101659702 A e.g., the antibody produced by hybridoma CGMCC No. 3240).

[0117] In some embodiments, the antigen-binding molecule which binds to a CNX-interacting molecule is an antibody, a lectin, or an aptamer. In some embodiments, the antigen-binding molecule is derived from an antibody, a lectin, or an aptamer.

[0118] In some embodiments, the antigen-binding molecule which binds to Tn is an antibody, a lectin, or an aptamer. In some embodiments, the antigen-binding molecule is derived from an antibody, a lectin, or an aptamer.

[0119] In some embodiments, the antigen-binding molecule which binds to Tn is a carbohydrate-binding protein. In some embodiments, the antigen-binding molecule which binds to Tn is a lectin.

[0120] The lectin may be any lectin which is capable of binding to Tn, such as those described by Bojar et al. (ACS Chemical Biology 2022 17 (11), 2993-3012, which is hereby incorporated by reference in its entirety). In some embodiments, the lectin is a lectin from Vicia Villosa, Helix pomatia, Cytisus scoparius, Dolichos biflorus, Glycine max, Wisteria floribunda, Bauhinia purpurea, Erythrina Cristagalli, Griffonia simplicifolia, Helix aspersa, Lycopersincon esculentum, and / or Soianumm tuberosum. In some embodiments the lectin is Vicia Villosa Lectin (VVL) or Helix pomatia lectin (HPL). In some embodiments the lectin is WL.

[0121] VVL is a GalNAc specific lectin which binds to Tn. The homotetrameric lectin B4 from V. villosa is a well- known lectin, and is described in the literature. For example, the structure of the tetrameric Vicia villosa isolectin B4 (VVLB4) in complex with Tn, has been determined at 2.7 A resolution by Babino et al. (FEBS Letters 536 (2003) 1873-3468), which is hereby incorporated by reference in its entirety.

[0122] In some embodiments, the antigen-binding molecule comprises the amino acid sequence of VVL B4 (Uniprot: P56625).

[0123] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 40% sequence identity, or more preferably one of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:21 .

[0124] In some embodiments, the antigen-binding molecule is an isoform, fragment, variant or homologue of VVL B4 (Uniprot: P56625).

[0125] Isoforms, fragments, variants or homologues of VVL B4 (Uniprot: P56625) according to the present disclosure may optionally be characterised as comprising an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:21 .

[0126] In some embodiments, the VVL is a homotetramer. In some embodiments, the VVL is a heterotetramer. In some embodiments, the VVL comprises the amino acid sequence of VVL B4. In some embodiments, the VVL comprises the amino acid sequence of VVL A4. In some embodiments, the VVL comprises the amino acid sequence of VVL A2B2.

[0127] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 40% sequence identity, or more preferably one of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of VVL subunit B.

[0128] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 40% sequence identity, or more preferably one of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of VVL subunit A.

[0129] Helix pomatia lectin (HPL), alternatively known as Helix pomatia agglutinin (HPA), is a GalNAc-binding lectin found in the albumen gland of the roman snail. HPL binds to Tn. HPL is well-known, and is described in the literature. For example, biochemical and structural analysis of HPL is reported by Sanchez et al. (J Biol Chem. 2006 Jul 21 ;281 (29):20171-80), which is hereby incorporated by reference in its entirety. HPL also been shown to recognize aberrant GalNAc glycosylation in cancer, including exposed Tn epitopes (Rambaruth et al. Glycobiology. 2012 Jun;22(6):839-48, which is hereby incorporated by reference in its entirety). HPL proteins are commercially available, for example HPL lyophilized powder is available from Sigma-Aldrich (Catalogue number L3382), and HPL conjugated to Alexa Fluor 488 is available from Thermo Fisher (Catalogue number L11271).

[0130] In some embodiments, the antigen-binding molecule comprises the amino acid sequence of HPL.

[0131] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 40% sequence identity, or more preferably one of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of HPL.

[0132] In some embodiments, the antigen-binding molecule is an isoform, fragment, variant or homologue of HPL.

[0133] In some embodiments, the lectin is biotinylated. In some embodiments, the VVL is biotinylated. In some embodiments, the lectin is biotinylated VVL (B-1235-2), as used in the examples herein.

[0134] In some embodiments, the antigen-binding molecule which binds to Tn comprises the CDRs of an antigen-binding molecule that binds to Tn. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to Tn. In some embodiments, the antigenbinding molecule comprises the CDRs and the FRs of an antigen-binding molecule that binds to Tn. That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule that binds to Tn.

[0135] In some embodiments, the antigen-binding molecule which binds to Tn comprises the CDRs, FRs and / or the VH and / or VL regions of an antibody which binds to Tn, described in the literature.

[0136] In some embodiments, the antigen-binding molecule which binds to Tn comprises the CDRs, FRs and / or the VH and / or VL regions of a Tn-binding antibody clone described in WO2021072244A1 , Zlocowski et al. (Scientific Reports volume 9, Article number: 8097. 2019), Matsumoto et al. (Glycobiology. 2020 May; 30(5): 282-300), Danussi et al. (Glycobiology, Volume 19, Issue 10, October 2009, Pages 1056-1067), or Akita et al. (Int J Gynecol Cancer. 2012 May;22(4):531-8)

[0137] In some embodiments, the antigen-binding molecule which binds to Tn comprises the CDRs, FRs and / or the VH and / or VL regions of ReBaGs6 (WO2021072244A1), Remab6 (WO2021072244A1), MA1-90544 (AB_1961080; Thermo Fisher), or MLS128 (FUJIFILM). In some embodiments, the antigen-binding molecule which binds to Tn is selected from: ReBaGs6 (WO2021072244A1), Remab6 (WO2021072244A1), MA1-90544 (AB_1961080; Thermo Fisher), and MLS128 (FUJIFILM).

[0138] In some embodiments, the antigen-binding molecule which binds to ERp57 comprises the CDRs of an antigen-binding molecule that binds to ERp57. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to ERp57. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule that binds to ERp57. That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule that binds to ERp57.

[0139] In some embodiments, the antigen-binding molecule which binds to ERp57 comprises the CDRs, FRs and / or the VH and / or VL regions of an antibody which binds to ERp57, described in the literature.

[0140] Antibodies which bind to ERp57 include monoclonal antibody clone MaP.Erp57 (Enzo Life Sciences), clone CL2444 (Sigma-Aldrich Cat. No. AMAB90988), clone OTI3E1 (ThermoFisher Scientific Cat. No. TA504995), clone OTI3D2 (ThermoFisher Scientific Cat. No. TA504990), clone OTI4D7 (ThermoFisher Scientific Cat. No. TA505008) and polyclonal antibodies ab13506 and ab13507 (Abeam).

[0141] Phage display techniques may also be employed in the identification of antibodies to a given target protein / protein complex, and are well known to the skilled person. The use of phage display for the identification of fully human antibodies to human target proteins is reviewed e.g. in Hoogenboom, Nat. Biotechnol. (2005) 23, 1105-1116 and Chan etal., International Immunology (2014) 26(12): 649-657, which are hereby incorporated by reference in their entirety.

[0142] In embodiments in accordance with the present disclosure, one or more amino acids are substituted with another amino acid. A substitution comprises substitution of an amino acid residue with a non-identical 'replacement' amino acid residue. A replacement amino acid residue of a substitution according to the present disclosure may be a naturally-occurring amino acid residue ( / .e. encoded by the genetic code) which is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie): leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Vai). In some embodiments, a replacement amino acid may be a non-naturally occurring amino acid residue - i.e. an amino acid residue other than those recited in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogues such as those described in Ellman, et al., Meth. Enzym. 202 (1991) 301-336, which is hereby incorporated by reference in its entirety. In some embodiments, a substitution may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row:

[0143] By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, lie, Trp, Tyr, Phe and Norleucine.

[0144] In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0145] That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non- identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid.

[0146] In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.

[0147] The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present disclosure comprises, or consists of, an Fv region that binds to CNX. In some embodiments, the VH and VL regions of the Fv are provided as single polypeptides joined by a linker region, i.e. a single chain Fv (scFv).

[0148] The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptides joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

[0149] In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody that binds to CNX. As used herein, ‘whole antibody’ refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.

[0150] Immunoglobulins of type G (i.e. IgG) are -150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chains comprise a VL followed by a CL. Depending on the heavy chain, immunoglobulins may be classed as IgG (e.g.

[0151] IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0152] In some embodiments, the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM that binds to CNX.

[0153] In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions (e.g. CH1 , CH2, CH3, etc.) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. Ig A1 , lgA2), IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG 1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. hlgA1 , hlgA2), hlgD, hlg E or hlgM. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human lgG1 allotype (e.g. G1 m1 , G1 m2, G1 m3 or G1 m17).

[0154] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:22, 27, 28 or 31 .

[0155] In some embodiments, the antigen-binding molecule comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:23 or 29. In some embodiments, the antigen-binding molecule comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:25. In some embodiments, the antigen-binding molecule comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:26 or 30.

[0156] In some embodiments, the antigen-binding molecule comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:24.

[0157] It will be appreciated that CH2 and / or CH3 regions may be provided with further substitutions in accordance with modification to an Fc region of the antigen-binding molecule as described herein. In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.

[0158] In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:35, 36, 37, 38, 39 or 40.

[0159] In some embodiments, the antigen-binding molecule is, or comprises, a monoclonal antibody, or an antigen-binding fragment thereof.

[0160] In some embodiments, the antigen-binding molecule is, or comprises, a fully human antibody / antibody fragment. A fully human antibody / antibody fragment may be encoded by human nucleic acid sequence(s). A fully human antibody / antibody fragment may be devoid of non-human amino acid sequences. Commonly employed techniques for the production of fully human antibodies include (i) phage display, in which human antibody genes are expressed in phage display libraries, and (ii) production of antibodies in transgenic mice engineered to have human antibody genes (described in Park and Smolen, Advances in Protein Chemistry (2001) 56: 369-421). Briefly, in the human antibody genephage display technique, genes encoding the VH and VL chains are generated by PCR amplification and cloning from ‘naive’ human lymphocytes, and are subsequently assembled into a library from which they can be expressed either as disulfide-linked Fab fragments or as single-chain Fv (scFv) fragments. The Fab- or scFv-encoding genes are fused to a surface coat protein of filamentous bacteriophage, and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab / scFv fragment. In the transgenic mouse technique, mice in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues are immunized with antigen, and monoclonal antibody is prepared by conventional hybridoma technology, to yield a fully human monoclonal antibody.

[0161] In some embodiments, the antigen-binding molecule of the present disclosure is a mouse antibody / antibody fragment. In some embodiments, the antibody / antibody fragment is obtained from phage display using a human naive antibody gene library.

[0162] In some embodiments, the antigen-binding molecule is a mouse / human chimeric antibody / antibody fragment ( / .e. an antigen-binding molecule comprising mouse antibody variable domains and human antibody constant regions). In some embodiments, the antigen-binding molecule is a humanised antibody / antibody fragment. In some embodiments, the antigen-binding molecule comprises mouse antibody CDRs and human antibody framework and constant regions. Mouse / human chimeric antigen-binding molecules can be prepared from mouse antibodies by the process of chimerization, e.g. as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), which is hereby incorporated by reference in its entirety, in Chapter 8 thereof, in particular section 3 of Chapter 8.

[0163] Humanised antigen-binding molecules can be prepared from mouse antibodies by the process of humanisation, e.g. as described in Human Monoclonal Antibodies: Methods and Protocols, Michael Steinitz (Editor), Methods in Molecular Biology 1060, Springer Protocols, Humana Press (2014), which is hereby incorporated by reference in its entirety, in Chapter 7 thereof, in particular section 3.1 of Chapter 7 entitled ‘Antibody Humanization’. Techniques for antibody humanisation are also described e.g. in Safdari et al., Biotechnol Genet Eng Rev (2013) 29:175-86, which is hereby incorporated by reference in its entirety.

[0164] Methods and assays

[0165] The present disclosure also provides methods and assays. For example, the disclosure provides a method of assessing whether a biological sample comprises disease-associated CNX.

[0166] The present disclosure also provides the articles of the present disclosure for use in methods for identifying, detecting, localizing or imaging a target molecule. The articles of the present disclosure are also provided for use in a method. In some embodiments, the method is a method of identifying, detecting, localizing or imaging a target molecule.

[0167] A method of assessing whether a sample comprises disease-associated CNX may alternatively be described as a method of detecting disease-associated CNX in a sample. In some embodiments, the method is a method of predicting whether disease-associated CNX is present in a sample. In some embodiments, the method is a method of predicting the amount of disease-associated CNX present in a sample. In some embodiments, the method is a method of predicting the relative amount of disease- associated CNX present in a sample.

[0168] In some embodiments, the target molecule is disease-associated CNX. In some embodiments, the target molecule is cell-surface CNX and / or glycosylated CNX. In some embodiments, the target molecule is cellsurface CNX. In some embodiments, the target molecule is glycosylated CNX.

[0169] The antigen-binding molecules described herein may be used in methods that involve detecting binding of an antigen-binding molecule to a target antigen. Such methods may involve detection of the bound complex of the antigen-binding molecule and target antigen.

[0170] As such, a method is provided, comprising contacting a sample containing, or suspected to contain, disease-associated CNX, and detecting the formation of a complex of the antigen-binding molecule and disease-associated CNX. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing disease-associated CNX, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing disease-associated CNX.

[0171] As such, a method is provided, comprising contacting a sample containing, or suspected to contain, CNX, and detecting the formation of a complex of the antigen-binding molecule and CNX. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing CNX, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing CNX.

[0172] A method is provided, comprising contacting a sample containing, or suspected to contain, Tn, and detecting the formation of a complex of the antigen-binding molecule and Tn. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing Tn, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing Tn.

[0173] A method is provided, comprising contacting a sample containing, or suspected to contain, ERp57, and detecting the formation of a complex of the antigen-binding molecule and ERp57. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing ERp57, and detecting the formation of a complex of the antigen-binding molecule and a cell expressing ERp57.

[0174] Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The methods may involve labelling the antigen-binding molecule, or target(s), or both, with a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label, radiolabel, chemical, nucleic acid or enzymatic label as described herein. Detection techniques are well known to those of skill in the art and can be selected to correspond with the labelling agent.

[0175] Methods comprising detecting disease-associated CNX, or cells expressing disease-associated CNX, include methods for diagnosing / prognosing a disease / condition described herein.

[0176] Methods of this kind may be performed in vitro on a patient sample, or following processing of a patient sample. Once the sample is collected, the patient is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body. In some embodiments, the method is performed in vivo.

[0177] Such methods may involve detecting or quantifying disease-associated CNX and / or cells expressing disease-associated CNX, e.g. in a patient sample. Where the method comprises quantifying the relevant factor, the method may further comprise comparing the determined amount against a standard or reference value as part of the diagnostic or prognostic evaluation. Other diagnostic / prognostic tests may be used in conjunction with those described herein to enhance the accuracy of the diagnosis or prognosis or to confirm a result obtained by using the tests described herein.

[0178] In some embodiments, the disease-associated CNX is cell-surface CNX, glycosylated CNX, and / or a CNX:ERp57 complex. In some embodiments, the disease-associated CNX is cell-surface CNX. In some embodiments, the disease-associated CNX is glycosylated CNX. Detection in a sample may be used for the purpose of diagnosis of a disease / condition (e.g. a cancer), predisposition to a disease / condition, or for providing a prognosis (prognosticating) for a disease / condition, e.g. a disease / condition described herein. The diagnosis or prognosis may relate to an existing (previously diagnosed) disease / condition.

[0179] A sample may be taken from any tissue or bodily fluid. The sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, the sample may be obtained or derived from a tissue or tissues which are affected by the disease / condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease / condition).

[0180] A tissue sample may be provided in any format known to the skilled person. In some embodiments, the tissue sample is a tissue section, fixed tissue, fresh tissue, frozen tissue, a tissue block (e.g., a FFPE Block), or a tissue microarray.

[0181] The sample may be a tissue sample in the form of a tissue section. Tissue sectioning is the process of cutting tissue into thin slices. Tissue is typically embedded with an additive such as paraffin or optimal cutting temperature (OCT) prior to being sectioned. Tissue section preparation may include additional steps, such as fixation, dehydration, embedding, antigen retrieval to unmask any epitopes altered by fixation, permeabilization to grant the antibody access to intracellular proteins, and blocking to prevent non-specific staining. Tissue sections can be mounted on slides, or stored in receptacles such as tubes or plates.

[0182] Fixation of a tissue sample can help preserve tissue morphology and antigenicity of target proteins. Fixation usually occurs before embedding, but after freezing.

[0183] In some embodiments, the tissue section is a fixed tissue section. In some embodiments, the tissue section is a formalin-fixed tissue section. In some embodiments, the tissue section is a paraffin- embedded tissue section. In some embodiments, the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section.

[0184] A subject may be selected for diagnostic / prognostic evaluation based on the presence of symptoms indicative of a disease / condition described herein, or based on the subject being considered to be at risk of developing a disease / condition described herein.

[0185] The present disclosure also provides methods for selecting / stratifying a subject for treatment with a CNX- targeted agent. In some embodiments a subject is selected for treatment / prevention in accordance with the methods of the present disclosure, or is identified as a subject which would benefit from such treatment / prevention, based on a method described herein. In some embodiments, the method is a method of assessing whether a sample comprises disease- associated CNX. In some embodiments, the disease-associated CNX is cell-surface CNX, glycosylated CNX, and / or a CNX:ERp57 complex. Therefore, in some embodiments, the method is a method of assessing whether a sample comprises cell-surface CNX, glycosylated CNX, and / or a CNX:ERp57 complex. In some embodiments, the method is a method of assessing whether a sample comprises cellsurface CNX. In some embodiments, the method is a method of assessing whether a sample comprises glycosylated CNX. In some embodiments, the method is a method of assessing whether a sample comprises a CNX:ERp57 complex.

[0186] In some embodiments, the method comprises contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule.

[0187] A CNX-interacting molecule is a molecule which associates ( / .e., interacts with CNX). In some embodiments, the CNX-interacting molecule is a molecule which binds to CNX.

[0188] In some embodiments, the CNX-interacting molecule is a molecule which conjugates with CNX. In some embodiments, the CNX-interacting molecule is a glycan which is capable of glycosylating CNX. In some embodiments, the CNX-interacting molecule is an O-glycan. In some embodiments, the CNX-interacting molecule is GalNAc. In some embodiments, the CNX-interacting molecule is Tn.

[0189] In some embodiments, the CNX-interacting molecule is a molecule which forms a disease-associated CNX complex. For example, ERp57 forms a CNX:ERp57 complex with CNX, and CNX:ERp57 complexes are associated with the development of diseases such as cancer. In some embodiments, the CNX- interacting molecule is ERp57.

[0190] The method may comprise comparing the positive output with an output of a control sample. In some embodiments, the method comprises quantitatively comparing the positive output with an output of a control sample.

[0191] The control may be a suitable control identified by the skilled person. In some embodiments, the control is a negative control. In some embodiments, the control does not comprise disease-associated CNX. In some embodiments, the control is a healthy sample. In some embodiments, the control has been taken from a healthy subject. In some embodiments, the control is taken from a healthy subject. In some embodiments, the healthy subject does not have a CNX-associated disease. In some embodiments, the healthy subject does not have cancer. In some embodiments, the healthy subject does not have arthritis. In some embodiments, the healthy subject does not have a disease characterised by ECM degradation.

[0192] In some embodiments, the method comprises performing a proximity assay. Different types of proximity assays are known to the skilled person, and are discussed in further detail in the subsequent sections of the disclosure. Proximity determination

[0193] Methods disclosed herein may comprise performing a proximity assay. Proximity assays can be used in the detection of proteins, protein modifications (e.g., glycosylation), and protein-protein interactions. Proximity assays are reviewed in the literature, for example, Greenwood et al. (Biomolecular Detection and Quantification. Vol. 4, 2015, 10-16, which is hereby incorporated by reference in its entirety) review the applications and a number of different types of proximity assays.

[0194] In general, proximity assays can be used to determine whether two molecules are colocalised - this information can be used to infer that the two molecules are interacting. For example, if the assay shows that two molecules (e.g., a protein and a glycan which are known to interact, such as CNX and Tn, or two proteins which are known to interact, such as CNX and ERp57) are frequently colocalised, this would indicate that the two molecules are interacting. In some embodiments, if an assay on a sample shows that CNX and Tn are frequently colocalised, this would indicate that glycosylated CNX is present within the sample. In some embodiments, if an assay on a sample shows that CNX and ERp57 are frequently colocalised, this would indicate that a CNX:ERp57 complex is present within the sample. In some embodiments, if an assay on a sample shows that CNX and Tn are frequently colocalised, this would indicate that cell-surface CNX is present within the sample.

[0195] Proximity assays are capable of providing an output (e.g., a positive output). In some embodiments, the proximity assay is capable of producing a positive output and a negative output. In some embodiments, a lack of a positive output is considered to be a negative output.

[0196] In some embodiments, the proximity assay produces a positive output when two molecules (e.g., CNX and a CNX-interacting molecule) are colocalised. In some embodiments, the proximity assay produces a positive output when two molecules are in close physical proximity. In some embodiments, the proximity assay produces a positive output when two molecules interact.

[0197] In some embodiments, the proximity assay produces a positive output when two antigen-binding molecules are colocalised. In some embodiments, the proximity assay produces a positive output when two antigen-binding molecules are in close physical proximity. In some embodiments, the proximity assay produces a positive output when the first and the second antigen-binding molecules are colocalised. In some embodiments, the proximity assay produces a positive output when the first and the second antigen-binding molecules are in close physical proximity.

[0198] In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds to CNX and the antigen-binding molecule which binds to a CNX-interacting molecule are colocalised. In some embodiments, the proximity assay produces a positive output when the antigenbinding molecule which binds to CNX and the antigen-binding molecule which binds to a CNX-interacting molecule are in close physical proximity.

[0199] In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds to CNX and the antigen-binding molecule which binds to Tn are colocalised. In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds to CNX and the antigen-binding molecule which binds to Tn are in close physical proximity.

[0200] In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds to CNX and the antigen-binding molecule which binds to ERp57 are colocalised. In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds to CNX and the antigen-binding molecule which binds to ERp57 are in close physical proximity.

[0201] The term “close physical proximity” may mean a physical proximity such that a positive output is produced by the assay.

[0202] In some embodiments, the term “close physical proximity” means a physical interaction between two molecules, e.g., the formation of a protein-protein complex or a glycosylated protein. A physical interaction may be the formation of a non-covalent complex formed of the two molecules, e.g. via electrostatic interaction (e.g. ionic bonding, hydrogen bonding) and / or Van der Waals forces. A physical interaction may be the formation of a covalent complex.

[0203] The term “close physical proximity” may mean a physical proximity such that hybridization of oligonucleotide tags is possible. The term “close physical proximity” may mean a physical proximity such that proximity ligation is possible. For example, in a PLA, close physical proximity may mean less than 40 nm, e.g. less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some PLAs, close physical proximity may mean between 1 and 40 nm, e.g. between 10 and 30 nm.

[0204] The term “close physical proximity” may mean a physical proximity such that a fluorescent output is emitted. The term “close physical proximity” may mean a physical proximity such that energy may be transferred between two molecules. For example, in FRET / BRET based systems, close physical proximity may mean less than 100 angstroms, e.g. less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20 or less than 10 angstroms. In some FRET / BRET based systems close physical proximity may mean between 10 and 100 angstroms, e.g. between 30 and 60 angstroms.

[0205] The positive output may be visual. For example, the positive output may be electromagnetic emission e.g. detectable light, fluorescence, luminescence. A positive output may be electromagnetic emission of a given wavelength.

[0206] The positive output may be an enzymatic activity e.g. luciferase, p-galactosidase, p-lactamase, antibiotic resistance. In some embodiments the reporter system is contacted with a substrate.

[0207] In some embodiments the enzymatic activity results in a coloured reaction product. In some embodiments the enzymatic activity results in product which fluoresces or luminesces. In some embodiments the enzymatic activity imparts antibiotic resistance. The method of determining an output can vary between different proximity assays. In some embodiments, the method of determining an output comprises the analysis of a fluorescent output. In some embodiments, the positive output is a fluorescent output.

[0208] In some embodiments, the fluorescent output is generated by a fluorescent-labeled oligonucleotide, Forster resonance energy transfer (FRET), bioluminescence Resonance Energy Transfer (BRET), bimolecular fluorescence complementation (BiFC) and / or dimerization-dependent fluorescent proteins (ddFP).

[0209] The method of determining / identifying / observing an output may comprise microscopy, PCR, nucleotide sequencing, and / or flow cytometry analysis.

[0210] Microscopy can be used to visually determine a positive output. Microscopy techniques are well known to the skilled person, and suitable microscopy techniques could be used to determine positive outputs from the disclosed methods. In some embodiments, the microscopy is fluorescence microscopy. Fluorescence microscopy (e.g., confocal microscopy) can be used to determine / observe a positive output comprising fluorescence, such as an output from PLA, FRET, BRET, BiFC, ddFP, and any other method comprising a fluorescent output. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high- resolution imaging in thick tissues, and is a good example of microscopy that could be used according to the present disclosure. Confocal microscopy is used in the Examples herein, and a positive output is shown in the Figures.

[0211] PCR is well known to the skilled person, and could be used to generate and observe a positive output in methods comprising the use of oligonucleotide tags (e.g., PLAs). Different types of PCR include real- time / qPCR, reverse transcriptase PCR (RT-PCR), nested PCR, digital PCR, and in situ PCR, all of which are well known to the skilled person. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995, which is hereby incorporated by reference in its entirety. Alternative amplification technology can also be exploited. For example, rolling circle amplification (Lizardi et al., 1998, Nat Genet 19:225, which is hereby incorporated by reference in its entirety) is an amplification technology which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. Nucleotide sequencing is also very well known to the skilled person, and could be used to generate and observe a positive output in methods comprising the use of oligonucleotide tags (e.g., PLAs, and PEAs). In some embodiments, the nucleotide sequencing is DNA sequencing. In some embodiments, the nucleotide sequencing (e.g., DNA sequencing) is next-generation sequencing (NGS). NGS technologies have been previously described (Levy et al. PLoS Biol 55, e254 (2007); Wheeler et al. Nature 452:872-876 (2008); Bentley et al., Nature 456:53-59 (2008), all of which are hereby incorporated by reference in their entirety).

[0212] Flow cytometry is well known to the skilled person and could be used to determine / observe a positive output comprising fluorescence, such as an output from PLA, FRET, BRET, BiFC, ddFP, and any other method comprising a fluorescent output. Flow cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells, and may be used to measure the intensity produced by fluorescently labelled markers (e.g., fluorescent tags such as flurorphores, fluorescent protein fragments, and fluorescent proteins). Flow cytometry is described in, for example, Landy et al. (eds.), Clinical Flow Cytometry, Annals of the New York Academy of Sciences Volume 677 (1993); Bauer et al. (eds.), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91 , Humana Press (1997); and Practical Shapiro, Flow Cytometry, 4th ed., Wiley-Liss (2003); all incorporated herein by reference.

[0213] In some embodiments, the positive output indicates that the sample comprises disease-associated CNX. In some embodiments, the positive output indicates that the sample comprises cell-surface CNX. In some embodiments, the positive output indicates that the sample comprises glycosylated CNX. In some embodiments, the positive output indicates that the sample comprises Tn glycosylated CNX. In some embodiments, the positive output indicates that the sample comprises a CNX:ERp57 complex. In some embodiments, the positive output indicates that the sample comprises a cell-surface CNX:ERp57 complex. In some embodiments, the positive output indicates that the sample comprises a glycosylated CNX:ERp57 complex.

[0214] In some embodiments, the positive output indicates that the sample comprises colocalised molecules. In some embodiments, the positive output indicates that the sample comprises interacting molecules. In some embodiments, the positive output indicates that the sample comprises bound molecules.

[0215] In some embodiments, the positive output indicates that the two target antigens (e.g., CNX and Tn, or CNX and ERp57) are within a proximity that indicates that the two target antigens are colocalised. In some embodiments, the positive output indicates that the two target antigens (e.g., CNX and Tn, or CNX and ERp57) are within a proximity that indicates that the two target antigens are interacting.

[0216] In some embodiments, the positive output indicates that the target antigens (e.g., CNX and Tn, or CNX and ERp57) are within a known proximity. The known proximity may depend on the type of proximity assay that is used.

[0217] In some embodiments, the proximity assay comprises the use of fluorescent probes. In some embodiments, the proximity assay comprises the use of proximity-driven fluorescent probes. Proximity- driven fluorescent probes are reviewed in detail by Poggio et al., (Get Closer to the World of Contact Sites: A Beginner’s Guide to Proximity-Driven Fluorescent Probes. Contact. 2022;5), which is hereby incorporated by reference in its entirety. In some embodiments, the proximity assay which comprises the use of fluorescent probes is a proximity ligation assay (PLA), a proximity extension assay (PEA), a Forster resonance energy transfer (FRET) assay, a bioluminescence Resonance Energy Transfer (BRET) assay, a bimolecular fluorescence complementation (BiFC) assay and / or a dimerizationdependent fluorescent protein (ddFP) assay. In some embodiments, the fluorescent probe is hybridised to a complementary oligonucleotide. In some embodiments, the fluorescent tag is a fluorescent probe. In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a Forster resonance energy transfer (FRET) assay, a bioluminescence Resonance Energy Transfer (BRET) assay, a bimolecular fluorescence complementation (BiFC) assay and / or a dimerizationdependent fluorescent protein (ddFP) assay.

[0218] In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a resonance energy transfer assay, a protein-fragment complementation assay, or a protein dimerization assay.

[0219] In some embodiments, the resonance energy transfer assay is a BRET assay or a FRET assay. In some embodiments, the protein-fragment complementation assay is a BiFC assay. In some embodiments, the protein dimerization assay is a ddFP assay.

[0220] In some embodiments, the proximity assay comprises the use of fluorescent-labeled oligonucleotide probes, Forster resonance energy transfer (FRET), bioluminescence Resonance Energy Transfer (BRET), bimolecular fluorescence complementation (BiFC) and / or dimerization-dependent fluorescent proteins (ddFP).

[0221] In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a resonance energy transfer assay, a protein-fragment complementation assay, or a protein dimerization assay. In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a resonance energy transfer assay, or a protein-fragment complementation assay. In some embodiments, the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), or a resonance energy transfer assay. In some embodiments, the proximity assay is a proximity ligation assay (PLA) or a proximity extension assay (PEA). In some embodiments, the proximity assay is a proximity ligation assay (PLA).

[0222] Direct and indirect proximity assays are known. In a direct proximity assay, a detectable tag (e.g., an oligonucleotide tag) is conjugated to the primary antigen-binding molecules ( / .e., first and second antigenbinding molecules). In an indirect proximity assay, a detectable tag (e.g., an oligonucleotide tag) is conjugated to secondary antigen-binding molecules (e.g., first, second, third, fourth, fifth, and / or sixth antigen-binding molecules). An exemplary indirect proximity assay is shown in Figure 3.

[0223] The method comprises assessment of the proximity of two molecules (e.g., two antigen binding molecules, or CNX and a CNX-interacting molecule.

[0224] The method may comprise assessing proximity of the first antigen-binding molecule and the second antigen-binding molecule. In some embodiments, the proximity of the first antigen-binding molecule and the second antigen-binding molecule is assessed directly (e.g., through a direct proximity assay). In some embodiments, the proximity of the first antigen-binding molecule and the second antigen-binding molecule is assessed indirectly (e.g., through an indirect proximity assay). In some embodiments, the method comprises: i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, and ii. performing a PLA, wherein the PLA comprises assessing proximity of the first antigenbinding molecule and the second antigen-binding molecule, wherein a positive output from the PLA indicates that the sample comprises disease-associated CNX.

[0225] In some embodiments, the method comprises: i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, and ii. contacting the sample with an additional antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule.

[0226] In some embodiments, the method comprises: i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, ii. contacting the sample with a third antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule, and

[0227] Hi. contacting the sample with a fourth antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule.

[0228] In some embodiments, the method comprises: i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, ii. contacting the sample with a third antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule,

[0229] Hi. contacting the sample with a fourth antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule, and iv. contacting the sample with a fifth antigen-binding molecule which binds to the third antigen binding molecule or the fourth antigen-binding molecule.

[0230] In some embodiments, the method comprises: i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to a CNX-interacting molecule, ii. contacting the sample with a third antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule,

[0231] Hi. contacting the sample with a fourth antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule, iv. contacting the sample with a fifth antigen-binding molecule which binds to the third antigen binding molecule or the fourth antigen-binding molecule, and v. contacting the sample with a sixth antigen-binding molecule which binds to the third antigen binding molecule or the fourth antigen-binding molecule.

[0232] In some embodiments, the second antigen binding molecule is a lectin, and the third antigen binding molecule is an antigen binding molecule which binds lectin. In some embodiments, the second antigen binding molecule is a tagged lectin ( / .e., a lectin comprising a tag), and the third antigen binding molecule is an antigen binding molecule which binds the tagged lectin (e.g., an antigen binding molecule which binds the tag). In some embodiments, the second antigen binding molecule is a biotinylated lectin, and the third antigen binding molecule is an antigen binding molecule which binds to biotin.

[0233] In some embodiments, an antigen binding molecule (e.g., the first and / or second antigen binding molecule) is tagged, and the third antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, an antigen binding molecule (e.g., the first, second, and / or third antigen binding molecule) is tagged, and the fourth antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, an antigen binding molecule (e.g., the first, second, third, and / or fourth antigen binding molecule) is tagged, and the fifth antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, an antigen binding molecule ((e.g., the first, second, third, fourth, and / or fifth antigen binding molecule) is tagged, and the sixth antigen binding molecule is an antigen binding molecule which binds to the tag.

[0234] In some embodiments, the second antigen binding molecule is biotinylated, and another antigen binding molecule is an antigen binding molecule which binds to biotin. In some embodiments, the second antigen binding molecule is biotinylated, and the third antigen binding molecule is an antigen binding molecule which binds to biotin. In some embodiments, the second antigen binding molecule is biotinylated, and the fourth antigen binding molecule is an antigen binding molecule which binds to biotin. In some embodiments, the second antigen binding molecule is biotinylated, and the fifth antigen binding molecule is an antigen binding molecule which binds to biotin. In some embodiments, the second antigen binding molecule is biotinylated, and the sixth antigen binding molecule is an antigen binding molecule which binds to biotin.

[0235] In some embodiments, the first antigen binding molecule is an antibody which binds to CNX, and the third antigen binding molecule is an antigen binding molecule which binds the antibody which binds to CNX. In some embodiments, the first antigen binding molecule is an antibody which binds to CNX, and the fourth antigen binding molecule is an antigen binding molecule which binds the antibody which binds to CNX.

[0236] In some embodiments, the antigen binding molecule comprises a detectable tag. In some embodiments, the first, second, third, fourth, fifth, and / or sixth antigen binding molecule comprises a detectable tag. In some embodiments, the first antigen binding molecule comprises a detectable tag. In some embodiments, the second antigen binding molecule comprises a detectable tag. In some embodiments, the third antigen binding molecule comprises a detectable tag. In some embodiments, the fourth antigen binding molecule comprises a detectable tag. In some embodiments, the fifth antigen binding molecule comprises a detectable tag. In some embodiments, the sixth antigen binding molecule comprises a detectable tag.

[0237] In some embodiments, the detectable tag comprises an oligonucleotide tag, a fluorophore, a fluorescent protein, or a fluorescent protein fragment.

[0238] Proximity ligation assays (PLAs)

[0239] A PLA is one example of a proximity assay. PLAs are suitable for quantitative studies of endogenous protein expression, protein modifications and close protein interactions (Soderberg O, et al. Methods. 2008;45(3):227-32; Weibrecht et al. Expert Rev Proteomics. 2010;7(3):401-9, which are hereby incorporated by reference).

[0240] The steps of a PLA can be adapted for different situations, but a brief example of an exemplary process it provided: a sample is contacted with a pair of antigen-binding molecules (sometimes referred to in the art as PLA probes), with each member of the pair having an oligonucleotide tag. When antigen-binding molecules bind to the target antigens, free ends of the oligonucleotide tags are brought into sufficiently close proximity to hybridize together. The hybridization of the oligonucleotide tags is facilitated by a common connector oligonucleotide which serves to bridge the oligonucleotide tags when they are positioned in sufficient proximity. Once the oligonucleotide tags are hybridized, the ends of the extensions are joined together by enzymatic DNA ligation. Each oligonucleotide extension comprises a primer site for DNA amplification. Once the oligonucleotide extensions are ligated, the oligonucleotides form a continuous DNA sequence which can be amplified for detection. Amplified DNA can then be evaluated through a number of different approaches to observe a positive output.

[0241] Antigen-binding molecules that do not bind the antigens of interest do not have the corresponding oligonucleotide extensions to be brought into proximity and thus no ligation or amplification can proceed, resulting in no positive output being produced.

[0242] The detectable tag may be an oligonucleotide tag. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is less than 40 nm. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is less than less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is between 0 and 40 nm, e.g. between 1 and 30 nm.

[0243] In some embodiments, a positive output is generated when the distance between oligonucleotide tags is less than 40 nm. In some embodiments, a positive output is generated when the distance between oligonucleotide tags is less than less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance between oligonucleotide tags is between 0 and 40 nm, e.g. between 1 and 30 nm. The above outlined exemplary process could be adapted, for example, by using an indirect PLA approach. There are alternative approaches for detecting the antigen-binding molecule / antigen interaction: one uses direct a primary antigen-binding molecule, the other indirect scheme uses a secondary antigen-binding molecule linked to DNA for detection (Greenwood et al. Biomolecular Detection and Quantification. Vol. 4, 2015, 10-16).

[0244] A further exemplary PLA procedure was utilised in the Examples (e.g., Example 1) herein, and this is summarised here: (i) a biotinylated antigen-binding molecule that binds to Tn (e.g., VVL lectin) and an antigen-binding molecule that binds to CNX (e.g., an anti-CNX antibody) are added to a sample (ii) an anti-biotin antibody is introduced to the sample, (iii) an antigen-binding molecule that binds the biotinylated antigen-binding molecule, and an antigen-binding molecule that binds the antigen-binding molecule that binds to CNX, are introduced into the sample, (iv) oligonucleotide tags present on the antigen binding molecules of step (iii) are hybridized and joined through enzymatic DNA ligation, (v) rolling circle amplification is performed to generate an amplified product, (vi) the amplified product is hybridized to a complementary fluorescent probe, and (vii) fluorescence microscopy is used to identify a positive output. The skilled person would be aware that certain steps of this process could be modified, for example, a direct PLA approach could be used, qPCR could be used to generate and evaluate the amplified DNA, or flow cytometry could be used to identify a positive output.

[0245] PCR (e.g., RT-PCR, qPCR, digital PCR), nucleotide sequencing (e.g., DNA sequencing), loop-mediated isothermal amplification, fluorescence microscopy (e.g., confocal microscopy) and flow cytometry can be used to generate / observe a positive output.

[0246] PCR amplification with fluorescent probes then permit visualization of spots of proximity by fluorescence microscopy. In some embodiments, PCR amplification is performed using fluorescent probes. In some embodiments, PCR amplification is performed using fluorescent probes, and a positive output is observed through fluorescence microscopy.

[0247] Rolling circle amplification can be used to generate an amplified product, before the amplified product is hybridized to a complementary fluorescent probe. The fluorescent probe can be observed as dots using microscopy or can be observed using other techniques, such as flow cytometry. In some embodiments, rolling circle amplification is performed to generate an amplified product. In some embodiments, an amplified product is hybridized to a complementary fluorescent probe. In some embodiments, fluorescence is investigated through fluorescence microscopy. In some embodiments, fluorescence is investigated through confocal microscopy.

[0248] In some embodiments, the oligonucleotide tag of an antigen binding molecule is hybridized with the oligonucleotide tag of another antigen binding molecule. In some embodiments, the oligonucleotide tag of an antigen binding molecule and the oligonucleotide tag of another antigen binding molecule is joined through enzymatic DNA ligation. In some embodiments, rolling circle amplification is performed to generate an amplified product. In some embodiments, the amplified product is hybridized to a complementary fluorescent probe. The positive output may be a fluorescent signal. In some embodiments, the fluorescent signal is identified through fluorescence microscopy. In some embodiments, the fluorescent signal is identified through confocal microscopy. In some embodiments, the fluorescent signal is identified through flow cytometry. In some embodiments, the positive output is identified through fluorescence microscopy. In some embodiments, the positive output is identified through confocal microscopy. In some embodiments, the positive output is identified through flow cytometry.

[0249] Proximity extension assays (PEAs)

[0250] A PEA is an example of a proximity assay. PEAs are similar to PLAs, but the main difference between the two is that in a PEA the ligation event is replaced by a DNA polymerisation step. PEAs are highly compatible with DNA sequencing (Wik etal., Technological Innovation and Resources Volume 20, 100168, 2021)

[0251] In its original design, one of the PEA probes consisted of a double stranded-oligonucleotide attached to the antibody at its 3'-end, with a nine nucleotide or so 3'-overhang at its 5'-end. This overhang was complementary to the 3'-end of the oligonucleotide bound to the other antibody partner. Following incubation of the proximity probes with a sample containing antigen recognised by the probes, the overhanging 3'-end could hybridise to the 5'-oligonucleotide and, following the addition of a DNA polymerase, the free 3'-OH was extended in the 5-3' direction towards the attachment site of the 5'- oligonucleotide. This generated a full-length amplicon and hybridisation site for the upstream primer and thus allows for the amplification and detection of the target antigen by PCR. This arrangement has now been replaced by a modification, where each of the two single-stranded oligonucleotides contains a complementary site for pair-wise annealing with the other oligonucleotide, allowing extension by a DNA polymerase. This obviates the requirement for a double-stranded oligonucleotide with a 3'-overhang (Greenwood et al. (Biomolecular Detection and Quantification. Vol. 4, 2015, 10-16).

[0252] In some embodiments, the proximity assay is a PEA. In some embodiments, the oligonucleotide tag of an antigen binding molecule is hybridized with the oligonucleotide tag of another antigen binding molecule. In some embodiments, a DNA polymerization step is performed after hybridization of oligonucleotides. In some embodiments, a DNA polymerization step is performed to generate DNA amplicons. In some embodiments, DNA amplicons are detected and quantified through PCR (e.g., qPCR) or DNA sequencing (e.g., next-generation DNA sequencing). In some embodiments, a positive output is determined through PCR. In some embodiments, a positive output is determined through DNA sequencing.

[0253] The detectable tag may be an oligonucleotide tag. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is less than 40 nm. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is less than less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is between 0 and 40 nm, e.g. between 1 and 30 nm. In some embodiments, a positive output is generated when the distance between oligonucleotide tags is less than 40 nm. In some embodiments, a positive output is generated when the distance between oligonucleotide tags is less than less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance between oligonucleotide tags is between 1 and 40 nm, e.g. between 10 and 30 nm.

[0254] Resonance energy transfer assays

[0255] Resonance energy transfer systems include e.g. FRET and BRET (e.g. NanoBRET) systems, which are described e.g. in Ciruela, Curr Opin Biotechnol. (2008) 19(4):338-43, which is hereby incorporated by reference. NanoBRET is described e.g. in Machleidt et al. ACS Chem. Biol. (2015) 10: 1797-1804, which is hereby incorporated by reference.

[0256] FRET is a collision-free, but distance-dependent photophysical process where radiationless transfer of energy occurs from an excited donor (D) fluorophore to a suitable acceptor (A) protein or fluorophore via a long-range dipole-dipole coupling mechanism. FRET is effective at a distance of 1-10 nm which is equivalent to the size of a typical macromolecule (Shrestha et al., I nt J Mol Sci. 2015 Apr; 16(4): 6718- 6756, which is hereby incorporated by reference).

[0257] In resonance energy transfer assays, the detectable tag may be a fluorophore. The fluorophore may be any suitable fluorophore known to the skilled person.

[0258] The fluorophore may be a fluorescent protein. A guide to fluorescent protein pairs is provided by Bajar et al. (Sensors (Basel). 2016 Sep; 16(9): 1488), which is hereby incorporated by reference. In some embodiments, the fluorophore is ECFP, EYFP, mTurquoise2, mVenus, EGFP, mCherry, Clover, mClover3, mRuby2, mRuby3, mNeonGreen, eqFP650, iRFP, mAmetrine, tdTomato, LSSmOrange, mKate2, sREACh, ShadowG, Phanta, mTagBFP, sfGFP, or mCardinal.

[0259] In some embodiments, the antigen-binding molecule is a fluorophore conjugated antigen-binding molecule. In some embodiments, the antigen-binding molecule is a fluorophore conjugated antibody. In some embodiments, the antigen-binding molecule is a fluorophore conjugated protein. In some embodiments, the antigen-binding molecule is a fluorophore conjugated lectin.

[0260] In some embodiments, the first antigen-binding molecule is a fluorophore conjugated antibody. In some embodiments, the second antigen-binding molecule is a fluorophore conjugated antibody. In some embodiments, the antigen-binding molecule that binds CNX is a fluorophore conjugated antibody. In some embodiments, the antigen-binding molecule that binds a CNX-interacting molecule is a fluorophore conjugated antibody.

[0261] In some embodiments, the antigen-binding molecule is a fluorophore conjugated lectin. In some embodiments, the antigen-binding molecule that binds a CNX-interacting molecule is a fluorophore conjugated lectin. Compatible donor and acceptor fluorophores are well known to the skilled person, and suitable pairs of fluorophores can be selected by the skilled person. Cyan-yellow fluorophore protein (CFP-YFP) pairs are commonly used fluorophore protein pairs. In some embodiments, one detectable tag comprises a CFP fluorophore. In some embodiments, one detectable tag comprises a YFP fluorophore. In some embodiments, the fluorophore is CFP, YFP, ECFP, EYFP, mTurquoise2, mVenus, EGFP, mCherry, Clover, mClover3, mRuby2, mRuby3, mNeonGreen, eqFP650, iRFP, mAmetrine, tdTomato, LSSmOrange, mKate2, sREACh, ShadowG, Phanta, mTagBFP, sfGFP, or mCardinal.

[0262] In some embodiments, resonance energy transfer occurs when the distance between the fluorophores is less than 10 nm. In some embodiments, oligonucleotide tags are capable of hybridizing when the distance between the tags is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, resonance energy transfer occurs when the distance between the fluorophores is between 1 and 10 nm, e.g. between 2 and 9 nm.

[0263] In some embodiments, a positive output is generated when the distance between fluorophores is less than 10 nm. In some embodiments, a positive output is generated when the distance between fluorophores is less than less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance between fluorophores is between 1 and 10 nm, e.g. between 2 and 9 nm.

[0264] Protein-fragment complementation assays

[0265] Protein-fragment complementation systems produce a detectable signal upon physical interaction between the constituent fragments. Protein-fragment complementation systems include e.g. yeast-two hybrid, split ubiquitin, split luciferase, biomolecular fluorescence complementation (BiFC; e.g. split Venus), and bimolecular luciferase complementation (BiLC; e.g. NanoLuc Binary Technology (NanoBiT) systems. BiFC and split Venus systems are described e.g. in Kodama et al., Biotechniques (2010) 49(5)793-805, Ohashi et al., BioTechniques (2012) 52: 45-50 and Shyu et al., Biotechniques (2006) 40(1 ):61 -6, all of which are hereby incorporated by reference. NanoLuc and NanoBiT are described e.g. in Thirukkumaran et al., Front Chem. (2020) 7:938 and Dixon et al., ACS Chem. Biol. (2016) 11 (2): 400- 408, both of which are hereby incorporated by reference.

[0266] The reporter components of a BiFC system (e.g. split Venus system) comprise a fluorescent protein split into two complementary fragments. Generally, fluorescent proteins consist of 11 antiparallel p-strands forming a p-barrel, with an a-helix inside and several short helical structures. The chromophore, located in the a-helix within the p-barrel structure, is chemically formed by three residues. In a BiFC system the fluorescent protein is split at a loop or within a p-stand. The two resulting non-fluorescent fragments can then be fused to proteins of interest. Interaction of the target proteins will bring the fragments into proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. Molecules (and their fluorophores) must be within 1 to 10 nm distance for FRET to occur, and BiFC has been found to occur over a distance of around 7 nm (Berendzen et al., Plant Methods volume 8, Article number: 25. 2012) Many different fluorescent proteins can be used in BiFC systems e.g. GFP, EGFP, YFP, EYFP, CFP, ECFP, Venus, Citrine, Cerulean, mRFP1-Q66T, mCherry, mLumin, mNeptune, mKG, Dronpa, iRFP.

[0267] In the split Venus system the fluorescent protein is Venus, a modified yellow fluorescent protein derived from Aequorea Victoria. Several combinations of fluorescent protein N- and C- terminal fragments support biomolecular fluorescence complementation. Fragments of Venus may be designated VNx and VCy, which correspond to the amino acid sequences of 1-x and y-238 of Venus, respectively. For example, VN173 comprises amino acids 1-173 of Venus, and VC155 comprises 155-238 of Venus.

[0268] Mutations can also be introduced into the fluorescent reporter proteins which reduce background fluorescence produced by spontaneous self-assembly of the fluorescent reporter protein, e.g. as described in Kodama & Hu, BioTechniques (2018) 53(5) 285-298, which is hereby incorporated by reference.

[0269] The detectable tag may comprise a fragment of a fluorescent protein. In some embodiments, one antigen-binding molecule comprises a fragment of a fluorescent protein and another antigen binding molecule comprises a complementary fragment of the same fluorescent protein.

[0270] In some embodiments, the antigen-binding molecule comprises a fragment of GFP, EGFP, YFP, EYFP, CFP, ECFP, Venus, Citrine, Cerulean, mRFP1-Q66T, mCherry, mLumin, mNeptune, mKG, Dronpa, iRFP. In some embodiments, one antigen-binding molecule comprises a fragment of GFP, EGFP, YFP, EYFP, CFP, ECFP, Venus, Citrine, Cerulean, mRFP1-Q66T, mCherry, mLumin, mNeptune, mKG, Dronpa, or iRFP, and another antigen binding molecule comprises a complementary fragment of GFP, EGFP, YFP, EYFP, CFP, ECFP, Venus, Citrine, Cerulean, mRFP1-Q66T, mCherry, mLumin, mNeptune, mKG, Dronpa, or iRFP.

[0271] In some embodiments, one antigen-binding molecule comprises a fragment of Venus, and another antigen binding molecule comprises a complementary fragment of Venus. In some embodiments, one antigen-binding molecule comprises a fragment of GFP, and another antigen binding molecule comprises a complementary fragment of GFP. In some embodiments, one antigen-binding molecule comprises a fragment of YFP, and another antigen binding molecule comprises a complementary fragment of YFP.

[0272] In some embodiments, a positive output is generated when the distance between the protein fragments is less than 10 nm. In some embodiments, a positive output is generated when the distance between the protein fragments is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance between the protein fragments is between 1 and 10 nm, e.g. between 1 and 7 nm.

[0273] In some embodiments, a positive output is generated when the distance between molecules (e.g., CNX and the CNX-interacting molecule, or the antigen binding molecule which binds to CNX and the CNX- interacting molecule) is less than 10 nm. In some embodiments, a positive output is generated when the distance is less than less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance is between 1 and 10 nm, e.g. between 1 and 7 nm.

[0274] Protein dimerization assays

[0275] Protein dimerization assays are an example of a proximity assay, and can be used for the detection of the interaction of proteins with other molecules. A protein dimerization assay comprises the use of two proteins which are known to dimerize, which produce a positive output (e.g., fluorescence) when dimerization occurs.

[0276] Dimerization-dependent fluorescent proteins (ddFP) can be utilised in protein dimerization assays. The ddFP method consists of two dark fluorescent proteins, weakly fluorescent in their monomeric form, which are able to interact with each other in a fluorescent heterodimeric complex (Ding et al., Nat Methods. 2015 Mar; 12(3): 195-198; hereby incorporated by reference in its entirety). Engineered versions of fluorescent proteins are required to limit their natural oligomerization tendency and to increase their fluorescent signal only upon dimerization (Poggio et al., Contact. 2022;5; hereby incorporated by reference in its entirety). Alford et al (ACS Synth Biol. 2012 Dec 21 ; 1 (12): 569-575; hereby incorporated by reference in its entirety) describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants.

[0277] The detectable tag may be a protein tag (e.g., a fluorescent tag). In some embodiments, one antigenbinding molecule comprises a protein tag and another antigen binding molecule comprises a complementary protein tag. In some embodiments, one antigen-binding molecule comprises a protein tag and another antigen binding molecule comprises a complementary protein tag, wherein dimerization of the complementary proteins results in a positive output. In some embodiments, one antigen-binding molecule comprises a protein tag and another antigen binding molecule comprises a complementary protein tag, wherein dimerization of the complementary proteins results in a fluorescent emission. In some embodiments, one antigen-binding molecule comprises a protein tag and another antigen binding molecule comprises a complementary protein tag, wherein dimerization of the complementary proteins results in an increase in fluorescent emission.

[0278] The protein tag may be any suitable protein tag known by the skilled person. In some embodiments, the protein tag is a suitable protein tag disclosed by Alford et al. (ACS Synth Biol. 2012 Dec 21 ; 1 (12): 569- 575, which is hereby incorporated by reference in its entirety). In some embodiments, the detectable tag is a ddFP. In some embodiments, the detectable tag is a ddGFP, a ddYFP, or a ddRFP.

[0279] In some embodiments, a positive output is generated when the distance between the proteins is less than 10 nm. In some embodiments, a positive output is generated when the distance between the proteins is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance between the proteins is between 1 and 10 nm, e.g. between 1 and 7 nm. In some embodiments, a positive output is generated when the distance between molecules (e.g., CNX and the CNX-interacting molecule, or the antigen binding molecule which binds to CNX and the CNX- interacting molecule) is less than 10 nm. In some embodiments, a positive output is generated when the distance is less than less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1 nm. In some embodiments, a positive output is generated when the distance is between 1 and 10 nm, e.g. between 1 and 7 nm.

[0280] Therapeutic and prophylactic applications

[0281] The methods described herein (e.g., methods of assessing whether a biological sample comprises disease-associated CNX) may find use in therapeutic and prophylactic methods.

[0282] Therapeutic and prophylactic methods may comprise the steps of a method disclosed herein (e.g., a method of assessing whether a biological sample comprises disease-associated CNX), before the administration of a therapeutic or prophylactic agent.

[0283] A method of treating or preventing a disease or condition is provided, comprising:

[0284] (i) the steps of a method described herein, and

[0285] (ii) administering to a subject a therapeutically or prophylactically effective amount of an antigenbinding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0286] In some embodiments, part (i) is a method of assessing whether a biological sample comprises disease- associated CNX. In some embodiments, the method of part (i) is a method of assessing whether a biological sample comprises cell-surface CNX, glycosylated CNX, and / or a CNX:ERp57 complex.

[0287] A method of treating or preventing a disease or condition is provided, comprising:

[0288] (i) selecting or stratifying a subject for treatment, and

[0289] (ii) administering to a subject a therapeutically or prophylactically effective amount of an antigenbinding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0290] A method of treating or preventing a disease or condition is provided, comprising:

[0291] (i) selecting or stratifying a subject for treatment with a CNX-targeted agent, and

[0292] (ii) administering to a subject a therapeutically or prophylactically effective amount of a CNX- targeted agent.

[0293] A CNX-targeted agent may be an antigen-binding molecule which binds CNX. In some embodiments, the antigen-binding molecule which binds CNX is an antibody which binds CNX. In some embodiments, the antigen-binding molecule which binds CNX comprises the CDRs, FRs and / or the VH and / or VL regions of 1 E1 , 1 D3, 1 D6, 1 E6, 2C6, 2H6, 3D1 , 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001 , C008, C010, C023, C025, C040, C046 or C117 (described in WG2024008960A1). A method of treating or preventing a disease or condition is provided, comprising:

[0294] (i) selecting or stratifying a subject for treatment with an antibody that binds CNX, and

[0295] (ii) administering to a subject a therapeutically or prophylactically effective amount of an antibody that binds CNX.

[0296] A CNX-targeted agent may be provided for use in a therapeutic and prophylactic method which comprises the steps of a method described herein (e.g., a method of assessing whether a biological sample comprises disease-associated CNX).

[0297] The present disclosure provides an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0298] The methods may be effective to reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition or reduce the pathology of a disease / condition. The methods may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the methods may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the methods may prevent development of the disease / condition to a later stage (e.g. a chronic stage or metastasis).

[0299] It will be appreciated that aspects of the present disclosure may be used in the treatment / prevention of a disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of CNX, or a reduction in the number or activity of cells comprising / expressing CNX.

[0300] For example, the disease / condition may be a disease / condition in which CNX, or cells expressing CNX are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of CNX or an increase in the number / proportion of cells comprising / expressing CNX is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of CNX or an increase in the number / proportion of cells comprising / expressing CNX may be a risk factor for the onset, development or progression of the disease / condition. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of CNX, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing CNX, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition.

[0301] The disease / condition may be a disease / condition in which disease-associated CNX, or cells expressing disease-associated CNX are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of disease-associated CNX or an increase in the number / proportion of cells comprising / expressing disease-associated CNX is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of disease-associated CNX or an increase in the number / proportion of cells comprising / expressing disease-associated CNX may be a risk factor for the onset, development or progression of the disease / condition.

[0302] The disease / condition may be a disease / condition in which glycosylated CNX, or cells expressing glycosylated CNX are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of glycosylated CNX or an increase in the number / proportion of cells comprising / expressing glycosylated CNX is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of glycosylated CNX or an increase in the number / proportion of cells comprising / expressing glycosylated CNX may be a risk factor for the onset, development or progression of the disease / condition.

[0303] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of glycosylated CNX, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing glycosylated CNX, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition.

[0304] The disease / condition may be a disease / condition in which cell-surface CNX, or cells expressing cellsurface CNX are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of cell-surface CNX or an increase in the number / proportion of cells comprising / expressing cell-surface CNX is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of cell-surface CNX or an increase in the number / proportion of cells comprising / expressing cell-surface CNX may be a risk factor for the onset, development or progression of the disease / condition. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of cellsurface CNX, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing cell-surface CNX, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition.

[0305] For example, the disease / condition may be a disease / condition in which a CNX:ERp57 complex, or cells expressing a CNX:ERp57 complex are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of a CNX:ERp57 complex or an increase in the number / proportion of cells comprising / expressing a CNX:ERp57 complex is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of a CNX:ERp57 complex or an increase in the number / proportion of cells comprising / expressing a CNX:ERp57 complex may be a risk factor for the onset, development or progression of the disease / condition.

[0306] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of a CNX:ERp57 complex, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing a CNX:ERp57 complex, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition.

[0307] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition described in WO 2020 / 159445 A1 (hereby incorporated by reference in its entirety). In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition described in PCT / EP2022 / 051297 (hereby incorporated by reference in its entirety).

[0308] Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of CNX; a reduction in the activity of CNX; and / or a reduction in the number / proportion of cells comprising / expressing CNX.

[0309] Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of disease-associated CNX; a reduction in the activity of disease- associated CNX; and / or a reduction in the number / proportion of cells comprising / expressing disease- associated CNX. Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of glycosylated CNX; a reduction in the activity of glycosylated CNX; and / or a reduction in the number / proportion of cells comprising / expressing glycosylated CNX.

[0310] Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of cell-surface CNX; a reduction in the activity of cell-surface CNX; and / or a reduction in the number / proportion of cells comprising / expressing cell-surface CNX.

[0311] Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of a CNX:ERp57 complex; a reduction in the activity of a CNX:ERp57 complex; and / or a reduction in the number / proportion of cells comprising / expressing a CNX:ERp57 complex.

[0312] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated GalNAc-transferase (GALNT) activity. For example, where the disease / condition is a cancer, the cancer may comprise cells having elevated GALNT activity. As used herein ‘GALNT activity’ refers to GALNT-mediated transfer of N-acetylgalactosamine (GalNAc) from UDP- GalNAc to the hydroxyl group of the side chain of e.g. a serine or threonine residue. An ‘elevated’ level of GALNT activity may refer to a level of GALNT activity which is greater than the level of GALNT activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Where the disease / condition is a cancer, the level of GALNT activity may be greater than the level of GALNT activity in equivalent non-cancerous cells / non-tumor tissue. A cancer / cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells / non-tumor tissue) causing upregulation of GALNT activity.

[0313] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated O-glycosylation. For example, where the disease / condition is a cancer, the cancer may comprise cells having an elevated level of O-glycosylation of a protein expressed by the cells. An ‘elevated’ level of O-glycosylation may refer to a level of O-glycosylation which is greater than the level of O-glycosylation in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Where the disease / condition is a cancer, the level of O-glycosylation may be greater than the level of O-glycosylation in equivalent non-cancerous cells / non-tumor tissue. A cancer / cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells / non-tumor tissue) causing upregulation of O-glycosylation.

[0314] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated Tn-glycosylation. For example, where the disease / condition is a cancer, the cancer may comprise cells having Tn-glycosylation of a protein expressed by the cells. An ‘elevated’ level of Tn-glycosylation may refer to a level of Tn-glycosylation which is greater than the level of Tn-glycosylation in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent nondiseased tissue). Where the disease / condition is a cancer, the level of Tn-glycosylation may be greater than the level of Tn-glycosylation in equivalent non-cancerous cells / non-tumor tissue. A cancer / cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells / non-tumor tissue) causing upregulation of Tn-glycosylation.

[0315] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by extracellular matrix (ECM) degradation. A disease / condition which is ‘characterised by ECM degradation’ may be a disease / condition in which ECM degradation is a symptom of the disease / condition.

[0316] The disease / condition to be treated / prevented in accordance with the present disclosure may be a disease / condition in which ECM degradation is pathologically-implicated. For example, the disease / condition may be a disease / condition in which ECM degradation, and / or an increased level of ECM degradation, is implicated in the pathology of the disease / condition.

[0317] Diseases / conditions characterised by ECM degradation include e.g. cancers, and diseases / conditions characterised by cartilage degradation (e.g., arthritis).

[0318] The involvement of ECM degradation in the development and progression of cancers is well known, and is reviewed e.g. in Walker et al., Int. J. Mol. Sci. (2018) 19(10): 3028, Najafi et al., J. Cell Biochem. (2019) 120(3):2782-2790 and Winkler et al., Nat. Commun. (2020) 11 (1):5120, all of which are hereby incorporated by reference in their entirety.

[0319] In some embodiments the disease / condition to be treated / prevented is a cancer. Cancer may refer to any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and / or white blood cells.

[0320] Tumors may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.

[0321] In some embodiments, the cancer is selected from: liver cancer, breast cancer, oral cancer (e.g. oral squamous cell carcinoma) sarcoma, lung cancer, prostate cancer, bladder cancer, renal cancer, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer and thyroid cancer. In some embodiments, the cancer is selected from liver cancer (e.g. hepatocellular carcinoma), breast cancer (e.g. triple-negative breast cancer), oral cancer (e.g. oral squamous cell carcinoma), sarcoma, lung cancer, prostate cancer, bladder cancer, renal cancer, melanoma, pancreatic cancer, endometrial cancer, colorectal cancer, thyroid cancer, gastric cancer, bile duct cancer (e.g. cholangiocarcinoma), ovarian cancer, and oesophageal cancer,

[0322] In some embodiments, the liver cancer is a primary liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar carcinoma, bile duct cancer (cholangiocarcinoma), angiosarcoma or hepatoblastoma.

[0323] In some embodiments, the breast cancer is a primary breast cancer. In some embodiments, the breast cancer is ductal carcinoma, lobular carcinoma, in situ breast cancer (e.g. ductal carcinoma in situ (DCIS) invasive carcinoma (e.g. invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), triple negative breast cancer or inflammatory breast cancer), Paget disease, angiosarcoma or Phyllodes tumor.

[0324] Treatment of a subject with an antigen-binding molecule in accordance with the present disclosure may: delay / prevent the onset of one or more symptoms of the cancer; reduce the severity of one or more symptoms of the cancer; increase survival of the subject; reduce / inhibit survival of cells of the cancer; reduce the number of cells of the cancer in the subject; reduce tumor size / volume; reduce cancer / tumor burden in the subject; reduce / inhibit growth of cells of the cancer; reduce / inhibit tumor growth; reduce / inhibit invasion by cells of the cancer; and / or reduce / inhibit metastasis of the cancer.

[0325] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is cartilage degradation, or a disease / condition characterised by cartilage degradation.

[0326] As used herein, ‘cartilage degradation’ refers to the degradation / degeneration / loss / destruction of cartilage tissue. Cartilage tissue is formed of chondrocytes, and extracellular matrix rich in glycosaminoglycans, proteoglycans, collagen and in some instances also elastin.

[0327] Cartilage is an avascular, aneural, alymphatic connective tissue found in the synovial joints, spine, ribs, external ears, nose, and airways, and in the growth plates of children and adolescents. There are three major types of cartilage found in humans: hyaline, fibrous and elastic (Wachsmuth et al., Histol Histopathol. 2006 May; 21 (5) :477-85) . Hyaline cartilage is the most widespread type of cartilage and is the type that makes up the embryonic skeleton. It persists in human adults at the ends of bones in free- moving joints as articular cartilage, at the ends of the ribs, and in the nose, larynx, trachea, and bronchi. Fibrocartilage is tough, strong tissue found predominantly in the intervertebral disks and at the insertions of ligaments and tendons; it is similar to other fibrous tissues but contains cartilage ground substance and chondrocytes. Elastic cartilage is more pliable than the other two forms because it contains elastin fibres in addition to collagen. In humans it makes up the external ear, the auditory tube of the middle ear, and the epiglottis.

[0328] In some embodiments, the cartilage degradation may be of hyaline cartilage, fibrous cartilage and / or elastic cartilage.

[0329] In most tissues, fibroblasts are the key cell type involved in producing extracellular matrices. However, fibroblasts can also degrade the matrix, allowing the turn-over of this essential component of tissues. How fibroblasts regulate these two opposite activities remains unclear. Synovial fibroblasts (SF) also called synoviocytes are the prototypical Janus-faced cells. In healthy individuals, SFs contribute to the viscosity of the synovial fluid by secreting proteins such as hyaluronic acid and lubricin (Jay et al., J. Rheumatol. 27, 594-600, 2000). In arthritic diseases, SFs adhere and degrade the cartilage, specifically the extracellular matrix (ECM) of the cartilage. Understanding this change in activity during arthritis has been a major focus of research in recent years (Ospelt. RMD Open. 3, e000471 , 2017). The GALNTs Activation pathway (GALA) regulates ECM degradation in cancer cells through glycosylation of MMP14 and CNX.

[0330] GALA induces matrix degradation through at least two mechanisms. First, it stimulates glycosylation of MMP14, which is required for its proteolytic activity (Nguyen et al., Cancer Cell. 32, 639-653. e6, 2017). Second, GALA induces the glycosylation of the ER-resident protein CNX, which forms a complex with ERp57 (Ros et al., Nat. Cell Biol. 22, 1371-1381. 2020). Following GALA-glycosylation, a fraction of the CNX:ERp57 complex is translocated to the surface of cancer cells. The CNX:ERp57 complex accumulates in invadosomes and reduces disulfide bridges in the ECM (Ros etal., Nat. Cell Biol. 22, 1371-1381. 2020). This reduction of disulfide bridges is essential for the effective degradation of ECM (Ros etal., Nat. Cell Biol. 22, 1371-1381 . 2020).

[0331] Aspects and embodiments of the present disclosure relate to the treatment / prevention of diseases / conditions characterised by cartilage degradation. A disease / condition which is ‘characterised by cartilage degradation’ is a disease / condition in which cartilage degradation is a symptom of the disease / condition. The disease / condition to be treated / prevented in accordance with the present disclosure may be a disease / condition in which cartilage degradation is pathologically-implicated. For example, the disease / condition may be a disease / condition in which cartilage degradation, and / or an increased level of cartilage degradation, is implicated in the pathology of the disease / condition.

[0332] Cartilage degradation can occur through, and / or lead to the development, progression or worsening of, disorders such as osteoarthritis, psoriasis arthritis, rheumatoid arthritis, juvenile arthritis, post-traumatic arthritis, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage damage, and polychondritis. Cartilage degradation can also occur as a consequence of physical trauma / mechanical damage, e.g. through sports injury (e.g. as a consequence of collision, or hyperextension) or surgery. Subjects having cartilage degradation commonly experience joint pain, stiffness, and inflammation, which can impact quality of life.

[0333] In some embodiments, a disease / condition characterised by cartilage degradation in accordance with the present disclosure may be selected from: a joint disorder, arthritis, osteoarthritis, psoriasis arthritis, rheumatoid arthritis juvenile arthritis, post-traumatic arthritis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, cartilage damage and polychondritis.

[0334] Arthritis is a group of diseases affecting joints (Barbour et al., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056). Rheumatoid arthritis (RA) and Osteoarthritis (OA) are two of the most common types (Murphy and Nagase. Nat. Clin. Pract. Rheumatol. 4, 128-135. 2008). It is thought that mechanical damage to the cartilage leads to a low-grade inflammatory condition that mediates progressive cartilage loss in arthritis (Kapoor et al., Nat. Rev. Rheumatol. 7, 33-42. 2011 ; Pap and Korb-Pap. Rheumatol. 11 , 606-615. 2015). Post-traumatic arthritis (PTA) develops after an acute direct trauma to the joints. PTA causes about 12% of all osteoarthritis cases, and a history of physical trauma may also be found in patients with chronic inflammatory arthritis.

[0335] In healthy synovial joints, the synovial membrane surrounds and isolates the joint cavity, secreting extracellular matrix proteins in the synovial fluid. Synovial fibroblasts are the main stromal cells of the synovial membrane, interspaced with resident macrophages (Barbour etal., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056). During the active phases of RA, SFs become activated, expressing the Fibroblast Activation Protein alpha, and proliferate. SF cells, as other stromal cells, express innate immune receptors such as Toll-Like Receptors. They can detect local pathogens and molecular damage, secreting cytokines that activate immune cells (Ospelt et al., Arthritis Rheum. 58, 3684-3692. 2008). During inflammation, SF proliferate, forming, together with infiltrating immune cells, an enlarged synovial membrane called a pannus (Choy. Rheumatology. 51 Suppl 5, v3-11. 2012). The pannus invades the joint cavity and degrades cartilage (Pap and Korb-Pap. Rheumatol. 11 , 606-615. 2015). In particular, SF in the synovial lining layer have been shown to mediate cartilage degradation, while SF in the sub-lining tend to mediate inflammation (Croft et al., Nature. 570, 246-251. 2019). The ECM degrading activity is due to increased production of matrix metalloproteinases (MMPs), Disintegrin, Metalloproteinases with Thrombospondin motifs (ADAMTs), and cathepsins (Rengel and Ospelt. Arthritis Res. Ther. 9, 221 2007). Arthritic synovial fibroblasts express both secreted (Jay et al., J. Rheumatol. 27, 594-600, 2000; Barbour et al., Morbidity and Mortality Weekly Report. 65. 2016, pp. 1052-1056; Smolen et al., Nature Reviews Disease Primers. 4. 2018. doi:10.1038 / nrdp.2018.1) and cell surface MMPs (Lange-Brokaar et al., Osteoarthritis Cartilage. 20, 1484-1499. 2012; Nygaard and Firestein. Nat. Rev. Rheumatol. 16, 316-333. 2020; Bauer et al., Arthritis Res. Ther. 8, R171 ; 2006) MMPs. MMP14 (MT1- MMP) in particular is essential for the invasive properties of SFs.

[0336] The acquisition of aberrant matrix degradation is also characteristic of SFs in OA (Fuchs et al., Osteoarthritis Cartilage. 12, 409-418. 2004). While the OA synovial membrane typically has fewer immune cells than in RA, it drives cartilage degradation as in RA. What controls the switch to ECM- degradation mode of SFs is not well understood. Changes in gene expressions are obviously suspected; similar transcriptional signatures have been detected in both diseases (Cai et al., J Immunol Res. 2019, 4080735. 2019). Epigenetic changes have been detected and proposed to drive the phenotype of arthritic SFs (Nakano eta!., Ann. Rheum. Dis. 72, 110-117. 2013).

[0337] The phenotype of SFs during arthritis has been compared to that of malignant cancer cells. Indeed, cancer growth requires a profound remodelling of the ECM in the tissue of origin, with degradation of the original tissue ECM (Hotary et al., Cell. 114, 33-45. 2003). MMPs and other matrix degradation enzymes are particularly active in malignant cells (Castro-Castro et al., Cell Dev. Biol. 32, 555-576. 2016).

[0338] Gout is an inflammatory type of arthritis, also known as gouty arthritis. Gout is the most common inflammatory arthritis with a prevalence of 2.5% in the UK. Although it has the potential to be cured, its treatment remains suboptimal (Abhishek et al., Clin Med (Lond). 2017 Feb; 17(1): 54-59). The ultrasonographic findings of gout include double contour sign (MSU crystal deposition on surface of hyaline articular cartilage). Normal adult articular cartilage is made up of an abundant ECM composed mainly of type II collagen fibrils interspersed with types IX and XI collagens. Cartilage loss tends to be a late feature of gouty arthropathy and, similar to bone erosion, is localized rather than diffuse. Cartilage damage is often associated with erosion and has been described as occurring in regions of biomechanical stress.

[0339] Chondrocalcinosis, or cartilage calcification, is calcification (accumulation of calcium salts) in hyaline cartilage and / or fibrocartilage. Build-up of calcium phosphate in the ankle joints 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 often found in weight bearing joints such as the hip, ankle and knee. The molecular structure of calcium pyrophosphate has the potential to trigger inflammatory responses. The presence of chondrocalcinosis has associations with the degradation of cartilage menisci and synovial tissue. It has been reported that presence of calcium-containing crystals, which are associated with chondrocalcinosis, is associated with higher prevalence of cartilage and meniscal damage (Gersing et al., Eur Radiol. 2017 Jun;27(6):2497-2506. doi: 10.1007 / s00330-016-4608-8. Epub 2016 Oct 4).

[0340] Fibromyalgia (FM) is a medical condition characterized by chronic widespread pain and a heightened pain response to pressure. FM is common in rheumatoid arthritis, axial spondyloarthritis and psoriatic arthritis, and could therefore influence management of these rheumatic conditions. FM is also associated with costochondritis.

[0341] Costochondritis is an inflammation of the cartilage in the rib cage. The condition usually affects the cartilage where the upper ribs attach to the breastbone, or sternum, an area known as the costosternal joint or costosternal junction. Costochondritis can be caused by mechanical stress, leading to cartilage loss and / or ECM degradation. Osteochondritis dissecans (OCD or OD) is a disorder in which cracks form in the articular cartilage and the underlying subchondral bone. OCD usually causes pain during and after sports. In later stages of the disorder there will be swelling of the affected joint which catches and locks during movement. Physical examination in the early stages can identify pain as symptom; in later stages there could be an effusion, tenderness, and a crackling sound with joint movement. Treatment to prevent, reduce, or reverse ECM degradation and / or cartilage loss would benefit patients with osteochondritis dissecans. In some cases, the osteochondritis dissecans to be treated is associated with ECM degradation and / or cartilage loss. Polychondritis, or relapsing polychondritis (RP), is an immune-mediated systemic disease characterized by recurrent inflammatory episodes of cartilaginous and proteoglycan-rich tissues, including the elastic cartilage of the ear and nose, the hyaline cartilage of peripheral joints, the fibrocartilage at axial sites and the cartilage of the tracheobronchial tree, which result in progressive anatomical deformation and functional impairment of the involved structures (Borgio et al., Biomedicines. 2018 Sep; 6(3): 84). Mono- or, more frequently, bilateral auricular chondritis is the most common feature of RP, which is observed in up to 90% of patients during the course of the disease, and is the inaugural symptom in 20% of cases. The onset is abrupt, with painful red to violaceous erythema and edema confined to the cartilaginous part of the ear, typically sparing the lobe as this lacks cartilage. Acute inflammatory episodes tend to resolve spontaneously within a few days or weeks, with recurrence at variable intervals. As a long-term consequence of repeated flares, the cartilage matrix is severely damaged and replaced by fibrous connective tissue (Borgio et al., Biomedicines. 2018 Sep; 6(3): 84).

[0342] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is a joint disorder. A joint is defined as a connection between two bones in the skeletal system. Joints can be classified by the type of the tissue present (fibrous, cartilaginous or synovial), or by the degree of movement permitted (synarthrosis, amphiarthrosis or diarthrosis). Therefore, a joint disorder is defined as a condition which affects a connection between two bones in the skeletal system. Definitions of specific joints and related aspects of anatomy can be found in “Netter, F. H. (2006). Atlas of human anatomy. Philadelphia, PA: Saunders / Elsevier”, which is incorporated by reference in its entirety. The joint disorder may affect a fibrous, cartilaginous or synovial joint.

[0343] Fibrous joints are connected by dense connective tissue consisting mainly of collagen. These joints are also called fixed or immovable joints because they do not move. Fibrous joints have no joint cavity and are connected via fibrous connective tissue. The skull bones are connected by fibrous joints called sutures.

[0344] Cartilaginous joints are a type of joint where the bones are entirely joined by cartilage, either hyaline cartilage or fibrocartilage. These joints generally allow more movement than fibrous joints but less movement than synovial joints.

[0345] A synovial joint is characterised by the presence of a fluid-filled joint cavity, contained within a fibrous capsule. It is the most common type of joint found in the human body and contains several structures which are not seen in fibrous or cartilaginous joints. The three main features of a synovial joint are: (i) an articular capsule, (ii) articular cartilage, and (iii) synovial fluid. The articular capsule surrounds the joint and is continuous with the periosteum of articulating bones. The articulating surfaces of a synovial joint (i.e. the surfaces that directly contact each other as the bones move) are covered by a thin layer of hyaline cartilage. The articular cartilage has two main roles: (a) minimising friction upon joint movement, and (b) absorbing shock. The synovial fluid is located within the joint cavity of a synovial jointSynovial joints can include accessory structures such as tendons, ligaments, bursae, and vasculature. There are numerous types of synovial joints. In some cases, the joint disorder is of a gliding joint, a hinge joint, a pivot joint, an ellipsoid joint, saddle joint, or a ball and socket joint. A gliding joint, also known as a plane joint or planar joint, is a common type of synovial joint formed between bones that meet at flat or nearly flat articular surfaces. Gliding joints allow the bones to glide past one another in any direction along the plane of the joint — up and down, left and right, and diagonally. Slight rotations can also occur at these joints, but are limited by the shape of the bones and the elasticity of the joint capsule surrounding them. A hinge joint (ginglymus) is a bone joint in which the articular surfaces are molded to each other in such a manner as to permit motion only in one plane. According to one classification system they are said to be uniaxial (having one degree of freedom) (Platzer, Werner (2008) Color Atlas of Human Anatomy, Volume 1). The direction which the distal bone takes in this motion is seldom in the same plane as that of the axis of the proximal bone; there is usually a certain amount of deviation from the straight line during flexion. The articular surfaces of the bones are connected by strong collateral ligaments. The best examples of ginglymoid joints are the I nterphalangeal joints of the hand and those of the foot and the joint between the humerus and ulna. The knee joints and ankle joints are less typical, as they allow a slight degree of rotation or of side-to-side movement in certain positions of the limb. The knee is the largest hinge joint in the human body. A pivot joint (trochoid joint, rotary joint or lateral ginglymus) is a type of synovial joint whose movement axis is parallel to the long axis of the proximal bone, which typically has a convex articular surface. According to one classification system, a pivot joint has one degree of freedom (Platzer, Werner (2008) Color Atlas of Human Anatomy, Volume 1). An ellipsoid joint (also called a condyloid joint) is an ovoid articular surface, or condyle that is received into an elliptical cavity. This permits movement in two planes, allowing flexion, extension, adduction, abduction, and circumduction, as seen in the wrist joint. A saddle joint is a type of synovial joint in which the opposing surfaces are reciprocally concave and convex. It is found in the thumb, the thorax, and the middle ear, and the heel. A ball and socket joint (or spheroid joint) is a type of synovial joint in which the ball-shaped surface of one rounded bone fits into the cup-like depression of another bone. The distal bone is capable of motion around an indefinite number of axes, which have one common centre. This enables the joint to move in many directions.

[0346] The joint disorder may affect a synarthrosis, amphiarthrosis or diarthrosis joint. The hip and shoulder are ball and socket joints. A synarthrosis is a type of joint which allows no movement under normal conditions. Sutures and gomphoses are both synarthroses. An amphiarthrosis is a joint that has limited mobility. An example of this type of joint is the cartilaginous joint that unites the bodies of adjacent vertebrae. A diarthrosis joint is a freely moveable joint. Sometimes the terms diarthrosis joints and synovial joints are used interchangeably.

[0347] The joint disorder may affect any joint. In some cases, the joint disorder affects the hip, knee, ankle, foot, toe, shoulder, elbow, wrist, hand, finger, neck, spine, ribs, or sacroiliac joint. In some embodiments, a joint disorder is selected from: osteoarthritis, psoriasis arthritis, rheumatoid arthritis juvenile arthritis, post-trauma arthritis, bursitis, gout, chondrocalcinosis, fibromyalgia, costochondritis, osteochondritis dissecans, polychondritis, cartilage damage, tendon damage, and ligament damage.

[0348] Bursitis is inflammation of a bursa, a small fluid-filled sac that acts as a cushion between bone and muscle, skin or tendon. The type of bursitis depends on where the affected bursa is located. This soft tissue condition commonly affects the shoulder, elbow, hip, buttocks, knees and calf. Athletes, the elderly and people who do repetitive movements like manual laborers and musicians are more likely to get bursitis. Bursitis is sometimes mistaken for arthritis because the pain can occur in a joint.

[0349] Tendon damage, or tendinopathy, can be caused in a number of ways, for example from overuse, aging, wear and tear, or a mechanical injury. Tendon damage may be tendinitis or tendinosis. Tendinitis refers to inflammation of a tendon, and tendinosis relates to tears in the tissue in and around the tendon. The tendon damage may be a strained tendon, a sprained tendon, a torn tendon, a partially ruptured tendon or a completely ruptured tendon.

[0350] Ligament damage can be caused in a number of ways for example from overuse, aging, wear and tear, or a mechanical injury. The ligament damage may be a strained ligament, a sprained ligament, a torn ligament, a partially ruptured ligament or a completely ruptured ligament.

[0351] Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being an amount sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered.

[0352] Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0353] Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral.

[0354] In some aspects and embodiments in accordance with the present disclosure there may be targeted delivery of articles of the present disclosure, i.e. wherein the concentration of the relevant agent in the subject is increased in some parts of the body relative to other parts of the body. In some embodiments, the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the relevant article is formulated in a targeted agent delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes and micro-sized silica rods. Such systems may comprise a magnetic element to direct the agent to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to one skilled in the art.

[0355] In some cases, the articles of the present disclosure are formulated for targeted delivery to specific cells, a tissue, an organ and / or a tumor.

[0356] Administration may be alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated. The antigen-binding molecule or composition described herein and a therapeutic agent may be administered simultaneously or sequentially.

[0357] In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment / prevention of a cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and / or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments, the therapeutic or prophylactic intervention comprises a stem cell transplant.

[0358] Simultaneous administration refers to administration of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the antigen-binding molecule / composition or therapeutic agent, followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

[0359] Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or y-rays). The drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.

[0360] The chemotherapy may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment. The treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc. For a co-therapy, a single treatment regime may be provided which indicates how each drug is to be administered.

[0361] Chemotherapeutic drugs may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE- PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S- Malate), Cabozantinib-S-Malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, lnterleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vi norelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-lntron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R- CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa- 2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (Abiraterone Acetate).

[0362] In some embodiments, the treatment may comprise administration of a corticosteroid, e.g. dexamethasone and / or prednisone.

[0363] In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment / prevention of cartilage degradation / a disease / condition characterised by cartilage degradation. Such intervention includes palliation (e.g., chondroplasty and debridement), repair e.g., drilling and microfracture [MF]), and restoration (e.g., autologous chondrocyte implantation [ACI], osteochondral autograft [OAT], and osteochondral allograft [OCA]) (Richter et al., Sports Health. Mar-Apr 2016;8(2):153-60. doi: 10.1177 / 1941738115611350. Epub 2015 Oct 12).

[0364] Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.

[0365] Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

[0366] Subjects

[0367] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use).

[0368] The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0369] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to a method described herein.

[0370] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of disease-associated CNX. For example, in some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cell-surface CNX and / or glycosylated CNX.

[0371] Kits

[0372] In some aspects of the present disclosure, a kit of parts is provided. In some embodiments, the kit may have at least one container having a predetermined quantity of an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0373] In some embodiments, the kit may comprise materials for producing an antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein. The kit may provide the antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions relating to a method described herein.

[0374] 5

[0375] The kit may provide the antigen-binding molecule, polypeptide, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions for administration to a patient in order to treat a specified disease / condition.

[0376] 10 In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition.

[0377] 15

[0378] Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0379] 20 Sequence identity

[0380] As used herein, ‘sequence identity’ refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining

[0381] 25 percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When

[0382] 30 using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0383] Sequences

[0384] ***

[0385] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0386] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0387] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0388] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0389] As used herein, an amino acid sequence or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960).

[0390] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0391] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0392] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0393] Brief Description of the Figures

[0394] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. Figure 1 . Glycosylated CNX is transported to the cell surface. (Left) Normal cells contain GalNAc- T enzyme in the golgi apparatus, separated from Calnexin, and no presence of Calnexin on cell surface, (right) In disease cells where GalNac-T activated pathway is present with activation of an oncogene such as Src or EGFR / PDGF, GalNAc-T enzymes are relocated to the endoplasmic reticulum by unbalanced retrograde traffic. This leads to aberrant Tn glycosylation of Calnexin, which leads to relocation of Calnexin to the cell membrane.

[0395] Figure 2. Schematic of the Sleeping Beauty inducible tet-on transposon plasmid system. The plasmid contains a tetracycline-responsive promoter (TCE), and a fusion of an ER-localized signal (derived from p33 MHC) to a GALNT1 cassette (ERG1). Doxycycline (Dox) addition triggers the expression of GALNT1 localized to the ER.

[0396] Figure 3. Schematic representation of an exemplary proximity assay, in this case a proximity ligation assay (PLA), for identifying glycosylated CNX in a biological sample by detecting the co-presence of Tn and Calnexin.

[0397] Figure 4. Results of a proximity assay, detecting glycosylated Calnexin in Huh7 inducible ERG1 cells. (Top) Representative images of Huh7 inducible ERG1 cells, fixed and permeabilized, in the presence (+Dox) or absence (-Dox) of Doxocycline, and Huh7 CNX- / - cells with Calnexin gene knockout achieved by Cas9 gene editing. Images show far-red PLA signals as spots and Hoechst staining for nuclei merged. Antibody test combination and cell condition are indicated for each image. Images are presented in grayscale. An inset is presented for ERG1+Dox with Calnexin antibody, VVL biotinylated, anti-biotin and PLA reagent to highlight PLA spots presence. Captured using a Phenix Opera laser spinning disk confocal microscope with a 63X water immersion objective. (Bottom) Automatic quantification of PLA spots per cell, derived from high-content imaging of 24 images per well and maximum projection across 3 planes. Quantification was performed with Columbus Imaging software, focusing on a cytoplasmic proximal ring relative to nuclei, where spots were detected using a thresholding method. The graph presents quantification from a single cell population per well. Statistical significance (p < 0.0001) is indicated for comparisons between Huh7 ERG1 + Dox vs. Huh7 ERG1 - Dox, or Huh7 ERG1 + Dox vs. Huh7 CNX- / - for PLA with Calnexin antibody, WL-biotinylated, anti-Biotin, and PLA reagents.

[0398] Figure 5. Results of a proximity assay detecting glycosylated Calnexin in various cell lines. (A) Representative images of various cell lines tested. The top image depicts nuclei / hoechst fluorescence. Bottom Images show respective far-red PLA fluorescent signal. Images are presented in grayscale with fixed settings. Images were captured using a Phenix Opera laser spinning disk confocal microscope with a 63X water immersion objective. (B) automatic quantification of intensity per cell, derived from high- content imaging of 10 images per well. Quantification was performed with Columbus Imaging software, focusing on a cytoplasmic proximal ring relative to nuclei where general intensity was quantified. The graph presents quantification from a single cell population per well as a boxplot. Figure 6. Tissue section analysis using a proximity assay. (Top) Representative images of liver section from a mouse injected with a Sleeping Beauty plasmid containing shp53, NRASG12V, and ERG1. Hoechst fluorescent channel is depicted. (Middle) The same tissue section with fluorescent far-red signal visualised using Calnexin antibody and VVL-biotinylated anti-biotin. The glycosylated-CNX-positive signal is localised in both large and small tumors generated by the plasmid. Imaging was performed with a Phenix Opera 20X spinning disk laser confocal microscope with a water immersion objective. (Bottom): Quantification of glycosylated-CNX-positive signal per cell from single-cell data across the tissue section shown in the top and middle panels. Data are classified into two groups: fields of view with glycosylated- CNX-positive signal (labeled as the liver tumor group) and fields of view without glycosylated-CNX- positive signal (labeled as the liver non-tumor group). Statistical analysis using a t-test is provided, with the p-value indicated for comparison between the two groups.

[0399] Figure 7. Results from analysis of liver cancer tissue microarray. (A) Representative images of liver cancer tissue microarray LV8011 from BioMax, displaying the Hoechst fluorescent channel. (B) The same liver cancer tissue microarray LV8011 showing fluorescent far-red signal, produced using Calnexin antibody, and VVL-biotinylated anti-biotin. Imaging was performed with a Phenix Opera 20X spinning disk laser confocal microscope with a water immersion objective. The tissue cores are classified as Normal (N), Inflamed (Ini), Hyperplasia (H), Cancer Grade 1 (1), Cancer Grade 2 (2), Cancer Grade 3 (3), and Metastatic (M). (C) Quantification of glycosylated-CNX-positive signal per core using single-cell data. Cells are classified based on their location within annotated fields of view. The number of positive cells is counted using a fixed threshold and related to the total number of cells in each core to determine the fraction of positive cells. Statistical analysis with a t-test is provided, with p-values indicated for comparisons with the Normal core group (N). (D) Quantification of glycosylated-CNX-positive signal per core using single-cell data, converted into an H-score. Positive cells are categorized into three groups based on intensity and with increased weight respectively: low, medium, and high. The H-score is calculated by relating these categories to the total number of cells in each core. Statistical analysis with a t-test is provided, with p-values indicated for comparisons with the Normal core group (N).

[0400] Figure 8. Results from analysis of Xenograph tissue microarray. (A) Representative images of cell derived xenograph (CDX) tissue microarray TMA-CP_MX-006 from Crown Biosciences, displaying Hoechst fluorescent channel on left and fluorescent far-red signal on right, produced using Calnexin antibody, and VVL-biotinylated anti-biotin. Imaging was performed with a Phenix Opera 20X spinning disk laser confocal microscope with a water immersion objective. (B) cell derived xenograph tissue microarray TMA-CP_MX-006 informative map. (C) Quantification of glycosylated-CNX-positive signal per core using single-cell data. Cells are classified based on their location within annotated fields of view. The number of glycosylated-CNX-positive cells is counted using a fixed threshold and related to the total number of cells in each core to determine the fraction of glycosylated-CNX-positive cells. 3 Cores are present for each CDX. Statistical analysis with a t-test is provided, with * indicating significant difference in comparisons with the healthy liver negative control. Positive control is derived from PLA on a tumor region from a mouse injected with a Sleeping Beauty plasmid containing shp53, NRASG12V, and ERG1 as explained in Figure 5. Figure 9. Representative images of mouse paws at baseline (A) and 14 days post-collagen antibody injection (B). Each panel shows nuclei stained with Hoechst on the left, and fluorescent far-red signal on right, produced using Calnexin antibody and WL-biotinylated anti-biotin.. Scale bar = 2 mm. Imaging was performed using a Phenix Opera 20X spinning disk laser confocal microscope with a water immersion objective. (C) Quantification of glycosylated-CNX-positive cells per field. Fields of view were selected, and the number of glycosylated-CNX-positive cells was counted using a fixed threshold. The fraction of glycosylated-CNX-positive cells was determined by relating the number of positive cells to the total number of cells in each field. Data points represent individual fields of view, with the boxplot showing the distribution. Statistical significance between Day 0 and Day 14 PLA-positive fractions is indicated by **** (p < 0.0001), as determined by t-test.

[0401] Figure 10. Specificity of the Calnexin Tn-Glycosylation PLA. (A) Representative immunofluorescence images of the Proximity Ligation Assay (PLA) detecting Calnexin (CNX) and the Tn antigen (via WL lectin) in the indicated cell lines. The cytoplasmic PLA signal and Hoechst-stained nuclei are shown in white / greyscale. Note the strong cytoplasmic signal in Huh7 ERG1 and Huh7 ERG1 CNX- / - +CNXwt rescue cells, which is absent in the knockout and mutant lines. (B) Quantification of the PLA signal intensity per cell from (A), displayed as violin and box plots. Statistical comparisons between indicated pairs were performed using a t-test.

[0402] Figure 11. Calnexin Tn-Glycosylation is Elevated in Multiple Cancers. The dot plot displays the H- score for individual tissue biopsies tested by PLA for Calnexin (CNX) and Tn antigen (using VVL lectin). The dotted line indicates the positivity threshold, defined as the upper 95% confidence interval of the healthy tissue group. Percentages reflect the proportion of cancer biopsies with scores above this baseline. Statistical significance is shown relative to the healthy group.aHealthy tissues correspond to a pool of liver, breast, pancreas, lung and ovarian biopsies.

[0403] Examples

[0404] In the following Examples, the inventors describe the development and use of exemplary methods of the identification of disease-associated CNX.

[0405] Example 1 : Disease-associated CNX in Huh7 ERG1 inducible cells identified through proximity assay

[0406] As an initial proof-of-concept, an exemplary proximity assay was developed and used to identify glycosylated CNX on the surface of Huh7 ERG1 inducible cells.

[0407] Methodology Huh7 ERG1 inducible cells

[0408] A hepatoma cell line (Huh7) was engineered to enable inducible GALNT1 expression. This modification permits controlled GALNT1 expression in the endoplasmic reticulum (ER) upon the addition of doxycycline to the growth medium (ERG1). Specifically, Huh7 cells were modified with a Sleeping Beauty inducible tet-on transposon plasmid system. The plasmid contains a tetracycline-responsive promoter (TCE) from Kowarz etal. (Biotechnology Journal, 2015. 10: 647-653), and a fusion of an ER-localized signal (derived from p33 MHC) to a GALNT1 cassette (ERG1) (Figure 2). Doxycycline (Dox) addition triggers the expression of GALNT1 localized to the ER. Huh7 cells were transfected with the pSBtet-ERG1 plasmid at a 19:1 ratio with SB100X transposase using Lipofectamine LTX (ThermoFisher). Seven days post-transfection, stable cell populations were selected using puromycin(1 pg / mL), which was expressed as a selection marker by the pSBtet-ERG1 construct. The resulting polyclonal stable cell population was then utilized for further analysis. ERG1 expression was induced by the addition of 1 pg / mL Doxycycline for 24 hours, after which the cells were fixed and permeabilized for subsequent processing with a proximity assay.

[0409] Proximity assay procedure

[0410] The exemplary proximity assay was performed according to the schematic of Figure 3.

[0411] Huh7 ERG1 inducible cells were seeded in a 384-well Greiner high-grade optical plate at a density appropriate for 24-hour incubation in DMEM supplemented with 10% fetal calf serum, with or without doxycycline (1 pg / mL), in a final volume of 50 pL.

[0412] Following incubation, the media was aspirated, and the cells were fixed with 50 pL of pre-warmed (37°C) 4% paraformaldehyde containing 2% sucrose for 15 minutes. After fixation, the cells were washed with 50 pL / well of phosphate-buffered saline (PBS) and permeabilized using 50 pL / well of 0.2% Triton X-100 in PBS for 10 minutes. After permeabilization, the cells were washed with 50 pL of PBS and incubated with 40 pL / well of Duolink blocking buffer for 60 minutes at room temperature. For primary antibody incubation, Duolink antibody diluent was prepared with rabbit anti-calnexin antibody (ab22595, Abeam) at a final concentration of 1 pg / mL and VVL-biotin (Vector Labs) at 2 pg / mL. This mixture was added at 40 pL / well and incubated overnight at 4°C. The next day, the cells were washed three times with 50 pL of Duolink Buffer A, each for 5 minutes. Next, the cells were incubated with 40 pL / well of Duolink antibody diluent premixed with anti-biotin mouse monoclonal antibody Z021 (ThermoFisher) at 1 pg / mL for 60 minutes at room temperature. After incubation, the cells were washed twice with 50 pL of Duolink© Buffer A for 5 minutes each. The cells were then incubated with Duolink antibody diluent containing Duolink© anti-rabbit and anti-mouse PLA probes, each at a 1X concentration (30 pL / well). Incubation was carried out for 1 hour at 37°C in a humidified chamber, followed by two washes with Duolink© Buffer A (50 pL / well) for 5 minutes each. A ligation mix was prepared according to Duolink© guidelines (1 :5 dilution of Duolink Ligation Buffer 5X and 1 :40 dilution of Duolink Ligase), and 30 pL / well was added to the cells. The plate was incubated at 37°C for 30 minutes in a humidified chamber, followed by two washes with 50 pL / well of Duolink Buffer A for 5 minutes each. For amplification, a mix was prepared (1 :5 dilution of Duolink Amplification Buffer Red 5X and 1 :80 dilution of Polymerase), and 30 pL / well was added. The plate was incubated at 37°C for 100 minutes in a humidified chamber. After amplification, cells were washed twice with Duolink Buffer B (50 pL / well) for 10 minutes each.

[0413] The cells were then stained with 50 pL / well of Hoechst 33342 (2 pg / mL) in Duolink Buffer B for 10 minutes, followed by a 10-minute wash with 50 pL / well of Duolink 0.01X Buffer B. Finally, the cells were washed with 50 pL of PBS. The plate was imaged using a Phenix high-content imager with sequential channel acquisition for Hoechst and red fluorescent signals, using a 63X water immersion objective. For each well, 24 fields were imaged with a Z-stack of 3 slices (1 pm apart). Image analysis was performed using Columbus Imaging software (Revvity). A maximum projection of the three slices was used. Nuclei were segmented using the default algorithm (Method B), and a proximal ring was extended by 75% from the nuclear object to define the cytoplasm. Cytoplasmic spots were detected using algorithm Method A, and the number of spots per cell was quantified.

[0414] Immunohistochemistry (IHC)

[0415] Formalin-fixed, paraffin-embedded (FFPE) tissue slides were dewaxed by incubating at 60°C for 30 minutes. The slides were then rehydrated following standard immunohistochemistry (IHC) protocols. Antigen retrieval was performed by incubating the slides overnight in citrate buffer (pH 6.0) at 60°C. The slides were washed twice for 5 minutes each with 0.1 M phosphate buffer (pH 7.4).

[0416] A hydrophobic barrier was drawn around the tissue sections using a DAKO pen. Slides were then permeabilized by incubating with 0.2% Triton X-100 in 0.1 M phosphate buffer (pH 7.3) for 30 minutes, followed by a wash with 1 mL of 0.1 M phosphate buffer (pH 7.4). To block endogenous biotin, the slides were incubated sequentially with streptavidin blocker (Molecular Probes) for 30 minutes, followed by two washes with 1 mL of 0.1 M phosphate buffer (pH 7.4) for 5 minutes each. Biotin blocker (Molecular Probes) was then applied for 30 minutes, followed by two additional washes with 1 mL of 0.1 M phosphate buffer (pH 7.4) for 5 minutes each. Next, Duolink blocking buffer was added and incubated for 60 minutes at room temperature. Primary antibody incubation was performed overnight at 4°C using a mixture of rabbit anti-calnexin antibody (ab22595, Abeam) at 1 pg / mL and VVL-biotin (Vector Labs) at 2 pg / mL, prepared in Duolink antibody diluent, with 150 pL / slide applied. The following day, the slides were washed three times with 1 mL of Duolink Buffer A for 5 minutes each. Subsequently, 150 pL / slide of Duolink antibody diluent premixed with anti-biotin mouse monoclonal antibody Z021 (ThermoFisher) at 1 pg / mL was applied and incubated for 60 minutes at room temperature. After incubation, the slides were washed twice with 1 mL of Duolink Buffer A for 5 minutes each. The slides were then incubated with Duolink antibody diluent containing Duolink anti-rabbit and anti-mouse PLA probes, each at a 1X concentration, for 1 hour at 37°C in a humidified chamber. This was followed by two washes with Duolink Buffer A (1 mL / slide) for 5 minutes each. A ligation mix was prepared according to Duolink guidelines (1 :5 dilution of Duolink Ligation Buffer 5X and 1 :40 dilution of Duolink Ligase), and 150 pL / slide was added. The slides were incubated at 37°C for 30 minutes in a humidified chamber, followed by two washes with Duolink Buffer A (1 mL / slide) for 5 minutes each. For amplification, a mix was prepared following Duolink guidelines (1 :5 dilution of Duolink Amplification Buffer Far Red 5X and 1 :80 dilution of Duolink Polymerase), and 150 pL / slide was applied. The slides were incubated at 37°C for 100 minutes in a humidified chamber. After amplification, the slides were washed twice with Duolink Buffer B (1 mL / slide) for 10 minutes each. The slides were then stained with 1 mL / slide of Hoechst 33342 (2 pg / mL) in Duolink Buffer B for 10 minutes, followed by a 10-minute wash with 1 mL / slide of Duolink 0.01X Buffer B. The slides were washed with 1 mL of PBS, and Fluorsave was applied before mounting with coverslips. Imaging was performed using a Phenix high-content imager with sequential channel acquisition for Hoechst and far-red fluorescence, utilizing a 20X water immersion objective. Image analysis was conducted using Columbus Imaging software (Revvity). Nuclei were segmented using the default algorithm (Method M), and a proximal ring extending 75% from the nuclear boundary was used to define the cytoplasm. Cytoplasmic intensity in the far-red channel was quantified within this cytoplasmic ring. Global images of the tissue sections were reconstructed, and additional quantification and cell counting were performed using an R-based pipeline using global CSV aggregated single cell data file generated by Columbus software.

[0417] Results

[0418] The combination of ERG1 + Dox with anti-Calnexin, anti-biotin, VVL-biotinylated, and PLA antibodies produced a statistically significant increase in the number of visible spots (P<0.0001 when compared to ERG1 - Dox), with a maximum of 150 spots per cell and an average of 17 spots per cell. These results were quantified reliably using a high content imager confocal microscope with a 63X magnification objective (see Figure 4). In contrast, negative controls — where the same conditions were applied to ERG1 -Dox or Huh7 cells with Calnexin (CNX) removed through gene editing — showed minimal spot formation, with an average of less than 2.25 spots per cell across all conditions tested (see Figure 4). A control Rabbit antibody in combination with anti-biotin, VVL-biotinylated and PLA antibodies produced background level of spots of less than 2.25 spots per cell across all conditions tested.

[0419] Example 2: Disease-associated CNX identified in cancer cell lines

[0420] The proximity assay used in Example 1 was further validated using a number of different cell lines. The methodology used was in line with that explained in detail in Example 1 .

[0421] This analysis confirmed that the assay could be used to quantitatively assess the relative amount of disease-associated CNX in a sample, as the assay effectively identified samples containing high levels of glycosylated CNX, and those containing low levels of glycosylated CNX. For example, the assay indicated that HT29, HeLa, BT549, BT474, and Capan-2 cells contained relatively high levels of glycosylated CNX, and A431 , Huh7, Huh6, SNU449, MHCCLM3, and HepG2 cells contained relatively low levels of glycosylated CNX (Figure 5).

[0422] Example 3: Tissue section analysis using a proximity assay

[0423] An exemplary protocol was further developed to perform immunohistochemistry on FFPE (formalin-fixed paraffin-embedded) tissue sections.

[0424] The FFPE tissue sections used in this study were obtained from a mouse model that had been hydrodynamically injected with a transposon plasmid. This plasmid co-expresses an shRNA targeting p53 tumor suppressor, the oncogene NRASG12V, and ERG1. The plasmid is taken up by liver hepatocytes, leading to tumor formation in the liver and ERG1 overexpression (Ros et al, Nat Cell Biol. 2020 Nov;22(11):1371-1381).

[0425] The sections were first dewaxed and rehydrated, followed by antigen retrieval and permeabilization using standard procedures. Due to the presence of endogenous biotin, biotin receptors, and streptavidin binding sites in many tissues, we implemented a pre-treatment step to minimize non-specific binding. Specifically, sections were incubated with excess unlabelled streptavidin to block biotin-binding sites, followed by incubation with excess unlabelled biotin to further reduce background signal. Additional assay steps were in line with those used in Example 1 .

[0426] The assay produced a very specific amplified positive output in tumor-regions of the liver while producing a background signal in healthy parts of the liver, all with a significant difference (P=<2.2e-16) (Figure 6).

[0427] Example 4: Analysis of stratified biological samples

[0428] Liver cancer microarray

[0429] To further expand the use of the proximity assay, a tissue microarray comprising various grades of liver cancer (a LV8011 (BioMax) array - see Figure 7A and B), was analysed.

[0430] The tissue cores were classified into the following categories: Normal (N), Inflamed (In / lnfl), Hyperplasia (H / Hyp), Cancer Grade 1 (1 / G1), Cancer Grade 2 (2 / G2), Cancer Grade 3 (3 / G3), and Metastatic (M).

[0431] Through global single-cell analysis, cells were assigned to their respective grade identities based on their location within each core. A fixed cutoff was established to classify cells as glycosylated-CNX-positive, allowing us to determine the fraction of glycosylated-CNX-positive cells in each core. The fraction of glycosylated-CNX-positive cells was significantly higher in Hyperplasia, Grade 2, Grade 3, and Metastatic cores compared to Normal cores (Figure 7C).

[0432] Additionally, a methodology was established for calculating a H score by classifying glycosylated-CNX- positive cells into three subtractions: low, medium, and high glycosylated-CNX-positive. The H score for each core was defined based on the following formula:

[0433] H-score-

[0434] The refined analysis using the H score revealed a statistically significant increase in H score for Hyperplasia, Grade 2, and Grade 3 cores compared to Normal cores (Figure 7D).

[0435] Xenograft tissue microarray

[0436] The proximity assay was also used to analyse a cell derived xenograft (CDX) tissue microarray from Crown Bioscience (Figure 8A and B).

[0437] Through cell level analysis, cells were assigned to their respective core identities based on their location. Fixed cutoff was established to classify cells as being glycosylated-CNX-positive, allowing the fraction of glycosylated-CNX-positive cells in each core to be determined.

[0438] A liver section from a mouse injected with a Sleeping Beauty plasmid containing shp53, NRASG12Vwas used to relatively calibrate the readout. The tumor region of this section produced a high fraction of glycosylated-CNX-positive cells (0.77), while healthy liver regions (negative control) produced a significantly lower fraction of glycosylated-CNX-positive cells (0.045) (Figure 8C). The fraction of glycosylated-CNX-positive cells was significantly higher than the healthy liver control for various samples, such as:

[0439] - Liver CDX - SNU-878, HuCCTI and PLC / PRF / 5, with levels >0.5, Esophagus CDX - KYSE-270 and OE19 ,with levels > 0.65, Large intestine CDX - HT-29, >0.9,

[0440] - Colon CDX - LOVO, >0.4.

[0441] Pancreas CDX - PANC 04.03, HPAF-II, Capan 1 and Capan 2, with levels >0.5,

[0442] - Stomach CDX - SNU-5 and SNU-620, with level >0.67.

[0443] Example 5: Disease-associated CNX identified in arthritis model

[0444] Paw tissues from a rheumatoid arthritis mouse model were next analysed. The paw tissues were generated by employing the collagen antibody-induced arthritis (CAIA) method. Mice were injected with collagen antibodies to induce acute arthritis, which developed within two weeks. The proximity assay outlined in Example 1 was used to assess CNX glycosylation.

[0445] Paw tissues were collected 14 days post-injection, and were analysed using the proximity assay outlined in Example 1. The results showed a marked increase in the fraction of glycosylated-CNX-positive cells compared to baseline levels (Figure 9 A-C). This indicates a significant upregulation of glycosylated calnexin at day 14, aligning with the peak inflammatory response in the rheumatoid arthritis model.

[0446] Example 6: Proximity assay specificity and Calnexin Tn-qlycosylation in various malignancies Methodology

[0447] For Huh7, custom CRIPSR / Cas9 guide were designed to target N terminal portion of CNX and were used to generate Huh7 CNX- / -. Gene Synthesis at vectorbuilder was used to created CNXwt or CNX11 mut plasmid which were used to perform rescue on CNX- / - cells. ER-GALNT1 transposon construct was as described in Example 1 herein. PLA was performed on cells as described in Example 1 herein.

[0448] For Tissue microarray: processing was performed as follows. FFPE (formalin-fixed paraffin-embedded) TMA tissue sections biopsies were used from the tissue array and comprised: st2084b (gastric cancer), PA2081-L64(pancreatic cancer), LV8011 (hepatocellular carcinomas), LV1004b (Cholangiocarcinomas), LC1203a (lung carcinomas), BR1509a (TNBC Breast cancer) OV20810a (ovarian Carcinomas). The sections were first dewaxed and rehydrated, followed by antigen retrieval and permeabilization using standard procedures. Sections were incubated with excess unlabeled streptavidin to block biotin-binding sites, followed by incubation with excess unlabeled biotin to further reduce background signal. This was followed by standard PLA (proximity ligation assay) blocking steps and the remaining PLA procedures as described in Example 1 herein. We used our methodology for calculating an H score by classifying PLA- positive cells into three subtractions: low, medium, and high PLA cells. The H score for each core was defined based on this formula. To ensure run-to-run consistency, the intensity thresholds for these categories were calibrated for each experiment using controls. A positive control was used to normalize the 'high' category to an H-score of approximately 290, while a negative control was confirmed to yield an H-score of less than 5. 100 PLA cells cti nt:' +(2X medium PLA cells coMifcs)+(lXkn*rPLA ceils cownt))

[0449] H-score= - - -: ;total cells count:— - -

[0450] Images were all acquired with Phenix confocal high content imager. Calculated Single Cell PLA cytoplasmic signal population data was used for all calculations derived with a custom R pipeline.

[0451] Results

[0452] To further validate the specificity of the Proximity Ligation Assay (PLA) for Calnexin Tn-glycosylation, we used CRISPR / Cas9 technology to generate a Calnexin knockout (CNX- / -) line from Huh7 ER_GALNT1 (ERG1) cells. Subsequently, we used these ERG1 CNX- / - cells to re-express either the wild-type Calnexin gene (CNXwt) or a mutated version (CNX11 mut). The CNX11 mut variant contains mutations at 11 O-glycosylation sites where Tn glycans would normally be attached. We then performed the PLA on these four Huh7 cell variants (parental ERG1 , Erg1 CNX- / -, ERG1 CNX- / - +CNXwt rescue, and ERG1 CNX- / - +CNX11 mut rescue) to detect cytoplasmic Calnexin Tn-glycosylation (Figure 10A & 10B). The results showed a strong PLA signal in the parental ERG1 cells, which was, as expected, absent in the CNX- / - knockout cells. Re-expression of CNXwt successfully in CNX- / - context restored the PLA signal. In contrast, the signal was nearly eliminated in CNX- / - cells expressing the CNX11 mut variant.

[0453] Quantification confirmed a significant reduction in the PLA signal in the CNX11 mut cells compared to the CNXwt rescue line (Figure 10B). These findings demonstrate that the PLA for CNX Tn specifically detects Tn glycans on the O-glycosylation sites of Calnexin itself. This confirms the signal is not an artifact of Calnexin interacting with other nearby Tn-glycosylated proteins.

[0454] To evaluate the prevalence of Calnexin Tn-glycosylation (CNX Tn) in various malignancies, we screened tissue microarrays (TMAs) containing biopsies from a range of human cancers. The expression of CNX Tn was quantified using a previously described H-score methodology. A healthy baseline was established by analyzing a pooled cohort of normal tissues, including liver, breast, pancreas, lung, and ovary (Figure 11). The positivity threshold was defined as the upper limit of the 95% confidence interval calculated from the H-scores of this healthy tissue group. Our analysis revealed that CNX Tn levels were significantly elevated across all cancer cohorts examined. A substantial percentage of tumors scored above the healthy baseline threshold, including: Gastric Cancer: 92%; Pancreatic Cancer: 66%; Hepatocellular Carcinoma: 67%; Cholangiocarcinoma: 77%; Triple-Negative Breast Cancer: 62%; Lung Cancer: 54%; Ovarian Cancer: 80%.

[0455] These results highlight the widespread and significantly increased expression of CNX Tn in diverse human cancers compared to normal tissue.

[0456] Together these data confirm the assay's molecular specificity and demonstrate that this marker is significantly amplified in cancer, highlighting the assay's potential clinical application

Claims

Claims:1 . A method of assessing whether a sample comprises disease-associated CNX, wherein the method comprises, i. contacting the sample with a first antigen-binding molecule which binds to CNX, and a second antigen-binding molecule which binds to Tn, and ii. performing a proximity assay, wherein the proximity assay comprises assessing proximity of the first antigen-binding molecule and the second antigen-binding molecule, wherein a positive output from the proximity assay indicates that the sample comprises disease- associated CNX.

2. The method of claim 1 , wherein the disease-associated CNX is cell-surface CNX.

3. The method according to claim 1 or claim 2, wherein the proximity assay comprises the use of fluorescent probes.

4. The method according to any one of claims 1 to 3, wherein the proximity assay is a proximity ligation assay (PLA), a proximity extension assay (PEA), a resonance energy transfer assay, a proteinfragment complementation assay, or a protein dimerization assay.

5. The method according to claim 4, wherein the resonance energy transfer assay is a BRET assay or a FRET assay.

6. The method according to claim 4, wherein the protein-fragment complementation assay is a bimolecular fluorescence complementation (BiFC), bimolecular luciferase complementation (BiLC), yeast- two hybrid, or a split ubiquitin assay.

7. The method according to claim 4, wherein the protein dimerization assay is a dimerizationdependent fluorescent protein (ddFP) assay.

8. The method according to any one of claims 1 to 7, wherein the positive output is determined through microscopy, PCR, nucleotide sequencing, and / or flow cytometry analysis.

9. The method according to any one of claims 1 to 8, wherein the antigen-binding molecule which binds to CNX is an antibody or an aptamer.

10. The method according to any one of claims 1 to 9, wherein the antigen-binding molecule which binds to Tn is a lectin, an antibody, or an aptamer.11 . The method according to any one of claims 1 to 10, wherein the first antigen-binding molecule which binds to CNX, and / or the second antigen-binding molecule which binds to Tn,(i) comprises a detectable tag, or72(ii) is bound by a further antigen-binding molecule, or a series of further antigen-binding molecules, comprising a detectable tag.

12. The method according to claim 11 , wherein the detectable tag comprises an oligonucleotide, a fluorophore, a fluorescent protein, and / or a fluorescent protein fragment.

13. The method according to any one of claims 1 to 12, wherein the sample is a tissue sample, a blood sample, or a serum sample.

14. The method according to any one of claims 1 to 13, wherein the sample is a fixed tissue section.

15. A method of determining the level of disease-associated CNX in a sample, the method comprising assessing whether a sample comprises disease-associated CNX according to any one of claims 1 to 14, and quantitatively comparing the positive output with an output of a control sample.

16. A method of selecting or stratifying a subject for treatment with a CNX-targeted agent, the method comprising assessing whether a sample comprises disease-associated CNX according to any one of claims 1 to 14, and quantitatively comparing the positive output with an output of a control sample.

17. A method of selecting or stratifying a subject which would benefit from treatment with a CNX- targeted agent, the method comprising assessing whether a sample comprises disease-associated CNX according to any one of claims 1 to 14, and quantitatively comparing the positive output with an output of a control sample.

18. The method according to claim 16 or claim 17, wherein the subject is suspected of having, at risk of developing, or has, a disease or disorder characterised by ECM degradation19. The method according to any one of claims 16 to 18, wherein the subject is suspected of having, at risk of developing, or has, cancer or arthritis.

20. The method according to any one of claims 16 to 19, wherein the method further comprises administering a therapeutically- or prophylactically-effective amount of a CNX-targeted agent to the subject.21 . The method according to any one of claims 16 to 20, wherein the CNX-targeted agent is an antigen-binding molecule which binds CNX.

22. A method of treating or preventing a disease or condition, the method comprising assessing whether a sample comprises disease-associated CNX according to any of claims 1 to 14, determining the level of disease-associated CNX in a sample according to claim 15, and / or selecting or stratifying a subject which would benefit from treatment with a CNX-targeted agent according to any one of claims 16to 19, and administering a therapeutically- or prophylactically-effective amount of an antigen-binding molecule which binds CNX.

23. An antigen-binding molecule which binds to CNX for use in a method of treating or preventing a disease or condition, the method comprising, assessing whether a sample comprises disease-associated CNX according to any of claims 1 to 14, determining the level of disease-associated CNX in a sample according to claim 15, and / or selecting or stratifying a subject which would benefit from treatment with a CNX-targeted agent according to any one of claims 16 to 19.

24. Use of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of a disease or condition, wherein the treatment or prevention of a disease or condition comprises assessing whether a sample comprises disease-associated CNX according to any of claims 1 to 14, determining the level of disease-associated CNX in a sample according to claim 15, and / or selecting or stratifying a subject which would benefit from treatment with a CNX-targeted agent according to any one of claims 16 to 19.

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