Inhibition of calnexin for the treatment of inflammatory bowel disease

Abstract

The present disclosure provides an antigen-binding molecule which binds to calnexin (CNX) for use in treating or preventing gastrointestinal disease or inflammatory bowel disease (IBD). Also provided is an in vitro complex of an antigen-binding molecule bound to glycosylated CNX.

Description

[0001] Inhibition of Calnexin for the treatment of Inflammatory Bowel Disease

[0002] This application claims priority from US 63 / 697,029 filed 20 September 2024 and US 63 / 757,446 filed 12 February 2025, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the fields of molecular biology, more specifically antibody technology. The present disclosure also relates to methods of medical treatment and prophylaxis.

[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 CNX and CNX-containing complexes (e.g. CNX:ERp57) 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] Ros et al. Nat. Cell Biol. (2020) 22(11 ):1371-1381 discloses anti-CNX antibodies, ab10286 and ab22595. Abcam’s ab10286 and ab22595 are each rabbit polyclonal antibody preparations to human CNX. Antigen binding molecules capable of binding to CNX, which are suitable for use in methods of medical treatment and prophylaxis, are disclosed in W0202 / 008960A1 .

[0009] Gastrointestinal diseases, such as inflammatory bowel disease (IBD), can be associated with considerable morbidity and mortality. In 2019, there were approximately 4.9 million cases of IBD worldwide, and it is predicted that IBD will continue to be a major public health burden due to increasing numbers of cases, and associated deaths. There is a clear need for alternative and improved treatments for such diseases.

[0010] Summary

[0011] In a first aspect, the present disclosure provides an antigen-binding molecule which binds to CNX for use in a method of treating or preventing a gastrointestinal disease.

[0012] The disclosure also provides a method of treating or preventing a gastrointestinal disease, the method comprising administering a therapeutically- or prophylactically-effective amount of an antigen-binding molecule which binds to CNX.

[0013] Use of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of a gastrointestinal disease, is also provided. In some embodiments, the gastrointestinal disease is characterised by the expression of glycosylated CNX.

[0014] In some embodiments, the gastrointestinal disease is characterised by intestinal inflammation.

[0015] In some embodiments, the gastrointestinal disease is characterised by the presence of a polyp. In some embodiments, the polyp is a colorectal polyp, and / or a colon polyp. In some embodiments, the polyp is an adenomatous polyp, a hyperplastic polyp, a sessile serrated lesion, an inflammatory polyp, a villous adenoma, and / or a hamartomatous polyp.

[0016] In some embodiments, the gastrointestinal disease is inflammatory bowel disease (IBD).

[0017] The present disclosure also provides an antigen-binding molecule which binds to CNX for use in a method of treating or preventing IBD.

[0018] The disclosure also provides a method of treating or preventing IBD, the method comprising administering a therapeutically- or prophylactically-effective amount of an antigen-binding molecule which binds to CNX.

[0019] Use of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of IBD, is also provided.

[0020] In some embodiments, the disease is characterised by extracellular matrix (ECM) degradation.

[0021] In some embodiments, the disease is characterised by expression of glycosylated CNX by a cell of the lamina propria. In some embodiments, the disease is characterised by expression of glycosylated CNX by a fibroblast. In some embodiments, the disease is characterised by expression of glycosylated CNX by an epithelial cell.

[0022] In some embodiments, the disease is characterised by expression of glycosylated CNX on the surface of a cell of the lamina propria. In some embodiments, the disease is characterised by expression of glycosylated CNX on the surface of a fibroblast. In some embodiments, the disease is characterised by expression of glycosylated CNX on the surface of an epithelial cell.

[0023] In some embodiments, the disease is characterised by presence of glycosylated CNX on the surface of a cell of the lamina propria. In some embodiments, the disease is characterised by presence of glycosylated CNX on the surface of a fibroblast. In some embodiments, the disease is characterised by presence of glycosylated CNX on the surface of an epithelial cell.

[0024] In some embodiments the glycosylated CNX is O-glycosylated CNX. In some embodiments the glycosylated CNX is Tn glycosylated CNX. An in vitro complex is also provided, optionally isolated, comprising an antigen-binding molecule bound to glycosylated CNX, wherein the glycosylated CNX is present on the surface of a cell disclosed herein.

[0025] An in vitro complex is also provided, optionally isolated, comprising an antigen-binding molecule bound to glycosylated CNX, wherein the glycosylated CNX is present on the surface of a cell of the lamina propria or on the surface of an epithelial cell.

[0026] In some embodiments, the antigen-binding molecule binds to CNX via contact with:

[0027] (a) one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:69, optionally wherein the antigen-binding molecule binds to CNX via contact with one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:67 or 68; or

[0028] (b) one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:77, optionally wherein the antigen-binding molecule binds to CNX via contact with one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NQ:70, 71 , 72, 73, 74, 75, 76, 78, or 79.

[0029] In some embodiments, the antigen-binding molecule comprises:

[0030] (i) a heavy chain variable (VH) region incorporating the following CDRs:

[0031] HC-CDR1 having the amino acid sequence of SEQ ID NO:3 HC-CDR2 having the amino acid sequence of SEQ ID NO:4 HC-CDR3 having the amino acid sequence of SEQ ID NO:5; and

[0032] (ii) a light chain variable (VL) region incorporating the following CDRs:

[0033] LC-CDR1 having the amino acid sequence of SEQ ID NO:11 LC-CDR2 having the amino acid sequence of SEQ ID NO: 12 LC-CDR3 having the amino acid sequence of SEQ ID NO: 13.

[0034] In some embodiments, the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:2; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:10.

[0035] A method comprising administering an antigen-binding molecule which binds to CNX to a subject having a gastrointestinal disease is provided, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

[0036] In some embodiments, the gastrointestinal disease is IBD. In some embodiments, the glycosylated CNX is present on the surface of a cell disclosed herein. In some embodiments, the glycosylated CNX is present on the surface of a cell of the lamina propria, or the surface of an epithelial cell.

[0037] An improved method comprising contacting calnexin (CNX) with an antigen-binding molecule which binds to CNX is provided, wherein the improvement comprises administering the antigen-binding molecule which binds to CNX to a subject having a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

[0038] A method of contacting calnexin (CNX) with an antibody is provided, wherein the antibody comprises a means for binding CNX, wherein the method comprises administering the antibody to a subject having a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

[0039] Description

[0040] The present disclosure provides antigen-binding molecules that bind to CNX, having novel biophysical and / or functional properties as compared to antigen-binding molecules disclosed in the prior art.

[0041] The inventors have developed a novel assay for the investigation of glycosylated CNX. This assay has numerous benefits over known methods, and has been used herein to highlight the unexpected finding that glycosylated CNX is expressed by cells in samples taken from subjects with gastrointestinal diseases (e.g., IBD) and in samples taken from other models of gastrointestinal diseases.

[0042] Antigen-binding molecules which bind to CNX are a promising novel therapy option for the treatment of gastrointestinal diseases, such as IBD. Gastrointestinal diseases can be associated with considerable morbidity and mortality. There is a clear need for alternative and improved treatments for such diseases.

[0043] Calnexin (CNX)

[0044] The present disclosure relates to CNX-specific antigen-binding molecules.

[0045] 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 ene yields three main CNX isoforms: isoform 1 (SEQ ID NO:39), isoform 2 (SEQ ID NO:40) and isoform 3 (SEQ ID NO:41 ). Isoform 2 differs from isoform 1 by insertion of a 35 amino acid sequence after position 1 of SEQ ID NO:39. Positions 1 to 108 of SEQ ID NO:39 are absent from isoform 3.

[0046] Human CNX isoform 1 comprises an N-terminal signal peptide (SEQ ID NO:42), followed by a calcium- binding lumenal domain (SEQ ID NO:43), a single-pass transmembrane domain (SEQ ID NO:44) and an acidic cytoplasmic domain (SEQ ID NO:45) at the C-terminus. The lumenal domain comprises a globular lectin domain (SEQ ID NO:46), followed by an arm-like, proline-rich P-domain (SEQ ID NO:47) and a second lectin domain (SEQ ID NO:48). The mature form of human CNX isoform 1 is shown in SEQ ID NO:49.

[0047] 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.

[0048] 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).

[0049] 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:39) and mouse CNX (UniProt: P35564-1 , v1 ; SEQ ID NQ:50) are homologues of one another. Homologues include orthologues.

[0050] 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.

[0051] 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.

[0052] 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:39, 40, 41 or 42. 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:50 or 58.

[0053] 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.

[0054] 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.

[0055] 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:49, 43, 44, 45, 46, 47 or 48.

[0056] 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:58, 52, 53, 54, 55, 56 or 57.

[0057] In some embodiments, the antigen-binding molecules of the present disclosure display binding to Calreticulin (CRT).

[0058] Inventors observed that CNX-depleted cells can compensate for CNX loss through the action of CRT. Therefore, the inventors haver identified antigen-binding molecules capable of binding both CNX and CRT.

[0059] In some embodiments, the antigen-binding molecule is cross-reactive for human CNX and CRT. In some embodiments, the antigen-binding molecule reduces an activity of CNX and an activity of CRT. In some embodiments, the antigen-binding molecule reduces CNX activity and CRT activity.

[0060] As used herein, a ‘cross-reactive’ antigen-binding molecule / domain binds to the target antigens for which the antigen-binding molecule / domain is cross-reactive. For example, an antigen-binding molecule / domain / polypeptide which is cross-reactive for CNX and CRT binds to CNX and is also capable of binding to CRT. Cross-reactive antigen-binding molecules / domains / polypeptides may display specific binding to each of the target antigens.

[0061] Human CRT (also known as calreticulin, calregulin or ERp60) is the protein identified by UniProt P27797. Human CRT has the amino acid sequence shown in SEQ ID NO:59. Human CRT comprises an N- terminal signal peptide (SEQ ID NQ:60), followed by a calcium-binding N-domain (SEQ ID NO:61 ), and an acidic C-domain (SEQ ID NO:62) at the C-terminus. The N-domain comprises a globular lectin domain (SEQ ID NO:63), followed by an arm-like, proline-rich P-domain (SEQ ID NO:65) and a second lectin domain (SEQ ID NO:64). The mature form of human CRT is shown in SEQ ID NO:66.

[0062] In this specification ‘CRT’ refers to CRT from any species, and includes isoforms, fragments, variants or homologues from any species. In some embodiments CRT is CRT from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, or primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the CRT is human CRT or mouse CRT.

[0063] Isoforms, fragments, variants or homologues of CRT 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 CRT isoform from a given species, e.g. human.

[0064] Isoforms, fragments, variants or homologues of CRT may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property / activity of the reference CRT (e.g. human CRT), as determined by analysis by a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of CRT may display binding to a monoglucosylated glycan- bearing N-glycoprotein, and / or association with ERp57, cyclophilin B and / or ERp29.

[0065] In some embodiments, the CRT 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:59 or 66.

[0066] A fragment of CRT may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0067] In some embodiments, a fragment of CRT 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:66, 61 , 62, 63, 64 or 65.

[0068] The structure and function of CNX and CRT is reviewed e.g. in Kozlov and Gehring, FEBS J. (2020) 287(20):4322-4340, which is hereby incorporated by reference in its entirety.

[0069] In healthy cells, CNX is typically an endoplasmic reticulum protein. However, in certain disease contexts, there is increased trafficking of GalNAc-Ts from the Golgi to 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 CNX glycosylation 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 (Ros et al, Nat Cell Biol. 2020 Nov;22(11):1371-1381), and arthritis (WO2022157281 A1 ).

[0070] 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.

[0071] Processing by glucosidase II removes the glucose residue of the monoglucosylated N-glycan required for interaction of the 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.

[0072] 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.

[0073] 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. 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).

[0074] Gastrointestinal disease

[0075] The physiology of the gastrointestinal system is reviewed by Ogobuiro et al. (Physiology, Gastrointestinal. 2023 Apr 8. Treasure Island. FL), which is hereby incorporated by reference in its entirety. The gastrointestinal system comprises the gastrointestinal tract and accessory organs. The gastrointestinal tract includes the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, and anal canal. The accessory organs include glandular organs such as salivary glands, liver, gallbladder, and pancreas. The main functions of the Gl system include ingestion and digestion of food, nutrient absorption, secretion of water and enzymes, and excretion of waste products.

[0076] Gastrointestinal diseases may affect any part of the gastrointestinal system. A gastrointestinal disease may alternatively be described as a disease or disorder of the gastrointestinal system.

[0077] In some embodiments, the gastrointestinal disease is a disease or a disorder of an organ or tissue of the gastrointestinal system, e.g., of the bowel, small intestine, esophagus, stomach, large intestine, colon, liver, or pancreas. In some embodiments, the gastrointestinal disease is a disease or a disorder of an organ or tissue of the gastrointestinal tract, e.g., of the bowel, small intestine, esophagus, stomach, large intestine, or colon. In some embodiments, the gastrointestinal disease is a disease or a disorder of the bowel. In some embodiments, the gastrointestinal disease is a disease or a disorder of the large intestine. In some embodiments, the gastrointestinal disease is a disease or a disorder of the small intestine. In some embodiments, the gastrointestinal disease is a disease or a disorder of the stomach.

[0078] The gastrointestinal disease may be characterised by inflammation. Many diseases are associated with an overactive inflammatory response (j.e. excessive inflammation and / or aberrantly activated inflammation), and / or chronic (prolonged) inflammation. Herein, excessive and / or chronic inflammation may be referred to as ‘pathological inflammation’. Pathological inflammation may refer to inflammation which is implicated in (i.e. which positively contributes to) the pathology of a disease. In some embodiments, the gastrointestinal disease is characterised by pathological inflammation. In some embodiments, inflammation may be of an organ or tissue of the gastrointestinal system, e.g. of the bowel, small intestine, large intestine, colon, liver, or pancreas. In some embodiments, inflammation may be of an organ or tissue of the gastrointestinal tract, e.g., of the bowel, small intestine, esophagus, stomach, large intestine, or colon. Inflammation may also occur in multiple tissues / organs at once.

[0079] Inflammation and its role in heath and disease is reviewed e.g. in Chen et al., Oncotarget (2018) 9(6): 7204-7218, which is hereby incorporated by reference in its entirety. Inflammation refers to the bodily response to cellular / tissue injury, and is characterised by edema, erythema (redness), heat, pain, and loss of function (stiffness and immobility) resulting from local immune, vascular, and inflammatory cell responses to infection or injury. The injury may result from e.g. of physical (e.g. mechanical) or chemical insult, trauma, infection, cancer, or overactive / aberrant immune responses (e.g. autoimmune disease). Inflammation forms part of the innate immune response and plays an important physiological role in wound healing and the control of infection and contributes to the restoration of tissue homeostasis.

[0080] The gastrointestinal disease may be characterised by fibrosis. Fibrosis is a form of pathologic tissue remodelling characterised by the formation of excess connective tissue as a consequence of the excess deposition of extracellular matrix (ECM) components (including collagen). ‘Excess connective tissue’ refers to an amount of connective tissue at a given location (e.g. a given tissue / organ, or part of a given tissue / organ) which is greater than the amount of connective tissue present at that location under normal, non-pathological conditions. Similarly, ‘excess deposition of ECM components’ refers to a level of deposition of one or more ECM components which is greater than the level of deposition under normal, non-pathological conditions.

[0081] The cellular and molecular mechanisms of fibrosis are described in Wynn, J. Pathol. (2008) 214(2): 199- 210, and Wynn and Ramalingam, Nature Medicine (2012) 18:1028-1040, both of which are hereby incorporated by reference in their entirety.

[0082] Damage to tissues can result from various stimuli, including infections, autoimmune reactions, toxins, radiation and mechanical injury. Repair typically involves replacement of injured cells by cells of the same type, and replacement of normal parenchymal tissue with connective tissue. Repair processes become pathologic when they are not controlled properly, resulting in excess deposition of ECM components in which normal parenchymal tissue is replaced with connective tissue. In diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, cardiovascular fibrosis, systemic sclerosis and nephritis, extensive tissue remodelling and fibrosis can ultimately lead to organ failure and death.

[0083] The gastrointestinal disease may be a disease which is associated with ECM degradation. In some embodiments, the gastrointestinal disease is a disease in which aberrant ECM degradation is pathologically-implicated. In some embodiments, the gastrointestinal disease is associated with an increase in ECM degradation (e.g. as compared to the activity in the absence of the disease / condition).

[0084] The gastrointestinal disease may be a disease which is associated with aberrant CNX activity. In some embodiments, the gastrointestinal disease is a disease in which CNX activity is pathologically-implicated. In some embodiments, the gastrointestinal disease is associated with an increase in CNX activity (e.g. as compared to the activity in the absence of the disease / condition).

[0085] The gastrointestinal disease may be a disease which is associated with increased levels of glycosylated CNX. In some embodiments, the gastrointestinal disease is a disease in which glycosylated CNX is pathologically-implicated. In some embodiments, the gastrointestinal disease is associated with an increase in the level of glycosylated CNX (e.g. as compared to the level in the absence of the disease / condition).

[0086] The gastrointestinal disease may be a disease in which fibroblast activity is pathologically-implicated. The development and biology of fibroblasts is described e.g. in Plikus et al. Cell (2021 ) 184(15):3852-3872 (hereby incorporated by reference in its entirety), and the role of fibroblasts in fibrosis is described e.g. in Kendall and Feghali-Bostwick, Front. Pharmacol. (2014) 5:123 (hereby incorporated by reference in its entirety). Fibroblasts have been shown to be involved in ECM deposition in models of colitis (Jasso et al., 2022. PLoS Biol 20(1 ): e3001532), and have also been shown to be associated with inflammation- induced remodelling in colitis (Cadinu et al., Cell. 2024 Apr 11 ;187(8):2010-2028.e30). Fibroblasts are essential components of parenchymal tissues, providing the framework that is necessary for tissue structure. However, emerging evidence has revealed critical functions for fibroblast cells that extend beyond their traditional roles as structural scaffolds, including roles in regulating cell survival, differentiation, and migration. For example, synovial fibroblasts have been shown to drive cartilage extracellular matrix degradation in arthritis (WO2022157281 A1 ).

[0087] The fibroblast may be a myofibroblast. Myofibroblasts are described e.g. in Baum and Duffy, J Cardiovasc Pharmacol. (2011 ) 57(4): 376-379 and Bagalad et al., J Oral Maxillofac Pathol. (2017) 21 (3): 462-463, both of which are hereby incorporated by reference in their entirety.

[0088] The gastrointestinal disease may be a disease in which macrophage activity is pathologically-implicated. Macrophages are key players for the maintenance of intestinal homeostasis (Ruder and Becker. Cells. 2020 Oct; 9(10): 2162, which is hereby incorporated by reference in its entirety). They play essential roles for the maintenance of epithelial integrity and tissue remodelling during wound healing processes.

[0089] Macrophages secrete immune-modulatory factors. Overwhelming activation or increased secretion of pro- inflammatory cytokines can contribute to the pathogenesis of gastrointestinal disease.

[0090] The gastrointestinal disease may be a disease or a disorder of the bowel. In some embodiments, the gastrointestinal disease is IBD.

[0091] Inflammatory bowel disease

[0092] Inflammatory Bowel Disease (IBD) is characterised by inflammation of the gastrointestinal tract. Crohn's disease and ulcerative colitis are the most common types of IBD. Crohn's disease can affect the small intestine and large intestine, as well as the mouth, esophagus, stomach and the anus, whereas ulcerative colitis primarily affects the colon and the rectum. IBDs are well known to the skilled person and are reviewed in detail, for example by Seyedian et al. (J Med Life. 2019 Apr-Jun;12(2):113-122), Guan (J Immunol Res. 2019; 2019: 7247238), and Wang et al. (United European Gastroenterol J. 2022 Dec; 10(10): 1179-1193), all of which are hereby incorporated by reference in their entirety.

[0093] Crohn's Disease is a chronic inflammatory intestinal disease, characterized by the alternation of periods of flares and remissions influenced by a complex pathogenesis in which inflammation plays a key role. Crohn's disease evolution is mediated by a complex alteration of the inflammatory response which is characterized by alterations of the innate immunity of the intestinal mucosa barrier, together with a remodelling of the extracellular matrix. Crohn's disease is reviewed by Petagna et al. (Biol Direct. 2020 Nov 7;15(1 ):23, which is hereby incorporated by reference in its entirety).

[0094] Ulcerative colitis is a long-term condition that results in inflammation and ulcers of the colon and rectum. Patients with ulcerative colitis have mucosal inflammation starting in the rectum that can extend continuously to proximal segments of the colon. Ulcerative colitis usually presents with bloody diarrhoea and is diagnosed by colonoscopy and histological findings. The aim of management is to induce and then maintain remission, defined as resolution of symptoms and endoscopic healing. Treatments for ulcerative colitis include 5-aminosalicylic acid drugs, steroids, and immunosuppressants. Some patients can require colectomy for medically refractory disease or to treat colonic neoplasia. Ulcerative colitis is reviewed by Ungaro et a / . (Lancet. 2017 Apr 29;389(10080): 1756-1770, which is hereby incorporated by reference in its entirety).

[0095] Microscopic colitis refers to two related medical conditions which cause diarrhoea: collagenous colitis and lymphocytic colitis. Microscopic colitis is characterized by an increase in inflammatory cells, particularly lymphocytes, in colonic biopsies with an otherwise normal appearance and architecture of the colon.

[0096] Microscopic colitis is reviewed by Farca§ et al. (Med Pharm Rep. 2022 Oct;95(4):370-376, which is hereby incorporated by reference in its entirety).

[0097] Diversion colitis is an inflammation of the colon which can occur as a complication of ileostomy or colostomy; symptoms may occur between one month and three years following surgery. Diversion colitis is reviewed by Tominaga et al. (World J Gastroenterol. 2018 Apr 28;24(16): 1734-1747, which is hereby incorporated by reference in its entirety).

[0098] The IBD may be Crohn's disease or colitis. In some embodiments, the colitis is ulcerative colitis, microscopic colitis, collagenous colitis, lymphocytic colitis, or diversion colitis. In some embodiments, the colitis is ulcerative colitis. In some embodiments, the IBD is Crohn's disease. In some embodiments, the IBD is ulcerative colitis.

[0099] Antigen-binding molecules

[0100] The present disclosure provides antigen-binding molecules capable of binding to CNX. An antigenbinding molecule that is capable of binding to a given target antigen may also be described as an antigenbinding molecule that binds to the given target antigen.

[0101] An ‘antigen-binding molecule' refers to a molecule that binds to a given target antigen. 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, 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.

[0102] 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).

[0103] 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. 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, which is hereby incorporated by reference in its entirety). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of a 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).

[0104] 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.

[0105] The antigen-binding molecules 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 antigenbinding domain formed by a VH and a VL may also be referred to herein as an Fv region.

[0106] 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). 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 CNX. 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 al., 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 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. 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 (e.g., an antibody clone 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).

[0113] By way of example, it is reported in W02024008960A1 that antibody clone 1 E1 comprises:

[0114] (I) a heavy chain variable (VH) region incorporating the following CDRs:

[0115] HC-CDR1 having the amino acid sequence of SEQ ID NO:3

[0116] (SEQ ID NO:33 of WG2024008960A1 )

[0117] HC-CDR2 having the amino acid sequence of SEQ ID NO:4

[0118] (SEQ ID NO:33 of WQ2024008960A1 )

[0119] HC-CDR3 having the amino acid sequence of SEQ ID NO:5

[0120] (SEQ ID NO:33 of WQ2024008960A1 ); and

[0121] (ii) a light chain variable (VL) region incorporating the following CDRs:

[0122] LC-CDR1 having the amino acid sequence of SEQ ID NO:11

[0123] (SEQ ID NO:33 of WQ2024008960A1 )

[0124] LC-CDR2 having the amino acid sequence of SEQ ID NO: 12 (SEQ ID NO:33 of WQ2024008960A1 )

[0125] LC-CDR3 having the amino acid sequence of SEQ ID NO: 13 (SEQ ID NO:33 of WQ2024008960A1 ).

[0126] It is also reported in WQ2024008960A1 that antibody clone 1 E1 comprises: a VH region comprising the amino acid sequence of SEQ ID NO:2

[0127] (SEQ ID NO:32 of WQ2024008960A1 ); and a VL region comprising the amino acid sequence of SEQ ID NO: 10

[0128] (SEQ ID NQ:40 of WQ2024008960A1 ).

[0129] The CDR, VH, and VL sequences of antibody clone 1 E1 are provided above by way of example, and further relevant sequences 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 and C117 are easily retrievable by the skilled person from WQ2024008960A1 in the same way.

[0130] 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 WQ2024008960A1 . In some embodiments, the antigen-binding molecule which binds to CNX is 1 E1 described in WQ2024008960A1 .

[0131] In some embodiments, the antigen-binding molecule comprises:

[0132] (1 ) a VH region incorporating the following CDRs:

[0133] HC-CDR1 having the amino acid sequence of SEQ ID NO:3 HC-CDR2 having the amino acid sequence of SEQ ID NO:4

[0134] HC-CDR3 having the amino acid sequence of SEQ ID NO:5, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0135] (2) a VH region incorporating the following FRs:

[0136] HC-FR1 having the amino acid sequence of SEQ ID NO:6

[0137] HC-FR2 having the amino acid sequence of SEQ ID NO:7

[0138] HC-FR3 having the amino acid sequence of SEQ ID NO:8

[0139] HC-FR4 having the amino acid sequence of SEQ ID NO:9, or a variant thereof in which 1 or 2 or 3 amino acids in HC-FR1 , and / or in which 1 or 2 or 3 amino acids in HC-FR2, and / or in which 1 or 2 or 3 amino acids in HC-FR3, and / or in which 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0140] (3) a VH region comprising the CDRs according to (1 ) and the FRs according to (2).

[0141] (4) a VH 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:2.

[0142] (5) a VL region incorporating the following CDRs:

[0143] LC-CDR1 having the amino acid sequence of SEQ ID NO:11

[0144] LC-CDR2 having the amino acid sequence of SEQ ID NO: 12

[0145] LC-CDR3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 , and / or in which 1 or 2 or 3 amino acids in LC-CDR2, and / or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0146] (6) a VL region incorporating the following FRs:

[0147] LC-FR1 having the amino acid sequence of SEQ ID NO:14

[0148] LC-FR2 having the amino acid sequence of SEQ ID NO: 15

[0149] LC-FR3 having the amino acid sequence of SEQ ID NO: 16

[0150] LC-FR4 having the amino acid sequence of SEQ ID NO:1 , or a variant thereof in which 1 or 2 or 3 amino acids in LC-FR1 , and / or in which 1 or 2 or 3 amino acids in LC-FR2, and / or in which 1 or 2 or 3 amino acids in LC-FR3, and / or in which 1 or 2 or 3 amino acids in LC- FR4 are substituted with another amino acid.

[0151] (7) a VL region comprising the CDRs according to (5) and the FRs according to (6). (8) a VL 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: 10.

[0152] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1 ) to (4) above, and a VL region according to any one of (5) to (8) above.

[0153] In some embodiments, the antigen-binding molecule comprises:

[0154] (I) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:3 HC-CDR2 having the amino acid sequence of SEQ ID NO:4 HC-CDR3 having the amino acid sequence of SEQ ID NO:5; and

[0155] (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:11 LC-CDR2 having the amino acid sequence of SEQ ID NO: 12 LC-CDR3 having the amino acid sequence of SEQ ID NO: 13.

[0156] In some embodiments, the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:2; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:10.

[0157] 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 WQ2024008960A1 ), 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).

[0158] 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 WQ2024008960A1 ), 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-lg) 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).

[0159] 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.

[0160] 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:

[0161] 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.

[0162] 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:

[0163] 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.

[0164] 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.

[0165] 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 polypeptide joined by a linker region, i.e. a single chain Fv (scFv).

[0166] 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 polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).

[0167] 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.

[0168] 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.

[0169] IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1, lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0170] 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.

[0171] 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 , Ig A2), IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG1 , hlgG2, hlgG3, hlgG4), hlgA (e.g. hlgA1 , hlgA2), hlgD, hlgE or hlgM. In some embodiments, the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of a human IgG 1 allotype (e.g. G1 ml , G1 m2, G1 m3 or G1 ml 7).

[0172] 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:17, 22, 23 or 26.

[0173] 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:18 or 24. 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 NQ:20. 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:21 or 25.

[0174] 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:19.

[0175] 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.

[0176] 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.

[0177] 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 NQ:30, 31 , 32, 33, 34 or 35.

[0178] In some embodiments, the antigen-binding molecule is, or comprises, a monoclonal antibody, or an antigen-binding fragment thereof.

[0179] 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 disu lfide-l inked 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.

[0180] 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.

[0181] In some embodiments, the antigen-binding molecule is a mouse / human chimeric antibody / antibody fragment (j.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.

[0182] Mouse / human chimeric antigen-binding molecules can be prepared from mouse antibodies by the process of chimerisation, 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.

[0183] 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

[0184] 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.

[0185] Aspects of the present disclosure relate to multispecific antigen-binding molecules. By ‘multispecific’ it is meant that the antigen-binding molecule displays specific binding to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).

[0186] In some embodiments, the antigen-binding molecule binds to CNX and another target (e.g. an antigen other than CNX), and so is at least bispecific. The term ‘bispecific’ means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.

[0187] It will be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific. For example, an antigen-binding molecule that binds to CNX and an antigen other than CNX may comprise: (i) an antigen-binding molecule that binds to CNX, and (ii) an antigen-binding molecule that binds to an antigen other than CNX.

[0188] It will also be appreciated that an antigen-binding molecule according to the present disclosure (e.g. a multispecific antigen-binding molecule) may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific.

[0189] In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g. a multispecific antigen-binding molecule) may be referred to e.g. as an ‘antigen-binding domain’ or ‘antigen-binding region’ of the larger antigen-binding molecule.

[0190] In some embodiments, the antigen other than CNX targeted by a multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, e.g. a cell surface receptor. In some embodiments, the antigen is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine. In some embodiments, the antigen is a growth factor or a hormone.

[0191] A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen’s expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (j.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular). The cancer cell antigen may be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and / or protein level) by cells of a cancer, e.g. as compared to the level of expression by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein. An immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell. In some embodiments, the part of the immune cell surface molecule which is bound by the antigen-binding molecule of the present disclosure is on the external surface of the immune cell (i.e. is extracellular). The immune cell surface molecule may be expressed at the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell).

[0192] In some embodiments, the antigen-binding molecule is an immune cell engager. Immune cell engagers are 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. Immune cell engager molecules comprise an antigen-binding region for a target antigen of interest, and an antigen-binding region for recruiting / engaging an immune cell of interest. Immune cell engagers recruit / engage immune cells through an antigen-binding region specific for an immune cell surface molecule.

[0193] The best studied immune cells engagers are bispecific T cell engagers (BiTEs), which comprise a target antigen binding domain, and a CD3 polypeptide (typically CD3e)-binding domain, through which the BiTE recruits T cells. Binding of the BiTE to its target antigen and to the CD3 polypeptide expressed by the T cell results in activation of the T cell, and ultimately directs T cell effector activity against cells expressing the target antigen. Other kinds of immune cell engagers are well known in the art, and include natural killer cell engagers such as bispecific killer engagers (BiKEs), which recruit and activate NK cells.

[0194] In some embodiments, multispecific antigen-binding molecules described herein display at least monovalent binding with respect to CNX, and also display at least monovalent binding with respect to a CD3 polypeptide (e.g. CD3E, CD36, CD3y or CD3 preferably CD3E, CD35 or CD3y; or more preferably CD3E). In some embodiments, the antigen-binding molecule comprises one binding site for CNX and one binding site for a CD3 polypeptide.

[0195] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g. CD3s, CD35, CD3y or CD3 ; preferably CD3s, CD36 or CD3y; or more preferably CD3s). In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g. CD3E, CD35, CD3y or CD3 preferably CD3E, CD35 or CD3y; or more preferably CD3E). In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule that binds to a CD3 polypeptide (e.g.

[0196] CD3E, CD35, CD3y or CD3 preferably CD3s, CD35 or CD3y; or more preferably CD3E). That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigenbinding molecule that binds to a CD3 polypeptide (e.g. CD3s, CD35, CD3y or CD3 ; preferably CD3s, CD35 or CD3y; or more preferably CD3E). In some embodiments, the antigen-binding molecule comprises the CDRs, FRs and / or the VH and / or VL regions of a CD3 polypeptide-binding antibody clone, or CDRs, FRs and / or VH and / or VL regions which are derived from those of a CD3 polypeptide-binding antibody clone.

[0197] In some embodiments, a CD3 polypeptide-binding antibody clone is selected from: OKT3 (in Kjer-Nielsen et a / ., PNAS (2004) 101 (20):7675-80), SP34 (described e.g. in WO 2014 / 122143 A1 ), UCHT1 (described e.g. in WO 2000 / 041474 A1 ) HIT3a (Invitrogen Cat # 16-0039-85), and clone SK7 (Invitrogen Cat # 16- 0036-81 ).

[0198] In some embodiments, the immune cell engaged by the immune cell engager is a T cell or an NK cell. In some embodiments, the immune cell engager is a T cell-engager.

[0199] Multispecific antigen-binding molecules according to the present disclosure may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1 / CL fusion proteins, e.g. scFv2-CH1 / CL, VHH2-CH1 / CL; ‘variable domain only’ bispecific antigenbinding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb / VHH, tertravalent dAb.VHH; Non-lg fusion proteins, e.g. scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g. scFv-Fc(kih), scFv- Fc(CH3 charge pairs), scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEEDbody), taFv-Fc(kih), scFv- Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc (SEEDbody), DART- Fc, scFv-CH3(kih), TriFabs; Fc fusions, e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-lg, scFv2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab- Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-lgG(kih), Fab-scFab-lgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1 / CL charge pairs, hinge / CH3 charge pairs, SEED-body, Duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*; appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgGs - HC fusions, e.g. IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CaCP) Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab), Fab-lgG(CaCp Fab), Fab-lgG(CR3), Fab-hinge-lgG(CR3); appended IgGs - LC fusions, e.g. IgG-scFv(LC), scFv(LC)-lgG, dAb-IgG; appended IgGs - HC and LC fusions, e.g. DVD-lg, TVD-lg, CODV-lg, scFv4-lgG, Zybody; Fc fusions, e.g. Fab-scFv- Fc, scFv4-lg; F(ab’)2 fusions, e.g. F(ab’)2-scFv2; CH1 / CL fusion proteins e.g. scFv2-CH1 -hinge / CL; modified IgGs, e.g. DAF (two-in one-IgG), DutaMab, Mab2; and non-lg fusions, e.g. DNL-Fab4-lgG.

[0200] The skilled person is able to design and prepare bispecific antigen-binding molecules. Methods for producing multispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001 . Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 — 2.13.16, which is hereby incorporated by reference in its entirety. For example, A / -succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.

[0201] Other methods for producing multispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001 . Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 — 2.13.16, which is hereby incorporated by reference in its entirety.

[0202] Multispecific antigen-binding molecules according to the present disclosure can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigenbinding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigenbinding molecules, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.

[0203] For example, a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding fragments ( .e. the light and heavy chain variable domains for the antigen-binding fragment capable of binding CNX, and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein), and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques. Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.

[0204] Fc regions

[0205] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region.

[0206] An Fc region is composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation. Modifications to antibody Fc regions that influence Fc-mediated functions are known in the art, such as those described e.g. in Wang et al., Protein Cell (2018) 9(1 ):63-73, which is hereby incorporated by reference in its entirety. Exemplary Fc region modifications known to influence antibody effector function are summarised in Table 1 of Wang et al., Protein Cell (2018) 9(1 ):63-73. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification to increase or reduce an Fc-mediated function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0207] Where an Fc region / CH2 / CH3 is described as comprising modification(s) ‘corresponding to’ reference substitution(s), equivalent substitution(s) in the homologous Fc / CH2 / CH3 are contemplated. By way of illustration, L234A / L235A substitutions in human lgG1 (numbered according to the EU numbering system as described in Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 ) correspond to L to A substitutions at positions 117 and 118 of the mouse Ig gamma-2A chain C region (UniProtKB: P01863-1 , v1 ).

[0208] Where an Fc region is described as comprising a modification, the modification may be present in one or both of the polypeptide chains which together form the Fc region.

[0209] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and / or CH3 regions.

[0210] In some embodiments, the Fc region comprises modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises modification to increase ADCC. In some embodiments, the Fc region comprises modification to increase ADCP. In some embodiments, the Fc region comprises modification to increase CDC. An antigen-binding molecule comprising an Fc region comprising modification to increase an Fc-mediated function (e.g. ADCC, ADCP, CDC) induces an increased level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0211] In some embodiments, the Fc region comprises modification to increase binding to an Fc receptor. In some embodiments, the Fc region comprises modification to increase binding to an Fey receptor. In some embodiments, the Fc region comprises modification to increase binding to one or more of FcyRI, FcyRlla, FcyRUb, FcyRllc, FcyRIHa and FcyRI I lb. In some embodiments, the Fc region comprises modification to increase binding to FcyRllla. In some embodiments, the Fc region comprises modification to increase binding to FcyRlla. In some embodiments, the Fc region comprises modification to increase binding to FcyRUb. In some embodiments, the Fc region comprises modification to increase binding to FcRn. In some embodiments, the Fc region comprises modification to increase binding to a complement protein. In some embodiments, the Fc region comprises modification to increase binding to C1q. In some embodiments, the Fc region comprises modification to promote hexamerisation of the antigen-binding molecule. In some embodiments, the Fc region comprises modification to increase antigen-binding molecule half-life. In some embodiments, the Fc region comprises modification to increase coengagement.

[0212] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions F243L / R292P / Y300L / V305I / P396L as described in Stavenhagen et al. Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S239D / I332E or S239D / I332E / A330L as described in Lazar et a / ., Proc Natl Acad Sci USA. (2006)103:4005-4010. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S298A / E333A / K334A as described in Shields et al., J Biol Chem. (2001 ) 276:6591-6604. In some embodiments, the Fc region comprises modification to one of heavy chain polypeptides corresponding to the combination of substitutions L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and modification to the other heavy chain polypeptide corresponding to the combination of substitutions D270E / K326D / A330M / K334E, as described in Mimoto et al., MAbs. (2013): 5:229-236. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions G236A / S239D / I332E as described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527.

[0213] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions K326W / E333S as described in Idusogie et al. J Immunol. (2001 ) 166(4):2571-5. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S267E / H268F / S324T as described in Moore et al. MAbs. (2010) 2(2): 181 -9. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions E345R / E430G / S440Y as described in Diebolder et al. Science (2014) 343(6176):1260-3.

[0214] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions M252Y / S254T / T256E as described in Dall’Acqua et al. J Immunol. (2002) 169:5171-5180. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions M428L / N434S as described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159.

[0215] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S267E / L328F as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions N325S / L328F as described in Shang et al. Biol Chem. (2014) 289:15309-15318. In some embodiments, the Fc region comprises modification to reduce / prevent an Fc-mediated function.

[0216] In some embodiments, the Fc region comprises modification to reduce / prevent ADCC. In some embodiments, the Fc region comprises modification to reduce / prevent ADCP. In some embodiments, the Fc region comprises modification to reduce / prevent CDC. An antigen-binding molecule comprising an Fc region comprising modification to reduce / prevent an Fc-mediated function (e.g. ADCC, ADCP, CDC) induces a reduced level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.

[0217] In some embodiments, the Fc region comprises modification to reduce / prevent binding to an Fc receptor.

[0218] In some embodiments, the Fc region comprises modification to reduce / prevent binding to an Fey receptor. In some embodiments, the Fc region comprises modification to reduce / prevent binding to one or more of FcyRI, FcyRlla, FcyRllb, FCYRIIC, FcyRllla and FcyRI lib. In some embodiments, the Fc region comprises modification to reduce / prevent binding to FcyRllla. In some embodiments, the Fc region comprises modification to reduce / prevent binding to FcvRIla. In some embodiments, the Fc region comprises modification to reduce / prevent binding to FcyRllb. In some embodiments, the Fc region comprises modification to reduce / prevent binding to a complement protein. In some embodiments, the Fc region comprises modification to reduce / prevent binding to C1q. In some embodiments, the Fc region comprises modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0219] In some embodiments, the Fc region is not able to induce one or more Fc-mediated functions ( / .e. lacks the ability to elicit the relevant Fc-mediated function(s)). Accordingly, antigen-binding molecules comprising such Fc regions also lack the ability to induce the relevant function(s). Such antigen-binding molecules may be described as being devoid of the relevant function(s).

[0220] In some embodiments, the Fc region is not able to induce ADCC. In some embodiments, the Fc region is not able to induce ADCP. In some embodiments, the Fc region is not able to induce CDC. In some embodiments, the Fc region is not able to induce ADCC and / or is not able to induce ADCP and / or is not able to induce CDC.

[0221] In some embodiments, the Fc region is not able to bind to an Fc receptor. In some embodiments, the Fc region is not able to bind to an Fey receptor. In some embodiments, the Fc region is not able to bind to one or more of FcyRI, FcyRlla, FcyRllb, FcyRIIc, FcyRllla and FcyRlllb. In some embodiments, the Fc region is not able to bind to FcyRllla. In some embodiments, the Fc region is not able to bind to FcyRlla. In some embodiments, the Fc region is not able to bind to FcyRllb. In some embodiments, the Fc region is not able to bind to FcRn. In some embodiments, the Fc region is not able to bind to a complement protein. In some embodiments, the Fc region is not able to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0222] In some embodiments, the Fc region comprises modification corresponding to N297A or N297Q or N297G as described in Leabman et al., MAbs. (2013) 5:896-903. In some embodiments, the Fc region comprises modification corresponding to L235E as described in Alegre et al., J Immunol. (1992) 148:3461-3468. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235A or F234A / L235A as described in Xu et al., Cell Immunol. (2000) 200:16-26. In some embodiments, the Fc region comprises modification corresponding to P329A or P329G as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235A / P329G as described in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S228P / L235E as described in Newman et al., Clin. Immunol. (2001 ) 98:164-174. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions H268Q / V309L / A330S / P331 S as described in An et al., MAbs. (2009) 1 :572-579. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., Methods. (2014) 65:114- 126. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331S as described in US 2015 / 0044231 A1.

[0223] The combination of substitutions ‘L234A / L235A’ and corresponding substitutions (such as e.g. F234A / L235A in human lgG4) are known to disrupt binding of Fc to Fey receptors and inhibit ADCC, ADCP, and also to reduce C1q binding and thus CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, hereby incorporated by reference in entirety). The substitutions ‘P329G’ and ‘P329A’ reduce C1q binding (and thereby CDC). Substitution of ‘N297’ with ‘A’, ‘G’ or ‘Q’ is known to eliminate glycosylation, and thereby reduce Fc binding to C1q and Fey receptors, and thus CDC and ADCC. Lo et al. J. Biol. Chem (2017) 292(9):3900-3908 (hereby incorporated by reference in its entirety) reports that the combination of substitutions L234A / L235A / P329G eliminated complement binding and fixation as well as Fey receptor dependent, antibody-dependent, cell-mediated cytotoxicity in both murine lgG2a and human lgG1.

[0224] The combination of substitutions L234A / L235E / G237A / A330S / P331 S in lgG1 Fc is disclosed in US 2015 / 0044231 A1 to abolish induction of phagocytosis, ADCC and CDC.

[0225] In some embodiments, the Fc region comprises modification corresponding to the substitution S228P as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The substitution S228P in lgG4 Fc reduces Fab-arm exchange (Fab arm exchange can be undesirable).

[0226] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235A. In some embodiments, the Fc region comprises modification corresponding to the substitution P329G. In some embodiments, the Fc region comprises modification corresponding to the substitution N297Q. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235A / P329G.

[0227] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235A / P329G / N297Q.

[0228] In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions L234A / L235E / G237A / A330S / P331 S.

[0229] In some embodiments, the Fc region comprises modification corresponding to the substitution S228P, e.g. in lgG4.

[0230] In some embodiments - particularly embodiments in which the antigen-binding molecule is a multispecific (e.g. bispecific) antigen-binding molecule - the antigen-binding molecule comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region. Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations. To improve the yield of the desired combinations of polypeptides in antigen-binding molecules in recombinant production, it is advantageous to introduce modification(s) into the Fc regions which promote association of the desired combination of heavy chain polypeptides. Modifications may promote e.g. hydrophobic and / or electrostatic interactions between CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety.

[0231] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of the following formats, as shown in Table 1 of Ha et al. (Front. Immnol. 2016. 7:394, which is hereby incorporated by reference in its entirety): KIH, KIHs-s, HA-TF, ZW1 , 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107.

[0232] Polypeptides and particular exemplary antigen-binding molecules

[0233] The present disclosure also provides polypeptide constituents of antigen-binding molecules. The polypeptides may be provided in isolated or substantially purified form.

[0234] The antigen-binding molecule of the present disclosure may be, or may comprise, a complex of polypeptides.

[0235] In the present specification, where a polypeptide comprises more than one domain or region, it will be appreciated that the plural domains / regions are preferably present in the same polypeptide chain. That is, the polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains / regions. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VL as described herein.

[0236] In some embodiments, the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and / or a CH3 region of an immunoglobulin (Ig).

[0237] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein.

[0238] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region as described herein.

[0239] In some embodiments, the polypeptide according to the present disclosure comprises a structure from N- to C-terminus according to one of the following:

[0240] (i) VH

[0241] (ii) VL

[0242] (ill) VH-CH1

[0243] (iv) VL-CL

[0244] (v) VL-CH1

[0245] (vi) VH-CL

[0246] (vii) VH-CH1-CH2-CH3

[0247] (viii) VL-CL-CH2-CH3

[0248] (ix) VL-CH1-CH2-CH3

[0249] (x) VH-CL-CH2-CH3

[0250] Also provided by the present disclosure are antigen-binding molecules composed of the polypeptides of the present disclosure. In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:

[0251] (A) VH + VL

[0252] (B) VH-CH1 + VL-CL

[0253] (C) VL-CH1 + VH-CL

[0254] (D) VH-CH1-CH2-CH3 + VL-CL

[0255] (E) VH-CL-CH2-CH3 + VL-CH1

[0256] (F) VL-CH1-CH2-CH3 + VH-CL

[0257] (G) VL-CL-CH2-CH3 + VH-CH1 (H) VH-CH1-CH2-CH3 + VL-CL-CH2-CH3

[0258] (I) VH-CL-CH2-CH3 + VL-CH1-CH2-CH3

[0259] In some embodiments, the antigen-binding molecule comprises more than one polypeptide of the combinations shown in (A) to (I) above. By way of example, with reference to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides comprising the structure VH- CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.

[0260] In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:

[0261] (J) VH (anti-CNX) + VL (anti-CNX)

[0262] (K) VH (anti-CNX)-CHI + VL (anti-CNX)-CL

[0263] (L) VL (anti-CNX)-CHI + VH (anti-CNX)-CL

[0264] (M) VH (anti-CNX)-CH1-CH2-CH3 + VL (anti-CNX)-CL

[0265] (N) VH (anti-CNX)-CL-CH2-CH3 + VL (anti-CNX)-CHI

[0266] (O) VL (anti-CNX)-CH1-CH2-CH3 + VH (anti-CNX)-CL

[0267] (P) VL (anti-CNX)-CL-CH2-CH3 + VH (anti-CNX)-CHI

[0268] (Q) VH (anti-CNX)-CH1-CH2-CH3 + VL (anti-CNX)-CL-CH2-CH3

[0269] Wherein: ‘VH (anti-CNX)’ refers to the VH of an antigen-binding molecule capable of binding to CNX (e.g., an antigen-binding molecule described in W02024008960A1 ); and ’VL(anti-CNX)’ refers to the VL of an antigen-binding molecule capable of binding to CNX (e.g., an antigen-binding molecule described in W02024008960A1 ).

[0270] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which 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:36, or 37.

[0271] In some embodiments, the antigen-binding molecule of the present disclosure comprises a polypeptide which 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:38.

[0272] Linkers and additional sequences

[0273] In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure comprise one or more linker sequences between amino acid sequences. A linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule / polypeptide. Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues.

[0274] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n (SEQ ID NO:81 and 82) or (GxS)nGm (SEQ ID NO:83 and 84); wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S (SEQ ID NO:82). In some embodiments, the linker sequence comprises or consists of (G4S)4 (SEQ ID NO:86) or (G4S)e (SEQ ID NO:87). In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.

[0275] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, antigenbinding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.

[0276] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.

[0277] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide, and may be present in the newly synthesised antigen-binding molecule / polypeptide. The signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule / polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding molecule / polypeptide secreted from the cell expressing the antigen-binding molecule / polypeptide.

[0278] Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176). Labels and conjugates

[0279] In some embodiments, the antigen-binding molecules of the present disclosure additionally comprise a detectable moiety.

[0280] In some embodiments, the antigen-binding molecule comprises a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The antigen-binding molecule may be covalently or non- covalently labelled with the detectable moiety.

[0281] Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Hydrogen3, Sulfur35, Carbon14, Phosphorus32, Iodine123, Iodine125, Iodine126, Iodine131, Iodine133, Bromine77, Technetiurn99m, Indium111, lndiurn113m, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rheniurn99m, Rhenium101, Rhenium105, Scandium47, Tellurium121171, Tellurium122171, Tellurium125111, Thulium165, Thuliuml167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217and Antimony211. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers.

[0282] In some embodiments, the antigen-binding molecule / polypeptide comprises an epitope tag, e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, calmodulin-binding protein (CBP), glutathione-s- transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, or haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule / polypeptide.

[0283] In some embodiments, the antigen-binding molecule / polypeptide comprises a moiety having a detectable activity, e.g. an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

[0284] In some embodiments, the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect, i.e. a drug moiety. A drug moiety may be a small molecule (e.g. a low molecular weight (< 1000 daltons, typically between -300-700 daltons) organic compound). Drug moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14 (hereby incorporated by reference in its entirety). In some embodiments, a drug moiety may be or comprise a cytotoxic agent. In some embodiments, a drug moiety may be or comprise a chemotherapeutic agent. Drug moieties include e.g. calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.

[0285] 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 antibodydrug conjugates (ADCs) comprising a (cytotoxic) drug moiety. 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) 154:102-1 17, both of which are hereby incorporated by reference in their entirety), including BiTEs, BiKEs and TriKEs. Antigenbinding 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).

[0286] In some embodiments, an antigen-binding molecule according to the present disclosure comprises a drug moiety. The antigen-binding molecule may be conjugated to the drug moiety. Antibody-drug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14 (hereby incorporated by reference in its entirety). FDA approved ADCs currently on the market are described in Tong et al., Molecules. 2021 Oct; 26(19): 5847 (hereby incorporated by reference in its entirety).

[0287] In some embodiments the antibody-drug conjugate comprises an antigen binding molecule moiety, a drug moiety (or payload moiety), and a linker to join the drug moiety to the antibody. In some embodiments the antibody-drug conjugate consists of an antibody moiety, a drug moiety (or payload moiety), and a linker to join the drug moiety to the antibody.

[0288] The antigen binding molecule moiety may be a molecule that binds to a given target antigen. Antigenbinding molecules include antibodies (i.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, single domain antibodies (e.g. VhH), etc.). Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments.

[0289] The linker may be cleavable or non-cleavable. The linker may be based on a chemical motifs such as disulfides, hydrazones or peptides (cleavable), or thioethers (non-cleavable). The type of linker, cleavable or noncleavable, lends specific properties to the cytotoxic drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic (anti-cancer) agent enter the targeted cancer cell where the antibody is degraded into an amino acid. The resulting complex - amino acid, linker and cytotoxic agent - is considered to be the active drug. In contrast, cleavable linkers are detached by enzymes in the cancer cell. The drug moiety (or payload) may be a small molecule or a nucleic acid drug. In some embodiments, the drug moiety (or payload), is or comprises a cytotoxic agent. In some embodiments, the drug moiety is or comprises a chemotherapeutic agent. In some embodiments, the drug moiety is or comprises an anti-arthritis drug. In some embodiments, the drug moiety is or comprises a steroid.

[0290] Functional properties of the antigen-binding molecules

[0291] The antigen-binding molecules described herein may be characterised by reference to certain functional properties. In some embodiments, the antigen-binding molecule described herein may possess one or more of the following properties: binds to CNX (e.g. human CNX and / or mouse CNX); binds to CRT (e.g. human CRT); binds cross-reactively to CNX (e.g. human CNX and / or mouse CNX) and CRT (e.g. human CRT); antagonises or inhibits CNX; reduces or inhibits CNX expression; reduces or inhibits CNX activity and / or activity of a complex comprising CNX; reduces a or inhibits a function of CNX and / or a function of a complex comprising CNX; reduces a or inhibits a function of a cell expressing glycosylated CNX; reduces or inhibits extracellular matrix degradation (e.g. collagen and / or gelatin degradation); reduces or inhibits extracellular matrix degradation activity of a cell characterised by CNX expression; reduces or inhibits extracellular matrix degradation activity of a cell characterised by glycosylated CNX expression; reduces or inhibits extracellular matrix degradation activity of a fibroblast; reduces or inhibits extracellular matrix degradation activity of an intestinal fibroblast; reduces or inhibits extracellular matrix degradation activity of a myofibroblast; reduces or inhibits extracellular matrix degradation by a cell characterised by CNX expression; reduces or inhibits extracellular matrix degradation by a cell characterised by glycosylated CNX expression; reduces or inhibits extracellular matrix degradation by an intestinal fibroblast; reduces or inhibits extracellular matrix degradation by a myofibroblast; reduces or inhibits extracellular matrix degradation by a fibroblast; reduces oxireductase activity; reduces disulfide bond reductase activity; increases killing of cells expressing CNX / CRT; reduces the pathology of a disease / condition described herein; reduces the pathology of a gastrointestinal disease; reduces the pathology of IBD; reduces the pathology of a disease / condition characterised by ECM degradation in a subject; reduces the pathology of a disease / condition characterised by the expression of glycosylated CNX (e.g. a gastrointestinal disease, such as IBD) in a subject.

[0292] It will be appreciated that a given antigen-binding molecule may display more than one of the properties recited in the preceding paragraph. A given antigen-binding molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject.

[0293] Where assays are cell-based assays, they may comprise treating cells with a given antigen-binding molecule in order to determine whether the antigen-binding molecule displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities / concentrations of a given antigen-binding molecule (e.g. a dilution series). It will be appreciated that the cells preferably express the target antigen for the antigen-binding molecule (i.e. CNX / CRT).

[0294] Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘ECso’. By way of illustration, the EC50 of a given antigen-binding molecule for binding to human CNX may be the concentration of the antigen-binding molecule at which 50% of the maximal level of binding to human CNX is achieved.

[0295] Depending on the property, the EC50 may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘IC50’, this being the concentration of the agent at which 50% of the maximal level of inhibition of a given property is observed.

[0296] The antigen-binding molecules described herein bind to CNX. In some embodiments, the antigen-binding molecules bind to CRT. The antigen-binding molecules and antigen-binding domains described herein preferably display specific binding to the relevant target antigen (e.g. CNX). As used herein, ‘specific binding’ refers to binding which is selective for the antigen, and which can be discriminated from nonspecific binding to non-target antigen. An antigen-binding molecule / domain that specifically binds to a target molecule preferably binds the target with greater affinity, and / or with greater duration than it binds to other, non-target molecules.

[0297] The ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.

[0298] In some embodiments, the extent of binding of the antigen-binding molecule to a non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA. Alternatively, binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1 x 10n, where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule. This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.

[0299] The affinity of binding to a given target antigen for an antigen-binding molecule described herein may be determined by Bio-Layer Interferometry, e.g. as described in the Examples of the present disclosure.

[0300] In some embodiments, the antigen-binding molecule described herein binds to CNX with an affinity in the micromolar range, i.e. KD = 9.9 x 104to 1 x 106M. In some embodiments, the antigen-binding molecule described herein binds to CNX with sub-micromolar affinity, i.e. KD < 1 x 106M. In some embodiments, the antigen-binding molecule described herein binds to CNX with an affinity in the nanomolar range, i.e. KD = 9.9 x 10'7to 1 x 109M. In some embodiments, the antigen-binding molecule described herein binds to CNX with sub-nanomolar affinity, i.e. KD < 1 x 10-9M. In some embodiments, the antigen-binding molecule described herein binds to CNX with an affinity in the picomolar range, i.e. KD = 9.9 x 10-10to 1 x 10-12M. In some embodiments, the antigen-binding molecule described herein binds to CNX with sub- picomolar affinity, i.e. KD < 1 x 10-12M.

[0301] In some embodiments, the antigen-binding molecule described herein binds to human CNX with a KD of 10 pM or less, preferably one of £5 pM, £2 pM, £1 pM, £500 nM, £100 nM, £75 nM, £50 nM, £40 nM, £30 nM, £20 nM, £15 nM, £12.5 nM, £10 nM, £9 nM, £8 nM, £7 nM, £6 nM, £5 nM, £4 nM £3 nM, £2 nM, £1 nM, £500 pM, £400 pM, £300 pM, £200 pM, £100 pM, £50 pM, £40 pM, £30 pM, £20 pM, £10 pM or £l pM (e.g. as determined by analysis as described in Example 2 herein). In some embodiments, the antigen-binding molecule described herein binds to human CNX with a KD of 100 nM or less, preferably one of £50 nM, £40 nM, £30 nM, £20 nM, £15 nM, £12.5 nM, £10 nM, £9 nM, £8 nM, £7 nM, £6 nM, £5 nM, £4 nM £3 nM, £2 nM, £1 nM, £500 pM, £400 pM, £300 pM, £200 pM, £100 pM, £50 pM, £40 pM, £30 pM, £20 pM, £10 pM or £1 pM (e.g. as determined by analysis as described in Example 2 herein).

[0302] In some embodiments, the antigen-binding molecule described herein binds to human CNX with an EC50 of 10 pM or less, preferably one of £5 pM, £2 pM, £1 pM, £500 nM, £100 nM, £75 nM, £50 nM, £40 nM, £30 nM, £20 nM, £15 nM, £12.5 nM, £10 nM, £9 nM, £8 nM, £7 nM, £6 nM, £5 nM, £4 nM £3 nM, £2 nM, <1 nM, <500 pM, <400 pM, <300 pM, <200 pM, <100 pM, <50 pM, <40 pM, <30 pM, <20 pM, <10 pM or <1 pM (e.g. as determined by analysis as described in Example 2 herein).

[0303] In some embodiments, the antigen-binding molecule is cross-reactive for human CNX and a homologue thereof (e.g. mouse CNX). In some embodiments, the antigen-binding molecule is cross-reactive for CNX and CRT. As used herein, a ‘cross-reactive’ antigen-binding molecule / domain binds to the target antigens for which the antigen-binding molecule / domain is cross-reactive. For example, an antigen-binding molecule / domain / polypeptide which is cross-reactive for human CNX and mouse CNX binds to human CNX, and is also capable of binding to mouse CNX. Similarly, an antigen-binding molecule / domain / polypeptide which is cross-reactive for human CNX and human CRT binds to CNX, and is also capable of binding to CRT. Cross-reactive antigen-binding molecules / domains / polypeptides may display specific binding to each of the target antigens.

[0304] In some embodiments, the antigen-binding molecule binds to human CNX (e.g. isoform 1 ), and mouse CNX. In some embodiments, the antigen-binding molecule binds to human CNX (e.g. isoform 1 ) and human CRT.

[0305] The antigen-binding molecules of the present disclosure may bind to a particular region of interest of CNX. Antigen-binding molecules according to the present disclosure may bind to a linear epitope of CNX, consisting of a contiguous sequence of amino acids ( / .e. an amino acid primary sequence). In some embodiments, an antigen-binding molecule may bind to a conformational epitope of CNX, consisting of a discontinuous sequence of amino acids of the amino acid sequence.

[0306] The region of a given target molecule to which an antigen-binding molecule binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety. In preferred embodiments, the region of a peptide / polypeptide to which an antigen-binding molecule binds is determined by hydrogendeuterium exchange analysis by mass spectrometry, performed essentially as described in Example 2 herein.

[0307] In some embodiments, the antigen-binding molecule of the present disclosure binds to a domain of CNX described herein, e.g. the lumenal domain (e.g. lectin domain 1 , P domain, lectin domain 2), transmembrane domain or cytoplasmic domain.

[0308] In some embodiments, the antigen-binding molecule of the present disclosure binds to the lumenal domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:43. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the antigen-binding molecule of the present disclosure binds to the lectin domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:46. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:46.

[0309] In some embodiments, the antigen-binding molecule of the present disclosure binds to the P domain of CNX. In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:47. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:47.

[0310] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:67. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:67. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:67. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:67.

[0311] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:68. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:68. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:68. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:68. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:68.

[0312] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:69. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:69. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:69. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:69. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:69.

[0313] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NQ:70. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NQ:70. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NQ:70. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NQ:70. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:70.

[0314] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:71 . In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:71 . In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:71. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:71 . In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:71 .

[0315] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:71 . In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:72. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:72. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:72. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:72.

[0316] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:73. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:73. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:73. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:73. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:73.

[0317] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:74. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:74. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:74. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:74. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:74.

[0318] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:75. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:75. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:75. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:75. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:75.

[0319] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:76. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:76. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:76. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:76. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:76.

[0320] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:77. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:77. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:77. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:77.

[0321] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:78. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:78. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:78. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:78.

[0322] In some embodiments, the antigen-binding molecule binds to the region of CNX shown in SEQ ID NO:79. In some embodiments, the antigen-binding molecule contacts the region of CNX shown in SEQ ID NO:79. In some embodiments, the antigen-binding molecule binds to CNX via contact with one or more amino acids of the region shown in SEQ ID NO:79. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising, or consisting of, the amino acid sequence shown in SEQ ID NO:79.

[0323] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, surface plasmon resonance and biolayer interferometry.

[0324] In some embodiments, the antigen-binding molecule is capable of binding the same region of CNX, or an overlapping region of CNX, as the region of CNX which is bound by an antibody comprising the VH and VL regions of one of clones 1 D3, 1 D6, 1 E1 , 1 E6, 2C6, 2H6, 3D1 , 2G9, 2G12, 2H5, 3F8, 3F9, 4G9, 5A3, 5E8, C001 , C008, C010, C023, C025, C040, C046 and C117. In some embodiments, the antigen-binding molecule is capable of binding the same region of CNX, or an overlapping region of CNX, as the region of CNX which is bound by an antibody comprising the VH and VL regions of C008 or 1 E1 .

[0325] Whether a test antigen-binding molecule binds to the same or an overlapping region of a given target as a reference antigen-binding molecule can be evaluated, for example, by comparative analysis of (i) interaction between the test antigen-binding molecule and the target in the absence of the reference binding molecule, and (ii) interaction between the test antigen-binding molecule and the target in the presence of the reference antigen-binding molecule, or following incubation of the target with the reference antigen-binding molecule. Determination of a reduced level of interaction between the test antigen-binding molecule and the target following analysis according to (ii) as compared to (i) might support an inference that the test and reference antigen-binding molecule bind to the same or an overlapping region of the target. Suitable assays for such analysis include e.g. competition ELISA assays and epitope binning assays.

[0326] In some embodiments, the antigen-binding molecule is an antagonist of CNX, CRT and / or an antagonist of a complex comprising CNX or CRT. In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process mediated by CNX and / or CRT, or mediated by complexes comprising CNX / CRT. In some embodiments, the antigen-binding molecule is capable of inhibiting a function or process mediated by a polypeptide complex comprising CNX or CRT. Herein, ‘inhibition’ refers to a reduction, decrease or lessening relative to a control condition. Suitable assays for investigating the function of CNX and / or CRT, and of complexes comprising CNX / CRT are well known to the skilled person.

[0327] In some embodiments, a complex comprising CNX may be selected from: a CNX:ERp57 complex, a CNX:ERp29 complex and a CNX:CypB complex. In some embodiments, a complex comprising CNX may comprise CNX and a glycopolypeptide. In some embodiments, a complex comprising CRT may be selected from: a CRT:ERp57 complex, a CRT:ERp29 complex and a CRT:CypB complex. In some embodiments, a complex comprising CRT may comprise CRT and a glycopolypeptide.

[0328] In some embodiments, a complex comprising CNX is a CNX:ERp57 complex. In some embodiments, a complex comprising CRT is a CRT:ERp57 complex.

[0329] Assays for the identification of antigen-binding molecules capable of reducing / inhibiting a function of CNX / CRT and / or of complexes comprising CNX / CRT may comprise treating cells / tissue expressing CNX / CRT and / or a complex comprising CNX / CRT with a test antigen-binding molecule, and subsequently comparing the level of the relevant function to the level observed in an appropriate control condition (e.g. untreated / vehicle-treated / control-treated cells / tissue). Antigen-binding molecules capable of reducing / inhibiting a function of CNX / CRT, and / or of a complex comprising CNX / CRT, may be identified using assays comprising detecting the level of a correlate of a function of CNX / CRT, and / or of a complex comprising CNX / CRT, (e.g. the gene and / or protein expression, and / or activity, of one or more proteins whose expression is directly / indirectly upregulated or downregulated as a consequence of a function of CNX / CRT and / or a complex comprising CNX / CRT). Such assays may comprise treating cells / tissue expressing CNX / CRT and / or a complex comprising CNX / CRT with the antigen-binding molecule, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for the functional consequences of an activity of CNX / CRT and / or a complex comprising CNX / CRT to be observed) comparing the level of the correlate of a function of CNX / CRT, and / or of a complex comprising CNX / CRT, in such cells / tissue to the level of the correlate of the relevant function in an appropriate control condition (e.g. untreated / vehicle-treated / control-treated cells / tissue).

[0330] In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a gastrointestinal cell. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is an intestinal cell. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a mucosal cell or an epithelial cell. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a cell of the lamina propria. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is an immune cell. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a fibroblast, macrophage, lymphocyte, plasma cell, or a mast cell. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a fibroblast or a macrophage. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a fibroblast. In some embodiments, the cell that expresses CNX (e.g., glycosylated CNX) is a macrophage.

[0331] In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing / inhibiting a function of CNX / CRT, or of a complex comprising CNX / CRT to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, SO.6 times, SO.55 times, SO.5 times, SO.45 times, S0.4 times, SO.35 times, SO.3 times, S0.25 times, S0.2 times, SO.15 times, S0.1 times, SO.05 times, or SO.01 times the level of the relevant function observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

[0332] In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring or involving Fc-mediated function. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, independently of Fc-mediated function. That is, in some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, in an Fc region-independent manner.

[0333] The ability of an antigen-binding molecule to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring / involving Fc-mediated function can be evaluated e.g. by analysing the ability of the antigen-binding molecule provided in a format lacking a functional Fc region to inhibit a function of CNX / CRT and / or a complex comprising CNX / CRT. For example, the effect on a function of CNX / CRT, and / or of a complex comprising CNX / CRT, can be investigated using an antigen-binding molecule comprising a ‘silent’ Fc region (e.g. comprising LALA PG substitutions), or using an antigen-binding molecule provided in a format lacking an Fc region (e.g. scFv, Fab etc.).

[0334] In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not involving ADCC. In some embodiments, the antigenbinding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not involving ADCP. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not involving CDC.

[0335] In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding of the antigen-binding molecule to an Fc receptor. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding of the antigen-binding molecule to an Fey receptor. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding of the antigen-binding molecule to one or more of FcyRI, FcyRlla, FcyRHb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding to FcyRllla. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding to FcyRlla. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding to FcyRHb. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding to a complement protein. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring binding to C1q. In some embodiments, the antigen-binding molecule is able to inhibit a function of CNX / CRT, and / or of a complex comprising CNX / CRT, by a mechanism not requiring N297 glycosylation.

[0336] It will be appreciated that in some embodiments, the antigen-binding molecule of the present disclosure achieves is functional effects via a mechanism not involving Fc-mediated function. In some embodiments, the antigen-binding molecule of the present disclosure achieves is functional effects via a mechanism not involving killing / depletion of cells expressing CNX / CRT, or of cells expressing complexes comprising CNX / CRT, e.g. Fc-mediated killing / depletion of such cells.

[0337] In some embodiments, a function of CNX / CRT, or a function of a complex comprising CNX / CRT, may be selected from: extracellular matrix (ECM) degradation, collagen degradation, gelatin degradation, oxireductase activity and disulfide bond reductase activity. A correlate of a function of CNX / CRT, or of a complex comprising CNX / CRT, may e.g. be a product of ECM / collagen / gelatin degradation, or oxireductase / disulfide bond reductase activity.

[0338] In some embodiments, the antigen-binding molecule reduces / inhibits extracellular matrix (ECM) degradation. In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation. In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation. In some embodiments, the antigen-binding molecule reduces / inhibits oxireductase activity. In some embodiments, the antigen-binding molecule reduces / inhibits disulfide bond reductase activity. In some embodiments, the antigen-binding molecule reduces / inhibits ECM degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g. a CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits collagen degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g. a CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits gelatin degradation mediated by CNX / CRT or a complex comprising CNX / CRT (e.g. a CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits oxireductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g. a CNX / CRT:ERp57 complex). In some embodiments, the antigen-binding molecule reduces / inhibits disulfide bond reductase activity mediated by CNX / CRT or a complex comprising CNX / CRT (e.g. a CNX / CRT:ERp57 complex).

[0339] The ability of an antigen-binding molecule to inhibit ECM / collagen / gelatin degradation can be determined for example by analysis of ECM / collagen / gelatin degradation in the presence of, or following incubation with, the antigen-binding molecule. An antigen-binding molecule which is capable of inhibiting ECM / collagen / gelatin degradation is identified by the observation of a reduction / decrease in the level of ECM / collagen / gelatin degradation in the presence of - or following incubation with - the antigen-binding molecule, as compared to the level of ECM / collagen / gelatin degradation in the absence of the antigenbinding molecule (or in the presence of an appropriate control antigen-binding molecule).

[0340] Antigen-binding molecules capable of reducing / inhibiting ECM / collagen / gelatin degradation (e.g. by CNX / CRT and / or a complex comprising CNX / CRT) may be identified using assays comprising detecting the level of ECM / collagen / gelatin, or the level of a correlate of ECM / collagen / gelatin degradation (e.g. a product of degraded ECM / collagen / gelatin), e.g. using antibody / reporter-based methods. Collagen / gelatin degradation assays are described e.g. in Hollander, Methods Mol. Biol. (2010) 622:367-78 and Vandooren et al., World J. Biol. Chem. (2011 ) 2(1 ): 14-24. In preferred embodiments, ECM / collagen / gelatin degradation can be evaluated in an assay performed essentially as described in Example 4 herein.

[0341] For example, a commercial solution of gelatin (2%) can be labeled with 5-Carboxy-X-Rhodamine, Succinimidyl Ester. The labeled gelatin can then be transferred onto sterile coverslips to create a thin layer, and stabilised by glutaraldehyde fixation. A solution of rat tail collagen can be used to coat the coverslips, creating a thin layer of collagen on top of the gelatin. The coverslips can then be transferred in culture vessels and cells with the appropriate degradative activity (e.g. human hepatocellular carcinoma Huh7 cells) can be seeded on the coverslips in the presence of test antigen-binding molecules, and incubated for 48h to allow degradation to occur. The coverslips can then be fixed, and subsequently stained with Hoescht to permit the counting of cells, and then analysed by confocal microscopy. The images acquired can be analysed using Imaged to determine the surface of degraded gelatin and the total area per field. In parallel, the number of nuclei can be calculated and the final result can be normalised to the number of cells in each field.

[0342] For example, a mixture of rat tail collagen and quenched fluorescent DQ collagen type I can be coated and polymerised on the bottom of a 384 well optical grade plate. Cells of the 3t3-vSrc mouse cell line can be seeded on top of the collagen layer in the presence of test antigen-binding molecules, and incubated for 48h to 72h. The fluorescent area of DQ signal from live cells can subsequently be evaluated by high content imaging, and normalised by nucleus count to determine the degraded area / cell.

[0343] In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing / inhibiting ECM degradation, collagen degradation or gelatin degradation to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, SO.15 times, <0.1 times, <0.05 times, or <0.01 times, the level of ECM degradation / collagen degradation / gelatin degradation observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

[0344] The ability of an antigen-binding molecule to inhibit oxireductase activity can be determined for example by analysis of oxireductase activity in the presence of, or following incubation with, the antigen-binding molecule. An antigen-binding molecule which is capable of inhibiting oxireductase activity is identified by the observation of a reduction / decrease in the level oxireductase activity in the presence of - or following incubation with - the antigen-binding molecule, as compared to the level of oxireductase activity in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[0345] Oxireductase activity can be evaluated using any one of a number of methods known to the person skilled in the art. For example, oxireductase activity can be evaluated in an insulin reduction assay, e.g. as described in Hirano et al., Eur J Biochem. (1995) 234(1 ):336-42.

[0346] In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing / inhibiting oxireductase activity to less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, SO.85 times, SO.8 times, SO.75 times, SO.7 times, SO.65 times, SO.6 times, SO.55 times, SO.5 times, SO.45 times, S0.4 times, SO.35 times, SO.3 times, S0.25 times, SO.2 times, SO.15 times, S0.1 times, SO.05 times, or SO.01 times, the level of oxireductase activity observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

[0347] The ability of an antigen-binding molecule to inhibit disulfide bond reductase activity can be determined for example by analysis of disulfide bond reductase activity in the presence of, or following incubation with, the antigen-binding molecule. An antigen-binding molecule which is capable of inhibiting disulfide bond reductase activity is identified by the observation of a reduction / decrease in the level disulfide bond reductase activity in the presence of - or following incubation with - the antigen-binding molecule, as compared to the level of disulfide bond reductase activity in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).

[0348] Disulfide bond reductase activity can be evaluated using any one of a number of methods known to the person skilled in the art. For example, disulfide bond reductase assays may employ antibodies for detecting reduced disulfide bonds in proteins, e.g. antibody clone 0X133, which recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues (see Holbrook et al., Mabs (2016) 8(4): 672-677).

[0349] In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing / inhibiting disulfide bond reductase activity to less than 1 times, e.g. 20.99 times, 20.95 times, 20.9 times, 20.85 times, 20.8 times, 20.75 times, 20.7 times, 20.65 times, 20.6 times, 20.55 times, 20.5 times, 20.45 times, 20.4 times, 20.35 times, 20.3 times, 20.25 times, 20.2 times, 20.15 times, 20.1 times, 20.05 times, or 20.01 times, the level of disulfide bond reductase activity observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

[0350] In some embodiments, an antigen-binding molecule according to the present disclosure reduces / inhibits cartilage degradation. Antigen-binding molecules capable of reducing / inhibiting cartilage degradation (e.g. by CNX / CRT and / or a complex comprising CNX / CRT) may be identified using assays comprising detecting the level of cartilage, or the level of a correlate of cartilage degradation (e.g. a product of degraded cartilage), e.g. using antibody / reporter-based methods. Cartilage degradation can be evaluated essentially as described in Example 6 herein. An ex vivo assay of cartilage degradation is also described e.g. in Neidlin et a / ., PLoS One (2019) 14(10):e0224231.

[0351] In some embodiments, the antigen-binding molecule of the present disclosure is capable of reducing / inhibiting cartilage degradation to less than 1 times, e.g. 20.99 times, 20.95 times, 20.9 times, 20.85 times, 20.8 times, 20.75 times, 20.7 times, 20.65 times, 20.6 times, 20.55 times, 20.5 times, 20.45 times, 20.4 times, 20.35 times, 20.3 times, 20.25 times, 20.2 times, 20.15 times, 20.1 times, 20.05 times, or 20.01 times, the level of cartilage degradation observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in a given assay.

[0352] In some embodiments an antigen-binding molecule according to the present disclosure is capable of reducing the number / proportion of cells expressing CNX / CRT, or a complex comprising CNX / CRT. In some embodiments, an antigen-binding molecule according to the present disclosure is capable of depleting / enhancing depletion of such cells. Antigen-binding molecules according to the present disclosure may comprise one or more moieties for potentiating a reduction in the number / proportion of cells expressing CNX / CRT, or a complex comprising CNX / CRT. For example, an antigen-binding molecule according to the present disclosure may e.g. comprise an Fc region and / or a drug moiety.

[0353] Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects. IgG Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about by Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells). Such functions occur through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0354] In some embodiments, an antigen-binding molecule according to the present disclosure comprises an Fc region capable of potentiating / directing one or more of (I) ADCC, ADCP, and / or CDC against; and / or (II) potentiating formation of a MAC on; and / or (ill) cell degranulation of, a cell expressing CNX / CRT, or a complex comprising CNX / CRT (e.g. a cell expressing CNX / CRT, or a complex comprising CNX / CRT at the cell surface).

[0355] In some embodiments, an antigen-binding molecule according to the present disclosure is capable of potentiating / directing ADCC against a cell expressing CNX / CRT, or a complex comprising CNX / CRT.

[0356] The ability of, and extent to which, a given antigen-binding molecule is able to induce ADCC of a given target cell type can be analysed e.g. according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporated by reference in its entirety), or by51Cr release assay as described e.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated by reference in its entirety). The ability of, and extent to which, a given antigen-binding molecule is able to induce ADCP can be analysed e.g. according to the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (hereby incorporated by reference in its entirety). The ability of, and extent to which, a given antigen-binding molecule is able to induce CDC can be analysed e.g. using a C1q binding assay, e.g. as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457- 466 (hereby incorporated by reference in its entirety).

[0357] In some embodiments, the antigen-binding molecule of the present disclosure does not induce ADCC of cells expressing CNX / CRT, or complexes comprising CNX / CRT, at the cell surface. In some embodiments, the antigen-binding molecule does not induce ADCP of cells expressing CNX / CRT, or complexes comprising CNX / CRT, at the cell surface. In some embodiments, the antigen-binding molecule does not induce CDC of cells expressing CNX / CRT, or complexes comprising CNX / CRT, at the cell surface. In some embodiments, the antigen-binding molecule does not induce ADCC, ADCP or CDC of cells expressing CNX / CRT, or complexes comprising CNX / CRT, at the cell surface.

[0358] Antigen-binding molecules which do not induce (i.e. are not able to induce) ADCC / ADCP / CDC elicit substantially no ADCC / ADCP / CDC activity against the relevant cell type, e.g. as determined by analysis in an appropriate assay for the relevant activity. “Substantially no ADCC / ADCP / CDC activity” refers to a level of ADCC / ADCP / CDC that is not significantly greater than ADCC / ADCP / CDC determined for an appropriate negative control molecule in a given assay (e.g. an antigen-binding molecule lacking an Fc region, or an antigen-binding molecule comprising a ‘silent’ Fc region (e.g. as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, which is incorporated by reference hereinabove)). “Substantially no activity” may be a level of the relevant activity which is s 5 times, e.g. < 4 times, 2 3 times, 22.5 times, S 2 times or S 1.5 times the level of activity determined for an appropriate negative control molecule in a given assay.

[0359] In some embodiments, an antigen-binding molecule according to the present disclosure comprises a drug moiety. The antigen-binding molecule may be conjugated to the drug moiety. Antibody-drug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14 (hereby incorporated by reference in its entirety). In some embodiments, the drug moiety is or comprises a cytotoxic agent, such that the antigen-binding molecule displays cytotoxicity to a cell expressing CNX / CRT, or a complex comprising CNX / CRT (e.g. a cell expressing CNX / CRT, or a complex comprising CNX / CRT at the cell surface). In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0360] In some embodiments, an antigen-binding molecule according to the present disclosure comprises an immune cell-engaging moiety. In some embodiments, the antigen-binding molecule comprises a CD3 polypeptide-binding moiety (e.g. an antigen-binding domain capable of binding to a CD3 polypeptide).

[0361] In some embodiments, an antigen-binding molecule according to the present disclosure is capable of potentiating / directing T cell-mediated cytolytic activity against a cell expressing CNX / CRT, or a complex comprising CNX / CRT.

[0362] Chimeric antigen receptors (CARs)

[0363] In some embodiments, the antigen-binding molecule is a Chimeric Antigen Receptors (CAR).

[0364] CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region. An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker. The CAR of the present disclosure comprises an antigen-binding region which comprises or consists of the antigen-binding molecule of the present disclosure, or which comprises or consists of a polypeptide according to the present disclosure.

[0365] The cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell. In some embodiments, the CAR comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CD3- , CD4, CD8 or CD28. As used herein, a region which is ‘derived from’ a reference amino acid sequence comprises an amino acid sequence having 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 reference sequence.

[0366] The signalling region of a CAR allows for activation of the T cell. The CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell. Signalling regions comprising sequences of other ITAM-containing proteins such as FcyRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1 ):182-187). Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27. In some cases, CARs are engineered to provide for co-stimulation of different intracellular signalling pathways. For example, signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, whereas 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling regions of CARs therefore sometimes contain co-stimulatory sequences derived from signalling regions of more than one co-stimulatory molecule. In some embodiments, the CAR of the present disclosure comprises one or more co-stimulatory sequences comprising, or consisting of, an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, 0X40, 4-1 BB, ICOS and CD27.

[0367] An optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Hinge regions may be derived from IgG 1 . In some embodiments, the CAR of the present disclosure comprises a hinge region comprising, or consisting of, an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of IgG 1 .

[0368] Also provided is a cell comprising a CAR according to the present disclosure. The CAR according to the present disclosure may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK cells. Engineering of CARs into immune cells may be performed during culture, in vitro. The antigen-binding region of the CAR of the present disclosure may be provided with any suitable format, e.g. scFv, scFab, etc.

[0369] Nucleic acids and vectors

[0370] The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule, polypeptide or CAR according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0371] In some embodiments, the nucleic acid(s) may be, or may be comprised in, a vector, or a plurality of vectors. That is, the nucleotide sequence(s) of the nucleic acid(s) may be contained in vector(s). The antigen-binding molecule, polypeptide or CAR according to the present disclosure may be produced within a cell by transcription from a vector encoding the antigen-binding molecule, polypeptide or CAR, and subsequent translation of the transcribed RNA.

[0372] Accordingly, the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure. The vector may facilitate delivery of the nucleic acid(s) encoding an antigen-binding molecule, polypeptide or CAR according to the present disclosure. The vector may be an expression vector comprising elements required for expressing nucleic acid(s) comprising / encoding an antigen-binding molecule, polypeptide or CAR according to the present disclosure.

[0373] Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0374] The nucleotide sequence may be contained in a vector, e.g. an expression vector. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0375] The term ‘operably linked’ may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s) / polypeptide(s). Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety.

[0376] In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0377] Constituent polypeptides of an antigen-binding molecule according to the present disclosure may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.

[0378] Cells comprisinq / expressinq the antigen-binding molecules and polypeptides

[0379] The present disclosure also provides a cell comprising or expressing an antigen-binding molecule, polypeptide or CAR according to the present disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure.

[0380] The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), or donkey).

[0381] In some embodiments, the cell is, or is derived from, a cell type commonly used for the expression of polypeptides for use in therapy in humans. Exemplary cells are described e.g. in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include e.g. CHO, HEK 293, PER.C6, NS0 and BHK cells. In preferred embodiments, the cell is, or is derived from, a CHO cell.

[0382] The present disclosure also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction). The present disclosure also provides a method for producing a cell expressing / comprising an antigenbinding molecule, polypeptide or CAR according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.

[0383] The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure.

[0384] Producing the antigen-binding molecules and polypeptides

[0385] Antigen-binding molecules and polypeptides according to the present disclosure may be prepared according to methods for the production of polypeptides known to the skilled person.

[0386] Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis. For example, peptides / polypeptides can be synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.

[0387] Alternatively, antigen-binding molecules and polypeptides may be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety.

[0388] In some cases, the antigen-binding molecules of the present disclosure are comprised of more than one polypeptide chain. In such cases, production of the antigen-binding molecules may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding molecule.

[0389] For recombinant production according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments, the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments, the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. a cell described hereinabove.

[0390] In some cases, the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. In addition, very high expression levels are possible in eukaryotes, and proteins can be easier to purify from eukaryotes using appropriate tags. Specific plasmids may also be utilised which enhance secretion of the protein into the media.

[0391] In some embodiments polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according to a system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431 , which is hereby incorporated by reference in its entirety.

[0392] Production may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s). Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0393] Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0394] Following culturing the cells that express the antigen-binding molecule / polypeptide(s), the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide, it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.

[0395] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.

[0396] Other methods for distinguishing different proteins are known in the art, for example ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation or may be performed subsequently to precipitation. Once the polypeptide(s) of interest have been isolated from culture it may be desired or necessary to concentrate the polypeptide(s). A number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.

[0397] Compositions

[0398] The present disclosure also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein.

[0399] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0400] The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0401] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0402] Compositions may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion. Suitable formulations may comprise the relevant article in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0403] In some embodiments, the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or tumor.

[0404] The present disclosure also provides methods for the production of pharmaceutically useful compositions. Such methods of production may comprise one or more steps selected from: producing an antigenbinding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0405] For example, a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0406] Therapeutic and prophylactic applications

[0407] The present disclosure provides an antigen-binding molecule, polypeptide, CAR, 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, CAR, 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, CAR, 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, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0408] The methods may be effective to reduce the development or progression of a disease / condition, alleviation of 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).

[0409] Articles of the present disclosure may be used for the treatment / prevention of a disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of CNX (or complexes comprising CNX), or a reduction in the number or activity of cells comprising / expressing CNX (or complexes comprising CNX).

[0410] In some embodiments, the disease / condition may be: a disease / condition in which CNX is pathologically-implicated, a disease / condition in which cells expressing CNX are pathologically-implicated, a disease / condition that would derive therapeutic or prophylactic benefit from a reduction of the level / activity of CNX, a disease / condition characterised by an increase in the level of expression of CNX, e.g. as compared to the level of a relevant control (e.g., expression of CNX in the absence of the disease / condition), a disease / condition characterised by an increase in the number / proportion / activity of cells expressing CNX, e.g. as compared to the level of a relevant control (e.g., expression of CNX in the absence of the disease / condition).

[0411] The CNX may be glycosylated CNX. Therefore, in some embodiments, the disease / condition may be: a disease / condition in which glycosylated CNX is pathologically-implicated, a disease / condition in which cells expressing glycosylated CNX are pathologically-implicated, a disease / condition that would derive therapeutic or prophylactic benefit from a reduction of the level / activity of glycosylated CNX, a disease / condition characterised by an increase in the level of expression of glycosylated CNX, e.g. as compared to the level of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition), a disease / condition characterised by an increase in the number / proportion / activity of cells expressing glycosylated CNX, e.g. as compared to the level of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition).

[0412] In some embodiments, the glycosylated CNX is O-glycosylated CNX. In some embodiments, the glycosylated CNX is Tn glycosylated CNX.

[0413] The disease / condition may be a disease / condition in which CNX, or cells expressing CNX, are pathologically-implicated. The disease / condition may be a disease / condition in which glycosylated CNX, or cells expressing glycosylated CNX, are pathologically-implicated. The disease / condition may be a disease / condition in which Tn glycosylated CNX, or cells expressing Tn glycosylated CNX, are pathologically-implicated.

[0414] The disease / condition may be a disease / condition in which a CNX-containing complex, or cells expressing a CNX-containing complex, are pathologically-implicated. In some embodiments, the CNX- containing complex is a CNX:ERp57 complex, CNX:ERp29 complex, or a CNX:integrin complex.

[0415] Integrins are alpha beta heterodimers that mediate cell-cell and cell-extracellular matrix (ECM) adhesion. It has been shown that integrin chains beta 1 and alpha 6 associate with the chaperone calnexin prior to integrin assembly (Lentin and Vestweber. J Biol Chem. 1994 Apr 22;269(16): 12263-8, which is hereby incorporated by reference in its entirety). In some embodiments, the integrin comprises a beta 1 (CD29) and / or an alpha 6 (CD49f) chain. In some embodiments, the CNX:integrin complex is a CNX:CD29 complex. In some embodiments, the CNX:integrin complex is a CNX:CD49f complex.

[0416] The disease / condition may be a disease / condition which is characterised by the expression of glycosylated CNX. A disease / condition which is characterised by the expression of glycosylated CNX may be any disease / condition which is associated with the expression of glycosylated CNX. For example, the disease / condition which is characterised by the expression of glycosylated CNX may be: a disease / condition in which glycosylated CNX is pathologically-implicated, a disease / condition in which cells expressing glycosylated CNX are pathologically-implicated, a disease / condition that would derive therapeutic or prophylactic benefit from a reduction of the level / activity of glycosylated CNX a disease / condition characterised by an increase in the level of expression of glycosylated CNX, e.g. as compared to the level of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition). a disease / condition characterised by an increase in the number / proportion / activity of cells expressing glycosylated CNX, e.g. as compared to the level of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition).

[0417] In some embodiments, the disease / condition which is characterised by the expression of glycosylated CNX is a disease / condition characterised by an increase in the level of expression of glycosylated CNX, e.g. as compared to the level of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition). In some embodiments, the disease / condition which is characterised by the expression of glycosylated CNX 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 of a relevant control (e.g., expression of glycosylated CNX in the absence of the disease / condition).

[0418] The disease may be a disease / condition in which an increased level / activity of CNX, of 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, of 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.

[0419] 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.

[0420] 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.

[0421] The CNX may be glycosylated CNX. In some embodiments, the glycosylated CNX is O-glycosylated CNX. In some embodiments, the glycosylated CNX is Tn glycosylated CNX.

[0422] 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.

[0423] 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.

[0424] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated O-glycosylation activity. For example, where the disease / condition is a gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) may comprise cells having elevated O-glycosylation activity. As used herein ‘O-glycosylation activity’ refers to addition 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. An ‘elevated’ level of O-glycosylation activity may refer to a level of O-glycosylation activity which is greater than the level of O-glycosylation 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 gastrointestinal disease (e.g., IBD), the level of O-glycosylation activity may be greater than the level of O-glycosylation activity in equivalent non-diseased tissue. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated Src activity. For example, where the disease / condition is a gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) may comprise cells having elevated Src activity. As used herein 'Src activity’ refers to Src-mediated phosphorylation of tyrosine residues. An ‘elevated’ level of Src activity may refer to a level of Src activity which is greater than the level of Src activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent nondiseased tissue). Where the disease / condition is a gastrointestinal disease (e.g., IBD), the level of Src activity may be greater than the level of Src activity in equivalent non-diseased tissue.

[0425] 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 gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) 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 gastrointestinal disease (e.g., IBD), the level of GALNT activity may be greater than the level of GALNT activity in equivalent non-diseased tissue.

[0426] 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 gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) 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 gastrointestinal disease (e.g., IBD), the level of O-glycosylation may be greater than the level of O-glycosylation in equivalent non-diseased tissue.

[0427] 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 gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) may comprise cells having Tn glycosylation of a protein expressed by the cells. As used herein ‘Tn glycosylation’ refers to the presence of N-acetylgalactosamine (GalNAc) linked to the hydroxyl group of the side chain of a serine or threonine residue of a protein by a glycosidic bond. A ‘Tn glycosylated’ protein comprises at least one Tn glycan, which may also be referred to as Tn antigen.

[0428] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated CNX glycosylation. For example, where the disease / condition is a gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) may comprise cells having an elevated level of glycosylation of CNX expressed by the cells. An ‘elevated’ level of glycosylation of CNX may refer to a level of CNX glycosylation which is greater than the level of CNX 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 gastrointestinal disease (e.g., IBD), the level of glycosylation may be greater than the level of glycosylation in equivalent non-diseased tissue.

[0429] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by an elevated level of glycosylated CNX (e.g., Tn glycosylated CNX). For example, where the disease / condition is a gastrointestinal disease (e.g., IBD), the gastrointestinal disease (e.g., IBD) may comprise cells having an elevated level of glycosylated CNX. An ‘elevated’ level of glycosylated CNX may refer to the number of CNX molecules which are glycosylated being greater than the number of CNX molecules which are glycosylated 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 gastrointestinal disease (e.g., IBD), the level of glycosylated CNX may be greater than the level of glycosylated CNX in equivalent non-diseased tissue.

[0430] Anti-CNX antibodies have been demonstrated to be useful to inhibit ECM degradation. Accordingly, 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.

[0431] 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.

[0432] One of the defining characteristics of inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, is the increased remodelling of the extracellular matrix (Derkacz et al., J Clin Med. 2021 Mar; 10(5): 1122, which is hereby incorporated by reference in its entirety). Research has shown that ECM degradation is associated with the pathology of gastrointestinal diseases. For example, Kirov et al (Mol. Omics, 2019, 15, 67-76, which is hereby incorporated by reference in its entirety) showed that degradation of the extracellular matrix is part of the pathology of ulcerative colitis. It has also been found that ECM markers correlate with disease activity, disease extent, and predict future disease outcome in ulcerative colitis (Poulsen et al. Journal of Crohn's and Colitis, 18, 2024, Pages i728— i729 , which is hereby incorporated by reference in its entirety).

[0433] The extracellular matrix is a highly dynamic structure, present in all tissues, which undergoes controlled remodelling. During this process, both quantitative and qualitative changes of its components take place in order to control homeostasis and tissue architecture [3]. Matrix components include: (1 ) collagen proteins: type I collagen, basement membrane collagens (type IV, VIII and X), type VI microfibrillar collagens, (fibril-associated collagens with interrupted triple helices (FACIT); (2) non-collagen ECM proteins (elastin, fibronectin, laminin, thrombospondin or tenascin; (3) proteoglycans (PG) and glycosaminoglycans (GAG); and (4) growth factors, enzymes, including matrix metalloproteinases. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by aberrant ECM remodelling. A disease / condition which is ‘characterised by aberrant ECM remodelling’ may be a disease / condition in which aberrant ECM remodelling is a symptom of the disease / condition.

[0434] Gastrointestinal tissue undergoes rapid cell turnover, with epithelial cells being replaced approximately every week. In this highly dynamic environment, the ECM is essential for maintaining tissue homeostasis, balancing tissue repair and degradation. However, in gastrointestinal diseases such as IBD, ECM remodeling becomes dysregulated (Shimshoni et al., Gut. 2015 Mar;64(3):367-72; Kirkegaard et al., Gut. 2004 May;53(5):701-9, Mortensen et al. Expert Rev Gastroenterol Hepatol. 2019 Oct;13(10):977-993, all of which are hereby incorporated by reference in their entirety). This imbalance contributes to chronic inflammation, tissue destruction, and impaired intestinal function.

[0435] The disease / condition to be treated / prevented in accordance with the present disclosure may be a disease / condition in which aberrant ECM remodelling is pathologically-implicated. The disease / condition may be a disease / condition in which ECM degradation is implicated in the pathology of the disease / condition. The disease / condition may be a disease / condition in which ECM formation, and / or an increased level of ECM formation, is implicated in the pathology of the disease / condition. The disease / condition may be a disease / condition in which ECM deposition, and / or an increased level of ECM deposition, is implicated in the pathology of the disease / condition.

[0436] In some embodiments the disease / condition to be treated / prevented is a gastrointestinal disease. A gastrointestinal disease may alternatively be described as a disease or disorder of the gastrointestinal system. In some embodiments, the gastrointestinal disease is a disease or a disorder of an organ or tissue of the gastrointestinal system. In some embodiments, the gastrointestinal disease is a disease or a disorder of an organ or tissue of the gastrointestinal tract. In some embodiments, the gastrointestinal disease is a disease or a disorder of the bowel. In some embodiments, the gastrointestinal disease is a disease or a disorder of the large intestine. In some embodiments, the gastrointestinal disease is a disease or a disorder of the small intestine. In some embodiments, the gastrointestinal disease is a disease or a disorder of the stomach.

[0437] The disease / condition may be characterised by inflammation. Many diseases are associated with an overactive inflammatory response ( / .e. excessive inflammation and / or aberrantly activated inflammation), and / or chronic (prolonged) inflammation. In some embodiments, the disease / condition to be treated / prevented is characterised by gastrointestinal inflammation. In some embodiments, the disease / condition to be treated / prevented is characterised by intestinal inflammation. In some embodiments, the disease / condition to be treated / prevented is characterised by inflammation of the colon. In some embodiments, the disease / condition to be treated / prevented is characterised by inflammation of the small intestine.

[0438] A polyp is a small growth of excess tissue that may grow on the lining of the gastrointestinal tract in subjects with gastrointestinal diseases, such as IBD. In some embodiments, the disease / condition to be treated / prevented is characterised by the presence of polyps. In some embodiments, the polyp is a colorectal polyp, and / or a colon polyp. In some embodiments, the polyp is an adenomatous polyp, a hyperplastic polyp, a sessile serrated lesion, an inflammatory polyp, a villous adenoma, and / or a hamartomatous polyp.

[0439] In some embodiments, the disease / condition to be treated / prevented is IBD. In some embodiments, the IBD is Crohn's disease or ulcerative colitis. In some embodiments, the disease / condition to be treated / prevented is Crohn's disease. In some embodiments, the disease / condition to be treated / prevented is ulcerative colitis.

[0440] The disease / condition may be characterised by the expression of CNX (e.g., glycosylated CNX) by a particular cell type. In some embodiments, the expression of CNX by a cell (e.g., an epithelial cell and / or a fibroblast) comprises the expression of CNX on the surface of the cell. In some embodiments, the CNX is glycosylated CNX (e.g., Tn glycosylated CNX).

[0441] In some embodiments, the disease / condition is characterised by the expression of CNX by gastrointestinal cells. In some embodiments, the disease / condition is characterised by the expression of CNX by intestinal cells. In some embodiments, the disease / condition is characterised by the expression of CNX by cells of the large intestine. In some embodiments, the disease / condition is characterised by the expression of CNX by cells of the small intestine.

[0442] The architecture of the gastrointestinal tract and its developmental features of different segments have been well defined and are well known to the skilled person. Intestinal architecture and development is reviewed by Rao and Wang (Regulation of Gastrointestinal Mucosal Growth. San Rafael (CA): Morgan & Claypool Life Sciences; 2010, which is hereby incorporated by reference in its entirety). All segments of the Gl tract are divided into four layers: the mucosa (epithelium, lamina propria, and muscular mucosae), the submucosa, the muscularis propria (inner circular muscle layer, intermuscular space, and outer longitudinal muscle layer), and the serosa.

[0443] The mucosa consists of three layers. The first layer facing the intestinal lumen is made up of epithelial cells, which is a single layer in the Gl tract and is attached to a basement membrane overlying the second layer, the lamina propria, which consists of subepithelial connective tissue and lymph nodes, underneath which is the third and deepest layer called muscularis mucosae. In some embodiments, a mucosal cell is an epithelial cell, a cell of the lamina propria, and / or a cell of the muscular mucosae. In some embodiments, the disease / condition is characterised by the expression of CNX by a mucosal cell. In some embodiments, the disease / condition is characterised by the expression of CNX by a gastrointestinal mucosal cell. In some embodiments, the disease / condition is characterised by the expression of CNX by an intestinal mucosal cell. In some embodiments, the disease / condition is characterised by the expression of CNX by a mucosal cell of the large intestine. In some embodiments, the disease / condition is characterised by the expression of CNX by a mucosal cell of the small intestine.

[0444] In some embodiments, the disease / condition is characterised by the expression of CNX by a cell of the lamina propria. In some embodiments, the disease / condition is characterised by the expression of CNX by a cell of the gastrointestinal lamina propria. In some embodiments, the disease / condition is characterised by the expression of CNX by cell of the intestinal lamina propria. In some embodiments, the disease / condition is characterised by the expression of CNX by a cell of the large intestine lamina propria. In some embodiments, the disease / condition is characterised by the expression of CNX by a cell of the small intestine lamina propria.

[0445] The lamina propria comprises the layer of loose connective tissue and interstitial matrix located just below the epithelium. Functionally, the lamina propria provides structural support for the lymphatics and vasculature. Furthermore, the lamina propria of the stomach and intestine is also particularly cell-rich, including fibroblasts, macrophages, lymphocytes, plasma cells, and mast cells. This large proportion of cells with immune function provides an effective secondary line of defence against potential invading microorganisms and aggregations of lymphoid nodules within the lamina propria of the small intestine give rise to the specialized areas known as Peyer's patches. In some embodiments, the cell of the lamina propria is a fibroblast, macrophage, lymphocyte, plasma cell, or a mast cell. In some embodiments, the cell of the lamina propria is a fibroblast or a macrophage.

[0446] In many 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. The fibroblast may be a specific type of fibroblast which is associated with a gastrointestinal disease / condition. The fibroblast may be a specific type of fibroblast which is associated with IBD. In some embodiments, the fibroblast is a myofibroblast, a mucosa-associated fibroblast, a interstitial fibroblast. In some embodiments, the fibroblast is an intestinal fibroblast.

[0447] In some embodiments, the disease / condition is characterised by the expression of CNX by a fibroblast. In some embodiments, the disease / condition is characterised by the expression of CNX by a gastrointestinal fibroblast. In some embodiments, the disease / condition is characterised by the expression of CNX by an intestinal fibroblast. In some embodiments, the disease / condition is characterised by the expression of CNX by a fibroblast of the large intestine. In some embodiments, the disease / condition is characterised by the expression of CNX by a fibroblast of the small intestine.

[0448] Macrophages are key players for the maintenance of intestinal homeostasis (Ruder and Becker.

[0449] Cells. 2020 Oct; 9(10): 2162, which is hereby incorporated by reference in its entirety). They belong to the group of mononuclear phagocytes, which exert bactericidal functions and help to clear apoptotic cells. Moreover, they play essential roles for the maintenance of epithelial integrity and tissue remodelling during wound healing processes. Macrophages secrete immune-modulatory factors, which are necessary to activate other intestinal immune cells and therefore to shape immune responses in the gut. However, overwhelming activation or increased secretion of pro-inflammatory cytokines can contribute to the pathogenesis of inflammatory bowel disease.

[0450] The macrophage may be a specific type of macrophage which is associated with a gastrointestinal disease / condition. The role of tissue-resident macrophages in the development and treatment of Inflammatory Bowel Disease is reviewed by Ma et al. (Front Cell Dev Biol. 2022; 10: 896591 , which is hereby incorporated by reference in its entirety). The macrophage may be a specific type of macrophage which is associated with IBD. In some embodiments, the macrophage is a tissue macrophage. In some embodiments, the macrophage is an intestinal macrophage. In some embodiments, the macrophage is a classically activated (M1 ) macrophage. In some embodiments, the macrophage is an alternatively activated (M2) macrophage.

[0451] In some embodiments, the disease / condition is characterised by the expression of CNX by a macrophage. In some embodiments, the disease / condition is characterised by the expression of CNX by a gastrointestinal macrophage. In some embodiments, the disease / condition is characterised by the expression of CNX by an intestinal macrophage. In some embodiments, the disease / condition is characterised by the expression of CNX by a macrophage of the large intestine. In some embodiments, the disease / condition is characterised by the expression of CNX by a macrophage of the small intestine.

[0452] 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 et al., Nat. Cell Biol. 22, 1371-1381 . 2020). This reduction of disulfide bridges is essential for the effective degradation of ECM (Ros et al., Nat. Cell Biol. 22, 1371-1381. 2020). The inventors have previously identified that synovial fibroblasts express glycosylated CNX, and further demonstrated the treatment of cartilage degradation using anti-CNX antibodies. Anti-CNX antibodies were additionally shown to reduce the pathology of arthritis in vivo, which is a disease characterised by cartilage degradation (WO2022157281A1 ).

[0453] In some embodiments, the disease / condition is characterised by the expression of CNX by an epithelial cell. In some embodiments, the disease / condition is characterised by the expression of CNX by a gastrointestinal epithelial cell. In some embodiments, the disease / condition is characterised by the expression of CNX by an intestinal epithelial cell. In some embodiments, the disease / condition is characterised by the expression of CNX by an epithelial cell of the large intestine. In some embodiments, the disease / condition is characterised by the expression of CNX by an epithelial cell of the small intestine. The epithelial cell may be a specific type of epithelial cell which is associated with a gastrointestinal disease / condition. The epithelial cell may be a specific type of epithelial cell which is associated with IBD. In some embodiments, the epithelial cell is an intestinal epithelial cell.

[0454] In some embodiments, the disease is a disease that would derive therapeutic or prophylactic benefit from a reduction in the expression or activity of CNX.

[0455] In some embodiments, the disease is a disease which is caused or exacerbated by expression / overexpression or activity of CNX. In some embodiments, the disease is a disease for which expression / overexpression or activity of CNX is a risk factor for the development or progression of the gastrointestinal disease.

[0456] As used herein, overexpression of a given protein / protein complex (e.g. CNX, or CNX containing complexes) refers to a level of gene or protein expression of the relevant protein / protein complex which is greater than the level of expression by equivalent non-diseased tissue.

[0457] CNX expression may be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined e.g. by detection of mRNA encoding CNX, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by detection of CNX, for example by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0458] In some embodiments, the cancer may be a cancer characterised by cell surface expression of CNX. In some embodiments, the cancer may comprise cells expressing CNX at the cell surface. CNX may be present in or at the cell membrane of cells of the cancer.

[0459] In some embodiments, the disease may be characterised by expression / overexpression of glycosylated CNX. The disease may comprise cells expressing / overexpressing glycosylated CNX. In some embodiments, the disease may be a disease characterised by expression of CNX having an elevated level of glycosylation. The cancer may comprise cells expressing CNX having an elevated level of glycosylation.

[0460] In some embodiments, the disease may be characterised by expression / overexpression of O- glycosylated CNX. The disease may comprise cells expressing / overexpressing O-glycosylated CNX. In some embodiments, the disease may be a disease characterised by expression of CNX having an elevated level of O-glycosylation. The cancer may comprise cells expressing CNX having an elevated level of O-glycosylation.

[0461] In some embodiments, the disease may be characterised by expression / overexpression of Tn glycosylated CNX. The disease may comprise cells expressing / overexpressing Tn glycosylated CNX. In some embodiments, the disease may be a disease characterised by expression of CNX having an elevated level of Tn glycosylation. The cancer may comprise cells expressing CNX having an elevated level of Tn glycosylation.

[0462] Glycosylated CNX (e.g., O-glycosylated CNX / Tn glycosylated CNX) may be identified through any method known in the art. For example, proximity assays (e.g., a proximity ligation assays (PLA)) and pulldown assays could be employed to identify / analyse / quantify glycosylated CNX and cells expressing glycosylated CNX.

[0463] An exemplary PLA procedure is utilised in Example 1 herein. Briefly, this exemplary assay comprises the following steps: (1 ) Initial Binding: Biotinylated WL lectin, which selectively binds the Tn antigen, is applied in combination with a rabbit-derived anti-Calnexin antibody (ab22595, Abeam). (2) Signal Conversion: A mouse-derived anti-biotin antibody (Z021 , ThermoFisher) is then introduced to convert the VVL lectin signal into a format suitable for PLA reagents. (3) PLA Reaction: Species-specific oligonucleotide-conjugated antibodies are then introduced (anti-rabbit "plus" and anti-mouse "minus" from Duolink PLA, Merck). Upon close proximity of the two target proteins, the oligonucleotides are brought together, allowing DNA ligation and the formation of a circular DNA molecule. (4) Amplification and Detection: The circular DNA is amplified using rolling circle amplification, followed by hybridization with a fluorescent probe complementary to the amplified DNA product. It is then possible to review the fluorescent output using methods such as flow cytometry and microscopy. Such techniques could be adapted to identify specific cell types comprising (e.g., expressing) glycosylated CNX.

[0464] Pulldown assays could also be utilised to identify glycosylated CNX. Exemplary methods are described in US20200264186A1.

[0465] 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 disease; reduce the severity of one or more symptoms of the disease; increase survival of the subject; reduce / inhibit survival of cells of the disease; and / or reduce the number of diseased cells in the subject.

[0466] 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 a gastrointestinal disease; delay / prevent the onset of one or more symptoms of IBD; delay / prevent the onset of one or more symptoms of Crohn's disease; delay / prevent the onset of one or more symptoms of ulcerative colitis; reduce the severity of one or more symptoms of a gastrointestinal disease; reduce the severity of one or more symptoms of IBD; reduce the severity of one or more symptoms of Crohn's disease reduce the severity of one or more symptoms of ulcerative colitis; increase colon length; inhibit the reduction of colon length; improve disease activity index (DAI) scores; reduce disease activity index (DAI) scores; improve stool consistency; reduce diarrhea frequency; reduce levels of blood in stools; reduce gastrointestinal bleeding severity; reduce gastrointestinal inflammation; reduce weight loss; preserve the integrity of the gastrointestinal tract; preserve colon integrity; and / or increase weight gain.

[0467] 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. 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.

[0468] 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.

[0469] 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 or 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.

[0470] 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. 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.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] 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).

[0475] Methods of detection

[0476] The present disclosure provides the articles of the present disclosure for use in methods for detecting, localizing or imaging CNX, or cells expressing CNX. The present disclosure also provides the articles of the present disclosure for use in methods for detecting, localizing or imaging glycosylated CNX, or cells expressing glycosylated CNX.

[0477] The antigen-binding molecules described herein may be used in methods that involve detecting binding of the antigen-binding molecule to CNX. Such methods may involve detection of the bound complex of the antigen-binding molecule and CNX.

[0478] 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.

[0479] 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.

[0480] Methods comprising detecting glycosylated CNX, or cells expressing glycosylated CNX, include methods for diagnosing / prognosing a gastrointestinal disease (e.g., IBD).

[0481] 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.

[0482] Such methods may involve detecting or quantifying glycosylated CNX and / or cells expressing glycosylated 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.

[0483] Detection in a sample may be used for the purpose of diagnosis of a disease / condition (e.g. a gastrointestinal disease), 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.

[0484] A sample may be taken from any tissue or bodily fluid. The sample may comprise or may be derived from: a tissue sample or biopsy, 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; 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).

[0485] 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. 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 detection / quantification of glycosylated CNX, or cells expressing glycosylated CNX, e.g. in a sample obtained from the individual.

[0486] 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) review the applications and a number of different types of proximity assays.

[0487] In general, proximity assays can be used to determine whether two molecules are co-localised - 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.

[0488] 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.

[0489] 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.

[0490] 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.

[0491] In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds 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 CNX and the antigen-binding molecule which binds to a CNX-interacting molecule are in close physical proximity. In some embodiments, the proximity assay produces a positive output when the antigen-binding molecule which binds 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 CNX and the antigen-binding molecule which binds to Tn are in close physical proximity.

[0492] 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. The term “close physical proximity” may mean a physical proximity such that a positive output is produced by the assay.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] The positive output may be an enzymatic activity e.g. luciferase, -galactosidase, p-lactamase, antibiotic resistance. In some embodiments the reporter system is contacted with a substrate.

[0497] 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.

[0498] 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 in a fluorescent output. 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).

[0499] The method of determining / identifying / observing an output may comprise microscopy, PCR, nucleotide sequencing, and / or flow cytometry analysis.

[0500] 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.

[0501] 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 (Lizard! et al., 1998, Nat Genet 19:225) 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).

[0502] 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 a / , (eds.), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et a / , (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.

[0503] 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.

[0504] 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.

[0505] In some embodiments, the positive output indicates that the two target antigens (e.g., CNX and Tn) 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) are within a proximity that indicates that the two target antigens are interacting. In some embodiments, the positive output indicates that the target antigens (e.g., CNX and Tn) are within a known proximity. The known proximity may depend on the type of proximity assay that is used.

[0506] 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.

[0507] 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.

[0508] 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 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.

[0509] 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).

[0510] 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).

[0511] 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.

[0512] The method comprises assessment of the proximity of two molecules (e.g., two antigen binding molecules, or CNX and a CNX-interacting molecule.

[0513] 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).

[0514] In some embodiments, the method comprises:

[0515] 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. 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.

[0516] In some embodiments, the method comprises:

[0517] 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 ill. contacting the sample with a fourth antigen-binding molecule which binds to the first antigen binding molecule or the second antigen-binding molecule.

[0518] In some embodiments, the method comprises:

[0519] 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, ill. 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.

[0520] In some embodiments, the method comprises:

[0521] 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, ill. 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.

[0522] 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.

[0523] In some embodiments, the antigen binding molecule is tagged, and the third antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, the antigen binding molecule is tagged, and the fourth antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, the antigen binding molecule is tagged, and the fifth antigen binding molecule is an antigen binding molecule which binds to the tag. In some embodiments, the antigen binding molecule is tagged, and the sixth antigen binding molecule is an antigen binding molecule which binds to the tag.

[0524] 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.

[0525] 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.

[0526] In some embodiments, the antigen binding molecule comprises a detectable tag.

[0527] 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.

[0528] 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.

[0529] In some embodiments, the detectable tag comprises an oligonucleotide tag, a fluorophore, a fluorescent protein, or a fluorescent protein fragment.

[0530] Subjects

[0531] 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). 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.

[0532] 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 the methods based on characterisation of one or more markers of such a disease / condition.

[0533] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells / tissue expressing CNX, or of cells / tissue overexpressing CNX, e.g. in a sample obtained from the subject.

[0534] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells / tissue expressing glycosylated CNX, or of cells / tissue overexpressing glycosylated CNX, e.g. in a sample obtained from the subject.

[0535] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells / tissue expressing CNX / CRT, or of cells / tissue overexpressing CNX / CRT, e.g. in a sample obtained from the subject.

[0536] Kits

[0537] 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, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0538] In some embodiments, the kit may comprise materials for producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.

[0539] The kit may provide the antigen-binding molecule, polypeptide, CAR, 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.

[0540] 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.

[0541] 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.

[0542] Sequence identity

[0543] 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 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 using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0544] Sequences 87 | Linker sequence 7 | GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS ~

[0545] ***

[0546] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0547] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0548] 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.

[0549] 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.

[0550] 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).

[0551] 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.

[0552] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0553] 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. Brief Description of the Figures

[0554] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0555] Figure 1 . (Top) Representative images of liver section from a mouse injected with a Sleeping Beauty plasmid containing shp53, NRASG12V, and ERG1 , wherein the hoechst fluorescent channel is depicted. (Bottom) The same tissue section with PLA fluorescent far red signal visualised using Calnexin antibody, VVL-biotinylated anti-biotin, and PLA reagents. The PLA 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.

[0556] Figure 2. (Top) Representative images of human colon section types at fixed image settings with hoechst fluorescent channel or PLA fluorescent far red signal visualised using Calnexin antibody, VVL- biotinylated anti-biotin, and PLA reagents. The PLA signal is well amplified and localised in Crohn disease sections and Colon chronic inflamed sections. Imaging was performed with a Phenix Opera 20X spinning disk laser confocal microscope with a water immersion objective. (Bottom) Quantification of PLA signal per core using single-cell data, converted into an H-score. PLA-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).

[0557] Figure 3 A-C. (A) Representative images of mouse colon Swiss roll sections from a control (no disease) animal, captured at fixed imaging settings. Hoechst staining was used to visualize nuclei (blue channel), and the PLA signal (far-red channel) was detected using a Calnexin antibody, biotinylated VVL lectin, anti-biotin antibodies, and PLA reagents. The inset on the left image is magnified and shown on the right. (B) Representative images of mouse colon Swiss roll sections from a chronic DSS-treated animal, also captured at fixed imaging settings. Hoechst staining highlights nuclei (blue channel), and the PLA signal (far-red channel) was detected using the same reagents as in panel A. The inset on the left image is magnified and shown on the right. (C) Spatial distribution of single cells in control (no disease) and chronic DSS-treated animals, based on two independent biological replicates. The positions of all cells are marked as grey dots (representing nuclei positions), while PLA-positive cells for Tn-glycosylated Calnexin are highlighted in black.

[0558] Figure 4 A-C. (A) Representative images of mouse colon Swiss roll sections from a control (no disease) animal, captured at fixed imaging settings. Hoechst staining was used to visualize nuclei (blue channel), and the PLA signal (far-red channel) was detected using a Calnexin antibody, biotinylated VVL lectin, anti-biotin antibodies, and PLA reagents. The inset on the left image is magnified and shown on the right. (B) Representative images of mouse colon Swiss roll sections from acute DSS-treated animal, also captured at fixed imaging settings. Hoechst staining highlights nuclei (blue channel), and the PLA signal (far-red channel) was detected using the same reagents as in panel A. The inset on the left image is magnified and shown on the right. (C) Spatial distribution of single cells in control (no disease) and acute DSS-treated animals, based on two independent biological replicates. The positions of all cells are marked as grey dots (representing nuclei positions), while PLA-positive cells for Tn-glycosylated Calnexin are highlighted in black.

[0559] Figure 5. Quantification and statistical analysis of PLA positive presence in mouse DSS model. Analysis is based on 3 technical replicates for each animal tested. Statistical analysis with a t-test is provided, with p-values indicated for comparisons with healthy control group (no disease).

[0560] Figure 6 A-E. (A) Study flow schematic. (B) Disease activity index (DAI) scores of mice from three treatment groups ((i) water control; (ii) DSS 3% + vehicle control; (iii) DSS 3% + anti-CNX antibody treatment) at different time points. (C) Area under curve for DAI score of mice from three treatment groups. (D) Decrease in colon length relative to mice without DSS-induced colitis. (E) Representative images of colon samples from three experimental groups.

[0561] Examples

[0562] In the following Examples, the inventors describe the development of a new assay for the investigation of CNX glycosylation, and the use of this assay to unexpectedly find that glycosylated CNX levels are elevated in IBD. Antigen-binding molecules that bind CNX are provided for the treatment of gastrointestinal diseases, such as IBD.

[0563] Example 1 : Assay to identify glycosylated calnexin

[0564] Elevated levels of glycosylated 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.

[0565] Inventors developed a proximity ligation assay (PLA) to assess the level of glycosylated calnexin (CNX) in a sample. This new method comprised the following steps: (1 ) Initial Binding: Biotinylated VVL lectin, which selectively binds the Tn antigen, was applied in combination with a rabbit-derived anti-Calnexin antibody (ab22595, Abeam). (2) Signal Conversion: A mouse-derived anti-biotin antibody (Z021 , ThermoFisher) was introduced in the second step to convert the WL lectin signal into a format suitable for PLA reagents. (3) PLA Reaction: Species-specific oligonucleotide-conjugated antibodies were then introduced (anti-rabbit "plus" and anti-mouse "minus" from Duolink PLA, Merck). Upon close proximity of the two target proteins, the oligonucleotides were brought together, allowing DNA ligation and the formation of a circular DNA molecule. (4) Amplification and Detection: The circular DNA was amplified using rolling circle amplification, followed by hybridization with a fluorescent probe complementary to the amplified DNA product. A detectable fluorescent signal confirmed the proximity of the target proteins, specifically reflecting the presence of Tn-glycosylated Calnexin.

[0566] To validate the protocol, the assay was used to identify glycosylated CNX in a control sample known to contain high levels of glycosylated CNX. The positive control sample was an FFPE liver section from a mouse model hydrodynamically injected with a transposon plasmid. This plasmid co-expressed an shRNA targeting the p53 tumor suppressor, the oncogene NRASG12V, and ER-localized GALNT1 . The plasmid was taken up by liver hepatocytes, leading to tumor formation and overexpression of ER- GALNT1 .

[0567] Specifically, 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).

[0568] 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.

[0569] Primary antibody incubation was performed overnight at 4°C using a mixture of rabbit anti-calnexin antibody (ab22595, Abeam) at 1 pg / mL and WL-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.

[0570] The slides were then incubated with Duolink© antibody diluent containing Duolink© anti-rabbit and antimouse PLA probes, each at a 1 X 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.

[0571] 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© 0.01 X 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.

[0572] PLA on the positive control FFPE liver sections produced a highly specific amplified signal in tumor regions of the liver, while only background signals were observed in healthy liver areas (Figure 1 ). The elevated PLA signal for CNX glycosylation in tumor tissues was consistent with results from biochemical pulldown assays on similar liver tissue types (Ros et al., 2020).

[0573] Example 2: Glycosylated Calnexin detected in human inflammatory bowel disease samples The newly developed and validated assay described in Example 1 was used to investigate the level of glycosylated CNX in human inflammatory bowel disease (IBD) samples.

[0574] An IBD tissue microarray (TissueArray.Com LLC, MD, United States) was prepared for analysis using a standard protocol for dewaxing, rehydration, antigen retrieval using citrate buffer, and permeabilization. To minimize non-specific binding caused by endogenous biotin, biotin receptors, and streptavidin binding sites present in many tissues, a pre-treatment step was used. Tissue sections were first incubated with excess unlabelled streptavidin to block biotin-binding sites, followed by incubation with excess unlabelled biotin to further reduce background signal. The steps provided in Example 1 were then followed to complete the PLA on the prepared samples.

[0575] Quantification of CNX Tn glycosylation in human IBD sections revealed significantly elevated levels in colon sections from Crohn's disease patients compared to control sections from the normal core group. Elevated levels of CNX Tn glycosylation were also observed in chronically inflamed colon sections, and in colon sections with polyps, compared to control sections from the normal core group. In contrast, tissues adjacent to tumors showed minimal changes in CNX Tn glycosylation levels compared to control sections from the normal core group.

[0576] Example 3: Glycosylated Calnexin detected in DSS-induced colitis samples

[0577] The level of glycosylated CNX was next investigated in Colon Swiss roll sections prepared from mice subjected to a dextran sulphate sodium (DSS) protocol. The DSS-induced colitis model is widely used because of its simplicity and many similarities with human ulcerative colitis (Chassaing et al. Curr Protoc Immunol. 20144;104: 15.25.1 -15). Standard procedures for the generation of mice with DSS-induced colitis were followed, and Colon Swiss roll sections were prepared in the typical way. The assay procedures used in Examples 1 and 2 were then used to complete the PLA on the Colon Swiss roll sections.

[0578] Swiss roll sections from control animals (no DSS treatment) displayed minimal PLA-positive cells, as shown in Figure 3A. In contrast, sections from animals treated with DSS for a chronic colitis state exhibited a substantial increase in PLA-positive cells. These cells were primarily localized beneath the epithelial layer and distributed across multiple sites along the length of the colon (Figure 3B).

[0579] Quantitative analysis of spatial cell distribution further validated these findings, showing minimal PLA- positive cell presence in control sections, while chronic colitis state DSS-treated animals demonstrated a pronounced accumulation of PLA-positive cells, particularly aligned with the epithelial lining of the colon, with considerable accumulation of PLA-positive cells being identified in the lamina propria (Figure 3C).

[0580] Similarly, sections from animals treated with DSS for acute colitis disease state displayed a comparable outcome, with a marked increase in PLA-positive cells aligned along the epithelial layer, with considerable accumulation of PLA-positive cells being identified in the lamina propria (Figures 4A-C). Statistical analysis across replicates revealed that both chronic and acute colitis induced through DSS treatment resulted in a statistically significant increase in PLA-positive cells compared to control (no disease) conditions (Figure 5).

[0581] Example 4: Anti-CNX antibodies reduce glycosylated calnexin

[0582] The effect of treatment with anti-CNX antibodies on the level of glycosylated CNX is investigated.

[0583] Standard procedures for the generation of mice with DSS-induced colitis are followed. Administration of anti-CNX antibodies is performed after colitis is induced. The assay procedures used in Examples 1 and 2 are then used to determine the level of glycosylated CNX in samples from mice treated with anti-CNX antibodies and control mice.

[0584] Anti-CNX antibodies are shown to reduce the level of glycosylated CNX. The comparison of the treatment and control groups show a decrease in PLA-positive cells in samples from mice treated with anti-CNX antibodies compared to control (no disease) conditions.

[0585] Example 5: Anti-CNX antibody treatment reduces disease symptoms in DSS mouse model

[0586] The effect of treatment with anti-CNX antibodies on disease symptoms in mice with DSS-induced colitis is investigated.

[0587] Standard procedures for the generation of mice with DSS-induced colitis are followed. Administration of anti-CNX antibodies is performed after colitis is induced. Disease symptoms (e.g., gastrointestinal remodelling, body weight, histological symptoms, and stool appearance) are analysed. Anti-CNX antibodies are shown to reduce disease symptoms in mice with DSS-induced colitis. The comparison of the treatment and control groups show a decrease in disease symptoms from mice treated with anti-CNX antibodies compared to control (no disease) conditions.

[0588] Example 6: ECM protective activities of CNX antibodies

[0589] The ECM protection assay measures cell-mediated degradation of fluorescent gelatin beneath a layer of cartilage ECM and rat tail Collagen I.

[0590] Fluorescently labelled gelatin is coated on sterile coverslips and stabilised with glutaraldehyde fixation. A mixture of ECM together with rat tail collagen I is then coated as a thin layer on top of the fluorescent gelatin. Gastrointestinal cells (e.g., fibroblasts) are then seeded on these coverslips. The cells are allowed to degrade the ECM gelatin coverslips, with and without presence of anti-CNX antibodies. After 48 hours, gelatin coverslips are fixed and stained with fluorescent Hoescht for nuclei counting. The coverslips are imaged on a confocal microscope. The images are analysed to quantify the area of gelatin degradation and the number of nuclei per field.

[0591] Anti-CNX antibodies are shown to inhibit ECM degradation.

[0592] Example 7: Anti-CNX antibody treatment improves disease activity index (DAI) scores and reduced colon shortening

[0593] Mouse studies were performed with MMD service unit in IMCB / A*STAR Singapore with an Institutional Animal Care and Use Committee (IACUC) approved protocol. On Day 0, male C57BL / 6 mice (8-10 weeks old) are randomly assigned into three experimental groups based on body weight stratification.

[0594] Group 1 served as a disease-free control and received standard drinking water without DSS ( / .e., no DSS-induced disease present). Group 2, the disease control group, received 3% DSS in drinking water for seven days (Days 1-8) and was administered the vehicle control, phosphate-buffered saline (PBS). Group 3 received 3% DSS in drinking water and was treated with the anti-CNX antibody at a dose of 20 mg / kg, administered via intraperitoneal injection on Days 0, 2, 4, and 6. The anti-CNX antibody 1 E1 was used as an exemplary anti-CNX antibody for this Example.

[0595] DSS solution at 3% w / v was provided ad libitum, and automated water nozzles are plugged to ensure uniform intake. DSS water was refreshed every two to three days, and mice were monitored closely for signs of dehydration. Cage cards with emergency contact information are placed for rapid intervention if necessary.

[0596] An overview of the study is provided in Figure 6A. Starting from Day 1 , mice were assessed daily for body weight changes, which were recorded and scored based on percentage change from baseline. Fecal consistency was evaluated for signs of diarrhoea, and stool samples were examined for occult or gross bleeding as an indicator of disease severity. On Day 8, mice were euthanized by cervical dislocation, and the peritoneal cavity was opened for tissue collection. The entire colon was excised and analyzed for colon shortening as a measure of inflammation. Colon length and weight were recorded, and tissues were processed for histopathological evaluation. A disease activity index (DAI) is calculated as a composite score based on weight loss, stool consistency, and bleeding severity (Yang et al., Inflammatory Bowel Diseases, Volume 30, Issue 5, May 2024, Pages 844-853).

[0597] DAI scoring was significantly reduced in mice treated with the anti-CNX antibody on Days 6, 7, and 8 compared to the disease control group (3% DSS + vehicle control) (Figure 6B). Area under curve for DAI score was also consistently and significantly reduced for 3% DSS + Anti-calnexin vs 3% DSS + vehicle control (Figure 6C). Treatment with the anti-CNX antibody also led to a notable improvement in colon length, with an approximate 28% increase compared to the disease control group (Figure 6D-E), indicating reduced colon shortening, reduced tissue damage, and reduced inflammation. These findings demonstrate the effects of anti-CNX antibodies in the context of gastrointestinal disease (e.g., IBD), specifically highlighting the ability of anti-CNX antibodies to mitigate disease severity and preserve colon integrity.

Claims

Claims:1 . An antigen-binding molecule which binds to CNX for use in a method of treating or preventing a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

2. A method of treating or preventing a gastrointestinal disease, the method comprising administering a therapeutically- or prophylactically-effective amount of an antigen-binding molecule which binds to CNX, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

3. Use of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

4. The antigen-binding molecule for use according to claim 1 , the method according to claim 2, or the use according to claim 3, wherein the gastrointestinal disease is further characterised by intestinal inflammation.

5. The antigen-binding molecule for use according to claim 1 or claim 4, the method according to claim 2 or claim 4, or the use according to claim 3 or claim 4, wherein the gastrointestinal disease is further characterised by the presence of polyps.

6. The antigen-binding molecule for use according to any one of claims 1 , 4, or 5 the method according to any one of claims 2, 4, or 5, or the use according any one of claims 3 to 5, wherein the gastrointestinal disease characterised by the expression of glycosylated CNX is an inflammatory bowel disease (IBD).

7. An antigen-binding molecule which binds to CNX for use in a method of treating or preventing IBD.

8. A method of treating or preventing IBD, the method comprising administering a therapeutically- or prophylactically-effective amount of an antigen-binding molecule which binds to CNX.

9. Use of an antigen-binding molecule which binds to CNX in the manufacture of a medicament for the treatment or prevention of IBD.

10. The antigen-binding molecule for use according to claim 6 or claim 7, the method according to claim 6 or claim 8, or the use according to claim 6 or claim 9, wherein the IBD is Crohn's disease or ulcerative colitis.9411 . The antigen-binding molecule for use according to any one of claims 1 , 4-7, or 10, the method according to any one of claims 2, 4-6, 8, or 10, or the use according any one of claims 3-6, or 9-10, wherein the disease is characterised by extracellular matrix (ECM) degradation.

12. The antigen-binding molecule for use according to any one of claims 1 , 4-7, or 10-11 , the method according to any one of claims 2, 4-6, 8, or 10-11 , or the use according any one of claims 3-6, or 9-11 , wherein the disease is characterised by expression of glycosylated CNX by a cell of the lamina propria.

13. The antigen-binding molecule for use according to any one of claims 1 , 4-7, or 10-12, the method according to any one of claims 2, 4-6, 8, or 10-12, or the use according any one of claims 3-6, or 9-12, wherein the disease is characterised by expression of glycosylated CNX by a fibroblast.

14. The antigen-binding molecule for use according to any one of claims 1 , 4-7, or 10-11 , the method according to any one of claims 2, 4-6, 8, or 10-11 , or the use according any one of claims 3-6, or 9-11 , wherein the disease is characterised by expression of glycosylated CNX by an epithelial cell.

15. An in vitro complex, optionally isolated, comprising an antigen-binding molecule bound to glycosylated CNX, wherein the glycosylated CNX is present on the surface of a cell of the lamina propria or on the surface of an epithelial cell.

16. The antigen-binding molecule for use according to any one of claims 1 , 4-7, or 10-14, the method according to any one of claims 2, 4-6, 8, or 10-14, the use according any one of claims 3-6, or 9-14, or the in vitro complex according to claim 15, wherein the antigen-binding molecule binds to CNX via contact with:(a) one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:69, optionally wherein the antigen-binding molecule binds to CNX via contact with one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:67 or 68; or(b) one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NO:77, optionally wherein the antigen-binding molecule binds to CNX via contact with one or more amino acid residues of the region of CNX corresponding to the region shown in SEQ ID NQ:70, 71 , 72, 73, 74, 75, 76, 78, or 79.

17. The antigen-binding molecule for use according to any one of claims 1 , 4-7, 10-14, or 16, the method according to any one of claims 2, 4-6, 8, 10-14, or 16, the use according any one of claims 3-6, 9- 14, or 16, or the in vitro complex according to claim 15 or 16, wherein the antigen-binding molecule comprises:(I) a heavy chain variable (VH) region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO:3 HC-CDR2 having the amino acid sequence of SEQ ID NO:4 HC-CDR3 having the amino acid sequence of SEQ ID NO:5; and(ii) a light chain variable (VL) region incorporating the following CDRs:LC-CDR1 having the amino acid sequence of SEQ ID NO:11 LC-CDR2 having the amino acid sequence of SEQ ID NO: 12 LC-CDR3 having the amino acid sequence of SEQ ID NO: 13.

18. The antigen-binding molecule for use according to any one of claims 1 , 4-7, 10-14, or 16-17, the method according to any one of claims 2, 4-6, 8, 10-14, or 16-17, the use according any one of claims 3- 6, 9-14, or 16-17, or the in vitro complex according to any one of claims 15-17, wherein the antigenbinding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:2; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:10.

19. A method comprising administering an antigen-binding molecule which binds to CNX to a subject having a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

20. The method according to claim 19, wherein the gastrointestinal disease is IBD.21 . The method according to claim 19 or claim 20, wherein the glycosylated CNX is present on the surface of a cell of the lamina propria, or the surface of an epithelial cell.

22. An improved method comprising contacting calnexin (CNX) with an antigen-binding molecule which binds to CNX, wherein the improvement comprises administering the antigen-binding molecule which binds to CNX to a subject having a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

23. A method of contacting calnexin (CNX) with an antibody, wherein the antibody comprises a means for binding CNX, wherein the method comprises administering the antibody to a subject having a gastrointestinal disease, wherein the gastrointestinal disease is characterised by the expression of glycosylated CNX.

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