Treatment / prevention of disease by LRG1 inhibition

Abstract

The present disclosure provides an LRG1 inhibitor for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, use of an LRG1 inhibitor in the manufacture of a medicament for use in the treatment or prevention of a disease or condition characterised by fibrosis and / or inflammation, and a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically- or prophylactically-effective amount of an LRG1 inhibitor to a subject.

Description

[0001] Treatment / Prevention of Disease by LRG1 Inhibition

[0002] This application claims priority from SG 10202402974V filed 25 September 2024, the contents and elements of which are incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present invention relates to the treatment and prevention of disease, via LRG1 inhibition.

[0005] Background

[0006] LRG1 is a serum protein primarily produced by hepatocytes and neutrophils. Reportedly, LRG1 can also be secreted from endothelial cells, epithelial cells, fibroblasts, and other types of myeloid cells in local tissue, such as the lung, kidney, heart, skin, brain, and testis. The physiological role of LRG1 remains poorly understood due to the lack of overt phenotypic abnormalities in Lrg1 ' mice. However, LRG1 is thought to be involved in a plethora of physiological processes including the modulation of various signalling cascades, mainly TGFp signalling.

[0007] LRG1 is believed to play beneficial roles in the acute response to infection and injury. In particular, upregulated LRG1 expression in response to bacterial infection can mediate the differentiation and infiltration of lymphocytes and promote the survival of circulating immune cells by neutralizing cytochrome c cytotoxicity. In addition, LRG1 promotes wound healing or tissue repair by stimulating the renewal of damaged epithelial cells, tissue vascularization, and peripheral nerve regeneration.

[0008] LRG1 has also been implicated in disease pathogenesis. Evidence points to LRG1 being a proangiogenic factor, that through local upregulation contributes to the formation of defective vessels. The expression of LRG1 is associated with a variety of malignancies, such as non-small-cell lung cancer (NSCLC), ovarian cancer, and bladder cancer, and is thought to promote tumour growth via the angiogenesis process. LRG1 has been indicated to promote neovascularization in mouse models of ocular disease by potentiating endothelial TGF-p / activin receptor-like kinase 1 (ALK1) signalling. Circulating LRG1 levels have also been reported to be raised in severe COVID-19 patients and in patients with vasculitis, where vascular damage is a primary feature.

[0009] LRG1 has also been implicated in fibrosis. LRG1 has been reported to promote fibrosis in several tissues (e.g. lung, kidney, dermal and ocular tissues). However, LRG1 has also been reported to protect against fibrosis. Reduced levels of LRG1 in fibroblasts following selective deletion of PPARp / 5, made the epidermis of mutant mice thicker and more susceptible to inflammation and dermal fibrosis. LRG1 has also been shown to prevent the activation of skin fibroblasts by inhibiting pro-fibrotic TGFp signalling. A similar protective role has been described in the heart, where LRG1 is constitutively expressed via PPARp / 5 in resident fibroblasts to counteract TGFp function and preserve tissue integrity. Accordingly, the role of LRG1 in fibrosis is not well understood, in view of the conflicting studies demonstrating both pro- and anti-fibrotic activities. LRG1 expression has also been observed to be increased in several inflammatory disorders such as Still’s disease, psoriasis, lupus nephritis, rheumatoid arthritis and vasculitis. However, the role of LRG1 in these conditions remains largely unclear.

[0010] LRG1 is known or speculated to be involved in modulation of many signalling pathways. LRG1 likely modifies cell behaviour both directly, by altering the cell transcriptome, and indirectly by interfering with intermediate steps of the signalling cascades. LRG1 has been mainly described as a modifier of the TGFp canonical pathway. LRG1 promotes pathogenic angiogenesis in endothelial cells through the ALK1-S mad 1 / 5 / 8 pathway. LRG1 binds directly to the TGF-p accessory receptor endoglin, which, in the presence of TGF-01 , results in promotion of the pro-angiogenic Smad1 / 5 / 8 signalling pathway. LRG1 may also modulate the ALK5-Smad2 / 3 arm to favour the formation of myofibroblasts and Th17 lymphocytes.

[0011] TGFp non-canonical signalling is also likely to mediate some LRG1-driven biological functions including neutrophil activation and wound healing via AKT. Additional transduction factors involved in LRG1 signalling include (i) EGFR which promotes pancreatic cancer cell malignancy through p38 / MAPK, dissemination of melanoma cells and cornea repair through STAT3; (ii) the IL-6 / STAT3 axis which modulates neutrophil chemotaxis; (iii) Wnt / pcatenin which, in the heart, inhibit fibroblast proliferation and migration.

[0012] Summary

[0013] In a first aspect the present disclosure provides an LRG1 inhibitor for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0014] The present disclosure also provides the use of an LRG1 inhibitor in the manufacture of a medicament for use in the treatment or prevention of a disease or condition characterised by fibrosis and / or inflammation.

[0015] The present disclosure also provides a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically- or prophylactically-effective amount of an LRG1 inhibitor to a subject.

[0016] In some embodiments, the LRG1 inhibitor: (i) inhibits interaction between LRG1 and an interaction partner for LRG1 ; or (ii) reduces the expression of LRG1 or an interaction partner of LRG1 .

[0017] In some embodiments, the LRG1 inhibitor binds to LRG1. In some embodiments, the LRG1 inhibitor binds to the LRRCT region of LRG1 . In some embodiments, the LRG1 inhibitor binds to the LRR region of LRG1.

[0018] In some embodiments, the LRG1 inhibitor is a peptide / polypeptide, nucleic acid or small molecule.

[0019] In some embodiments, the LRG1 inhibitor is an antibody or antigen-binding fragment thereof. In some embodiments, the LRG1 inhibitor is an inhibitory nucleic acid capable of reducing expression of LRG1 by RNA interference (RNAi).

[0020] In some embodiments, the LRG1 inhibitor is capable of modifying a gene encoding LRG1 to reduce its expression.

[0021] In some embodiments, the LRG1 inhibitor comprises a site-specific nuclease (SSN) targeting a gene encoding LRG1 .

[0022] In some embodiments, the disease or condition is characterised by: (i) fibrosis and / or inflammation of the eye; or (ii) fibrosis and / or inflammation of the pancreas. In some embodiments, the disease or condition is characterised by: (i) fibrosis and / or inflammation of the eye; (ii) fibrosis and / or inflammation of the pancreas; (iii) fibrosis and / or inflammation of the joints; (iv) fibrosis and / or inflammation of the skin; (v) fibrosis and / or inflammation of the bowel; (vi) fibrosis and / or inflammation of the kidney; or (vii) fibrosis and / or inflammation of the lungs.

[0023] In some embodiments, the disease is characterised by pathological angiogenesis.

[0024] In some embodiments, the disease or condition is selected from: macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, diffuse intralobular fibrosis, pancreatitis, cystic fibrosis or pancreatic cancer. In some embodiments, the disease or condition is selected from: macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), diabetic macular edema (DME), diabetic retinopathy, proliferative diabetic retinopathy (PDR), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis, preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, diffuse intralobular fibrosis, pancreatitis, cystic fibrosis, pancreatic cancer, skin fibrosis, kidney fibrosis, pulmonary fibrosis, rheumatoid arthritis, inflammatory bowel disease, diabetic nephropathy, or a diabetic wound.

[0025] In some embodiments, the disease or condition is selected from: subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis, preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, and diffuse intralobular fibrosis.

[0026] In some embodiments, the disease or condition is subretinal fibrosis.

[0027] In some embodiments, the disease or condition is a cancer. In some embodiments, the cancer is selected from: a cancer comprising cells expressing / overexpressing LRG1 , a solid tumor, metastatic tumor, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, liver cancer, hepatocellular carcinoma, skin cancer, melanoma, lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, non-small cell lung cancer, breast cancer, breast carcinoma, ductal carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, cervical cancer, cervical squamous cell carcinoma, esophageal cancer, esophageal adenocarcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

[0028] Description

[0029] LRG1 ’s role in diseases associated with fibrosis and / or inflammation is not well understood, especially in view of the conflicting studies demonstrating both pro- and anti-fibrotic activities for LRG1 . The present invention is based on the inventors’ unexpected finding that inhibition of LRG1 reduces fibrosis and inflammation in fibroinflammatory disorders. Thus demonstrating the utility of LRG1 inhibitors in the treatment of fibroinflammatory disorders.

[0030] LRG1

[0031] The present disclosure relates to LRG1 -specific antigen-binding molecules.

[0032] Leucine-rich a-2 glycoprotein 1 (LRG1) is a secreted member of the family of leucine-rich repeat proteins.

[0033] Human LRG1 is the protein identified by UniProt P02750 (SEQ ID NO:172). Human LRG1 comprises an N-terminal signal peptide (SEQ ID NO:174), 8 leucine-rich repeats (LRRs) (SEQ ID NO:175, 176, 177, 178, 179, 180, 181 , and 182) and a leucine-rich C-terminal domain (LRRCT) (SEQ ID NO:183). Human LRG1 contains 5 glycosylation sites (T37, N79, N186, N269 and N325) and two disulfide bonds (amino acid 43 to amino acid 56, and amino acid 303 to amino acid 329) (numbering is with reference to SEQ ID NO:172). Upon cleavage of the N-terminal signal peptide, LRG1 is released in the extracellular space. The mature form of human LRG1 is shown in SEQ ID NO:173.

[0034] In this specification ‘LRG1 ’ refers to LRG1 from any species, and includes isoforms, fragments, variants or homologues from any species. In some embodiments LRG1 is LRG1 from a mammal (e.g. a therian, placental, epitherian, preptotheria, archontan, primate (rhesus, cynomolgous, non-human primate or human)). In some embodiments, the LRG1 is human LRG1 or mouse LRG1 .

[0035] As used herein, a ‘fragment’, ‘variant’, ‘isoform’ or ‘homologue’ of a given protein may optionally be characterised as having at least 60% (e.g. one of >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98% or >99%) amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform).

[0036] 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. 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 LRG1 (UniProt P02750, v2; SEQ ID NO:172) and mouse LRG1 (UniProt: Q91XL1, v1 ; SEQ ID NO:184) are homologues of one another. Homologues include orthologues.

[0037] Isoforms, fragments, variants or homologues of LRG1 according to the present disclosure may optionally be characterised as having at least 60% (e.g. one of >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >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 LRG1 isoform from a given species, e.g. human.

[0038] Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property / activity of the reference LRG1 (e.g. human LRG1), as determined by analysis by a suitable assay for the functional property / activity. For example, an isoform, fragment, variant or homologue of LRG1 may display binding to and / or an association with a TGF-beta- receptor-ll / endoglin / ALK1 complex and / or a TGF-beta-receptor-ll / ALK5 complex.

[0039] In some embodiments, the LRG1 comprises, or consists of, an amino acid sequence having at least 70%, (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:172 or 173. In some embodiments, the LRG1 comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:184 or 185.

[0040] In some embodiments, the LRG1 comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:188.

[0041] 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.

[0042] A fragment of LRG1 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.

[0043] In some embodiments, a fragment of LRG1 comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, or 183.

[0044] In some embodiments, a fragment of LRG1 comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:185, 186, or 187. In some embodiments, a fragment of LRG1 comprises, or consists of, an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:189.

[0045] As used herein, ‘LRRCT region’ or ‘leucine-rich C-terminal domain’ refers to an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:183, 187 or 189. In some embodiments, the LRRCT region has an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:183.

[0046] The structure and function of LRG1 is reviewed e.g. in Camilli et al., Journal of Biomedical Science (2022) 29(1 ):6, which is hereby incorporated by reference in its entirety. LRG1 inhibition

[0047] The present invention is concerned with LRG1 inhibition. As used herein, ‘LRG1 inhibition’ refers to inhibition of one or more activities / functions of LRG1 .

[0048] In some embodiments, the activity / function of LRG1 is selected from promoting: fibrotic gene expression, ECM protein expression, cell migration, cell proliferation, epithelial-mesenchymal transition (EMT) (e.g. by perivascular cells or retinal epithelial cells), fibroblast generation, myofibroblast generation, fibroblast activation, angiogenesis, fibrosis, ALK1 / S mad 1 / 5 / 8 signalling, ALK5 / Smad2 / 3 signalling, regulation of neutrophil phenotype, and regulation of macrophage phenotype.

[0049] In some embodiments, LRG1 inhibition comprises reducing / preventing interaction between LRG1 and an interaction partner for LRG1 . In some embodiments, LRG1 inhibition comprises reducing the level / amount of LRG1. In some embodiments, LRG1 inhibition comprises one or more of: inhibiting interaction between LRG1 and an interaction partner for LRG1 ; inhibiting the gene or protein expression of nucleic acid encoding LRG1 ; modifying a gene encoding LRG1 to reduce / prevent its expression; reducing the level of RNA encoding LRG1 ; inhibiting transcription of nucleic acid encoding LRG1 ; increasing degradation of RNA encoding LRG1 ; reducing the level of LRG1 ; disrupting normal post-translational processing (e.g. splicing, translation, post-translational processing) of RNA encoding LRG1 ; and increasing degradation of LRG1.

[0050] Herein, an interaction partner for LRG1 may be any molecule (e.g. protein / nucleic acid) with which LRG1 interacts. An interaction partner for LRG1 may be a protein capable of forming a complex with LRG1 through protein-protein interaction. An interaction partner for LRG1 may be a molecular complex (e.g. a multiprotein complex) in which one or more constituent components of the complex are capable of forming a complex with LRG1 through protein-protein interaction. In some embodiments, an interaction partner for LRG1 may be e.g. endoglin (ENG), cytochrome C, TGF-01 , TGFpRII / ALK1 / ENG complex, TGFpRII / ALK5 complex, BMP, BMP / BMPR complex, TNFR, or EGFR.

[0051] Interaction between LRG1 and interaction partners of LRG1 can be analysed using techniques well known to the skilled person such as co-immunoprecipitation, surface plasmon resonance (e.g. Biacore™ SPR Systems), mass spectrometry, and resonance energy transfer (RET) assays using appropriately labelled species. Inhibition of interaction can be determined in such assays by detection of a reduction in the level of interaction as compared to a control, uninhibited condition.

[0052] LRG1 and interactions partners of LRG1 may be detected e.g. using methods well known to the skilled person such as antibody-based methods including western blot, immunohisto / cytochemistry, flow cytometry, ELISA, or by reporter-based methods.

[0053] Gene expression can be determined by means well known to the skilled person. The level of RNA encoding LRG1 can be determined e.g. by techniques such as RT-qPCR, northern blot, etc. A reduction in the level of RNA encoding LRG1 may e.g. be the result of reduced transcription of nucleic acid encoding LRG1 , or increased degradation of RNA encoding LRG1 .

[0054] Reduced transcription of nucleic acid LRG1 may be a consequence of inhibition of assembly and / or activity of factors required for transcription of the DNA encoding LRG1. Increased degradation of RNA encoding LRG1 may be a consequence of increased enzymatic degradation of RNA encoding LRG1 , e.g. as a consequence of RNA interference (RNAi), and / or reduced stability of RNA encoding LRG1 .

[0055] Protein expression can be determined by means well known to the skilled person. The level of LRG1 can be determined e.g. by antibody-based methods including western blot, immunohisto / cytochemistry, flow cytometry, ELISA, or by reporter-based methods.

[0056] Protein degradation can be evaluated e.g. by detection of, or analysis of the level / proportion of, ubiquitinated protein, association with ubiquitin ligase and / or proteosomal localisation.

[0057] A reduction in the level of LRG1 may e.g. be the result of a reduced level of RNA encoding LRG1 , reduced post-transcriptional processing of RNA encoding LRG1 , or increased degradation of LRG1 .

[0058] Disruption to normal post-transcriptional processing of LRG1 may e.g. be reduced / altered splicing of pre- mRNA to mature mRNA encoding LRG1 , reduced translation of mRNA encoding LRG1 , or reduced / altered post-translational processing of LRG1 .

[0059] Reduced / altered splicing of pre-mRNA to mature mRNA encoding LRG1 may be a consequence of inhibition of assembly and / or activity of factors required for normal splicing. Reduced translation of mRNA encoding LRG1 may be a consequence of inhibition of assembly and / or activity of factors required for translation. Reduced / altered post-translational processing (e.g. enzymatic processing, folding) may be a consequence of inhibition of assembly and / or activity of factors required for normal post-translational processing of LRG1. Increased degradation of LRG1 may be a consequence of increased enzymatic (e.g. protease-mediated) degradation of the protein, which may e.g. be associated with misfolding.

[0060] LRG1 inhibition may be characterised by a reduced level of function of LRG1. In some embodiments, LRG1 inhibition may be determined by detection of a reduced level of a correlate of LRG1 function. A correlate of a function of LRG1 may be e.g. fibrotic gene expression, ECM protein expression, cell migration, cell proliferation, epithelial-mesenchymal transition (EMT) (e.g. by perivascular cells or retinal epithelial cells), fibroblast generation, myofibroblast generation, fibroblast activation, angiogenesis, fibrosis, ALK1 / Smad1 / 5 / 8 signalling, or ALK5 / Smad2 / 3 signalling.

[0061] In particular embodiments contemplated herein, LRG1 inhibition is achieved by inhibiting interaction between LRG1 and an interaction partner of LRG1 . In some embodiments, inhibiting interaction between LRG1 and an interaction partner of LRG1 is achieved using an antibody or antigen-binding fragment thereof which is capable of binding to LRG1 . LRG1 inhibitors

[0062] Aspects of the present invention comprise LRG1 inhibition using an LRG1 inhibitor.

[0063] As used herein, a ‘LRG1 inhibitor’ refers to any agent capable of achieving LRG1 inhibition. LRG1 inhibitors include agents capable of inhibiting one or more functions of LRG1 , inhibiting the interaction of LRG1 and an interaction partner of LRG1 , or reducing the level / amount of LRG1 .

[0064] Such agents may be effectors of ( / .e. may directly or indirectly cause) LRG1 inhibition as described hereinabove. LRG1 inhibitors may also be referred to as LRG1 antagonists.

[0065] In some embodiments, an LRG1 inhibitor may: inhibit a function of LRG1 ; inhibit interaction between LRG1 and an interaction partner for LRG1 ; inhibit the gene or protein expression of nucleic acid encoding LRG1 ; modify a gene encoding LRG1 to reduce / prevent its expression; reduce the level of RNA encoding LRG1 ; inhibit transcription of nucleic acid encoding LRG1 ; increase degradation of RNA encoding LRG1 ; reduce the level of LRG1 ; disrupt normal post-translational processing (e.g. splicing, translation, post- translational processing) of RNA encoding LRG1 ; and increase degradation of LRG1 .

[0066] It will be appreciated that a given LRG1 inhibitor may display more than one of the properties recited in the preceding paragraph. A given agent may be evaluated for the properties recited in the preceding paragraph using suitable assays. The assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays.

[0067] Where assays are cell-based assays, they may comprise treating cells with the test agent in order to determine whether the agent displays one or more of the recited properties. Assays may employ endogenously- or recombinantly-expressed proteins, and may use species labelled with detectable entities in order to facilitate their detection.

[0068] Agents capable of inhibiting a function of LRG1 may be identified using assays comprising detecting the level of a correlate of LRG1 function. A correlate of a function of LRG1 may be e.g. fibrotic gene expression, ECM protein expression, cell migration, cell proliferation, epithelial-mesenchymal transition (EMT) (e.g. by perivascular cells or retinal epithelial cells), fibroblast generation, myofibroblast generation, fibroblast activation, angiogenesis, fibrosis, ALK1 / Smad1 / 5 / 8 signalling, ALK5 / Smad2 / 3 signalling, BMP signalling, TNFa, or EGF signalling.

[0069] Agents capable of inhibiting interaction between a LRG1 and an interaction partner for LRG1 may be identified using assays comprising detecting the level of interaction between LRG1 and an interaction partner for LRG1 , e.g. using antibody / reporter-based methods. The level of interaction between LRG1 and an interaction partner for LRG1 can be analysed e.g. using resonance energy transfer techniques (e.g. FRET, BRET), co-immunoprecipitation or methods analysing a correlate of interaction (e.g. a function of LRG1). Assays may comprise treating cells / tissue with the agent, and subsequently comparing the level of interaction in such cells / tissue to the level of interaction in cells / tissue of an appropriate control condition (e.g. untreated / vehicle-treated cells / tissue). Interaction between LRG1 and an interaction partner for LRG1 can also be analysed e.g. using techniques such as ELISA, surface plasmon resonance or biolayer interferometry analysis. Assays may comprise comparing the level of interaction in the presence of the agent to the level of interaction in an appropriate control condition (e.g. the absence of the agent).

[0070] Agents capable of reducing gene expression of LRG1 (e.g. reducing the level of RNA encoding LRG1 , reducing transcription of nucleic acid encoding LRG1 and / or increasing degradation of RNA encoding LRG1) may be identified using assays comprising detecting the level of RNA encoding the relevant protein, e.g. by RT-qPCR. Such assays may comprise treating cells / tissue with the agent, and subsequently comparing the level of RNA encoding the relevant protein in such cells / tissue to the level of RNA encoding the relevant protein in cells / tissue of an appropriate control condition (e.g. untreated / vehicle-treated cells / tissue). Assays for detecting reduced / altered splicing of pre-mRNA of a given protein may comprise detecting and / or quantifying one or more isoforms of the relevant protein, or RNA encoding one or more of said isoforms.

[0071] Agents capable of reducing protein expression of LRG1 (e.g. reducing the level of LRG1 , increasing degradation of LRG1) may be identified using assays comprising detecting the level of the relevant protein, e.g. using antibody / reporter-based methods (western blot, ELISA, immunohisto / cytochemistry, etc.). Such assays may comprise treating cells / tissue with the agent, and subsequently comparing the level of the relevant protein in such cells / tissue to the level of the relevant protein in cells / tissue of an appropriate control condition (e.g. untreated / vehicle-treated cells / tissue). Assays of protein degradation may comprise evaluating e.g. ubiquitination or proteosomal localisation of the relevant protein, and / or the proportion of the relevant protein that is ubiquitinated or localised to the proteasome.

[0072] An LRG1 inhibitor according to the present disclosure may be any agent / plurality of agents achieving the desired inhibitory activity.

[0073] In some embodiments, an LRG1 inhibitor is capable of inhibiting interaction between LRG1 and an interaction partner of LRG1 . In some embodiments, an LRG1 inhibitor is capable of inhibiting LRG1 function.

[0074] The ability of a candidate LRG1 inhibitor to inhibit interaction between LRG1 and an interaction partner for LRG1 can be evaluated e.g. by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the candidate LRG1 inhibitor. An example of a suitable assay to determine whether a given binding agent is capable of inhibiting interaction between LRG1 and an interaction partner for LRG1 is a competition ELISA.

[0075] In some embodiments, an LRG1 inhibitor is capable of binding to LRG1 or an interaction partner of LRG1 . In preferred embodiments, an LRG1 inhibitor is capable of binding to LRG1 . In some embodiments an LRG1 inhibitor behaves as a competitive inhibitor of interaction between LRG1 and an interaction partner of LRG1 . The LRG1 inhibitor may occupy, or otherwise reduce access to, a region of LRG1 required for binding to an interaction partner for LRG1 , or may occupy, or otherwise reduce access to, a region of an interaction partner for LRG1 required for binding to LRG1 .

[0076] LRG1 inhibitors may display specific binding to the relevant factor ( / .e. LRG1 or an interaction partner of LRG1). As used herein, “specific binding” refers to binding which is selective, and which can be discriminated from non-specific binding to non-target molecules. LRG1 inhibitors that specifically bind to LRG1 or an interaction partner of LRG1 preferably bind to the relevant factor with greater affinity, and / or with greater duration than other, non-target molecules; such LRG1 inhibitors may be described as being “specific for” the relevant factor.

[0077] The ability of a candidate LRG1 inhibitor to bind to LRG1 or an interaction partner of LRG1 can be evaluated e.g. 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.

[0078] In some embodiments, an LRG1 inhibitor targets / binds to the LRRCT domain of LRG1 . As used herein, ‘LRRCT region’ or ‘leucine-rich C-terminal domain’ refers to an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:183, 187 or 189. In some embodiments, the LRRCT region has an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:183.

[0079] In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:183.

[0080] As used herein, an LRG1 inhibitor ‘contacts’ a given region refers to an LRG1 inhibitor binding to LRG1 , or a polypeptide comprising the LRRCT region, via contact with one or more amino acids of the given region.

[0081] In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NQ:190. In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NQ:190. In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NQ:190. In some embodiments, the epitope of the LRG1 inhibitor comprises or consists of the amino acid sequence shown in SEQ ID NO:190. In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:190.

[0082] In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO:191 . In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NO:191 . In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:191. In some embodiments, the epitope of the LRG1 inhibitor comprises or consists of the amino acid sequence shown in SEQ ID NO:191 . In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:191 .

[0083] In some embodiments, the LRG1 inhibitor does not bind to the region of LRG1 shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor does not contact the region of LRG1 shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor does not bind to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:183. In some embodiments, the LRG1 inhibitor does not bind to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:183.

[0084] In some embodiments, the LRG1 inhibitor does not bind to the region of LRG1 shown in SEQ ID NO:192. In some embodiments, the LRG1 inhibitor does not contact the region of LRG1 shown in SEQ ID NO:192. In some embodiments, the LRG1 inhibitor does not bind to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:192. In some embodiments, the LRG1 inhibitor does not bind to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:192.

[0085] In some embodiments, an LRG1 inhibitor targets / binds to the LRR region of LRG1. As used herein, ‘LRR region’ refers to an amino acid sequence having at least 70% (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) amino acid sequence identity to SEQ ID NO:195. The LRR region comprises 8 leucine-rich repeats (LRRs) (SEQ ID NO:175, 176, 177, 178, 179, 180, 181 , and 182). In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO:175, 176, 177, 178, 179, 180, 181 or 182.

[0086] In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO:195. In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NO:195. In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:195. In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:195.

[0087] In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO:196. In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NO:196. In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO:196. In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:196. In some embodiments, the LRG1 inhibitor binds to the region of LRG1 shown in SEQ ID NO: 175, 176, 177, 178, 179, 180, 181 or 182. In some embodiments, the LRG1 inhibitor contacts the region of LRG1 shown in SEQ ID NO: 175, 176, 177, 178, 179, 180, 181 or 182. In some embodiments, the LRG1 inhibitor binds to LRG1 via contact with one or more amino acids of the region shown in SEQ ID NO: 175, 176, 177, 178, 179, 180, 181 or 182. In some embodiments, the LRG1 inhibitor binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 175, 176, 177, 178, 179, 180, 181 or 182.

[0088] The region of a given target molecule to which an LRG1 inhibitor binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of inhibitor-target (e.g. antibody-antigen) complexes, peptide scanning, mutagenesis mapping, hydrogendeuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysisbased ‘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 LRG1 inhibitor binds is determined by hydrogendeuterium exchange analysis by mass spectrometry, performed essentially as described in Example 2 herein.

[0089] In some embodiments, LRG1 inhibitors which are capable of binding to LRG1 or an interaction partner of LRG1 and inhibiting interaction between LRG1 and an interaction partner for LRG1 and / or inhibiting LRG1 function may e.g. be peptides / polypeptides.

[0090] In some embodiments, peptide / polypeptide LRG1 inhibitors capable of binding to LRG1 / interaction partner for LRG1 include e.g. antibodies or antigen binding fragments thereof, peptide aptamers, thioredoxins, monobodies, anticalin, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies ( / .e. single-domain antibodies (sdAbs)) affilins, armadillo repeat proteins (ArmRPs), OBodies and 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).

[0091] In some embodiments, peptide / polypeptide LRG1 inhibitors capable of binding to LRG1 or an interaction partner of LRG1 may be antibodies (immunoglobulins) such as monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and fragments and derivatives thereof (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.).

[0092] 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.

[0093] 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.

[0094] 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.

[0095] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et a!., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDRs and FRs of antibodies or antigen-binding fragments thereof referred to herein are defined according to the IMGT information system.

[0096] In some embodiments, the LRG1 inhibitor is an antibody or antigen-binding fragment thereof capable of binding to LRG1.

[0097] Antibodies capable of binding to LRG1 include e.g. LRG1 monoclonal antibody clones D2, C5, C4 (Invitrogen), LRG1 recombinant rabbit monoclonal antibody clones 7A1 and JB34-31 (Invitrogen), LRG1 monoclonal antibody LRG1 / 4882 and LRG1 / 4883 (NeoBiotechnologies), anti-LRG1 / LRG1 antibody EPR12362 and EPR12363 (Abeam), Magacizumab and antibodies disclosed in WO 2016 / 135462 A1. Antibodies capable of binding to LRG1 also include the particular exemplary LRG1 binding agents described herein below.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises the CDRs of an antibody that binds to LRG1 . In some embodiments, the antibody, or antigen-binding fragment thereof, comprises the FRs of an antibody that binds to LRG1 . In some embodiments, the antibody, or antigenbinding fragment thereof, comprises the CDRs and the FRs of an antibody that binds to LRG1 . That is, in some embodiments, the antibody, or antigen-binding fragment thereof, comprises the VH region and the VL region of an antibody that binds to LRG1 . In some embodiments, the antibody, or antigen-binding fragment thereof, comprises the CDRs, FRs and / or the VH and / or VL regions of a LRG1 -binding antibody clone described herein, or CDRs, FRs and / or VH and / or VL regions which are derived from those of a LRG1 -binding antibody clone described herein. In some embodiments, a LRG1-binding antibody clone is selected from: EBC-58, EBC-59, EBC-

[0099] 60, EBC-61 , EBC-95, EBC-97, EBC-103, EBC-107, EBC-1191 , EBC-1192, EBC-1193, EBC-1194, EBC- 1195, EBC-1196, EBC-1197, EBC-1198, EBC-1199, EBC-1200, EBC-1201 , EBC-1202, EBC-1203, EBC- 1204, EBC-1205, EBC-1206, EBC-1207, EBC-1208, EBC-1209, EBC-1210, EBC-1211 and EBC-1212.

[0100] In some embodiments, a LRG1 -binding antibody clone is selected from: EBC-58, EBC-59, EBC-60, EBC-

[0101] 61 , EBC-103, EBC-1191 , EBC-1192, EBC-1193, EBC-1194, EBC-1195, EBC-1196, EBC-1197, EBC- 1198, EBC-1199, EBC-1200, EBC-1201 , EBC-1202, EBC-1206, EBC-1207, EBC-1208 and EBC-1212.

[0102] In some embodiments, a LRG1-binding antibody clone is selected from: EBC-59, EBC-60, EBC-61 , EBC- 103, EBC-1191 , EBC-1192, EBC-1195, EBC-1196, EBC-1199, EBC-1200 and EBC-1212.

[0103] In some embodiments, a LRG1 -binding antibody clone is selected from: EBC-95, EBC-97, EBC-103 and EBC-107.

[0104] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region according to one of (1) to (6) below:

[0105] (1) a VH region incorporating the following CDRs:

[0106] HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:4, 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.

[0107] (2) a VH region incorporating the following CDRs:

[0108] HC-CDR1 having the amino acid sequence of SEQ ID NO:18 HC-CDR2 having the amino acid sequence of SEQ ID NO:19 HC-CDR3 having the amino acid sequence of SEQ ID NQ:20, 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.

[0109] (3) a VH region incorporating the following CDRs:

[0110] HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:32 HC-CDR3 having the amino acid sequence of SEQ ID NO:33, 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.

[0111] (4) a VH region incorporating the following CDRs:

[0112] HC-CDR1 having the amino acid sequence of SEQ ID NO:43

[0113] HC-CDR2 having the amino acid sequence of SEQ ID NO:44

[0114] HC-CDR3 having the amino acid sequence of SEQ ID NO:45, 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.

[0115] (5) a VH region incorporating the following CDRs:

[0116] HC-CDR1 having the amino acid sequence of SEQ ID NO:53

[0117] HC-CDR2 having the amino acid sequence of SEQ ID NO:54

[0118] HC-CDR3 having the amino acid sequence of SEQ ID NO:55, 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.

[0119] (6) a VH region incorporating the following CDRs:

[0120] HC-CDR1 having the amino acid sequence of SEQ ID NO:69

[0121] HC-CDR2 having the amino acid sequence of SEQ ID NQ:70

[0122] HC-CDR3 having the amino acid sequence of SEQ ID NO:71 , 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.

[0123] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region according to one of (7) to (19) below:

[0124] (7) a VH region incorporating the following FRs:

[0125] HC-FR1 having the amino acid sequence of SEQ ID NO:5

[0126] HC-FR2 having the amino acid sequence of SEQ ID NO:6

[0127] HC-FR3 having the amino acid sequence of SEQ ID NO:7

[0128] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0129] (8) a VH region incorporating the following FRs:

[0130] HC-FR1 having the amino acid sequence of SEQ ID NO:21 HC-FR2 having the amino acid sequence of SEQ ID NO:22

[0131] HC-FR3 having the amino acid sequence of SEQ ID NO:23

[0132] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0133] (9) a VH region incorporating the following FRs:

[0134] HC-FR1 having the amino acid sequence of SEQ ID NO:34

[0135] HC-FR2 having the amino acid sequence of SEQ ID NO:35

[0136] HC-FR3 having the amino acid sequence of SEQ ID NO:23

[0137] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0138] (10) a VH region incorporating the following FRs:

[0139] HC-FR1 having the amino acid sequence of SEQ ID NO:46

[0140] HC-FR2 having the amino acid sequence of SEQ ID NO:35

[0141] HC-FR3 having the amino acid sequence of SEQ ID NO:23

[0142] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0143] (11) a VH region incorporating the following FRs:

[0144] HC-FR1 having the amino acid sequence of SEQ ID NO:56

[0145] HC-FR2 having the amino acid sequence of SEQ ID NO:57

[0146] HC-FR3 having the amino acid sequence of SEQ ID NO:58

[0147] HC-FR4 having the amino acid sequence of SEQ ID NO:59, 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.

[0148] (12) a VH region incorporating the following FRs:

[0149] HC-FR1 having the amino acid sequence of SEQ ID NO:72

[0150] HC-FR2 having the amino acid sequence of SEQ ID NO:73

[0151] HC-FR3 having the amino acid sequence of SEQ ID NO:74

[0152] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0153] (13) a VH region incorporating the following FRs:

[0154] HC-FR1 having the amino acid sequence of SEQ ID NO:83

[0155] HC-FR2 having the amino acid sequence of SEQ ID NO:84

[0156] HC-FR3 having the amino acid sequence of SEQ ID NO:85

[0157] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0158] (14) a VH region incorporating the following FRs:

[0159] HC-FR1 having the amino acid sequence of SEQ ID NO:83

[0160] HC-FR2 having the amino acid sequence of SEQ ID NO:84

[0161] HC-FR3 having the amino acid sequence of SEQ ID NQ:104

[0162] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0163] (15) a VH region incorporating the following FRs:

[0164] HC-FR1 having the amino acid sequence of SEQ ID NO:83

[0165] HC-FR2 having the amino acid sequence of SEQ ID NO:84

[0166] HC-FR3 having the amino acid sequence of SEQ ID NQ:106

[0167] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0168] (16) a VH region incorporating the following FRs:

[0169] HC-FR1 having the amino acid sequence of SEQ ID NQ:108

[0170] HC-FR2 having the amino acid sequence of SEQ ID NQ:109

[0171] HC-FR3 having the amino acid sequence of SEQ ID NO:110

[0172] HC-FR4 having the amino acid sequence of SEQ ID NO:111 , 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.

[0173] (17) a VH region incorporating the following FRs: HC-FR1 having the amino acid sequence of SEQ ID NO:123

[0174] HC-FR2 having the amino acid sequence of SEQ ID NO:84

[0175] HC-FR3 having the amino acid sequence of SEQ ID NO:124

[0176] HC-FR4 having the amino acid sequence of SEQ ID NO:111 , 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.

[0177] (18) a VH region incorporating the following FRs:

[0178] HC-FR1 having the amino acid sequence of SEQ ID NO:126

[0179] HC-FR2 having the amino acid sequence of SEQ ID NO:127

[0180] HC-FR3 having the amino acid sequence of SEQ ID NO:128

[0181] HC-FR4 having the amino acid sequence of SEQ ID NO:111 , 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.

[0182] (19) a VH region incorporating the following FRs:

[0183] HC-FR1 having the amino acid sequence of SEQ ID NO:83

[0184] HC-FR2 having the amino acid sequence of SEQ ID NO:84

[0185] HC-FR3 having the amino acid sequence of SEQ ID NO:85

[0186] HC-FR4 having the amino acid sequence of SEQ ID NO:8, 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.

[0187] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region comprising the CDRs according to any one of (1) to (6) above, and the FRs according to any one of (7) to

[0188] (19) above.

[0189] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region according to one of (20) to (32) below:

[0190] (20) a VH region comprising the CDRs according to (1) and the FRs according to (7).

[0191] (21) a VH region comprising the CDRs according to (2) and the FRs according to (8).

[0192] (22) a VH region comprising the CDRs according to (3) and the FRs according to (9).

[0193] (23) a VH region comprising the CDRs according to (4) and the FRs according to (10).

[0194] (24) a VH region comprising the CDRs according to (5) and the FRs according to (11). (25) a VH region comprising the CDRs according to (6) and the FRs according to (12).

[0195] (26) a VH region comprising the CDRs according to (2) and the FRs according to (13).

[0196] (27) a VH region comprising the CDRs according to (2) and the FRs according to (14).

[0197] (28) a VH region comprising the CDRs according to (2) and the FRs according to (15).

[0198] (29) a VH region comprising the CDRs according to (2) and the FRs according to (16).

[0199] (30) a VH region comprising the CDRs according to (2) and the FRs according to (17).

[0200] (31) a VH region comprising the CDRs according to (2) and the FRs according to (18).

[0201] (32) a VH region comprising the CDRs according to (2) and the FRs according to (19).

[0202] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region according to one of (33) to (44) below:

[0203] (33) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:1 .

[0204] (34) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity)to the amino acid sequence of SEQ ID NO:17.

[0205] (35) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:31 .

[0206] (36) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:42.

[0207] (37) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:52. (38) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:68.

[0208] (39) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:82.

[0209] (40) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NQ:103.

[0210] (41) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NQ:105.

[0211] (42) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NQ:107.

[0212] (43) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:122.

[0213] (44) a VH region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:125.

[0214] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL region according to one of (45) to (50) below:

[0215] (45) a VL region incorporating the following CDRs:

[0216] LC-CDR1 having the amino acid sequence of SEQ ID NQ:10

[0217] LC-CDR2 having the amino acid sequence of SEQ ID NO:11

[0218] LC-CDR3 having the amino acid sequence of SEQ ID NO:12, 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.

[0219] (46) a VL region incorporating the following CDRs:

[0220] LC-CDR1 having the amino acid sequence of SEQ ID NO:25

[0221] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0222] LC-CDR3 having the amino acid sequence of SEQ ID NO:27, 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.

[0223] (47) a VL region incorporating the following CDRs:

[0224] LC-CDR1 having the amino acid sequence of SEQ ID NO:37

[0225] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0226] LC-CDR3 having the amino acid sequence of SEQ ID NO:38, 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.

[0227] (48) a VL region incorporating the following CDRs:

[0228] LC-CDR1 having the amino acid sequence of SEQ ID NO:25

[0229] LC-CDR2 having the amino acid sequence of SEQ ID NO:26

[0230] LC-CDR3 having the amino acid sequence of SEQ ID NO:48, 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.

[0231] (49) a VL region incorporating the following CDRs:

[0232] LC-CDR1 having the amino acid sequence of SEQ ID NO:61

[0233] LC-CDR2 having the amino acid sequence of SEQ ID NO:62

[0234] LC-CDR3 having the amino acid sequence of SEQ ID NO:63, 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.

[0235] (50) a VL region incorporating the following CDRs:

[0236] LC-CDR1 having the amino acid sequence of SEQ ID NO:76

[0237] LC-CDR2 having the amino acid sequence of SEQ ID NO:77

[0238] LC-CDR3 having the amino acid sequence of SEQ ID NO:78, 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.

[0239] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL region according to one of (51) to (64) below:

[0240] (51) a VL region incorporating the following FRs:

[0241] LC-FR1 having the amino acid sequence of SEQ ID NO:13

[0242] LC-FR2 having the amino acid sequence of SEQ ID NO:14

[0243] LC-FR3 having the amino acid sequence of SEQ ID NO:15

[0244] LC-FR4 having the amino acid sequence of SEQ ID NO:16, 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. (52) a VL region incorporating the following FRs:

[0245] LC-FR1 having the amino acid sequence of SEQ ID NO:28

[0246] LC-FR2 having the amino acid sequence of SEQ ID NO:29

[0247] LC-FR3 having the amino acid sequence of SEQ ID NQ:30

[0248] LC-FR4 having the amino acid sequence of SEQ ID NO:16, 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.

[0249] (53) a VL region incorporating the following FRs:

[0250] LC-FR1 having the amino acid sequence of SEQ ID NO:39

[0251] LC-FR2 having the amino acid sequence of SEQ ID NQ:40

[0252] LC-FR3 having the amino acid sequence of SEQ ID NO:41

[0253] LC-FR4 having the amino acid sequence of SEQ ID NO:16, 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.

[0254] (54) a VL region incorporating the following FRs:

[0255] LC-FR1 having the amino acid sequence of SEQ ID NO:49

[0256] LC-FR2 having the amino acid sequence of SEQ ID NQ:50

[0257] LC-FR3 having the amino acid sequence of SEQ ID NO:51

[0258] LC-FR4 having the amino acid sequence of SEQ ID NO:16, 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.

[0259] (55) a VL region incorporating the following FRs:

[0260] LC-FR1 having the amino acid sequence of SEQ ID NO:64

[0261] LC-FR2 having the amino acid sequence of SEQ ID NO:65

[0262] LC-FR3 having the amino acid sequence of SEQ ID NO:66

[0263] LC-FR4 having the amino acid sequence of SEQ ID NO:67, 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.

[0264] (56) a VL region incorporating the following FRs:

[0265] LC-FR1 having the amino acid sequence of SEQ ID NO:79

[0266] LC-FR2 having the amino acid sequence of SEQ ID NQ:80

[0267] LC-FR3 having the amino acid sequence of SEQ ID NO:81

[0268] LC-FR4 having the amino acid sequence of SEQ ID NO:67, 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.

[0269] (57) a VL region incorporating the following FRs:

[0270] LC-FR1 having the amino acid sequence of SEQ ID NO:87

[0271] LC-FR2 having the amino acid sequence of SEQ ID NO:88

[0272] LC-FR3 having the amino acid sequence of SEQ ID NO:89

[0273] LC-FR4 having the amino acid sequence of SEQ ID NQ:90, 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.

[0274] (58) a VL region incorporating the following FRs:

[0275] LC-FR1 having the amino acid sequence of SEQ ID NO:92

[0276] LC-FR2 having the amino acid sequence of SEQ ID NO:93

[0277] LC-FR3 having the amino acid sequence of SEQ ID NO:94

[0278] LC-FR4 having the amino acid sequence of SEQ ID NQ:90, 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.

[0279] (59) a VL region incorporating the following FRs:

[0280] LC-FR1 having the amino acid sequence of SEQ ID NO:96

[0281] LC-FR2 having the amino acid sequence of SEQ ID NO:93

[0282] LC-FR3 having the amino acid sequence of SEQ ID NO:97

[0283] LC-FR4 having the amino acid sequence of SEQ ID NQ:90, 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.

[0284] (60) a VL region incorporating the following FRs:

[0285] LC-FR1 having the amino acid sequence of SEQ ID NO:99

[0286] LC-FR2 having the amino acid sequence of SEQ ID NQ:100

[0287] LC-FR3 having the amino acid sequence of SEQ ID NQ:101

[0288] LC-FR4 having the amino acid sequence of SEQ ID NQ:102, 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.

[0289] (61) a VL region incorporating the following FRs: LC-FR1 having the amino acid sequence of SEQ ID NO:113

[0290] LC-FR2 having the amino acid sequence of SEQ ID NO:88

[0291] LC-FR3 having the amino acid sequence of SEQ ID NO:89

[0292] LC-FR4 having the amino acid sequence of SEQ ID NQ:102, 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.

[0293] (62) a VL region incorporating the following FRs:

[0294] LC-FR1 having the amino acid sequence of SEQ ID NO:115

[0295] LC-FR2 having the amino acid sequence of SEQ ID NO:116

[0296] LC-FR3 having the amino acid sequence of SEQ ID NO:117

[0297] LC-FR4 having the amino acid sequence of SEQ ID NQ:102, 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.

[0298] (63) a VL region incorporating the following FRs:

[0299] LC-FR1 having the amino acid sequence of SEQ ID NO:119

[0300] LC-FR2 having the amino acid sequence of SEQ ID NQ:120

[0301] LC-FR3 having the amino acid sequence of SEQ ID NO:121

[0302] LC-FR4 having the amino acid sequence of SEQ ID NQ:102, 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.

[0303] (64) a VL region incorporating the following FRs:

[0304] LC-FR1 having the amino acid sequence of SEQ ID NO:99

[0305] LC-FR2 having the amino acid sequence of SEQ ID NQ:130

[0306] LC-FR3 having the amino acid sequence of SEQ ID NO:131

[0307] LC-FR4 having the amino acid sequence of SEQ ID NQ:102, 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.

[0308] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL region comprising the CDRs according to any one of (45) to (50) above, and the FRs according to any one of (51) to (64) above.

[0309] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL region according to one of (65) to (78) below: (65) a VL region comprising the CDRs according to (45) and the FRs according to (51).

[0310] (66) a VL region comprising the CDRs according to (46) and the FRs according to (52).

[0311] (67) a VL region comprising the CDRs according to (47) and the FRs according to (53).

[0312] (68) a VL region comprising the CDRs according to (48) and the FRs according to (54).

[0313] (69) a VL region comprising the CDRs according to (49) and the FRs according to (55).

[0314] (70) a VL region comprising the CDRs according to (50) and the FRs according to (56).

[0315] (71) a VL region comprising the CDRs according to (46) and the FRs according to (57).

[0316] (72) a VL region comprising the CDRs according to (46) and the FRs according to (58).

[0317] (73) a VL region comprising the CDRs according to (46) and the FRs according to (59).

[0318] (74) a VL region comprising the CDRs according to (46) and the FRs according to (60).

[0319] (75) a VL region comprising the CDRs according to (46) and the FRs according to (61).

[0320] (76) a VL region comprising the CDRs according to (46) and the FRs according to (62).

[0321] (77) a VL region comprising the CDRs according to (46) and the FRs according to (63).

[0322] (78) a VL region comprising the CDRs according to (46) and the FRs according to (64).

[0323] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VL region according to one of (79) to (92) below:

[0324] (79) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:9.

[0325] (80) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:24.

[0326] (81) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:36. (82) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:47.

[0327] (83) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:60.

[0328] (84) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:75.

[0329] (85) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:86.

[0330] (86) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:91 .

[0331] (87) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:95.

[0332] (88) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:98.

[0333] (89) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:112.

[0334] (90) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:114.

[0335] (91) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:118. (92) a VL region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:129.

[0336] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a VH region according to any one of (1) to (44) above, and a VL region according to any one of (45) to (92) above.

[0337] 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.

[0338] 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:

[0339] 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.

[0340] 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:

[0341] 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.

[0342] 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 antibody or antigen-binding fragment thereof comprising the substitution as compared to the equivalent unsubstituted molecule.

[0343] The VH and VL region of an antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antibody, or antigen-binding fragment thereof, according to the present disclosure comprises, or consists of, an Fv region that binds to LRG1 . 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).

[0344] 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 antibody, or antigen-binding fragment thereof, 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).

[0345] In some embodiments, the antibody described herein comprises, or consists of, a whole antibody that binds to LRG1 . 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.

[0346] 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. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM. The light chain may be kappa (K) or lambda (A).

[0347] Herein, a ‘CH1 domain’ refers to an amino acid sequence corresponding to the CH1 domain of an immunoglobulin (Ig). The CH1 domain is the region of an Ig formed by positions 118 to 215 of the immunoglobulin constant domain, according to the EU numbering system (described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85). A ‘hinge domain’ refers to an amino acid sequence corresponding to the hinge domain of an Ig. The hinge domain is the region of an Ig formed by positions 216 to 230 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH2 domain’ refers to an amino acid sequence corresponding to the CH2 domain of an Ig. The CH2 domain is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH3 domain’ refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). The CH3 domain is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant domain, according to the EU numbering system. A ‘CH2-CH3 region’ refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant domain, according to the EU numbering system.

[0348] In some embodiments, the antibody, or antigen-binding fragment thereof, 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 LRG1.

[0349] In some embodiments, the antibody, or antigen-binding fragment thereof, 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. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM, e.g. a human IgG (e.g. hlgG 1 , 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 m17).

[0350] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:132 or 137. In some embodiments, the antigenbinding molecule comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:193. In some preferred embodiments, the antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:132.

[0351] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:133. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:135. In some embodiments, the antibody, or antigenbinding fragment thereof, comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:136.

[0352] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:134.

[0353] 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 antibody, or antigen-binding fragment thereof, as described herein. In some embodiments, the antibody, or antigen-binding fragment thereof, 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.

[0354] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:138, 139, 140, 141 , 142, 143. In some embodiments, the antigen-binding molecule comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 194. In some preferred embodiments, the antibody, or antigen-binding fragment thereof, comprises an amino acid sequence having at least 70% sequence identity (e.g. one of >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:138

[0355] In some embodiments, the antibody, or antigen-binding fragment thereof, is or comprises a monoclonal antibody, or an antigen-binding fragment thereof.

[0356] In some embodiments, the antibody, or antigen-binding fragment thereof, 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 assembled into a library from which they can be expressed either as disulfide-linked Fab fragments or as single-chain Fv (scFv) fragments. The Fab- or scFv- encoding genes are fused to a surface coat protein of filamentous bacteriophage and Fab or scFv capable of binding to the target of interest can then be identified by screening the library with antigen. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab / scFv fragment. In the transgenic mouse technique, mice in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues are immunized with antigen, and monoclonal antibody is prepared by conventional hybridoma technology, to yield a fully human monoclonal antibody. In some embodiments, the antibody, or antigen-binding fragment thereof, 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.

[0357] In some embodiments, the antibody, or antigen-binding fragment thereof, is a mouse / human chimeric antibody / antibody fragment ( / .e. an antibody, or antigen-binding fragment thereof, comprising mouse antibody variable domains and human antibody constant regions). In some embodiments, the antibody, or antigen-binding fragment thereof, is a humanised antibody / antibody fragment. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises mouse antibody CDRs and human antibody framework and constant regions.

[0358] Mouse / human chimeric antibodies / antibody fragments 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), in Chapter 8 thereof, in particular section 3 of Chapter 8.

[0359] Humanised antibodies / antibody fragments 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), in Chapter 7 thereof, in particular section 3.1 of Chapter 7 entitled ‘Antibody Humanization’. Techniques for antibody humanisation are also described e.g. in Safdari et al., Biotechnol Genet Eng Rev (2013) 29:175- 86.

[0360] Antibodies may be produced by a process of affinity maturation in which a modified antibody is generated that has an improvement in the affinity of the antibody for antigen, compared to an unmodified parent antibody. Affinity-matured antibodies may be produced by procedures known in the art, e.g., Marks et al.,Rio / Technology 10:779-783 (1992); Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0361] In some embodiments, peptide / polypeptide LRG1 inhibitors capable of binding to LRG1 / an interaction partner for LRG1 may e.g. be a peptide aptamer. Peptide aptamers and methods for their generation and identification are reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12):1082-101 , which is hereby incorporated by reference in its entirety.

[0362] In some embodiments, peptide / polypeptide LRG1 inhibitors capable of binding to LRG1 / interaction partner for LRG1 may e.g. be based on an interaction partner for the relevant factor ( / .e. LRG1 or an interaction partner for LRG1) to which the inhibitor binds.

[0363] As used herein, a peptide / polypeptide which is “based on” a reference protein comprises or consists of an amino acid sequence having high sequence identity (e.g. at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to all or part of the amino acid of the reference protein.

[0364] For example, an LRG1 inhibitor which binds to LRG1 may comprise / consist of a peptide / polypeptide fragment of an interaction partner for LRG1 . Similarly, an LRG1 inhibitor which binds to an interaction partner for LRG1 may comprise / consist of a peptide / polypeptide fragment of LRG1 .

[0365] Such inhibitors preferably possess the ability to bind to the relevant factor, but lack or display a reduced level of one or more other properties of the protein on which they are based. For example, an LRG1 inhibitor may comprise the amino acid sequence(s) required for binding to the relevant factor, and may lack the amino acid sequence(s) required for one or more other properties of the protein on which it is based.

[0366] Such peptide / polypeptide LRG1 inhibitors may be referred to as ‘decoy’, ‘dominant-negative’ or ‘mimetic’ versions of the proteins on which they are based, and preferably display competitive inhibition of interaction between LRG1 and an interaction partner for LRG1 protein. In this way, such peptide / polypeptide LRG1 inhibitors inhibit the formation of LRG1 -containing complexes and / or disrupt existing LRG1-containing complexes via displacement of endogenous interaction partners, forming nonfunctional complexes / complexes having a reduced level of function.

[0367] Such peptide / polypeptide LRG1 inhibitors can be identified by screening of libraries of the relevant peptides / polypeptides for LRG1 inhibition.

[0368] In some embodiments an LRG1 inhibitor is a small molecule inhibitor of LRG1 . As used herein, a “small molecule” refers to a low molecular weight (< 1000 daltons, typically between -300-700 daltons) organic compound.

[0369] A small molecule LRG1 inhibitor may bind to LRG1 or an interaction partner for LRG1 . A small molecule LRG1 inhibitor may inhibit interaction between LRG1 and an interaction partner for LRG1 . A small molecule LRG1 inhibitor may bind to LRG1 and inhibit LRG1 function.

[0370] Suitable small molecule LRG1 inhibitors may be identified e.g. by screening of small molecule libraries.

[0371] In some embodiments an LRG1 inhibitor is, or comprises, a nucleic acid.

[0372] The nucleic acid may bind to LRG1 or an interaction partner for LRG1 . The nucleic acid may inhibit interaction between LRG1 and an interaction partner for LRG1. The nucleic acid may bind to LRG1 and inhibit LRG1 function.

[0373] Nucleic acid aptamers are reviewed e.g. in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202. They may be identified and / or produced by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEX), or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) PLoS ONE 5(12):e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968):505-10, and in WO 91 / 19813.

[0374] Nucleic acid aptamers may comprise DNA and / or RNA, and may be single-stranded or double-stranded. They may comprise chemically modified nucleic acids, for example in which the sugar and / or phosphate and / or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.

[0375] Nucleic acid aptamers may be chemically synthesised, e.g. on a solid support. Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detritylated, then coupled with a suitably activated nucleoside phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer (e.g., see Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539; and Beaucage, S. L.; Lyer, R. P. (1992). Tetrahedron 48 (12): 2223).

[0376] In some embodiments, an LRG1 inhibitor is capable of reducing expression (e.g. gene and / or protein expression) of LRG1 or an interaction partner of LRG1 . In preferred embodiments, an LRG1 inhibitor is capable of reducing expression (e.g. gene and / or protein expression) of LRG1.

[0377] Inhibition of expression of LRG1 will result in a decrease in the quantity of LRG1 in a cell / tissue / organ / organ system / subject. For example, in a given tissue the inhibition of expression of LRG1 will result in a decrease in the level of LRG1 relative to an untreated cell.

[0378] Inhibition may be partial. Preferred degrees of inhibition are at least 50% (e.g. one of >50%, >60%, >70%, >75%, >80%, >85, or >90%). A level of inhibition between 90% and 100% is considered a ‘silencing’ of expression or function. Gene and protein expression may be determined as described herein or by methods in the art that are well known to a skilled person.

[0379] In some embodiments the LRG1 inhibitor is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an antisense nucleic acid. In some embodiments the inhibitory nucleic acid is an antisense oligonucleotide (ASO). Antisense oligonucleotides may be single-stranded, and may bind by complementary sequence binding to a target oligonucleotide, e.g. mRNA.

[0380] ASOs may be designed to inhibit / prevent expression of LRG1 or an interaction partner of LRG1 .

[0381] Oligonucleotides designed to inhibit / prevent expression of LRG1 or an interaction partner of LRG1 , may have substantial sequence identity to a portion of nucleic acid encoding LRG1 / an interaction partner of LRG1 , or the complementary sequence thereto. In some embodiments, the inhibitory nucleic acid reduces expression of LRG1 / an interaction partner of LRG1 by RNA interference (RNAi). RNAi involves inhibition of gene expression and translation by targeted neutralisation of mRNA molecules. In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA).

[0382] A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has been demonstrated. Double-stranded RNA (dsRNA)- dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA. RNAi based therapeutics have been progressed into Phase I, II and III clinical trials for a number of indications (Nature 2009 Jan 22; 457(7228) :426-433).

[0383] In the art, such RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences. siRNAs are typically double stranded and, in order to optimise the effectiveness of RNA mediated downregulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response. miRNAs are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reversecomplement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed e.g. in John etal, PLoS Biology, 11 (2), 1862-1879, 2004.

[0384] Typically, the oligonucleotides intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double-stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such the Ambion siRNA finder. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors). In some embodiments the siRNA is synthesized synthetically. siRNA-mediated knockdown of LRG1 and siRNAs for achieving the same are described e.g. in Wang et al., Nature (2013) 499:306-311 and Zhang et al., Journal of Experimental & Clinical Cancer Research (2016) 35:29, which are hereby incorporated by reference in their entirety.

[0385] Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21 :324-328). The longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two (ribo)nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

[0386] Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In some embodiments the shRNA is produced within a cell by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human H1 or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously ( / n vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA molecule comprises a partial sequence of a gene encoding a LRG1 protein. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure. shRNA-mediated knockdown of LRG1 and shRNAs for achieving the same are described e.g. in Zhong et al., (2015) 36:4271-4278, which is hereby incorporated by reference in its entirety.

[0387] In some embodiments, the inhibitory nucleic acid is a splice-switching oligonucleotide (SSO). Splice switching oligonucleotides are reviewed e.g. in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549-6563, which is hereby incorporated by reference in its entirety. SSOs disrupt the normal splicing of target RNA transcripts by blocking the RNA-RNA base-pairing and / or protein-RNA binding interactions that occur between components of the splicing machinery and pre-mRNA. SSOs may be employed to alter the number / proportion of mature mRNA transcripts encoding a LRG1 protein or particular isoform(s) thereof. SSOs may be designed to target a specific region of the target transcript, e.g. to effect skipping of exon(s) of interest, e.g. exons encoding domains / regions of interest.

[0388] SSOs generally comprise alterations to oligonucleotide sugar-phosphate backbones to prevent RNAse H degradation, and may comprise include e.g. phosphorodiamidate morpholino (PMOs), peptide nucleic acid (PNA), locked nucleic acid (LNA), and / or 2'0-methyl (2'0Me) and 2'-O-methoxyethyl (MOE) ribose modifications.

[0389] Inhibitory nucleic acids may be made recombinantly by transcription of a nucleic acid sequence, e.g. contained within vector. Transcription may be performed in cell-free transcription reactions, or in a cell comprising nucleic acid encoding the inhibitory nucleic acid. In some embodiments inhibitory nucleic acids are produced within a cell, e.g. by transcription from a vector. Vectors encoding such molecules may be introduced into cells in any of the ways known in the art. Optionally, expression of the nucleic acid can be regulated using a cell / tissue (e.g. heart, muscle, etc.) specific promoter.

[0390] Inhibitory nucleic acids may also be synthesized using standard solid or solution phase synthesis techniques which are known in the art.

[0391] In some embodiments, the LRG1 inhibitor is a molecule / plurality of molecules capable of modifying nucleic acid encoding LRGI / an interaction partner for LRG1 to reduce / prevent expression of LRG1 / an interaction partner for LRG1 . In preferred embodiments, the LRG1 inhibitor is a molecule / plurality of molecules capable of modifying nucleic acid encoding LRG1 to reduce / prevent expression of LRG1.

[0392] Modifying nucleic acid encoding LRG1 / an interaction partner for LRG1 may comprise modifying a gene encoding LRG1 / an interaction partner of LRG1. In some embodiments, modifying nucleic acid encoding LRG1 / an interaction partner of LRG1 comprises introducing an insertion, substitution or deletion into a nucleic acid sequence encoding the LRG1 / an interaction partner of LRG1 .

[0393] In some embodiments modifying nucleic acid encoding LRG1 / an interaction partner for LRG1 comprises modifying the nucleic acid to introduce a premature stop codon in the sequence transcribed from the nucleic acid. In some embodiments modifying nucleic acid encoding LRG1 / an interaction partner for LRG1 comprises modifying the nucleic acid to encode a truncated and / or non-functional version of the LRG1 protein. In some embodiments modifying nucleic acid encoding LRG1 / an interaction partner for LRG1 comprises modifying the nucleic acid to encode a version of LRG1 / an interaction partner for LRG1 which is misfolded and / or degraded.

[0394] The modification may be of nucleic acid comprised in a cell, e.g. endogenous nucleic acid encoding LRG1 / an interaction partner. The modification causes the cell to have a reduced level of gene and / or protein expression of LRG1 / an interaction partner as compared to an equivalent unmodified cell. In some embodiments the modification is performed in vitro or ex vivo. In some embodiments the modification is performed in vivo.

[0395] Methods for modifying nucleic acids encoding proteins of interest and agents for achieving the same are well known in the art, and include e.g. including modification of the target nucleic acid by homologous recombination, and target nucleic acid editing using site-specific nucleases (SSNs).

[0396] Suitable methods may employ targeting by homologous recombination, which is reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4:Unit 4.29 and Vasquez et al., PNAS 2001 , 98(15): 8403-8410 both of which are hereby incorporated by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequence through crossover events guided by homologous sequences.

[0397] In some embodiments the methods employ target nucleic acid editing using SSNs. Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.

[0398] SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems.

[0399] ZFN systems are reviewed e.g. in Umov et al., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence.

[0400] TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8): 1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “Nl” binds to A, “NG” binds to T and “NN” or “NK” binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501 .). CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the singleguide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0401] In some embodiments the SSN system is a ZFN system, a TALEN system, CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system or a CRISPR / C2c3 system.

[0402] SSN-mediated knockout of LRG1 (e.g. CRISPR / Cas9-mediated knockout of LRG1) and agents for achieving the same are described e.g. in Choi et al., eLife (2022) 11 :e81559, which is hereby incorporated by reference in its entirety.

[0403] Therapeutic and prophylactic applications

[0404] The present invention provides methods and articles (agents and compositions) for the treatment and / or prevention of diseases through LRG1 inhibition. Treatment / prevention of disease is achieved by LRG1 inhibition in e.g. a cell, tissue / organ / organ system / subject. The LRG1 inhibitors described herein find use in therapeutic and prophylactic methods.

[0405] The present disclosure provides an LRG1 inhibitor described herein for use in a method of medical treatment or prophylaxis. Also provided is an LRG1 inhibitor described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of an LRG1 inhibitor 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 LRG1 inhibitor described herein.

[0406] The methods may be effective to reduce the development or progression of a disease / condition, alleviation of the symptoms of a disease / condition or reduction in 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 a later stage of the disease / condition.

[0407] It will be appreciated that the articles of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of LRG1 .

[0408] For example, the disease / condition may be a disease / condition in which LRG1 is pathologically- implicated, e.g. a disease / condition in which an increased level / activity of LRG1 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 LRG1 may be a risk factor for the onset, development or progression of the disease / condition.

[0409] In some embodiments, the disease / condition to be treated in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of LRG1 , e.g. as compared to the level of expression / activity in the absence of the disease / condition.

[0410] Treatment in accordance with the methods of the present disclosure may achieve a reduction in the level of expression / activity of LRG1 in a subject (compared to an equivalent untreated subject, or a subject treated with an appropriate control).

[0411] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by elevated LRG1 expression / activity. Diseases / conditions associated with LRG1 dysfunction are reviewed in Camilli et al., Journal of Biomedical Science (2022) 29(1):6, which is hereby incorporated by reference in its entirety.

[0412] LRG1 inhibitors are useful in the treatment diseases characterised by upregulation in LRG1 expression / activity.

[0413] Diseases characterised by upregulation in LRG1 expression include: cancer such as glioblastoma, retinoblastoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, non small cell lung cancer, squamous cell lung carcinoma, breast cancer, clear cell renal cell carcinoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, biliary tract carcinoma, colorectal carcinoma, ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer, leukaemia; Alzheimer’s disease, Parkinson’s disease, progressive supranuclear palsy, stroke, hydrocephalus, neovascular AMD, diabetic retinopathy, corneal neovascularisation, idiopathic pulmonary fibrosis, asthma, bronchopulmonary dysplasia, SARS-CoV-2 acute respiratory distress syndrome, emphysema, myocardial infarction, atherosclerosis, idiopathic pulmonary hypertension, diabetic kidney disease, idiopathic nephrotic syndrome, lupus nephritis, vasculitides, colitis, rheumatoid arthritis, osteoarthritis, Still’s disease, viral and bacterial infection, diabetes, acute pancreatitis and psoriasis.

[0414] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a fibroinflammatory disease / condition.

[0415] As used herein, a ‘fibroinflammatory disease’ or ‘fibroinflammatory condition’ refers to a disease / condition associated with fibroinflammation ( / .e. associated with fibrosis and / or inflammation). A fibroinflammatory disease / condition may be associated with fibroinflammatory processes / pathways ( / .e. fibrotic and / or inflammatory processes / pathways). Fibroinflammatory disorders are reviewed in e.g. Systemic Fibroinflammatory disorders, 2017 (Springer, DOI: 10.1007 / 978-3-319-41349-5), which is hereby incorporated by reference in its entirety.

[0416] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure may be fibroinflammation, a fibroinflammatory condition, or a disease / disorder characterised by fibroinflammation.

[0417] In some embodiments, the disease / disorder may be fibrosis, a fibrotic condition, or a disease / disorder characterised by fibrosis. In some embodiments, the disease / disorder may be inflammation, an inflammatory condition, or a disease / disorder characterised by inflammation. In some embodiments, the disease / disorder may be fibrosis and inflammation, a fibrotic and inflammatory condition, or a disease / disorder characterised by fibrosis and inflammation.

[0418] Inflammatory reactions play an important part in triggering fibrosis in many different organ systems. Inflammation can lead to excess in deposition of ECM components in the affected tissues. Low-grade but persistent inflammation is also thought to contribute to the progression of fibrosis in cardiovascular disease and hypertension. In many fibrotic disorders, a persistent inflammatory trigger is crucial to upregulation of production of growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture.

[0419] As used herein, “fibrosis” refers to the formation of excess fibrous connective tissue as a result of the excess deposition of extracellular matrix components, for example collagen. Fibrous connective tissue is characterised by having extracellular matrix (ECM) with a high collagen content. The collagen may be provided in strands or fibers, which may be arranged irregularly or aligned. The ECM of fibrous connective tissue may also include glycosaminoglycans.

[0420] As used herein, “excess fibrous connective tissue” refers to an amount of connective tissue at a given location (e.g. a given tissue or organ, or part of a given tissue or organ) which is greater than the amount of connective tissue present at that location in the absence of fibrosis, e.g. under normal, non- pathological conditions. As used herein, “excess deposition of extracellular matrix components” refers to a level of deposition of one or more extracellular matrix components which is greater than the level of deposition in the absence of fibrosis, e.g. under normal, non-pathological conditions.

[0421] 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, which are hereby incorporated by reference in their entirety.

[0422] 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.

[0423] The main cellular effectors of fibrosis are myofibroblasts. In response to tissue injury, damaged cells and leukocytes produce pro-fibroinflammafory factors such as TGFp, IL-13 and PDGF, which activate fibroblasts (and other myofibroblast precursor cells) to become aSMA-ex pressing myofibroblasts, and recruit myofibroblasts to the site of injury. Myofibroblasts produce large amounts of extracellular matrix components such as collagen and periostin for wound contracture and closure, and also produce proinflammatory cytokines such as IL-6, and tissue remodelling factors such as MMP2 and TIMP1 . Persistent / chronic infection and / or inflammation can result in the generation of too many myofibroblasts, and consequently the over-production of extracellular matrix, resulting in fibrosis. In many diseases and conditions characterised by fibrosis, a persistent inflammatory trigger is crucial to upregulation of production of growth factors, proteolytic enzymes, angiogenic factors and fibrogenic cytokines, which stimulate the deposition of connective tissue elements that progressively remodel and destroy normal tissue architecture.

[0424] Fibrosis can be triggered by pathological conditions, e.g. conditions, infections or disease states that lead to production of pro-fibrotic factors such as TGFpl . In some embodiments, fibrosis may be caused by physical injury / stimuli, chemical injury / stimuli or environmental injury / stimuli. Physical injury / stimuli may occur during surgery, e.g. iatrogenic causes. Chemical injury / stimuli may include drug induced fibrosis, e.g. following chronic administration of drugs such as bleomycin, cyclophosphamide, amiodarone, procainamide, penicillamine, gold and nitrofurantoin (Daba etal., Saudi Med J 2004 Jun; 25(6): 700-6). Environmental injury / stimuli may include exposure to asbestos fibres or silica.

[0425] Fibrosis can be of any tissue / organ of the body. In some embodiments, fibrosis is of the lung (e.g. bronchioles, alveoli), airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi), heart, kidney, liver, skeletal muscle, blood vessels, eye, skin, pancreas, bowel, small intestine, large intestine, colon, joints, brain, or bone marrow. Fibrosis may also occur in multiple tissues / organs at once.

[0426] In some embodiments, fibrosis may be of an organ or tissue of the respiratory system, e.g. the lung (e.g. bronchioles, alveoli), or airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, fibrosis may be of an organ or tissue of the cardiovascular system, e.g. the heart or blood vessels. In some embodiments, fibrosis may be of an organ or tissue of the gastrointestinal system, e.g. of the liver, bowel, small intestine, large intestine, colon, or pancreas. In some embodiments, fibrosis may be of the eye. In some embodiments, fibrosis may be of the skin. In some embodiments, fibrosis may be of an organ or tissue of the nervous system, e.g. the brain. In some embodiments, fibrosis may be of the bone marrow. In some embodiments, fibrosis may be of the joints. In some embodiments, fibrosis may be of an organ or tissue of the urinary system, e.g. the kidneys. In some embodiments, fibrosis may be of an organ or tissue of the musculoskeletal system, e.g. muscle tissue. In some embodiments, fibrosis may be of an organ or tissue of one or more organ systems. In some embodiments, fibrosis may be of the eye.

[0427] As used herein, a disease / condition which is ‘characterised by fibrosis’ is a disease / condition in which fibrosis is a symptom of the disease / condition. Diseases and conditions characterised by fibrosis include, but are not limited to:

[0428] Diseases / conditions affecting the respiratory system such as pulmonary fibrosis, fibrothorax, radiation-induced lung injury, interstitial lung disease (ILD), idiopathic interstitial pneumonia (IIP), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, progressive massive fibrosis, scleroderma, obliterative bronchiolitis, Hermansky-Pudlak syndrome, asbestosis, silicosis, sarcoidosis, tumor stroma in lung disease, chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis and asthma;

[0429] Diseases / conditions affecting the liver such as chronic liver disease, liver fibrosis, bridging fibrosis, cirrhosis, non-alcoholic fatty liver disease (NAFLD), steatohepatitis, non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), alcoholic fatty liver (AFL), alcoholic hepatitis, alcoholic steatohepatitis (ASH), primary biliary cirrhosis (PBC), schistosomal liver disease and hepatocellular carcinoma (HCC);

[0430] Diseases / conditions affecting the cardiovascular system such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, interstitial fibrosis, replacement fibrosis Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrotic vascular disease, hypertension, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), atherosclerosis, arterial stiffness, chronic pulmonary hypertension, AIDS-associated pulmonary hypertension, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), varicose veins and cerebral infarcts;

[0431] Diseases / conditions affecting the kidneys such as tubulointerstitial fibrosis, glomerular fibrosis, renal fibrosis, nephritic syndrome, Alport's syndrome, HIV-associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic glomerulonephritis and nephritis associated with systemic lupus;

[0432] Diseases / conditions affecting the pancreas such as pancreatic fibrosis, cystic fibrosis and pancreatitis (e.g. chronic pancreatitis, acute pancreatitis);

[0433] Diseases / conditions affecting the nervous system such as gliosis, Alzheimer's disease and multiple sclerosis;

[0434] Diseases / conditions affecting the musculoskeletal system such as muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker’s muscular dystrophy (BMD) and fibrotic myopathy;

[0435] Diseases / conditions affecting the gastrointestinal system such as inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, microscopic colitis and primary sclerosing cholangitis (PSC);

[0436] Diseases / conditions affecting the skin such as scleroderma, nephrogenic systemic fibrosis, Dupuytren’s contracture, cutis keloid and diabetic wounds;

[0437] Diseases / conditions affecting the eye such as macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), diabetic macular edema (DME), diabetic retinopathy, proliferative diabetic retinopathy (PDR), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis and preretinal fibrosis;

[0438] Diseases / conditions affecting the joints such as arthrofibrosis, arthritis (e.g. rheumatoid arthritis) and adhesive capsulitis;

[0439] Diseases / conditions affecting multiple tissues / organ systems, including progressive systemic sclerosis (PSS), chronic graft versus host disease (GVHD); fibrotic pre-neoplastic and fibrotic neoplastic disease, and fibrosis induced by chemical or environmental insult (e.g., cancer chemotherapy, pesticides, radiation / cancer radiotherapy);

[0440] Cancers, such as liver cancer (e.g. hepatocellular carcinoma), skin cancer (e.g. melanoma), lung cancer, breast cancer, gastric cancer, oesophageal cancer, head and neck cancer, colorectal cancer, pancreatic cancer, cervical cancer, and vulvar cancer;

[0441] Mediastinal fibrosis, retroperitoneal fibrosis, myelofibrosis and Peyronie’s disease.

[0442] It will be appreciated that many of the diseases / conditions listed above are interrelated.

[0443] In some embodiments, the fibrosis is fibrosis of the eye ( / .e. ocular fibrosis). In some embodiments, the fibrosis is selected from: subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis and preretinal fibrosis.

[0444] In some embodiments, the fibrosis is associated with one or more of: macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), diabetic macular edema (DME), diabetic retinopathy, proliferative diabetic retinopathy (PDR), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis and preretinal fibrosis.

[0445] As used herein “early AMD” refers to a stage of AMD characterised by the presence of medium-sized drusen, commonly having a width of up to -200 pm, within the Bruch’s membrane adjacent to the RPE layer. Subjects with early AMD typically do not present significant vision loss. As used herein “intermediate AMD” refers to a stage of AMD characterised by large drusen and / or pigment changes in the retina. Intermediate AMD may be accompanied by some vision loss. As used herein “late AMD” refers to a stage of AMD characterised by the presence of drusen and vision loss due to damage to the macula. In all stages of AMD, ‘reticular pseudodrusen’ (RPD) or ‘reticular drusen’ may be present, referring to the accumulation of extracellular material in the subretinal space between the neurosensory retina and RPE. “Late AMD” encompasses ‘dry’ and ‘wet’ AMD. In ‘dry’ AMD (also known as geographic atrophy), there is a gradual breakdown of the light-sensitive cells in the macula that convey visual information to the brain and of the supporting tissue beneath the macula. In ‘wet’ AMD (also known as choroidal neovascularization AMD (nAMD) and exudative AMD), abnormal blood vessels grow underneath and into the retina. These vessels can leak fluid and blood which can lead to swelling and damage of the macula and subsequent fibrosis / scar formation. The damage may be rapid and severe.

[0446] Fibrosis of the eye and fibrotic diseases of the eye are reviewed e.g. in Mallone et al., Int J Mol Sci. (2021) 22:11748 and Friedlander, J Clin Invest. (2007) 117(3):576-586 which are hereby incorporated by reference in their entirety. Neovascular AMD (nAMD) is reviewed e.g. in Pugazhendhi et al., Int J Mol Sci. (2021) 22(3):1170, which is hereby incorporated by reference in its entirety.

[0447] In some embodiments, the fibrosis is fibrosis of the pancreas ( / .e. pancreatic fibrosis). In some embodiments, the fibrosis is selected from: interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis and diffuse intralobular fibrosis. In some embodiments, the pancreatic fibrosis is associated with pancreatitis (e.g. chronic pancreatitis, acute fibrosis), cystic fibrosis or pancreatic cancer (e.g. pancreatic ductal adenocarcinoma). Fibrosis of the pancreas and fibrotic diseases of the pancreas are reviewed e.g. in Huang et al., Int J Mol Sci. (2021) 22(9): 4970 and Kloppel et al., Virchows Archiv (2004) 445:1-8, which are hereby incorporated by reference in their entirety.

[0448] The methods of the present invention may be effective to reduce the development or progression of fibrosis. The methods may be effective to prevent progression of fibrosis, e.g. to prevent worsening of, or to slow the rate of development of, fibrosis. In some embodiments, the methods may lead to an improvement in the disease / condition, e.g. a reduction fibrosis or reduction in some correlate of the severity of fibrosis. In some embodiments, the methods may prevent development of a later stage of fibrosis.

[0449] It will be appreciated that diseases / conditions characterised by fibrosis may also exhibit further symptoms / pathologies. For example, a disease / condition characterised by fibrosis may further be characterised by inflammation and / or pathological angiogenesis. In some embodiments, the methods may reduce the development or progression of one or more of: fibrosis, inflammation and pathological angiogenesis. In some embodiments, the methods may be effective to prevent the progression (e.g. to prevent worsening, or the slow the rate of development) of one or more of: fibrosis, inflammation and pathological angiogenesis. In some embodiments, the methods may lead to an improvement in the disease / condition, e.g. a reduction in one or more of: fibrosis, inflammation and pathological angiogenesis; or a reduction in some correlate of the severity of one or more of: fibrosis, inflammation and pathological angiogenesis. In some embodiments, the methods may prevent development of a later stage of one or more of: fibrosis, inflammation and pathological angiogenesis. For example, the methods may reduce / prevent the development / progression of fibrosis and inflammation. The methods may reduce / prevent the development / progression of fibrosis and pathological angiogenesis. The methods may reduce / prevent the development / progression of inflammation and pathological angiogenesis. The methods may reduce / prevent the development / progression of fibrosis, inflammation and pathological angiogenesis.

[0450] 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. 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.

[0451] However, many diseases are associated with an overactive inflammatory response ( / .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 ( / .e. which positively contributes to) the pathology of a disease.

[0452] Inflammation to be treated / prevented in accordance with the present disclosure can be of any tissue / organ of the body. In some embodiments, the inflammation is of the lung (e.g. bronchioles, alveoli), airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi), heart, kidney, liver, skeletal muscle, blood vessels, eye, skin, pancreas, bowel, small intestine, large intestine, colon, joints, brain, or bone marrow. Inflammation may also occur in multiple tissues / organs at once.

[0453] In some embodiments, inflammation may be of an organ or tissue of the respiratory system, e.g. the lung (e.g. bronchioles, alveoli), or airways (e.g. nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, inflammation may be of an organ or tissue of the cardiovascular system, e.g. the heart or blood vessels. In some embodiments, inflammation may be of an organ or tissue of the gastrointestinal system, e.g. of the liver, bowel, small intestine, large intestine, colon, or pancreas. In some embodiments, inflammation may be of the eye. In some embodiments, inflammation may be of the skin. In some embodiments, inflammation may be of an organ or tissue of the nervous system, e.g. the brain. In some embodiments, inflammation may be of the bone marrow. In some embodiments, inflammation may be of the joints. In some embodiments, inflammation may be of an organ or tissue of the urinary system, e.g. the kidneys. In some embodiments, inflammation may be of an organ or tissue of the musculoskeletal system, e.g. muscle tissue. In some embodiments, inflammation may be of the eye. In some embodiments, inflammation may be of an organ or tissue of one or more organ systems. As used herein, a disease / condition which is ‘characterised by inflammation’ is a disease / condition in which inflammation is a symptom of the disease / condition. Diseases / conditions characterised by inflammation include, but are not limited to:

[0454] Diseases / conditions affecting the respiratory system, such as sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis and mediastinitis;

[0455] Diseases / conditions affecting the accessory digestive organs such as hepatitis, ascending cholangitis, cholecystitis, pancreatitis (e.g. acute pancreatitis) and peritonitis;

[0456] Diseases / conditions affecting the cardiovascular system such as carditis, endocarditis, myocarditis, cardiogenic shock, pericarditis, vasculitis, arteritis, phlebitis and capillaritis; Diseases / conditions affecting the urinary system such as nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis and urethritis;

[0457] Diseases / conditions affecting the nervous system such as encephalitis, myelitis, meningitis, arachnoiditis and neuritis;

[0458] Diseases / conditions affecting the musculoskeletal system such as arthritis (e.g. rheumatoid arthritis), dermatomyositis, soft tissue, myositis, synovitis / tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendinitis, panniculitis, osteochondritis: osteitis / osteomyelitis, spondylitis, periostitis and chondritis;

[0459] Diseases / conditions affecting the oral cavity and throat such as stomatitis, gingivitis, gingivostomatitis, periodontitis, glossitis, tonsillitis, sialadenitis, parotitis, cheilitis, pulpitis and gnathitis;

[0460] Diseases / conditions affecting the gastrointestinal system such as esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, caecitis, appendicitis, proctitis and Peutz- Jeghers syndrome;

[0461] Diseases / conditions affecting the skin such as dermatitis, folliculitis, cellulitis and hidradenitis;

[0462] Diseases / conditions affecting the eye such as dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis and uveitis;

[0463] Diseases / conditions affecting the ear such as otitis externa, otitis media, labyrinthitis and mastoiditis;

[0464] Diseases / conditions of the reproductive system such as oophoritis, salpingitis, endometritis, endometriosis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, posthitis, balanoposthitis, chorioamnionitis, funisitis and omphalitis;

[0465] Diseases / conditions of the endocrine system such as insulitis, hypophysitis, thyroiditis, parathyroiditis and adrenalitis;

[0466] Diseases / conditions of the lymphatic system such as lymphangitis and lymphadenitis;

[0467] Cancers, including inflammation-induced and inflammation-associated cancers, such as lung cancer (e.g. lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer), breast cancer, prostate cancer, hematological malignancies (e.g. multiple myeloma), pancreatic cancer (e.g. pancreatic ductal adenocarcinoma), cervical cancer, stomach cancer, oesophageal cancer, head and neck cancer, colorectal cancer, colon cancer, liver cancer (e.g. hepatocellular carcinoma), skin cancer (e.g. melanoma) and bile duct cancer. In some embodiments, the inflammation may be acute inflammation. In some embodiments, the inflammation may be chronic inflammation.

[0468] Inflammation can promote angiogenesis ( / .e. the growth and development of new blood vessels from existing vasculature), through multiple different pathways as described e.g. in Granger and Senchenkova, ‘Chapter 6: Angiogenesis’, in ‘Inflammation and the Microcirculation’, Morgan & Claypool Life Sciences; 2010. For example, inflammation can lead to hypoxic conditions in inflamed tissue, which in turn upregulates the expression of the potent angiogenic factor vascular endothelial growth factor (VEGF) which induces the growth of new blood vessels. Inflammatory cells such as macrophages, lymphocytes, mast cells and also fibroblasts produce angiogenic factors such as VEGF and FGF. Increased blood flow to inflamed tissue can stimulate angiogenesis through shear stresses on the endothelium of existing vessels, and extravasated plasma proteins, such as fibrinogen products, may also stimulate neovascularisation. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by angiogenesis, (e.g. pathological angiogenesis). In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by inflammation-induced angiogenesis.

[0469] As used herein, ‘pathological angiogenesis’ refers to angiogenesis ( / .e. the growth of new blood vessels from an existing vascular plexus), wherein the angiogenesis contributes to the development and / or progression of a disease.

[0470] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease characterised by pathological angiogenesis.

[0471] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is characterised by one or more of fibrosis, inflammation and pathological angiogenesis. In some embodiments, the disease / condition to be treated / prevented is characterised by fibrosis and inflammation. In some embodiments, the disease / condition to be treated / prevented is characterised by fibrosis and pathological angiogenesis. In some embodiments, the disease / condition to be treated / prevented is characterised by inflammation and pathological angiogenesis. In some embodiments, the disease / condition to be treated / prevented is characterised by fibrosis, inflammation, and pathological angiogenesis.

[0472] Fibrosis, inflammation and pathogenic angiogenesis are hallmarks of AMD. This is reviewed in e.g. Helotera and Kaarniranta, Cells (2022) 11 (21):3453, which is hereby incorporated by reference in its entirety.

[0473] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease characterised by tumorigenesis. As used herein, “tumorigenesis” refers to the formation or development of a tumour. Tumorigenesis may involve the transformation of a normal cell(s) into cancerous cell(s). In some embodiments, the disease characterised by tumorigenesis is a cancer. In some embodiments, the disease or condition to be treated is a cancer. The cancer may be any cancer as described hereinabove.

[0474] As used herein, a ‘cancer’ may be or comprise any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant. The cancer may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, biliary tract, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.

[0475] Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.

[0476] In some embodiments the cancer is a cancer in which LRG1 is pathologically-implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of LRG1 , a cancer for which expression of LRG1 is a risk factor and / or a cancer for which expression of LRG1 is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by expression of LRG1 , e.g. the cancer may comprise cells (e.g. cells of tumor tissue) expressing LRG1 . Such cancers may be referred to as being positive for LRG1 . A cancer which is ‘positive’ for LRG1 may be a cancer comprising cells expressing LRG1 . A cancer which is ‘positive’ for LRG1 may overexpress LRG1.

[0477] 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 LRG1 , for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA. In some embodiments, the cancer to be treated is selected from: a cancer comprising cells expressing / overexpressing LRG1 , a solid tumor, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, liver cancer, hepatocellular carcinoma, skin cancer, melanoma, lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, breast cancer, breast carcinoma, ductal carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, cervical cancer, cervical squamous cell carcinoma, esophageal cancer, esophageal adenocarcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

[0478] In some embodiments, the cancer to be treated is selected from: pancreatic cancer, liver cancer, skin cancer (e.g. melanoma), lung cancer, breast cancer, and colorectal cancer. In some embodiments, the cancer to be treated is selected from: pancreatic cancer, liver cancer, skin cancer (e.g. melanoma) and lung cancer. In some embodiments, the cancer to be treated is selected from: pancreatic cancer, skin cancer (e.g. melanoma), colorectal cancer, and lung cancer. In some embodiments, the cancer to be treated is pancreatic cancer (e.g. pancreatic ductal adenocarcinoma). In some embodiments, the cancer to be treated is skin cancer (e.g. melanoma). In some embodiments, the cancer to be treated colorectal cancer. In some embodiments, the cancer to be treated is lung cancer (e.g. non-small cell lung cancer). The treatment / prevention may be aimed at one or more of: delaying / preventing the onset / progression of symptoms of the cancer, reducing the severity of symptoms of the cancer, reducing the survival / growth / invasion / metastasis of cells of the cancer, reducing the number of cells of the cancer and / or increasing survival of the subject. In some embodiments, treatment with an LRG1 inhibitor (e.g. an LRG1 inhibitor as described herein) achieves one or more of the following: reducing the survival / growth / proliferation of cells of the cancer, reducing migration / invasion of cells of the cancer, and reducing epithelial to mesenchymal transition (EMT) of cells of the cancer.

[0479] In some embodiments, the disease or condition to be treated is pancreatitis, e.g. acute pancreatitis.

[0480] Acute pancreatitis is a common clinical condition, the incidence of which has been increasing over recent years. Mild acute pancreatitis may be self-limiting and not requiring any treatment, but up to 25% of patients suffer a severe attack and between 30 and 50% of these will die. Most cases are secondary to biliary disease or excess alcohol consumption. Acute pancreatitis is an inflammatory disorder, which develops a complex cascade of immunological events, which not only affect the pathogenesis but also the course of the disease. It will be appreciated that acute pancreatitis is a fibroinflammatory disease according to the present disclosure. Acute pancreatitis and the role of inflammation in the disease is reviewed e.g. in Bhatia et al., (2005) 5:132-144, which is hereby incorporated by reference in its entirety.

[0481] The treatment may be aimed at one or more of: reducing the severity of the symptoms of acute pancreatitis, reducing inflammation in acute pancreatitis, and increasing pancreatic regeneration in acute pancreatitis.

[0482] In some embodiments, the disease or condition to be treated is arthritis, e.g. rheumatoid arthritis.

[0483] Rheumatoid arthritis (RA) is a chronic autoimmune and inflammatory condition often affecting symmetrical joints of the body. As the disease progresses, symptoms can spread from smaller joints to weight-bearing joints, such as ankles, knees, and hips. Over time, the joints may deform and eventually lose function. Besides the articular presentation, around 40% of RA patients also experience systemic manifestations affecting all aspects of patients’ organ systems, and these complications are often more fatal to people with RA than those without. The primary goal of RA therapy is to minimize disease activity and control joint damage. However, the disease symptoms persists in a substantial number of patients despite active treatment and a variety of side effects have been reported for existing antirheumatic drugs. Given the limitations of current treatment for RA, there is a need for more effective treatment for RA.

[0484] It will be appreciated that rheumatoid arthritis is a fibroinflammatory disease according to the present disclosure. Rheumatoid arthritis and the role of inflammation in rheumatoid arthritis are reviewed in e.g. Shrivastava & Pandey, Journal of Physiology and Biochemistry (2012) 69:335-347, which is hereby incorporated by reference in its entirety.

[0485] The treatment / prevention may be aimed at delaying / preventing the onset / progression of symptoms of rheumatoid arthritis, reducing the severity of symptoms of rheumatoid arthritis, and / or reducing inflammation in rheumatoid arthritis.

[0486] In some embodiments, the disease or condition to be treated is inflammatory bowel disease (e.g. Crohn’s disease (CD), ulcerative colitis (UC)), In some embodiments, the disease or condition to be treated is Crohn’s disease. In some embodiments, the disease or condition to be treated is ulcerative colitis.

[0487] The treatment / prevention may be aimed at delaying / preventing the onset / progression of symptoms of inflammatory bowel disease, reducing the severity of symptoms of inflammatory bowel disease, and / or reducing inflammation in inflammatory bowel disease.

[0488] In some embodiments, the disease or condition to be treated is associated with diabetes. In some embodiments, the disease or condition to be treated is associated with diabetic vascular complications. Diseases / conditions associated with diabetes include diabetic retinopathy, diabetic macular edema diabetic nephropathy and diabetic wound(s). In some embodiments, the disease or condition to be treated is selected from: diabetic retinopathy, diabetic macular edema, diabetic nephropathy and diabetic wound(s). In some embodiments, the disease or condition to be treated is selected from: diabetic nephropathy and diabetic wound(s). In some embodiments, the disease or condition to be treated is diabetic nephropathy. In some embodiments, the disease or condition to be treated is a diabetic wound. In some embodiments, the disease or condition to be treated is diabetic retinopathy. In some embodiments, the disease or condition to be treated is a diabetic macular edema.

[0489] Diabetic nephropathy (DN) is a common renal disease associated with long-term diabetes mellitus. Over a lifetime, diabetic nephropathy occurs in approximately 30-35% of patients with type 1 and type 2 diabetes. It is one of the leading causes of end-stage renal disease (ESRD) globally. Chronic high blood sugar levels lead to structural and functional changes in the kidneys. This includes thickening of the glomerular basement membrane, mesangial expansion, and podocyte injury. Its early stage is often asymptomatic, with microalbuminuria as the first detectable sign. With the disease progression, persistent proteinuria (macroalbuminuria), hypertension, and declining glomerular filtration rate (GFR) start to show up. Significant reduction in GFR indicates the advanced stage of DR, which leads to chronic kidney disease (CKD) and eventually ESRD, requiring dialysis or kidney transplantation. With the exception of kidney dialysis and renal replacement therapy, few therapeutic strategies have been found to be effective in treating diabetes nephropathy. Early detection allows time for the intensive treatment of glycaemic control, blood pressure, and other cardiovascular risk factors.

[0490] Diabetic wounds are a serious complication of diabetes, primarily resulting from a combination of poor blood circulation, nerve damage (neuropathy), and elevated blood sugar levels that impair the body's ability to heal. These wounds most commonly manifest as diabetic foot ulcers (DFUs), which affect up to 25% of people with diabetes during their lifetime. Diabetic foot ulcers typically appear as open sores on the feet, especially in pressure-bearing areas like the heel or ball of the foot, and are often painless due to nerve damage, making early detection challenging. Other types of diabetic wounds include venous stasis ulcers, arterial (ischemic) ulcers, pressure injuries, and traumatic wounds — all of which are slow to heal and prone to infection.

[0491] Complications from diabetes also occur in the eye. Diabetic retinopathy (DR) is classically characterized by gradually progressing changes that occur in the microvasculature. These changes include alterations in the retinal permeability, macular edema, retinal ischemia and neovascularization. Diabetic macular edema (DME) is a major cause of vision impairment in diabetic individuals, characterized by fluid accumulation in the macula due to a breakdown of the blood-retinal barrier (BRB).

[0492] The treatment / prevention may be aimed at delaying / preventing the onset / progression of symptoms of a disease / condition associated with diabetes, reducing the severity of symptoms of a disease / condition associated with diabetes, reducing inflammation in a disease / condition associated with diabetes, and / or reducing fibrosis in a disease / condition associated with diabetes. The treatment / prevention may be aimed at delaying / preventing the onset / progression of diabetic nephropathy, reducing the severity of symptoms of diabetic nephropathy, reducing inflammation in diabetic nephropathy, and / or reducing fibrosis in diabetic nephropathy. The treatment / prevention may be aimed at delaying / preventing the onset / progression of a diabetic wound, reducing the severity of symptoms of a diabetic wound, reducing inflammation in a diabetic wound, and / or reducing fibrosis in a diabetic wound.

[0493] In some embodiments, the disease or condition is characterised by:

[0494] (i) angiogenesis, fibrosis and / or inflammation of the eye;

[0495] (ii) fibrosis and / or inflammation of the pancreas;

[0496] (iii) vascular abnormality, fibrosis and / or inflammation of the joints;

[0497] (iv) vascular abnormality, fibrosis and / or inflammation of the skin;

[0498] (v) fibrosis and / or inflammation of the bowel;

[0499] (vi) vascular abnormality, fibrosis and / or inflammation of the kidney;

[0500] (vii), vascular abnormality, tumorigenesis, fibrosis and / or inflammation of a cancer (e.g. melanoma, pancreatic cancer, colorectal cancer, and lung cancer); or

[0501] (viii) fibrosis and / or inflammation of the lungs.

[0502] As used herein, “vascular abnormality” refers to an irregularity or disorder in the structure or function of blood vessels. For example, vascular abnormality / vascular abnormalities can be found in tumours due to the abnormal growth of blood vessels.

[0503] Administration / delivery of LRG1 inhibitors

[0504] The present disclosure also provides compositions comprising an LRG1 inhibitor as described herein.

[0505] The LRG1 inhibitor described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0506] 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).

[0507] 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.

[0508] 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.

[0509] 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.

[0510] In some embodiments, the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or tumor.

[0511] In some embodiments, an LRG1 inhibitor results in modification of a cell or cells to comprise an LRG1 inhibitor as described herein.

[0512] LRG1 inhibitors may be formulated to facilitate delivery to and / or uptake by a cell / tissue. LRG1 inhibitors may be linked to a moiety in order to facilitate delivery to and / or uptake by a cell / tissue. Strategies for facilitating intracellular delivery of molecular cargo are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.

[0513] In some embodiments an LRG1 inhibitor is formulated with a cationic polymer. In some embodiments an LRG1 inhibitor is encapsulated in a nanoparticle or a liposome.

[0514] In some embodiments, a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a PLGA, polypeptide, poly(p-amino ester), DOPE, p-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle.

[0515] In some embodiments an LRG1 inhibitor is (covalently or non-covalently) associated with a cellpenetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier. In some embodiments an LRG1 complex inhibitor is associated with a peptide / polypeptide (e.g. antibody, peptide aptamer, ligand for a cell surface molecule / fragment thereof) or a nucleic acid (e.g. nucleic acid aptamer) capable of binding to a target cell of interest or an antigen thereof.

[0516] In some embodiments, an LRG1 complex inhibitor is administered in the form of nucleic acid encoding the LRG1 inhibitor. For example, the LRG1 inhibitor may be administered in the form of nucleic acid encoding a peptide / polypeptide or nucleic acid LRG1 inhibitor, or an SSN system targeting LRG1 .

[0517] In some embodiments, an LRG1 inhibitor is administered in the form of nucleic acid encoding the factors required for production of an LRG1 inhibitor (e.g. nucleic acid encoding a precursor of the LRG1 inhibitor and / or nucleic acid encoding factors required for production / assembly of the LRG1 inhibitor). For example, the LRG1 inhibitor may be administered in the form of nucleic acid encoding factors required for production of a small molecule or biomolecular LRG1 inhibitor.

[0518] The nucleic acid may be, or may be comprised in, a vector. A “vector” as used herein is a nucleic acid 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 target cell. Such vectors may include a promoter sequence operably linked to the nucleic acid sequence to be expressed. A vector may also include a termination codon and expression enhancers. In this specification 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 the nucleotide 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. Where appropriate, the resulting transcript may then be translated into a desired polypeptide.

[0519] Any suitable vectors, promoters, enhancers and termination codons known in the art may be used.

[0520] Suitable vectors include viral vectors, e.g. retroviral vectors, 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.

[0521] In some embodiments, a vector is selected based on tropism for a cell type / tissue / organ to which it is desired to deliver the nucleic acid, e.g. a cell type / tissue / organ affected by the disease to be treated / prevented in accordance with the invention ( / .e. cells / tissue / an organ in which the symptoms of the disease manifest).

[0522] For example, in some embodiments it is desired to deliver nucleic acid encoding an LRG1 inhibitor to muscle cells / tissue (e.g. eye and / or pancreatic cells / tissue), and vectors having a tropism for such cells / tissue may be employed in such embodiments. In some embodiments, a vector may be an adeno-associated viral vector. In some embodiments, a vector may be an adeno-associated viral vector of one of AAV serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 , or hybrids and / or mutants thereof.

[0523] In some embodiments a vector comprises modification to increase binding to and / or transduction of a cell-type of interest ( / .e. as compared to the level of binding / transduction by the unmodified vector). In some embodiments modification is to a capsid protein.

[0524] In some embodiments the nucleic acid / vector comprises one or more sequences for controlling expression of the nucleic acid. Accordingly, in some embodiments the nucleic acid / vector comprises a control element for inducible expression of the nucleic acid. A sequence for controlling expression of the nucleic acid may provide for expression of the nucleic acid by cells of a particular type or tissue. For example, expression may be under the control of a cell type- or tissue-specific promoter.

[0525] Promoters for cell type- or tissue-specific expression of a nucleic acid in accordance with the present invention can be selected in accordance with the disease to be treated / prevented. For example, the promoter may drive expression in cells / tissue / an organ affected by the disease ( / .e. cells / tissue / an organ in which the symptoms of the disease manifest). In some embodiments, a promoter may provide for expression in eye cells / tissue (e.g. retinal epithelial cells). In some embodiments, a promoter may provide for expression in pancreatic cells / tissue.

[0526] Multiple doses of the LRG1 inhibitor may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent. The accompanying therapeutic agent may be any agent suitable for the treatment of the disease to be treated. In some embodiments, the accompanying therapeutic agent is an antiangiogenic therapy, e.g. a VEGF inhibitor, e.g. aflibercept (Eylea).

[0527] 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).

[0528] 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 LRG1 inhibitor described herein; isolating an LRG1 inhibitor described herein; and / or mixing an LRG1 inhibitor described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0529] 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 LRG1 inhibitor described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[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).

[0532] 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.

[0533] 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 for one or more markers of such a disease / condition.

[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 LRG1 , or of cells / tissue overexpressing LRG1 , e.g. in a sample obtained from the subject.

[0535] Kits

[0536] 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 LRG1 inhibitor or composition described herein.

[0537] In some embodiments, the kit may comprise materials for producing an LRG1 inhibitor or composition described herein.

[0538] The kit may provide the LRG1 inhibitor or composition together with instructions for administration to a patient in order to treat a specified disease / condition.

[0539] 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. 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.

[0540] Numbered statements

[0541] The following numbered paragraphs (paras) describe particular aspects and embodiments of the present invention:

[0542] 1 . An LRG1 inhibitor for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

[0543] 2. Use of an LRG1 inhibitor in the manufacture of a medicament for use in the treatment or prevention of a disease or condition characterised by fibrosis and / or inflammation.

[0544] 3. A method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically- or prophylactically-effective amount of an LRG1 inhibitor to a subject.

[0545] 4. The LRG1 inhibitor for use according to para 1 , the use according to para 2, or the method according to para 3, wherein the LRG1 inhibitor: (i) inhibits interaction between LRG1 and an interaction partner for LRG1 ; or (ii) reduces the expression of LRG1 or an interaction partner of LRG1 .

[0546] 5. The LRG1 inhibitor for use, the use, or the method according to para 4, wherein the LRG1 inhibitor binds to LRG1 , optionally wherein the LRG1 inhibitor binds to the LRRCT region of LRG1 .

[0547] 6. The LRG1 inhibitor for use, the use, or the method according to para 5, wherein the LRG1 inhibitor is a peptide / polypeptide, nucleic acid or small molecule.

[0548] 7. The LRG1 inhibitor for use, the use, or the method according to para 6, wherein the LRG1 inhibitor is an antibody or antigen-binding fragment thereof.

[0549] 8. The LRG1 inhibitor for use, the use, or the method according to para 4, wherein the LRG1 inhibitor is an inhibitory nucleic acid capable of reducing expression of LRG1 by RNA interference (RNAi).

[0550] 9. The LRG1 inhibitor for use, the use, or the method according to para 4, wherein the LRG1 inhibitor is capable of modifying a gene encoding LRG1 to reduce its expression.

[0551] 10. The LRG1 inhibitor for use, the use, or the method according to para 9, wherein the LRG1 inhibitor comprises a site-specific nuclease (SSN) targeting a gene encoding LRG1 . 11 . The LRG1 inhibitor for use according to any one of paras 1 or 4 to 10, the use according to any one of paras 2 or 4 to 10, or the method according to any one of paras 3 or 4 to 10, wherein the disease or condition is characterised by: (i) fibrosis and / or inflammation of the eye; (ii) fibrosis and / or inflammation of the pancreas; or (iii) fibrosis and / or inflammation of the joints.

[0552] 12. The LRG1 inhibitor for use according to any one of paras 1 or 4 to 11 , the use according to any one of paras 2 or 4 to 11 , or the method according to any one of paras 3 or 4 to 11 , wherein the disease or condition is characterised by pathological angiogenesis.

[0553] 13. The LRG1 inhibitor for use according to any one of paras 1 or 4 to 12, the use according to any one of paras 2 or 4 to 12, or the method according to any one of paras 3 or 4 to 12, wherein the disease or condition is selected from: macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, diffuse intralobular fibrosis, pancreatitis, cystic fibrosis, pancreatic cancer, or rheumatoid arthritis.

[0554] 14. The LRG1 inhibitor for use according to para 13, the use according to para 13, or the method according to para 13, wherein the disease or condition is selected from: subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis, preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, and diffuse intralobular fibrosis.

[0555] 15. The LRG1 inhibitor for use according to para 14, the use according to para 14, or the method according to para 14, wherein the disease or condition is subretinal fibrosis.

[0556] 16. The LRG1 inhibitor for use according to any one of paras 1 or 4 to 12, the use according to any one of paras 2 or 4 to 12, or the method according to any one of paras 3 or 4 to 12, wherein the disease or condition is a cancer.

[0557] 17. The LRG1 inhibitor for use according to para 14, the use according to para 14, or the method according to para 14, wherein the cancer is selected from: a cancer comprising cells expressing / overexpressing LRG1 , a solid tumor, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, liver cancer, hepatocellular carcinoma, skin cancer, melanoma, lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, breast cancer, breast carcinoma, ductal carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal

[0558] 5 cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, cervical cancer, cervical squamous cell carcinoma, esophageal cancer, esophageal adenocarcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma. 0

[0559] Sequence identity

[0560] 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 5 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. 0 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. 5 Sequences

[0561]

[0562]

[0563] 72

[0564] able B

[0565] Table C

[0566] Table D

[0567] ***

[0568] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0569] 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.

[0570] 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.

[0571] 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 >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to the amino acid sequence of 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).

[0572] 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.

[0573] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0574] 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.

[0575] Brief Description of the Figures

[0576] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures. Figure 1. Graph showing serum LRG1 levels are elevated in nAMD patients with baseline fibrosis and incident fibrosis. Luminex analysis of serum LRG1 levels in nAMD patients without subretinal fibrosis at the baseline and in 12 months (NF), without subretinal fibrosis at the baseline but developing subretinal fibrosis in 12 months (IF), with subretinal fibrosis at the baseline (BF). Data are expressed as mean ± S.E.M of n > 45. Statistical significance was determined by one-way ANOVA. ***P<0.001 , **P<0.01 .

[0577] Figure 2. Graph showing LRG1 levels are induced in the RPE / choroid compartment of the eye of C57BL / 6 mice subjected to laser-induced subretinal fibrosis. qRT-PCR analysis of LRG1 mRNA levels in the RPE / choroid fraction of the eye of C57BL / 6 mice 4, 7, 14, 21 and 35 days following laser treatment. Data are expressed as mean ± S.E.M of n =3.

[0578] Figure 3. Images and graph showing Lrg1 deletion leads to reduced subretinal fibrosis in vivo. Representative images (left) and quantification (right) of total collagen I positive area in wild-type and Lrg1 knockout mice 35 days following the laser treatment. All images are representative. Data are expressed as mean ± S.E.M of n =6. Statistical significance was determined by the student’s t-test. ***P, 0.001.

[0579] Figure 4. Images showing Lrg1 is required for corneal fibrosis. Representative H&E images of cross section of mouse eyes collected from wild-type and Lrg1 mice following the treatment with 1M NaOH.

[0580] Figure 5. LRRCT, but not LRR, promotes CTGF expression in HRPC. Representative Western Blot shows an increase in basal and TGFp-induced CTGF expression in LRRCT treated-HRPCs

[0581] Figures 6A to 6F. Graphs and images showing that LRG1 promotes human RPE cells activation in vitro. (6A) MTS assay demonstrating increased cell survival when RPE is subjected to continuous rhLRGI treatment for 3 days. (6B) Representative IF staining and quantification of Ki67+ RPE following the treatment with rhLRGI . (6C) Representative IF staining of aSMA, Ki67 and DAPI of untreated and LRG1 -treated mouse foetal metatarsal explants. (6D) Representative image and quantification of scratch area as a marker of RPE migratory potential following rhLRGI treatment. (6E). Bar graph demonstrating the fold change in migrated RPE cells across the Transwell following the LRG1 or vehicle treatment. (6F) Representative images and quantification demonstrating the TGFp and TNFa-induced RPE cell contraction following the LRG1 or vehicle treatment. All images are representative. Data are expressed as average ± S.E.M of n=3. Statistical significance was determined by the student’s t-test. *P<0.05, **P<0.01.

[0582] Figures 7A to 7D. LRG1 promotes the expression of fibrotic molecules in human RPE cells cells. (7A) Representative Western Blot and densitometry quantification demonstrating a promoting effect of recombinant human LRG1 (rhLRGI) on the expression of ECM proteins and the proteins involved in RPE activation. (7B) Quantification of gene transcripts FN, CTGF, TGFB and Col1a1 suggesting increased expression of ECM markers in rhLRGI -treated cells. (7C) Representative IF staining and quantification of fibronectin accumulation in human RPE cultures following rhLRGI treatment. (7D) Representative immunoblots and quantification of pSmad2 / 3 following the treatment of rhLRGI (20 pg / ml) and SB431542 (10 pM). Data are expressed as average ± S.E.M of n=3. Statistical significance was determined by the student’s t-test. *P<0.05, **P<0.01 , ***P<0.001 .

[0583] Figure 8. Images showing Mouse species cross-reactivity of LRG1 monoclonal antibodies using mouse liver and retina extracts by immunoblots. Recombinant human LRG1 (rhLRGI) is used as positive control.

[0584] Figure 9. Graphs showing binding kinetics of chimerised EBC-58, EBC-59, EBC-60 and magacizumab (MGZ) against native LRG1 protein measured by BLI. Recombinant LRG1 protein was immobilised on ARG2 biosensors. Ligand-loaded sensors were incubated with different concentrations of analyte in BLI buffer (100, 50, 25, 12.5 nM of LRG1 antibodies). The concentration of each curve is orientated as top-down, with top concentration 100 nM presented at the top, followed by 2-fold serial dilutions concentrations down. Binding curves were fitted using a 1 :1 binding model, and fitted lines are shown in red.

[0585] Figure 10. Peptide mapping of chimeric EBC-58, EBC-59, EBC-60, EBC-61 and magacizumab (MGZ) antibodies by ELISA binding assay using biotinylated 15-mer overlapping peptides covering LRR6 region (peptide 1 - 3) and LRR8 and LRRCT regions (peptide 4 -19).

[0586] Figure 11. Graphs showing chimeric LRG1 blocking antibodies inhibit TGFp-induced fibrotic gene expression in RPE cells. RT-PCR analysis of fibrotic gene expression in RPE cells subjected to the treatment rhLRGI with or without the presence of LRG1 blocking antibodies EBC-59, EBC-60, and EBC- 61 (n=3 except for CTGF). Statistical significance was determined by One-Way ANOVA with *p < 0.05, **P< 0.01 , and *** P< 0.001.

[0587] Figure 12. Images and graph showing that chimeric LRG1-blocking antibodies inhibit TGFp-induced RPE cell migration. Representative images (left) and quantification (right) of RPE cell migration over Transwell following the treatment with TGFp with or without the presence of LRG1 blocking antibodies EBC-59, EBC-60 and EBC-61. Data are expressed as average ± S.E.M of n=6. Statistical significance was determined by one way ANOVA. *P<0.05, **P<0.01 , ***P<0.001 .

[0588] Figure 13. Images and graph showing chimeric LRG1-blocking antibodies inhibit TGFp-induced fibronectin expression in RPE cells. Representative immunofluorescence images (left) and quantification (right) of fibronectin expression in RPE cells following the treatment with TGFp with or without the presence of LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61 . Data are expressed as average ± S.E.M of n=3. Statistical significance was determined by one-way ANOVA. ***P<0.001 .

[0589] Figure 14. Images and graph showing chimeric LRG1-blocking antibodies inhibit LRG1-induced RPE cell proliferation. Representative immunofluorescence images (left) and quantification (right) of Ki67 positive RPE cells following the treatment with rhLRGI with or without the presence of LRG1 blocking antibodies EBC-59, EBC-60, and EB-C61. Data are expressed as average ± S.E.M of n=3. Statistical significance was determined by one way ANOVA. *P<0.05, **P<0.01 ,***P<0.001 .

[0590] Figure 15. Images and graph showing chimeric LRG1-blocking antibodies inhibit metatarsal angiogenesis and fibrosis. Representative immunofluorescence images (left) and quantification (right) of CD31 positive vessel outgrowth from metatarsal bones following the treatment with rhLRGI with or without the presence of LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61. Data are expressed as average ± S.E.M of n=6 (for CD31 staining) and n=3 (for CD31 and aSMA co-staining). Statistical significance was determined by one way ANOVA. *P<0.05, **P<0.01 ,***P<0.001 .

[0591] Figures 16A and 16B. Images and graphs showing chimeric LRG1-blocking antibodies inhibit laser- induced CNV and subretinal fibrosis. (16A) Representative images (left) and quantification (right) of fundus fluorescein angiography following the treatment with LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61. (16B) Representative immunofluorescence images (left) of aSMA and Collagen I stained RPE flatmount following the treatment with LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61. Quantification of total Collagen 1 positive subretinal fibrosis area (right). Data are expressed as average ± S.E.M of n=6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 , ***P<0.001.

[0592] Figure 17. Graphs showing binding kinetics of EBC-59 (parental chimeric mAb) and its humanised variants (EBC-1191 , EBC-1192, EBC-1196, EBC-1199, EBC-1200, EBC-1212) to human LRG1 protein as determined by BLI. Recombinant LRG1 protein was immobilised on ARG2 biosensors. Ligand-loaded sensors were incubated with different concentrations of analyte in BLI buffer (100, 50, 25, 12.5 nM of LRG1 antibodies). The concentration of each set of curves is orientated as top-down, with top concentration 100 nM presented at the top, followed by 2-fold serial dilutions concentrations down. Binding curves were fitted using a 1 :1 binding model, and fitted lines are shown in red.

[0593] Figure 18A and 18B: Images and graphs showing the inhibitory effect of humanised variants of EBC-59 (EBC-1191 , EBC-1192, EBC-1195, EBC-1196, EBC-1199, EBC-1200, and EBC-1212) on RPE cell proliferation. Representative immunofluorescence images (18A) and quantification (18B) of Ki67+ (proliferating RPE cells) to nuclei (total RPE cell count). Data are expressed as average ± S.E.M of n=3. Statistical significance was determined by one-way ANOVA. L: recombinant LRG1 ; n.s: not significant; *P<0.05, ****P<0.0001

[0594] Figure 19. Humanized LRG1 antibody inhibits laser-induced choroidal neovascularization (CNV) in mice in a dose-dependent manner. Representative FFA and IF images (top) and quantification (bottom) demonstrating a dose-dependent inhibition of laser-induced CNV in mice. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 , ****p<0.0001 Figure 20. Representative FFA images demonstrating an immediate and prolonged anti-angiogenic effect of humanised LRG1 antibody EBC-1212 either on its own or in combination with the standard of care Eylea, in comparison to Eylea mono-therapy in Kimba mice.

[0595] Figures 21 A to 21 B. LRG1 blocking antibodies demonstrated a comparative anti-angiogenic efficacy and an additional anti-fibrotic property to the standard of care VEGF inhibitor, Avastin. (21 A) HREC Matrigel tube formation assay demonstrated potent inhibitory efficacy of parental (EBC-59) and humanized LRG1 blocking antibody (EBC-1212), which is comparable to the standard of care Avastin. (21 B) Scratch assay showed that EBC-59 and EBC-1212 but not Avastin inhibit the migration of RPE cells. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 .

[0596] Figure 22. Representative IF images (top) and quantification (bottom) of Collagen I staining and psOCT demonstrating a dose-dependent inhibition of laser-induced subretinal fibrosis in mice by humanized LRGIantibody EBC-1212. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 .

[0597] Figure 23. Representative IF images (top) and quantification (bottom) of Fn, aSMA and IB4 I staining demonstrating a comparable anti-angiogenic efficacy of EBC-59 and EBC-1212 to a comparator LRG1 blocking antibody magacizumab (EBC-78) and the standard of care. However, EBC-59 and EBC- 1212 offer an additional antifibrotic efficacy. EBC-146 serves as a negative IgG control. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 .

[0598] Figure 24. Representative IF images (top) and quantification (bottom) of Fn, aSMA and IB4 I staining demonstrating an additive anti-angiogenic efficacy and an additional anti-fibrotic benefit of EBC- 1212 to the standard of care Eylea as a combination treatment with Eylea. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA, **P<0.01 , ***P<0.001 , ****P<0.0001 .

[0599] Figure 25. Representative IF images (top) and quantification (bottom) of Fn, aSMA and IB4 I staining demonstrating the ability of EBC-1212 to slow down the progression of the laser-induced subretinal fibrosis. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA, *P<0.05 **P<0.01 , ***P<0.001 , ****P<0.0001.

[0600] Figure 26. Immunofluorescence staining images of mouse pancreatic tissue showing colocalization of LRG1 and CD31. For the staining, paraffin (6pm) or cryosections (8pm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 , CD31 and AN2 followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies. Figures 27A to 27C. Graphs and images showing an increase LRG1 expression in human patients with acute pancreatitis. (27A) Concentration of LRG1 in serum of patients with acute pancreatitis and a healthy control. (27B) Correlation between serum LRG1 concentration and inflammation marker C- reactive protein. (27C) Immunofluorescence staining for LRG1 in CD31+ vascular cells, Amy+ (amylase) acinar cells and MPO+ (myeloperoxidase) inflammatory cells in human acute pancreatis (Figure 27C).

[0601] Figure 28. Schematic showing the experimental protocol using cerulean to induce acute pancreatitis in mice.

[0602] Figures 29A and 29B. Graphs showing an increase in serum LRG1 concentration (29A) and pancreatic LRG1 mRNA expression (29B) in the caerulein-induced acute pancreatitis mouse model. mRNA quantity was determined by quantitative RT-PCR. The expression level of LRG1 was normalized to RPLPO, and relative gene expressions were calculated using standard 2AACT.

[0603] Figure 30A and 30B. Graphs and images showing an increase in expression of LRG1 protein in pancreatic tissue during cerulean-induced acute pancreatitis. (30A) Western Blot analysis of pancreatic LRG1 protein levels following the onset of cerulean-induced acute pancreatitis. (30B) Immunofluorescence staining for LRG1 , CD31 , amylase (AMY) and myeloperoxidase (MPO). Staining was performed 24 hours after caerulein treatment.

[0604] Figure 31A to 31C. Graphs showing LRG1 expression during acute pancreatitis. (31A) Pancreatic LRG1 expression as measured by qRT-PCR following bone marrow transplantation and caerulein induction of acute pancreatitis. Three donor / recipient combinations were evaluated: wild-type to wild-type, LrgT / _knock-out to wild-type, and wild-type to LrgT / _knock-out. Acute pancreatitis was induced in recipient mice 5 weeks post-transplantation. (31 B) qRT-PCT analysis of LRG1 mRNA, and Western Blot analysis of LRG1 protein in isolated acinar cells following caerulein induction of acute pancreatitis.

[0605] Figures 32A and 32B. Images and graphs showing accelerated pancreatic recovery in the LrgTAknockout compared to Wild-type. (32A) Immunofluorescence staining of proliferation marker Ki67 and AMY in acinar cells from wild-type and Lrg1 knock outs following caerulein induction of acute pancreatitis. (32B) Expression of cyclins CCNB, CCND1 and CCNE 'n the pancreas as determined by qRT-PCR in wild-type and Lrg1 knock out mice following caerulein induction of acute pancreatitis.

[0606] Figures 33A to 33G. Images and graphs showing that acinar cell function is regulated by LRG1 through AKT-mediated CCKAR expression. (33A and 33B) qRT-PCT analysis of pancreatic CCKAR mRNA expression, and Western Blot analysis of pancreatic CCKAR protein expression in wild-type and Lrg1!knock out mice. (33C) Western blot analysis of TGFp associated non-canonical signalling proteins AKT, ALK5 and ERK in wild-type and Lrg1'- knock out mice. (33D) Western blot analysis of AKT and ALK5 in acinar cells in wild-type, Lrg1'- knock out and Lrg1'- knock out treated with recombinant LRG1 (Lrg1'- + rhLRGI). (33E) qRT-PCT analysis of CCKAR mRNA expression, and Western Blot analysis of CCKAR protein expression in isolated acinar cells of wild-type, Lrg1'- and Lrg / ^treated with rhLRGI . (33F) Western Blot analysis of AKT and ALK5 in Lrgl1- acinar cells following treatment with ALK5 / Smad2 / 3 inhibitor SB431542 and AKT inhibitor MK2206. (33G) qRT-PCT analysis of CCKAR mRNA expression, and Western Blot analysis of CCKAR protein in Lrgl1- acinar cells following treatment with inhibitors SB431542 and MK2206.

[0607] Figures 34A to 34H. Images and graphs showing that LRG1 blocking antibody EBC-103 can alleviate the effects of acute pancreatitis. (34A) Schematic outlining the experimental protocol to test the effect of LRG1 blocking antibodies on acute pancreatitis induced by caerulein. (34B) Western blot analysis of TGFp associated signalling proteins AKT and ALK5 following caerulein induced acute pancreatitis and subsequent treatment with EBC-103 or control IgG. (34C) qRT-PCR analysis of CCK1R mRNA, and Western Blot analysis of CCK1 R protein, following caerulein induced acute pancreatitis and subsequent treatment with EBC-103 or control IgG. (34D) H&E staining and pathology evaluation of pancreatic tissue following caerulein induced acute pancreatitis and subsequent treatment with EBC-103 or control IgG. (34E, 34F) qRT-PCR analysis of anti-inflammatory NFKBIA and 1110 mRNA (34E) or amylase AMY2) and cell cycle markers (CCNB and CCNE) (34F) following caerulein induced acute pancreatitis and subsequent treatment with EBC-103 or control IgG. (34G) Immunofluorescence staining for proliferation marker Ki67 and amylase (AMY) following caerulein induced acute pancreatitis and subsequent treatment with EBC-103 or control IgG. All images are representative. Data are expressed as average ± S.E.M of n>6. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01 , ****P<0.0001

[0608] Figure 35A to 35C. Graphs and images showing upregulation of LRG1 in PDAC cells. (35A; left panel) RNA sequencing expression data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project showing differential mRNA expression of LRG1 in PDAC compared to normal pancreas tissue. (35A; right panel) Data extracted from the Clinical Proteomic Tumour Analysis Consortium (CPTAC) database to show LRG1 protein abundance in normal versus human PDAC tumour tissues. (35B) Kaplan-Meier survival analyses of PDAC patients with differential expression of LRG1 obtained from the Human Protein Atlas database. (35C) Immunofluorescence staining of PDAC low- and high-grade tumour ductal cells for LRG1 and KRT19 to establish colocalization.

[0609] Figure 36A to 36H. Images and graphs showing LRG1 promotes tumour growth, epithelial-mesenchymal transition (EMT) and metastasis in-vivo. (36A) Schematic detailing experimental protocol to introduce PDAC cells transformed with a pCDH-LRG1 expression vector into a mouse model. (36B) Analysis of weight and volume of PDAC tumours expressing either a control or pCDH-LRG1 expression vector. (36C) Representative immunofluorescence images (left) and quantification (right) of Ki67 positive cells in PDAC tumours overexpressing LRG1 (pCDH-LRG1) and a vector control (pCDH). (36D) qRT-PCR analysis of cyclin Ccnd mRNA expression in PDAC tumours overexpressing LRG1 (pCDH-LRG1) and a vector control (pCDH). (36E) Semi-quantitative scoring of neoplastic cell and neoplastic nuclear pleomorphisms following hematoxylin-eosin staining and histopathological grading of in PDAC tumours overexpressing LRG1 (pCDH-LRG1) and a vector control (pCDH). In general, a score of 0 indicated that there were no abnormalities detected; 1 : minimal; 2: mild; 3: moderate; 4: marked; 5: severe. For pleomorphism, a semi- quantitative score of 1 -fold, 2-fold, or 3-fold change compared to normal cells was determined. (36F) Staining of PDAC tumour tissue overexpressing LRG1 (pCDH-LRG1) (right) or a vector control (pCDH) (left) for mesenchymal N-cadherin. (36G) qRT-PCR analysis of cadherin (CDH2) mRNA expression in PDAC tumours overexpressing LRG1 (pCDH-LRG1) or a vector control (pCDH). (36H) Analysis of liver weight and metastatic area of livers from mice having a PDAC tumour overexpressing LRG1 (pCDH- LRG1) or a vector control (pCDH).

[0610] Figure 37A to 37F. Images and graphs showing LRG1 promotes cell proliferation, migration and invasion in-vitro. (37A) Cell viability of human PDAC cells (PANC-1) transformed with either an empty control vector (pcDNA) or an LRG1 expressing vector (pLRG1) over 3 days as a measure of cell proliferation. (37B) Cell viability of PDAC cells (PANC-1) treated with siRNA (siLRGI) to knock down LRG1 expression, or a control siRNA (siCtrl) over 3 days as a measure of cell proliferation. (37C and 37D) Representative images of cell migration and invasion assays of PDAC cells transformed with pLRG1 or pcDNA control (37C), and siLRGI or siCtrl control (37D). (37E) Western Blot analysis and quantification of proliferation marker cyclin D (CCND) protein and EMT marker N-Cadherin (CDH2) protein expression in PDAC cells (PANC-1) transformed with an empty control vector (pcDNA) or an LRG1 expressing vector (pLRG1). (37F) Western Blot analysis and quantification of proliferation marker cyclin D (CCND) protein and EMT marker N-Cadherin (CDH2) protein in PDAC cells (PANC-1) treated with siRNA (siLRGI) to knock down LRG1 expression, or a control siRNA (siCtrl).

[0611] Figure 38A to 38E. Images and graphs showing LRG1 promotes tumorigenesis via EGFR / ErbB / AKT signalling. (38A) Dot blot profiler assay measuring the protein expression of EGFR ligand amphiregulin in mouse KPC cells overexpressing LRG1 (pCDH-LRG1) and control KPC cells (pCDH). (38B) qRT-PCR analysis of amphiregulin (Areg) mRNA expression in KPC cells (left) and tumour (right) overexpressing LRG1 (pCDH-LRG1) and control KPC cells (pCDH). (38C) Dot blot profiler assay measuring expression of EGFR, ErBb2, AKT and GSK signalling transducers in KPC cells overexpressing LRG1 (pCDH-LRG1) and control KPC cells (pCDH). (38D) Western Blot analysis of EGFR, ErBb2, PDK1 , AKT and GSK signalling protein expression in KPC cells overexpressing LRG1 (pCDH-LRG1) and control KPC cells (pCDH). (38E) Western Blot analysis of EGFR / ErbB / AKT associated signalling proteins in KPC cells overexpressing LRG1 following treatment with inhibitors Tucatinib (0.5pM) and MK2206 (10pM) or DMSO control. (38F) Cell viability assay results of mouse KPC cells overexpressing LRG1 and treated with Tucatinib or MK2206 or DMSO control as a measure of cell proliferation. (38G) Representative images and quantification of cell migration and invasion assays of KPC cells overexpressing LRG1 treated with Tucatinib, MK2206, or DMSO control.

[0612] Figure 39A to 39C. Graphs and images showing administration of LRG1 neutralizing antibody (EBC-61) inhibits tumour growth in vitro and in vivo. (39A) Cell viability assays of KPC cells overexpressing LRG1 (KPC pCDH-Lrg1 (also referred to herein as “pLrgl)) following treatment with anti-LRG1 antibody EBC-61 or an lgG1 isotype control antibody as a measure of cell proliferation. (39B) Representative images of cell migration assays measuring migration of KPC cells overexpressing LRG1 following treatment with EBC- 61 or an IgG 1 isotype control antibody. Immunofluorescence staining (left) and quantitative analysis (right) of migrated or invaded of control IgG or LRG1 antibody-treated pCDH-LRG1 KPC cells, Scale bar: 100|jm. (39C) Western blot analysis and quantification of CCND and CDH2 protein expression in KPC cells overexpressing LRG1 following treatment with EBC-61 or an lgG1 isotype control antibody. (39D) Representative images of KPC pLrgl pancreatic tumours in IgG- or EBC 61- treated wild-type mice. (39E) Endpoint KPC pLrgl tumour volumes following treatment with IgG or EBC 61 -antibodies. All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by unpaired, two-tailed Student’s t-test of n > 3 mice or independent experiments; *: p< 0.05, **: p< 0.01 .

[0613] Figure 40A and 40B. LRG1 is highly expressed in rheumatoid arthritis (RA) patients. (40A; left panel) ELISA analysis of LRG1 expression in serum of rheumatoid arthritis (RA) and age-matched osteoarthritis (OA) patients (n=3). (40A; middle panel) Quantitative real-time PCR analysis of LRG1 in synovial tissues collected from RA and age-matched OA patients (n=3). (40A; right panel) ELISA analysis of LRG1 expression in synovial fluid of RA and age-matched OA patients (n=3). (40B) Western blot and quantitative real-time PCR (qRT-PCR) analysis of LRG1 expression in primary fibroblast-like synoviocytes (FLS) isolated from RA and age-matched OA patients (n=3). Data are expressed as mean ± SEM. Statistical significance was determined by two-tailed, unpaired student’s t-test. *P<0.05.

[0614] Figure 41 A to 411. LRG1 regulates the function of multiple cell types in the synovium. (41 A) Representative images of immunofluorescence staining of adherent junction protein VE-cadherin in EC subjected to the treatment with rhLRGI . Paracellular gaps and disrupted intercellular contacts are marked by arrows. DAPI is used to label the nucleus and F-Actin highlights the stress fibres. (41 B) Mile’s assay shows that Lrg 1 - / - mice are resistant to VEGF-induced vessel permeability. (41 C) Representative images (left) and quantification (right) of Ki67 positive synovial endothelial cell (EC) in control and rhLRGI treatment conditions. (41 D) Representative images (left) and quantification (right) of the number of synovial ECs migrated across the Transwell in control and rhLRGI treatment conditions. (41 E) Representative images (left) and quantification (right) of synovial EC tube formation in control and rhLRGI treatment conditions. (41F) Representative images (left) and quantification (right) of the number of neutrophils adhered to the synovial ECs in control and rhLRGI treatment conditions. (41G) Representative images (left) and quantification (right) of the number of neutrophils migrate across the synovial ECs in control and rhLRGI treatment conditions. (41 H) FLS proliferation as demonstrated by MTS assay of control and rhLRGI treatment conditions. (411) qRT-PCR analysis of the MMP9 gene expression in FLS in control and following the treatment of rhLRGI . All images are representative. Data are expressed as average ± SEM, n>3. Statistical significance was determined by two-way ANOVA or two-tailed, unpaired student’s t-test, *p<0.05, **p<0.01 , ***p<0.001

[0615] Figure 42A and 42B. Collagen-induced arthritis is alleviated in Lrg1- / - mice. Wild-type and Lrg1- / - mice were subjected to CIA. (42A) Representative images on day 53 post collagen treatment (left) and clinical scoring (right) showing macroscopic joint symptoms in wild-type or Lrg1- / - mice post collagen treatment. Arrow indicates swelling of affected limbs. Severity scores were based on the symptoms in WT (n=4) and Lrg1- / - mice (n=5). (42B) Representative H&E images of hind paw joints of wild-type and Lrg1- / - mice at day 60 post collagen treatment. Data are expressed as average ± SEM. Statistical significance was determined by two-tailed, unpaired student’s t-test, *p<0.05. Figure 43A to 43E. LRG1 inhibition in Melanoma. (43A) Representative images of B16F10 tumours dissected from IgG- or EBC- treated wild-type mice. (43B) B16F10 tumour volume over time in IgG- or EBC-treated wild-type mice. (43C) Endpoint tumour volume following treatment with IgG- or EBC antibodies. (43D) qRT-PCR analysis of Ki67 mRNA levels in tumours treated with IgG- or EBC antibodies. (43E) qRT-PCR analysis of N-Cadherin mRNA levels of in tumours treated with IgG- or EBC antibodies. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test n > 3 mice; *: p < 0.05, **: p < 0.01 .

[0616] Figure 44A to 44F. LRG1 inhibition in CRC. (44A) Representative images of HT-29 tumours dissected from IgG- or EBC-treated Balb / c nude mice. (44B) HT-29 tumour volume over time in IgG- or EBC-treated Balb / c nude mice. (44C) Endpoint tumour volume following treatment with IgG- or EBC antibodies. (44D) MTS assay demonstrating the cell viability of HT-29 CRC cells treated with control IgG or LRG1 antibodies, EBC 103, EBC 107 and EBC78 over 3 days. (44E) Day 3 endpoint MTS assay results demonstrating cell viability of HT-29 CRC cells treated with control IgG or LRG1 antibodies, EBC 103, EBC 107 and EBC78. (44F) Representative Western blot images showing increased E-cadherin (reduced epithelial -mesenchymal transition) and reduced Cyclin D1 (reduced cell proliferation) in EBC103 treated HT-29 cells compared to IgG- treated controls. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test n > 3 mice or independent repeats.

[0617] Figure 45A to 45C. LRG1 inhibition in primary human glomerular mesangial cells. (45A) Representative Western blot and densitometry analysis of (45B) CTGF and (45C) FN1 protein levels following treatment with IgG- or EBC antibodies in the presence of high glucose and TGF-01 . All images are representative. Data are presented as the mean ± s.e.m. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test n > 3 independent repeats.

[0618] Figure 46. Representative immunoblots showing EBC107 and EBC103 binding to endogenous LRG1 extracted from mouse liver lysate. Expected size of LRG1 monomer: ~45kDa (marked by black arrow). Observed tissue LRG1 protein: (marked by white triangle)

[0619] Figure 47A and 47B. Metatarsal angiogenesis assay with EBC107 and EBC103. (47A) Representative immunofluorescence images of CD31 -stained fetal metatarsal bone explants and (47B) quantitative analysis of vascular density normalized to the explant size. Data are expressed as mean ± S.E.M., n=6 / group. Statistical significance was determined by one-way ANOVA, * p<0.05, ** p<0.01 , *** p<0.001 .

[0620] Figure 48A to 48E. (48A) Schematic of DSS-induced acute colitis model and LRG1 antibody treatment scheme. (48B) Tabulated daily body weight loss and (48C) disease activity index (DAI). (48D) Representative endpoint gross necropsy images of whole intestinal tract and (48E) tabulated colon length at endpoint, day 10. Data are expressed as mean ± S.E.M., n=6 / group. Statistical significance was determined by one-way ANOVA, * p<0.05, “ p<0.01 , “* p<0.001 . Figures 49A to 49C. (49A and B) Histological scoring of disease severity following LRG1 -neutralizing antibody treatment. (49A) Representative H&E staining of treatment groups. (49B) Tabulated histological scores evaluating epithelial morphology, immune cell infiltration and global architecture. Data are expressed as mean ± S.E.M., n=6 / group. (49C) Quantification of functional indicators, presumptive toxicity and systemic indicators in the DSS colitis model following LRG1-neutralizing antibody treatment. Statistical significance was determined by one-way ANOVA, * p<0.05, ** p<0.01 , *** p<0.001 .

[0621] Figure 50. Schematic of experiments in the collagen antibody induced arthritis (CAIA) mice model.

[0622] Figures 51A to 51G. LRG1 neutralizing antibodies in CAIA mice model. (51A) Change in ankle (hind paw) measurements throughout CAIA model in CAIA mice treated with LRG1 neutralizing antibodies or isotype control. (51 B) Change in wrist (front paw) measurements throughout CAIA model in CAIA mice treated with LRG1 neutralizing antibodies or isotype control. (51 C) Change in clinical scores for CAIA mice treated with LRG1 neutralizing antibodies and isotype control. (51 D) Clinical score for CAIA mice treated with LRG1 neutralizing antibodies and isotype control at day 7 post induction (Man-Whitney test). (51 E) Percentage reduction in grip strength of CAIA mice treated with LRG1 neutralizing antibodies and isotype control (t test). (51 F) Endpoint spleen mass of CAIA mice treated with LRG1 neutralizing antibodies and isotype control. (51 G) Change in mass of CAIA mice treated with LRG1 neutralizing antibodies and isotype control.

[0623] Figures 52A to 52C. LRG1 inhibition in NSCLC. (52A) Representative images of LL / 2 tumours dissected from IgG- or EBC-treated wild-type mice. (52B) LL / 2 tumour volume over time in IgG- or EBC-treated wild-type mice. (52C) Endpoint tumour volume following treatment with IgG- or EBC antibodies. Data are presented as mean ± s.e.m. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test n > 3 mice; p < 0.01 , ***: p < 0.001 .

[0624] Figures 53A to 53M. LRG1 inhibition in AR mouse model of DN. (53A) qRT-PCR analysis of Lrg1 mRNA levels in the kidneys of wild-type and AR mice. (53B) Changes in urinary albumin excretion rates (UAE) in untreated or EBC-107 treated AR mice from 12-weeks to 18-weeks. (53C) Urinary albumin excretion rates (UAE) in untreated or EBC-107 treated AR mice at 18-weeks endpoint. (53D) Representative Masson’s Trichrome staining of glomerular and tubulo-interstitial regions in untreated and EBC-107 treated AR mouse kidney sections. (53E) Quantitative analysis of trichrome positive area over total kidney area in untreated and EBC-107 treated AR mice. (53F) Representative Western blot and densitometry analysis of (53G) COL1 and (53H) COL4 protein levels following treatment with EBC-107 compared to untreated counterparts. qRT-PCR analysis of (53I) Mmp2, (53J) Kimi, (53K) Cc / 2, (53L) Cc / 5, (53M) 116. Data are presented as mean ± s.e.m. Significance was determined by unpaired two-tailed Student’s t-test of n > 3 mice; *: p < 0.05, **: p < 0.01 . Figure 54. Hematoxylin and Eosin (H&E) staining of liver sections of RA CAIA treated with LRG1 neutralizing antibodies and isotype control showed no overt differences, highlighting no toxicity related to LRG1 antibody treatments. Original magnification is 20x.

[0625] Figure 55. Representative images of Hematoxylin and Eosin (H&E) staining of sagittal sections of the hind paws of CAIA mice treated with respective neutralizing LRG1 ab or isotype control. White stars indicate areas of synovial inflammation, black triangles indicate areas with bone inflammation and dotted lines indicate the regions of bone erosion. Original magnification is at 4x and 10x respectively.

[0626] Figure 56. Hematoxylin and Eosin (H&E) staining of transverse sections of the hind paws of CAIA mice treated with respective neutralizing LRG1 ab or isotype control. White stars indicate areas of synovial inflammation, black triangles indicate areas with bone inflammation and dotted lines indicate the regions of bone erosion. Original magnification is at 4x and 10x respectively.

[0627] Figure 57. Safranin O (SafO)ZFast green staining of sagittal sections of the hind paws of CAIA mice treated with respective neutralizing LRG1 ab or isotype control. White block arrows indicate areas of destaining of superficial cartilage while black arrows indicate areas with erosion of superficial cartilage. Original magnification is 20x.

[0628] Figure 58. Safranin O (SafO)ZFast green staining of transverse sections of the hind paws of CAIA mice treated with respective neutralizing LRG1 ab or isotype control. White block arrows indicate areas of destaining of superficial cartilage while black arrows indicate areas with erosion of superficial cartilage. Original magnification is 20x.

[0629] Figure 59. Skin thickness was measured weekly using a caliper over a 4-week period in the bleomycin- induced skin fibrosis mouse model.

[0630] Figures 60A and 60B. Dermis thickness in the bleomycin-induced skin fibrosis mouse model. (60A) Representative images of H & E staining (60B) Quantification of dermis thickness.

[0631] Figures 61 A and 61 B. Collagen stained by Mason’s trichrome staining in the bleomycin-induced skin fibrosis mouse model. (61A) Representative images of Masson’s trichrome staining of skin sections. (61 B) Quantification of collagen area percentage compared with controls.

[0632] Figures 62A to 62C. RT-PCR analysis of the bleomycin-induced skin fibrosis model showing that EBC treatment reduces fibrosis. Several fibrosis-associated markers were examined: (62A) Relative expression of Col1a1. (62B) Relative expression of fibronectin. (62C) Relative expression of a-SMA.

[0633] Figures 63A and 63B. Western blot analysis of the bleomycin-induced skin fibrosis model showing that EBC treatment reduces fibrosis. (63A) Representative Western blots and (63B) densitometric Analysis. Figures 64A and 64B. Wound size healing in a mouse model of diabetic wound healing. (64A) Representative wound images. (64B) Quantification of wound size.

[0634] Figures 65A and 65B. (65A) Table showing epitope binning with EBC107, EBC103 and EBC78. (65B) Graph showing epitope mapping with MGZ(EBC78), EBC-107 (chM14P2-H5) & EBC-103 (chM2P1-E8).

[0635] Examples

[0636] Example 1 : LRG1 level is closely associated with subretinal fibrosis in both disease animal model and humans

[0637] LRG1 was previously shown to be expressed at high levels in the vitreous of human patients with neovascular age-related macular degeneration (nAMD). To establish the association between LRG1 and subretinal fibrosis, serum LRG1 levels were analysed in human nAMD patients without subretinal fibrosis at the baseline and in 12 months (NF), without subretinal fibrosis at the baseline but develop subretinal fibrosis in 12 months (IF), and with subretinal fibrosis at the baseline (BF). Results showed that serum LRG1 levels are significantly higher in IF and BF patients compared to that in patients without fibrosis at all (Figure 1). This observation was further confirmed in an animal model of laser-induced choroidal neovascularization (CNV). In this model, subretinal fibrosis becomes apparent on day 35 post-laser injury. Results showed that the expression of LRG1 in the retinal pigmented epithelium (RPE)Zchoroidal fraction of the eye is significantly induced at day 35 post-laser (Figure 2). Together, these data demonstrate a close association between LRG1 and subretinal fibrosis in both diseased mice and humans.

[0638] Example 2: Lrg1 deletion leads to reduced subretinal and corneal fibrosis

[0639] Following the establishment of the association between elevated LRG1 levels and subretinal fibrosis in human patients and disease mice, the impact of Lrg1 deletion in subretinal fibrosis was investigated. Wild-type and Lrg1 knockout mice were subjected to laser treatment. RPE flatmount collected from wildtype and Lrg1 knockout mice 35 days after the laser treatment were subjected to immunofluorescence staining with a fibrotic marker collagen I . Results showed that the total area of collagen l-positive lesions is significantly reduced in Lrg1 knockout mice as compared to that in wild-type controls (Figure 3), demonstrating that LRG1 is required for subretinal fibrosis in vivo.

[0640] To further expand the cause-effect relationship between LRG1 and ocular fibrosis at the anterior of the eye such as corneal, the extent of the corneal fibrosis was examined in wild-type and Lrg1 mice following the exposure to 1 M NaOH. Results showed that there is a significant reduction in corneal fibrosis in the absence of LRG1 (Figure 4).

[0641] Example 3: LRG1 promotes the transformation of retinal vascular pericytes Perivascular cells, such as pericytes contribute to ocular fibrosis. Once activated, pericytes will become proliferative, and migratory and start to secrete extracellular matrix (ECM) proteins. To understand LRG1 ’s role in pericyte activation, human retinal vascular pericytes (HRPC) were treated with recombinant human LRG1 (rhLRGI). Western blot analysis showed that rhLRGI promotes the expression of ECM components, including connective tissue growth factor (CTGF), fibronectin, and alpha-smooth muscle actin (ASMA), as well as proteins involved in the transformation of pericytes, including N-Cadherin (N-Cad) and Plasminogen activator inhibitor-1 (PAI-1) (Figure 7A). Immunofluorescence staining with a cell proliferation marker, Ki67, was used to determine proliferating pericytes following rhLRGI treatment. Results showed that rhLRGI significantly promotes HRPC proliferation (Figure 6C). A transwell assay was used to evaluate the impact of rhLRGI on HRPC migration. Results showed HRPC treated with rhLRGI are more motile (Figure 6E). Overall, the data show that LRG1 promotes HRPC activation.

[0642] Example 4: The LRRCT region of LRG1 is responsible for its pro-fibrotic effect.

[0643] To further understand the role of LRG1 in pericyte activation and ocular fibrosis, truncated LRG1 proteins were generated containing the leucine-rich-repeat (LRR) or leucine-rich C terminal (LRRCT) domain of LRG1 . Western blot analysis showed that LRRCT, but not LRR, is able to promote basal and TGFpl- induced CTGF expression in HRPCs (Figure 5).

[0644] Example 5: LRG1 promotes transdifferentiation of human retinal pigment epithelium

[0645] Retinal pigment epithelial (RPE) cells maintain the health and functional integrity of both photoreceptors and the choroidal vasculature. During the development of subretinal fibrosis in nAMD, RPE lose their epithelial-like characteristics cells and subseguently undergo epithelial-mesenchymal transition (EMT) and differentiate into 9s, with increased migratory, proliferative and ECM synthesis capacity.

[0646] Assays were performed in which RPE cells were exposed to rhLRGI . Results show that rhLRGI significantly promotes RPE survival (Figure 6A), proliferation (Figure 6B), migration (Figure 6D), ECM gene transcripts (including fibronectin; FN, Connective tissue growth factor; CTGF, Transforming growth factor beta; TGFB and alpha-1 type I Collagen; COL1a1) (Figure 7B), as well as fibronectin protein accumulation (Figure 7C). Western blot studies revealed that rhLRGI promotes transdifferentiation of RPE through activation of the TGFB-Smad2 / 3 signalling, and showed abolishment of Smad2 / 3 phosphorylation using SB431542, a selective inhibitor of the ALK5 / Smad2 / 3 signalling cascade (Figure 7D). This demonstrates a potential mode of action of LRG1 -mediated RPE activation.

[0647] Example 6: Identification of a panel of monoclonal chimeric antibodies with strong binding activities against distinct epitope of LRG1 and with cross-species reactivity

[0648] Having established a promoting role of LRG1 in HRPC activation, LRG1 blocking antibodies were generated using mouse immunisation followed by hybridoma and single B-cell cloning antibody discovery campaigns. More than 3,000 clones were screened and a panel of monoclonal antibodies against LRG1 was identified. Selected antibodies were further expressed as recombinant chimeric antibodies and characterised for their binding EC50 to LRG1 protein of different species as well as the LRC region (LRRCT) of LRG1 by ELISA assay. In total six chimeric anti-LRG1 antibodies showed good binding EC50 (sub-nM to double digit pM) to human and cynomolgus monkey (Macaca fascicularis) LRG1 (Table 1). Magacizumab (MGZ), a reported antibody against LRG-1 (WO 2016 / 135462 A1) shows similar binding EC50 to human LRG1 but demonstrated weaker binding activity towards monkey LRG1 and no binding activity against LRC region.

[0649] Table 1. Binding profiles of chimeric anti-LRG1 antibodies to full length of human and monkey LRG1 protein as well as the LRRCT region of human LRG1 .

[0650] The mouse species cross-reactivity was examined by Western blot in mouse retina and liver extracts. Immunoblot shows all antibodies detect LRG-1 protein band (~50kDa) in mouse liver extracts, with EBC- 59 showing the highest sensitivity in mouse retina and liver extracts (Figure 8). MGZ exhibited the weakest immunostaining against mouse liver extracts. This confirmed the mouse and monkey crossreactivity of these antibodies of invention and the suitability of characterising the blocking potency and safety of these antibodies in in vivo animal preclinical model.

[0651] Binding kinetics of chimeric EBC-58, EBC-59, EBC-61 and MGZ on native human LRG1 were further evaluated using Biolayer interferometry (BLI). Briefly, the LRG1 protein was immobilised using amine- coupling to ARG2 (Amine Reactive 2nd Generation) biosensors. Ligand-loaded sensors were incubated with different concentrations of LRG1 antibody (analytes) during the association step and returned to the baseline well for the dissociation step. The EBC-58, EBC-59 and EBC-61 exhibited strong binding for native human LRG1 protein (Figure 9). The dissociation (Kdis) of these antibodies is slow, suggesting tight binding of the antibodies towards human LRG1 . The KD could not be determined accurately due to the tight binding kinetics of the antibodies which exceeded the binding kinetics limit of BLI instrument.

[0652] EBC-58, EBC-59, EBC-60 and EBC-61 demonstrated binding activity towards the LRC region, whereas MGZ shows negligible binding, suggesting the specificity of these antibodies toward the leucine-rich C terminal domain of LRG1 (Table 1). The epitope of these antibodies was further examined by linear peptide mapping using ELISA. EBC-58, EBC-59, EBC-60 and EBC-61 recognise a linear peptide spanning the region of LRRCT (ICDQNLSDLYRWLQAQ). This is distinct from MGZ which recognises the peptide spanning the LRR6 region (Figure 10). This suggests EBC-58, EBC-59, EBC-60 and EBC-61 bind to an epitope region that is distinct from MGZ. Alignment of EBC-58, EBC-59, EBC-60 and EBC-61 target human LRRCT epitope with the orthologous sequences revealed 100% monkey and 65% mouse LRG1 sequence identity. This shows higher sequence identity across species than for MGZ’s LRR6 epitope with 87% monkey and 60% mouse LRG1 identity. This is consistent with the observed better cross-species binding reactivity of EBC-58, EBC-59, EBC-60 and EBC-61 as compared to MGZ.

[0653] Example 7: LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61 attenuate TGFB-induced fibrotic gene expression, cell migration, and fibronectin expression in RPE cells

[0654] RPE cells were treated with EBC-59, EBC-60, and EBC-61 antibodies and their impact on rhLRGI- induced fibrotic gene expression was evaluated. Results showed that EBC-59, EBC-60, and EBC-61 antibodies attenuate the rhLRGI-induced expression of collagen 1A1 , and collagen 4a2, fibronectin and CTGF expression in RPE cells (Figure 11).

[0655] In subretinal fibrosis, RPE cells are activated and become migratory. A transwell migration assay was used to evaluate the impact of LRG1 blocking antibody on TGFp-induced RPE cell migration. Results showed that EBC-59, EBC-60, and EBC-61 antibodies strongly inhibited the TGFp-induced RPE cell migration (Figure 12).

[0656] RPE cells express a large amount of extracellular matrix protein (ECM), including fibronectin, during subretinal fibrosis. TGFp is a key stimulator of ECM protein expression in different types of cells. To evaluate the impact of LRG1 blocking antibodies on TGFp-induced ECM protein expression, RPE cells were subjected to the treatment with different LRG1 blocking antibodies or an isotype control in the presence of TGFp. As demonstrated by immunofluorescence staining, each of EBC-59, EBC-60, and EBC-61 strongly attenuate the TGFp-induced fibronectin expression in RPE cells (Figure 13).

[0657] Example 8: LRG1 blocking antibodies EBC-59, EBC-60, EBC-61 attenuate the LRG1-induced RPE cell proliferation

[0658] During subretinal fibrosis, RPE cells become hyperproliferative. Example 5 showed that recombinant human LRG1 (rhLRGI) promotes RPE cell proliferation. To evaluate the impact of LRG1 -blocking antibodies on RPE cell proliferation, RPE cells were treated with rhLRGI in the presence or absence of EBC-59, EBC-60, and EBC-61 antibodies. RPE cell proliferation was evaluated by immunofluorescence staining with cell proliferation marker Ki67. The results show that LRG1 -blocking antibodies significantly inhibit LRG1-induced RPE cell proliferation (Figure 14).

[0659] Example 9: LRG1 blocking antibodies EBC-59, EBC-60, and EBC-61 attenuate the LRG1 -induced vessel outgrowth from metatarsal explants

[0660] To evaluate the impact of newly generated LRG1 blocking antibodies on neovascularization, metatarsal explants were treated with rhLRGI with the presence or absence of EBC-59, EBC-60, and EBC-61 antibodies. Vessel outgrowth from metatarsal explants was visualised by immunofluorescence staining with an endothelial cell marker (CD31) and guantified accordingly (Figure 15). EBC-59, EBC-60, and EBC-61 significantly supressed the LRG1-induced aSMA+ area, demonstrating these antibody candidates could play a role in inhibiting fibroblast activation and fibrosis. Example 10: LRG1 -blocking antibodies EBC-59, EBC-60, and EBC-61 attenuate the laser-induced CNV and subretinal fibrosis in vivo

[0661] The anti-angiogenic and anti-fibrotic roles of LRG1 -blocking antibodies were evaluated in vivo using a mouse model of laser-induced choroidal neovascularization (CNV) and subretinal fibrosis. C57BL / 6 mice were treated with LRG1 -blocking antibodies on the day of the laser treatment. CNV was visualized by fundus fluorescein angiography (FFA) on day 7 and day 14 following the laser treatment (Figure 16A). Mice were sacrificed on day 35 for RPE flatmount preparation followed by immunofluorescence staining with the fibrotic marker aSMA and Collagen I (Figure 16B). The results show that each of EBC-59, EBC- 60, and EBC-61 strongly suppress CNV and subretinal fibrosis in vivo.

[0662] Example 11 : Humanised EBC-59 antibody retains binding kinetics towards native recombinant human LRG1 protein

[0663] 22 humanised variants of parental EBC-59 were constructed (Table 2). Out of 22 variants, 19 variants were expressed successfully and were screened for binding KD at 3 titrated concentrations (150nM, 75nM and 37.5nM) by BLL Complete dose response binding kinetics were performed for the top 6 humanised variants. All selected humanised variants (EBC-1191 , EBC-1192, EBC-1196, EBC-1199, EBC-1200 and EBC-1212) demonstrated comparable binding kinetics with parental chimeric EBC-59 antibody (Figure 17). The affinity constant (KD) and rate constants (association constant, [ka], dissociation constant, [kdis]) of these humanised antibody variants were similar to parental chimeric EBC- 59 with no binding of isotype control to human LRG1 protein (Table 3).

[0664] Table 2. Binding KDs of different constructed humanised LRG1 antibody variants by 3 titrated concentrations (150nM, 75nM and 37.5nM) using BLL

[0665] Table 3. Comparable affinity constant (KD) and rate constants (association constant, [ka], dissociation constant, [Kdis]) of humanised antibody variants (EBC-1191 , EBC-1192, EBC-1196, EBC- 1199, EBC-1200 and EBC-1212) and parental EBC-59 with no binding of isotype control to human LRG1 protein.

[0666] Example 12: Humanised antibodies targeting LRG1 exhibited effective functional potency in antiangiogenesis and anti-fibrosis

[0667] Humanised variants of EBC-59 (EBC-1191 , EBC-1192, EBC-1195, EBC-1196, EBC-1199, EBC-1200 and EBC-1212) were further evaluated for functional potency in an ex vivo metatarsal explants assay.

[0668] Humanised variants of EBC-59 antibodies demonstrate anti-angiogenic and anti-fibrotic properties in the ex vivo angiogenesis assay.

[0669] Using human lgG1 (EBC-146) as an isotype control, a statistically significant reduction in rhLRGI- induced fibroblast activation was observed in the presence of EBC-1191 , EBC-1192, EBC-1195 and EBC-1212 (Figure 19A and Figure 19B). Consistent with angiogenesis, humanised EBC-1212 demonstrated the most effective anti-fibrosis potency. Humanised variants of EBC-59 antibodies are effective in attenuating LRG1 -induced fibroblast activation.

[0670] Figure 19 shows that EBC-1212 inhibits laser-induced choroidal neovascularization (CNV) in mice in a dose-dependent manner. Figure 22 shows that EBC-1212 exhibits dose-dependent inhibition of laser- induced subretinal fibrosis in mice. Figure 25 shows that EBC-1212 slows down the progression of laser- induced subretinal fibrosis compared to an isotype control. Figure 21 A and Figure 21 B show that EBC-59 and EBC-1212 exhibit a comparable anti-angiogenic effect to Avastin. The results also show that EBC-59 and EBC-1212 exhibit an anti-fibrotic effect compared to an isotype control, while Avastin does not exhibit an anti-fibrotic effect. Figure 23 shows that EBC-59 and EBC- 1212 exhibit a comparable anti-angiogenic effect to Eylea and a comparator anti-LRG1 antibody (magacizumab; EBC-78). The results also show that EBC-59 and EBC-1212 exhibit an anti-fibrotic effect compared to an isotype control, while Eylea and magacizumab (EBC-78) do not exhibit an anti-fibrotic effect.

[0671] Figure 20 shows that EBC-1212 exhibits an immediate and prolonged anti-angiogenic effect of humanised LRG1 antibody EBC-1212, either on its own or in combination with Eylea. Figure 24 shows an additive anti- angiogenic effect of the combination of EBC-1212 + Eylea compared to either agent as a monotherapy. The results also show that the combination of EBC-1212 + Eylea exhibits an anti-fibrotic effect, while Eylea monotherapy does not.

[0672] Example 13: Materials and methods relating to Examples 14 to 17

[0673] 13. 1 Human Pancreatitis

[0674] Formalin-fixed, paraffin-embedded (PFFE) human pancreatitis tissues (#PA691) were obtained from TissueArray.com.

[0675] 13.2 Animals and AP induction

[0676] C57BL / 6N mice were purchased from InVivos (Singapore). Lrg1 — / — mice on a C57BL / 6N background were purchased from the University of California, Davis, Knockout Mouse Project (KOMP) (http: / / www.komp.org). AP was induced in 10 to 12-week-old female mice via 7 times hourly intraperitoneal injections of supramaximal concentrations of Caerulein (50pg / kg, #HY-A0190, MedChemExpress, USA). Saline (0.9% NaCI) was used as a vehicle control. Pancreatic duct ligation (PDL) surgery was performed as previously described [1 , 2]. The pancreas was visualized using a stereomicroscope and the tail region of the main pancreatic duct was ligated with 7-0 nonabsorbable, polypropylene suture (#M8703, Ethicon, USA). Mice were sacrificed at relevant time points by CO2 asphyxiation.

[0677] 13.3 Bone marrow transplantation

[0678] 10 to 12-week-old wild-type or LrgTAmice were lethally irradiated at a fractionated dose of 5.5Gy, twice in a 4-hour interval using a BIOBEAM GM y irradiation device (Gamma-Service Medical, Germany). Bone marrow cells were harvested from the bilateral tibia and femur bones of isogeneic donor mice and filtered through a 70pm cell strainer. 5x106bone marrow cells were delivered to irradiated recipient mice through the tail vein 24 hours after the irradiation. AP was induced in recipient mice 5 weeks post-transplantation as described above.

[0679] 13.4 Histology and Immunofluorescence staining Mouse pancreatic tissues were fixed in 4% paraformaldehyde (#158127, Sigma-Aldrich, USA) overnight before being embedded in paraffin or O.C.T compound following a standard protocol. Paraffin sections (6pm) were subjected to hematoxylin-eosin staining followed by histopathological grading by an independent pathologist (Advanced Molecular Pathology Laboratory, Institute of Molecular and Cell Biology, A*Star, Singapore). A score of 0 indicated that there were no abnormalities detected; 1 : minimal; 2: mild; 3: moderate; 4: marked; 5: severe [3].

[0680] For immunofluorescence staining, paraffin (6pm) or cryosections (8pm) were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224- 1-AP, Proteintech, USA), CD31 (#550274, BD Biosciences, USA or #ab28364, Abeam, UK), AN2 (#130- 100-468, Miltenyi Biotec, Germany), Glucagon (#ab92517, Abeam, UK), Insulin (#ab ab7842, Abeam, UK), Amylase (#sc-46657, Santa-Cruz Biotechnology, USA), Cytokeratin 19 (#PAB12676, Abnova, USA), Myeloperoxidase (MPO) (#ab9535; Abeam, UK or #AF3667, R&D Systems, USA) and Ki67 (#ab15580; Abeam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA). Images were captured using Leica DM5500 microscope (Leica Microsystems, USA) or Carl Zeiss LSM 710 confocal microscopy (Zeiss, Germany) and analysed using Adobe Photoshop software (Adobe Inc, USA).

[0681] 13.5 Cells and cell culture

[0682] Mouse primary acinar cells were isolated using a standard collagenase IA (#C9891 , Sigma-Aldrich, USA) digestion protocol as previously described [4]. Human promyelocytic leukemia cell line, HL-60 (#CCL-240, ATCC, USA) and human pancreatic microvascular endothelial cells, HPaMEC (#3800, ScienCell Research Laboratories, USA) were maintained according to the supplier’s instructions. Cells were treated with Caerulein (1 pM, #HY-A0190, MedChemExpress, USA), recombinant human IL-6 (100ng / mL, #200- 06, PeproTech, USA), recombinant human LRG1 (200ng / mL, #7890-LR-025, R&D systems, USA), ALK5 inhibitor, SB431542 (10pM, Sigma-Aldrich, USA) and AKT inhibitor, MK-2206 (10pM, MedChemExpress, USA) as indicated.

[0683] 13.6 Quantitative RT-PCR

[0684] Pancreatic tissues were stored in RNAIater™ Stabilization Solution (#AM7020, Thermo Fisher Scientific, USA). Total RNA was isolated and purified from the mouse pancreas or cultured cells using RNAeasy kit (#74106, Qiagen, USA) and NucleoSpin RNA kit (#740955, Macherey-Nagel, Germany) respectively, before being converted to cDNA using qScript cDNA Supermix (#157031 , Quanta Biosciences, USA). PCR was conducted with PrecisionFAST qPCR MasterMix (PPLUS-machine type-1 ML, Primer Design, UK) using Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies, USA). The expression levels of respective target genes were normalized to RPLP0, and relative gene expressions were calculated using standard 2AACT.

[0685] 13.7 SDS-PAGE and Western Blotting

[0686] Cells or tissues were lysed on ice in radioimmunoprecipitation assay buffer containing 1x protease inhibitor (1 tablet in 500uL, #04693116001 , Roche, Switzerland), 1 mmol / L dithiothreitol (#10197777001 , Sigma-Aldrich, USA), and 1 mmol / L phenylmethylsulfonyl fluoride (#P7626, Sigma-Aldrich, USA). Additional 100 mmol / L phosphatase inhibitors (#07575-51 , Nacalai Tesque, Japan) were used when detecting cell signalling transducers. Proteins were separated by 10% SDS-PAGE before being transferred onto an Immobilon-PSQ PVDF Membrane (#IPVH00010, Merck Millipore, USA). Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1 -AP, Proteintech, USA), phospho-PKC 5 antibody (mouse monoclonal (#sc-365969, Santa-Cruz Biotechnology, USA), phospho-PKC epsilon antibody (rabbit polyclonal, #ab63387, Abeam, UK), PKC antibody (mouse monoclonal, #sc-17769, Santa-Cruz Biotechnology, USA), phospho-Stat3 (rabbit monoclonal, #9145, Cell Signaling Technology, USA), Stat3 (rabbit monoclonal, #12640, Cell Signaling Technology, USA), phospho-SAPK / JNK (rabbit monoclonal, #9255, Cell Signaling Technology, USA), SAPK / JNK (rabbit monoclonal, #9145, Cell Signaling Technology, USA), cleaved caspase 3 antibody (rabbit monoclonal, #9664, Cell Signaling Technology, USA), phospho-PRKD3 antibody (rabbit polyclonal, #orb4440, Biorbyt, UK), PRKD3 antibody (rabbit polyclonal, #bs-4157R, Bioss Inc, USA), CCKAR antibody (rabbit polyclonal, #bs-11514R, Bioss Inc, USA), phosphor-TGFBRI antibody (rabbit polyclonal, # A50933, Antibodies.com, UK), phospho-Akt antibody (rabbit monoclonal, #4060; Cell Signaling Technology), Akt antibody (rabbit monoclonal, #9272; Cell Signaling Technology), phospho-p44 / 42 MAPK (ERK1 / 2) antibody (rabbit monoclonal, #4370, Cell Signaling Technology, USA), p44 / 42 MAPK (ERK1 / 2) antibody (rabbit monoclonal, #4695, Cell Signaling Technology, USA), phospho-Smad2 antibody (rabbit monoclonal, #3108, Cell Signaling Technology, USA), Smad2 antibody (rabbit monoclonal, #5339, Cell Signaling Technology, USA), HSP60 antibody (rabbit monoclonal, #12165, Cell Signaling Technology, USA), RPLPO antibody (rabbit polyclonal, #11290-2-AP, Proteintech, USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, USA). Densitometry was performed using Imaged.

[0687] 13.8 Molecular Biological Methods siRNA oligonucleotides (#L-015179-01 ; Dharmacon, USA) were used for knocking down LRG1 with nontargeting siRNA (#D-001810-01 ; Dharmacon, USA) as a negative control. Transfection in dHL-60 cells was performed using RNAiMAX (#13778150, Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

[0688] 13.9 Statistics

[0689] Data are presented as mean ± standard error of the mean (s.e.m.). Statistical analyses were performed by an unpaired, two-tailed Student’s t-test or one-way ANOVA followed by an appropriate post-hoc test using Prism 5 (GraphPAD Software Inc.). Statistical details for each experiment, including n values, are provided in figure legends.

[0690] 13.10 Study approval

[0691] All animal experiments were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of Nanyang Technological University (IACUC ARF-LKC / A18026, A19110) and SingHealth (2020 / SHS / 1593, 1594), Singapore, and the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health. Example 14: Evaluation of LRG-1 expression in the pancreas

[0692] Having established that humanised antibodies targeting LRG-1 block LRG-1 function, expression of LRG- 1 in the pancreas was investigated using immunofluorescence staining of healthy mouse pancreas (Figure 26). LRG-1 is expressed in normal mouse pancreas and LRG-1 colocalises with the vascular marker CD31 (Figure 26, upper panel). However, LRG1 does not colocalise with the perivascular marker AN2 / NG2 (Figure 26, lower panel).

[0693] Expression of LRG1 was then evaluated in human patients with acute pancreatis (Figure 27A, Figure 27B and Figure 27C). Results show that levels of LRG1 are increased in patients with acute pancreatitis compared to levels in healthy controls (Figure 27A). The correlation between serum LRG1 concentration and C-reactive protein, which is a marker of inflammation severity in patients was then assessed (Figure 27B). This analysis showed that the increase in serum LRG1 concentration correlates with the serum concentration of C-reactive protein. Figure 27C shows LRG1 is present in Amy+ acinar cells.

[0694] Example 15: LRG1 expression is elevated in acute pancreatitis

[0695] LRG1 expression was assessed in an acute pancreatis mouse model. In the mouse model, acute pancreatis was induced by administering caerulein, which is an analogue of the pancreas trophic hormone cholecystokinin (Figure 28). Hyperstimulation of mouse pancreas by supramaximal concentrations of caerulein replicates mild clinical pancreatitis (especially histopathological / molecular changes in early phases of human disease).

[0696] The concentration of LRG1 in serum following induction of acute pancreatitis in the mouse model was then assessed (Figure 29A), as was LRG1 mRNA expression in the pancreas (Figure 29B). These data show that induction of acute pancreatis causes an increase LRG1 serum concentration and pancreatic LRG1 mRNA expression. Western blot analysis shows that pancreatic LRG1 protein expression also increased following induction of acute pancreatis (Figure 30A).

[0697] 24 h after the first caerulein injection, CD31 + vascular cells, Amy+ acinar cells and MPO+ inflammatory cells were subjected to immunofluorescence staining (Figure 30B). The staining shows that LRG1 colocalisation with CD31 does not change following the onset of acute pancreatitis and that additional sources of LRG1 in acute pancreatitis include Amy+ acinar cells and MPO+ myeloid cells.

[0698] Bone marrow transplantation assays using wild-type and LRG1 knock out (Lrgl ) donors were performed and levels of LRG1 mRNA expression were assessed (Figure 31 A). Bone marrow was donated from either a wild-type mouse and transplanted into a wild-type or LrgT / _recipient, or donated from a LrgTAmouse and transplanted into a wild-type host. Actuate pancreatis was then induced in the hosts using caerulein injection.

[0699] LRG1 mRNA expression increased in response to the onset of acute pancreatitis in recipient mice with a wild-type background, while LRG1 mRNA expression did not increase in host mice lacking LRG1 (LrgT / _; Figure 31 A). These data show that the majority of pancreatic LRG1 expression after acute pancreatitis induction is derived from the parenchyma background (e.g., acinar cells, etc) rather than the transplanted immune cells.

[0700] LRG1 mRNA and protein expression was assessed in isolated acinar cells following induction of acute pancreatitis (Figure 31 B). These data show that both LRG1 mRNA and LRG1 protein expression increase in acinar cells following the onset of actuate pancreatitis.

[0701] Example 16: LRG1 contributes to recovery from acute pancreatitis

[0702] The contribution of LRG1 to recovery from acute pancreatitis was then evaluated. Immunofluorescence staining was used to assess levels of proliferation marker Ki67 and amylase (AMY) in acinar cells from wild-type and Lrg1 knock out lines (Figure 32A). This assay revealed that knocking out LRG1 results in an increase in Ki67 positive proliferating acinar cells on Day 3 following the induction of acute pancreatitis.

[0703] Cyclin (Ccnb, Ccndl, Ccne) mRNA expression in the pancreas was also measured following acute pancreatitis induction in wild-type and Lrg1 knock out mice (Figure 32B). The results show that the increase in cyclin expression at Day 3 following acute pancreatitis induction is greater in the knock-out than in the wild-type.

[0704] These results show that knock-out of LRG1 leads to increased recovery following induction of acute pancreatitis.

[0705] Example 17: TGFP associated non-canonical signalling proteins contribute to increased CCKAR expression in the Lrgl1- knock out

[0706] To investigate the relationship between LRG1 and acinar cell function further, expression of CCKAR mRNA (Figure 33A) and CCKAR protein (Figure 33B) was also evaluated in wild-type and Lrgl1- knock out pancreatic tissue. The results show CCKAR mRNA and protein expression are increased in Lrgl1- knock out tissue.

[0707] Phosphorylation of TGFp associated non-canonical signalling proteins, including ALK5 / TGFp receptor 1 and AKT, was also evaluated in the pancreas of wild-type and Lrgl1- knock out mice (Figure 33C). Results show an increase TGFp associated non-canonical signalling (ALK5 / TGFB receptor 1) in the pancreas of Lrgl1- knock out mice.

[0708] Phosphorylation of TGFp associated signalling proteins was also measured in wild-type or Lrgl1- knock out acinar cells. Figure 33D shows an increase TGFp associated non-canonical signalling (ALK5 / TGFB receptor 1) in Lrgl1- knock out acinar cells compared to wild-type, and that this phenotype is reversed by the addition of recombinant LRG1 . Expression of CCKAR mRNA and CCKAR protein were also measured in wild-type or Lrgl1- knock out acinar cells. Figure 33E shows increased CCKAR mRNA and CCKAR protein expression in in Lrg1'- knock out acinar cells compared to wild-type, and that this phenotype is reversed by the addition of recombinant LRG1 .

[0709] Together these data show that LRG1 has a suppressive effect on of TGFp associated signalling proteins, and downstream expression of CCKAR.

[0710] To investigate this signalling pathway further, the effect of inhibiting TGFp associated signalling proteins on the expression of signalling proteins and CCKAR in acinar cells from Lrgl1- knock out mice was assessed (Figure 33F and Figure 33G). SB431542, a selective inhibitor of the ALK5 / Smad2 / 3 signalling cascade, reduced levels of phosphorylated ALK5 and AKT (Figure 33F), and also reduced CCKAR mRNA and protein expression in Lrgl1- compared to a DMSO control (Figure 33G). MK2206, an inhibitor of AKT, reduced levels of phosphorylated AKT (Figure 33F) and reduced levels of CCKAR mRNA and protein expression in Lrgl1- compared to a DMSO control (Figure 33G).

[0711] Example 18: Anti-LRG1 antibodies promotes ALK5 / AKT signalling to induce CCKAR expression To assess the therapeutic potential of LRG1 blocking antibodies, acute pancreatitis was induced in subject mice through injection of caerulein before administration of anti-LRG1 antibodies (Figure 34A).

[0712] Figure 34B and Figure 34C show that anti-LRG1 antibody EBC-103 treatment led to increased levels of phosphorylated ALK5 and AKT, CCKAR mRNA and CCKAR protein expression compared to treatment with a control antibody.

[0713] Figure 34D shows treatment with EBC-103 reduced pancreatic inflammation / overall damage compared to treatment with a control antibody, as analysed by H&E staining and pathology evaluation of pancreatic tissue. Figure 34E shows treatment with EBC-103 increased expression of anti-inflammatory NFKBIA and 1110 mRNA compared to treatment with a control antibody.

[0714] Figure 34F shows expression of amylase (AMY2) and cell cycle marker (CCNB and CCNE) mRNA was increased following treatment with ECB-103 compared to treatment with a control antibody. Figure 34G shows an increase in the number of Ki67+ AMY+ cells following treatment with ECB-103 compared to treatment with a control antibody. These results show that treatment with EBC-103 promotes pancreatic regeneration following induction of acute pancreatitis.

[0715] In summary, these results show that inhibition of LRG1 promotes pancreatic regeneration following induction of acute pancreatitis.

[0716] Example 19: Materials and methods relating to example 20 to 24

[0717] Human PDAC specimens Formalin-fixed, paraffin-embedded (PFFE) human pancreatitis tissues were obtained from TissueArray.com (#BIC14011 b) or SingHealth Tissue Repository (Singapore).

[0718] Animals and animal models

[0719] C57BL / 6N mice were purchased from InVivos (Singapore). Lrg1~ ~ mice on a C57BL / 6N background were purchased from the University of California, Davis, Knockout Mouse Project (KOMP) (http: / / www.komp.org). The orthotopic mouse model of PDAC was performed as previously described [5]. In brief, a left laparotomy was performed to expose the pancreatic body and to allow for direct injection of PDAC cells. The mouse model of liver metastasis was carried out as described [6], where a right laparotomy was performed to reveal the portal vein and PDAC cells were injected at approximately 10mm below the liver at an angle less than 5° to the vein. CP was induced in 8-week-old male mice via 7 times hourly intraperitoneal injections of supramaximal concentrations of caerulein (50pg / kg, #HY-A0190, MedChemExpress, USA), three times a week for a total of 6 weeks.

[0720] Histology and Immunofluorescence staining

[0721] Mouse pancreatic tissues were fixed in 4% paraformaldehyde (#158127, Sigma-Aldrich, USA) overnight before being embedded in paraffin following a standard protocol. Paraffin sections were subjected to hematoxylin-eosin staining, Masson’s Trichrome or Picrosirius Red staining followed by histopathological grading by an independent pathologist (Advanced Molecular Pathology Laboratory, Institute of Molecular and Cell Biology, A*Star, Singapore). In general, a score of 0 indicated that there were no abnormalities detected; 1 : minimal; 2: mild; 3: moderate; 4: marked; 5: severe. For pleomorphism, a semi-quantitative score of 1-fold, 2-fold, or 3-fold change compared to normal cells was determined. For immunofluorescence staining, paraffin sections were subjected to antigen retrieval using sodium citrate buffer before being stained with primary antibodies against LRG1 (#13224-1-AP, Proteintech, USA), CD31 (#ab28364, Abeam, UK), Cytokeratin 19 (#PAB12676, Abnova, USA), alpha-smooth muscle actin (a-SMA, #ab32575, Abeam, UK), N-Cadherin (CDH2, #13116, Cell SignalingTechnology, USA) and Ki67 (#ab15580; Abeam, UK) followed by staining with DAPI, Alexa 488, Alexa 594 and Alexa 657 secondary antibodies (Thermo Fisher Scientific, USA). Images were captured using EVOS M5000 Imaging System (Thermo Fisher Scientific, USA) or Nikon N-STORM with Andor CSU-W1 spinning disk (Nikon, Japan) and analysed using Adobe Photoshop software (Adobe Inc, USA).

[0722] Cells and cell culture

[0723] Murine PDAC cell line, KPC (#153474, Ximbio, UK) and human PDAC cell line, PANC-1 (#CRL-1469™, ATCC, USA) were maintained in high glucose DMEM (#11995, Life Technologies, USA) and further supplemented with 10% FBS (Gibco, USA). Cells were treated with ERBB2 inhibitor, Tucatinib (0.5pM, #HY-16069A, MedChemExpress, USA), AKT inhibitor, MK-2206 (10pM, #HY-10358 , MedChemExpress, USA), recombinant human TGF-01 (10ng / mL, #100-21-10, PeproTech, USA), PDGF-BB (20ng / mL, #100- 14B-10, PeproTech, USA), recombinant human LRG1 (200ng / mL, #7890-LR-025, R&D systems, USA) or LRG1 antibody (40ug / mL) as indicated.

[0724] Cell viability assay Cell viability was determined via 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay using CellTiter 96® AQueous One Solution reagent (#G3582, Promega, USA) as per manufacturer’s instructions. Absorbance readings at 490nm were measured using the Synergy H1 multi-mode reader (Biotek, USA) 3 hours post-incubation.

[0725] Cell Migration & Invasion assay

[0726] Transwell migration assay was carried out in 24-well (6.5mm in diameter, 8pm pore size) Transwell Permeable Support inserts (#3422, Corning, USA) coated overnight with 100pg / ml rat tail collagen I (#354236, Corning, USA). Transwell invasion assay was carried out in 24-well Transwell Permeable Support inserts precoated with Matrigel (#354234, Corning, USA) diluted to 300ug / mL in 0.01 M Tris (pH 8.0) and 0.7% NaCI coating buffer. Cells were seeded onto the upper chamber in serum free media and allowed to migrate through the membrane. Following which, migrated cells were fixed and stained with DAPI dye and then visualized using EVOS M5000 Imaging System (Thermo Fisher Scientific, USA). Image segmentation using appropriate intensity threshold values and particle analysis was then conducted on the images using Imaged software (National Institutes of Health, USA) to determine the total number of migrated cells.

[0727] Quantitative RT-PCR

[0728] Pancreatic tissues were stored in RNAIater™ Stabilization Solution (#AM7020, Thermo Fisher Scientific, USA). Total RNA was isolated and purified from the mouse pancreas or cultured cells using RNAeasy kit (#74106, Qiagen, USA) and NucleoSpin RNA kit (#740955, Macherey-Nagel, Germany) respectively, before being converted to cDNA using qScript cDNA Supermix (#157031 , Quanta Biosciences, USA). PCR was conducted with PrecisionFAST qPCR MasterMix (PPLUS-machine type-1 ML, Primer Design, UK) using Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies, USA). The expression levels of respective target genes were normalized to GAPDH, and relative gene expressions were calculated using standard 2-AACT.

[0729] SDS-PAGE and Western Blotting

[0730] Cells or tissues were lysed on ice in radioimmunoprecipitation assay buffer containing 1x protease inhibitor (1 tablet in 500uL, #04693116001 , Roche, Switzerland), 1 mmol / L dithiothreitol (#10197777001 , Sigma-Aldrich, USA), and 1 mmol / L phenylmethylsulfonyl fluoride (#P7626, Sigma-Aldrich, USA). Additional 100 mmol / L phosphatase inhibitors (#07575-51 , Nacalai Tesque, Japan) were used when detecting cell signalling transducers. Proteins were separated by 10% SDS-PAGE before being transferred onto an Immobilon-PSQ PVDF Membrane (#IPVH00010, Merck Millipore, USA). Blots were probed with LRG1 antibody (rabbit monoclonal, #13224-1 -AP, Proteintech), Cyclin D1 antibody (rabbit monoclonal, #2922, Cell Signaling Technology, CST), N-Cadherin antibody (rabbit monoclonal, #13116, CST), AREG antibody (rabbit polyclonal, #16036-1 -AP, Proteintech), phospho-EGFR antibody (rabbitmonoclonal, #3777; CST), EGFR antibody (rabbit monoclonal, #4267; CST), phospho-ErBb2 antibody (rabbit monoclonal, #2243; CST), ErBb2 antibody (rabbit monoclonal, #2165; CST), phospho- PDK1 antibody (rabbit monoclonal, #3438; CST), PDK1 antibody (rabbit polyclomal, #3062; CST), phospho-GSK3p antibody (rabbit monoclonal, #5558; CST), GSK3p antibody (rabbit polyclonal, #9315; CST), phospho-Akt antibody (rabbit monoclonal, #4060; CST), Akt antibody (rabbit monoclonal, #9272; CST), a-SMA Antibody (rabbit polyclonal, #ab5694, Abeam, UK), RPLPO antibody (rabbit polyclonal, #11290-2-AP, Proteintech, USA), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, USA). Densitometry was performed using Imaged.

[0731] Proteom e Profiler A ssays

[0732] Whole cell protein lysates were subjected to Proteome Profiler Mouse XL Cytokine Array (#ARY028, R&D systems, USA), Proteome Profiler Mouse Phospho-RTK Array Kit (#ARY014, R&D systems, USA) and Proteome Profiler Human Phospho-Kinase Array Kit (#ARY003C, R&D systems, USA) according to supplier’s instructions. Densitometry was performed using Imaged.

[0733] Molecular Biological Methods

[0734] The 6xHis-tagged coding sequence of human LRG1 (NM_052972) was cloned into the pcDNA 3.1 + plasmid backbone (Invitrogen, USA) to generate LRG1 expression vector, pLRG1. Small interfering RNA (siRNA) oligonucleotides (#L-015179-01 -0005, Dharmacon, USA) were used for LRG1 gene knockdown and control siRNA (#D-001810-01-05, Dharmacon, USA) was used as a negative control. Transfection was performed using Lipofectamine 3000 (#L3000015, Invitrogen, USA) according to the manufacturer’s protocol. For lentiviral stable cell transduction, KPC target cells were incubated overnight with lentiviral particles carrying pCDH or pCDH-LRG1 expression vector. Successfully transduced cells were subjected to fluorescent-activated cell sorting (FACS) using FACSMelody™ cell sorter (BD, USA). Cells successfully transduced with pCDH (or pCDH-LRG1) expressed CopGFP. GFP-positive KPC cells were collected and resuspended in complete growth medium for further cell population expansion. 2 rounds of cell sorting were conducted to ensure maximum enrichment of pCDH (or pCDH-LRG1) expressing KPC cells.

[0735] Bioinformatics

[0736] The GEPIA2 web server database (http: / / gepia2.cancer-pku.en / #index) featuring RNA sequencing expression data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project was used to determine differential mRNA expression of LRG1 in PDAC compared to normal pancreas tissue; and also used to validate the identified target gene signature unique to human PDAC compared to other gastroenterology associated cancers. LinkedOmicsKB (https: / / kb.linkedomics.org / ) portal containing data extracted from the Clinical Proteomic Tumour Analysis Consortium (CPTAC) database was used to determine LRG1 protein abundance in normal versus human PDAC tumour tissues. Kaplan-Meier survival analyses of PDAC patients with differential expression of LRG1 , AREG and IL1 RN were obtained from the Human Protein Atlas database (http: / / www.proteinatlas.org). Correlation AnalyzeR tool (https: / / gccri.bishop-lab.uthscsa.edu / shiny / correlation-analyzer / ) was used to determine the association of LRG1 with AREG and IL1 RN in human PDAC.

[0737] Statistics Data are presented as mean ± standard error of the mean (s.e.m.). Statistical analyses were performed by an unpaired, two-tailed Student’s t-test or one-way ANOVA followed by an appropriate post-hoc test using Prism 5 (GraphPAD Software Inc.). Statistical details for each experiment, including n values, are provided in figure legends.

[0738] Study approval

[0739] All animal experiments were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of Nanyang Technological University (IACUC ARF-LKC / A18026, A18094, A19109) and SingHealth (2020 / SHS / 1595, 1596), Singapore, and the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health.

[0740] Example 20: LRG1 is uprequlated in human Pancreatic Ductal Adenocarcinoma (PDAC)

[0741] To evaluate the role of LRG1 in human pancreatic ductal adenocarcinoma (PDAC), LRG1 mRNA and protein expression was analysed in PDAC tumour tissue and normal tissue. Figure 35A shows increased LRG1 mRNA and protein expression in PDAC tumour tissue compared to normal tissue. Figure 35B shows higher levels of serum LRG1 concentration are associated with worse survival probability.

[0742] LRG1 localisation in PDAC low- and high-grade tumour tissue was then evaluated using immunofluorescence staining (Figure 35C). The staining revealed that LRG1 colocalises with KRT19+ tumour ductal cells in both low and high grade tumour tissue.

[0743] Example 21 : LRG1 promotes tumour growth, epithelial-mesenchymal transition (EMT) and metastasis in vivo.

[0744] To establish the contribution of LRG1 to PDAC tumour growth, an orthotopic mouse model of PDAC was used (Figure 36A). Tumour weight and volume were then assessed (Figure 36B). PDAC tumours overexpressing LRG1 had increased weight and volume compared to those tumours transduced with a control vector.

[0745] Figure 36C shows that LRG1 -overexpressing tumours had increased Ki67+ cell proliferation compared to control tumours. Figure 36D shows cyclin D (Ccnd) mRNA was increased in LRG1 -overexpressing tumours compared to control tumours.

[0746] Neoplastic cell and neoplastic nuclear pleomorphism in PDAC tumours overexpressing LRG1 was then assessed following staining of tumour tissue (Figure 36E). Results show a greater fold change in the level of neoplastic cell and neoplastic nuclear pleomorphism in LRG1 -overexpressing tumours compared to control tumours.

[0747] PDAC tumour tissue was then stained for mesenchymal N-cadherin and the quantity of cadherin (CDH2) mRNA was evaluated by qRT-PCR (Figure 36F and Figure 36G). The results of both assays show that cadherin expression is increased in LRG1 -overexpressing tumours compared to control tumours. Figure 36H shows that liver weight relative to body wight, and metastatic area as a proportion of total liver are increased in LRG1 -overexpressing tumours compared to control tumours.

[0748] Example 22: LRG1 promotes cell proliferation, migration and invasion in-vitro.

[0749] Call viability assays were conducted to assess cell proliferation in human PDAC cells (PANC1-) transformed with either an empty control vector (pcDNA) or an LRG1 expressing vector (pLRG1). Results show an increase in cell proliferation in PDAC cells overexpressing LRG1 compared to the control (Figure 37A). Cell viability was also assed in PDAC cells in which LRG1 expression was knocked down by siRNA (siLRGI). The results show that knocking down LRG1 using siRNA decreases cell proliferation compared to cells treated with control siRNA (siCtrl) (Figure 37B).

[0750] Figure 37C shows that LRG1 -overexpressing PDAC cells (pLRG1) display increased migration and invasion compared to control PDAC cells (pcDNA). Figure 37D shows that PDAC cells in which LRG1 expression was knocked down by siRNA (siLRGI) display reduced cell migration and invasion compared to control cells (siCtrl).

[0751] Figure 37E shows increased expression of proliferation marker, cyclin D (CCND) protein and epithelial- mesenchymal transition (EMT) marker, N-Cadherin (CDH2) was increased in LRG1 -overexpressing PDAC cells (pLRG1) compared to control PDAC cells (pcDNA). Figure 37F shows expression of cyclin D and N-Cadherin was decreased in PDAC cells in which LRG1 expression was knocked down by siRNA (siLRGI) compared to control cells (siCtrl).

[0752] Example 23: LRG1 promotes tumoriqenesis via EGFR / ErbB / AKT signalling.

[0753] To establish the mechanism through which LRG1 overexpression promotes tumour growth, dot blot profiler assays were performed to measure the expression of amphiregulin (an EGFR ligand) in LRG1- overexpressing KPC cells (a mouse PDAC cell line). Figure 38A shows that expression of amphiregulin is increased in KPC cells overexpressing LRG1 compared to control cells (KPC cells transduced with a control vector). Levels of amphiregulin (Areg) mRNA was also assessed by qRT-PCT in KPC cells and tumour overexpressing LRG1. Figure 38B shows that expression of amphiregulin is increased in both in LRG1 -overexpressing KPC cells and in LRG1 -overexpressing KPC tumour compared to controls.

[0754] Further dot blot profiler assays were performed to assess EGFR, ErBb2, AKT and GSK associated signalling. Figure 38C and Figure 38D show increased pEGFR, pErBb2, pAKT and pGSK in LRG1- overexpressing cells compared to control cells. The results show that LRG1 overexpression activates EGFR, ErBb2, AKT and GSK and associated signalling pathways.

[0755] To evaluate EGFR / ErbB / AKT signalling further, the ErBb2 inhibitor Tucatinib and the AKT inhibitor MK2206 were used to treat KPC cells overexpressing LRG1 and the phosphorylation level of EGFR / ErbB / AKT associated signalling proteins was analysed by Western blot. Figure 38E shows that the addition of the ErBb2 and AKT inhibitors inhibits EGFR / AKT associated signalling in KPC cells overexpressing LRG1 Cell viability in KPC cells overexpressing LRG1 treated with Tucatinib and MK2206 was then measured to assess KPC proliferation. Figure 38F shows that treatment with either Tucatinib or MK2206 reduces proliferation of KPC cells overexpressing LRG1 compared to a control treated with DMSO. The effect of Tucatinib and MK2206 on KPC cell migration and invasion was also evaluated. Figure 38G shows that treatment with either Tucatinib or MK2206 inhibitors also reduces migration and invasion of KPC cells overexpressing LRG1 compared to control cells treated with DMSO.

[0756] Taken together these results show that LRG1 is promoting tumorigenesis via EGFR / ErbB / AKT associated signalling pathways.

[0757] Example 24: Administration of LRG1 neutralising antibodies inhibits tumour growth

[0758] Having established that LRG1 expression promotes tumorigenesis, the effect of neutralising LRG1 in PDAC cells was evaluated using anti-LRG1 antibodies.

[0759] Figure 39A shows treatment of LRG1 -overexpressing PDAC cells with anti-LRG1 antibody EBC-61 was reduced compared to treatment with a control antibody. Figure 39B shows treatment of LRG1- overexpressing PDAC cells with EBC-61 reduced cell migration and invasion compared to treatment with a control antibody. Figure 39C shows treatment of LRG1 -overexpressing PDAC cells with EBC-61 reduced expression of cyclin D (CCND) and N-Cadherin (CDH2) compared to treatment with a control antibody.

[0760] In summary, these results show that LRG1 blocking antibodies can reduce proliferation, migration and invasion of PDAC cells.

[0761] Example 25: LRG1 is elevated in Rheumatoid Arthritis (RA)

[0762] Methods: Collagen Induced Arthritis (CIA) mouse model

[0763] A collagen-induced arthritis (CIA) mouse model was produced according to published protocols [7,8]. In brief, complete Freud Adjuvant (CFA) was prepared by adding 100mg Heat-Killed Mycobacterium Tuberculosis (H37Ra; BD Biosciences) to 20ml of Incomplete Freud Adjuvant (IFA; InVivoGen). Type II Chicken Collagen (Sigma) was dissolved in acetic acid overnight at 4°C. Egual volume of dissolved type II chicken collagen was then added drop by drop to the CFA over ice to form a white emulsion. 10- to 13- week-old wildtype (WT) or LRG1 knockout (KO) mice were then injected at 2 different sites intra-dermally at the base of the tail with 10Oul of emulsion containing 100 pg of type II chicken collagen and 250 pg of M. tuberculosis. The same injection was repeated on day 14 as a booster.

[0764] Circulating and synovial LRG1 levels are significantly higher in RA patients

[0765] Results showed that LRG1 is expressed at significantly higher levels in the serum (Figure 40A; left panel), synovial tissue (Figure 40A; middle panel), and synovial fluid (Figure 40A; right panel) of human RA patients as compared to those of age-matched osteoarthritis (OA) patients. Primary fibroblast-like synoviocytes (FLS) were isolated from RA and OA patients and examined. Although no significant changes in cell morphology were observed, FLS isolated from RA patients express higher LRG1 than those from OA patients at both protein and mRNA levels (Figure 40B). Together, these data established a strong association between elevated LRG1 levels and RA.

[0766] LRG1 regulates the function of multiple cell types present in the synovium

[0767] Disruption in endothelial barrier function and integrity leads to increased blood vessel permeability which is essential for immune cell infiltration into the injured tissue. Results shows that rhLRGI treatment leads to abnormal distribution of EC adherens junction protein VE-cadherin as demonstrated by immunofluorescence staining (Figure 41 A). Consistent with this observation, Lrg1 - / - mice are resistant to the VEGF-induced increase in vascular permeability as demonstrated by Mile’s assay (Figure 41 B).

[0768] Moreover, rhLRGI promotes synovial EC proliferation, as demonstrated by immunofluorescence staining with a cell proliferation marker Ki67 (Figure 41 C), migration across the Transwell (Figure 41 D), and the ability to form tube-like structure in Matrigel (Figure 41 E).

[0769] Furthermore, rhLRGI promotes neutrophil adhesion (Figure 41 F) to and migration (Figure 41 G) across the synovial EC monolayer. In RA, FLS become hyperproliferative. An MTS assay show that rhLRGI treatment leads to a significant increase in the FLS proliferation rate (Figure 41 H). The invasion of hypertrophied synovium involves the destruction of cartilage and non-osseous support structure in the joint tissue. This process is mainly caused by the increased production of matrix-degrading enzymes MMPs by FLS. qRT-PCR study show that the expression of MMP9 is significantly increased in FLS following the treatment with rhLRGI (Figure 411). Together, these data show that LRG1 can regulate the function of multiple cell types present in the synovial microenvironment.

[0770] Lrg1 deficient mice are resistant to Collagen Induced Arthritis (CIA)

[0771] CIA is the most widely used animal model of RA. It shares several pathological features with RA in humans. To access the role of LRG1 in the development and progression of RA, CIA was established in Lrg 1 - / - and age-matched control mice. Images show that the development of RA, as indicated by the level of swelling of the affected limb, was significantly alleviated in Lrg1 - / - mice (Figure 42A, left). The development and progression of RA were also evaluated by the total severity score of each paw over the time course of 60 days. 0 indicates normal, 1 indicates erythema, 2 indicates erythema and swelling, and 3 indicates extension / loss of function. The results show that the onset of RA in Lrg1 - / - mice is 12 days later than that in wild-type controls and Lrg 1 - / - mice demonstrate much milder symptoms (Figure 42A, right). This observation was further supported by immunohistochemistry staining which demonstrated reduced inflammation in the joint of Lrg 1 - / - mice following CIA as compared to that in wild-type control mice (Figure 42B). Together, the data show a causative role of LRG1 in RA pathology and that inhibition / reduction of LRG1 activity reduces RA pathology. Example 26: LRG1 inhibition in cancers

[0772] 26.1 Materials and Methods

[0773] Mouse model for Pancreatic Ductal Adenocarcinoma (PDAC)

[0774] The orthotopic mouse model of PDAC was performed (Qiu et al, Methods Mol Biol, 2013). In brief, a left laparotomy was performed to expose the pancreatic body and to allow for direct injection of 1 .5 x 105LRG1 -overexpressing KPC (pLrgl) cells suspended in 50pL of saline. 2 weeks after the establishment of PDAC tumour, mice were randomized into treatment groups and 50mg / kg of control lgG1 isotype or EBC 61 were injected intraperitoneally in mice every other day for another 2 weeks. Mice were sacrificed 4 weeks post-tumour implantation, and thereafter, the tumour-bearing pancreas was excised, and tumour dimensions were measured using a calliper. Tumour volume was calculated using the formula: V= 0.5 x [_ x W2, where V is the tumour volume, L is the tumour length, and W is the tumour width.

[0775] Timeline for PDAC mouse model: wkO, tumour induction 1.5x105pLrgl cells; wk2, EBC61 injection I.P, 50mg / kg every other day; wk4, endpoint.

[0776] Mouse model for melanoma

[0777] 2 x 106B16F10 melanoma cells suspended 1 OOpI PBS were injected subcutaneously into the left flank of six- to eight-week-old wild-type C57BL / 6 mice. Mice were monitored daily. Once tumours became palpable, mice were randomized into treatment groups and 200ug / 50pL of control lgG1 isotype or EBC antibodies (EBC103, EBC107, EBC78) were injected intratumorally every other day. Tumours were measured using a caliper and tumor volume was calculated using the formula: V= 0.5 x L x VV2, where V is the tumor volume, L is the tumor length, and W is the tumor width. Mice were sacrificed at the end of the experiment after 9 days following the first injection. B16F10 cells were selected based on highest LRG1 expression among various melanoma cancer cell lines (A375, B16F10, SK-MEL2, SK-MEL28).

[0778] Timeline for melanoma mouse model: day -5 to day -7, tumour induction 2x10A6 cells; day 1 , antibody injection (once tumour palpable) 200ug / 50pL, intratumoural, every other day; day 9, endpoint.

[0779] Mouse model for colorectal cancer (CRC)

[0780] 2.5 x 106HT-29 colorectal cancer cells suspended 1 OOpI PBS were injected subcutaneously into the left flank of six- to eight-week-old Balb / c Nude mice. Mice were monitored daily. Once tumours became palpable, mice were randomized into treatment groups and 200ug / 50pL of control lgG1 isotype or EBC antibodies (EBC103, EBC107, EBC78) were injected intratumorally every other day. Tumours were measured using a caliper and tumor volume was calculated using the formula: V= 0.5 x L x VV2, where V is the tumor volume, L is the tumor length, and W is the tumor width. Mice were sacrificed at the end of the experiment after 18 days following the first injection. HT-29 cells were selected based on highest LRG1 expression among various CRC cancer cell lines (Caco-2, HCT116, HT-29, SW480, RKO).

[0781] Timeline for CRC mouse model: day -5 to day -7, tumour induction 2.5x10A6 cells; day 1 , antibody injection (once tumour palpable) 200ug / 50pL, intratumoural, every other day; day 18, endpoint. Mouse model for Non-Small Cell Lung Cancer (NSCLC)

[0782] 1 .5 x 106Lewis lung carcinoma (LL2) cancer cells suspended 10OpI PBS were injected subcutaneously into the left flank of six- to eight-week-old wild-type C57BL / 6 mice. Mice were monitored daily. Once tumours became palpable, mice were randomized into treatment groups and 200ug / 50pL of control lgG1 isotype or EBC antibodies (EBC103, EBC107, EBC78) were injected intratumorally every other day. Tumours were measured using a caliper and tumor volume was calculated using the formula: V= 0.5 x L x W2, where V is the tumor volume, L is the tumor length, and W is the tumor width. Mice were sacrificed at the end of the experiment after 9 days following the first injection.

[0783] Timeline for NSCLC mouse model: day -5 to day -6, tumour induction 1.5x10A6 LL / 2 cells; day 1 , antibody injection (once tumour palpable) 200ug / 50pL, intratumoural, every other day; day 9, endpoint.

[0784] Cell viability assay

[0785] Cell viability / growth was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay with the CellTiter 96® AQueous One Solution reagent (#G3582, Promega, USA) according to the manufacturer’s instructions. Absorbance was measured at 490nm using the Synergy H1 multi-mode reader (Biotek, USA) 3 hours post-incubation.

[0786] Cell Migration & Invasion assay

[0787] The transwell migration assay was performed using 24-well Transwell Permeable Support inserts (6.5mm in diameter, 8pm pore size, #3422, Corning, USA) coated overnight with 100 pg / ml rat tail collagen I (#354236, Corning, USA). The transwell invasion assay was conducted in 24-well Transwell Permeable Support inserts precoated with Matrigel (#354234, Corning, USA) diluted to 300ug / mL in 0.01 M Tris (pH 8.0) and 0.7% NaCI coating buffer. Cells were seeded onto the upper chamber in serum-free media and allowed to migrate through the membrane. After incubation, the migrated cells were fixed, stained with DAPI, and visualized using the EVOS M5000 Imaging System (Thermo Fisher Scientific, USA). Image segmentation was performed using appropriate intensity threshold values and particle analysis was conducted on the images using Imaged software (National Institutes of Health, USA) to quantify the total number of migrated cells.

[0788] Quantitative RT-PCR

[0789] Total RNA was extracted from tumour tissues using the RNAeasy kit (#74106, Qiagen, USA) and NucleoSpin RNA kit (#740955, Macherey-Nagel, Germany) respectively. The RNA was then converted to cDNA using qScript cDNA Supermix (#157031 , Quanta Biosciences, USA). PCR was performed with PrecisionFAST qPCR MasterMix (PPLUS-machine type-1 ML, Primer Design, UK) using Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies, USA). Gene expression levels were normalized to GAPDH, and relative expressions were calculated using standard 2-AACT. PCR primer sequences are listed in Table 4.

[0790] Table 4. qRT-PCR primer sequences

[0791] SDS-PAGE and Western Blotting

[0792] Cells or tissues were lysed on ice in radioimmunoprecipitation assay buffer containing 1x protease inhibitor (1 tablet in 500uL, #04693116001 , Roche, Switzerland), 1 mmol / L dithiothreitol (#10197777001 , Sigma-Aldrich, USA), and 1 mmol / L phenylmethylsulfonyl fluoride (#P7626, Sigma-Aldrich, USA).

[0793] Additional 100 mmol / L phosphatase inhibitors (#07575-51 , Nacalai Tesque, Japan) were added for detecting cell signalling transducers. Proteins were separated by 10% SDS-PAGE before being transferred onto an Immobilon-PSQ PVDF Membrane (#IPVH00010, Merck Millipore, USA). Blots were probed with Cyclin D1 antibody (rabbit monoclonal, #2922, Cell Signaling Technology, CST), N-Cadherin antibody (rabbit monoclonal, #13116, CST), E-Cadherin (rabbit monoclonal antibody, #3195, CST), GAPDH antibody (mouse monoclonal, #sc-32233, Santa-Cruz Biotechnology, USA), followed by horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, USA). Densitometry was performed using Imaged.

[0794] 26.2 Results

[0795] Leucine-rich-alpha 2 glycoprotein 1 (LRG1) is a multifunctional protein known to be upregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDAC), melanoma, colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). The following results validate the efficacy of LRG1 blockage as a broad-spectrum oncology therapeutic tool.

[0796] Pancreatic Ductal Adenocarcinoma (PDAC)

[0797] Mice implanted with Lrg1 -overexpressing (pCDH-LRG1) KPC tumours were intraperitoneally injected with EBC-61 . Importantly, we showed EBC-61 treatment resulted in a significant reduction of PDAC tumour volume compared to IgG controls (Figure 39D & 39E). Consistently, treatment with EBC-61 antibody also significantly inhibited PDAC cell growth in-vitro (Figure 39A), migration and invasion (Figure 39B). Levels of proliferative marker, cyclin D, CCND and marker of epithelial-mesenchymal transition, N-cadherin, CDH2 were also reduced following antibody treatment (Figure 39C). These results demonstrate that targeting LRG1 attenuates PDAC cell tumorigenesis and hence cancer development.

[0798] Melanoma

[0799] Mice subcutaneously inoculated with B16F10 melanoma cells were subjected to intratumoral injection of IgG control, EBC-78 (Magacizumab), EBC-103 or EBC-107. We showed that both EBC-103 and EBC- 107 treatment resulted in a comparable and significant reduction in melanoma tumour growth (Figure 43A & 43B) and tumour volume (Figure 43C) compared to IgG controls. EBC-103 and EBC-107 were also superior to EBC-78 in its anti-tumoral effect (Figure 43C). Consistently, gene expression levels of Ki67, a marker of cell proliferation, were markedly reduced in tumours treated with EBC-103 and EBC-107 compared to IgG controls or EBC-78 (Figure 43D). Additionally, EBC-107 treated tumours also demonstrated a significant reduction in gene expression levels of N-Cadherin, a marker of epithelial- mesenchymal transition (Figure 43E). These results provide evidence that targeting LRG1 is beneficial in attenuating melanoma development.

[0800] Colorectal Cancer (CRC)

[0801] Mice subcutaneously inoculated with HT-29 CRC cells were subjected to intratumoral injection of IgG control, EBC-78 (Magacizumab), EBC-103 or EBC-107. We showed that EBC-103 treatment inhibited CRC tumour growth (Figure 44A & 44B) and tumour volume (Figure 44C) compared to IgG controls. EBC-103 was also superior to EBC-107 and EBC-78 in its anti-tumoral effect (Figure 44C). Consistently, treatment with EBC-103 antibody also significantly inhibited HT-29 cell growth in-vitro (Figure 44D & 44E). Levels of E-cadherin, an epithelial marker were increased, and levels of proliferative marker, cyclin D, CCND1 were reduced following antibody treatment (Figure 44F). These results demonstrate that targeting LRG1 attenuates CRC cell tumorigenesis and hence cancer development.

[0802] Non-small cell lung cancer (NSCLC)

[0803] Mice subcutaneously inoculated with LL / 2 Lewis lung carcinoma cells were subjected to intratumoral injection of IgG control, EBC-78 (Magacizumab), EBC-103 or EBC-107. We showed that both EBC-103 and EBC-107 treatment resulted in a comparable and significant reduction in NSCLC tumour growth (Figure 52A & Figure 52B) and tumour volume (Figure 52C) compared to IgG controls. EBC-103 and EBC-107 were also superior to EBC-78 in its anti-tumoral effect (Figure 52C). These results demonstrate that targeting LRG1 attenuates NSCLC development.

[0804] Example 27: LRG1 inhibition in diabetic nephropathy (DN)

[0805] 27.1 Materials and Methods

[0806] Cell culture

[0807] All cells used were cultured in a humidified incubator kept at 5% CO2 and 37°C. Primary human glomerular mesangial cells (HMCs) (#4200, ScienCell Research Laboratories, USA) were maintained in Mesangial Cell Medium supplemented with 2% FBS, 1% Mesangial Cell Growth Supplement and 1% Penicillin-Streptomycin as specified (#4201 , ScienCell Research Laboratories, USA). Cells were further supplemented with 30mmol / L D-glucose to mimic high glucose conditions. Only cells between passages 5 to 10 were used for the study. Recombinant human TGF-01 (#100-21-10, PeproTech, USA) was dissolved in 10mM citric acid and used at a concentration of 10ng / mL. IgG or EBC- antibodies were used at a concentration of 40ug / mL.

[0808] Animal model

[0809] 12-week-old '\29S6-Akita-ReninTg mice (AR mice), a mouse model of human diabetic nephropathy (DN) carrying the lns2Akitamutation and renin transgene [ReninTg] activation on a susceptible 129S background, were obtained from Professor Thomas Coffman. Mice were subjected to once a week, intraperitoneal administration of 40mg / kg EBC-107 antibody for 6 weeks. During the experimental period, body weights were measured weekly. Blood glucose was measured using a glucose monitoring system (AlphaTRAK, Abbott) and 24-hour urine samples were collected in metabolic cages prior to the start of injection at 12 weeks and before endpoint (18 weeks). Urine albumin levels were measured using an enzyme-linked immunosorbent assay (Albuwell M, Exocell). Kidney tissues were collected at endpoint for further histological analysis.

[0810] Masson’s Trichrome staining and quantification

[0811] Tissue sections were stained with Masson’s Trichrome (#ab150686, Abeam, UK) according to manufacturer’s instructions with slight modifications. Briefly, slides were preheated in Bouin’s fluid at 60°C for 1 hour before being rinsed in tap water for 10 minutes. Slides were then stained in Weigert’s Iron Haematoxylin solution for 5 minutes followed by another rinse in running tap water for 2 minutes. Biebrich Scarlet-acid Fuchsin solution was then applied to the slides for 1 minute before differentiating in phosphomolybdic phosphotungstic acid solution for another 15 minutes. Aniline blue was then added to the slides for 20 minutes. Slides were then briefly rinsed in distilled water. Finally, 1% acetic acid was applied to slides for 5 minutes before routine dehydration using xylene.

[0812] For quantification of Masson’s Trichrome staining, up to 7 random field of view of kidney sections were imaged. The colour deconvolution plugin in Imaged software (National Institutes of Health, USA) was then used for pigment separation. In brief, a rectangular region of interest (ROI) was manually drawn over an area in the image where only one colour of the histological stain was represented. This process was repeated for the other colours present. The algorithm within the plugin then defined a colour matrix and unmixed the initial RGB image to generate 3 separate channels. Image threshold was then applied to the channel of interest (i.e., blue channel for Masson’s Trichrome) and used to quantify Masson’s Trichrome positive area. The resultant value obtained was then divided by the total area of the tissue section to determine the ratio of the Masson’s Trichrome positive area over the total kidney area.

[0813] Quantitative RT-PCR

[0814] Total RNA was extracted from kidney tissues using the RNAeasy kit (#74106, Qiagen, USA) and NucleoSpin RNA kit (#740955, Macherey-Nagel, Germany) respectively. The RNA was then converted to cDNA using qScript cDNA Supermix (#157031 , Quanta Biosciences, USA). PCR was performed with PrecisionFAST qPCR MasterMix (PPLUS-machine type-1 ML, Primer Design, UK) using Applied Biosystems StepOnePlus™ Real-Time PCR System (Life Technologies, USA). Gene expression levels were normalized to p-actin, and relative expressions were calculated using standard 2-AACT. PCR primer sequences are listed in Table 5.

[0815] Table 5. qRT-PCR primer sequences SDS-PAGE and Western Blotting

[0816] Cells or tissues were lysed on ice in radioimmunoprecipitation assay buffer containing 1x protease inhibitor (1 tablet in 500uL, #04693116001 , Roche, Switzerland), 1 mmol / L dithiothreitol (#10197777001 , Sigma-Aldrich, USA), and 1 mmol / L phenylmethylsulfonyl fluoride (#P7626, Sigma-Aldrich, USA). Additional 100 mmol / L phosphatase inhibitors (#07575-51 , Nacalai Tesque, Japan) were added for detecting cell signalling transducers. Proteins were separated by 10% SDS-PAGE before being transferred onto an Immobilon-PSQ PVDF Membrane (#IPVH00010, Merck Millipore, USA). Blots were probed with Collagen 1A1 antibody (mouse monoclonal, #H00001277-M01 , Abnova, USA), Collagen IV antibody (rabbit polyclonal, #PA1-28534, Thermofisher Scientific, USA), p-actin antibody (HRP conjugated, #HRP-60008, Proteintech, USA), followed by horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, USA). Densitometry was performed using Imaged.

[0817] 27.2 Results

[0818] Diabetes nephropathy (DN) is a common renal disease associated with long-term diabetes mellitus. DN occurs in approximately 30-35% of patients with type 1 and type 2 diabetes, and it is one of the leading causes of end-stage renal disease (ESRD) globally. LRG1 has been implicated in the pathogenesis of DN. Many studies demonstrated that LRG1 levels were elevated in urine, serum, or kidney tissues of individuals with DN. The following results validate the efficacy of LRG1 blockage in the management of DN.

[0819] Mesangial cell expansion or an increased deposition of extracellular matrix proteins or fibrosis is a key pathological feature of DN. Here, we showed that the addition of high glucose and transforming growth factor-p1 (TGF-01) resulted in a significant deposition of pro-fibrotic factors, connective tissue growth factor (CTGF) and fibronectin (FN1) (Figure 45A-45C) in primary human glomerular mesangial cells. Importantly, treatment with LRG1 neutralizing antibodies, EBC-58, EBC-59, EBC-60, EBC-61 , EBC-103 and EBC-107 resulted in a significant reduction in CTGF expression compared to IgG treated controls (Figure 45B). EBC-59, EBC-103 and EBC-107 treatment further reduced the expression levels of FN1 compared to controls (Figure 45C). These results suggest that targeting LRG1 inhibits mesangial cell expansion in DN. Amongst the LRG1 neutralizing antibodies tested, EBC-107 demonstrated the greatest overall anti-fibrotic effect on mesangial cell expansion and was subjected to further in-vivo testing.

[0820] To evaluate the therapeutic potential of LRG1 inhibition in DN, we made use of the AR mouse model which effectively recapitulates human DN. Compared to its wild-type counterparts, AR mice demonstrated an increased expression of Lrg1 in the kidneys (Figure 53A). Albuminuria, as determined by urinary albumin excretion (UAE) rates, is a key indicator of the extent of kidney damage in DN. Importantly, while UAE increased in untreated AR mice from 12- to 18-weeks, mice treated with EBC-107 demonstrated a robust reduction in UAE from 12- to 18-weeks (Figure 53B). Indeed, at 18-weeks old, there was a significant decrease in UAE in EBC-107 treated AR mice compared to the untreated controls (Figure 53C), thus suggesting an improvement in kidney function following LRG1 inhibition. Additionally, the extent of glomerular and tubulointerstitial fibrosis (Figure 53D) as determined by the ratio of Masson’s Trichrome-stained positive area over total kidney area (Figure 53E) were also reduced in EBC-107 treated AR mice compared to their respective controls. Consistently, treatment with EBC-107 also resulted in a significant decrease in expression of fibrotic proteins, COL1A1 , COL4 and FN1 (Figures 53F, 53G and 53H), as well as an increased expression of matrix-degrading enzymes like MMP-2 (Figure 53I). Notably, EBC-107 treatment also resulted in a significant decrease in kidney expression of a Kidney Injury Molecule 1 (KIM-1) (Figure 53J), a tubular cell marker associated with kidney damage as well as markers of kidney inflammation including monocyte chemoattractant protein-1 (MCP-1 / CCL2) (Figure 53K), CCL5 (Figure 53L), and interleukin-6, (IL-6) (Figure 53M). Taken together, these results demonstrate that LRG1 inhibition is protective in DN.

[0821] Example 28: Characterization of non-LRC targeting LRG1 antibody (EBC-103, EBC-107)

[0822] Our previous data validated the anti-angiogenic and anti-fibrosis capacities of LRC targeting neutralizing antibodies. Now, we guery if the non-LRC targeting neutralizing antibodies could also exert such effect. Hence, non-LRC targeting antibodies, EBC 107 and 103, were generated and assessed. We first conducted western blot analysis to examine the binding capacity of these antibodies (Figure 46). Recombinant human LRG1 protein and mouse liver lysates were first denatured and resolved using SDS- PAGE, then the cross-reactivity of various antibodies were assessed using western blots. Results suggest that all the tested antibodies bind to both human and mouse-derived LRG1 protein.

[0823] Next, metatarsal angiogenesis assay was conducted (Figure 47). Explants were treated with rhLRGI and LRC-targeting (EBC 58) as well as non-LRC targeting antibodies (EBC 107 and EBC 103). Endpoint CD31 -staining confirmed that all antibodies tested showed an antiangiogenic effect. Explants treated with EBC 107 and EBC 103 demonstrates more modest reduction vascularity when compared to the explants that were treated with EBC 58. LRC-targeting antibodies reduced vascularity to a greater extent than non- LRC targeting antibodies.

[0824] Example 29: LRG1 -neutralizing antibodies in inflammatory bowel disease

[0825] Inflammatory bowel disease (IBD), which comprises Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic inflammation associated with impaired epithelial barrier function and intestinal immune dysregulation. LRG1 has been previously reported to be implicated in IBD, elevation of LRG1 has been associated with worse disease progression [9, 10]. As such, antibody blockade of LRG1 may present as a novel therapeutic strategy. Here, we adopted the widely used dextran sodium sulfate (DSS)- induced experimental colitis model to elucidate the therapeutic efficacy of LRG1-neutralizing antibodies in mitigating the pathogenesis of IBD (Figure 48 for schematic illustration of acute colitis model). Male 6-10- week-old C57BL / 6 wild-type mice were fed with 2.5 % DSS (MW ~30 KDa)-spiked drinking water for 7 days and subseguently switched back to normal drinking water for another 3 days. The mice were randomly assigned to different experimental groups and 10mg / kg of LRG1 -neutralizing antibody was administered intraperitoneally daily as preventive treatment against acute intestinal inflammation. Mice were monitored daily, disease activity / severity index tabulated according to published guide

[0011] . DSS- induced IBD is fundamentally associated with damage of the colon epithelial integrity, thus leading to a reduction in its length and function over time. Our data demonstrated attenuated acute colitis following the preventive treatment of EBC 107 or 103 (10mg / kg). We observed overall lower DAI (Figure 48C) and concomitant preservation of colon length (Figure 48E) following the daily injections of EBC 107 or 103 when compared to the EBC146 isotype control group.

[0826] At experimental endpoint, the colonic tissues were immediately fixed in 4% formaldehyde solution and made into wax blocks. Then wax bl...

Claims

Claims:1 . An LRG1 inhibitor for use in a method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation.

2. Use of an LRG1 inhibitor in the manufacture of a medicament for use in the treatment or prevention of a disease or condition characterised by fibrosis and / or inflammation.

3. A method of treating or preventing a disease or condition characterised by fibrosis and / or inflammation, comprising administering a therapeutically- or prophylactically-effective amount of an LRG1 inhibitor to a subject.

4. The LRG1 inhibitor for use according to claim 1 , the use according to claim 2, or the method according to claim 3, wherein the LRG1 inhibitor: (i) inhibits interaction between LRG1 and an interaction partner for LRG1 ; or (ii) reduces the expression of LRG1 or an interaction partner of LRG1 .

5. The LRG1 inhibitor for use, the use, or the method according to claim 4, wherein the LRG1 inhibitor binds to LRG1 , optionally wherein the LRG1 inhibitor binds to the LRRCT region of LRG1 or the LRR region of LRG1 .

6. The LRG1 inhibitor for use, the use, or the method according to claim 5, wherein the LRG1 inhibitor is a peptide / polypeptide, nucleic acid or small molecule.

7. The LRG1 inhibitor for use, the use, or the method according to claim 6, wherein the LRG1 inhibitor is an antibody or antigen-binding fragment thereof.

8. The LRG1 inhibitor for use, the use, or the method according to claim 4, wherein the LRG1 inhibitor is an inhibitory nucleic acid capable of reducing expression of LRG1 by RNA interference (RNAi).

9. The LRG1 inhibitor for use, the use, or the method according to claim 4, wherein the LRG1 inhibitor is capable of modifying a gene encoding LRG1 to reduce its expression.

10. The LRG1 inhibitor for use, the use, or the method according to claim 9, wherein the LRG1 inhibitor comprises a site-specific nuclease (SSN) targeting a gene encoding LRG1 .

11. The LRG1 inhibitor for use according to any one of claims 1 or 4 to 10, the use according to any one of claims 2 or 4 to 10, or the method according to any one of claims 3 or 4 to 10, wherein the disease or condition is characterised by: (i) fibrosis and / or inflammation of the eye; (ii) fibrosis and / or inflammation of the pancreas; (iii) fibrosis and / or inflammation of the joints; (iv) fibrosis and / or inflammation of the skin; (v) fibrosis and / or inflammation of the bowel; (vi) fibrosis and / or inflammation of the kidney; or (vii) fibrosis and / or inflammation of the lungs.

12. The LRG1 inhibitor for use according to any one of claims 1 or 4 to 11 , the use according to any one of claims 2 or 4 to 11 , or the method according to any one of claims 3 or 4 to 11 , wherein the disease or condition is characterised by pathological angiogenesis.

13. The LRG1 inhibitor for use according to any one of claims 1 or 4 to 12, the use according to any one of claims 2 or 4 to 12, or the method according to any one of claims 3 or 4 to 12, wherein the disease or condition is selected from: macular degeneration, Age-related Macular Degeneration (AMD), Geographic Atrophy (‘dry’ or non-exudative AMD), early AMD, early onset macular degeneration (EOMD), intermediate AMD, late / advanced AMD, ‘wet’ (neovascular or exudative) AMD, choroidal neovascularisation (CNV), retinal dystrophy, glaucoma (open-angle or closed-angle), neuromyelitis optica (neuromyelitis optica spectrum disorder (NMOSD)), diabetic macular edema (DME), diabetic retinopathy, proliferative diabetic retinopathy (PDR), polypoidal choroidal vasculopathy, proliferative vitreoretinopathy (PVR), macular edema, drusen formation, Grave's ophthalmopathy, corneal opacification, subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis, preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, diffuse intralobular fibrosis, pancreatitis, cystic fibrosis, pancreatic cancer, skin fibrosis, kidney fibrosis, pulmonary fibrosis, rheumatoid arthritis, inflammatory bowel disease, diabetic nephropathy, or a diabetic wound.

14. The LRG1 inhibitor for use according to claim 13, the use according to claim 13, or the method according to claim 13, wherein the disease or condition is selected from: subretinal fibrosis, corneal fibrosis, epiretinal fibrosis, post-surgical fibrosis (e.g. of the posterior capsule following cataract surgery, or the bleb following trabeculectomy for glaucoma), conjunctival fibrosis, subconjunctival fibrosis, macular fibrosis, preretinal fibrosis, interlobular fibrosis, periductal fibrosis, diffuse interlobular fibrosis, and diffuse intralobular fibrosis.

15. The LRG1 inhibitor for use according to claim 14, the use according to claim 14, or the method according to claim 14, wherein the disease or condition is subretinal fibrosis.

16. The LRG1 inhibitor for use according to any one of claims 1 or 4 to 12, the use according to any one of claims 2 or 4 to 12, or the method according to any one of claims 3 or 4 to 12, wherein the disease or condition is a cancer.

17. The LRG1 inhibitor for use according to claim 14, the use according to claim 14, or the method according to claim 14, wherein the cancer is selected from: a cancer comprising cells expressing / overexpressing LRG1 , a solid tumor, metastatic tumor, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, liver cancer, hepatocellular carcinoma, skin cancer, melanoma, lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, non-small cell lung cancer, breast cancer, breast carcinoma, ductal carcinoma, colorectal cancer, colorectal carcinoma,colorectal adenocarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, cervical cancer, cervical squamous cell carcinoma, esophageal cancer, esophageal adenocarcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma and thymoma.

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