Agents and methods directed to disease-associated cells

Abstract

Provided herein are agents and methods suitable for the treatment of inflammatory diseases of the intestine. For instance, agents and methods are provided for the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, Celiac disease, and colitis-associated colorectal cancer.

Description

[0001] AGENTS AND METHODS DIRECTED TO DISEASE-ASSOCIATED CELLS

[0002] FIELD OF THE INVENTION

[0003] Provided herein are agents and methods suitable for the treatment of inflammatory diseases of the intestine. For instance, agents and methods are provided for the treatment of inflammatory bowel disease, Crohn’s disease, ulcerative colitis, Celiac disease, and colitis-associated colorectal cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Inflammatory Bowel Disease (IBD), encompassing Crohn's disease and ulcerative colitis (UC), involves chronic inflammation of the gastrointestinal (GI) tract. A critical aspect of IBD pathophysiology is the role of the epithelial barrier. The epithelial layer contains specialized intestinal epithelial cells that are linked together by tight junctions. The layer is regenerated by intestinal epithelial cells located at the base of the crypts that divide and differentiate to absorptive enterocytes, Paneth cells, Goblet cells, enteroendocrine, and microfold cells. The epithelial barrier serves multiple functions: it acts as a physical blockade to prevent the entry of harmful pathogens and toxins, plays a vital role in immune signaling to maintain mucosal immunity, and interacts with the gut microbiome to regulate its composition and function. In IBD, the integrity of the epithelial barrier is compromised, leading to increased intestinal permeability that triggers and perpetuates immune responses. The resulting chronic inflammation further damages the epithelial barrier, creating a vicious cycle that exacerbates the disease. Therapies for IBD encompass both broad spectrum immunosuppressants (e.g. 5-ASA or corticosteroids) and immune system targeting biologies (e.g. TNF, IL-12 / 23 antagonists)1>2. Currently, there are no approved therapies targeting intestinal epithelium. Therefore, understanding the molecular changes behind epithelial barrier dysfunction in IBD can lead to the development of targeted therapies aimed at alleviating disease symptoms.

[0006] The success in monoclonal antibodies has driven the development of various types of engineered therapeutic antibodies, particularly in the cancer field. Multiple mechanisms of action for antibody-based therapeutics are being developed, including receptor agonists and antagonists, receptor or ligand blocking antibodies, and ADCCs. One emerging strategy for antibody-based therapeutics is to harness the immunomodulatory effects of cytokine family proteins, such as TNF, IFN, IL-2 or IL- 15, to enhance the properties of monoclonal antibodies or to use the antibody moiety to deliver cytokine payloads to specific cells. For example, cancer-targeting monoclonal antibodies have been fused with engineered IL-2 variants with enhanced activity in activating expansion and function of effector T cells3,4

[0007] SUMMARY OF THE INVENTION

[0008] In a first aspect, there is provided a composition comprising: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74: and ii) a therapeutic moiety.

[0009] The first antigen-binding domain may be specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1. The first antigen-binding domain may be specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DUOX2, GPRC5A, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74, and the composition binds to MUC6-expressing epithelial cells and / or is selective for MUC6-expressing epithelial cells. The MUC6- expressing epithelial cells may be mucous gland neck cells. The MUC6-expressing epithelial cells may be present at the base of intestinal crypts. The MUC6-expressing epithelial cells may express PGC, AQP5, and BPIFB1. The first antigen-binding domain may be specific for any one of TSP ANT , DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74, and the composition binds to MUC5AC-producing cells and / or is selective for MUC5AC-producing cells. The MUC5AC-producing cells may be surface foveolar cells. The MUC5AC-producing cells may be present at intestinal villus tips. The MUC5AC-producing cells may express CEACAM7, CEACAM1 , DUOX2, and LCN2. The therapeutic moiety may be a protein domain. The therapeutic moiety may be biologically active. The therapeutic moiety may be immunosuppressive. The therapeutic moiety may enhance regulatory T cell activity and / or cell number. The therapeutic moiety may enhance regulatory T cell suppressive function, proliferation, and / or survival.

[0010] The therapeutic moiety may be a cytokine, or a mutein and / or portion thereof. The therapeutic moiety may be IL- 2, or a mutein and / or portion thereof. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells. The therapeutic moiety may be an immunosuppressive cytokine, or a mutein and / or portion thereof. The therapeutic moiety may be IL- 10, or a mutein and / or portion thereof. The therapeutic moiety may be IL-4, or a mutein and / or portion thereof.

[0011] In an embodiment, the first antigen-binding domain is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and the therapeutic moiety is IL-2, or a mutein and / or portion thereof.

[0012] The first antigen-binding domain may be an antigen-binding portion of an antibody. The first antigen-binding domain may be a single-chain fragment variable (scFv). The first antigen-binding domain may be human or humanized. The therapeutic moiety may be derived from a human protein.

[0013] The composition may be or may comprise a polypeptide or a fusion protein.

[0014] In a second aspect, there is provided one or more nucleic acid molecules encoding a composition of the first aspect.

[0015] In a third aspect, there is provided one or more vectors comprising the one or more nucleic acid molecules of the second aspect.

[0016] In a fourth aspect, there is provided a cell comprising one or more nucleic acid molecules as disclosed herein or one or more vectors as disclosed herein. The cell may be a cell suitable for commercial production of recombinant polypeptides and proteins.

[0017] In a fifth aspect, there is provided a method of producing a composition of the first aspect, comprising culturing a cell disclosed herein under conditions that result in expression and / or production of the composition, and isolating the composition thereof from the cell or culture.

[0018] In a sixth aspect, there is provided a cell comprising a composition of the first aspect, one or more nucleic acid molecules of the second aspect, or one or more vectors of the third aspect. The cell may be suitable for administration to subjects in order to deliver the composition in vivo.

[0019] In a seventh aspect, there is a provided a pharmaceutical composition comprising a composition of the first aspect, one or more nucleic acid molecules of the second aspect, one or more vectors of the third aspect, or a cell of the sixth aspect.

[0020] In an eighth aspect of the invention, there is provided a composition of the first aspect, one or more nucleic acids of the second aspect, one or more vectors of the third aspect, a cell of the sixth aspect, or a pharmaceutical composition of the seventh aspect for use as a medicament.

[0021] In a ninth aspect of the invention, there is provided a composition of the first aspect, one or more nucleic acids of the second aspect, one or more vectors of the third aspect, a cell of the sixth aspect, or a pharmaceutical composition of the seventh aspect for use in a method of treating inflammation of the intestine. The inflammation may be chronic inflammation. The inflammation may be inflammation of the ileum, duodenum, or colon. The method may be for the treatment of inflammatory bowel disease. The method may be for the treatment of Crohn’s disease, ulcerative colitis, Celiac disease, or colitis-associated colorectal cancer (CRC).

[0022] In a tenth aspect, there is provided a method of targeting a therapeutic moiety to a site of intestinal inflammation, the method comprising the delivery of a composition of the first aspect to a subject in need thereof.

[0023] In an eleventh aspect, there is provided a method of identifying a subject with a disease affecting the intestine, the method comprising determining the proportion and / or location of disease-associated epithelial cells within intestinal tissue of said subject.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1: Single cell RNA sequencing identifies epithelial cells in the small intestine of inflammatory bowel disease patients, a) UMAP plot showing cells from small intestinal epithelium of both healthy individuals and patients with inflammatory bowel disease. Mucous gland neck and Surface foveolar cells that appear in disease affected ileum are highlighted with dashed circles, b) Dot plot displaying genes expressed in Mucous gland neck and Surface foveolar cells. MUC6, PGC, AQP5 and BPIFB1 are selectively expressed in Mucous gland cells, while MUC5AC, CEACAM7 and SLC26A3 are selectively expressed in Surface foveolar cells, c) Proportion of Mucous gland neck cells in small intestines, highlighting the presence of Mucous gland neck cells in ileum only in patients with IBD.

[0026] Figure 2: Mucous gland neck cells (MUC6+) are located at the base of the intestinal crypt and Surface foveolar cells (MUC5AC+) at the top of the villus, a) Representative protein staining of MUC6 and MUC5AC in Crohn’s disease ileum, b) Protein staining of MUC6 and MUC5AC in Crohn’s disease ileum showing association with tertiary lymphoid structures (dense nuclei and CD3 / CD20+ regions), c) Protein staining of MUC6 and MUC5AC in colon resection tissue from ulcerative colitis (UC) patients. Upper and lower panels are images from two different patients, d) Protein staining of MUC6 in duodenum of celiac disease patients from two separate donors and, distinguishing Mucous gland neck cells in inflamed epithelium from cells in Brunner’s glands.

[0027] Figure 3: Mucous gland neck cells found in inflammatory bowel diseases are transcriptionally distinct from similar cells in the healthy stomach, a) UMAP plot showing subclustered MUC6-expressing cells from across the single cell datasets including cells from various tissue locations, patient ages and conditions. MUC6-expressing cells from different regions and / or developmental stages (ie. in utero) occupy separate coordinates in the UMAP plot. Inflammatory epithelial cells present in Crohn’s disease and paediatric IBD tissue are circled with dashed line and cluster seperately from the cells found in the stomach, b) Overlap of MUC6-expressing cell-selective genes across different tissue regions. Marker genes of MUC6-expressing cells were calculated by differential gene expression (wilcoxon rank-sum test) of other stomach and small intestine epithelial cells separately for healthy adult stomach MUC6-expressing cells, healthy adult duodenum MUC6-expressing cells, ileum Crohn’s disease MUC6-expressing inflammatory cells and duodenum celiac disease MUC6-expressing inflammatory cells. Overlapping marker genes show greater similarity of inflammatory cells to healthy adult stomach MUC6- expressing cells, than to healthy adult duodenum MUC6-expressing cells. There are 205 genes expressed in MUC6-expressing cells in IBD patients that are not found in MUC6-expressing cells in the stomach and duodenum of healthy patients. Similarly, MUC6-expressing cells in celiac patients show expression of 259 genes not present in the same cells from the stomach or duodenum of control patients.

[0028] Figure 4: Mucous gland neck cells are expanded in a) human UC colon and in human Crohn’s disease colon and small intestinal tissue, b) celiac disease and c) colorectal cancers. The results are generated using bulk deconvolution analysis (BayesPrism) by using disease intestinal epithelium as a reference. Colorectal tumour tissue is stratified by microinstability status. Micro satellite instability (MSI)-high tumours are predicted to have higher levels of Mucous gland neck cells.

[0029] Figure 5: Mucous gland neck cells in IBD ileum are likely derived from epithelial LGR5+ stem cells, a) UMAP plot of small intestine epithelial cells coloured by pseudotime values inferred with Monocle3 analysis on epithelial cells from ileum only, highlighting trajectory from stem cells to inflammatory Mucous gland neck cells. Small UMAP alongside indicates cell types, b) Expression of Stem and inflammatory Mucous gland neck cell markers along pseudotime, along with cell type abundance along the Stem - TA (Transit amplyfmg cell) - inflammatory Mucous gland neck cell trajectory, c) Proliferation (MKI67) and Stem (LGR5) marker gene expression by smFISH in inflammatory Mucous gland neck cells (MUC6) from Crohn’s disease ileum and duodenum.

[0030] Figure 6: Disease-associated epithelial cells express chemokines and MHC II genes with linked to inflammatory properties, a) Violin plot show the score of chemokine and MHCII gene expression per cell comparing small intestine epithelial cells in the atlas in healthy control and disease (IBD and celiac) samples showing specificity of upregulated chemokine and MHCII related gene expression across epithelial cells. Mucous gland neck cells vs surface foveolar-like cells are highlighted by boxes, b) Gene score of chemokines across Mucous gland neck cells from disease (IBD and celiac) and MUC6-producing cells from stomach, c) Dot plot showing the expression of chemokines by disease-associated epithelial cells, d) Protein staining of inflammatory mucous gland neck-like cells (MUC6), macrophages (CD68) and MHCII (HLA-DR) in ileum from Crohn’s disease resection showing high MHCII expression in inflammatory mucous gland neck-like cells.

[0031] Figure 7: Mucous gland neck cells are located in proximity to vasculature. smFISH staining of Mucous gland neck cells (solid line, MUC6, BPIFB1), Surface foveolar (dashed line, MUC5AC) and activated endothelial cells (arrows, ACKR1) showing proximity of vessels to metaplastic glands in Crohn’s disease duodenum.

[0032] Figure 8: Disease-associated epithelial cells are in proximity to T cell and Treg subsets. Protein staining to visualise Mucous gland neck cells (MUC6) in Crohn’s disease ileum (top) or in celiac disease duodenum tissue (bottom) and CD4, CD8 or yST T cell subsets (CD4+CD3+, CD8+CD3+, TCRy5+CD3+ T cells). Intraepithelial T cells are found in MUC6+ epithelial glands, b) Protein expression in Crohn’s disease ileum of HLA-DR (MHCII) in inflammatory mucous gland neck-like cells (MUC6) along with localisation of CD3+ T cells and regulatory T cells (FoxP3+CD3+).

[0033] Figure 9: Key identified surface receptors are expressed in disease-associated epithelial cells in disease tissue. Spatial plots showing disease associated epithelial cell presence and key receptor gene expression in Crohn’s disease tissue using the spatial transcriptomics technology (lOx Genomics Xenium).

[0034] Figure 10: Identification of key organs and tissues, beyond gastrointestinal tract, where target genes are expressed. Bar plots with TPM expression of selected target genes across human bulk RNA-seq datasets from GTEx resource.

[0035] Figure 11: The target surface receptors are mostly expressed in epithelial lineages across the organs, beyond gastrointestinal tract. Dot plot with mean expression and cell fraction in single cell data from healthy human organs and cell types from CZI Cellxgene Discover dataset.

[0036] DETAILED DESCRIPTION

[0037] Understanding of the diversity of epithelial cells and their interactions with resident immune system has been greatly enhanced by the recent adoption of single cell and spatial RNA sequencing technologies on intestinal tissues ’ Described herein is the identification of epithelial cells that emerge in intestinal inflammation, such as active IBD, using single cell and spatial technologies. The inventors have identified two cell types that are present in the intestines of patients with inflammatory diseases but are present at a lower level, differently distributed, or not present in the intestine of patients without said pathology. These cells in disease tissue are referred to herein as inflammatory epithelial cells akin to Mucous gland neck cells and surface foveolar cells in healthy tissues and are described fully below.

[0038] The inventors have made use of these data to identify relevant surface receptors for targeting of agents to these cells. Thus, the inventors have identified markers of disease-associated epithelial cells that allow the specific or selective targeting of these cells by agents.

[0039] Furthermore, the inventors have developed therapeutic agents for the targeting of the identified disease-associated epithelial cells and hence for treatment of diseases associated with the identified cells. As an example of a mechanism by which these agents can operate, the agents may have a moiety capable of binding to one of said markers of the disease-associate epithelial cells, and a second moiety that has a therapeutic effect. Thus, the disease-associated epithelial cell acts as an anchor for the therapeutic moiety and hence increases the local concentration of the therapeutic moiety at the site of inflammation.

[0040] A purely illustrative example is an antibody-cytokine therapeutic. For instance, provided herein is an antibodycytokine therapeutic for the treatment of IBD, wherein the antibody-cytokine therapeutic comprises one arm that is specific for one of said markers of disease-associated epithelial cells and wherein the antibody-cytokine therapeutic comprises IL-2 or a biologically active fragment or mutein of IL-2.

[0041] Thus, in a first aspect, there is provided a composition comprising: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74: and ii) a therapeutic moiety.

[0042] In particular embodiments, the first antigen-binding domain is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIGl .

[0043] The first antigen-binding domain may be specific for AQP5.

[0044] The first antigen-binding domain may be specific for CLDN2.

[0045] The first antigen-binding domain may be specific for CLDN18.

[0046] The first antigen-binding domain may be specific for DUOX2.

[0047] The first antigen-binding domain may be specific for V SIGI .

[0048] As discussed herein, the markers above allow the targeting of disease-associated epithelial cells. In particular, the epithelial cells may be Mucous gland neck-like cells and / or surface foveolar-like cells, and which marker is relevant to which cell is described in Table 1. Thus, the composition may bind to Mucous gland neck-like cells and / or may be selective for Mucous gland neck- like cells if the first antigen-binding domain is specific for a relevant marker as listed in Table 1. The composition may bind to surface foveolar-like cells and / or may be selective for surface foveolar-like cells if the first antigen-binding domain is specific for a relevant marker as listed in Table 1. Exemplary Ensembl Gene IDs are also provided in Table 1.

[0049] As used herein, the term “selective” means that the compositions bind to a higher proportion of target cells in comparison to the binding to a population of non-target cells from the same organism. In some embodiments, the compositions bind to a higher proportion of targeted disease-associated epithelial cells in comparison to the binding to a population of non-targeted epithelial cells. In some embodiments, the compositions bind to a higher proportion of Mucous gland neck-like cells and / or surface foveolar-like cells in comparison to the binding to other epithelial cells from the intestine. The non-target cells for the aforementioned embodiments may be otherwise unsorted cells from the organism in question, from the intestine, or that are epithelial cells from the intestine or other organs.

[0050] The composition of the first aspect may bind to MUC6-expressing epithelial cells and / or may be selective for MUC6-expressing epithelial cells. More particularly, these cells may be referred to as mucous gland neck (MGN) cells or MGN-like cells. Thus, the composition may bind to MUC6-expressing MGN cells and / or may be selective for MUC6-expressing MGN cells. The composition may bind to MUC6-expressing MGN-like cells and / or may be selective for MUC6-expressing MGN-like cells. These cells may be present at the base of intestinal crypts. The composition of the first aspect may bind to disease-associated inflammatory MGN-like cells and / or may be selective for disease-associated inflammatory MGN-like cells . These cells may also express PGC, AQP5, and BPIFB1 (Fig. lb). The relevant markers for targeting the cells discussed in this paragraph are disclosed in Table 1.

[0051] The composition of the first aspect may bind to MUC5AC-producing epithelial cells and / or may be selective for MUC5AC-producing epithelial cells. The composition may bind to MUC5AC-producing surface foveolar cells and / or may be selective for MUC5AC-producing surface foveolar cells. The composition may bind to MUC5AC- producing surface foveolar-like cells and / or may be selective for MUC5AC-producing surface foveolar-like cells. These cells may be present at intestinal villus tips. These cells may also express CEACAM7, CEACAM1, DUOX2, and LCN2. The relevant markers for targeting the cells discussed in this paragraph are disclosed in Table 1.

[0052] Table 1 : Top receptors expressed in inflamed epithelial cells

[0053] The first antigen-binding domain may be based on an antibody scaffold. The first antigen-binding domain may be or may comprise an antigen-binding portion of an antibody. In some examples, the first antigen-binding domain is a murine, human, humanized, or chimeric antibody, or antigen-binding fragments thereof. The antigen-binding domains may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post- translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. The antigen-binding domains may be mutated to alter such post-translational modifications, for example by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site. The antigen-binding domains may be modified for example by amino acid substitution to remove potential proteolytic sites. The antigen-binding domains may comprise one or more substitutions, deletions and / or insertions which remove a post-translational modification (PTM) site, for example a glycosylation site (N-linked or O-linked), a deamination site, a phosphorylation site or an isomerisation / fragmentation site.

[0054] Examples of suitable formats for antigen-binding domains that are antibody fragments include a Fab, a Fab', an F(ab')2, an Fd, an Fv, an scFv, a single domain antibody (sdAb), a VHH domain, a maxibody, a minibody, an intrabody, a diabody, a triabody, a tetrabody, a v-NAR, or a bis-scFv.

[0055] In a particular embodiment, the first antigen-binding domain is an antibody or antigen-binding fragment thereof specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74. The first antigen-binding domain may be an antibody or antigen-binding fragment thereof specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIGl .

[0056] In another embodiment, the first antigen-binding domain is an scFv specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1 , CD24, PIGR, and CD74. The first antigen-binding domain may be an scFv specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1.

[0057] The composition may comprise a second or further antigen-binding domain. The second antigen-binding may be specific for a different antigen, for instance another antigen in Table 1. The second antigen-binding domain may contribute to composition’s binding to or selectivity for the disease-associated epithelial cells disclosed herein.

[0058] The second antigen-binding domain may be based on an antibody scaffold and may have any of the properties or attributes of such scaffolds as discussed for the first antigen-binding domain.

[0059] The first and / or second antigen-binding domains may be an antibody mimetic, for instance a designed ankyrin repeat protein (DARPin) (See chapter 5. "Designed Ankyrin Repeat Proteins (DARPins); From Research to Therapy", Methods in Enzymology, vol 503: 101— 134 (2012); or "Efficient Selection of DARPins with Sub- nanomolar Affinities using SRP Phage Display", J. Mol. Biol. (2008) 382, 1211-1227).

[0060] As used herein, “specific for” a target means that the domain is capable of binding to a particular target to a greater extent than the domain would bind to dissimilar targets. Specificity can be tested by comparison with a control domain that has not been designed or selected for specificity to the target in question. In the context of an antibody, portion thereof, or antibody scaffold, the term “specific for” means that the antibody binds to an antigen whereas an isotype control antibody would not bind or would only minimally bind. An “isotype control antibody” is an antibody, portion thereof, or antibody scaffold that matches the antibody in question but has an irrelevant specificity. For instance, the isotype control may be the same as the antibody or portion in question but may have a different variable domain, a different set of CDRs, or may have been raised against an irrelevant antigen.

[0061] The therapeutic moiety may be a moiety that has an effect on cells with which it comes into contact. It may be referred to as biologically active. For instance, the therapeutic moiety may cause a change in the activity, function, survival, differentiation, or proliferation of a cell with which it comes into contact. In some embodiments, the composition of the first aspect binds to particular cells in inflamed tissues (disease-associated epithelial cells) and the therapeutic moiety affects other cell types present in the vicinity. Thus, the disease- associated epithelial cells (Mucous gland neck-like or surface foveolar-like) may act as anchors such that the composition of the first aspect may present the therapeutic moiety in the tissue-to-be-treated.

[0062] In particular, the therapeutic moiety may have an immunosuppressive effect.

[0063] The therapeutic moiety with an immunosuppressive effect may be any suitable that can be included in a composition of the first aspect. Examples that may be part of the composition of the first aspect include steroids, such as glucocorticoids. It is known in the art that glucocorticoid receptor modulator pay loads can be, for instance, conjugated to antibodies for targeted delivery. An example is disclosed in McPherson et al. (Science Translational Medicine, 20 Mar 2024, Vol 16, Issue 739). Thus, the composition of the first aspect may comprise an immunosuppressive steroid. The composition of the first aspect may comprise an immunosuppressive glucocorticoid. The composition of the first aspect may comprise a glucocorticoid receptor modulator. The composition of the first aspect may comprise an immunosuppressive steroid, glucocorticoid, glucocorticoid receptor modulator conjugated to a polypeptide comprising the first antigen-binding domain. The composition of the first aspect may comprise an immunosuppressive steroid, glucocorticoid, glucocorticoid receptor modulator as a part of an antibody-drug conjugate. The immunosuppressive moiety may be linked to the rest of the composition in a manner such that it retains activity or it may be linked via a linker than allows release in the relevant tissue or microenvironment.

[0064] The immunosuppressive effect of the therapeutic moiety may be due to effects on cells that have an immunological role. In particular, the therapeutic moiety may affect T cells in the relevant tissue, for instance regulatory T cells or other immunosuppressive T cells. The regulatory T cells may be CD25+ regulatory T cells. The T cells may express Foxp3.

[0065] The effect on the regulatory T cells may be to enhance their immunosuppressive effect in the relevant tissue. The effect may be to increase the number or proportion of regulatory T cells in the relevant tissue. The effect may be to enhance the suppressive activity of the regulatory T cells, to enhance their proliferation, and / or to enhance their survival.

[0066] The effect of the therapeutic moiety may be tested on regulatory T cells, for instance T cells that are CD25+ CD4+ FOXP3+ T cells. The CD25+ CD4+ T cells may be FOXP3+. The effects of the therapeutic moiety on these cells may be measured by conventional techniques, such as measuring the number of Ki67+ cells or cell tracker dilution to determine effects on proliferation.

[0067] The therapeutic moiety may be a cytokine or may be a fragment of a cytokine that retains the relevant biological activity. The cytokine may be of a natural sequence or may have been mutated. Mutated cytokines can be referred to as muteins. A mutein may retain at least one biological effect of the parent cytokine, and this effect may be the same or enhanced compared to the parent. A mutein may be mutated to reduce or remove undesired biological effects or may be mutated to improve other properties (such as stability, immunogenicity, pharmacokinetics, suitability for conjugation, etc). The therapeutic moiety may be a mutein or may be a fragment of a mutein that retains the relevant biological activity.

[0068] The therapeutic moiety may be IL-2 or a biologically active fragment thereof. The IL-2 may be a mutein that retains the relevant biological activity of IL-2, or a biologically active fragment thereof. In particular, the IL-2 may be suitable for enhancing the immunosuppressive effect of regulatory T cells in the relevant tissue. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells. The IL-2 may be a mutein that has a larger effect on regulatory T cells in comparison to the effect on effector CD4+ T cells or conventional naive CD4+ T cells. The regulatory T cells may be CD25+ regulatory T cells. The T cells may express Foxp3.

[0069] The IL-2 may be a mutein with reduced potency and enhanced regulatory T cell-selectivity due to increased dependence on the IL-2-receptor component CD25. The IL-2 may be mutated at the CD122 contact interface to reduce CD122 binding. The IL-2 may be a mutein as disclosed in Shanafelt et al. (Nat Biotechnol. 2000 Nov;18(l 1 ): 1197-202. doi: 10.1038 / 81199) or Khoryati et al. (Sci Immunol. 2020 Aug 14; 5(50): eaba5264), each herein incorporated by reference. The mutein may be a human version of a mutein disclosed in Khoryati et al. The IL-2 may be human IL-2 or a derivative thereof. The IL-2 may be at least 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 in US 6,955,807 Bl, SEQ ID NO: 1 as disclosed in AU 2015301936 B2, or SEQ ID NO: 2 in US 2021 / 0308222 Al . Any mutations disclosed herein may be applied to said IL-2.

[0070] The IL-2 may comprise anN88 mutation (relative to SEQ ID NO: 1 in US 6,955,807 Bl). In some examples, the N88 mutation is N88D, N88R, N88I, or N88G. In particular, the mutation may be N88D or N88R. The IL-2 may be a mutein as disclosed or as claimed in US 6,955,807 Bl, herein incorporated by reference.

[0071] The IL-2 may comprise a D109 mutation (relative to SEQ ID NO: 1 in AU 2015301936 B2). In examples, the IL- 2 comprises the mutation D109C. The IL-2 may have at least 95% identity to SEQ ID NO: 1 as disclosed in AU 2015301936 B2. The IL-2 may be a mutein as disclosed or as claimed in AU 2015301936 B2, herein incorporated by reference.

[0072] The IL-2 may comprise a T3, C125, D20, N88, and / or Q126 mutation relative to SEQ ID NO: 2 in US 2021 / 0308222 Al (herein incorporated by reference). The IL-2 may comprise a C125 mutation, such as C125S, relative to SEQ ID NO: 2 in US 2021 / 0308222 Al . The IL-2 may comprise a D20 mutation, such as D20H, relative to SEQ ID NO: 2 in US 2021 / 0308222 Al . The IL-2 may comprise a substitution selected from the group consisting of T3A and C125S relative to the amino acid sequence of SEQ ID NO: 2 in US 2021 / 0308222 Al, and a substitution selected from the group consisting of D20H, N88I, N88G, N88R, Q126L, and Q126F relative to the amino acid sequence of SEQ ID NO: 2 in US 2021 / 0308222 Al . The IL-2 may be a human IL-2 variant protein domain as disclosed in US 2021 / 0308222 Al or as defined in the claims of said document.

[0073] The IL-2 may comprise the mutations C126S, N88R / N88D, and / or D20H (relative to human IL-2). The IL-2 may comprise the mutations C126S, N88R / N88D, and D20H. The IL-2 may comprise only mutations selected from C126S, N88R / N88D, and / or D20H. The IL-2 may comprise the mutations C126S, N88R / N88D, and / or D20H and be at least 80%, 90%, 95%, 99%, or 100% identical (not including the mutations) to SEQ ID NO: 1 in US 6,955,807 Bl, SEQ ID NO: 1 as disclosed in AU 2015301936 B2, or SEQ ID NO: 2 in US 2021 / 0308222 Al . The IL-2 may comprise the mutations C126S, N88R / N88D, and D20H and be at least 80%, 90%, 95%, 99%, or 100% identical (not including the mutations) to SEQ ID NO: 1 in US 6,955,807 Bl, SEQ ID NO: 1 as disclosed in AU 2015301936 B2, or SEQ ID NO: 2 in US 2021 / 0308222 AL The IL-2 may comprise the mutations C126S, N88R / N88D, and D20H and be at least 80%, 90%, 95%, 99%, or 100% identical (not including the mutations) to SEQ ID NO: 1 in US 6,955,807 Bl, SEQ ID NO: 1 as disclosed in AU 2015301936 B2, or SEQ ID NO: 2 in US 2021 / 0308222 Al . The IL-2 may comprise only mutations selected from C126S, N88R / N88D, and D20H and be at least 80%, 90%, 95%, 99%, or 100% identical (not including the mutations) to SEQ ID NO: 1 in US 6,955,807 Bl, SEQ ID NO: 1 as disclosed in AU 2015301936 B2, or SEQ ID NO: 2 in US 2021 / 0308222 Al .

[0074] The therapeutic moiety may be an immunosuppressive cytokine or may be a fragment of an immunosuppressive cytokine that retains the relevant biological activity. The immunosuppressive cytokine may be of a natural sequence or may have been mutated.

[0075] The immunosuppressive cytokine may be IL- 10 or a biologically active fragment thereof. The IL- 10 may be a mutein that retains the relevant biological activity of IL- 10, or biologically active fragment thereof. In particular, the IL- 10 may have an immunosuppressive effect in the relevant tissue.

[0076] IL-10 exists naturally as a homodimer. In the compositions of the first aspect, the IL-10 subunits may be joined by a linker. Exemplary publications disclosing single-chain IL-10 include: Josephson K, DiGiacomo R, Indelicate SR, lyo AH, Nagabhushan TL, Parker MH, et al. Design and analysis of an engineered human interleukin- 10 monomer. J Biol Chem. (2000) 275:13552-7. 10.1074 / jbc.275.18.13552 or Front Immunol. 2020; 11 : 1794. Published online 2020 Aug 11. doi: 10.3389 / fimmu.2020.01794 (both herein incorporated by reference). The IL-10 may retain one or more natural biological activity. For instance, the IL-10 may downregulate the expression of Thl cytokines, MHC class II antigens, and / or co-stimulatory molecules on macrophages. The IL-10 may inhibit lipopolysaccharide and bacterial-product-mediated induction of the pro-inflammatory cytokines TNFa, IL-ip, IL-12, and / or IFNy secretion from Toll-Like Receptor triggered myeloid lineage cells. The IL-10 may induce IL- 10 production by contacted cells, for instance monocytes / macrophages. The IL- 10 may affect the epithelial integrity in the small intestine. Further information about IL- 10 and its therapeutic effects can be found in Wang eta / . (Cold Spring Harb Perspect Biol. 2019 Feb; 11(2): a028548, herein incorporated by reference).

[0077] The therapeutic moiety may be IL-4 or a biologically active fragment thereof. The IL-4 may be a mutein that retains the relevant biological activity of IL-4, or biologically active fragment thereof.

[0078] The IL-4 may comprise a R121, Y124, or S125 mutation, such as R121E (as described in Shanafelt etal., Proc Natl Acad Sci U S A., 1998 doi: 10.1073 / pnas.95.16.9454 - herein incorporated by reference). The IL-4 may be a selective agonist disclosed in Shanafelt et al.

[0079] IL-4 has been reported to have wound repair properties (Bosurgi et al., Science, 2017 https: / / www.science.org / doi / 10.1126 / science.aai8132; Allen Annual Rev Immunol 2023, DOI: 10.1146 / annurev- immunol-101921-041206). And so, although IL4 may have adverse effects if delivered systemically, positive effects on repair could be achieved if IL-4 were delivered to a tissue microenvironment (Benstein et al., Immunol Review, 2023 (DOI: 10.1111 / imr.l 3230)). The compositions of the present invention allow such localised delivery or, to phrase this another way, allows the local concentration of administered IL-4 to be raised in the targeted tissue or microenvironment.

[0080] The composition of the first aspect may be or may comprise a polypeptide. For instance, the first antigen-binding domain may be or may comprise a polypeptide. The therapeutic moiety may be or may comprise a polypeptide.

[0081] The first antigen-binding domain may be linked to the therapeutic moiety via a linker. The linker may be any suitable linking moiety. For instance, the linker may comprise a covalent or non-covalent bond. Linkers are well known in the art, for instance for the construction of antibody-drug conjugates. The linker may be a synthetic linker, for instance an unnatural moiety and / or a chemically synthesised linker. The linker may be via a bifunctional linker. The linkage may be via two moieties capable of forming a non-covalent interaction, such as streptavidin and biotin.

[0082] The linker may be a polypeptide. The composition of the first aspect may be or may comprise a single-chain polypeptide. The single-chain polypeptide may comprise the first antigen-binding domain and the therapeutic moiety. The single-chain polypeptide may comprise the first antigen-binding domain, the therapeutic moiety, and a second antigen-binding domain. In such embodiments, the composition of the first aspect may be referred to as a fusion protein or as comprising a fusion protein. In some embodiments, the composition of the first aspect may be referred to as an antibody-cytokine conjugate or may be referred to as comprising an antibody-cytokine conjugate. In some embodiments, the composition of the first aspect may be referred to as a bispecific or multispecific antibody-cytokine conjugate or may be referred to as comprising a bispecific or multispecific antibody-cytokine conjugate.

[0083] The composition of the first aspect may be an isolated molecule. The composition may be linked to a payload or agent. The composition may be or may be a part of an immunoconjugate. The immunoconjugate may further comprise another agent, such as a label or a second therapeutic moiety.

[0084] The composition of the first aspect may comprise an immunoglobulin constant region. The composition may comprise an immunologically inert constant region. The composition may comprise a constant region capable of binding to Fc receptors, or a specific pattern of Fc receptors such as activating Fc receptors. The constant region may be modified to have an increase or decrease in one or more effector activities. In some examples, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2. In additional examples, the immunoglobulin constant region is IgGl, IgG2, IgG3, IgGlnull, IgG4(S228P), IgAl or IgA2.

[0085] The composition of the first aspect may comprise other domains capable of binding or being bound. For instance, as discussed above the molecules may comprise an Fc region and may be capable of interacting with Fc receptors. In this situation, it is common to refer to such molecules as comprising an additional functionality rather than an additional specificity. For instance, bispecific antibodies comprising a functional Fc domain are sometimes referred to as trifunctional.

[0086] In a particular embodiment, there is provided a composition that comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74: and ii) a cytokine or biologically active fragment thereof. Exemplary cytokines include IL-2, IL-4, and IL- 10, including muteins of said cytokines. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0087] In a particular embodiment, there is provided a polypeptide that comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and 0)74: and ii) a cytokine or biologically active fragment thereof. Exemplary cytokines include IL-2, IL-4, and IL- 10, including muteins of said cytokines. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0088] In another embodiment, there is provided a composition that comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and ii) an IL-2 or a biologically active fragment thereof. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0089] In another embodiment, there is provided a polypeptide that comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and ii) an IL-2 or a biologically active fragment thereof. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0090] In another embodiment, there is provided a composition that comprises: i) a first antigen-binding antibodyfragment specific for AQP5, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0091] In another embodiment, there is provided a composition that comprises: i) a first antigen-binding antibodyfragment specific for CLDN2, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0092] In another embodiment, there is provided a composition that comprises: i) a first antigen-binding antibodyfragment specific for CLDN18, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0093] In another embodiment, there is provided a composition that comprises: i) a first antigen-binding antibodyfragment specific for DUOX2, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue. In another embodiment, there is provided a composition that comprises: i) a first antigen-binding antibodyfragment specific for VSIG1, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0094] In a second aspect, there is provided one or more nucleic acid molecules encoding a composition of the first aspect.

[0095] The composition may be a single polypeptide chain, in which case it may be encoded by a single nucleic acid molecule. In other embodiments, the composition may comprise two or more polypeptide chains and one nucleic acid molecule may encode the two or more polypeptide chains. The genes encoding these chains may be under the control of separate promoters. In other examples, the genes encoding these chains may under the control of the same promoter, but separated by a region that can induce ribosomal skipping or by a self-cleaving peptide sequence. Examples of such features include P2A, an internal ribosome entry site (IRES), or an intein.

[0096] In some embodiments, the composition may comprise two or more polypeptide chains and each polypeptide chain may be encoded on a different nucleic acid molecule. Thus, in an embodiment, there is provided two or more nucleic acid molecules encoding the composition.

[0097] In a third aspect, there is provided one or more vectors comprising one or more nucleic acid molecules of the second aspect. The one or more vectors may be expression vectors or may be vectors designed for delivery to a subject, such as a human patient.

[0098] In a fourth aspect, there is provided a cell comprising one or more nucleic acid molecules as disclosed herein or one or more vectors as disclosed herein. The cell may be a recombinant cell. The cell may be a cell suitable for commercial production of recombinant polypeptides and proteins. In some examples, the cell is mammal, bacteria, yeast, or plant derived. For instance, the cells may be CHO cells, NS0 cells, HEK cells, E. coli cells, Pichia pastoris cells, or other suitable cells.

[0099] In a fifth aspect, there is provided a method of producing a composition of the first aspect, comprising culturing a cell disclosed herein under conditions that result in expression and / or production of the composition, and isolating the composition thereof from the cell or culture.

[0100] In a sixth aspect, there is provided a cell comprising a composition of the first aspect, one or more nucleic acid molecules of the second aspect, or one or more vectors of the third aspect. The cell may be a mammalian or human cell. The cell may be an immune cell, such as a B cell. The cell may be suitable for administration to subjects in order to deliver the composition in vivo. Of course, this aspect is relevant to embodiments where the composition is or comprises a polypeptide (e.g. a post-translationally modified polypeptide).

[0101] The composition of the first aspect may be in a lyophilised form for reconstitution prior to administration. For example, lyophilised proteins may be re- constituted in sterile water and mixed with saline prior to administration to an individual. A nucleic acid of the second aspect or a vector of the third aspect may be in a lyophilised form. A cell as disclosed herein may be provided in a stable form for storage, for instance, the cells may be frozen in the presence of a cryoprotectant. In other embodiments, all of these products may be provided in a form suitable for administration.

[0102] Thus, in a seventh aspect, there is a provided a pharmaceutical composition comprising a composition of the first aspect, one or more nucleic acid molecules of the second aspect, one or more vectors of the third aspect, or a cell of the sixth aspect. The compositions will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the composition of the first aspect. Thus, pharmaceutical compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the composition. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route.

[0103] In an eighth aspect of the invention, there is provided a composition of the first aspect, one or more nucleic acids of the second aspect, one or more vectors of the third aspect, a cell of the sixth aspect, or a pharmaceutical composition of the seventh aspect for use as a medicament.

[0104] In particular, there is provided a composition of the first aspect for use as a medicament.

[0105] There is provided a method of treatment comprising administering a therapeutically effective amount of a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0106] There is provided use of a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for the manufacture of a medicament.

[0107] As disclosed herein, the cells targeted by the compositions of the first aspect are present or enriched in inflamed intestinal tissues. Thus, in a ninth aspect of the invention, there is provided a composition of the first aspect, one or more nucleic acids of the second aspect, one or more vectors of the third aspect, a cell of the sixth aspect, or a pharmaceutical composition of the seventh aspect for use in a method of treating inflammation of the intestine. The inflammation of the intestine may be inflammation of the ileum, duodenum, or colon.

[0108] In particular, there is provided a composition of the first aspect for use in a method of treating inflammation of the intestine.

[0109] There is provided a method of treating inflammation of the intestine, the method comprising administering a therapeutically effective amount of a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure to a subject in need thereof.

[0110] There is provided use of a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for the manufacture of a medicament for the treatment of inflammation of the intestine.

[0111] In a particular embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74; and ii) a cytokine or biologically active fragment thereof. Exemplary cytokines include IL-2, IL-4, and IL- 10, including muteins of said cytokines. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0112] In a particular embodiment, there is provided a polypeptide for use in a method of treating inflammation of the intestine, wherein the polypeptide comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74: and ii) a cytokine or biologically active fragment thereof. Exemplary cytokines include IL-2, IL-4, and IL- 10, including muteins of said cytokines. The IL-2 may be a mutein with selectivity for regulatory T cells over conventional T cells. In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and ii) an IL-2 or a biologically active fragment thereof. The IL- 2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0113] In another embodiment, there is provided a polypeptide for use in a method of treating inflammation of the intestine, wherein the polypeptide comprises: i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and ii) an IL-2 or a biologically active fragment thereof. The IL- 2 may be a mutein with selectivity for regulatory T cells over conventional T cells.

[0114] In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding antibody-fragment specific for AQP5, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0115] In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding antibody-fragment specific for CLDN2, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0116] In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding antibody-fragment specific for CLDN18, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0117] In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding antibody-fragment specific for DUOX2, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0118] In another embodiment, there is provided a composition for use in a method of treating inflammation of the intestine, wherein the composition comprises: i) a first antigen-binding antibody-fragment specific for VSIG1, and ii) an IL-2 domain that is a mutein, or biologically active fragment thereof, with selectivity for regulatory T cells over conventional T cells. The IL-2’s biological activity leads to an increase in the immunosuppression mediated by regulatory T cells in the relevant tissue.

[0119] The inflammation may be caused by a chronic condition and so may be chronic inflammation. For instance, the inflammation may be caused by inflammatory bowel disease (IBD). Thus, in an embodiment, there is provided a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use in a method of treating IBD. In the Examples section, it is demonstrated that cells targeted by the compositions of the first aspect are enriched in the ileum of patients with IBD.

[0120] In another embodiment, there is provided a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use in a method of treating Crohn's disease. In an embodiment, there is provided a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use in a method of treating ulcerative colitis.

[0121] In an embodiment, there is provided a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use in a method of treating Celiac disease.

[0122] In an embodiment, there is provided a composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition of the present disclosure for use in a method of treating colitis-associated colorectal cancer (CRC). The CRC may be adenocarcinoma. The CRC may have microsatellite instability (MSI -high). The CRC may be MSI-high adenocarcinoma. The treatment may be a preventative treatment.

[0123] In a tenth aspect, there is provided a method of targeting a therapeutic moiety to a site of intestinal inflammation, the method comprising the delivery of a composition of the first aspect to a subject in need thereof.

[0124] The delivery may be administration of the composition of the first aspect or a pharmaceutical composition of the seventh aspect. The delivery may comprise the administration of one or more nucleic acids of the second aspect, one or more vector of the third aspect, or a cell of the sixth aspect for the production of a composition of the first aspect in vivo.

[0125] The method of the tenth aspect may be the targeting of a composition of the first aspect to the ileum of a subject with intestinal inflammation. The method of the tenth aspect may be the targeting of a composition of the first aspect to the duodenum or colon of a subject with intestinal inflammation. Further details of the presence of disease-associated epithelial cells in various intestinal tissues in patients with intestinal diseases are provided herein, and inform the nature of the targeting.

[0126] In an eleventh aspect, there is provided a method of identifying a subject with a disease affecting the intestine, the method comprising determining the proportion and / or location of disease-associated epithelial cells within intestinal tissue of said subject.

[0127] The disease-associated epithelial cells are as disclosed herein for the first aspect. The method may comprise determining the proportion and / or location of MUC6-expressing epithelial cells. More particularly, these cells may be referred to as MGN cells or MGN-like cells. The cells may be disease-associated inflammatory MGN- like cells. The method may comprise determining the proportion and / or location of MUC5AC-producing epithelial cells. The cells may be referred to as surface foveolar cells or surface foveolar-like cells.

[0128] The method may identify subjects with inflammatory bowel disease, Crohn’s disease, ulcerative colitis, or Celiac disease. The method may identify subjects that are at risk of colitis-associated colorectal cancer.

[0129] The method may be a method of identifying subjects suitable for treatment according to the eighth, ninth, or tenth aspects. The method of the eleventh aspect may further comprise treating the subject by performing the steps of the eighth, ninth, or tenth aspects.

[0130] The method may comprise identification of enrichment of mucous gland neck-like cells, as discussed herein, in the inflamed ileum. Such subjects may have IBD. The method may comprise identification of enrichment of mucous gland neck-like cells, as discussed herein, in the inflamed duodenum. Such subjects may have Celiac disease. The method may comprise identification of enrichment of mucous gland neck-like cells, as discussed herein, in the inflamed colon. Such subjects may have ulcerative colitis. The method may comprise identification of enrichment of mucous gland neck-like cells, as discussed herein, in the inflamed duodenum and / or ileum. Such subjects may have Crohn’s disease. The method may comprise identification of enrichment of surface foveolar-like cells, as discussed herein, in the inflamed ileum. Such subjects may have IBD. The method may comprise identification of enrichment of surface foveolar-like cells, as discussed herein, in the inflamed colon. Such subjects may have ulcerative colitis. The method may comprise identification of enrichment of surface foveolar-like cells, as discussed herein, in the inflamed ileum. Such subjects may have Crohn’s disease.

[0131] The method may involve the examination of a biopsy or a tissue resection from the intestine of the subject. In some embodiments, the methods involve the examination of a biopsy or tissue resection but not the taking of the biopsy or tissue resection.

[0132] The methods may be used for the treatment of the human or animal body. The methods may be for treatment of a mammalian subject. The methods may be for treatment of a mouse, rat, rabbit, dog, cat, horse, or pig. Preferably, the methods are for the treatment of a human patient.

[0133] Administration is normally in a “therapeutically effective amount”, this being sufficient to show benefit to a subject. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of medical professionals and may depend on the severity of the symptoms and / or progression of a disease being treated. A therapeutically effective amount or suitable dose of a medicament may be determined by comparing in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the medicament is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the medicament and the nature of any detectable label or other molecule attached to the composition.

[0134] The methods of treatment include prophylactic or preventative treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset).

[0135] The terms “prophylactic”, “preventative”, or “preventing” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. Prevention and the like do not mean preventing a subject from ever getting the specific disease or disorder. Prevention may require the administration of multiple doses. Prevention can include the prevention of a recurrence of a disease in a subject for whom all disease symptoms were eliminated, or prevention of recurrence in a relapsing-remitting disease.

[0136] Administration may be via any suitable method, such as intravenous or subcutaneous administration, e.g. by injection. Administration may be of a single dose or may be repeated periodically. The agents disclosed herein may be administered as a monotherapy or in combination with other agents.

[0137] Sequence comparisons can be conducted with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0138] The skilled technician will appreciate how to calculate the percentage identity between two nucleic sequences or two amino acid sequences. In order to calculate the percentage identity, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, the Needleman- Wunsch algorithm (e.g. as applied by Needle(EMBOSS) or Stretcher(EMBOSS), the Smith- Waterman algorithm (e.g. as applied by Water(EMBOSS)), or the LALIGN application (e.g. as applied by Matcher(EMBOSS); and (ii) the parameters used by the alignment method, for example, local versus global alignment, the matrix used, and the parameters applied to gaps. In a particular embodiment, the sequence identities disclosed herein may be calculated based on a global alignment of the relevant feature. The comparison may be of a complete length of an IL-2 domain in a composition to the relevant part of a reference sequence.

[0139] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length-dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0140] A calculation of percentage identities between two nucleic acid sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.

[0141] The sequence alignment may be a pairwise sequence alignment. Suitable services include Needle (EMBOSS), Stretcher (EMBOSS), Water (EMBOSS), Matcher (EMBOSS), LALIGN, or Gene Wise. In an example, the identity between two amino acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two amino acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (BLOSUM62), gap open (14), gap extend (4), alternative matches (1). In an example, the identity between two nucleic acid sequences may be calculated using the service Needle(EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (10), gap extend (0.5), end gap penalty (false), end gap open (10), and end gap extend (0.5). In another example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0143] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0144] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way.

[0145] EXAMPLES

[0146] Example 1 - Outline

[0147] We curated, integrated, and harmonized healthy cells across the GI tract from 24 scRNAseq datasets, covering the oral mucosa, esophagus, stomach, small and large intestines, and mesenteric lymph nodes. To process the data uniformly, we remapped raw sequencing data and processed gene counts, removing low-quality cells using scAutoQC pipeline. We used sc VI10for data integration. Our final integrated data was annotated into 7 broad lineages, subclustered, and further annotated into 136 fine-grained cell types, creating a reference atlas of ~1.1 million cells from 175 donors.

[0148] Next, we projected disease data from UC, Crohn’s disease, pediatric IBD, celiac disease (unpublished), CRC, and gastric cancer patients onto the healthy reference using scANVI and scArches11>12. Overall, we added -500,000 cells to our atlas, totaling 1.6 million cells. To annotate disease cells, we projected disease data onto our subclustered, lineage- and region- specific views of the atlas.

[0149] Example 2 - Identification of transcriptional profile of a metaplastic intestinal epithelial cell type in human IBD tissues

[0150] In the single cell data from small intestine, we identified two distinct epithelial populations with unique signatures in both healthy and diseased samples. Mucous gland neck cells expressed MUC6, PGC, AQP5, and BPIFB1, while Surface foveolar cells showed selective expression of MUC5AC, CEACAM7 (Figure 1 a,b). In healthy people, we observed these cells in healthy stomach and duodenum (likely Brunner's glands). In patients with IBD, Mucous gland neck-like cells were enriched in inflamed ileum. In untreated celiac disease patients, the duodenum had more Mucous gland neck cells than in controls (Figure 1c).

[0151] In healthy tissue, Brunner’s glands reside primarily in the submucosal layer of the proximal duodenum and function to guard the epithelium by secreting gel-forming mucins (including MUC6 and MUC5AC), and factors involved in immune defence, pH regulation and cell proliferation and differentiation. These cells are highly abundant in the healthy stomach epithelium, with surface foveolar cells (MUC5AC expressing, also known as pit or surface mucous cells) residing at the top of the glands and mucous gland neck cells (MUC6 expressing) residing in the lower half of the pyloric gland, with similar mucous-secreting and barrier functions13. Importantly, these cells are normally absent in healthy jejunum, ileum or large intestine and their presence in ileum (usually identified histologically via pyloric gland morphology in H&E or MUC6 / MUC5AC IHC) indicates pyloric metaplasia.

[0152] Example 3 - MUC6-expressing cells are located at the crypt and MUC5AC- producing cells located at the villus tips

[0153] To validate the presence of Mucous gland neck-like cells and Surface foveolar-like cells in patients with IBD and celiac disease, we performed IHC in patient samples. We located Mucous gland neck-like cells (MUC6+AQP5+BPIFB1+) at the crypt base and Surface foveolar-like cells (MUC5AC+) at the crypt top of metaplastic glands in Crohn’s disease mucosa (Figure 2a-c). We observed their close association with ulcerated regions and tertiary lymphoid structures (Figure 3b). We also validated Mucous gland neck-like cells in disease tissue from untreated celiac (duodenum) and UC patients (colon) (Figure 2c, d). In untreated celiac patients, MUC6-expressing metaplastic glands were distinguished from healthy MUC6+ Brunner’s gland cells by their mucosal localisation (Figure 2d). Importantly, MUC6+ or MUC5AC+ cells were not found in healthy ileum.

[0154] Thus, inflammatory mucous gland neck-like cells are found across the intestines during chronic inflammation and share transcriptional similarities to healthy mucous gland neck cells, which are restricted to the stomach and duodenum (with important differences discussed below).

[0155] Example 4 - Expansion of Mucous gland neck-like cells in human Crohn’s disease ileum and UC colon as well as in colorectal cancers

[0156] We also investigated bulk RNA-seq datasets of mucosal biopsies from pediatric and adult IBD patients. Using bulk deconvolution with our single cell data as a reference, we found significantly higher proportions of Mucous gland neck-like cells in Crohn’s disease and UC patient samples (Figure 4a). Mucous gland neck-like cells were present across the intestines in Crohn’s disease but only in the large intestines of UC patients, consistent with the aetiology and site of inflammation (Figure 4a), and detected in patients with celiac disease (Figure 4b). CRC patients with microsatellite instability (MSI-high), display higher mutational burden and increased infiltration of immune cells14. Intriguingly, there is a potential link between inflammatory Mucous gland neck-like cells and CRC. We observed inflammatory Mucous gland neck-like cells in tissue sections and bulk RNAseq data of UC patients, who have an increased risk of CRC15. Bulk deconvolution of TCGA data suggested that inflammatory Mucous gland neck-like cells are present in colon adenocarcinoma, particularly in MSI-high tumours (Figure 4c). In two independent studies, MUC6 expression in the colon of UC patients was significantly associated with neoplasms, suggesting that inflammatory Mucous gland neck-like cells may play a direct role in colitis-associated CRC16,17. These results are consistent with recent identification of gastric metaplasia related gene expression (including TFF2, AQP5 along with reduced CDX2) in serrated polyps, which are pre-cancerous lesions associated with MSI high CRC18.

[0157] Example 5 - Pseudotime analysis suggests that MUC6-expressing cells have a progenitor in common with MUC5AC- producing cells

[0158] To interrogate the origin of inflammatory Mucous gland neck-like cells, we performed trajectory analysis using Monocle 3 on small intestine epithelial cells (Figure 5a). Inflammatory Mucous gland neck-like cells branched from LGR5+ stem cells (Figure 5a) and retained expression of sternness genes along the trajectory (Figure 5b). Using smFISH, we found LGR5 and MKI67 expression in inflammatory Mucous gland neck-like cells in tissue from Crohn’s disease ileum (Figure 5c), validating a sternness and proliferative phenotype.

[0159] To identify potential intercellular networks impacting on stem cell and Mucous gland neck-like cell trajectories and function, we performed cell-cell communication analysis with LIANA+19, which combines different ligandreceptor databases and analysis methods. Using the output of LIANA+ for NMF analysis, we identified a factor representing signalling from fibroblast subsets to stem cells and inflammatory Mucous gland neck-like cells. Predicted interactions among these cell types included the ligands NGR1, AREG and EREG, which were upregulated in oral mucosa / inflammatory fibroblasts and potentially signal to stem cells and inflammatory Mucous gland neck-like cells via EGFR / ERBB2 / ERBB3.

[0160] Together, our data suggest that metaplasia can arise from inflammation- induced changes within crypt-based stem cells giving rise to Mucous gland neck-like cells, the major lineage of pyloric metaplasia. Moreover, Mucous gland neck-like cells retain stem-like properties in intestinal disease, representing a plastic population.

[0161] Example 6 - Inflammatory intestinal epithelial cells have mucosal healing as well as pro-inflammatory properties and are located in close proximity to the immune infiltrates and vasculature.

[0162] Inflammatory Mucous gland neck-like cells possess antimicrobial and inflammatory properties (Figure 6). These cells expressed genes involved in the antibacterial response (Figure 6a), including LCN2, which limits bacterial growth, and BPIFB1 , which binds bacterial LPS and modulates cellular responses. SLPI protects epithelial surfaces from proteolytic enzymes and has broad antimicrobial activity. CXCL2 is an antimicrobial chemokine expressed during inflammation. LTF, an iron-binding protein, has antimicrobial properties. CFH regulates complement activation, targeting microbial infections, and CRISP3, although its exact role is unclear, is related to innate immunity. Additionally, the expression of DUOX2 and DUOXA2 was detected, with roles in generating reactive oxygen species for microbial killing.

[0163] This cell type expressed genes involved in mucus production and gastric function (Figure 6a), including MUC6, MUC5AC, ZG16B, MUC5B, and WFDC2. TESC regulates cell pH by controlling Na(+) / H(+) exchange. TCN1 binds and protects vitamin Bl 2 in the acidic stomach environment. PGC encodes a digestive enzyme in the gastric mucosa, and LIPF encodes gastric lipase, crucial for fat digestion. A4GNT is necessary for type III mucin synthesis, protecting against gastric inflammation. KCNN4 encodes a potassium channel protein involved in calcium influx. TFF2 and TFF3 stabilizes the mucus layer and may aid in mucosal protection and healing, while inhibiting gastric acid secretion. Inflammatory Mucous gland neck cells also may contribute to chronic intestinal inflammation by upregulating cytokine- induced inflammatory programs and IFNy-mediated pathways (Figure 6a-d). They overexpress chemokines like CXCL16, CXCL2 / 3 / 5, and CXCL17, recruiting various immune cells. CXCL2 / 3 / 5 on inflammatory Mucous gland neck-like cells interacts with the ACKR1 receptor on venous endothelial cells, axis that is known to be needed for the recruitment of neutrophils into the tissue. Using smFISH, we found a close association of ACKR1 + endothelial vascular cells with MUC6-expressing cells in CD tissue (Figure 7).

[0164] These cells also show elevated MHC-II gene expression and HLA-DR protein levels in Crohn’s disease patients, suggesting a role in antigen presentation (Figure 6 a, b, e). In addition, we observed CD8+, CD4+ and y5 T cells surrounding MUC6-expressing cells in CD and celiac disease tissue, in contrast to low numbers of T cells surrounding healthy Brunner’s glands (Figure 8a). Inflammatory Mucous gland neck cells may attract CD4+ and CD8+ T cells, acting as non- conventional antigen-presenting cells, exacerbating chronic inflammation in IBD and celiac disease. Overall, in addition to the mucosal healing properties due to antimicrobial and mucus production functions, MUC6-expressing cells can exacerbate chronic inflammation through interactions with immune cells and vasculature.

[0165] Example 7 - Selected receptors upregulated in inflammatory intestinal epithelial cells

[0166] Mucins are mostly secreted protein (e.g. MUC6), therefore identifying other surface receptors that are expressed on the surface of inflamed epithelial cells can provide therapeutic strategies to target these cells. We analysed single cell and spatial transcriptomics data and identified several surface receptors expressed in inflamed epithelial cells (Table 1). We identified a selection of surface receptors that are found in either Mucous gland neck- like cells, surface foveolar-like cells or both. While some receptors were upregulated compared to similar cell types in the stomach (e.g. AQP5, CLDN2), others were equally expressed in disease-associated cells and their healthy counterparts (e.g. VSIG1, CLDN18). Validation of receptor expression was done using lOx Genomics Xenium spatial transcriptomics technology (Figure 9). Some expression od selected surface receptors are present in other organs beyond gastrointestinal tract, as showed in bulk RNA-sequencing data from GTEx (Figure 10). Most receptor expression is restricted to epithelial cells (e.g. AQP5, VSIG1, CLDN2, CLDN18, DUOX2) across healthy human organs, as identified through analysis of single cell data across multiple human organs and cell lineages (Figure 11).

[0167] References

[0168] 1. Verstockt, B. et al. IL-12 and IL-23 pathway inhibition in inflammatory bowel disease. Nat. Rev. Gastroenterol. Hepatol. 20, 433-446 (2023).

[0169] 2. Neurath, M. F. Current and emerging therapeutic targets for IBD. Nat. Rev. Gastroenterol. Hepatol. 14, 269-278 (2017).

[0170] 3. Overwijk, W. W., Tagliaferri, M. A. & Zalevsky, J. Engineering IL-2 to give New Life to T cell immunotherapy. Annu. Rev. Med. 72, 281-311 (2021).

[0171] 4. Neri, D. Antibody-cytokine fusions: Versatile products for the modulation of anticancer immunity. Cancer Immunol. Res. 7, 348-354 (2019).

[0172] 5. Elmentaite, R. et al. Cells of the human intestinal tract mapped across space and time. Nature 597, 250- 255 (2021).

[0173] 6. Martin, J. C. et al. Single-cell analysis of Crohn’s disease lesions identifies a pathogenic cellular module associated with resistance to anti-TNF therapy. Cell 178, 1493-1508. e20 (2019).

[0174] 7. Elmentaite, R. et al. Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn’s disease. bioRxiv (2020) doi: 10.1101 / 2020.02.06.937110.

[0175] 8. Smillie, C. S. et al. Intra- and inter-cellular rewiring of the human colon during ulcerative colitis. Cell 178, 714-730. e22 (2019).

[0176] 9. Parikh, K. et al. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 567, 49-55 (2019).

[0177] 10. Lopez, R., Regier, J., Cole, M. B., Jordan, M. I. & Yosef, N. Deep generative modeling for single-cell transcriptomics. Nat. Methods 15, 1053-1058 (2018).

[0178] 11. Xu, C. et al. Probabilistic harmonization and annotation of single-cell transcriptomics data with deep generative models. Mol. Syst. Biol. 17, e9620 (2021). 12. Lotfoilahi, M. et al. Mapping single-cell data to reference atlases by transfer learning. Nat. Biotechnol.

[0179] 40, 121-130 (2022).

[0180] 13. Willet, S. G. & Mills, J. C. Stomach organ and cell lineage differentiation: From embryogenesis to adult homeostasis. Cell. Mol. Gastroenterol. Hepatol. 2, 546-559 (2016). 14. Mlecnik, B. IntegrativeAnalysesofColorectalCancerShowImmunoscore Is a Stronger Predictor of Patient

[0181] Survival Than Microsatellite Instability. Immunity 44, 698-711 (2016).

[0182] 15. Lakatos, P. L. & Lakatos, L. Risk for colorectal cancer in ulcerative colitis: Changes, causes and management strategies. World J. Gastroenterol. 14, 3937 (2008).

[0183] 16. Borralho, P., Vieira, A., Freitas, J., Chaves, P. & Soares, J. Aberrantgastric apomucin expression in ulcerative colitis and associated neoplasia. J. Crohns. Colitis 1, 35-40 (2007).

[0184] 17. Tatsumi, N. et al. Cytokeratin 7 / 20 and mucin core protein expression in ulcerative colitis-associated colorectal neoplasms. Virchows Arch. 448, 756-762 (2006).

[0185] 18. Chen, B. et al. Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell 184, 6262-6280. e26 (2021). 19. Dimitrov, D. et al. LIANA+: an all-in-one cell-cell communication framework. bioRxiv

[0186] 2023.08.19.553863 (2023) doi:10.1101 / 2023.08.19.553863.

Claims

CLAIMS1. A composition comprising : i) a first antigen-binding domain that is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74; and ii) a therapeutic moiety.

2. The composition of claim 1 , wherein the first antigen-binding domain is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1.

3. The composition of claim 1 or claim 2, wherein the first antigen-binding domain is specific for any one of AQP5, CLDN2, PROMI, BACE2, MSLN, TM4SF1, TSPAN1, DUOX2, GPRC5A, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74, and wherein the composition binds to MUC6-expressing epithelial cells and / or is selective for MUC6-expressing epithelial cells.

4. The composition of claim 3, wherein the MUC6-expressing epithelial cells: are mucous gland neck cells; and / or are present at the base of intestinal crypts; and / or express PGC, AQP5, and BPIFB1.

5. The composition of claim 1 or claim 2, wherein the first antigen-binding domain is specific for any one of TSPAN1, DSG2, CEACAM5, DUOX2, GPRC5A, VSIG1, VSIG2, CLDN18, MUC1, CD24, PIGR, and CD74, and wherein the composition binds to MUC5AC-producing cells and / or is selective for MUC5AC-producing cells.

6. The composition of claim 5, wherein the MUC5AC-producing cells: are surface foveolar cells; and / or are present at intestinal villus tips; and / or express CEACAM7, CEACAM1 , DUOX2, and LCN2.

7. The composition of any preceding claim, wherein the therapeutic moiety is a protein domain.

8. The composition of any preceding claim, wherein the therapeutic moiety is biologically active.

9. The composition of any preceding claim, wherein the therapeutic moiety: i) is immunosuppressive, and / or ii) enhances regulatory T cell activity and / or cell number; and / or iii) enhances regulatory T cell suppressive function, proliferation, and / or survival.

10. The composition of any preceding claim, wherein the therapeutic moiety is a cytokine, or a mutein and / or portion thereof.11 The composition of any preceding claim, wherein the therapeutic moiety is IL-2, or a mutein and / or portion thereof.

12. The composition of claim 11 , wherein the IL-2 is a mutein with selectivity for regulatory T cells over conventional T cells.

13. The composition of any preceding claim, wherein the therapeutic moiety is an immunosuppressive cytokine, or a mutein and / or portion thereof.

14. The composition of claim 13, wherein the therapeutic moiety is IL-10, or a mutein and / or portion thereof.

15. The composition of claim 8, wherein the therapeutic moiety is IL-4, or a mutein and / or portion thereof.

16. The composition of claim 1 , wherein the first antigen-binding domain is specific for any one of AQP5, CLDN2, CLDN18, DUOX2, and VSIG1 ; and the therapeutic moiety is IL-2, or a mutein and / or portion thereof.

17. The composition of any preceding claim, wherein the first antigen-binding domain is an antigen-binding portion of an antibody.

18. The composition of any preceding claim, wherein the first antigen-binding domain is a single-chain fragment variable (scFv).

19. The composition of claim 17 or claim 18, wherein the first antigen-binding domain is human or humanized.

20. The composition of any preceding claim, wherein the therapeutic moiety is derived from a human protein.

21. The composition of any preceding claim, wherein the composition is or comprises a polypeptide or a fusion protein.

22. One or more nucleic acid molecules encoding a composition of any one of claims 1 to 21.

23. One or more vectors comprising the one or more nucleic acid molecules of claim 22.

24. A cell comprising the one or more nucleic acid molecules of claim 22 or the one or more vectors of claim 23.

25. A pharmaceutical composition comprising a composition of any one of claims 1 to 21, one or more nucleic acid molecules of claim 22, one or more vectors of claim 23, or a cell of claim 24.

26. A composition of any one of claims 1 to 21 , one or more nucleic acid molecules of claim 22, one or more vectors of claim 23, a cell of claim 24, or a pharmaceutical composition of claim 25 for use in a method of treatment.

27. The composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition for use of claim 26, wherein the method is for the treatment of inflammation of the intestine.

28. The composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition for use of claim 27, wherein the inflammation is chronic inflammation.

29. The composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition for use of claim 27 or claim 28, wherein the inflammation is inflammation of the ileum, duodenum, or colon.

30. The composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition for use of any one of claims 26 to 29, wherein the method is for the treatment of inflammatory bowel disease.

31. The composition, one or more nucleic acids, one or more vectors, cell, or pharmaceutical composition for use of any one of claims 26 to 29, wherein the method is for the treatment of Crohn’s disease, ulcerative colitis, Celiac disease, or colitis-associated colorectal cancer (CRC).

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