WO2022073038A1 - Interleukin-22 (il-22) fusion proteins and uses thereof - Google Patents

Abstract

The disclosure provides a fusion protein comprising human IL-22, or an active fragment thereof, conjugated to an anti-tenascin scFv, and uses thereof.

Description

INTERLEUKIN-22 (IL-22) FUSION PROTEINS AND USES THEREOF RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63/086,967, filed on October 2, 2020. The content of the foregoing priority application is incorporated by reference herein in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 24, 2021, is named A103017_1520WO_SL.txt and is 15,808 bytes in size. BACKGROUND OF THE INVENTION Interleukin-22 (IL-22) is a 17 kDa globular cytokine belonging to the IL-10 family. IL-22 is mainly secreted by NK cells, dendritic cells and T-cells (Murphy (2012) Janeway’s Immunobiology Garland Science). IL-22 contains two intramolecular disulfide bonds and three N-linked glycosylation sites. IL-22 is associated with inflammatory bowel disease (IBD) susceptibility genes that are crucial for regulating tissue responses during inflammation (Mizoguchi (2012) Inflamm Bowel Dis.18:1777–1784). These processes play important roles in the pathogenesis of IBD. In the intestine, IL22 induces mucin production, antimicrobial, proliferative and antiapoptotic pathways, which prevent tissue damage and promote epithelial repair. (Li et al. (2014) World J Gastroenterol 20(48):18177-18188). While IL-22 is associated with decreasing intestinal inflammation, targeting IL-22 to the intestine as a therapeutic presents a unique challenge. SUMMARY OF THE INVENTION Provided herein are fusion proteins comprising human IL-22, or a functional fragment thereof, fused to an anti-tenascin C domain D single chain variable fragment (scFv). In one aspect, the invention provides a fusion protein comprising human interleukin-22 (hIL-22) and a single chain variable fragment (scFv) that specifically binds to human tenascin C domain D, wherein the hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, wherein the scFv comprises a heavy chain variable (VH) domain comprising the amino acid sequence set forth as SEQ ID NO: 5 and a light chain variable (VL) domain comprising the amino acid sequence set forth as SEQ ID NO: 7, and wherein the hIL-22 is conjugated to the N- terminus of the scFv via a peptide linker. In certain embodiments, the peptide linker is 10 to 20 amino acids long. In another embodiment, the peptide linker comprises the amino acid sequence set forth as SEQ ID NO: 3. In certain embodiments, the VH domain and the VL domain of the scFv are conjugated via a peptide linker comprising the amino acid sequence set forth as SEQ ID NO: 6. In one aspect, the invention provides a fusion protein comprising human interleukin-22 (hIL-22) and a single chain variable fragment (scFv) that specifically binds to human tenascin C domain D, wherein the hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, wherein the scFv comprises the amino acid sequence set forth as SEQ ID NO: 4 , and wherein the hIL-22 is conjugated to the N-terminus of the scFv via a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence set forth as SEQ ID NO: 3. In a further aspect, the invention provides a fusion protein comprising human interleukin- 22 (hIL-22) and an anti- human tenascin C domain D single chain variable fragment (scFv), wherein the fusion protein comprises the amino acid sequence set forth as SEQ ID NO: 1. In certain embodiments, the invention pertains to a diabody comprising two of the fusion proteins disclosed herein. In other embodiments, the invention provides a method of treating a human subject having inflammatory bowel disease (IBD), the method comprising administering a therapeutically effective dose of a fusion protein or a diabody disclosed herein, to the human subject having IBD. In one embodiment, the IBD is Crohn’s disease or ulcerative colitis. In certain other aspects, the invention provides a nucleic acid encoding a fusion protein disclosed herein; a vector comprising the nucleic acid; or a host cell comprising the vector. In other aspects the invention provides a method of producing a fusion protein disclosed herein, the method comprising culturing a host cell comprising a vector comprising a nucleic acid encoding the fusion protein, under conditions suitable for production of the fusion protein. In yet other aspects, the invention pertains to a pharmaceutical composition comprising a fusion protein disclosed herein or a diabody disclosed herein, and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic representation of the fusion protein IL-22-CPR01 containing from amino to carboxy terminus the following elements: human IL-22 (hIL-22) with a signal peptide (SP) connected via a peptide linker (L1 (GGGGS)3) to an anti-tenascin single chain variable fragment (scFv) comprising the VH and VL domains of antibody CPR01. The VH and VL domains of the scFv are linked by a peptide linker (L2 GGSGG). FIG.1 also describes cloning sites of the fusion protein into EcoR1 and Not1 of the pCDAN3.1 vector using primers a, b, c, d, e and f. Sequences of the primers are described in the Sequence Summary Table. FIG.2 graphically depicts results of an in vitro experiment showing phosphorylation of STAT3 in human colon epithelial cell line T-84 by increasing concentrations of either human IL- 22-CPR01 (square in key) or recombinant human IL-22 protein (rhIL-22; circle in key). FIGS 3A-3D provide micrographs showing the localization of mouse IL-22-CPR01 (muIL-22-CPR01; surrogate of human IL-22-CPR01) in colon tissues from a dextran sodium sulfate (DSS)-induced colitis animal model. FIG.3A provides near-infrared (NIR) fluorescence image of muIL-22-CPR01 localization in colon tissues from the DSS-induced colitis model. FIG. 3B provides a magnified image of the inset shown in FIG.3A. FIG.3C provides a micro- autoradiography (MARG) image of muIL-22-CPR01 localization (black specks) in colon tissue of a DSS-treated mouse that was dosed with ¹²⁵I-radiolabeled muIL-22-CPR01. FIG.3D provides a magnified image of the inset shown in FIG.3C. FIG.4 graphically depicts results of an experiment showing protein levels (expressed as mean fluorescence intensity) of murine-IL-22-CPR01 (muIL-22-CPR01) and a non-specific antibody huIL-22 protein fusion (-huIL-22-Ab) (untargeted antibody linked to human Il-22 in the same orientation as IL-22-CPR01) in whole colon of naïve C57/B6 mice, whole colon of 7-day DSS-treated mice, and colon lesions of 7-day DSS-treated mice that were dosed with 150 µg of 800CW-labeled huIL-22-Ab or 800CW-labeled muIL-22-CPR01. FIGS 5A-5D graphically depicts data showing the advantage offered by targeted delivery of murine IL-22-CPR01 (muIL-22-CPR01) over a control murine IL-22-Fc fusion protein (muIL- 22-Fc) in DSS-treated mice that were administered 150 µg of 800CW-labeled muIL-22-CPR01 or 800CW-labeled muIL-22-Fc on day 3 following DSS initiation. Concentrations for muIL-22- CPR01 or muIL-22-Fc in whole blood or tissue (colon) homogenates were calculated by extrapolation of fluorescence intensities using calibration curves obtained with serial dilutions of the 800CW-conjugated proteins. FIG.5A graphically depicts results of an experiment showing whole blood pharmacokinetic (PK) profile for muIL-22-CPR01 or muIL-22-Fc in DSS-treated mice that were dosed with 150 µg of 800CW-labeled muIL-22-CPR01 or muIL-22-Fc at 8h, 24h and 72h post-dosing. n=3 for each time-point. FIG.5B graphically depicts results of an experiment showing concentration of muIL-22-CPR01 or muIL-22-Fc in colon of DSS-treated mice that were dosed with 150 µg of 800CW-labeled muIL-22-CPR01 or muIL-22-Fc at 8h, 24h and 72h post-dosing. n=3 for each time-point. FIG.5C graphically depicts results of an experiment showing colon:blood ratio (colon/blood ratio) of muIL-22-CPR01 or muIL-22-Fc in DSS-treated mice that were dosed with 150 µg of 800CW-labeled muIL-22-CPR01 or muIL-22- Fc at 8h, 24h and 72h post-dosing. n=3 for each time-point. FIG.5D provides representative NIR microscopy images of muIL-22-CPR01 localization in lesional regions of DSS colon tissue from DSS-treated mice that were dosed with 150 µg of 800CW-labeled muIL-22-CPR01 at 8h, 24h and 72h post-dosing. FIG.6A and 6B provide representative NIR fluorescence endoscopy images showing NIR intensity map superimposed on white light images and demonstrates accumulation of muIL- 22-CPR01 in inflamed mucosa of DSS-treated undosed mice (Fig 6B) or DSS-treated mice that were dosed with 150 µg of 800CW-labeled muIL-22-CPR01 at 24h post-dosing (Fig.6A). FIGS 7A-7B provide results from experiments showing prevention of epithelial erosion and suppression of inflammation in mice from a DSS-induced colitis model that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-CPR01 fusion protein (muIL- 22-CPR01) every other day beginning at the initiation of DSS. PBS was used as a negative control. FIG 7A graphically depicts results of an experiment showing decrease in epithelial damage (expressed as decrease in erosion length) in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-CPR01 every other day beginning at the initiation of DSS. FIG 7B graphically depicts results from an experiment showing decrease in macrophage infiltration (expressed as decrease in % of IBA1) in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-CPR01 every other day beginning at the initiation of DSS. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 ANOVA with Dunnett multiple comparison vs vehicle. PBS was used as a negative control. FIGS 8A-8D provide results from experiments comparing in vivo activity of a murine IL- 22 CPR01 fusion protein (muIL-22-CPR01) (Figures 8A and 8B) and muIL-22-Fc (Figures 8C and 8D) in a mouse model of DSS colitis that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-CPR01 or muIL-22-Fc on days 0 and 3 beginning at the time of DSS initiation (day 0). On day 7, colons, feces and serum were collected for analyses. FIG.8A is a graph showing decrease in erosion length (circle) and increase in fecal Reg3β (square) level in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22- CPR01 on days 0 and 3 beginning at the time of DSS initiation (day 0). FIG.8B is a graph showing decrease in erosion length (circle) and unchanged serum SAA level (square) in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-CPR01 on days 0 and 3 beginning at the time of DSS initiation (day 0). FIG.8C is a graph showing decrease in erosion length (circle) and increase in fecal Reg3β level (square) in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-Fc on days 0 and 3 beginning at the time of DSS initiation (day 0). FIG.8D is a graph showing decrease in erosion length (circle) and increase in serum SAA level (square) in DSS mice that were treated with increasing doses (5, 15, 50, and 150 µg/mouse) of muIL-22-Fc on days 0 and 3 beginning at the time of DSS initiation (day 0). DETAILED DESCRIPTION OF THE INVENTION The invention relates to a fusion protein containing human IL-22 fused to a single chain variable fragment (scFv) that recognizes the D domain of human tenascin C, and use of such fusion protein, e.g., in the treatment of inflammatory bowel disease (IBD). I. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The term “fusion protein”, as used herein, refers to a recombinant protein created by linking two or more polypeptides where the components of the fusion protein are linked to each other by peptide-bonds. A fusion protein may be produced by joining two or more polynucleotides that originally coded for separate proteins into a single polynucleotide. In certain embodiments, the components of the fusion protein are connected via a peptide linker. In other embodiments, domains within a fusion protein are directly fused without a linker. In one embodiment, a fusion protein comprises human IL-22 (hIL-22 or huIL-22) and an anti-tenascin C scFv where the hIL-22 and the scFv are connected via a peptide linker. The term "linker" is used herein to denote an amino acid or polypeptide comprising two or more amino acid residues joined by peptide bonds, that is used to connect two or more protein moieties, e.g., two domains of a fusion protein. For example, a VH and a VL of an scFv may be connected via a linker. Further, human IL-22 and an scFv described herein may be conjugated via a linker. As used herein, the term “conjugated” or “linked” refers to covalent attachment or linkage of one molecule to another molecule, e.g., a cytokine, or active fragment thereof, to an scFv. In one embodiment, two polypeptides are conjugated to form a fusion protein. The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Also contemplated are antibody binding fragments. The binding fragments of the disclosure include those that are capable of specifically binding to tenascin C, or more specifically to the D domain of tenascin C. Examples of antibody binding fragments include by way of example and not limitation, Fab, Fab', F(ab')₂, Fv fragments, and single domain fragments. A Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab' fragments are produced by cleavage of the ^{disulfide bond at the hinge cysteines of the F(ab')} ₂ ^{pepsin digestion product. Additional chemical} couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F(ab') ₂ fragments lack the Fragment crystallizable (Fc) region of an intact antibody, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than an intact antibody (see, e.g., Wahl et al., 1983, J. Nucl. Med.24:316). As is commonly understood in the art, an "Fc" region is the Fragment crystallizable constant region of an antibody not comprising an antigen-specific binding region. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of an antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain. An "Fv" fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single domain fragments" are composed of a single VH or VL domains which exhibit sufficient affinity to tenascin. In a specific embodiment, the single domain fragment is camelized (See, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38). A "single-chain Fv" or "scFv" comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form a structure favorable for target binding. As used herein, the term “diabody” refers to a dimer comprising two scFv molecules that are covalently or noncovalently attached. The scFv molecule may further be conjugated to another protein(s), e.g., human IL-22 or an active fragment thereof. In such an instance, a diabody would contain a dimer of a fusion protein, each containing an scFv. The term "subject" refers to a human which is to be the recipient of a particular treatment. The terms "subject" and "patient" are used interchangeably herein in reference to a human subject. The term "therapeutically effective amount" refers to an amount of a fusion protein effective to "treat" a disease or disorder in a human. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result. Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen one or more symptoms of, and/or slow or halt progression of a diagnosed pathologic condition or disorder (“therapeutic treatment”). Thus, those in need of therapeutic treatment include those already diagnosed with or suspected of having the disorder. Prophylactic or preventative measures refer to measures that prevent the development of a targeted pathological condition or disorder (“prophylactic treatment”). Thus, those in need of prophylactic treatment include those prone to have the disorder and those in whom the disorder is to be prevented. As used herein, the term "specifically binds" refers to the ability of a binding polypeptide to bind to an antigen with an Kd of at least about 1x10^-6 M, 1x10^-7 M, 1x10^-8 M, 1x10^-9 M, 1x10- ¹⁰ M, 1x10^-11 M, 1x10^-12 M, or more, and/or bind to an antigen with an affinity that is at least two- fold greater than its affinity for a nonspecific antigen. The term "vector", as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Various aspects of the invention are described in further detail in the following subsections. II. IL-22 / Anti-Tenascin C Fusion Protein and Uses Thereof Disclosed herein are fusion proteins comprising human IL-22 conjugated to an anti- tenascin C scFv that binds to human tenascin C domain D. The disclosed fusion proteins provide for localized delivery of IL-22 to a target tissue, e.g., inflamed intestinal tissue, that would benefit from IL-22 activity. Localized delivery is achieved by fusing human IL-22 to an antibody fragment that binds to human tenascin C, specifically the alternatively spliced D domain of tenascin C. In one embodiment, a fusion protein comprises an scFv comprising the variable heavy (VH) and variable light (VL) regions of the anti-tenascin C antibody CPR01 conjugated to human IL-22. An exemplary fusion protein disclosed herein comprises the amino acid sequence set forth as SEQ ID NO: 1 in the Sequence Summary Table below. Additional details regarding human IL-22 / anti-tenascin C scFv fusion proteins are provided below. IL-22 is a 17 kDa globular cytokine belonging to the IL-10 family, and is secreted by NK cells, dendritic cells and T-cells (Murphy, Janeway’s Immunobiology, Garland Science (2012)). IL-22 is also referred to as IL-10-related T-cell-derived inducible factor, TIFa, IL-21, ILTIF, IL- TIF, IL-D110, zcyto18, TIFIL-23. The gene sequence of human IL-22 is described at reference sequence NCBI Gene NO. 50616, and the nucleotide mRNA reference sequence can be found at reference sequence NM_020525.5. The amino acid sequence of precursor human IL-22 is provided below as SEQ ID NO: 20 (see also GenBank accession / version no. AAH70261.1):

Provided herein is a fusion protein comprising human IL-22, or an active fragment thereof, where the fusion protein is designed to target IL-22 to tissue or cells that could benefit from human IL-22 activity. In certain embodiments, the fusion protein comprises an active fragment of IL-22 linked to an anti-tenascin C domain D scFv, e.g., scFv comprising the amino acid sequence set forth as SEQ ID NO: 4. Thus, in one embodiment, the fusion protein of the invention comprises human IL-22 comprising the amino acid sequence of SEQ ID NO: 2. Also included in the invention are fusion proteins comprising active fragments of SEQ ID NO: 20 Active fragments of human IL-22 can be identified according to standard methods known in the art where the active fragment retains one or more biological activities associated with human IL-22. For example, human IL-22 activates intracellular kinase JAK which in turn induces phosphorylation of STAT3. Thus, an active fragment of human IL-22 can be identified as a fragment that is able to induce phosphorylation of STAT3 in an in vitro assay. An example of such an assay is provided in Example 2 using T-84 human colon epithelial cells. Other examples of assays that could be used to determine whether a fragment of human IL-22 (a fragment of SEQ ID NO: 20) is active include the ability of the fragment to activate JAK/STAT, ERK, JNK, and p38 MAP kinase pathways; induce activation of JAK1 and Tyk2; or induce phosphorylation of STAT1, STAT3, and STAT5 on tyrosine residues. In certain embodiments, a fusion protein of the invention comprises human IL-22 comprising SEQ ID: 2 or 20, or an active fragment thereof, comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to either SEQ ID NO: 20 or 2. Also contemplated herein are fusion proteins comprising human IL-22 comprising the amino acid sequence set forth as SEQ ID NO: 2, or an active fragment thereof, where the amino acid sequence contains conservative amino acid substitutions that do not substantially impact the activity of human IL-22, or the fragment thereof. Activities of human IL-22 that can be tested are described above in the context of fragments and are also relevant to testing variants of IL- 22. Conservative amino acid substitutions may be defined by substitutions within the classes of amino acids, i.e., acidic residues Asp (D) and Glu (E); basic residues Lys (K), Arg (R), and His (H); hydrophilic uncharged residues Ser (S), Thr (T), Asn (N), and Gln (Q); aliphatic uncharged residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I); non-polar uncharged residues Cys (C), Met (M), and Pro (P); and aromatic residues Phe (F), Tyr (Y), and Trp (W). In certain embodiments a fusion protein of the invention comprises human IL-22, or an active fragment thereof, having at least 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1, conservative amino acid residue replacements, e.g., 9, 8, 7, 6, 5, 4, 3, 2 or 1, conservative amino acid residue substitutions within SEQ ID NO: 2. As disclosed herein, targeted delivery of human IL-22 is achieved by conjugating human IL-22, or an active fragment thereof, to an antibody, or an antigen binding fragment thereof, that binds to human tenascin C, specifically the D domain of human tenascin C. By conjugating human IL-22 to an anti-tenascin C (D domain of tenascin C) antibody or an antigen-binding fragment thereof, human IL-22 is directed to tissues expressing the D domain of tenascin C. Tenascin C modulates cellular adhesion and is a large hexameric glycoprotein of the extracellular matrix. Tenascin C is involved in cell proliferation and cell migration and is associated with changes in tissue architecture as occurring during morphogenesis and embryogenesis as well as under tumorigenesis or angiogenesis. Several isoforms of tenascin C are generated as a result of alternative splicing, which leads to the inclusion of any one of multiple domains in the central part of the protein, ranging from domain A1 to domain D (Borsi et al., Int J Cancer 52:688-692 (1992); Carnemolla et al., Eur J Biochem 205:561-567 (1992); WO 2006/050834). In particular embodiments, a fusion protein disclosed herein comprises an anti-tenascin C antibody or a fragment thereof (e.g., an antigen-binding fragment thereof) that specifically binds to the D domain of tenascin C, conjugated to human IL-22, or an active fragment thereof. In certain embodiments of the present disclosure, the anti-tenascin C antibody, or antigen-binding fragment thereof, is CPR01, or an antigen binding fragment of CPR01. CPR01 and CPR01 variants are described, for example, in WO 2017/097990 and US Patent No. 10,647,760, the contents of which are incorporated herein by reference in its entirety. CPR01 cross reacts with the D domain of murine and human tenascin C. The amino acid sequence of the variable heavy (VH) chain region of CPR01 is as set forth as SEQ ID NO: 5, and the amino acid sequence of the variable light chain region of CPR01 is as set forth as SEQ ID NO: 7. An anti-tenascin C scFv for use in the compositions and methods described herein may comprise the variable heavy and light chains of antibody CPR01, i.e., the amino acid sequences set forth as SEQ ID NOs: 5 and 7, respectively. Thus, in certain embodiments of the invention, a fusion protein comprises human IL-22, or an active fragment thereof, conjugated to an anti-tenascin C scFv comprising a variable heavy chain region comprising an amino acid sequence set forth as SEQ ID NO: 5 and a variable light chain region comprising an amino acid sequence set forth as SEQ ID NO: 7. In another embodiment, the fusion protein comprises human IL-22, or an active fragment thereof, conjugated to an anti-tenascin C scFv comprising a heavy chain variable region comprising a CDR1 domain as set forth as amino acid residues 26-35 of SEQ ID NO: 5, a CDR2 domain as set forth as amino acid residues 50-66 of SEQ ID: 5, and a CDR3 domain as set forth as amino acid residues 99-105 of SEQ ID NO: 5; and a light chain variable region comprising a light chain variable region comprising a CDR1 domain as set forth as amino acid residues 23-33 of SEQ ID NO: 7, a CDR2 domain as set forth as amino acid residues 49-55 of SEQ ID: 7, and a CDR3 domain as set forth as amino acid residues 88-98 of SEQ ID NO: 7. In other embodiments of the invention, a fusion protein comprises human IL-22, or an active fragment thereof, conjugated to an anti-tenascin C scFv comprising the amino acid sequence set forth as SEQ ID NO: 4. In some embodiments of the present disclosure, the fusion protein comprises human IL- 22, or an active fragment thereof, conjugated to an anti-tenascin C antibody, or an antigen- binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR set as set forth as SEQ ID NO: 5, and a light chain variable region comprising a light chain CDR set as described as SEQ ID NO: 7. In one embodiment of the present disclosure, the fusion protein comprises human IL-22, or an active fragment thereof, conjugated to an anti-tenascin C antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 domain as set forth as amino acid residues 26-35 of SEQ ID NO: 5, a CDR2 domain as set forth as amino acid residues 50-66 of SEQ ID: 5, and a CDR3 domain as set forth as amino acid residues 99-105 of SEQ ID NO: 5; and a light chain variable region comprising a light chain variable region comprising a CDR1 domain as set forth as amino acid residues 23-33 of SEQ ID NO: 7, a CDR2 domain as set forth as amino acid residues 49-55 of SEQ ID: 7, and a CDR3 domain as set forth as amino acid residues 88-98 of SEQ ID NO: 7In certain embodiments, a fusion protein disclosed herein comprises human IL-22, or an active fragment thereof, and an anti-tenascin C antibody, an antigen-binding fragment thereof, or an scFv, comprising a VH region having the CDRs set forth as SEQ ID NO: 5 and having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 other than the CDRs. In certain embodiments, a fusion protein disclosed herein comprises human IL-22, or an active fragment thereof, and an anti-tenascin C antibody, an antigen-binding fragment thereof, or an scFv, comprising a VL region having the CDRs set forth as SEQ ID NO: 7 and having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 other than the CDRs. Generally, sequence identity may be determined using methods known in the art. For example, sequence identity of two sequences may be determined using the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred; however, other algorithms may be used, e.g., BLAST (which uses the method of Altschul et al., J Mol Biol 215: 405-410 (1990)), FASTA (which uses the method of Pearson and Lipman, PNAS USA 85:2444- 2448 (1988)), or the Smith-Waterman algorithm (Smith and Waterman, J Mol Biol 147: 195-197 (1981)), or the TBLASTN program of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl Acids Res 25:3389-3402 (1997)) may be used. In some embodiments of the present disclosure, an anti-tenascin C antibody, an antigen- binding fragment thereof, or an scFv comprises a heavy chain and/or a light chain variable region set forth as SEQ ID NOs: 5 and 7, respectively, where the heavy and/or light chain contains conservative amino acid substitutions. As described above, conservative amino acid substitutions may be defined by substitutions within the classes of amino acids. In certain embodiments, a fusion protein of the invention comprises an anti-tenascin C scFv comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 5 and having at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1, conservative amino acid residue substitutions, and comprising a variable light chain region comprising the amino acid sequence set forth as SEQ ID NO: 7 and having 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1, conservative amino acid residue substitutions. In certain embodiments, a fusion protein of the invention comprises an anti-tenascin C scFv comprising the amino acid sequence set forth as SEQ ID NO: 4, and having 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1, conservative amino acid residue substitutions. An anti-tenascin scFv included in the fusion protein of the disclosure comprises a heavy chain variable region and a light chain variable region that are connected by a linker. In one embodiment, an anti-tenascin C scFv (e.g., scFv-CPR01) included in the fusion protein of the invention comprises a VH domain comprising the amino acid sequence set forth as SEQ ID NO: 5 and a VL domain comprising the amino acid sequence set forth as SEQ ID NO: 7, where the VH and VL domains are linked by a peptide linker. In some embodiments, the length of the peptide linker between the VH and VL chains is less than 15 amino acids, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids. In one embodiment, the peptide linker contains 5- 12, 5-11, 6-12, 7-12, 4-10 amino acids, 4-9 amino acids, or 4-8 amino acids. In one embodiment, the peptide linker comprises the amino acid sequence GGSGG (SEQ ID NO: 6). As described throughout, the present disclosure provides a fusion protein comprising an anti-tenascin C domain D antibody, an antigen-binding fragment thereof, or an scFv conjugated to human IL-22, or an active fragment of human IL-22. The anti-tenascin antibody, fragment, or scFv, and human IL-22, or fragment thereof, may be conjugated via a linker. In one embodiment, an anti-tenascin C domain D scFv and human IL-22, or an active fragment thereof, are conjugated via a peptide linker. In one embodiment, the peptide linker comprises 10-20 amino acids, 11-19 amino acids, 12-18 amino acids, 13-17 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, and so forth. In one embodiment, an anti-tenascin C scFv and human IL-22, or an active fragment thereof, are conjugated via a peptide linker comprising the amino acid sequence (GGGGS)₃ (SEQ ID NO: 3). In some embodiments, a fusion protein of the disclosure comprises human IL-22, or an active fragment thereof, conjugated either through a peptide linker or directly, to the N-terminus of an anti-tenascin C scFv. Such an orientation is described in Figure 1. In other embodiments, human IL-22, or an active fragment thereof, is conjugated to the C terminus of an anti-tenascin C scFv. In some embodiments, human IL-22 is conjugated, either through a peptide linker or directly without a linker, via its C-terminus or N-terminus to an anti-tenascin C domain D antibody molecule to form a targeted fusion protein. In certain embodiments, human IL-22 (or an active fragment thereof) is conjugated to an scFv, that binds human tenascin C domain D via the C-terminus of huIL-22 (or an active fragment thereof). In particular embodiments, huIL-22 (or an active fragment thereof) is connected via its C-terminus to an scFv comprising an amino acid sequence set forth as SEQ ID NO: 4. In one embodiment of the invention, a fusion protein comprises an active fragment of human IL-22 as set forth as SEQ ID NO: 2 conjugated via a peptide linker (e.g., SEQ ID NO: 3) to the amino terminus of an anti-tenascin C scFv comprising a heavy chain variable region (VH) comprising an amino acid sequence set forth as SEQ ID NO: 5 and a light chain variable region (VL) comprising the amino acid sequence set forth as SEQ ID NO: 7. In one embodiment, the VH and the VL are connected via a peptide linker that is 10-20 amino acids in length. In one embodiment, the VH and the VL are connected via a peptide linker comprising the amino acid sequence set forth as SEQ ID NO: 6. In one embodiment of the disclosure, a fusion protein comprises an active fragment of human IL-22 as set forth as SEQ ID NO: 2 conjugated via a peptide linker to the amino terminus of an anti-tenascin C scFv comprising the amino acid sequence set forth as SEQ ID NO: 4. In one embodiment, the peptide linker connecting the anti-tenascin scFv and human IL-22, or an active fragment thereof, comprises the amino acid sequence set forth as SEQ ID NO: 3. In certain embodiments, a fusion protein described herein comprises human interleukin- 22 (hIL-22) and an scFv that specifically binds to human tenascin C domain D, wherein hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, and wherein the scFv comprises a heavy chain variable (VH) domain comprising the amino acid sequence set forth as SEQ ID NO: 5 and a light chain variable (VL) domain comprising the amino acid sequence set forth as SEQ ID NO: 7, and wherein the hIL-22 is conjugated to the N-terminus of the scFv via a peptide linker. In other embodiments, a fusion protein of the invention comprises human interleukin-22 (hIL-22) and an scFv that specifically binds to human tenascin C domain D, wherein the hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, wherein the scFv comprises the amino acid sequence set forth as SEQ ID NO: 4 , and wherein the hIL-22 is conjugated to the N-terminus of the scFv via a peptide linker. In one embodiment of the invention, a fusion protein comprises human interleukin-22 (hIL-22) and an scFv that specifically binds to human tenascin C domain D, wherein the fusion protein comprises the amino acid sequence set forth as SEQ ID NO: 1. While, the fusion proteins described herein are described in the context of a monomer, i.e., a single chain protein comprising human IL-22 (huIL-22) or an active fragment thereof (e.g., SEQ ID NO: 2) conjugated to an anti-tenascin C domain D scFv (e.g., scFv comprising the amino acid sequence set forth as SEQ ID NO: 4) via a linker, it should be noted that monomers form diabodies in solution In solution, fusion proteins described herein form diabodies comprising two fusion proteins that associate via their respective scFv regions. This association is non-covalent. Thus, diabodies comprising fusion proteins described herein are also contemplated in the invention. In one embodiment, a diabody comprises two fusion proteins that are non- covalently associated, wherein each of the fusion proteins comprises the amino acid sequence set forth as SEQ ID NO: 1. In one embodiment, a diabody comprises two fusion proteins that are non-covalently associated, where each of the fusion proteins comprises an human IL-22 (huIL-22) or an active fragment thereof (e.g.,the amino acid sequence set forth as SEQ ID NO: 2) conjugated to an anti-tenascin C domain D scFv (e.g., scFv comprising the amino acid sequence set forth as SEQ ID NO: 4), where the carboxy terminus of the huIL-22 is conjugated via a linker to the amino terminus of the scFv. In one embodiment, the disclosure provides a solution comprising a diabody disclosed herein. The solution is characterized as a solution in which fusion proteins of the invention can form diabodies, including, for example, phosphate buffered saline (PBS). Fusion proteins of the disclosure may be produced using nucleic acid molecules corresponding to the amino acid sequences disclosed herein. In one embodiment, nucleic acid molecules comprising nucleic acid sequences set forth as SEQ ID NOs: 14, 15, 16, 17, 18, or 19, or combinations thereof, are used in the production of a fusion protein. Further provided are constructs in the form of plasmids, vectors (e.g., expression vectors), transcription or expression cassettes which comprise such nucleic acids. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids e.g., phagemid, or viral e.g., phage, as appropriate. For further details, see, for example, Sambrook & Russell (2001) Molecular Cloning: a Laboratory Manual: 3rd edition, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al., (1999) 4^th eds., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons. Vectors useful for the expression of nucleic acid molecules comprising polynucleotide sequences described in the Sequence Summary Table are also included in the invention. Such vectors can be introduced into an appropriate host cell for expression. Fusion proteins may be produced by any of a number of techniques. For example, expression from host cells, wherein expression vector(s) encoding the fusion protein is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection and the like. Preferred host cells for expressing the fusion proteins of the invention include mammalian cells. When recombinant expression vectors encoding fusion proteins are introduced into mammalian host cells, the fusion proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the fusion protein in the host cells or, more preferably, secretion of the fusion protein into the culture medium in which the host cells are grown.. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. The selected transformant host cells are cultured to allow for expression of the fusion protein and fusion protein is recovered from the culture medium. Once a fusion protein of the invention has been produced by recombinant expression or any other of the disclosed techniques, it may be purified or isolated by methods known in the art in that it is identified and separated and/or recovered from its natural environment and separated from contaminants that would interfere with diagnostic or therapeutic uses for the fusion protein. These isolated preparations may be purified using various art-recognized techniques. Fusion proteins described herein may be used for therapeutic purposes, including for the treatment of inflammatory bowel disease (IBD). Fusion proteins of the present disclosure can thus be used in treatment of IBD in patients, preferably human patients. Examples of IBD that can be treated using the fusion proteins described herein include Crohn’s Disease (CD) and ulcerative colitis (UC). In one embodiment, a human subject having IBD (e.g., CD or UC) is treated by administering to a patient suffering from IBD a therapeutically effective amount of a fusion protein of the present disclosure. In accordance with the present disclosure, compositions provided herein may be administered to mammals, preferably humans. Administration may be in a therapeutically effective amount, which is an amount sufficient to show benefit to a patient (e.g., an IBD patient (e.g., CD patient, UC patient, etc.)). Such benefit may be at least amelioration of at least one symptom (e.g., at least one symptom of IBD (e.g., CD, UC, etc.)). Fusion proteins and diabodies according to the present disclosure can be administered in the form of a pharmaceutical composition, which may contain at least one pharmaceutically acceptable excipient and/or carrier in addition to the fusion protein or diabody. Further aspects and embodiments of the disclosure will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification. EXAMPLES The following Examples may be used for illustrative purposes and should not be deemed to narrow the scope of the invention. Example 1. Cloning and expression of IL-22 anti-tenascin C scFv fusion protein The following example describes the cloning and expression of a fusion protein (referred to as IL-22-CPR01) that contains human IL-22 fused to the N-terminus of scFv-CPR01. CPR01 is an antibody that binds to the D domain of human tenascin C (see WO 2017/097990). The amino acid sequence of IL-22-CPR01 is set forth as SEQ ID NO: 1. As described in SEQ ID NO: 1 below, a 15 amino acid peptide linker (L1:(GGGGS)3) (SEQ ID NO: 3) (shown in bold) links the cytokine IL-22 (italicized in SEQ ID NO: 1) with scFv-CPR01. The VH and VL domains of scFv-CPR01 are linked by a short 5 amino acid peptide linker (L2:(GGSGG) (SEQ ID NO: 6) (underlined below). The short 5 amino acid peptide linker prevents the VH and VL of a single scFv from folding together. The diabody is thus formed as a noncovalent dimer of two scFv molecules.

A cloning map and orientation of IL-22-CPR01 is shown in Figure 1, which describes a signal peptide (SP) fused to the amino terminus of human IL-22 protein. Cloning of IL-22-CPR01 was performed by standard molecular biology techniques using primers a, b, c, d, e, and f (SEQ ID NOs 8 to 13, respectively), as described in Figure 1. The IL- 22-CPR01 nucleic acid sequence within the plasmid was confirmed by Sanger sequencing. The nucleic acid sequences of the signal peptide, hIL-22, L1 linker, CPR01 VH, L2 linker, and CPR01 VL are provided as SEQ ID NOs: 14 to 19, respectively. Example 2. IL-22-CPR01 induces STAT3 phosphorylation in vitro IL-22 signal transduction involves the activation of JAK, which subsequently induces phosphorylation of STAT3. The ability of IL-22-CPR01 to induce STAT3 phosphorylation was assessed in order to evaluate the bioactivity of IL-22-CPR01. A STAT3 phosphorylation assay in the T-84 human colon epithelial cell line that endogenously expresses IL-22 receptor complex IL-22R1 and IL-10R2 was used to test the bioactivity of IL-22-CPR01 in vitro. On day 1, T-84 cells (human colon epithelial cell line) were seeded in collagen I-coated 96-well plates (0.3x10⁵ cells/well) in 100 µl/well of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), 5% Fetal Bovine Serum (FBS), 100 U/ml Penicillin-Streptomycin, 10 mM HEPES and 2 mM L-glutamine. On day 2, medium was removed, and cells were washed with phosphate-buffered saline (PBS) to remove FBS. 100 µl assay medium (containing DMEM/F12, 100 U/ml Penicillin-Streptomycin, 10 mM HEPES and 2 mM L-glutamine) was then added to all wells. On day 3, recombinant human IL-22 protein (R&D SYSTEMS; Cat # 782-IL) and IL-22-CPR01 were titrated into different concentrations in assay medium and added on top of the cells (50 µl/well). After incubating the cells for 45 minutes at 37°C, medium was removed, cells were lysed with 50 µl ice cold lysis buffer and phosphorylation of STAT3 was detected, as described by standard techniques (CISBIO Phospho-STAT3 (Tyr705) HTRF kit; Cat # 62AT3PEG). Briefly, the cells were lysed for 30 min at room temperature under shaking and then 16 ^L of lysate per well was added to 96-well half-area plates (COSTAR; Cat # 3642). Next, 4 µl of HTRF pre-mixed phospho-STAT3 antibodies was added to the wells and the plates were incubated for 16 h at room temperature. Plates were then read with an PHERASTAR plate reader at ex 320, em 620 and 665 nm. The EC50 values were calculated using a four parameter fit (GRAPHPAD PRISM). In vitro experiments were performed to determine whether IL-22-CPR01 was active in the JAK pathway. Recombinant human IL-22 (rhIL-22) was used as a positive control. Results from the T84 colon epithelial cell STAT3 phosphorylation in vitro experiments are described in Figure 2. IL-22-CPR01 increased STAT3 phosphorylation with an average EC50 value of 0.536 ± 0.218 nM (experiment was performed twice). rhIL-22 increased STAT3 phosphorylation with an average EC50 value of 0.042 ± 0.008 nM (experiment was performed three times). In conclusion, IL-22-CPR01 exhibited bioactivity, demonstrated by its ability to induce STAT3 phosphorylation in an in vitro STAT3 phosphorylation assay. Example 3. In vivo imaging shows muIL-22-CPR01 localization to gut tissue In order to evaluate the ability of IL-22-CPR01 to target specific tissues, such as the inflamed gut, in vivo imaging using surrogate molecule CPR01-murine IL-22 (CPR01-muIL-22) was performed following administration of the protein into mice with induced colitis. Imaging analysis was performed using both IL-22-CPR01 and its murine surrogate muIL-22- CPR01(murine IL-22 fused to CPR01 scFv). Materials and methods Near infrared labeling of muIL-22-CPR01 Fluorescently labeled CPR01 fusion proteins containing murine IL-22 (muIL-22) were generated using heterogeneous lysine labeling. 1 mg of fusion protein in 500 µl PBS was labeled with 5 nmol of 800CW NHS ester (929-70020, LICOR) according to the following protocol. The fusion protein was thawed at 4°C and desalted 2x using ZEBA 7kDa MWCO (5 ml size) desalting columns following manufacturer’s instructions. Desalting columns were washed using PBS. Post desalting, A280 for the protein was collected to account for potential protein loss. Based on A280, the amount of 800CW NHS ester for the reaction was determined in accordance to a 1:0.8 protein:fluorophore stoichiometric ratio. To a solution of desalted protein, 10% V/V of 7.5% sodium bicarbonate buffer was added, followed by 800CW NHS ester in a sterile polypropylene EPPENDORF tube. The reaction was gently inverted upon addition of dye to ensure mixing and then left at room temperature without mixing for 2 h. Unreacted 800CW NHS ester and hydrolyzed 800CW COOH were removed via 2x ZEBA 7kDa MWCO desalting columns following manufacturer’s protocols. Protein degree of labeling (DOL) was determined using a nanodrop spectrophotometer (THERMOFISHER SCIENTIFIC) that can detect up to 850 nm. Using manufacturer’s extinction coefficients for the fluorophore and protein A280, the average DOL was determined with a target range between 0.5 - 1. The CPR01 binding capacity of the 800CW-labeled antibody was validated using surface plasmon resonance (BIACORE). In vivo targeting in colitis mouse model In vivo targeting performance of muIL-22-CPR01 was assessed by quantitative bio- distribution studies in a dextran sodium sulfate (DSS)-induced colitis model. As described above, muIL-22-CPR01 was used as a surrogate to CPR01-huIL-22 in the murine colitis model. Acute murine colitis was induced in C57BL/6 mice with 3% DSS in drinking water, and on day 7, 150 μg of 800CW-labeled muIL-22-CPR01 was administered intravenously (i.v.) via the tail vein. Mice were sacrificed 24 h post-injection, perfused with saline and the organs were excised to acquire fluorescence images across different tissues using a LICOR Pearl imaging system. The organs were then weighed and homogenized, and the fluorescence intensities measured on a LICOR Odyssey CLx plate scanner were converted to concentrations using a standard curve created with the dosing solution. To perform microscopic assessment of targeting, a cohort of DSS mice were dosed and perfused as described above, and colons from these mice were resected and flushed thoroughly with saline to remove residual fecal material, followed by fixation in 4% paraformaldehyde for 24 h. The next day, tissues were placed in 30% cold sucrose for 24 h at 4°C. The samples were then positioned in molds containing OCT (TISSUE TEK embedding medium, SAKURA, Cat #4583) and frozen by slowly immersing the mold in cold 2-methylbutane (JT BAKER; Cat #Q223-08) chilled in dry ice bucket. Frozen blocks were then stored at -80^C. Colon sections (10 µm) were cut using a cryostat and mounted on a glass slide, following which the slides were incubated in 1 µg/mL Hoechst nuclear stain for 10 min. The slides were then imaged using a wide-field LEICA epifluorescence microscope equipped with an ANDOR EMCCD camera (OXFORD TECHNOLOGIES) and LED excitation at 740 nm to visualize 800CW fluorescence in the tissue sections. Results In order to evaluate targeting of CPR01-huIL22 to the inflamed gut of a human subject, surrogate muIL-22-CPR01 was labeled with a near-infrared (NIR) fluorophore 800CW (as described above) and tested in a murine colitis model. As described above, DSS was administered to mice in order to induce epithelial damage resulting in areas of epithelial erosions in the colon and inflammation of the underlying lamina propria. When added to drinking water of mice for 7 days, DSS induces tissue damage that triggers deposition of new extracellular matrix including tenascin-C containing the D domain isoform. Labeled muIL-22- CPR01 was administered after 7 days of DSS treatment and bio-distribution across multiple tissues was assayed 24 hours later. Profiles of mean fluorescence intensity (MFI) across different organs exhibited significantly increased levels of muIL-22-CPR01 retained in diseased DSS colon relative to the other tissues, indicating localization of the fusion protein to the inflamed gut tissue (data not shown). In addition, NIR images of DSS colon tissue allowed visualization of the uptake of muIL- 22-CPR01 in focal regions, and fluorescence microscopic analysis of these tissue sections demonstrated that this specific distribution was associated with inflamed regions of the colon. These images are provided in Figures 3A and 3B. Figure 3A provides an NIR fluorescent image of DSS colon illustrating focal distribution of muIL-22-CPR01, as described in Figure 3A (see, e.g., box region), while Figure 3B provides an NIR fluorescent microscopy image of a colon tissue section indicating that the NIR fluorescence signal originated from an inflamed region of the colon. These observations with 800CW-labeled muIL-22-CPR01 were further validated with micro-autoradiography (MARG) of colon tissue from a cohort of DSS mice dosed with ¹²⁵I- radiolabeled muIL-22-CPR01. MARG enables spatial assessment of radiolabeled reagents at the cellular level in histological samples, wherein the location of the drug-derived radioactivity is made apparent by the black specks that appear over the tissue section. The MARG images revealed a distribution pattern consistent with that observed with NIR microscopy, and the co- registration of radioactive signal with hematoxylin and eosin (H&E) confirmed that the localization of muIL-22-CPR01 was in focal regions of colonic inflammation (see Figures 3C and 3D). As described in Figure 3C, MARG image of DSS colon from mice dosed with 40 µCi of ¹²⁵I-radiolabeled muIL-22-CPR01 showed that the lesion-associated distribution (black specks) is consistent with the NIR image of Figure 3A. As shown in Figure 3D, magnified image of the inset from Figure 3C confirmed that the radioactive signal, superimposed on the H&E stained tissue, was localized to inflammation sites. This imaging data showed that surrogate muIL-22-CPR01 was able to localize to inflamed gut tissue using a mouse colitis model. To more specifically quantitate the targeting ability of IL-22-CPR01 and assess the contribution from nonspecific tissue binding, additional imaging bio-distribution studies were conducted in naïve C57/B6 and 7-day DSS mice dosed with 150 µg of 800CW-labeled huIL-22- Ab (control huIL-22 fusion protein containing a non-targeted scFv) or muIL-22-CPR01. Colons were imaged for MFI-based comparative analysis at 24 h post-administration. The results are described in Figure 4. As described in Figure 4, similar levels of both surrogate muIL-22-CPR01 and huIL-22- Ab were observed in naïve mice, and a 2.5-fold higher amount of muIL-22-CPR01 in comparison to control huIL-22-Ab in DSS mice Interestingly, this differential was markedly increased to about 8-fold when levels of muIL-22-CPR01 was assayed on the basis of NIR signal only in the colon lesions rather than the average value of NIR signal observed over the whole colon sample. This data suggests that levels of muIL-22-CPR01 in inflammatory lesions in the colon are significantly higher than levels of muIL-22-CPR01 fusion protein in non-lesional tissue, which is consistent with targeting and retention of muIL-22-CPR01 fusion protein in inflamed lesions. Example 4. Evaluation of colon concentration vs. blood exposure for muIL-22-CPR01 The relationship of colon concentration vs. blood exposure was examined in order to determine whether targeted delivery of IL-22 to the gut provides an advantage. On day 3 following DSS initiation, mice were dosed 150 µg of either 800CW-labeled surrogate muIL-22-CPR01 or labeled muIL-22-Fc (see Example 3 for protocol).^^Blood samples were obtained at multiple time-points post-dosing up to 72 hours (h), and the 800CW fluorescence signal was converted to whole blood concentration by comparing the intensity to a calibration curve of known concentrations. The whole blood PK profile was then fitted to a biexponential decay using PRISM. As described in Figure 5A, muIL-22-CPR01 showed a faster decay or a shorter circulation half-life compared to muIL-22-Fc. At 8, 24, and 72 hours post-dosing, colons were excised from the DSS mice (n=3 for each time-point) and homogenized to quantitate tissue concentration of muIL-22-CPR01 and muIL-22-Fc. Concentration of muIL-22-CPR01 in tissue homogenates was calculated by extrapolation of fluorescence intensities using calibration curves obtained with serial dilutions of the 800CW-conjugated proteins. As described in Figure 5B, concentration of muIL-22-CPR01 in the colon was higher than that of muIL-22-Fc at both 8 hours and 24 hours post-dosing. The decreased concentration of muIL-22-CPR01 at 72 hours post-dosing was consistent with its shorter half-life compared to muIL-22-Fc. The colon:blood ratio of muIL-22-CPR01 and muIL-22-Fc was also calculated. As described in Figure 5C, muIL-22-CPR01 exhibited a higher colon:blood ratio at 24 hours and 72 hours post-dosing compared to muIL-22-Fc. The higher colon:blood ratio of muIL-22-CPR01 at 24 hours and 72 hours shows the benefit of targeted delivery offered by the diabody format of a IL-22-CPR01 fusion protein even with its the shorter circulation half-life compared to that of IL- 22-Fc. NIR microscopy images of DSS colon tissue were obtained at 24 hours, 48 hours, and 72 hours following dosing with 800CW-labeled CPR01-muIL-22; the images are provided in Figure 5D. As described in Figure 5D, sustained targeting with muIL-22-CPR01 was also reflected in the NIR microscopy images that showed significantly detectable signal above background in colon lesions up to 72 hours post-dosing. Example 5. NIR fluorescence endoscopy analysis of CPR01-muIL-22 An NIR fluorescence endoscope was used to visualize the uptake of surrogate muIL-22- CPR01 in the gastrointestinal tract of DSS mice. NIR fluorescence images captured with an NIR fluorescence endoscope are provided in Figures 6A and 6B. As described in Figure 6A, the NIR intensity map superimposed on white light images clearly demonstrated accumulation muIL-22-CPR01 in inflamed mucosa at 24 hours post- administration of 800CW-labeled CPR01-muIL-22. Results from an un-dosed negative control are provided in Figure 6B and show no fluorescence. This platform is similar to that adopted in the clinic. Preclinical endoscopy data not only illustrated IL-22-CPR01 targeting in a live animal but also demonstrated the potential for this modality to be adopted in an early phase human clinical trial to visualize targeted delivery of IL- 22-CPR01. Summary of Examples 3 to 5 In conclusion, the data described in Examples 3 to 5 and corresponding Figures 3 to 6 show that IL-22-CPR01 (via surrogate CPR01-muIL-22) can be targeted to lesion regions of inflamed colon tissues and that the fusion protein shows sustainable localization there. Example 6. In vivo efficacy of muIL-22-CPR01 in colitis model The following study was conducted to evaluate the in vivo efficacy of IL-22-CPR01 (via surrogate) in a mammalian disease model for inflammatory bowel disease (IBD). Materials and methods On Day 0, 3% DSS dissolved in water was provided in 500 ml bottles ad libitum to female C57Bl/6N mice (6-8 wks, 5 per cage). Clinical signs of disease (change in body weight, change in stool consistency, occult blood in the stool, bleeding per rectum) began about 5 days post-initiation of DSS and progressed as long as DSS continued. Animals were monitored at least every other day for changes in these parameters until study termination on Day 7. Mice were dosed i.v. with 100 µL of muIL-22-CPR01 every other day starting on Day 0 (4 doses total). muIL-22-CPR01 was dosed at concentration of 5, 15, 50, and 150 µg/mouse in PBS (n=10 mice per group). For exposure levels, mice were tail nicked and 10 µL of whole blood was placed in 40 µL assay buffer with EDTA. Blood was collected at 0 min, 5 min, 1 hour, 8 hours, 24 hours, 48 hours, 72 hours, and 96 hours post last dose. With respect to histology, colons were excised and the distal most 5 cm portions were cut into 2 segments. Colons were collected and fixed in 10% neutral buffered formalin for a minimum of 24 hours. Samples were processed on an automated tissue processor and then embedded in paraffin wax. All samples were sectioned at 5 ^m and stained with hematoxylin and eosin (H&E). Colons slides were scanned using a PANNORAMIC whole slide scanner at a magnification of 20x. The H&E slides were measured for erosion and severe distal damage (SDD) lengths. The SDD endpoint measures mucosal gland loss. The SDD measurement starts at the rectal mucosal junction and continues along the inflamed colon proximally until glands recover to a point greater than 50% of the mucosal surface. Erosions were measured from the rectum proximally in both colon segments. Erosions were defined as loss of surface epithelium and were measured on either side of the lumen up to 3 cm proximal from the rectum. For both readouts, length measurements in microns were drawn on the image and exported to a spreadsheet for analysis. Line measurements started at the rectal mucosal junction and continued along the colon to a point of about 3 cm. Image analysis of the macrophage marker IBA-1 was used as a quantitative endpoint of mucosal inflammation. For immunohistochemical (IHC) evaluation of IBA-1, 5 µm sections from paraffin embedded tissues were prepared. IHC staining for IBA-1 was performed using the LEICA BOND RX automated stainer (LEICA BIOSYSTEMS). Sections were deparaffinized, and antigens were retrieved with proprietary buffer, ED2 (EDTA buffer, LEICA BIOSYSTEMS). Sections were incubated with primary antibody for IBA-1 (Rabbit Polyclonal, WAKO, Cat # 019- 19741) at 0.15 ^g/ml in PBS; optimized concentration of the primary antibody was previously determined on control tissues. Next, a proprietary goat anti-rabbit HRP polymer (LEICA BIOSYSTEMS) was applied to sections followed by a peroxidase blocking reagent. The detection was performed with the LEICA DAB kit (LEICA BIOSYSTEMS) and slides were counterstained with hematoxylin. IBA-1 image analysis was performed using Visiopharm. Prior to analysis, a region of interest (ROI) around the distal colon was circled. The tertiary lymphoid organs (TLO) external to the muscularis mucosa and the rectum were removed from this ROI. VISIOPHARM software was used to run two algorithms for the complete analysis: Step (1) algorithm to identify mucosa versus submucosa and muscularis; and step (2) algorithm to identify 3,3’-diaminobenzidine (DAB)+ IBA-1+ macrophages in that area. These algorithms report total IBA-1 in the outlined mucosal tissue ROI excluding TLO. These data were then reported as IBA-1+ percent of total mucosal area. Results The ability of IL-22-CPR01 to provide a therapeutic benefit for the intestinal disorders was tested using the surrogate CPR01-mu-IL-22 in a mouse colitis model. DSS was delivered to mice via their drinking water in order induce colitis characterized by epithelial cell death and mucosal inflammation. Disease, as measured by histology, demonstrated frank erosions (epithelial cell loss), loss of crypts, squamous metaplasia and infiltration of inflammatory cells, including neutrophils and macrophages. muIL-22-CPR01 was administered intravenously every other day beginning at the initiation of DSS and inhibited development of erosions in acute DSS-induced colitis in a range of doses, as described in Figure 7A. For example, at a dose of 150 µg close to 80% inhibition of erosions was observed. muIL-22-CPR01 also inhibited influx of macrophages into the lamina propria, as described in Figure 7B. This effect was secondary to improving epithelial barrier function as macrophages do not express IL-22R. This data indicates that IL-22-CPR01 is effective in preventing epithelial damage associated with colitis, thereby, decreasing inflammatory cell burden in the lamina propria. Example 7. Comparison study of IL-22-CPR01 vs. IL-22-Fc A study was performed to compare the activity of IL-22-CPR01 (via surrogate muIL-22- CPR01) to muIL-22-Fc. In vivo studies were performed using the murine DSS colitis model. Mice were treated with a dose response (150, 50, 15 and 5 µg/mouse) of muIL-22- CPR01 or muIL-22-Fc on days 0 and 3 beginning at the time of DSS initiation (day 0). On day 7, colons, feces and serum were collected for analyses. Experiments in the literature have shown that serum Reg3α (human homolog of mouse Reg3β) and serum amyloid A (SAA; a major acute phase protein produced predominantly by the liver) are dose-dependently induced by muIL-22-Fc in healthy volunteers (Eckhardt et al., BMC Gastroenterol, 10:133 (2010); Rothenberg et al., Clin Pharmacol Ther, 105:177-189 (2019)). Thus, to compare the gut bioactivity of muIL-22-CPR01 with that of muIL-22-Fc, Reg3β was measured in feces of DSS mice that were treated with muIL-22-CPR01 or muIL-22-Fc. Efficacy was determined by the effect of drug (muIL-22-CPR01 or muIL-22-Fc) treatment on epithelial erosions in the colon. The results form the study are shown in Figures 8A and 8C. As shown in Figure 8A, muIL-22-CPR01 decreased erosion lengths and induced expression of fecal Reg3β in a range of doses, suggesting that Reg3 expression correlated with therapeutic response in this model. Similar to CPR01-muIL-22, treatment with muIL-22-Fc also showed a dose-responsive effect on epithelial damage and erosion length and increased fecal Reg3β levels (Figure 8C). The magnitude of Reg3β induction was greater with muIL-22-Fc treatment compared to CPR01-muIL-22, possibly because muIL-22-Fc is not targeted to inflamed sites but stimulates Reg3β production throughout the colon. Next, to compare the systemic activity of muIL-22-CPR01 with that of muIL-22-Fc, serum SAA was monitored in DSS mice that were treated with muIL-22-CPR01 or muIL-22-Fc. Efficacy was determined by the effect of drug (muIL-22-CPR01 or muIL-22-Fc) treatment on epithelial erosions in the colon. The results are shown in Figures 8B and 8D. As shown in Figure 8B, muIL-22-CPR01 had essentially no effect on serum SAA levels. This data is consistent with limited systemic exposure of muIL-22-CPR01 owing to its targeted delivery to inflamed colon. Unlike muIL-22-CPR01, muIL-22-Fc produced a dose responsive increase in serum SAA (Figure 8D), which was consistent with the longer serum half-life for this drug and the reported systemic bioactivity of muIL-22-Fc. Thus, the data described in Figure 8 shows that muIL-22-CPR01 is efficacious in mouse DSS model, exhibits local bioactivity, and demonstrates a benefit over muIL-22-Fc by sparing extra-intestinal bioactivity as measured by serum SAA. By targeting muIL-22 to the inflamed gut and by virtue of the short half-life, muIL-22-CPR01 has the potential to limit systemic muIL-22 exposure. Treatment with muIL-22-Fc also showed a dose-responsive effect on epithelial damage and erosion length and increased fecal Reg3β levels (Figures 8C and 8D). The magnitude of Reg3β induction was greater with IL-22-Fc treatment vs. muIL-22-CPR01 potentially due to the fact that muIL-22-Fc is not targeted to inflamed sites but stimulates Reg3β production throughout the colon. Unlike muIL-22-CPR01, muIL-22-Fc produced a dose responsive increase in serum SAA consistent with the longer serum half-life for this drug and the reported systemic bioactivity of muIL-22-Fc (Figures 8C and 8D). Thus, muIL-22-CPR01 is efficacious in mouse DSS, exhibits local bioactivity, and demonstrates a benefit over muIL-22-Fc by sparing extra-intestinal bioactivity as measured by serum SAA. The observation that both IL-22-CPR01 and muIL-22-Fc induce fecal Reg3β, but only muIL-22-Fc induces serum SAA points to the fact that muIL-22-CPR01 may have tissue-specific biological responses that can be harnessed to monitor gut versus extra-intestinal IL-22 activity directly in the serum. In conclusion, the data described in Figures 7A-7B and 8A-8D support a finding that IL- 22-CPR01 can inhibit epithelial erosion and suppress inflammation in IBD using a murine colitis model. Sequences disclosed herein and/or relevant to the present invention are listed in the Sequence Summary Table.

Incorporation by reference All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMS What is claimed: 1. A fusion protein comprising human interleukin-22 (hIL-22) and a single chain variable fragment (scFv) that specifically binds to human tenascin C domain D, wherein the hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, and wherein the scFv comprises a heavy chain variable (VH) domain comprising the amino acid sequence set forth as SEQ ID NO: 5 and a light chain variable (VL) domain comprising the amino acid sequence set forth as SEQ ID NO: 7, and wherein the hIL-22 is conjugated to the N-terminus of the scFv via a peptide linker.

2. The fusion protein of claim 1, wherein the peptide linker is 10 to 20 amino acids long.

3. The fusion protein of claim 2, wherein the peptide linker comprises the amino acid sequence set forth as SEQ ID NO: 3.

4. The fusion protein of any one of claims 1-3, wherein the VH domain and the VL domain of the scFv are conjugated via a peptide linker comprising the amino acid sequence set forth as SEQ ID NO: 6.

5. A fusion protein comprising human interleukin-22 (hIL-22) and a single chain variable fragment (scFv) that specifically binds to human tenascin C domain D, wherein the hIL-22 comprises the amino acid sequence set forth as SEQ ID NO: 2, wherein the scFv comprises the amino acid sequence set forth as SEQ ID NO: 4 , and wherein the hIL-22 is conjugated to the N-terminus of the scFv via a peptide linker.

6. The fusion protein of claim 5, wherein the peptide linker comprises the amino acid sequence set forth as SEQ ID NO: 3.

7. A fusion protein comprising human interleukin-22 (hIL-22) and an anti-human tenascin C domain D single chain variable fragment (scFv), wherein the fusion protein comprises the amino acid sequence set forth as SEQ ID NO: 1.

8. A diabody comprising two of the fusion proteins of any one of claims 1-7.

9. A method of treating a human subject having inflammatory bowel disease (IBD), the method comprising administering a therapeutically effective dose of the fusion protein of any one of claims 1-7 or the diabody of claim 8 to the human subject having IBD.

10. The method of claim 9, wherein the IBD is Crohn’s disease or ulcerative colitis.

11. A nucleic acid encoding the fusion protein of any one of claims 1-7.

12. A vector comprising the nucleic acid of claim 11.

13. A host cell comprising the vector of claim 12.

14. A method of producing the fusion protein of any one of claims 1-7, the method comprising culturing the host cell of claim 13 under conditions suitable for production of the fusion protein.

15. A pharmaceutical composition comprising a fusion protein of any one of claims 1-7 or the diabody of claim 8, and a pharmaceutically acceptable carrier.