WO2018215614A1

WO2018215614A1 - Fusion protein for enhancing intestinal regeneration

Info

Publication number: WO2018215614A1
Application number: PCT/EP2018/063708
Authority: WO
Inventors: Willibrordus Barend Maria DE LAU; Johannes Carolus Clevers
Original assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
Priority date: 2017-05-24
Filing date: 2018-05-24
Publication date: 2018-11-29
Also published as: GB201708330D0

Abstract

The present invention provides fusion proteins and related compounds for use in improving the regenerative capacity of tissues.

Description

FUSION PROTEIN FOR ENHANCING INTESTINAL REGENERATION

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to fusion proteins for use in treatment of cancer, and to kits, vectors, and nucleic acid sequences thereof. The invention relates particularly to adjuvant therapies that improve the regenerative capacity of tissues affected by chemotherapy or radiotherapy.

BACKGROUND

Modern regimens for the treatment of cancer typically involve chemotherapy and/or

radiotherapy. Though recent advances in the field have increased the efficacy of these treatments, the severity of their side-effects remains an obstacle to their safe application. In particular, treatments targeted towards rapidly dividing cancer cells often damage healthy cells that also rapidly divide. Such cells include those of the intestinal epithelium, which exhibits intense cell division due to a weekly complete self-renewal (Beumer, J. & Clevers, H.

Regulation and plasticity of intestinal stem cells during homeostasis and regeneration.

Development 143, 3639-3649 (2016)). There is therefore a need to enhance the regenerative capacity of the intestine during chemotherapy and/or radiotherapy of cancers associated with the intestines.

The Wnt (beta-catenin) pathway is known to act in a diverse range of tissues to regulate cell proliferation and tissue expansion. The intestinal epithelium represents a well-understood example of the role of Wnt signaling in homeostatic self-renewal (Gregorieff, A., & Clevers, H., Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes & Development, 19(8), 877-890 (2005)). Not only are intestinal epithelial cells dependent on Wnt (beta-catenin) signaling for their growth, but the Wnt pathway is also central in tumorigenesis of the intestine in at least two scenarios.

In one scenario, mutations downstream of the Wnt pathway - typically in APC (80% of patients) or beta-catenin (10-15% of patients) - can give rise to colon cancers by inducing strong, uncontrolled activation of the pathway, independent from any stimulatory interactions at the cell membrane caused by extracellular Wnt agonists (Clevers, Hans, and Roel Nusse. "Wnt/β- Catenin Signaling and Disease." Cell 149, 6: 1192-205 (2012)). In a second scenario, mutations in extracellular agonists of the Wnt pathway can also induce strong, uncontrolled activation of the pathway, but without concomitant mutations in APC or beta-catenin. An example of such extracellular Wnt agonists is the R-spondin (Rspo or 'roof plate-specific spondin') family of secreted proteins, which mediate enhancement of Wnt signaling by acting at the cell membrane (De Lau, W., et al., The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes & Development 28, 4:305-16 (2014)). Their persistent inappropriate expression by intestinal stem cells (e.g. as a consequence of gene fusions) has been shown to initiate colon cancer (Seshagiri, S., et al., Recurrent R-spondin fusions in colon cancer. Nature 488, 660-664 (2012)). One approach to enhancement of the regenerative capacity of the intestine during chemotherapy and/or radiotherapy is to expose non-cancerous cells of the intestinal epithelium to extracellular Wnt agonists, added exogenously (Zhou, W., et al., Induction of intestinal stem cells by

R-spondin 1 and Slit2 augments chemoradioprotection. Nature, 501(7465), 107-111 (2013)).

In patients with colon cancer arising from aberrantly constitutively activated Wnt signaling (e.g. in either of the scenarios mentioned above), exogenous addition of extracellular Wnt agonists will not necessarily affect the tumor, because the tumor already has maximal expression of Wnt genes. The normal epithelium will, however, exhibit a growth response from an exogenous Wnt agonist such as the R-spondins. Nevertheless, a major limitation of therapeutic approaches utilizing exogenous Wnt agonists, such as the R-spondins, is that high systemic concentrations of agonist are required. As Wnt agonists like the R-spondins can activate virtually all cells (such as those of the liver and pancreas), the potential systemic side-effects preclude effective

enhancement of intestinal regenerative capacity using exogenous R-spondins.

Therefore, there is a need for a means of enhancing intestinal regenerative capacity that also mitigates the risk of systemic side-effects associated with agonists of Wnt signaling, such as R-spondins.

SUMMARY OF INVENTION

The inventors have developed a means of enhancing intestinal regenerative capacity that advantageously mitigates the risk of systemic side-effects associated with agonists of Wnt signaling. This involves in some embodiments selectively targeting R-spondins to the intestinal epithelia by developing fusion proteins containing an 'agonist portion' comprising in some embodiments the signaling domains of R-spondins, and an 'antibody portion' targeted in some embodiments to a cell surface protein specific to the intestinal epithelia. Accordingly, the invention provides among other things a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells.

In a particular embodiment, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion comprises one or more of R- spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4 (also referred to herein as "R-spondin 1-4") or a biologically active fragment or variant thereof, and/or the antibody portion selectively targets Gpa33.

The invention also provides a nucleic acid encoding the amino acid sequence of a fusion protein of the invention.

The invention also provides a vector comprising a nucleic acid of the invention, or comprising a nucleic acid encoding the amino acid sequence of a fusion protein of the invention.

The invention also provides a pharmaceutical composition comprising a fusion protein, nucleic acid or vector of the invention. The invention also provides a kit comprising a pharmaceutical composition of the invention.

The invention also provides a method for the treatment of intestinal epithelial cellular damage in a subject, wherein the method comprises administration to the subject of a fusion protein or a pharmaceutical composition of the invention.

The invention also provides a fusion protein or a pharmaceutical composition of the invention for use in therapy, for example, for use in regenerative therapy and/or for treating cellular damage.

The invention also provides a method for the treatment of deleterious effects associated with therapy of a subject for one or more cancers and/or for one or more precancerous lesions, wherein the method comprises administration to the subject of the fusion protein or the pharmaceutical composition of the invention.

The invention also provides a fusion protein or a pharmaceutical composition according of the invention, for use in a method for the treatment of deleterious effects associated with therapy of a subject for one or more cancers and/or for one or more precancerous lesions.

The invention also provides a method for treating cancer, wherein the method comprises co- administering to a subject a cancer therapy in combination with a fusion protein or a

pharmaceutical composition of the invention. The invention also provides a fusion protein or a pharmaceutical composition of the invention, for use in a method for treating cancer, wherein the method comprises co-administering to a subject a cancer therapy in combination with the fusion protein or the pharmaceutical composition. The invention also provides the use of a fusion protein of the invention for the manufacture of a medicament for treatment of a disease.

The invention also provides a method of producing a fusion protein, comprising expressing the fusion protein in a host cell.

The invention also provides a method of producing a nucleic acid sequence or vector encoding a fusion protein of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Wnt activating-potential of fusion proteins is preferentially delivered to HEK cells expressing human Gpa33.

Figure 1A. Wnt signaling (TOP) was measured for HEK293T cells transiently expressing human Gpa33 or mouse Gpa33, upon exposure to identical ranges of conditioned media (CM) containing Wnt (open bars), Wnt with wildtype fusion protein (black bars), or Wnt with

CDPv2-mutant fusion protein (grey bars). The Figure is explained in detail in Example 2.

Figure IB. Wnt signaling (TOP) was measured for HEK293T cells expressing doxycycline (DOX)-inducible human Gpa33, with CM containing Wnt (open bar), or CM containing Wnt and intact fusion protein. The same conditions were applied in the presence and absence of DOX. The Figure is explained in detail in Example 3.

Figure 2. Fusion protein targets R-spondin activity to human colon organoids.

Proliferative activity was measured by Luminescent Cell Viability Assay for human colon- derived organoids grown in the presence of Wnt alone, or in combination with two-fold dilutions of either wildtype fusion protein or a CDR2-mutant anti-Gpa33 fusion protein. The Figure is explained in detail in Example 4.

Figure 3. Insertion of the human Gpa33-specific antibody epitope in the analogous mouse locus.

Figure 3A. Exon structure of the Gpa33 gene, encoding the extracellular part of this

transmembrane molecule. The Figure is explained in detail in Example 5. Figure 3B. Immuno-fluorescent Gpa33-Rspo4 fusion protein staining of HEK293T cells transiently trans fected with cDNA for one of human Gpa33; mouse Gpa33; or mouse Gpa33 in which exon-II or exon-III encoding peptides were replaced for the human counterparts. The Figure is explained in detail in Example 5.

Figure 3C. Comparison of exon-III encoded peptides for mouse Gpa33 and human Gpa33. Differences in amino acid composition of human versus mouse peptides are shown in bold. The Figure is explained in detail in Example 5.

Figure 3D. Schematic representation of the homologous recombination procedure designed to substitute exon-III. The Figure is explained in detail in Example 5.

Figure 4. Southern blot analysis for homologous recombination at the Gpa33 locus.

Genomic DNA was isolated from 96 Neomycin-resistant ES cell clones and Hind-III digested. The digestion products were agarose electrophoresed, blotted, and incubated with a 32P-dCTP- labelled DNA probe. After stringent washing, radioactivity was visualized using phospho- imaging. These data are shown in in Figure 4 (clones that properly recombined are indicated *). The Figure is explained in detail in Example 6.

Figure 5. Fusion protein interacts with intestinal epithelium of a human

Gpa33-expressing mouse.

Intestinal crypts were isolated from a mouse homozygous for human Gpa33, and from a wildtype littermate. Both isolates were incubated with intact fusion protein, followed by a labelled anti- huIgGl antibody. Fluorescence is indicative of binding. The Figure is explained in detail in Example 7.

Figure 6. Selective targeting of R-spondin activity to human Gpa33-expressing mouse small intestinal organoids.

Small- intestinal organoids were produced from either mice homozygous for humanized Gpa33 or wildtype littermates. Organoids were cultured in various dilutions of either human Rspo3-CM or purified fusion protein. Cell growth was quantified after 10 days by an ATP-driven luciferase cell viability assay. The experiment was performed in triplo. The Figure is explained in detail in Example 8. Figure 7. Time course of intestinal growth at high dose 5FU.

Mice (n = 2) received a single high dose (450 mg kg^"1) of 5FU at day 0. Mice were sacrificed on each of days 1 , 2, 3 and 4 and immunohistochemical analysis of growth performed based on BrdU incorporation. Representative images of intestinal paraffin sections are shown, and dark nuclei indicate BrdU incorporation and cell proliferation. The Figure is explained in detail in Example 9.

Figure 8. Fusion protein improves regenerative capacity of intestinal epithelium in vivo.

Three groups of twelve mice each - two groups of wildtype mice and one group of mice homozygous for humanized (chimeric m/h) gpa33 - received one intraperitoneal injection of 5FU on day 0. One group of wildtype mice, and the m/h gpa33 mice, also received injections of fusion protein at 2 μg (25 g)^"1 on each of days 0, 1 , 2 and 3.

At days 1 , 2, 3, and 4 three mice of each group received intraperitoneal injections of BrdU prior to sacrifice. Intestines were subject to paraffin immunohistochemistry and BrdU

immunostaining. The Figure is explained in detail in Example 10.

Figure 9. Structure of R-spondins 1, 2, 3 and 4.

Amino acid sequences and domain organizations of the four human R-spondins are displayed. Figure 10. Exclusive Gpa33-driven Wnt enhancement by Lgr-dead fusion protein.

Stable DOX-inducible hGpa33 expressing HEK293 cells (prepared according to Example 3) were incubated with Wnt3a alone or Wnt3a combined with dilutions of CM containing the Lgr- dead fusion protein. Wnt pathway activity, in the absence and presence of DOX, was measured 48 hrs upon transient transfection with a TOP luciferase reporter (RL-Normalized). The Figure is explained in detail in Example 12.

Figure 11. Site of mutation for Lgr-dead R-spondin 4.

The residue mutated in the Lgr-dead fusion protein of Example 12 is located in the furin-2 domain, shown bolded and underlined. The Figure is explained in detail in Example 12.

Figure 12. Fusion protein improves regenerative capacity of methotrexate-treated intestinal epithelium in vivo as measured by number of BrdU⁺ crypt cells. Images of BrdU⁺ nuclei in representative crypt regions from two wildtype and two chimeric mice. Mice were exposed to methotrexate, followed by fusion protein, then BrdU staining and sacrifice. The data are quantified as percentage positive nucleic (with standard deviation) in the inset table. The Figure is explained in detail in Example 13. DETAILED DESCRIPTION OF INVENTION

Definitions

"Approximately" or "about", as used in this application, are equivalent. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by the person skilled in the art. As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%), 6%), 5%, 4%, 3%, 2%>, 1%), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

"Biologically active" refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a peptide is biologically active, a portion of that peptide that shares at least one biological activity of the peptide is typically referred to as a "biologically active" portion.

"Carrier" or "diluent" refers to a pharmaceutically acceptable (e.g. safe and non-toxic for administration to a human) carrier or diluting substance useful for the preparation of a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

"Conservative mutation" refers to an amino acid addition, substitution or deletion that has no significant change on the functional or structural properties of a protein. It may in particular be a substitution of an amino acid in one class with a different amino acid in the same class. The classes of amino acids are:

Class Name of the amino acids

Aliphatic

Hydroxyl or Sulfur/Selenium-containing Serine, Cysteine, Selenocysteine, Threonine, Methionine Cvclic Proline

Aromatic Phenylalanine, Tyrosine, Tryptophan

Basic Histi linc, Lysine, Argininc

Acidic and their Amide Aspartate, Glutamate, Asparagine, Glutamine

"Non-conservative mutation" refers to an amino acid addition, substitution or deletion that has a significant change on the functional or structural properties of a protein. It may in particular be a substitution of an amino acid in one class with a different amino acid in a different class (see table above). "Comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

"DNA damage" refers broadly to the features of nucleic acids with impaired structure or function, such as those produced by exposure of DNA to mutagens. "DNA damage" may refer more specifically to structural or chemical alterations in DNA moieties including the bases and/or deoxyribose backbone. Examples of DNA damage include abasic nucleotides, alkylated bases, deaminated bases, depurinated bases, depyrimidinated bases, DNA adducts, DNA double- strand breaks, DNA single-strand breaks methylated bases, mismatched base pairing, and oxidized bases. "Dose" refers to a specified quantity of a pharmaceutical agent provided in a single

administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In certain embodiments, a dose is administered as a slow infusion.

"Furin" may refer to both or either of two cysteine-rich repeat domains common to R-spondin family members (commonly referred to as "furin-like domains"), and may in particular refer to those encoded by exon II and/or exon III of R-spondin4. "Fusion protein" refers to an amino acid polymer comprising or consisting of two or more originally separate amino acid polymers (or biologically active fragments thereof). The fusion protein is typically produced by recombinant methods, i.e. the fusion protein is typically created through the joining of two or more polynucleotide sequences encoding the originally separate amino acid polymers (or biologically active fragments thereof) and the subsequent expression of the polynucleotide sequence in a host cell. The fusion protein may consist of a single polypeptide chain expressed from a single nucleic acid, or it may comprise multiple polypeptide chains (expressed from the same or from different vectors) that are covalently linked, e.g. a fusion protein may comprise heterodimeric portions such as covalently- linked heavy and light chains of an antibody. The two originally separate amino acid polymers (or biologically active fragments thereof) may be joined directly or joined across a linker.

"Human antibody" refers to an antibody in which the variable and/or constant domain sequences are derived from human sequences.

"Humanized antibody" refers to an antibody from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. Methods for producing humanized antibodies and examples of humanized antibodies are known in the art.

"Immunoglobulin Fc region" refers to an amino acid polymer comprising the heavy-chain constant region 2 (CH2) and the heavy-chain constant region 3 (CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CHI) and the light-chain constant region 1 (CL1) of the immunoglobulin.

Fc regions may further include a hinge region at the heavy-chain constant region. The Fc region may also have a deletion in a relatively long portion of the amino acid sequence of CH2 and/or CH3. That is, the immunoglobulin Fc region may include 1) a CHI domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CHI domain and a CH2 domain, 3) a CHI domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer of each domain of the heavy-chain constant regions and the light-chain constant region.

Further, immunoglobulin Fc region refers to native amino acid sequences and/or sequence derivatives (mutants) thereof. "Intestine" and "intestinal" refer to the gastrointestinal tract, including the mouth, oral cavity, oesophagus, stomach, large intestine, small intestine, rectum, and anus.

"Linker" refers to, in a fusion protein, an amino acid polymer not appearing at a particular position in one or more originally separate amino acid polymers. The linker is generally designed to be flexible or to interpose a structure, such as an a-helix, between two other portions. A linker is also referred to as a spacer. A linker or a spacer typically does not have biological function on its own. "Portion" may refer to an amino acid polymer or nucleotide polymer which forms part of a fusion protein. Individual portions may be distinguished from other portions in the fusion protein based on structural and/or functional differences between portions.

"Regenerative capacity" and "self-renewal" may refer to the ability of an organ/tissue/cell to develop in the body. Specific examples of regenerative capacity include the rearrangement of pre-existing tissue, cell proliferation, the differentiation of adult somatic stem cells, the dedifferentiation and/or transdifferentiation of cells, cellular pattern formation, tissue polarity establishment, and replacement of body parts lost by injury. Regenerative capacity may be measured according to the ability of the organ/tissue/cell to form three-dimensional organoid structures ex vivo.

"Selective targeting" refers to the complete or substantial co- localisation of a 'targeting' component with a 'target' component. With respect to fusion proteins, a portion of the protein may co-localise with one or more particular organs/tissues/cells or with one or more molecular markers specific to one or more particular organs/tissues/cells. "Selective" or "specific" may also refer to molecules having a measurably higher affinity for one 'target' molecule than for related 'non-target' molecules.

"Sequence identity" or "sequence homology" with respect to a reference protein or nucleic acid sequence (e.g. a reference fusion protein or portion thereof) identified herein may refer to the percentage of amino acid residues or nucleotide residues in a candidate sequence that are identical with the amino acid residues or nucleotide residues in the reference sequence - for example after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity.

It is well known by the person skilled in the art that certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution.

Amino acid sequence identity between polypeptide sequences is preferably determined by pairwise alignment algorithm using the Needleman-Wunsch global alignment algorithm

(Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453), using default parameters (e.g. with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice et al. (2000) Trends Genet 16:276-277). Sequence identity should be calculated over the entire length of the polypeptide sequence of the invention.

"Variant" in the context of an amino acid sequence refers to an amino acid sequence having at least 60% amino acid sequence identity with the original amino acid sequence, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the original amino acid sequence, including for example 80%>, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

"Variant" in the context of a nucleic acid sequence refers to a nucleic acid sequence having at least 60% nucleic acid sequence identity with the original nucleic acid sequence, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% nucleic acid sequence identity with the original nucleic acid sequence, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% nucleic acid sequence identity. "Side effect" or "deleterious effect" refers to a physiological response attributable to a treatment other than desired effects.

"Subject" may refer to a human or any non- human animal (such as any mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). In preferred embodiments, the subject is a mammal, more preferably a human. "Human" may refer to pre- and/or post-natal forms. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

"Suffering from" refers to an individual who has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.

"Susceptible to" refers to an individual who has not been diagnosed with a disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event may be characterized by one or more of the following: (1) a genetic mutation associated with

development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, condition, and/or (5) having undergone, planning to undergo, or requiring a transplant. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

"Therapeutically effective amount" refers to an amount of a therapeutic agent that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by the skilled person that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

"Treating", "treat", "treatment" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

Fusion Protein

Structure. Fusion proteins of the invention generally have structures as shown in formula I below: NH₂-Ab-L„-Ag-COOH, wherein: Ag is an agonist portion; L is an optional linker; n is an integer, including zero; and Ab is an antibody portion. Usually n is 1.

Alternatively, fusion proteins of the invention may have a structure shown in formula II below (using the same nomenclature as outlined for formula I): NH₂-Ag-L„-Ab-COOH.

Fusion proteins of the invention most preferably have the structure shown in formula I.

For cellular production of the fusion protein, the signal sequence should be at the N-terminal end of the fusion protein. This ensures proper introduction of the protein into the ER, required for secretion of the fusion protein into the extracellular space. Typically the antibody portion comprises a signal sequence at the N-terminal of its heavy chain. In other embodiments, a separate signal sequence may be included at the N-terminal of the fusion protein (in the context of either formula I or formula II above).

As explained in more detail below, the Ab portion may consist of a single polypeptide chain, or it may further comprise one or more additional polypeptide chains linked to the first polypeptide chain by covalent bonding (typically disulphide bridges).

Functional properties. In some embodiments, fusion proteins of the invention may enhance Wnt signaling in a cell by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400% or 500% compared to the Wnt signaling induced by the original agonist amino acid polymer (not joined in a fusion protein), by a neutral substance, or by a negative control. This can be measured in a Wnt activity assay using Renilla luciferase (RL)-normalized TOP luciferase, e.g. as described in Example 2.

In some embodiments, fusion proteins of the invention may equal the Wnt signaling induced by the original agonist amino acid polymer (not joined in a fusion protein) when used at about 2- fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold lower concentration than the original agonist amino acid polymer. This can be measured in a Wnt activity assay using Renilla luciferase (RL)-normalized TOP luciferase, e.g. as described in Example 2.

In some embodiments, fusion proteins of the invention may increase the cell proliferation induced by the original agonist amino acid polymer (not joined in a fusion protein) by about 2- fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold or 200-fold. This can be measured in a cell proliferation assay, e.g. an ATP-driven luciferase cell viability assay as described in

Example 8.

Sequence. In some embodiments, the fusion protein of the invention consists of two polypeptide chains with amino acid sequences identical to SEQ ID NO: 7 and SEQ ID NO: 12. In some embodiments, variants of SEQ ID NO: 7 and SEQ ID NO: 12 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 12, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 12, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

Half-life. In some embodiments, fusion proteins of the invention have longer half-lives than agonist portions alone. Without wishing to be bound by theory, the joining of an agonist portion to a suitable antibody portion can reduce clearance of agonist from a subject, thereby advantageously increasing the half-life of the agonist portion. One advantageous result of increased half-life is reduced dosage frequency.

For example, a fusion protein of the invention may have an in vitro and/or in vivo half-life of or greater than about 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In some embodiments, a fusion protein of the invention has an in vitro and/or in vivo half- life of between 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days, between 2 days and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between 3.5 days and 10 days, between 3.5 days and 9 days, between 3.5 days and 8 days, between 3.5 days and 7 days, between 3.5 days and 6 days, between 3.5 days and 5 days, between 3.5 days and 4 days, between 4 days and 10 days, between 4 days and 9 days, between 4 days and 8 days, between 4 days and 7 days, between 4 days and 6 days, between 4 days and 5 days, between 4.5 days and 10 days, between 4.5 days and 9 days, between 4.5 days and 8 days, between 4.5 days and 7 days, between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, between 9 days and 10 days.

Half-life may be measured by any means known to the person skilled in the art. For instance, the half- life may be measured by Western blot, ELISA or RIA over an appropriate period of time. The half-life may be measured in any appropriate animal, such as a primate, e.g. cynomolgus monkey, or a human.

Agonist Portion General. Fusion proteins of the invention comprise an agonist portion. The biological activity of the agonist portion is enhancing signaling via the Wnt/beta-catenin signaling pathway.

R-spondins. In some embodiments, the agonist portion is an R-spondin protein, or a biologically active fragment or variant thereof. The biological activity of R-spondin is to enhance signaling via the Wnt/beta-catenin signaling pathway. Therefore, a biologically active fragment or variant of R-spondin can be easily identified using a Wnt activity assay using Renilla luciferase

(RL)-normalized TOP luciferase, e.g. as described in Example 2. In a preferred embodiment, a biologically active fragment of variant of R-spondin enhances Wnt signaling by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400% or 500% compared to the Wnt signaling induced by wild-type full-length R- spondin in the same assay. In some embodiments, a biologically active fragment or variant of R- spondin is able to enhance signaling via the Wnt/beta-catenin signaling pathway only when included in the fusion protein of the invention (see comments under "Lgr-dead R-spondin" below). Such fragments or variants can also be easily identified by testing a fusion protein of the invention comprising said biologically active fragment or variant of R-spondin in a Wnt activity assay using Renilla luciferase (RL)-normalized TOP luciferase, e.g. as described in Example 2.

The mammalian family of R-spondin proteins includes four gene products: R-spondin 1 (mRNA accession number NM 001242908 as in SEQ ID NO: 23, protein accession number

NP_001229837 as in SEQ ID NO: 24), R-spondin 2 (mRNA accession number NM_178565 as in SEQ ID NO: 25, protein accession number NP 848660 as in SEQ ID NO: 26), R-spondin 3 (mRNA accession number NM_032784 as in SEQ ID NO: 27, protein accession number NP l 16173 as in SEQ ID NO: 28) and R-spondin-4 (mRNA accession number NM_ 001029871 as in SEQ ID NO: 29, protein accession number NP 001025042 as in SEQ ID NO: 30). Thus in some embodiments, the agonist portion is R-spondin 1-4, or a biologically active fragment or variant thereof. In a preferred embodiment, the agonist portion is R-spondin 1 or R-spondin 4, or a biologically active fragment or variant thereof. In a further preferred embodiment, the agonist portion is R-spondin 4, or a biologically active fragment or variant thereof.

The R-spondin genes share a common organization each consisting of five coding exons corresponding to structural domains, including a leading signal peptide (exon 1), two furin-like cysteine rich domains (exons 2 and 3, also referred to herein as the "furin domains"), a thrombospondin domain (exon 4) and a C-terminal basic region (exon 5). This organization is displayed in Figure 9 for each of R-spondin 1-4. The furin-like cysteine rich domains have been shown to be sufficient for enhancing signaling via the Wnt/beta-catenin signaling pathways. In particular embodiments, therefore, the agonist consists of or comprises one or more furin domains of an R-spondin protein (e.g. as shown in any of SEQ ID NOs: 15-22), or a biologically active fragment or variant thereof. In a particularly preferred embodiment, the biologically active fragment of R-spondin consists of or comprises a furin- 1 domain of an R-spondin protein (e.g. SEQ ID NOs: 15, 16, 17 or 18) or a biologically active fragment or variant thereof, and a furin-2 domain of an R-spondin protein (e.g. SEQ ID NOs: 19, 20, 21 or 22) or a biologically active fragment or variant thereof, for example as set out in SEQ ID NOs: 11, 39, 40 or 41.

In some embodiments, the agonist consists of or comprises a furin- 1 domain from one R-spondin protein (e.g. R-spondin 1, 2, 3 or 4), and a furin-2 domain from a different type of R-spondin (e.g. R-spondin 1, 2, 3 or 4). These chimeric R-spondin fragments may be "Superspondins" with improved properties relative to wildtype R-spondins (see Warner, M. L., et ah, "Engineering High-Potency R-spondin Adult Stem Cell Growth Factors." Molecular Pharmacology 87, 3:410- 20 (2014)).

In some embodiments, the agonist consists of or comprises one or more furin domains of R- spondin-4 as set out in SEQ ID NOs: 18 and 22, preferably two furin domains of R-spondin-4, e.g. as set out in SEQ ID NO: 11, or biologically active fragment or variant thereof.

In some embodiments, the fusion protein of the invention has a human agonist portion, for example human R-spondin 1 or its furin domains, human R-spondin2 or its furin domains, human R-spondin3 or its furin domains, or human R-spondin4 or its furin domains. Lgr-dead R-spondin. In some embodiments, the biologically active variant is an Lgr-dead R- spondin. As described in more detail in Example 12, an Lgr-dead R-spondin is an R-spondin which has been mutated so that it no longer interacts with Lgr4, Lgr5 or Lgr6 but which is still able to enhance Wnt signaling activity when included in a fusion protein of the invention.

Suitable mutations are known in the art and include, but are not limited to mutations (particularly non-conservative mutations) in the phenylalanine clamp.

The phenylalanine clamp is located primarily in the furin 2 domain and normally arranges around Alal90 of Lgr5 (or an equivalent residue in Lgr4 or Lgr6) (see Peng et al, Cell Reports 3, 1885-1892, 2013). There are two conserved phenylalanine (F) residues present in the phenylalanine clamp in R-spondin 1-4: F 106 and Fl 10 of R-spondin 1 (see SEQ ID NO: 24); F105 and F109 of R-spondin 2 (see SEQ ID NO: 26); F106 and Fl 10 of R-spondin 3 (see SEQ ID NO: 28); and F99 and F103 of R-spondin 4 (see SEQ ID NO: 30).

In some embodiments, the Lgr-dead R-spondin comprises mutations (particularly non- conservative mutations) in one or two phenylalanine residues present in the phenylalanine clamp. In some embodiments, the phenylalanine residue(s) are mutated to glutamate (E), aspartate (D), asparagine (N) or glutamine (Q). Any combination of suitable mutations is envisaged. In a particular embodiment, the phenylalanine (F) residue(s) are mutated to glutamate (E). The F to E mutants for each of the conserved phenylalanine residues have been shown to be sufficient to abolish Lgr binding and Wnt signaling (see more details in Example 12).

The inventors have surprisingly shown that an Lgr-dead R-spondin, which cannot bind Lgrs and which therefore has no Wnt signaling activity when used alone, can nevertheless enhance Wnt signaling when included in a fusion protein of the invention. The inventors hypothesise that the antibody portion, which selectively targets the fusion protein to intestinal epithelial cells, replaces the function of the Lgr-R-spondin interaction in recruiting the R-spondin to the intestinal epithelial cells for enhancing signaling via the Wnt/beta-catenin signaling pathway. These Lgr-dead R-spondins have advantages for use with the fusion proteins of the invention because off-target effects on cells expressing Lgr but not expressing the molecule selectively targeted by the antibody portion of the fusion protein are reduced and/or eliminated. It is therefore also envisaged that these Lgr-dead R-spondins would be useful in fusion proteins selectively targeting tissues other than the intestine.

Thus a "biologically active fragment or variant of R-spondin " specifically includes (but is not limited to) an Lgr-dead R-spondin, which is able enhance signaling via the Wnt/beta-catenin signaling pathway when included in a fusion protein of the invention. In some embodiments of the invention, the agonist of the fusion protein is any Lgr-dead Rspondin described herein.

Sequence. In some embodiments, the agonist portion of the fusion protein of the invention has an amino acid sequence identical to one or more of SEQ ID NOs: 11 or 39-41. In some embodiments, variants of one or more of SEQ ID NOs: 11 or 39-41 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of one or more of SEQ ID NOs: 11, 15-22 or 39-41, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%), 99%) or 100% amino acid sequence identity with the amino acid sequence of one or more of SEQ ID NOs: 11, 15-22 or 39-41, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity. In a particular embodiment, the agonist portion of the fusion protein of the invention has any of the R-spondin 1-4 or fragment or variant amino acid sequences described above, and further comprises a non-conservative mutation at position F106 and/or Fl 10 of R-spondin 1 (or equivalent positions in R-spondin 1-4 or fragments or variants thereof). Disclaimer. In some embodiments, the agonist portion does not comprise Slit2. For the avoidance of any doubt, Slit2 is a ligand for Robo receptors and does not enhance signaling via the Wnt/beta-catenin signaling pathways.

Linker

General. In some embodiments, fusion proteins of the invention comprise one or more linker portions, for example one linker portion.

A linker is generally designed to be flexible or to interpose a rigid structure between other portions of the fusion protein. The linker of the invention is preferably a rigid linker. Suitable rigid linkers are known in the art and include linkers containing an alpha-helix or a proline-rich sequence (e.g. see Chen et al, Adv Drug Deliv Rev. 2013 65(10): 1357-1369, and Yan, W., et al. , Alpha-helical linker of an artificial 6-zinc finger peptide contributes to selective DNA binding to a discontinuous recognition sequence. Biochemistry 46, 8517-8524 (2007)). Thus in some embodiments, the linker is an alpha-helical linker or a proline-rich linker, most preferably an alpha-helical linker.

In some embodiments, the linker sequence comprises or consists of: (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu, and/or

(EEEAK)n, or (EAAAR)n, wherein n is any integer, preferably wherein n is any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, more preferably wherein n is between 2 and 5, more preferably wherein n is between 3 and 5, more preferably wherein n is 4. A particularly advantageous linker of the invention consists of or comprises the sequence (EAAAR)n, wherein n is 2-5, preferably wherein n is 4. This is an alpha- helical linker.

Typically, a linker of the invention consists of 3-100 (e.g. 5-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10- 25, 10-20) amino acids. In some embodiments, a linker is equal to or longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. Typically, a longer linker may decrease steric hindrance. In a preferred embodiment, the linker is about 10-30 amino acids, more preferably about 20 amino acids.

Orientation. The agonist portion may be linked to the linker portion or the antibody portion at the C terminus of the agonist portion; or at the N terminus of the agonist portion. In a preferred embodiment, the linker is linked to the C-terminus of the antibody portion and the N-terminus of the agonist portion. In some embodiments, the linker is linked directly to the agonist portion and to the antibody portion (wherein "directly" means without any intervening amino acid

sequences).

Sequence. In some embodiments, a fusion protein of the invention has a linker portion with an amino acid sequence identical to SEQ ID NO: 10. In other embodiments, variants of SEQ ID NO: 10 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100%) amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

Antibody Portion

General. Fusion proteins of the invention comprise an antibody portion. The biological activity of the antibody portion is selectively targeting the fusion protein to intestinal epithelial cells, by association with one or more molecules specific to intestinal epithelial cells. The molecules specific to the intestinal epithelial cells are preferably surface glycoproteins; have an

extracellular domain; have high expression; not have an apical subcellular distribution; and/or have baso-lateral subcellular distribution. A preferred surface glycoprotein is Gpa33 (see further comments below).

Functional properties. In some embodiments, the antibody portion has the effect of increasing the affinity and/or selectivity of the agonist activity for the target cells. An agonist used alone will diffuse throughout its environment (e.g. the body or cell culture) where it is used. This means that it will activate all agonist-responsive cells. Where the agonist might be used for treatment purposes and is administered to a subject, the dose would have to be kept low in order to minimize adverse effects on non-target cells caused by non-selective binding of agonist.

Agonists in fusion proteins that bind to a target cell, require lower dosages to achieve similar, equal or better activation of the Wnt pathway in a target organ/tissue/cell whilst minimizing the adverse effects on the non-target cells.

In some embodiments, the antibody portion of the invention binds to its target antigen or epitope with an affinity of at least 10^Λ-14 M KD, at least 10^Λ-13 M KD, at least 10^Λ-12 M KD, at least 10^Λ-11 M KD, at least 10^Λ-10 M KD, at least 10^Λ-9 M KD, at least 10^Λ-8 M KD, at least 10^Λ-7 M KD, at least 10^Λ-6 M KD, at least 10^Λ-5 M KD, or at least 10^Λ-4 M KD as determined using Biacore™ (GE Healthcare) or a F AC Scan assay. For example, the antibody portion of the invention may bind to its target antigen or epitope with an affinity of at least about 10^A-9 M KD.

Antibody characteristics, such as on-rates (k_a), off-rates (kd), and affinities (KD) can be determined in competitive binding assays using known platforms such as Octet™ (ForteBio), ProteOn™ (Bio-Rad), and Biacore™ (GE Healthcare).

Structure. The antibody portion of the fusion protein may comprise or consist of any antibody type described herein.

A conventional antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three CDRs which are primarily responsible for binding an epitope of a target antigen. They are referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, of which the CDR3 region comprises the most variable region and normally provides a substantial part of the contact residues to a target. The more highly conserved portions of the variable regions are called the "framework regions". In some embodiments, the antibody portion comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion consists of one heavy and one light chain.

The term antibody is used herein in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD and IgE, polyclonal antibodies including recombinant polyclonal antibodies, Oligoclonics, multispecific antibodies, chimeric antibodies, nanobodies, diabodies, BiTE's, Tandabs, mimetobodies, bispecific antibodies, humanized antibodies, human antibodies, deimmunised antibodies and antibody fragments. In addition, scaffolds will be covered under this term, such as Anticalins, Ankarins, etc. An antibody reactive with a specific epitope of the target molecule can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the target nucleic acid encoding the target epitopes. A fusion protein of the invention comprises at least one antibody chain in the same polypeptide chain as the agonist portion of the fusion protein (e.g. that is expressed as part of the same polypeptide chain).

In some embodiments, the antibody portion of the fusion protein comprises a first antibody chain (e.g. the heavy chain) that is in the same polypeptide chain as the agonist portion of the fusion protein, and a second antibody chain (e.g. the light chain) that is not in the same polypeptide chain as the agonist portion, and which is instead linked to the first antibody chain by covalent bonding (preferably disulphide bonding).

In some embodiments, an antibody portion according to the invention consists of or comprises a single domain antibody (also termed a sdAb or Nanobody), a F(ab')2, Fab, Fab', Facb, or single chain Fv (scFv) fragment. A scFv fragment is an epitope-binding fragment that contains at least one fragment of an antibody heavy chain variable region (VH) linked to at least one fragment of an antibody light chain variable region (VL). The linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the VL and VH regions occurs once they are linked so as to maintain the target molecule binding-specificity of the whole antibody from which the single-chain antibody fragment is derived. The carboxyl terminus of the VL or VH sequence may be covalently linked by a linker to the amino acid terminus of a complementary VL or VH sequence.

In some embodiments, the antibody portion of the fusion protein comprises a constant region or Fc region. Fc regions may be obtained from native forms isolated from humans and other animals including cows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms.

They may be obtained from a native immunoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native immunoglobulin into Fab and Fc regions, and pepsin treatment results in the production of pF'c and F(ab)2 fragments. These fragments may be subjected, for example, to size-exclusion chromatography to isolate Fc.

In some embodiments, the Fc region is modified. For example, the immunoglobulin Fc region of the present invention may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in a deglycosylated form. The increase, decrease or removal of the immunoglobulin Fc sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method and a genetic engineering method using a microorganism. The removal of sugar chains from an Fc region results in a sharp decrease in binding affinity to the complement (clq) and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement- dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. In this regard, an immunoglobulin Fc region in a deglycosylated or aglycosylated form (the latter produced for example by a prokaryote, preferably E. coli) may be used according to the present invention.

In addition, the immunoglobulin Fc region may be an Fc region that is derived from IgG, IgA, IgD, IgE and IgM, or that is made by hybrids thereof (sequences encoding two or more immunoglobulin Fc regions of different origin, present in a single-chain immunoglobulin Fc region). In the present invention, various types of hybrids are possible. That is, domain hybrids may be composed of one to four domains selected from the group consisting of CHI, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include a hinge region.

Therefore, in some embodiments, the Fc region is a hybrid. Preferably, the Fc region is derived from IgG or IgM, which are among the most abundant proteins in the human blood, and most preferably from IgG, which is known to enhance the half- life of ligand-binding proteins. Further, IgG is divided into IgGl, IgG2, IgG3 and IgG4 subclasses, and the present invention includes combinations or hybrids thereof. Preferred are IgG2 and IgG4 subclasses.

In some embodiments, the Fc region is IgG in isotype, for example IgGl, preferably human IgG, or human IgGl . In some embodiments, the Fc region is modified to silence or reduce ADCC and/or complement effector functions. In some embodiments, the Fc region is modified for increased FcRn affinity (which can extend half- life). Suitable modifications are described in Monnet, Celine, et al., "Selection of IgG Variants with Increased FcRn Binding Using Random and Directed

Mutagenesis: Impact on Effector Functions." Frontiers in Immunology 6:39 (2015). In some embodiments, the modified Fc region is an IgG variant with one or more, or preferably all, of the mutations: E294Del, T307P, and N434Y (e.g. see Table 6, entry "C6A-66" of Monnet et al.,). These particular mutations are expected to silence ADCC and complement effector functions and increase half-life.

In some embodiments, antibodies from which the antibody portions of fusion proteins of the invention are derived may be human, or humanized, antibodies. In a preferred embodiment, the antibody portion comprises a humanized antibody.

Gpa33. In some embodiments, the antibody portion is an anti-Gpa33 antibody. Gpa33 (A33 glycoprotein), or human Gpa33 (hGpa33), is a cell surface glycoprotein expressed strictly in the intestine, with homogenous and rich baso-lateral expression (e.g. see Johnstone, Cameron N., et al., Characterization of mouse A33 antigen, a definitive marker for baso lateral surfaces of intestinal epithelial cells, American Journal of Physiology - Gastrointestinal and Liver

Physiology 279(3), G500-G510 (2000)). Structurally, Gpa33 is a 43-kDa protein, containing two immunoglobulin-like domains, a hydrophobic transmembrane domain, and a short intracellular tail (Heath, J.K., et al., The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily. Proceedings of the National Academy of Sciences of the United States of America 94, 469-474 (1997)). Genomic organization as well as amino acid-composition are fairly well conserved between human and mouse. Most of the extracellular domain is encoded by exon-II to -V (see Figure 3A). Therefore, antibodies targeting any one of exons II to V are expected to be particularly useful in the context of the invention.

In some embodiments, therefore, the antibody portion of the invention binds to an epitope of Gpa33, in (or at least partially in) the polypeptide region encoded by exons II to V. In some embodiments, the antibody portion of the invention binds to an epitope of Gpa33, in (or at least partially in) the polypeptide region encoded by exon II, exon III, exon IV or exon V. In a preferred embodiment, the antibody portion of the invention binds to an epitope of Gpa33, in (or at least partially in) the polypeptide region encoded by exon III.

In some embodiments, the antibody portion of the invention binds to Gpa33 with an affinity of at least 10^Λ-14 M KD, at least 10^Λ-13 M KD, at least 10^Λ-12 M KD, at least 10^Λ-11 M KD, at least 10^A-10 M KD, at least 10^Λ-9 M KD, at least 10^Λ-8 M KD, at least 10^Λ-7 M KD, at least 10^Λ-6 M KD, at least 10^Λ-5 M KD, or at least 10^Λ-4 M KD as determined using Biacore™ (GE

Healthcare) or a FACScan assay. For example, the antibody portion of the invention may bind to Gpa33 with an affinity of at least about 10^A-9 M KD, or about 10^A-9 M KD. Sequence. In some embodiments, the antibody portion comprises the heavy chain CDRs shown in SEQ ID NOs: 34-36, and the light chain CDRs shown in SEQ ID NOs: 31-33. In other embodiments, variants of SEQ ID NOs: 34-36 and SEQ ID NOs: 31-33 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NOs: 34-36 and SEQ ID NOs: 31-33, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99%) or 100%) amino acid sequence identity with the amino acid sequence of SEQ ID NOs: 34-36 and optionally SEQ ID NOs: 31-33, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

In some embodiments, the antibody portion comprises the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 13. In other

embodiments, variants of SEQ ID NO: 8 and SEQ ID NO: 13 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 13, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 13, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

In some embodiments, the antibody portion comprises or consists of the full-length (i.e.

comprising both variable and constant regions) heavy chain as shown in SEQ ID NO: 14 and the full-length light chain as shown in SEQ ID NO: 12. In other embodiments, variants of SEQ ID NO: 14 and SEQ ID NO: 12 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 12, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 12, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

In some embodiments, the antibody portion of the fusion protein comprises a first antibody chain comprising SEQ ID NO: 14 (or a variant as described above) that is in the same polypeptide chain as the agonist portion of the fusion protein, and a second antibody chain comprising SEQ ID NO: 12 (or a variant as described above) that is not in the same polypeptide chain as the agonist portion, and which is instead linked to the first antibody chain by covalent bonding (e.g. disulphide bonding). Disclaimer. In some embodiments, the antibody portion is not an anti-EpCam (epithelial call adhesion molecule) antibody, i.e. the fusion protein does not selectively target EpCam. For the avoidance of doubt, EpCam is expressed in all epithelial tissues and so is not a selective marker for intestinal epithelial cells. Exemplary fusion proteins of the invention

A fusion protein of the invention may comprise or consist of any combination of features described above, in particular any combination of agonist portion, antibody portion and linker portion. There are no known technical incompatibilities between the portions provided herein. Some non-limiting examples are described below.

In some embodiments, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells, wherein the agonist portion is selected from Rspondin 1-4 or a biologically active fragment or variant thereof, wherein the antibody portion comprises an anti-Gpa33 antibody, and wherein optionally a linker joins the C-terminal of the antibody portion to the N-terminal of the agonist portion.

In some embodiments, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells, wherein the agonist portion is selected from Rspondin 1-4 or a biologically active fragment or variant thereof and wherein the agonist portion is an Lgr-dead Rspondin, wherein the antibody portion comprises an anti-Gpa33 antibody, and wherein optionally a linker joins the C-terminal of the antibody portion to the N-terminal of the agonist portion.

In some embodiments, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells, wherein the agonist portion comprises or consists of one or more furin domain of R-spondin 1-4 or a biologically active fragment or variant thereof, wherein the antibody portion comprises an anti- Gpa33 antibody, and wherein optionally a linker joins the C-terminal of the antibody portion to the N-terminal of the agonist portion.

In some embodiments, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells, wherein the agonist portion is selected from Rspondin 1-4 or a biologically active fragment or variant thereof, wherein the antibody portion comprises an anti-Gpa33 antibody comprising a heavy chain and a light chain, wherein the heavy chain is in the same polypeptide chain as the agonist portion, and wherein the light chain is linked to the heavy chain by covalent bonding, and wherein optionally a linker joins the C-terminal of the antibody portion to the N-terminal of the agonist portion.

In some embodiments, the invention provides a fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells, wherein the agonist portion comprises or consists of one or more furin domain of R-spondin 1-4 or a biologically active fragment or variant thereof, wherein the antibody portion comprises an anti- Gpa33 antibody comprising a heavy chain and a light chain, wherein the heavy chain is in the same polypeptide as the agonist portion, and wherein the light chain is linked to the heavy chain by covalent bonding, and wherein optionally a linker joins the C-terminal of the antibody portion to the N-terminal of the agonist portion.

Any one of the fusion proteins above may comprise an optional linker. The linker is preferably a rigid linker, preferably of about 15 to about 30 amino acids in length.

Lgr-dead R-spondin fusion protein. In some embodiments, the invention provides a fusion protein comprising an agonist portion of Lgr-dead R-spondin or biologically active fragment or variant thereof (for example comprising or consisting of one or more Lgr-dead furin domains), and an antibody portion selectively targeting an epithelial organ, tissue or cell type. The organ, tissue or cell type may be intestinal, or it may be from another tissue. Other tissues that may be targeted by the Lgr-dead R-spondin fusion protein particularly include tissues that contain epithelial cells expressing Rnf43 and/or ZnrO. Exemplary tissues include, but are not limited to, the liver, pancreas, breast, lung, skin, hair follicle, prostate, testes, kidney, gall bladder, tongue, trachea and uterus. Suitable antibody targets exclusively expressed in these tissues are known in the art. In some embodiments, the Lgr-dead R-spondin fusion protein comprises an antibody portion selectively targeting liver epithelial stem cells. Anti-hGpa33/Rspo4. An exemplary fusion protein of the invention designated

"anti-hGpa33/Rspo4" comprises an agonist portion that itself comprises human R-spondin 4 (hRspo4) furin domains identical to the full length of SEQ ID NO: 11. Anti-hGpa33/Rspo4 further comprises a linker portion identical to the full length of SEQ ID NO: 10.

Anti-hGpa33/Rspo4 further comprises an antibody portion that itself comprises human IgGl (hlgGl) isotype anti-hGpa33 antibody heavy chain region, which has the linker and furin domain attached at its C-terminus, and a covalently-linked human IgGl (hlgGl) isotype anti-hGpa33 antibody light chain region, identical to the full lengths of SEQ ID NOs: 14 (full-length heavy chain) and 12 (full-length light chain), respectively. The arrangement of each portion in anti-hGpa33/Rspo4 may be identical to that shown in the full length of SEQ ID NO: 7 with SEQ ID NO: 12.

Methods of Treatment and Medical Uses

General. The invention provides methods of treating, preventing and/or palliating diseases associated with cellular damage and/or impairment of the regenerative capacity of intestinal epithelia, and fusion proteins and compositions for use in such methods. For example, in some embodiments, fusion proteins of the invention may find use in the treatment of inflammatory bowel disease, as discussed in more detail below.

Fusion proteins of the invention may in particular find use in enhancing the regenerative capacity of intestinal epithelia in order to mitigate the side-effects of cancer treatment (including chemotherapy and/or radiotherapy). One common side-effect of cancer treatment is mucositis, which is discussed in more detail below.

The methods of the invention may comprise administering a therapeutically effective amount of a fusion protein, nucleic acid, vector, cell, or pharmaceutical composition of the invention to a subject, particularly to a subject who is suffering from or susceptible to one or more diseases specified below.

In addition, or alternatively, methods of the invention may comprise administering a

therapeutically effective amount of a fusion protein, nucleic acid, vector, cell, or pharmaceutical composition of the invention to a subject in combination with a cancer therapy. Any combination therapies or co-administered therapies described herein, may be administered simultaneously or sequentially and/or separately or in a single composition.

Any method of the invention may be in vivo or in vitro.

Subjects. Fusion proteins, compounds, and compositions of the invention may be administered to one or more subjects as defined herein. In some embodiments, the one or more subjects are human.

Diseases. The methods of treatment and/or medical uses provided herein may find use in any disease in which the regenerative capacity of intestinal epithelia is impaired or in which intestinal epithelial cells have or may be damaged.

In some embodiments, the disease is inflammatory bowel disease, mucositis, gastrointestinal disease, Crohn's disease, irritable bowel syndrome, colitis, gastroenteritis, celiac disease, enteropathy, malabsorption, bowel obstruction, flatulence, functional gastrointestinal disorder, fistula, enteritis, colorectal polyp, short bowel syndrome, gastrointestinal perforation, hirschsprung's disease, intestinal pseudo-obstruction, small intestinal bacterial overgrowth, giardiasis, adhesion, gastrointestinal bleeding, lymphangiectasia, parasitic disease, volvulus, Whipple's disease, necrotizing enterocolitis, intussusception, ischemic colitis, cryptosporidiosis, ileus, amoebiasis, eosinophilic gastroenteritis, tropical sprue, angiodysplasia, or intestinal permeability. In some embodiments, the disease is inflammatory bowel disease (IBD).

Improvements in a mouse model of IBD have been demonstrated (data not shown). Cellular and molecular mechanisms of the epithelial repair in IBD are described in Okamoto & Watanabe, Digestive Diseases and Sciences 50(1) S34-S38 (2005). In some embodiments, the disease is mucositis. Mucositis typically results from damaged barrier function in the intestine, which allows resident bacteria to enter the surrounding tissues and induce an inflammatory reaction.

In some embodiments, the disease is any disease, including any cancer, in which intestinal epithelia are directly or indirectly exposed to chemotherapy and/or radiotherapy. Exemplary cancers or precancerous lesions include appendix cancer, bile duct cancer, bladder cancer, bowel cancer, cancer of the small intestine, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, myeloma, esophageal cancer, pancreatic cancer, rectal cancer, stomach cancer, and tumors of the torso. In some embodiments, the cancer is any intestinal cancer, including bowel cancer, cancer of the small intestine, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer and stomach cancer. In some embodiments, the cancer is colon cancer.

In some embodiments, the cancer is characterized by constitutive activation of the Wnt/p-catenin signaling pathway. A constitutively active pathway is a pathway which is constantly active, e.g. which is not under the control of any regulatory signals or is no longer under the control of normal regulatory signals. In some embodiments, the cancer is characterized by deactivating APC and/or GSK3 mutations and/or stabilizing CTNNB1 (encoding β-catenin) mutations. There are several such mutations known in the art that are associated with cancers and with the constitutive activation of the Wnt/p-catenin signaling pathway. Further, in some embodiments the cancer is characterized by the presence of R-spondin fusion genes. These gene fusions are persistently and inappropriately expressed, causing saturation of Wnt signaling in the tumour (Seshagiri, S., et ah, Recurrent R- spondin fusions in colon cancer. Nature 488, 660-664 (2012)).

In some embodiments, the cancer is an intestinal cancer (such as a colon cancer) characterized by constitutive activation of the Wnt/p-catenin signaling pathway, optionally resulting from deactivating APC and/or GSK3fi mutations and/or stabilizing CTNNB1 (encoding β-catenin) mutations.

Clinical symptoms. Side-effects or deleterious effects of cancer and cancer treatment are well known to the person skilled in the art. A given side-effect may be diagnosed by a clinician, and it is to be expected that new side-effects are observed from time-to-time that are ameliorable by the methods of treatment and/or medical uses provided herein.

Exemplary side-effects of cancer treatment, which are ameliorable by the methods of treatment and medical uses provided herein, include acute enteritis, constipation, cellular damage to non-cancerous cells, diarrhoea, fatigue, fecal incontinence, GI syndrome, hair loss, histological abnormalities, increased duodenal permeability, increased intestinal permeability, increased susceptibility to infections, inflammation, leucocyte recruitment, loss of appetite, loss of crypt base columnar cells (CBCs), morphological changes in mucosa, mouth sores, mucositis, nausea, necrosis, obliterative endarteritis, painful bowel movements, radiation enteropathy,

radiation-induced gastrointestinal syndrome (RIGS), rectal bleeding, stomatitis, ulceration, vascular injury, vomiting, and weight loss. In some embodiments, the invention relates to treatment of "radiation- or chemotherapy- induced" versions of these diseases or disorders.

In a particular embodiment, the invention relates to treatment of radiation- or chemotherapy- induced mucositis. The mucositis may be oral or intestinal. Causes of oral and intestinal mucositis and accompanying symptoms are described in (i) Duncan, M & Grant, G., Oral and intestinal mucositis - causes and possible treatments, Alimentary Pharmacology & Therapeutics 18(9) 853-874, 2003 and (ii) Gibson & Dorothy, Cancer chemotherapy- induced diarrhoea and constipation: mechanisms of damage and prevention strategies, Supportive Care in Cancer 14(9), 890-900, 2006.

For example, the invention provides a fusion protein of the invention for use in a method of treating chemotherapy- or radiotherapy-induced mucositis. The invention also provides a method for treating chemotherapy- or radiotherapy- induced mucositis, wherein the method comprises administration of a fusion protein of the invention to a subject suffering from or susceptible to chemotherapy- or radiotherapy- induced mucositis.

Cellular damage. Exemplary forms of cellular damage, which are ameliorable by the methods of treatment and medical uses provided herein, include altered levels of cellular markers, aneuploidy, apoptosis, cell cycle arrest, cell death, DNA damage, endoreduplication, genomic instability, growth inhibition, impaired self-renewal, increased micronuclei, inhibition of DNA repair, loss of clonogenicity, mitochondrial DNA damage, mitochondrial oxidative damage, oxidative stress, reduced stem cell differentiation, reduced tissue regeneration, senescence, and sister chromatid exchange. Assays for testing for cellular damage are known in the art and available commercially.

Cellular damage may be a clinical symptom associated with side-effects of cancer treatment. Cellular markers. Cellular damage and/or clinical symptoms associated with side-effects of cancer treatment, may be measured according to the absence, presence, downregulation, upregulation, or deregulation, of certain cellular markers.

Exemplary upregulated cellular markers include Bcl-2 family proteins, clastogenic factors, cleavage of PARP, COX-2, DNA damage response proteins, HSF1, ICAM-1, LTB4, p21, p53, PAF, PERK, pro -inflammatory genes, P-selectins, PUMA, reactive radical species, and VCAM- 1.

Target tissues. In some embodiments, the methods of treatment and medical uses of the invention comprise enhancing the regenerative capacity and/or treating cellular damage of targeted organs and/or tissues, such as epithelial tissue and/or intestinal epithelial tissue. In some embodiments, the methods of the invention do not enhance the regenerative capacity and/or treat cellular damage of healthy or cancerous non-target organs/tissues/cells, including organs/tissues/cells of one or more of the adrenal gland, appendix, bladder, blood, bone, brain, cancerous and/or precancerous tissues, ear, esophagus, eye, gall bladder, heart, kidney, large intestine, liver, lung, mouth, muscle, nose, pancreas, parathyroid gland, pineal gland, pituitary gland, skin, small intestine, spleen, stomach, thymus, thyroid gland, trachea, uterus, and vermiform appendix.

In some embodiments, the methods of treatment and medical uses of the invention enhance the regenerative capacity and/or treats cellular damage of intestinal epithelial tissue only.

Co-administration. Provided herein are methods of treatment, and compositions for use in treatment, for which fusion proteins of the invention are co-administered with one or more additional agents and/or treatments in a co-therapy (also referred to as a combination therapy).

The one or more additional agents and/or treatments may impair the regenerative capacity of epithelia, such as intestinal epithelia, particularly cancerous epithelia. In these embodiments, the fusion protein of the invention is useful for treating the undesirable side-effects of the additional agents and/or treatments.

For example, it is contemplated that the fusion protein of the invention would be beneficial in protecting against any additional agent that results in intestinal damage and/or that selectively target rapidly proliferating cells. Radiation and chemotherapy remain the most effective and widely used cancer treatments. These treatments selectively target rapidly proliferating cells such as cancer cells, as well as inevitably cause damage to normal tissues, particularly those undergoing rapid self-renewal, such as the intestine (Yu, Intestinal stem cell injury and protection during cancer therapy, Translational Cancer Research Vol 2, No 5, 2013). Therefore, in some embodiments, the additional agent and/or treatment is any additional agent and/or treatment that results in intestinal damage and/or that selectively target rapidly proliferating cells. In some embodiments, the one or more additional agent and/or treatment is a chemotherapeutic agent or a radiation therapy. In some embodiments, the one or more additional agents may be DNA damaging agents, such as one or more of l,3-bis(2-chloroethyl)-l -nitrosourea, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide, or topotecan. In some embodiments, the one or more additional agents may be chemotherapeutic agents, such as one or more of 5-fluorouracil (5FU), carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin, etoposide, gemcitabine, ifosfamide, irinotecan, methotrexate, mitomycin c, oxaliplatin, sorafenib, and vinblastine. In a preferred embodiment, the chemotherapeutic agent is 5FU and/or doxorubicin. The one or more additional agents and/or treatments include compounds used in cancer treatment, including chemotherapeutic reagents and radiotherapeutic reagents, well known to the person skilled in the art.

In animal models, methotrexate can be more severely inhibitory of intestinal regeneration than 5FU, and this can result in earlier weight loss in methotrexate-treated animals. The fusion protein of the invention has been shown to reduce weight loss in mice exposed to methotrexate (see Example 13) even more effectively in the early stages of exposure than for other

chemotherapeutics such as 5FU. Thus, in a further embodiment, the chemotherapeutic agent is methotrexate.

In some embodiments, the one or more additional agents and/or treatments may also provide protection against side-effects of cancer therapy. For example, in some embodiments, the methods of treatment and medical uses of the invention comprise co-administration of

Granulocyte Colony Stimulating Factor (G-CSF) and/or Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF). G-CSF and GM-CSF are used widely to promote the production of granulocytes or antigen presenting cells (APC), and are sometimes administered to ameliorate wider side-effects of chemotherapy. It is envisaged that the fusion proteins of the invention might advantageously be administered in combination with G-CSF and GM-CSF.

The co-administration may be simultaneous or sequential, and the two or more agents may be administered separately or as part of the same pharmaceutical. Modes of administration. The products provided herein, and the methods of treatment and/or medical uses provided herein, may involve administration of a product or composition of the invention to a subject.

The administration of a product or composition of the invention may comprise or consist of one or more of auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis,

endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, dental, intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and vaginal administration. The administration can be continuous (e.g. by infusion) or acute (e.g. by bolus injection). Some formulations encompass slow release systems.

Dose. In certain embodiments, a pharmaceutical composition is administered in the form of a dosage unit (e.g. tablet, capsule, bolus, etc.). In some embodiments, a pharmaceutical composition comprises a fusion protein, nucleic acid, cell or vector of the invention at a dose within a range selected from 1 mg to 800 mg, 1 mg to 700 mg, 1 mg to 600 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, 1 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg.

In certain embodiments, such pharmaceutical compositions comprise a fusion protein, nucleic acid, cell or vector of the invention in a dose selected from 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg,

65 mg, 70 m g, 75 mg 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, l lO mg, 1 15 mg, 120 mg,

125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg,

180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg,

235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg,

290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg,

345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg,

400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg,

455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 505 mg,

510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg,

565 mg, 570 mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg,

620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg,

675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg,

730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg,

785 mg, 790 mg, 795 mg, and 800 mg. In certain such embodiments, a pharmaceutical composition of the comprises a dose of a fusion protein, nucleic acid, cell or vector of the invention selected from 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800 mg.

In some embodiments, the dose is provided per unit mass of the subject. In such embodiments, a pharmaceutical composition comprises a fusion protein, nucleic acid, cell or vector of the invention at a dose within a range selected from 0.01 mg g^"1, 0.02 mg g^"1, 0.05 mg g^"1, 0.1 mg g^" 0.5 mg g^"1, 1 mg g^"1, 2 mg g^"1, 3 mg g^"1, 4 mg g^"1, 5 mg g^"1, 6 mg g^"1, 7 mg g^"1, 8 mg g^"1, 9 mg g^"1, 10 mg g^"1, 15 mg g^"1, 20 mg g^"1, 30 mg g^"1, 40 mg g^"1, 50 mg g^"1, 60 mg g^"1, 70 mg g^"1, 80 mg g-^O mg g-¹, 100 mg g ¹.

Additional Methods and Products of the Invention

Nucleic acids. The invention provides nucleic acids encoding one or more fusion proteins of the invention or a portion thereof. These nucleic acids may be deoxyribonucleic acid (DNA) or ribonucleic acid (R A). In some embodiments, these nucleic acids are non-modified nucleic acids. In other embodiments, these nucleic acids feature one or more modifications. In some embodiments of the invention, different chains of the antibody portion of the fusion protein of the invention may be provided on the same or on different nucleic acids.

In some embodiments, a nucleic acid of the invention comprises or consists of a nucleotide sequences identical to one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 25, 27, and 29. In other embodiments, variants of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 25, 27, and 29 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of nucleotides. Ordinarily, these nucleotide sequence variants will have a nucleotide sequence having at least 60% nucleotide sequence identity with the nucleotide sequence of one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 25, 27, and 29, preferably having at least 65%, 70%, 75%, 80%, 90%), 95%), 99%) or 100% nucleotide sequence identity with the nucleotide sequence of one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 23, 25, 27, and 29, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and 100% nucleotide sequence identity. In some embodiments, the nucleic acids of the invention comprise or consist of nucleotide sequences encoding an amino acid polymer identical to one or more of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41. In other embodiments, nucleic acids encoding variants of one or more of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41 useable in a method of the invention may be produced by the addition, deletion or substitution (preferably conservative) of nucleotides encoding certain amino acids. Ordinarily, these nucleotide sequence variants will encode an amino acid sequence having at least 60%> amino acid sequence identity with the amino acid sequences of any one of one or more of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41 preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequences of one or more of SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity.

Vectors. The invention provides vectors comprising any nucleic acid of the invention, and vectors encoding any fusion protein of the invention. In some embodiments of the invention, different chains of the antibody portion of the fusion protein of the invention may be provided on the same or on different vectors. Cells. The invention provides a cell or a population of cells comprising one or more nucleic acids or vectors of the invention, or expressing one or more fusion proteins of the invention.

In certain embodiments, the cell is in vivo. In certain embodiments, the cell is in vitro. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the cell is a HEK293 cell.

Pharmaceutical compositions. The invention provides pharmaceutical compositions comprising any fusion protein, nucleic acid, cell and/or vector of the invention. In some embodiments, the pharmaceutical composition additionally comprises one or more diluents, excipients and/or pharmaceutically acceptable carriers.

Kits. The invention provides kits comprising any fusion protein, nucleic acid, vector, cell and/or pharmaceutical composition of the invention.

In some embodiments, the kit comprises one or more of the following: syringe, alcohol swab, cotton ball, gauze pad, instructions for administering the fusion protein, nucleic acid, cell, vector and/or pharmaceutical composition of the invention.

Methods of manufacture. The invention also provides a method for manufacturing a fusion protein of the invention, wherein the method comprises: (a) providing one or more nucleic acid comprising a nucleotide sequence encoding the fusion protein of the invention expressible in a mammalian cell; (b) expressing the nucleic acid in a cell or organism to form the fusion protein; and optionally (c) purifying the fusion protein.

Sequences. Sequences provided by the invention include the following:

A) hlgGl Heavy chain anti-hGpa33/Rspo4 fusion

GAAGTGCAGCTGCTGGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGATCTCTGAG ACTGAGCTGTGCCGCCAGCGGCTTCGCCTTCAGCACCTACGATATGAGCTGGGTGCG CCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCACAATCAGCAGCGGCGGCAGCT ACACCTACTACCTGGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAGCAGC AAGAACACCCTGTACCTGCAGATGAACTCCCTGCAGGCCGAGGACAGCGCCATCTA CTACTGTGCCCCTACCACCGTGGTGCCCTTCGCCTATTGGGGCCAGGGAACACTCGT GACCGTGTCCTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGCAGTACAACAGCACGTACCGT GTGGTCAGCGTCCTCCCCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACTACCACTACACGCAGAAAAGCTTAAGCCTGAGCCCCGGCAAAGAGGCCGCTG CCAGAgaagctgccgctagagaagccgctgctcgggaagctgctgcaagagtgggaacaggcctgggcggcaattgcaccggctg catcatctgcagcgaggaaaacggctgcagcacctgtcagcagagactgttcctgttcatcagaagagagggcatccggcagtacggcaa gtgcctgcacgattgcccccctggctacttcggcatcagaggccaggaagtgaaccgctgcaagaagtgcggcgccacttgcgagagct gcttcagccaggacttctgcatcaggtgcaagcggcagttctacctgtacaagggcaagtgtctgcctacctgccctcctggcacactggcc caccagaacaccagagaatgccagggcgaa (formatting as in Example 1) (SEQ ID NO: 1)

B) Variable region hlgGl Heavy chain anti hGpa33

GAAGTGCAGCTGCTGGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGATCTCTGAG ACTGAGCTGTGCCGCCAGCGGCTTCGCCTTCAGCACCTACGATATGAGCTGGGTGCG CCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCACAATCAGCAGCGGCGGCAGCT ACACCTACTACCTGGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAGCAGC AAGAACACCCTGTACCTGCAGATGAACTCCCTGCAGGCCGAGGACAGCGCCATCTA CTACTGTGCCCCTACCACCGTGGTGCCCTTCGCCTATTGGGGCCAGGGAACACTCGT GACCGTGTCCTCT (capitalized and underscored in A) (SEQ ID NO: 2)

C) CDR2-Mutated Variable region hlgGl Heavy chain anti hGpa33

GAAGTGCAGCTGCTGGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGATCTCTGAG ACTGAGCTGTGCCGCCAGCGGCTTCGCCTTCAGCACCTACGATATGAGCTGGGTGCG CCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCACAATCAGCAACAAGGACAAC AGCTACGCAACCGCATACGCAGTGAAGGGCCGGTTCACCATCAGCCGGGACAGCAG CAAGAACACCCTGTACCTGCAGATGAACTCCCTGCAGGCCGAGGACAGCGCCATCT ACTACTGTGCCCCTACCACCGTGGTGCCCTTCGCCTATTGGGGCCAGGGAACACTCG TGACCGTGTCCTCT (CDR2 mutations underscored, here, and in B) (SEQ ID NO: 3)

D) Linker

GAAGCTGCCGCTAGAGAAGCCGCTGCTCGGGAAGCTGCTGCAAGA (lower case and underscored in A) (SEQ ID NO: 4)

E) Rspondin4 furin domains

GTGGGAACAGGCCTGGGCGGCAATTGCACCGGCTGCATCATCTGCAGCGAGGAAAA CGGCTGCAGCACCTGTCAGCAGAGACTGTTCCTGTTCATCAGAAGAGAGGGCATCC GGCAGTACGGCAAGTGCCTGCACGATTGCCCCCCTGGCTACTTCGGCATCAGAGGCC AGGAAGTGAACCGCTGCAAGAAGTGCGGCGCCACTTGCGAGAGCTGCTTCAGCCAG GACTTCTGCATCAGGTGCAAGCGGCAGTTCTACCTGTACAAGGGCAAGTGTCTGCCT ACCTGCCCTCCTGGCACACTGGCCCACCAGAACACCAGAGAATGCCAGGGCGAA

(lower case in A) (SEQ ID NO: 5)

F) hlgG anti-Gpa33 L-chain

GACATCCAGATGACCCAGTCCCCCTCCTCCCTGTCCGTGTCCGTGGGCGACCGCGTG ACCATCACCTGCAAGGCCTCCCAGAACGTGCGCACCGTGGTGGCCTGGTACCAGCA GAAGCCCGGCCTGGCCCCCAAGACCCTGATCTACCTGGCCTCCAACCGCCACACCG GCGTGCCCTCCCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCTTCACCATCTC CTCCCTGCAGCCCGAGGACATCGCCACCTACTTCTGCCAGCAGCACTGGTCCTACCC CCTGACCTTCGGCCAGGGCACCAAGGTGGAGGTGAAGCGCCGTACGGTGGCTGCAC CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAA AGAGCTTCAACAGGGGAGAGTGTTAGGCGGCCGC (SEQ ID NO: 6)

G) hlgGl Heavy chain anti-Gpa33/Rspo4 fusion

EVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPG GLEWVATISSGGSYT YYLDSV GRFTISRDSSK TLYLQMNSLQAEDSAIYYCAPTTVVPFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREQY NSTYRVVSVLPVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTK QVSLTCLVKGFYPSDIAVEWESNGQPE NYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHYHYTQ SLSLSPG eaaareaaareaaareaaarVGTGLGGNCTGC IICSEENGCSTCQQRLFLFIRREGIRQYGKCLHDCPPGYFGIRGQEVNRCK CGATCESCF SQDFCIRCKRQFYLYKGKCLPTCPPGTLAHQNTRECQGE (SEQ ID NO: 7)

H) Variable region hlgGl Heavy chain anti hGpa33

EVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPGKGLEWVATISSGGSYT YYLDSVKGRFTISRDSSK TLYLQMNSLQAEDSAIYYCAPTTVVPFAYWGQGTLVTVSS

(region underscored in G) (SEQ ID NO: 8)

I) CDR2-Mutated Variable region hlgGl Heavy chain anti hGpa33

EVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPGKGLEWVATISNKDNSY ATAYAVKGRFTISRDSSK TLYLQMNSLQAEDSAIYYCAPTTVVPFAYWGQGTLVTVSS

(CDR2 underscored, here, and in H) (SEQ ID NO: 9)

J) Linker

EAAAREAAAREAAAREAAAR (lower case and underscored in G) (SEQ ID NO: 10)

K) hRspondin4 furin domains

VGTGLGGNCTGCIICSEENGCSTCQQRLFLFIRREGIRQYGKCLHDCPPGYFGIRGQEVN

RCK CGATCESCFSQDFCIRCKRQFYLYKGKCLPTCPPGTLAHQNTRECQGE (lower case in G) (SEQ ID NO: 11)

L) Amino acid sequence hlgG anti-Gpa33 L-chain

DIQMTQSPSSLSVSVGDRVTITCKASQNVRTVVAWYQQKPGLAP TLIYLASNRHTGVP SRFSGSGSGTDFTFTISSLQPEDIATYFCQQHWSYPLTFGQGT VEVKRRTVAAPSVFIFP

PSDEQLKSGTASVVCLL NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (formatting as in Example 1) (SEQ

ID NO: 12)

M) Variable region hlgGl Light chain anti hGpa33

DIQMTQSPSSLSVSVGDRVTITCKASQNVRTVVAWYQQKPGLAP TLIYLASNRHTGVP SPvFSGSGSGTDFTFTISSLOPEDIATYFCOOHWSYPLTFGOGTKVEVKR (region underscored as in L) (SEQ ID NO: 13)

N) Amino acid sequence hlgG anti-Gpa33 H-chain

EVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPG GLEWVATISSGGSYT YYLDSV GRFTISRDSSK TLYLQMNSLQAEDSAIYYCAPTTVVPFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREQY NSTYRVVSVLPVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTK QVSLTCLVKGFYPSDIAVEWESNGQPE NYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK (region underscored as in G) (SEQ ID NO: 14)

O) Rspo-1 furin 1

AEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYF (SEQ ID NO:15)

P) Rspo-2 furin 1 YVSNPICKG-CLSCSKDNGCSRCQQKLFFFLRREGMRQYGECLHSCPSGYY (SEQ ID NO: 16)

Q) Rspo-3 furin 1

PNVSQGCQGGCATCSDYNGCLSCKPRLFFALERIGMKQIGVCLSSCPSGYY (SEQ ID NO: 17)

R) Rspo-4 furin 1

TGLGGNCTG-CIICSEENGCSTCQQRLFLFIRREGIRQYGKCLHDCPPGYF (SEQ ID NO: 18)

S) Rspo-1 furin 2

MNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSS (SEQ ID NO: 19)

T) Rspo-2 furin 2

MNRCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFA (SEQ ID NO: 20)

U) Rspo-3 furin 2

DINKCTKCKADCDTCFNK FCTKCKSGFYLHLGKCLDNCPEGLE (SEQ ID NO: 21)

V) Rspo-4 furin 2

VNRCK CGAT-CESCFSQDFCIRCKRQFYLYKGKCLPTCPPGTL (SEQ ID NO: 22)

W) Human R-spondin 1 mRNA NM 001242908

1 attccctccc tggtgctcgc agaggactgg cccctctccg ggctgggagc tccggccgag

61 cggaggcgcg acggagagca ccagcgcagg gcagagagcc cggagcgacc ggccagagta

121 gggcatccgc tcgggtgctg cggagaacga gggcagctcc gagccgcccc ggaggaccga

181 tgcgccgggt ggggcgctgg ccccgagggc gtgagccgtc cgcagattga gcaacttggg

241 aacgggcggg cggagcgcag gcgagccggg cgcccaggac agtcccgcag cgggcgggtg

301 agcgggccgc gccctcgccc ctcccgggcc tgcccccgtc gcgactggca gcacgaagct

361 gagattgtgg tttcctggtg attcaggtgg gagtgggcca gaagatcacc gctggcaagg

421 actgggttgg tttttggagt agtccctgct gacgtgacaa aaagatctct catatgatat

481 tccgaggtat ctttgaggaa gtctctcttt gaggacctcc ctttgagctg atggagaact

541 gggctcccca caccctctct gtccccagct gagattatgg tggatttggg ctacggccca

601 ggcctgggcc tcctgctgct gacccagccc cagaggtgtt agcaagagcc gtgtgctatc

661 caccctcccc gagaccaccc ctccgaccag gggcctggag ctggcgcgtg actatgcggc

721 ttgggctgtg tgtggtggcc ctggttctga gctggacgca cctcaccatc agcagccggg

781 ggatcaaggg gaaaaggcag aggcggatca gtgccgaggg gagccaggcc tgtgccaaag

841 gctgtgagct ctgctctgaa gtcaacggct gcctcaagtg ctcacccaag ctgttcatcc

901 tgctggagag gaacgacatc cgccaggtgg gcgtctgctt gccgtcctgc ccacctggat

961 acttcgacgc ccgcaacccc gacatgaaca agtgcatcaa atgcaagatc gagcactgtg

1021 aggcctgctt cagccataac ttctgcacca agtgtaagga gggcttgtac ctgcacaagg

1081 gccgctgcta tccagcttgt cccgagggct cctcagctgc caatggcacc atggagtgca

1141 gtagtcctgc gcaatgtgaa atgagcgagt ggtctccgtg ggggccctgc tccaagaagc

1201 agcagctctg tggtttccgg aggggctccg aggagcggac acgcagggtg ctacatgccc

1261 ctgtggggga ccatgctgcc tgctctgaca ccaaggagac ccggaggtgc acagtgagga

1321 gagtgccgtg tcctgagggg cagaagagga ggaagggagg ccagggccgg cgggagaatg

1381 ccaacaggaa cctggccagg aaggagagca aggaggcggg tgctggctct cgaagacgca

1441 aggggcagca acagcagcag cagcaaggga cagtggggcc actcacatct gcagggcctg

1501 cctagggaca ctgtccagcc tccaggccca tgcagaaaga gttcagtgct actctgcgtg

1561 attcaagctt tcctgaactg gaacgtcggg ggcaaagcat acacacacac tccaatccat

1621 ccatgcatac atagacacaa gacacacacg ctcaaacccc tgtccacata tacaaccata

1681 catacttgca catgtgtgtt catgtacaca cgcagacaca gacaccacac acacacatac

1741 acacacacac acacacacac ctgaggccac cagaagacac ttccatccct cgggcccagc

1801 agtacacact tggtttccag agctcccagt ggacatgtca gagacaacac ttcccagcat

1861 ctgagaccaa actgcagagg ggagccttct ggagaagctg ctgggatcgg accagccact

1921 gtggcagatg ggagccaagc ttgaggactg ctggtgacct gggaagaaac cttcttccca

1981 tcctgttcag cactcccagc tgtgtgactt tatcgttgga gagtattgtt acccttccag 2041 gatacatatc agggttaacc tgactttgaa aactgcttaa aggtttattt caaattaaaa

2101 caaaaaaatc aacgacagca gtagacacag gcaccacatt cctttgcagg gtgtgagggt

2161 ttggcgaggt atgcgtagga gcaagaaggg acagggaatt tcaagagacc ccaaatagcc

2221 tgctcagtag agggtcatgc agacaaggaa gaaaacttag gggctgctct gacggtggta

2281 aacaggctgt ctatatcctt gttactcaga gcatggcccg gcagcagtgt tgtcacaggg

2341 cagcttgtta ggaatgagaa tctcaggtct cattccagac ctggtgagcc agagtctaaa

2401 ttttaagatt cctgatgatt ggcatgttac ccaaatttga gaagtgctgc tgtaattccc

2461 cttaaaggac gggagaaagg gccccggcca tcttgcagca ggagggattc tggtcagcta

2521 taaaggagga ctttccatct gggagaggca gaatctatat actgaagggc tagtggcact

2581 gccaggggaa gggagtgcgt aggcttccag tgatggttgg ggacaatcct gcccaaaggc

2641 agggcagtgg atggaataac tccttgtggc attctgaagt gtgtgccagg ctctggacta

2701 ggtgctaggt ttccagggag gagccaaaca cgggccttgc tcttgtggag cttagaggtt

2761 ggtggggaag aaaataggca tgcaccaagg aattgtacaa acacatatat aactacaaaa

2821 ggatggtgcc aagggcaggt gaccactggc atctatgctt agctatgaaa gtgaataaag

2881 cagaataaaa ataaaatact ttctctcagg (SEQ ID NO: 23)

X) Human R-spondinl protein NP 001229837

1 MRLGLCWAL VLSWTHLT I S SRGIKGKRQR RI SAEGSQAC AKGCELCSEV NGCLKCSPKL 61 FI LLERNDIR QVGVCLPSCP PGYFDARNPD MNKCIKCKIE HCEACFSHNF CTKCKEGLYL 12 1 HKGRCYPACP EGSSAANGTM ECSSPAQCEM SEWSPWGPCS KKQQLCGFRR GSEERTRRVL 1 81 HAPVGDHAAC SDTKETRRCT VRRVPCPEGQ KRRKGGQGRR ENANRNLARK ESKEAGAGSR

24 1 RRKGQQQQQQ QGTVGPLTSA GPA (SEQ ID NO: 24)

Y) Human R-Spondin 2 mRNA N M l 78565

1 agcctagact tagatgcctt ggaccacagc accacctact tatagaagca tcccaagcct

61 cagccggtct gcatctccat cggaaagtgc gcttgccaca tcccttcgga tcacttcgtc

121 ctcccgagag cgttctgcct tctacagctc ggaaagaaag aaatcttagc tgtgaagtga

181 ccgtggagaa agcgcaggaa gcgacacaat tggttaggga ggcagagagt gtgagcgggc

241 gcaccccttg cctggggacc gcgctcgcgg gcggggacgg agcatcccag tggctgcacc

301 cgccgctccg cgctcctgcc tggcgtcgcc aaccccgcgg cggccgctgg aattccagag

361 ctgccaggcg ctcccagccg gtctcggcaa acttttcccc agcccacgtg ctaaccaagc

421 ggctcgcttc ccgagcccgg gatggagcac cgcgcctagg gaggccgcgc cgcccgagac

481 gtgcgcacgg ttcgtggcgg agagatgctg atcgcgctga actgaccggt gcggcccggg

541 ggtgagtggc gagtctccct ctgagtcctc cccagcagcg cggccggcgc cggctctttg

601 ggcgaaccct ccagttccta gactttgaga ggcgtctctc ccccgcccga ccgcccagat

661 gcagtttcgc cttttctcct ttgccctcat cattctgaac tgcatggatt acagccactg

721 ccaaggcaac cgatggagac gcagtaagcg agctagttat gtatcaaatc ccatttgcaa

781 gggttgtttg tcttgttcaa aggacaatgg gtgtagccga tgtcaacaga agttgttctt

841 cttccttcga agagaaggga tgcgccagta tggagagtgc ctgcattcct gcccatccgg

901 gtactatgga caccgagccc cagatatgaa cagatgtgca agatgcagaa tagaaaactg

961 tgattcttgc tttagcaaag acttttgtac caagtgcaaa gtaggctttt atttgcatag

1021 aggccgttgc tttgatgaat gtccagatgg ttttgcacca ttagaagaaa ccatggaatg

1081 tgtggaagga tgtgaagttg gtcattggag cgaatgggga acttgtagca gaaataatcg

1141 cacatgtgga tttaaatggg gtctggaaac cagaacacgg caaattgtta aaaagccagt

1201 gaaagacaca atactgtgtc caaccattgc tgaatccagg agatgcaaga tgacaatgag

1261 gcattgtcca ggagggaaga gaacaccaaa ggcgaaggag aagaggaaca agaaaaagaa

1321 aaggaagctg atagaaaggg cccaggagca acacagcgtc ttcctagcta cagacagagc

1381 taaccaataa aacaagagat ccggtagatt tttaggggtt tttgtttttg caaatgtgca

1441 caaagctact ctccactcct gcacactggt gtgcagcctt tgtgctgctc tgcccagtat

1501 ctgttcccag taacatggtg aaaggaagca ccaccagcat ggcccctgtg ttatttatgc

1561 tttgatttga atctggagac tgtgaaggca ggagtaagtg cacagcccgt gacttggctc

1621 agtgtgtgct gagagaatcc gtccccggca ccatggacat gctagaggtg tgaggctgca

1681 gaacaccgct ggaggacgga cttgtgccta tttatgtgaa agaagatgct tggcaggcaa

1741 tgcgctactc actcgtgacc tttatttctc acattgtgca ttttcaagga tatgtttgtg 1801 tggatatctg cttagtgtta ccacatggta ttctcagcat gttaccttca cactgttgtg

1861 cgatgaaact gcttttagct gaggatatgc tctggaaatt cctgctcagt ttcactgcag

1921 ccctaatatg tacatatact gcaggagcta catataaagc tcttatttac tgtatattta

1981 tgctttcttg tgggtaacaa gtcatacctg attaatatga tgccactttg tttctagtgg

2041 ttcctaaccc attgtctgat aaatgacttt tctagtttgg ggaattgaca cttgttttgt

2101 tgcctcttga aacttttttt ttttcccctc attgtgggct tatttctcat tgtaagggta

2161 ggataaacta gtttttgtat atagagtcaa atgaccagtg tcaaagagtt tgcatattgg

2221 gtagaccttc tccactccac atgtcccaca catatagata aagcagcagg cggcatctgg

2281 caatcagaag cccaaactgc ctttgagtct aagatgtgat gactttgatg aaacacaact

2341 gaaaacatga gggactatat ccagtcactt gtagccagtt tcacaggcca gctacagaat

2401 tgtccaaaca aacattattt ctgactgcaa tttttttccc ccaaatttaa agcaatccct

2461 ggctttaaat gacaaggcac ctaccaatgt tcttgggtca ctgaagaagc tactaccatg

2521 agcctgggca tagaatttta ggagataaaa ggatgaattt ctgtgactgc cagtcagatc

2581 ttaacaggtt tctgttgagc cagaatctgt ttcagatcca agatggagag gaacactatg

2641 gaaacttccc aggtgacttt cagagcagtt gtttcaaaca catcattgtc cttttagggg

2701 aaccagtttt tagaaggttg tgaattggct ttttcacaaa gcatgattat cttcctggct

2761 gatccaggag aaaattagaa cagaaaaata atggttgtgg attttgaaac aaagcaaggt

2821 aaagcctttt ttttttcacc ttgcattggc aaaactacct cttcagtgtt tttaactttt

2881 gattcaaaag catcttacca ataaggataa atatcatata catcgttatg aaaatattgc

2941 tatgagataa taagccacat atgaatgttg tatacaactt tagggtttac atttaatcct

3001 gaagtgttac ctcctttcat gtctatttac actattttcc catttactaa gtggggaggg

3061 ggtctcctta tatagtgctt catcgttaat aagtcaatac ctgttgttcc tgggatgttc

3121 ttttttgtgc attaaaaact tcaaaatta (SEQ ID NO: 25)

Z) Human R-Spondin 2 protein NP 848660

1 MQFRLFSFAL I I LNCMDYSH CQGNRWRRSK RASYVSNPIC KGCLSCSKDN GCSRCQQKLF

61 FFLRREGMRQ YGECLHSCPS GYYGHRAPDM NRCARCRIEN CDSCFSKDFC TKCKVGFYLH

12 1 RGRCFDECPD GFAPLEETME CVEGCEVGHW SEWGTCSRNN RTCGFKWGLE TRTRQIVKKP

1 81 VKDT I LCPT I AESRRCKMTM RHCPGGKRTP KAKEKRNKKK KRKLIERAQE QHSVFLATDR 24 1 ANQ (SEQ ID NO: 26)

AA) Human R-Spondin 3 mRNA NM 032784

1 gcggccgccc cggcggctcc tggaaccccg gttcgcggcg atgccagcca ccccagcgaa

61 gccgccgcag ttcagtgctt ggataatttg aaagtacaat agttggtttc cctgtccacc

121 cgccccactt cgcttgccat cacagcacgc ctatcggatg tgagaggaga agtcccgctg

181 ctcgggcact gtctatatac gcctaacacc tacatatatt ttaaaaacat taaatataat

241 taacaatcaa aagaaagagg agaaaggaag ggaagcatta ctgggttact atgcacttgc

301 gactgatttc ttggcttttt atcattttga actttatgga atacatcggc agccaaaacg

361 cctcccgggg aaggcgccag cgaagaatgc atcctaacgt tagtcaaggc tgccaaggag

421 gctgtgcaac atgctcagat tacaatggat gtttgtcatg taagcccaga ctattttttg

481 ctctggaaag aattggcatg aagcagattg gagtatgtct ctcttcatgt ccaagtggat

541 attatggaac tcgatatcca gatataaata agtgtacaaa atgcaaagct gactgtgata

601 cctgtttcaa caaaaatttc tgcacaaaat gtaaaagtgg attttactta caccttggaa

661 agtgccttga caattgccca gaagggttgg aagccaacaa ccatactatg gagtgtgtca

721 gtattgtgca ctgtgaggtc agtgaatgga atccttggag tccatgcacg aagaagggaa

781 aaacatgtgg cttcaaaaga gggactgaaa cacgggtccg agaaataata cagcatcctt

841 cagcaaaggg taacctgtgt cccccaacaa atgagacaag aaagtgtaca gtgcaaagga

901 agaagtgtca gaagggagaa cgaggaaaaa aaggaaggga gaggaaaaga aaaaaaccta

961 ataaaggaga aagtaaagaa gcaatacctg acagcaaaag tctggaatcc agcaaagaaa

1021 tcccagagca acgagaaaac aaacagcagc agaagaagcg aaaagtccaa gataaacaga

1081 aatcggtatc agtcagcact gtacactaga gggttccatg agattattgt agactcatga

1141 tgctgctatc tcaaccagat gcccaggaca ggtgctctag ccattaggac cacaaatgga

1201 catgtcagtt attgctctgt ctaaacaaca ttcccagtag ttgctatatt cttcatacaa

1261 gcatagttaa caacaaagag ccaaaagatc aaagaaggga tactttcaga tggttgtctt 1321 gtgtgcttct ctgcattttt aaaagacaag acattcttgt acatattatc aataggctat 1381 aagatgtaac aacgaaatga tgacatctgg agaagaaaca tcttttcctt ataaaaatgt 1441 gttttcaagc tgttgtttta agaagcaaaa gatagttctg caaattcaaa gatacagtat 1501 cccttcaaaa caaataggag ttcagggaag agaaacatcc ttcaaaggac agtgttgttt 1561 tgaccgggag atctagagag tgctcagaat tagggcctgg catttggaat cacaggattt 1621 atcatcacag aaacaactgt tttaagatta gttccatcac tctcatcctg tatttttata 1681 agaaacacaa gagtgcatac cagaattgaa tataccatat gggattggag aaagacaaat 1741 gtggaagaaa tcatagagct ggagactact tttgtgcttt acaaaactgt gaaggattgt 1801 ggtcacctgg aacaggtctc caatctatgt tagcactatg tggctcagcc tctgttaccc 1861 cttggattat atatcaacct gtaaacatgt gcctgtaact tacttccaaa aacaaaatca 1921 tacttattag aagaaaattc tgattttata gaaaaaaaat agagcaagga gaatataaca 1981 tgtttgcaaa gtcatgtgtt ttctttctca atgagggaaa aacaatttta ttacctgctt 2041 aatggtccac ctggaactaa aagggatact attttctaac aaggtatatc tagtagggga 2101 gaaagccacc acaataaata tatttgttaa tagtttttca agttttgttc actctgtttt 2161 attgtttgtt ttattgagaa attcttactc ttagagactc atgaattaag aaagagaatt 2221 ctgctaactc agagaacctg gttcctatgt aattcagaat atattacatt tctcagtaat 2281 atttgttttt tgaatccacc tttatctgag ccaatggaga tttacttata gcgtattagg 2341 agatatttat tccattttct tattttaatc aacattctaa ttatagacac atgggcctcc 2401 ctagctgatt tcactgctcc cccttcattg cttagaaatg ggcatcattt cttgtatgtc 2461 agatccccct gcatcttcaa catttagtct tttcttctcc atattttcta tctgtggatc 2521 tctttagggg attgaagtca ccctagctga aggcctcacc agtgtttcac agaggacaca 2581 gcccacccct tgcaggagga ggtatctctg agtgtgcagc acagaatcgc atgacccacc 2641 ttaaccttcc tgttgtcatg gaaggatgca cggctgctct gtccactgtg attcctagcc 2701 ctctcaagat cactgctttc tgaagaattt gcaatgactc tggcttctgg ctgcttatct 2761 ctggacaccc gttctccacc agttgtacag ttcatgtaat ctacttggct taattgattt 2821 tccacttctc tcttcctctt ctaagatata aacattttaa atgatttatt cctgtttctt

2881 attctggtgt ttctttcctt gtccctatga gataagtgtc tcaactcact aaatctattc 2941 ccaatgtata aaataattct aattccattt tcagctaaaa catatattac caagaagaaa 3001 caaactttat cctacagaat gatgttaggt agaaatatgt ccccaggttt gagacctttc 3061 ggatgatttc atataccatc tttcttctga gtgttaccca gtcaagtata agtagccaaa 3121 ttatttttgc acatctttct gtttctcatg tcttcattta ttcaacaagc acttactggg 3181 aaggtctaca cctgcatagg caatgctgga aaaagggtta agtaaaccag gacatgacaa 3241 tggtggcaaa tgactatcag gtcttcccat gtgtttgact caaacttatt accctatggt 3301 ccttctgaca atggcagaag gtctgaatcc ttgatgctaa acttatataa aagtagaatt 3361 attacaaagg aaaaagaaat aaaaactaac attcattttc atatgttgga tgaaatataa 3421 atgaagaaaa agataacatc aattttaact gtaattctcc atccaccagt aacagatcct 3481 taagacaata gaatcataca gtattcaaac cagcagcctt ctcaaatttg agcaaaaact 3541 ctatcaacct ctggtaaagt tcctacacta gtcacagaag gtgttaactt tctactctga 3601 ttctgtctcc ataatggggt aaactgttga tagtttaccc catcaacaga tggtcggtaa 3661 attattgatt cgaagaatcg agagagtgca gcaacataaa tctgttaatg tctgatcaag 3721 ctcctgccct gttctccgaa ttcagcttca taattaaggg aaggcctgtt ttctatcctc 3781 agatttaggt tctagtagca gttgtgtaac cactagtgag tcacttaact cctctgggtc 3841 cccatttctc atgtgcaaca agaaagaggg gaactggaga tgatcactct agttccagac 3901 aagggaacat ttcacacttt gtttacttca gggtgatgtc cctgagtcct cattagtgac 3961 tgcgtccttt ggaagttatc ccaaccctgc ttttctcaaa agtgaaaatg tataggctct 4021 cagaggagac agatttaact ctgcttctct aatgttattg aattaaaagc tgttcacatt 4081 agtggttatt aaatattgaa ataacactgg gaagaaaaag catatataaa tacagctaaa 4141 aacaagaata gatattcatt ctcacaaagg gagacagcaa agaaaatgga aagtgcactg 4201 gtgctagcgt tagacagctt gtgttaatgt ctcaattctg ctactaactg gttgcagctt 4261 gtgtgacctt gggcacattg tatgatctcg cagaatatca tcccaaatct gcaaaatgga 4321 attggcatca tctcttttgc aagattgtta tgagaattaa aaggttcttc attcaatata 4381 ataataaata ttttgtatat aaatgaatat caattaaaag ttatgactaa ttccacaagt 4441 caaacatata aattttattt cttgattcat gatatgtgat agtattcata aaaatgtaca

4501 tgcatgataa tttcaaggaa taagtatata tgtgagaatc atggaaatga aattaataat

4561 attaactagt aattaaattg taa (SEQ ID NO: 27)

BB) Human R-Spondin 3 protein NP l 16173

1 MHLRLI SWLF I I LNFMEYIG SQNASRGRRQ RRMHPNVSQG CQGGCATCSD YNGCLSCKPR

61 LFFALERIGM KQIGVCLSSC PSGYYGTRYP DINKCTKCKA DCDTCFNKNF CTKCKSGFYL

12 1 HLGKCLDNCP EGLEANNHTM ECVS IVHCEV SEWNPWSPCT KKGKTCGFKR GTETRVRE I I

1 81 QHPSAKGNLC PPTNETRKCT VQRKKCQKGE RGKKGRERKR KKPNKGESKE AI PDSKSLES 24 1 SKE I PEQREN KQQQKKRKVQ DKQKSVSVST VH (SEQ ID NO: 28)

CC) Human R-Spondin 4 mRNA NM_ 001029871

1 gcccacagca gcccccgcgc ccgccgtgcc gccgccggga cgtggggccc ttgggccgtc

61 gggccgcctg gggagcgcca gcccggatcc ggctgcccag atgcgggcgc cactctgcct

121 gctcctgctc gtcgcccacg ccgtggacat gctcgccctg aaccgaagga agaagcaagt

181 gggcactggc ctggggggca actgcacagg ctgtatcatc tgctcagagg agaacggctg

241 ttccacctgc cagcagaggc tcttcctgtt catccgccgg gaaggcatcc gccagtacgg

301 caagtgcctg cacgactgtc cccctgggta cttcggcatc cgcggccagg aggtcaacag

361 gtgcaaaaaa tgtggggcca cttgtgagag ctgcttcagc caggacttct gcatccggtg

421 caagaggcag ttttacttgt acaaggggaa gtgtctgccc acctgcccgc cgggcacttt

481 ggcccaccag aacacacggg agtgccaggg ggagtgtgaa ctgggtccct ggggcggctg

541 gagcccctgc acacacaatg gaaagacctg cggctcggct tggggcctgg agagccgggt

601 acgagaggct ggccgggctg ggcatgagga ggcagccacc tgccaggtgc tttctgagtc

661 aaggaaatgt cccatccaga ggccctgccc aggagagagg agccccggcc agaagaaggg

721 caggaaggac cggcgcccac gcaaggacag gaagctggac cgcaggctgg acgtgaggcc

781 gcgccagccc ggcctgcagc cctgaccgcc ggctctcccg actctctggt cctagtcctc

841 ggcccctgca cacctcctcc tgctccttct cctcctctcc tcttactctt tctcctctgt

901 cttctccatt tgtcctctct ttctttccac ccttctatca tttttctgtc agtctacctt

961 ccctttcttt ttctttttta tttcctttat ttcttccacc tccattctcc tctcctttct

1021 ccctccctcc ttcccttcct tcctcttctt tctcacttat cttttatctt tccttttctt

1081 tcttcctgtg tttcttcctg tccttcaccg catccttctc tctctccctc ctcttgtctc

1141 cctctcacac acactttaag agggaccatg agcctgtgcc ctcccctgca gctttctcta

1201 tctacaactt aaagaaagca aacatctttt cccaggcctt tccctgaccc catctttgca

1261 gagaaagggt ttccagaggg caaagctggg acacagcaca ggtgaatcct gaaggccctg

1321 cttctgctct gggggaggct ccaggaccct gagctgtgag cacctggttc tctggacagt

1381 ccccagaggc catttccaca gccttcagcc accagccacc ccgaggagct ggctggacaa

1441 ggctccaggg cttccagagg cctggcttgg acacctcccc cagctggccg tggagggtca

1501 caacctggcc tctgggtggg cagccagccc tggagggcat cctctgcaag ctgcctgcca

1561 ccctcatcgg cactccccca caggcctccc tctcatgggt tccatgcccc tttttcccaa

1621 gccggatcag gtgagctgtc actgctgggg gatccacctg cccagcccag aagaggccac

1681 tgaaacggaa aggaaagctg agattatcca gcagctctgt tccccacctc agcgcttcct

1741 gcccatgtgg ggaaacaggt ctgagaagga aggggcttgc ccagggtcac acaggaagcc

1801 ttcaggctct gcttctgcct gatggctctg ctcagcacat tcacggtgga gaggagaatt

1861 tgggggtcac ttgagggggg aaatgtaggg aattgtgggt ggggagcaag ggaagatccg

1921 tgcactcgtc cacacccacc accacactcg ctgacaccca cccccacacg ctgacaccca

1981 cccccacact tgcccacacc catcaccgca ctcgcccaca cccaccacca cactgcccca

2041 cacccaccac cacactcccc cacacccacc accacactcg cccacaccca ccaccagtga

2101 cttgagcatc tgtgcttcgc tgtgacgccc ctcgccctag gcaggaacga cgctgggagg

2161 agtctccagg tcagacccag cttggaagca agtctgtcct cactgcctat ccttctgcca

2221 tcataacacc cccttcctgc tctgctcccc ggaatcctca gaaacgggat ttgtatttgc

2281 cgtgactggt tggcctgaac acgtagggct ccgtgactgg gacaggaatg ggcaggagaa

2341 gcaagagtcg gagctccaag gggcccaggg gtggcctggg gaaggaagat ggtcagcagg

2401 ctgggggaga ggctctaggt gatgaaatat tacattcccg accccaagag agcacccacc 2461 ctcagacctg ccctccacct ggcagctggg gagccctggc ctgaaccccc ccctcccagc

2521 aggcccaccc tctctctgac ttccctgctc tcacctcccc gagaacagct agagccccct

2581 cctccgcctg gccaggccac cagcttctct tctgcaaacg tttgtgcctc tgaaatgctc

2641 cgttgttatt gtttcaagac cctaactttt ttttaaaact ttcttaataa agggaaaaga

2701 aacttgtaaa aaaaaaaaaa aaaaa (SEQ ID NO: 29)

DD) Human R-Spondin 4 protein NP 001025042

1 MRAPLCLLLL VAHAVDMLAL NRRKKQVGTG LGGNCTGCI I CSEENGCSTC QQRLFLFIRR 61 EGIRQYGKCL HDCPPGYFGI RGQEVNRCKK CGATCESCFS QDFCIRCKRQ FYLYKGKCLP 12 1 TCPPGTLAHQ NTRECQGECE LGPWGGWSPC THNGKTCGSA WGLESRVREA GRAGHEEAAT 1 81 CQVLSESRKC PIQRPCPGER SPGQKKGRKD RRPRKDRKLD RRLDVRPRQP GLQP (SEQ ID

NO: 30)

EE) L-chain Gpa33 antibody CDR1

KASQNVRTVVA (SEQ ID NO: 31)

FF) L-chain Gpa33 antibody CDR2

LASNRHT (SEQ ID NO: 32)

GG) L-chain Gpa33 antibody CDR3

QHWSYPLT (SEQ ID NO: 33)

HH) H-chain Gpa33 antibody CDR1

FAFSTYDMS (SEQ ID NO: 34)

II) H-chain Gpa33 antibody CDR2

TISSGGSYTYYLDSVKG (SEQ ID NO: 35)

JJ) H-chain Gpa33 antibody CDR3

PTTVVPFAY (SEQ ID NO: 36)

KK) hRspondinl furin domains

ISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDM NKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECSSPA (SEQ ID NO: 39)

LL) hRspondin2 furin domains

ASYVSNPICKGCLSCSKDNGCSRCQQKLFFFLRREGMRQYGECLHSCPSGYYGHRAPD MN

RCARCRIENCDSCFSKDFCTKCKVGFYLHRGRCFDECPDGFAPLEETMECVEG (SEQ ID NO: 40)

MM) hRspondin3 furin domains

MHPNVSQGCQGGCATCSDYNGCLSCKPRLFFALERIGMKQIGVCLSSCPSGYYGTRYPD I

NKCTKCKADCDTCFNK FCTKCKSGFYLHLGKCLDNCPEGLEANNHTMECVSIV (SEQ ID NO: 41)

EXAMPLES

Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples.

Example 1. Generation of Fusion Protein. The hlgGl Heavy chain part of the fusion protein "anti-hGpa33/Rspo4", and the corresponding human kappa light chain, of sequences as shown in SEQ ID NOs: 7 and 12 respectively, were generated by recombinant DNA technology known and available in the art.

Briefly, Furinl and Furin2 repeats of R-spondin4 were inserted at the C-terminus of the H-chain proteins, connected by an alpha-helical linker (Yan, W., et al., Alpha-helical linker of an artificial 6-zinc finger peptide contributes to selective DNA binding to a discontinuous recognition sequence. Biochemistry 46, 8517-8524 (2007)). The expression constructs encoding these extended H-chain products were combined with the Gpa33 -specific L-chain construct, to produce the full "anti-hGpa33/Rspo4" fusion protein by transfection. Unless indicated otherwise, this "anti-hGpa33/Rspo4" fusion protein is used in the following

Examples. It may be described as "intact" or "wildtype" fusion protein, in the sense that it is not mutated so as to alter Gpa33 recognition.

It is envisaged that these results could be extrapolated to other fusion proteins within the scope of the invention. Heavy chain/R-spondin fusion:

EVOLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVROAPGKGLEWVATISSGGSYT YYLDSVKGRFTISRDSSKNTLYLQMNSLQAEDSAIYYCAPTTWPFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREQ YNSTYRVVSVLPVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWOOGNVFSCSVMHEALHYHYTOKSLSLSPGKeaaareaaareaaareaaarVGTGLGGNCTG CIICSEENGCSTCQQRLFLFIRREGIRQYGKCLHDCPPGYFGIRGQEVNRCKKCGATCESC FSQDFCIRCKRQFYLYKGKCLPTCPPGTLAHQNTRECQGE (SEQ ID NO: 7, ordered to show hGpa33 -specific Variable region (EVQLL VTVSS), Constant region

(ASTKG LSPGK), linker (eaaar....eaaar), and R-spondin4 Furin domains

(VGTGL ECQGE), with CDRs 1-3 bolded and in the order CDR1, CDR2 and CDR3).

Light chain: DIQMTQSPSSLSVSVGDRVTITCKASONVRTWAWYQQKPGLAP TLIYLASNRHTGV PSRFSGSGSGTDFTFTISSLQPEDIATYFCQOHWSYPLTFGQGT VEVKRRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12, ordered to show

Variable-region (DIQMT VEVKR and Constant region (RTVAA NRGEC), with CDRs bolded and in the order CDR1, CDR2, CDR3).

Conditioned media used in the following examples are detailed in Table 1.

Table 1 : Culture conditions for maintenance.

Example 2. Induction of Wnt Signalling by Fusion Protein in Cells with Transient

Expression of Human Gpa33 or Mouse Gpa33.

This example illustrates that intact fusion protein (as described in Example 1) induces Wnt signaling in hGpa33 -expressing cells, where weaker signaling is observed in the absence of fusion protein, in the presence of a mutated fusion protein, and in mouse Gpa33 (mGpa33)- expressing cells.

HEK293 cells were transiently transfected (Tx) with C-terminal Flag-tagged versions of either hGpa33 or mGpa33. HEK293 cells do not exhibit endogenous Gpa33 expression. PEI (linear Polyethylenimine: Polysciences) was used as a vector for transient introduction of expression constructs. The cells were grown in the presence of one of three conditioned media (CM) at a number of dilutions: Wnt-only, Wnt with intact fusion protein, or (as a negative control) Wnt with the fusion protein mutated in CDR2 of the Heavy chain V-region (mutated region shown in SEQ ID NO: 9) to alter Gpa33 recognition. Purified fusion protein with intact Gpa33 recognition, or the CDPv2-mutated version, were produced by transient co-transfection of the relevant H-chain/R- spondin4 plasmid with the hGpa33-specific L-chain (r-PEX protein expression platform at U- Protein Express BV, Utrecht, The Netherlands).

EVQLLESGGGLVQPGGSLRLSCAASGFAFSTYDMSWVRQAPG GLEWVATISNKDNSY ATAYAVKGRFTISRDSSKNTLYLOMNSLQAEDSAIYYCAPTTVVPFAYWGOGTLVTVSS

(SEQ ID NO: 9, underlined corresponding to SEQ ID NO: 7 above and bolded in the mutated region)

Cells transfected with (and expressing) human Gpa33 exhibited an enhanced response to the presence of intact fusion protein in a Wnt activity assay using Renilla luciferase (RL)-normalized TOP luciferase (first described by Korinek, V. et al., Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science 275, 1784-1787 (1997); variants of the assay reviewed in Barolo, S., Transgenic Wnt/TCF pathway reporters: all you need is Lef? Oncogene 25(57), 7505-7511 (2006)), relative to the cells transfected with (and expressing) mouse Gpa33. No response was detected upon incubation of either transfected mouse type with the protein mutated in CDR2 of the Heavy chain V-region. These data are shown in Figure 1 A. Example 3. Induction of Wnt Signalling by Fusion Protein in Cells with Inducible

Gpa33 Expression.

This example illustrates that intact fusion protein (described in Example 1) induces Wnt signaling in cells induced to express Gpa33, where weaker signaling is observed in cells not induced to express human Gpa33. This ensures that any confounding effects of transient transfection from Example 2 are excluded.

A doxycycline (DOX)-inducible HEK293 cell line was produced by PEI-transfection of cells with a pcDNA4 based expression construct for human Gpa33 with C-terminal Flag tag and the selection marker for Neomycin-resistance. Three days after plasmid introduction, selection for stable (random) integration was tested by adding Neomycin (500 μg ml^"1) to the culture medium. Individual NeomycinR clones were tested for expression by addition of DOX and subsequent staining for the Flag-tag (Sigma mouse antibody M2) or the hGpa33 protein by incubating with the intact fusion protein. Doxycycline (DOX)-inducible cells were then grown in the presence of one of three conditioned media (CM): Wnt-only, Wnt with intact fusion protein at a number of concentrations without DOX, or Wnt with intact fusion protein at a number of concentrations with DOX.

Cells transfected with (and DOX- induced to express) human Gpa33 exhibited an enhanced response to the presence of intact fusion protein in a Wnt activity assay using Renilla luciferase (RL)-normalized TOP luciferase, relative to the cells not induced to express human Gpa33 with DOX. The magnitude of response was independent of the concentration of fusion protein over the range tested. These data are shown in Figure IB.

Example 4. Fusion proteins targets R-spondin activity to human colon organoids. This examples illustrates that the antibody portion of the fusion protein selectively targets the agonist portion activity to human colon organoids. This is demonstrated by testing the growth-promoting potential of the fusion protein, by titrating fusion protein into a medium of cultured human colon organoids.

Normal adult human colon organoids were established from a tumor-free colon segment of a patient diagnosed with CRC as previously described (Sato T., et ah, Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141 : 1762-1772 (2011)). Mouse wildtype (wt) and humanized Gpa33 small intestinal organoids were isolated and propagated essentially as described in Sato T., et ah, Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459: 262-265 (2009). 3D cultures were performed using Matrigel (BD Biosciences) and overlaid with a liquid medium consisting of DMEM/F12 advanced medium (Invitrogen), supplemented with additional factors as outlined in table below. mWnt3a-conditioned medium was home-made (produced using stably transfected L cells in the presence of DMEM/F12 advanced medium supplemented with 10% FBS). Conditioned media used are detailed in Table 1. The human colon-derived organoids were then grown in the presence of Wnt alone, or in combination with two-fold dilutions of either wildtype fusion protein or a CDR2 -mutant anti- Gpa33 fusion protein (SEQ ID NO: 9). Proliferative activity was measured in a Luminescent Cell Viability Assay fueled by available cellular ATP.

These data are shown in Figure 2. In the presence of Wnt alone, little luciferase activity is seen. However, across the full range of intact fusion protein concentrations - even as the concentration of fusion protein decreases - luciferase activity can be seen in normal colon organoids but not in organoids with the CDR2-mutant fusion protein. This suggests that targeting of R-spondin using anti-Gpa33 antibody in a fusion protein increases effectively Wnt expression.

Example 5. Exon III of Human Gpa33 is Important for Binding of Fusion Protein.

This example illustrates construction of a humanized Gpa33 mouse model to enable in vivo testing of fusion protein, using a strategy of homologous recombination in embryonic stem (ES) cells. The example also shows that human exon III is the Ab-epitope, as an Rspo-antibody fusion protein will bind to mouse Gpa33 in which human Exon III has been inserted but not in the absence of human Exon III. cDNA construct production. To trace the Ab-epitope, mouse Gpa33 cDNA constructs were made in which the peptide sequences encoded by these exons were replaced by the human versions. ES cells were transfected with cDNA for one of human Gpa33, mouse Gpa33, mouse Gpa33 in which human Exon II had been substituted, or mouse Gpa33 in which human Exon III had been substituted ('exon III knock-in mice' containing exon III in "chimeric m/h gpa33"). These data are shown in Figure 3B.

Test variant binding by immunofluorescence. ES cells were incubated with Rspo-antibody fusion protein and immunofluorescence was used to detect binding of the fusion protein to the cells expressing one of the four Gpa33 proteins. Fluorescence was observed for human Gpa33, and for mouse Gpa33 in which human Exon III had been substituted. Binding was not seen for mouse Gpa33 or mouse Gpa33 in which human Exon II had been substituted. Therefore, immunofluorescent detection of fusion protein-binding to transient transfectants revealed the presence of the epitope in exon-III. The differences in amino acid composition of the human and mouse EXON-III variants are indicated in Figure 3C.

Production of exon-III knock-in mice. Human Gpa33-EXON III knock-in mice were generated through homologous recombination in ES cells. Homology arms (4 kb each) were created by high fidelity PCR, using mouse ES cell DNA as a template. Human Gpa33 exon-III was PCR- amplified similarly, using HEK293 cell-derived DNA. These three components were cloned into PL451. The targeting construct was Sall-linearized and electroporated (300V, 3F) into cultured ES cells. Recombinant clones were selected by adding Neomycin (250 μg ml^"1) to the culture medium. Resistant clones (>200) were subjected to Southern blot analysis using a 500-bp 32P- dCTP DNA probe. Two clones showing homologous recombination, were injected into blastocysts derived from C57BL/6 mice based on standard procedures. Both clones gave germline transmission. The Frt-flanked neomycin cassette was excised in vivo by crossing the mice with the general FLP deleter strain (Jackson Laboratories). Mouse tail-derived genomic DNA was used for PCR-based genotyping by PCR. All experiments were approved by the Animal Experimentation Committee of the Royal Dutch Academy of Science. These

experiments are shown schematically in Figure 3D.

Genotyping to identify exon-III knock-in mice. PCR was used to routinely score the genotypes of mice for the presence of the exon-III recombined allele. The wildtype (wt) allele was amplified with primers Fori + Rev, the allele carrying human exon-III with primers FOR 2+REV.

FOR 1 CCTGGAATTTTGTGACAAAGAAA

FOR 2 TCTGGCCGTTTTCAAACAAAAAC

REV GAGTCCAACAACTACCACATACA

Example 6. Southern blot analysis to identify ES cell clones that have undergone correct recombination at the Gpa33 locus.

This example illustrates that the strategy of homologous recombination in embryonic stem (ES) cells described in Example 5, produces correct recombination at the Gpa33 locus in ± 10% of clones.

Genomic DNA was isolated from 96 Neomycin-resistant ES cell clones (prepared according to Example 5), and Hind-III digested. The digestion products were agarose electrophoresed, blotted, and incubated with a 32P-dCTP-labelled DNA probe. After stringent washing, radioactivity was visualized using phospho-imaging. These data are shown in in Figure 4 (clones that properly recombined are indicated *). Southern blotting performed on neomycin¹¹ clones revealed successful recombination in ± 10%> of clones.

Example 7. Binding of the Fusion Protein to Intestinal Epithelia Demonstrated by Immunolabelling.

This example shows that fusion protein binds specifically to human Gpa33, with negligible non-specific binding in the absence of human Gpa33.

Intestinal crypts were isolated from a mouse homozygous for human Gpa33, and from a wildtype littermate. Both isolates were incubated with intact fusion protein, followed by a labelled anti-huIgGl antibody. Additional detail in Sato, T., et ah, "Single Lgr5 stem cells build crypt- villus structures in vitro without a mesenchymal niche." Nature 459, 7244:262-65 (2009).

Fluorescence is indicative of binding, shown in Figure 5. Example 8. Selective Targeting of Intestinal Epithelia and Enhanced Cell

Proliferation Demonstrated by Luciferase Cell Viability Assay.

This example shows that the effects of Rspo are enhanced in intestinal epithelia when present as a fusion protein containing a human Gpa33 -targeting region relative to Rspo alone. It provides an ex vivo indication of the therapeutic potential of fusion proteins of the invention.

Human colon organoids were produced according to the methods of Sato T., et al, Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141 : 1762-1772 (2011). Mouse intestinal organoids were produced according to the methods of Sato T, et al, Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459: 262-265 (2009).

Small- intestinal organoids were produced from either mice homozygous for humanized Gpa33 or wildtype littermates. Selective binding to humanized Gpa33 epithelial cells was ascertained, in advance, by staining isolated crypts from both animal types. Organoids were cultured in various dilutions of either human Rspo3-CM or purified fusion protein. Cell growth was quantified after 10 days by an ATP-driven luciferase cell viability assay. The experiment was performed in triplo.

These data are shown in Figure 6. As can be seen, both genetically modified organoids (black bars) and wildtype organoids (grey bars) respond similarly to incubation with a range of Rspo3- CM dilutions. However, genetically modified organoids demonstrate a consistently greater cell proliferation response to incubation with fusion protein than do wildtype organoids. This is especially true over the range of 0.4 nM to 0.2 nM, where 25-fold and 200-fold differences in cell viability are exhibited (respectively).

Example 9. Intestinal Renewal is Strongly Inhibited by 5FU Administration.

This example illustrates that the cytostatic drug 5-fluorouracine (5FU) completely inhibits intestinal renewal two days after administration. Chemotherapy with 5FU remains a widely used therapy for late stage metastatic colon cancer. It selectively targets fast dividing cancer cells by blocking DNA replication during the S-phase.

Mice (n = 2) received a single high dose (450 mg kg^"1) of 5FU at day 0. Mice were sacrificed on each of days 1, 2, 3 and 4 and immunohistochemical analysis of growth (proliferation) performed based on BrdU incorporation. For growth analysis by BrdU incorporation, the following protocol was performed. Two hours prior to sacrificing, mice were intra-peritoneally injected with 250 μΐ of a 5mg ml^"1 BrdU (Sigma- Aldrich) solution. For detection on paraffin sections a BrdU-specific mouse antibody (Sigma B2531) was used according to

recommendations of the manufacturer.

Representative images of intestinal paraffin sections are shown in Figure 7. Dark nuclei indicate BrdU incorporation and cell proliferation. No S-phase activity is detected on day 1 or day 2 after 5FU administration. The first sign of spontaneous recovery was visible on day three, culminating in strong growth on day four.

Example 10. Fusion Protein Improves the Regenerative Capacity of the Intestinal Epithelium in vivo.

This example illustrates that, after exposure to 5FU as described in Example 9, administration of fusion protein to mice expressing humanized Gpa33 accelerates regeneration of the intestinal epithelium as represented by increased length of the BrdU⁺ compartment crypt.

Three groups of twelve mice each - two groups of wildtype mice and one group of mice homozygous for humanized (chimeric m/h) gpa33 - received one intraperitoneal injection of 5FU on day 0. One group of wildtype mice, and the m/h gpa33 mice, also received injections of fusion protein at 2 μg (25 g)^"1 on each of days 0, 1, 2 and 3.

At days 1, 2, 3, and 4 three mice of each group received intraperitoneal injections of BrdU prior to sacrifice. Intestines were subject to paraffin immunohistochemistry and BrdU immunostaining as described in Example 9.

In both groups of wildtype mice (receiving 5FU, or 5FU plus fusion protein), intestinal growth was inhibited on days 1-2 but recovered on days 3-4. In the m h gpa33 group, recovery began on day 2 and by days 3-4 had surpassed the highest compartment crypt length seen in either wildtype group. These data are shown in Figure 8, and indicate that fusion protein administered to mice expressing humanized gpa33 exhibit improved regenerative capacity after treatment with a chemotherapeutic agent. Example 11. In vivo administration of fusion protein assists in tolerating a high dose of SFU.

This example illustrates that mice expressing human Gpa33 and receiving fusion protein are better able to tolerate exposure to high-dose 5FU as in Examples 9 and 10, than mice lacking human Gpa33 and mice not receiving fusion protein. Homozygous (horn) Gpa33 -humanized mice and wildtype (wt) littermates (aged 12-13 weeks) received a single dose of 5FU (450 mg kg^"1). At day 0, 1, 2 and 3, wt and homozygous mice were injected with fusion protein at a concentration of 2 μg (25 g)^"1. Relative body weight (%) with respect to day 0 was monitored daily. Two 5FU-injected wt animals, without fusion protein injection, served as controls.

These data are shown in Table 2. As can be seen, mice expressing human Gpa33 and receiving fusion protein maintained their bodyweight relative to mice not receiving fusion protein and to mice receiving fusion protein but expressing human Gpa33.

Table 2: Bodyweight relative to day 0 (%) measured in days following high-dose 5FU administration for wildtype (wt) mice not receiving fusion protein; wt mice receiving fusion protein; and human Gpa33-expressing mice receiving fusion protein.

Fusion

genotype Day l Day 2 Day 3 Day 4 DayS Day 6 Day ? Day 8 Day 9 Day 10 Day 11 protein wt No 39 93,7 87,3 89,7 90,5 81,6 94,4 84,5 80,6 73,8 t wt No 100 97,2 91,6 93,2 95,6 97,5 97,2 98,0 95,2 91,6 89,2

wt Yes 102,7 109,4 91,8 83,2 83,9 89,7 80,8 78,4 76,5 74,5 t wt Yes 37,3 99,2 90,9 92,1 35,9 95,9 38,3 99,6 95,0 89,3 82,6

hom Yes 102,1 98,5 95,4 92,3 91,3 89,7 90,3 100,5 100,5 94,9 97,9 yes 105,5 99,1 93,2 95,0 93,2 95,9 98,6 100,9 100,0 102,3 102,3

Example 12. Exclusive Gpa33-driven Wnt enhancement by Lgr-dead fusion protein.

Lgr 4, 5 and 6 (Lgr 4-6) bind R-spondins with high affinity, thus mediating R-spondin input into the Wnt/beta-catenin signaling pathways. The Lgr/R-spondin complex is thought to enhance Wnt/beta-catenin signaling by neutralizing Rnf 3 and ZnrO, two transmembrane E3 ligases that remove Wnt receptors from the stem cell surface. Rnf43/Znrf3 are themselves encoded by Wnt target genes and constitute a negative Wnt feedback loop (De Lau et al, Genes Dev. 2014 Feb 15; 28(4): 305-316).

This example illustrates that R-spondin, mutated to lack affinity for Lgr (herein termed "Lgr- dead"), is surprisingly able to enhance Wnt signaling when the R-spondin portion is linked to an anti-Gpa33 antibody portion in a fusion protein. The inventors hypothesise that the interaction between anti-Gpa33 antibody portion and Gpa33 on the intestinal epithelial cells, effectively replaces the normal interaction between R-spondin and Lgrs. The antibody portion of the fusion protein, thus allows recruitment of the Lgr-dead Rspondin to the intestinal epithelial cells, where it is able to interact with other cell surface proteins, such as Rnf43 and ZnrO, to enhance Wnt/beta-catenin signaling. A fusion protein containing Lgr-dead Rspondin has advantages in that it cannot have off-target effects on cells that do not express Gpa33 (or any other target moiety that may be selectively targeted by the antibody portion of a fusion protein of the invention).

In this example, an Lgr-dead fusion protein was prepared, comprising an R-spondin4 furin-2 domain containing the mutation F99E (see the residue bolded and underlined in Figure 11). The equivalent R-spondin 1 mutation at F106E, occurring at the "phenylalanine clamp", was described in Peng, Weng Chuan, et al., "Structure of Stem Cell Growth Factor R-spondin 1 in Complex with the Ectodomain of Its Receptor LGR5." Cell Reports 3, 6: 1885-892 (2013), where it was shown to abolish Wnt signaling (see Figure 2 (D) of Peng et al, 2013). Another mutation, Fl 10E, was also shown to have the same effect (again, see Figure 2 (D) of Peng et al, 2013).

These mutations are thought to disrupt the interaction between the conserved phenylalanines in the phenylalanine clamp in the Rspondin furin 2 domain, with Ala 190 of Lgr5 and residues surrounding Ala 190 of Lgr5 (and equivalent conserved sites in Lgr4 and Lgr6). Mutations equivalent to F106E and Fl 10E in Rspondin 1 (or equivalent positions in Rspondin 1-4 and fragments and variants thereof) would be expected to have the same effect, and also result in

"Lgr-dead" R-spondin. Any other mutations that disrupt the phenylalanine clamp, e.g. any non- conservative mutation at F106 and/or Fl 10 of R-spondinl (or equivalent positions), would be expected to result in a similar Lgr-dead Rspondin. Stable DOX-inducible hGpa33 expressing HEK293 cells (prepared according to Example 3) were incubated with Wnt3a alone or Wnt3a combined with dilutions of CM containing the Lgr- dead fusion protein. Wnt pathway activity, in the absence and presence of DOX, was measured 48 hrs upon transient transfection with a TOP luciferase reporter (RL-Normalized). These data are shown in Figure 10.

The data show that in the absence of DOX (i.e. in the absence of hGpa33 protein expression), the Lgr-dead fusion protein does not enhance Wnt signalling beyond the presence of Wnt3a alone. Strikingly, the presence of DOX (i.e. expression of the hGpa33 target protein) causes a sharp increase in Wnt3a enhancement that is sustained through all dilutions of fusion protein tested. Without wishing to be bound by theory, it may be interpreted that the expression of the target protein (hGpa33) in the presence of an anti-Gpa33 antibody portion effectively replaces defective targeting in an Lgr-dead R-spondin, allowing the R-spondin to enhance Wnt signalling. These results also highlight the efficacy of targeting by fusion proteins of the invention.

Example 13. Fusion protein improves regenerative capacity of methotrexate-treated intestinal epithelium in vivo as measured by number of BrdU⁺ crypt cells.

In this example, wildtype and chimeric mice were treated with methotrexate (as opposed to e.g. 5FU) to more rigorously block regeneration of the intestinal epithelium. Mice were then treated with fusion protein to assess whether the fusion protein could overcome the more rigorous effects of methotrexate. Specifically, one group of four wildtype mice and one group of four mice homozygous for humanized (chimeric m/h) Gpa33 received intraperitoneal injections of 100 mg kg^"1

methotrexate on day 0 and 50 mg kg^"1 methotrexate on day 1. Both groups also received injections of fusion protein (as described in Example 1) at 2 μg (25 g)^"1 on each of days 0, 1, and 2. The bodyweight of each mouse was monitored daily. At day 3 the mice received

intraperitoneal injections of BrdU prior to sacrifice. Intestines were subject to paraffin immunohistochemistry and BrdU immunostaining as described in Example 9.

Table 3 shows the bodyweights of the mice across days 0-3. Wildtype mice (lacking the epitope targeted by the fusion protein antibody portion) lose more weight than chimeric mice receiving the fusion protein, similar to the results in Table 2 for 5FU exposure. Thus the Gpa33 -targeting effect of the fusion protein enhances protection against weight loss, presumably by more effectively supporting intestinal regeneration. Images of BrdU⁺ nuclei in representative crypt regions from two wildtype and two chimeric mice are shown in Figure 12 and quantified as percentage positive nucleic (with standard deviation) in the inset table.

Upon administration of fusion protein to mice treated with methotrexate, the number of BrdU⁺ cells (i.e. dividing cells) was considerably greater in the chimeric mice than in the wildtype mice. As chimeric mice possess the human Gpa33 epitope targeted by the antibody portion of the fusion protein (where wildtype mice do not), these data provide further support that the intestinal targeting of the fusion protein results in more effective intestinal regeneration. This is true even in the presence of the more rigorous regeneration inhibition induced by methotrexate. Weight loss was reduced even more potently in the presence of methotrexate (Table 3) than 5FU (Table 2)·

The effects of the fusion protein have been demonstrated in the context of 5FU and

methotrexate, but it is contemplated that similar benefits would be observed when the fusion protein is administered in combination with any other chemotherapeutic agents or any other treatments that have deleterious effects on the intestine.

Table 3: Absolute bodyweight (g) and bodyweight relative to day 0 (%) measured in days following methotrexate administration for wildtype (wt) mice receiving fusion protein; and human Gpa33-expressing mice (chimeric mice) receiving fusion protein.

Weight (gram) Relative weight (%)

Average

Day O Day l Day 2 Day 3 Day 1/0 Day 2/0 Day 3/0 Day 1/0 Day 2/0 Day 3/0

Animal 1 25,4 24,6 24,5 24.5 96,9 96,5 96,5 99,8 98,3 98,8

2 24,2 24,5 24,2 24,3 101,2 100,0 100.4 Mouse/human

3 17,4 17,3 17,2 17,0 99,4 98,9 97,7 Gpa33

4 19,0 19,3 18,6 19,1 101,6 97,9 100,5

1 32,1 31,8 31,1 30,5 99,1 96,9 95,0 97,0 93,9 94,2

2 31,4 30,3 28,2 30,6 96,5 89,8 97,5 W tvpe

3 23,9 23.3 23,0 23,1 97,5 96,2 96,7

4 23,6 22,4 21,9 20,7 94,9 92,8 87,7

Claims

1) A fusion protein comprising an agonist portion and an antibody portion, wherein the agonist portion enhances Wnt signaling and/or beta-catenin signaling, and the antibody portion selectively targets intestinal epithelial cells. 2) A fusion protein, comprising an agonist portion and an antibody portion, optionally according to claim 1, wherein the agonist portion comprises one or more of R-spondin 1-4 or a biologically active fragment or variant thereof, and/or the antibody portion selectively targets Gpa33.

3) The fusion protein of either claim 1 or claim 2, wherein the agonist portion comprises or consists of one or more, preferably two, furin domains of R-spondin 1-4 or a biologically active fragment or variant thereof; and/or comprises an Lgr-dead R-spondin, for example comprising a mutation in the

phenylalanine clamp of furin-2 of the R-spondin.

4) The fusion protein of any one of claims 1 to 3, wherein the agonist portion has at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 11, 39, 40 or

41, and preferably has at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 11, 39, 40 or 41.

5) The fusion protein of any one of claims 1 to 4, wherein the antibody portion binds to its target with an affinity of at least 10^A-14 M KD, at least 10^A-13 M KD, at least 10^A-12 M KD, at least 10^A-11 M KD, at least 10^A-10 M KD, at least 10^A-9 M KD, at least 10^A-8 M KD, at least 10^A-7 M KD, at least 10^A-6 M KD, at least 10^A-5 M KD, or at least 10^A-4 M KD as determined using Biacore™ (GE Healthcare).

6) The fusion protein of any one of the preceding claims, wherein the antibody portion of the fusion protein comprises a first antibody chain that is in the same polypeptide as the agonist portion of the fusion protein, and a second antibody chain that is not in the same polypeptide chain as the agonist portion, and which is instead linked to the first antibody chain by disulphide bonding.

7) The fusion protein of any one of the preceding claims, wherein the antibody portion of the invention binds to an epitope of Gpa33 in the polypeptide region encoded by exons II to V, preferably to an epitope of Gpa33 in the polypeptide region encoded by exon III. 8) The fusion protein of any one of the preceding claims, wherein the antibody portion of the fusion protein of the invention comprises

(a) heavy chain CDRs as shown in SEQ ID NOs: 34-36, and light chain CDRs as shown in SEQ ID NOs: 31-33; or (b) a heavy chain variable region having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 8, preferably having at least 65%, 70%, 75%, 80%, 90%), 95%), 99%) or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 8; and a light chain variable region having at least 60% amino acid sequence identity with amino acid sequence of SEQ ID NO: 13, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%), 99%) or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 13; or

(c) a heavy chain having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14; and a light chain having at least 60% amino acid sequence identity with amino acid sequence of SEQ ID NO: 12, preferably having at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 12.

9) The fusion protein of any one of the preceding claims, wherein the fusion protein comprises a linker portion.

10) The fusion protein of claim 9, wherein the linker portion is linked to the C-terminus of the antibody portion and to the N-terminus of the agonist portion; is a rigid linker; comprises an alpha- helix, optionally with the sequence (EAAAR)n, wherein n is 2-5, preferably wherein n is 4; and/or is about 10-30 amino acids, optionally about 20 amino acids.

11) The fusion protein of claim 9 or claim 10, wherein the linker portion has at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10, and preferably has at least 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 10. 12) The fusion protein of any one of the preceding claims, wherein the fusion protein comprises a first polypeptide chain comprising an anti-Gpa33 antibody heavy chain, a linker and two R-spondin furin domains, and having at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7, preferably having 65%, 70%, 75%, 80%, 90%, 95%, 99%) or 100%) amino acid sequence identity with the amino acid sequence of SEQ ID NO: 7, and wherein optionally, the fusion protein additionally comprises a second polypeptide chain comprising an anti-Gpa33 antibody light chain having at least 60% amino acid sequence identity with amino acid sequence of SEQ ID NO: 12, preferably having at least 65%, 70%, 75%), 80%), 90%), 95%), 99% or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 12, wherein the second polypeptide is linked to the first by one or more disulphide bond.

13) A nucleic acid encoding the amino acid sequence of the fusion protein of any one of the preceding claims.

14) The nucleic acid of claim 13, wherein the nucleic acid sequence has at least 60% polynucleotide sequence identity with the polynucleotide sequence of SEQ ID NO: 1 , preferably having 65%, 70%, 75%, 80%, 90%, 95%, 99% or 100% polynucleotide sequence identity with the amino acid sequence of SEQ ID NO: 1.

15) A vector comprising (a) a nucleic acid according to claim 13 or claim 14, or (b) a nucleic acid encoding the amino acid sequence of the fusion protein of any one of claims 1 to 12. 16) A pharmaceutical composition comprising the fusion protein, nucleic acid or vector of any one of the preceding claims.

17) The pharmaceutical composition of claim 16, further comprising a pharmaceutically acceptable carrier and/or diluent.

18) A kit comprising the pharmaceutical composition of any one of claims 16 to 17. 19) A method for the treatment of intestinal cellular damage in a subject, wherein the method comprises administration to the subject of a fusion protein according to claims 1 to 12, or a pharmaceutical composition according to claim 16 or claim 17.

20) A fusion protein according claims 1 to 12, or a pharmaceutical composition according to claim 16 or claim 17 for use in therapy, for example, for use in regenerative therapy and/or for treating intestinal cellular damage.

21) A method for the treatment of deleterious effects associated with therapy of a subject for one or more cancers and/or for one or more precancerous lesions, wherein the method comprises administration to the subject of the fusion protein of any one of claims 1 to 12 or the pharmaceutical composition of claim 16 or claim 17.

22) A fusion protein according to claims 1 to 12, or a pharmaceutical composition according to claim 16 or claim 17 for use in a method for the treatment of deleterious effects associated with therapy of a subject for one or more cancers and/or for one or more precancerous lesions.

23) A method for treating cancer, wherein the method comprises co-administering to a subject a cancer therapy in combination with a fusion protein according to claims 1 to 12, or a pharmaceutical composition according to claim 16 or claim 17.

24) A fusion protein according to claims 1 to 12 or a pharmaceutical composition according to claim 16 or claim 17, for use in a method for treating cancer, wherein the method comprises co-administering to a subject a cancer therapy in combination with the fusion protein or the pharmaceutical composition.

25) Use of the fusion protein of any one of claims 1 to 12 for the manufacture of a medicament for treatment of a disease. 26) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the subject is human.

27) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for the treatment comprises administering the fusion protein of pharmaceutical composition to the subject by one or more of intravenous,

intraperitoneal, buccal, dental, periodontal, endocervical, enteral, extracorporeal,

intraabdominal, intrabiliary, intradermal, intraduodenal, intralesional, intramuscular, intraprostatic, intratesticular, intratumoral, nasogastric, parenteral, oral, percutaneous, and transdermal administration.

28) The method of claims 21 or 23, or the fusion protein for use according to any one of claims 22 or 24, wherein the therapy for one or more cancers or the cancer therapy comprises chemotherapy and/or radiotherapy.

29) The method of claims 21 or 23, or the fusion protein for use according to any one of claims 22 or 24, wherein the deleterious effects are associated with intestinal epithelial tissues.

30) The method of claims 21 or 23, or the fusion protein for use according to any one of claims 22 or 24, wherein the deleterious effects comprise one or more of acute enteritis, cellular damage to non-cancerous cells, constipation, diarrhea, fatigue, fecal incontinence, GI syndrome, hair loss, histological abnormalities, increased duodenal permeability, increased intestinal permeability, increased susceptibility to infections, inflammation, leucocyte recruitment, loss of appetite, loss of crypt base columnar cells (CBCs), morphological changes in mucosa, mouth sores, mucositis, nausea, necrosis, obliterative endarteritis, painful bowel movements, radiation enteropathy, radiation induced gastrointestinal syndrome (RIGS), rectal bleeding, stomatitis, ulceration, vascular injury, vomiting, and weight loss.

31) The method of claims 21, 23, 29 or 30, or the fusion protein for use according to any one of claims 22 or 24, wherein the deleterious effects comprise or consist of weight loss.

32) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the cellular damage comprises one or more of altered levels of cellular markers, aneuploidy, apoptosis, cell cycle arrest, cell death, DNA damage,

endoreduplication, genomic instability, growth inhibition, impaired self-renewal, increased micronuclei, inhibition of DNA repair, loss of clonogenicity, mitochondrial DNA damage, mitochondrial oxidative damage, oxidative stress, reduced stem cell differentiation, reduced tissue regeneration, senescence, and sister chromatid exchange. 33) The method of claim 32 or the fusion protein for use according to claim 32, wherein the altered levels of cellular markers comprise upregulation of one or more of Bcl-2 family proteins, clastogenic factors, cleavage of PARP, COX-2, DNA damage response proteins, HSF1, ICAM- 1, LTB4, p21, p53, PAF, PERK, pro-inflammatory genes, P-selectins, PUMA, reactive radical species, and VCAM-1. 34) The method of claims 21 or 23, or the fusion protein for use according to any one of claims 22 or 24, wherein the one or more cancers comprises bowel cancer, cancer of the small intestine, colon cancer, colorectal cancer, gastrointestinal cancer, oesophageal cancer, rectal cancer, and/or stomach cancer.

35) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for treatment comprises enhancing the regenerative capacity of intestinal epithelial tissue.

36) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for treatment does not enhance the regenerative capacity of hepatic and/or pancreatic tissue. 37) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for treatment enhances the regenerative capacity only of intestinal epithelial tissues. 38) The method of claims 19 or 21, or the fusion protein for use according to any one of claims 20 or 22 , wherein the method for treatment further comprises co-administration of one or more cancer therapies.

39) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for treatment further comprises co-administration of

GM-CSF or G-CSF.

40) The method of claims 19, 21 or 23, or the fusion protein for use according to any one of claims 20, 22 or 24, wherein the method for treatment is applied before, during and/or after the therapy for one or more cancers and/or for one or more precancerous lesions. 41) A method of producing the fusion protein, nucleic acid sequence or vector of any one of the preceding claims, comprising expressing the fusion protein in a host cell.

42) A new fusion protein, pharmaceutical composition, preparation process, or use in the manufacture of a medicament, substantially as herein described.