Recombinant variants of R-spondin proteins and uses thereof

Abstract

The present invention relates to recombinant variants of R-spondin proteins and their use as medicaments, in particular for the treatment of diabetes.

Description

Detailed Description of the Invention

[0001] The present disclosure relates to recombinant variants of R-spondin proteins and their use as medicaments, particularly for the treatment of diabetes.

[0002] 〔background〕 During the past decades, diabetes has become one of the most widespread metabolic disorders in epidemic dimension, affecting almost 9% of the world's population (WHO, 2016). By 2049, the number of people affected by diabetes is expected to reach 600 million. Diabetes is characterized by high blood glucose levels and is most often due to the inability of the pancreas to secrete sufficient amounts of insulin. Type 1 diabetes (T1D) is caused by autoimmune-mediated destruction of insulin-producing beta cells, while type 2 diabetes (T2D) is due to resistance to insulin action and eventual beta cell failure / loss over time.

[0003] Current treatments for diabetes fail to restore euglycemia precisely, and in the case of T1D, substituting defective insulin secretion with exogenous insulin injections may even appear to be symptom-alleviating. Thus, replenishing the pancreas with new functioning β-cells and / or maintaining the health of the remaining β-cells represents an important strategy for the treatment of both conditions. However, to date, there is no available treatment to prevent pancreatic β-cell loss or induce proliferation, particularly in human patients suffering from type 1 diabetes.

[0004] Rspo1 belongs to a family of cysteine-rich secreted proteins, which also includes Rspo2, Rspo3 and Rspo4. They share a common structural organization, including four structurally and functionally distinct domains, as shown in Figure 1. At the N-terminus, a signal peptide sequence ensures the correct entry of R-spondin proteins into the canonical secretory pathway. The mature secreted form contains two amino-terminal cysteine-rich furin-like repeats (FU1 and FU2) that are important for interaction with the R-spondin-specific receptor LGR (leucine-rich repeat-containing G protein-coupled receptor)4-6 (de Lau, WB, Snel, B. & Clevers, HC Genome Biol 13, 242, doi:10.1186 / gb-2012-13-3-242 (2012)). The central part of the protein contains a single thrombospondin type 1 repeat domain (TSP1) involved in interactions with specific components of the extracellular matrix, followed by a carboxy-terminal basic amino acid-rich domain, the function of which remains unclear. R-spondin protein is involved in cell proliferation (Kim, KA et al. Science 309, 1256-1259, doi:10.1126 / science.1112521 (2005). Da Silva, F. et al. Dev Biol 441, 42-51, doi:10.1016 / j.ydbio.2018.05.024 (2018)), cell specification (Vidal, V. et al. Genes Dev 30, 1389-1394, doi: 10.1101 / gad.277756.116 (2016)), and sex determination (Chassot, AA et al. Hum Mol Genet 17, 1264-1277, doi:10.1093 / hmg / ddn016 (2008)) and other processes.It has been reported as a central regulator of the canonical WNT signaling pathway (also known as the WNT / β-catenin or cWNT pathway) (Jin, YR & Yoon, JK The R-spondin family of proteins: emerging regulators of WNT signaling. Int J Biochem Cell Biol 44, 2278-2287, doi:10.1016 / j.biocel.2012.09.006 (2012)).

[0005] Despite the great interest raised by the possible involvement of the cWNT pathway in pancreatic maturation and function (Scheibner et al. 2019, Curr Opin Cell Biol. 61:48-55), the role and contribution of R-spondin proteins remains poorly studied in this organ.

[0006] In vitro analysis reported that in the presence of Rspo1, beta-cell proliferation and function were increased in the Min6 tumor-derived cell line (Wong, VS, Yeung, A., Schultz, W. & Brubaker, PL R-spondin-1 is a novel beta-cell growth factor and insulin secretagogue. J Biol Chem 285, 21292-21302, doi:10.1074 / jbc.M110.129874 (2010)). However, more recent studies from the same group reported contradictory statements: Rspo1 deficiency in mice is associated with increased β-cell mass and enhanced glycemic control (Wong, VS, Oh, AH, Chassot, AA, Chaboissier, MC & Brubaker, PL Diabetologia 54, 1726-1734, doi:10.1007 / s00125-011-2136-2 (2011) and Chahal et al 201, Pancreas Vol 43(1) pp 93-102).

[0007] In contrast to the latter study, PCT / EP2021 / 050289 reports that treatment with recombinant Rspo1 protein induces in vivo proliferation of functional pancreatic β-cells, improves glucose tolerance, and increases glucose-stimulated insulin secretion (GSIS) in a mouse model of diabetes. Furthermore, it was shown that treatment with recombinant Rspo1 protein induces near-complete β-cell ablation, suggesting that Rspo1 protein administration can induce proliferation and reconstitution of functional β-cell mass in the remaining β-cells capable of maintaining normoglycemia. Finally, it was shown that Rspo1 can also induce β-cell proliferation in humans, opening new and unexpected avenues for the treatment and prevention of human diabetes with recombinant Rspo1 protein.

[0008] RSPO1 residues important for receptor binding and biological activity are reported, for example, in Wang et al. GENES & DEVELOPMENT 27:1339-1344, 2013; Xie et al. EMBO reports VOL 14 | NO 12 | 2013; Xu et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 4, pp. 2455-2465, January 23, 2015. In FIG. 1, the key residues for binding and activity in both the ZNRF3 and LGR4 receptors are shown and reported.

[0009] There remains a need to further improve Rspo1-derived proteins to optimize their exploitable and pharmacological properties for use as biological pharmaceuticals.

[0010] It is therefore an object of the present disclosure to provide novel recombinant variants of R-spondin proteins for use as drugs, for example in the treatment of diabetes.

[0011] Detailed Description definition In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set out throughout the detailed description.

[0012] The term "amino acid" refers to naturally occurring and non-natural amino acids (also referred to herein as "non-naturally occurring amino acids"), including amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, such as an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. The analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

[0013] Substitution refers to the replacement of a naturally occurring amino acid with another naturally occurring or non-natural amino acid. For example, during chemical synthesis of synthetic peptides, a natural amino acid can be easily replaced by another naturally occurring or non-natural amino acid. Alternatively, substitution can be performed by mutating a gene coding sequence to change a codon into a codon that codes for another amino acid residue. The polypeptide obtained by expression of the mutated coding sequence has one amino acid substitution (one amino acid replaced with another different amino acid).

[0014] As used herein, the term "protein" refers to any organic compound made up of amino acids arranged in one or more linear chains (also referred to as "polypeptide chains") and folded into a globular form. This includes proteinaceous substances or fusion proteins. The amino acids in the polypeptide chain may be linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" further includes, but is not limited to, peptides, single-chain polypeptides, or any composite protein consisting primarily of two or more amino acid chains. It further includes, but is not limited to, glycoproteins or other known post-translational modifications. It further includes, but is not limited to, known natural or artificial chemical modifications of natural proteins, such as, for example, glycoengineering, pegylation, hesylation, PASylation, etc., incorporation of non-natural amino acids, amino acid modifications for chemical conjugates or other molecules, etc.

[0015] As used herein, the term "recombinant protein" includes proteins that are prepared, expressed, produced or isolated by recombinant means, such as fusion proteins isolated from a host cell that has been transformed to express the corresponding protein (e.g., from a transfectoma, etc.).

[0016] As used herein, the term "fusion protein" refers to a recombinant protein comprising at least one polypeptide chain that is obtained or can be obtained by genetic fusion, for example by genetic fusion of at least two gene fragments encoding separate functional domains of separate proteins. Thus, the protein fusion of the present disclosure comprises at least one R-spondin or variant thereof as described below, and at least one other moiety. The other moiety is a polypeptide other than the R-spondin polypeptide or variant thereof as described below. In certain embodiments, the other moiety may also be a non-protein moiety (e.g., a polyethylene glycol (PEG) moiety, or other chemical moiety or conjugate). In a preferred embodiment, the second moiety may be the Fc region of an antibody, and thus such a fusion protein is indicated as "Fc fusion protein".

[0017] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain (including native sequence Fc regions and variant Fc regions), preferably including insertions, deletions, or substitutions of 5, 10, 15, or 20 or less amino acids relative to a native human Fc region. A native human Fc region can be of any of the IgG1, IgG2, IgG3, IgG4, IgA, IgA, IgD, IgE, or IgM isotypes. A human IgG heavy chain Fc region is generally defined as including amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody. The numbering of residues in the Fc region is the EU numbering of Kabat (EU index). The C-terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the Fc fusion protein.

[0018] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (i.e., % identity = number of identical positions / total number of positions x 100), and the number of gaps and the length of each gap must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm described below.

[0019] The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm.

[0020] Percent identity between two nucleotide or amino acid sequences may be determined using algorithms such as, for example, EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk, Rice et al 2000 Trends Genet 16:276-277). For example, EMBOSS Needle uses a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, and a "gap alignment penalty" of 0.5. A "zero penalty", a false "end gap penalty", an "end gap open penalty" of 10, and an "end gap extend penalty" of 0.5 may be used. In general, "percent identity" is a function of the number of matching positions divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. Percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules that have the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.

[0021] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" preferably includes mammals (e.g., non-human primates, sheep, dogs, cats, horses, etc.).

[0022] As used herein, the term "R-spondin protein" refers to the naturally occurring R-spondin1, R-spondin2, R-spondin3, or R-spondin4 proteins (also referred to herein as Rspo1, Rspo2, Rspo3, Rspo4 proteins) encoded by the corresponding Rspo1, Rspo2, Rspo3, Rspo4 genes, respectively.

[0023] A native R-spondin protein typically contains, from its N-terminus to its C-terminus, a signal peptide (SP), two cysteine-rich furin-like domains (FU1 and FU2), an N-glycosylation site between the FU2 domain and the TSP domain, a thrombospondin (TSP1) motif (TSP), and a basic amino acid-rich (BR) domain, which contains a potential O-glycosylation site.

[0024] FIG. 1 provides an alignment of the Rspo1, Rspo2, Rspo3 and Rspo4 amino acid sequences along with a schematic of the different domains FU1, FU2, TSP and BR. Throughout this specification, when referring to an amino acid position of Rspo1, or the "equivalent amino acid position" of Rspo2, Rspo3 and Rspo4, reference should be made to the alignment in FIG. 1 to identify the "equivalent" amino acid position. Similarly, the "equivalent (or corresponding)" domain of Rspo1 in Rspo2, Rspo3 or Rspo4 can be identified by reference to the alignment in FIG. 1. The full-length amino acid sequences of human Rspo1, Rspo2, Rspo3 and Rspo4 (with their signal peptides) are set forth in SEQ ID NOs: 1-4, respectively. The amino acid sequences of the FU1, FU2, TSP and BR domains of human Rspo1 are set forth in SEQ ID NOs: 5-8, respectively. The amino acid sequences of the FU1, FU2, TSP, and BR domains of human Rspo2 are set forth in SEQ ID NOs: 9 to 12, respectively. The amino acid sequences of the FU1, FU2, TSP, and BR domains of human Rspo3 are set forth in SEQ ID NOs: 13 to 16, respectively. The amino acid sequences of the FU1, FU2, TSP, and BR domains of human Rspo4 are set forth in SEQ ID NOs: 17 to 20, respectively.

[0025] As used herein, the term "chimeric protein" refers to a protein that contains the replacement of one or more domains in a particular protein with the equivalent domains of a protein of the same family. For example, a chimeric protein of Rspo1 has the FU1 domain of Rspo1 replaced by the corresponding FU1 domain of Rspo2, and contains other domains (such as the FU2, TSP, or BR domains) that are identical to the native Rspo1 protein.

[0026] As used herein, the term "hybrid domain" refers to a domain of a protein in which some amino acid residues are mutated to the equivalent residues of another member of the same family.For example, the hybrid FU1 domain of R-spondin protein can correspond to the FU1 domain of Rspo2 with some mutations, which are made to replace one or more amino acid residues with the equivalent residues of the FU1 domain of Rspo2.

[0027] Variants of the present disclosure The present disclosure relates to recombinant variants of native R-spondin proteins, including the following FU1, FU2, TSP and BR domains. FU1 is a domain having at least 80% identity to any of the FU1 domains of human Rspo1, Rspo2, Rspo3 or Rspo4 as set forth in SEQ ID NOs: 5, 9, 13 and 17, respectively; FU2 is a domain having at least 80% identity to any of the FU2 domains of human Rspo1, Rspo2, Rspo3 or Rspo4 as set forth in SEQ ID NOs: 6, 10, 14, 18, respectively; The TSP is a domain having at least 80% identity to any of the TSP domains of human Rspo1, Rspo2, Rspo3, or Rspo4 as set forth in SEQ ID NOs: 7, 11, 15, 19, and BR is a domain having at least 80% identity to any of the BR domains of human Rspo1, Rspo2, Rspo3, or Rspo4 as set forth in SEQ ID NOs: 8, 12, 16, 20.

[0028] The recombinant variants of the native R-spondin proteins defined above are hereinafter referred to as "variants of the present disclosure" or "Rspo1 variants".

[0029] For ease of reading, the sequences of the variants of the present disclosure described below are always described without the signal peptide sequence. However, all variants disclosed herein may or may not include an N-terminal signal peptide (SP) sequence, in particular one of the (SP) sequences of Rspo1, Rspo2, Rspo3 or Rspo4, typically amino acid residues 1-20 of the Rspo1 protein. A variant of the present disclosure may also lack its normal signal sequence and have a different signal sequence instead. The choice of signal sequence depends on the type of host cell in which the recombinant protein is produced, and a different signal sequence may take the place of the native signal sequence. In addition, a variant of the present disclosure may include an additional peptide sequence at its C-terminus, for example for purification. All variants disclosed herein may also include a C-terminal tag, such as a polyhistidine tag of SEQ ID NO: 90, which contains six histidine residues.

[0030] In a specific embodiment of said variant, the domains FU1, FU2, TSP and BR have at least 80% identity with the FU1, FU2, TSP and BR domains of the human Rspo1 domain of SEQ ID NO:1, respectively.

[0031] In a specific embodiment, the TSP domain and BR domain have 100% identity to the corresponding TSP domain and BR domain of human Rspo1, respectively, and the amino acid sequences of the FU1 domain and FU2 domain are at least 80% identical to the corresponding FU1 and FU2 amino acid sequences of human Rspo1, with the differences being due to amino acid substitutions.

[0032] In a specific embodiment, the variants of the present disclosure have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in each of the FU1 domain or FU2 domain when aligned with the FU1 domain of human Rspo1 of SEQ ID NO: 5 and the FU2 domain of Rspo1 of SEQ ID NO: 6, respectively.

[0033] In a specific embodiment, the variant of the present disclosure is a chimeric protein comprising: (i) a hybrid Rspo2 FU1 domain having no more than 1, 2, 3, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the Rspo2 FU1 of SEQ ID NO: 9; and (ii) The FU2 domain of Rspo1, which is identical to SEQ ID NO:6 or which contains no more than 1, 2, 3, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the FU2 domain compared to SEQ ID NO:6.

[0034] Preferably, the variants of the present disclosure, or their functional equivalents, described herein exhibit one or more of the following properties, at least at a level similar to the Rspo1 protein of SEQ ID NO: 41: (i) induces the proliferation of functional beta cells to a level at least comparable to that of the reference human Rspo1 of SEQ ID NO: 41, e.g., as determined in an in vitro Min6 beta cell proliferation assay; and / or (ii) induces the proliferation of functional beta cells to a level at least comparable to that of the reference human Rspo1 of SEQ ID NO: 41, e.g., as determined in an in vivo beta cell proliferation assay; (iii) optionally binds to the LGR4 receptor with at least the same affinity as the human Rspo1 of reference SEQ ID NO:41, as measured in an Rspo1 / LGR4 binding affinity in vitro assay, e.g., as determined by an SPR or ELISA assay; (iv) optionally binds to the ZNRF3 receptor with at least the same affinity as human Rspo1 of reference SEQ ID NO: 41, as measured in an Rspo1 / ZNRF3 binding affinity in vitro assay, e.g., as determined by an SPR or ELISA assay; (v) optionally, enhances the Wnt / β-catenin pathway in vitro, for example as determined by a Top Flash assay.

[0035] The disclosed variants may be advantageously used as pharmaceutical agents, particularly for the treatment of diabetes in humans and / or for inducing proliferation of pancreatic beta cells in vivo or in vitro.

[0036] Amino acid deletion at the A N terminal residue The inventors have surprisingly found that deletion of residues 21-31 of the Rspo1 protein results in a more active protein in a Min6 proliferation assay compared to the native Rspo1 protein of SEQ ID NO: 41. Thus, in a specific embodiment, a variant of the disclosure comprises a deletion of the first 10-14 N-terminal amino acid residues within region 21-33 of Rspo1, or within the equivalent region in Rspo2, Rspo3, or Rspo4. Typically, a variant of the disclosure is an Rspo1 protein having a deletion of 10-14 N-terminal amino acid residues within region 21-33 of Rspo1.

[0037] More specifically, the variant of the present disclosure comprises or consists of the protein of SEQ ID NO: 24. The protein corresponds to human Rspo1 in which amino acid residues 21-31 of Rspo1 are deleted (see variant #009 disclosed in the Examples).

[0038] B. Amino acid substitutions in the FU1 domain that increase β-cell proliferation.

[0039] The mutation R66A in the FU1 domain of Rspo1 reduces or abolishes the binding of Rspo1 to ZNRF3 as described by Xie et al. (EMBO reports VOL 14 | NO 12 | 2013).

[0040] ZNRF3, as used herein, refers to an E3 ubiquitin protein ligase that acts as a negative regulator of the Wnt signaling pathway by mediating the ubiquitination and subsequent degradation of Frizzled and LRP6, components of the Wnt receptor complex. Rspondin acts on both the canonical and non-canonical Wnt signaling pathways. Rspondin proteins, particularly Rspo1, have been reported to bind to ZNRF3 and have been described as required for R-spondin signaling activity (Xie et al. EMBO reports VOL 14 | NO 12 | 2013). ZNRF3 protein is listed in the Uniprot database under accession number Q9ULT6 and NCBI Entrz Gene number 84133.

[0041] The present inventors found that the Rspo1 mutation R66A not only does not eliminate the function of Rspo1 in the Min6 proliferation assay, but surprisingly enhances its activity.

[0042] In a specific embodiment, the variants of the present disclosure advantageously comprise at least an amino acid substitution that reduces or eliminates ZNRF3 binding of residue R66 in the FU1 domain of human Rspo1 of SEQ ID NO:5, or the equivalent arginine residue in the FU1 domain sequence of human Rspo2, Rspo3, or Rspo4.

[0043] The binding affinity of any variant to ZNFR3 can be compared with the corresponding binding affinity of the reference human Rspo1 protein of SEQ ID NO: 41 to ZNRF3. Methods for measuring binding affinity to ZNRF3 are described in the Examples, including, for example, the ZNRF3 binding assay as disclosed in the Examples below. As used herein, reduced ZNRF3 binding means that the binding affinity is significantly lower, preferably at least 20% lower, more preferably at least 30%, 40%, 50% lower than the corresponding binding affinity measured with the reference Rspo1 of SEQ ID NO: 41. Binding is abolished when binding is below detectable levels or similar to non-specific binding proteins.

[0044] Typically, the variant of the disclosure comprises a FU1 domain of Rspo1 of SEQ ID NO: 5 with a single amino acid substitution at residue R66, typically R66A, that reduces or eliminates ZNRF3 binding. In a specific embodiment, the variant of the disclosure comprises a FU1 domain of Rspo1 of SEQ ID NO: 5 with a single amino acid substitution at residue R66, typically R66A, that reduces or eliminates ZNRF3 binding, and the FU2, TSP and BR domains are 95%, preferably 100%, identical to the FU2, TSP and BR domains of Rspo1, respectively.

[0045] For example, a variant of the present disclosure comprises or consists essentially of SEQ ID NO:23 (corresponding to variant #008 disclosed in the Examples below).

[0046] C. Amino acid substitutions in the FU1 and FU2 domains to increase binding to LGR4 and / or ZNRF3 Variants of the present disclosure may further comprise amino acid substitutions in the FU1 and / or FU2 domains to increase binding affinity to LGR4 and / or ZNRF3 compared to the native Rspo1 of SEQ ID NO:41.

[0047] The binding affinity of any variant to LGR4 and / or ZNFR3 can be compared to the corresponding binding affinity of the reference human Rspo1 protein of SEQ ID NO: 41 to ZNRF3 or LGR4. Methods for measuring binding affinity to ZNRF3 or LGR4 are described in the Examples, including, for example, the ZNRF3 binding assay as disclosed in the Examples below. As used herein, increased ZNRF3 or LGR4 binding means that the binding affinity is significantly higher, preferably at least 10% higher, preferably at least 20%, 30%, 40%, 50% higher than the corresponding binding affinity measured with the reference Rspo1 of SEQ ID NO: 41.

[0048] Amino acid residues relevant for binding affinity to LGR4 and / or ZNRF3, and possible amino acid mutations, are described, inter alia, in Wang et al. GENES & DEVELOPMENT 27:1339-1344, 2013; Xie et Al. EMBO reports VOL 14 | NO 12 | 2013; Xu et Al. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 4, pp. 2455-2465, January 23, 2015.

[0049] In a specific embodiment, the variant of the disclosure comprises one or more amino acid substitutions at positions H108, N109, E116, L118, P127, A128, S133, A136, G138, or S143 of Rspo1, or at the corresponding residues of Rspo2, Rspo3, or Rspo4, to enhance binding affinity to LGR4. In a more specific embodiment, the variant of the disclosure comprises one or more of the following amino acid substitutions at FU2 of Rspo1, or at the corresponding residues of Rspo1, Rspo3, or Rspo4 (preferably Rspo1): H108K, H108R, N109D, N109E, E116V; L118F; P127D; A128E; S133F; A136L; G138E; S143V. More specifically, the variants of the present disclosure comprise or consist essentially of SEQ ID NO:66 (corresponding to variant #049 as described in the Examples), SEQ ID NO:70 (corresponding to variant #054 as described in the Examples), SEQ ID NO:72 (corresponding to variant #056 as described in the Examples), SEQ ID NO:67 (corresponding to variant #050 as described in the Examples), SEQ ID NO:28 (corresponding to variant #051 as described in the Examples), SEQ ID NO:74 (corresponding to variant #068 as described in the Examples), or SEQ ID NO:75 (corresponding to variant #069 as described in the Examples).

[0050] In a specific embodiment, the variant of the present disclosure comprises one or more amino acid substitutions at positions E45, L46, E49, V50, N51, K55, S57, I62, L63, D68, P77, F84, D85, N88, or I95 of Rspo1, or at the corresponding residues of Rspo2, Rspo3, or Rspo4, to increase binding affinity to ZNRF3. In a specific embodiment, the variant of the present disclosure comprises one or more of the following amino acid substitutions L46S, E49K, V50D, K55R, S57Q, I62F, L63F, D68G, P77H, F84Y, D85Y, N88A, or I95A at FU1 of Rspo1, or at the corresponding residues of Rspo1, Rspo3, or Rspo4 (preferably Rspo1). In more specific embodiments, the variants of the present disclosure comprise or consist essentially of SEQ ID NO:65 (corresponding to variant #048 as described in the Examples), SEQ ID NO:67 (corresponding to variant #50 as described in the Examples), SEQ ID NO:68 (corresponding to variant #052 as described in the Examples), SEQ ID NO:69 (corresponding to variant #053 as described in the Examples), SEQ ID NO:71 (corresponding to variant #055 as described in the Examples), SEQ ID NO:73 (corresponding to variant #057 as described in the Examples).

[0051] In certain embodiments, variants of the present disclosure include: (i) one or more amino acid substitutions at positions E45, L46, E49, V50, N51, K55, S57, I62, L63, D68, P77, F84, D85, N88 or I95 of Rspo1, or at the corresponding residues of Rspo2, Rspo3 or Rspo4, to enhance binding affinity to ZNRF3; for example, one or more of the following amino acid substitutions at FU1 of Rspo1, or at the corresponding residues of Rspo1, Rspo3 or Rspo4, preferably at the corresponding residues of Rspo1: L46S, E49K, V50D, K55R, S57Q, I62F, L63F, D68G, P77H, F84Y, D85Y, N88A or I95A; (ii) one or more amino acid substitutions at positions H108, N109, E116, L118, P127, A128, S133, A136, G138, or S143 of Rspo1, or the corresponding residues of Rspo2, Rspo3 or Rspo4, to enhance binding affinity to LGR4; for example, one or more of the following amino acid substitutions at FU2 of Rspo1, or the corresponding residues of Rspo1, Rspo3 or Rspo4, preferably the corresponding residues of Rspo1: H108K, H108R, N109D, N109E, E116V; L118F; P127D; A128E; S133F; A136L; G138E; S143V.

[0052] Examples of such variants include variant #050 (SEQ ID NO: 67), variant #057 (SEQ ID NO: 73), and variants #108 to #112.

[0053] Amino acid deletions or substitutions at DN-glycosylation sites Also, advantageously, the variants of the present disclosure may include amino acid deletions or substitutions to remove N-glycosylation sites in the FU2 domain, such that the resulting variants are not N-glycosylated, even when produced in eukaryotic expression systems, e.g., in mammalian cell lines, such as CHO cell lines or human cell lines.

[0054] Preferably, the variants of the disclosure comprise an amino acid substitution at residue N137 of Rspo1, or the equivalent residue in Rspo2, Rspo3, or Rspo4, that prevents N-glycosylation at this site, e.g., the variants comprise the amino acid substitution N137Q or Rspo1, or the equivalent amino acid substitution in Rspo2, Rspo3, or Rspo4. In specific embodiments, the variants of the disclosure comprise or consist essentially of SEQ ID NO:22 (corresponding to variant #005 in the Examples below), SEQ ID NO:54 (corresponding to variant #030 in the Examples below).

[0055] E. Chimeric or Hybrid R-spondin Proteins The variants of the present disclosure also include chimeric R-spondin proteins, optionally further comprising one or more modifications (deletions or amino acid substitutions) as described in B to D above.

[0056] In a specific embodiment, a chimeric variant of the present disclosure comprises a FU1 domain that is 100% identical to the FU1 domain of Rspo2 of SEQ ID NO: 9, and a FU2 domain selected from among the FU2 domain of Rspo1, the FU2 domain of Rspo3, the FU2 domain of Rspo4, or functional variants thereof with amino acid substitutions that maintain at least the same binding affinity to LGR4 (compared to Rspo1 of SEQ ID NO: 41 as a reference). Preferably, the FU2 domain is 100% identical to the FU2 domain of Rspo1 of SEQ ID NO: 6.

[0057] Thus, in a specific embodiment, said variant is a chimeric protein combining the FU1 domain of Rspo2 with other domains of Rspo1, Rspo3, Rspo4, or their functional equivalents.

[0058] In a specific embodiment, the variant is a chimeric protein comprising at least the amino acid residues 144-263 to the C-terminal region of Rspo1 (SEQ ID NO: 49).

[0059] Typically, an example of such a chimeric variant is a variant comprising the FU1 domain of Rspo2 of SEQ ID NO: 9 and the FU2 domain of Rspo1 of SEQ ID NO: 6, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably a variant of SEQ ID NO: 25 (corresponding to variant #034 disclosed in the Examples) or a variant of SEQ ID NO: 26 (corresponding to variant #035 disclosed in the Examples).

[0060] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo1 and the FU2 domain of Rspo3, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably a variant of SEQ ID NO: 50 or 51 (corresponding to variant #026 or variant #027 disclosed in the Examples).

[0061] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo3 and the FU2 domain of Rspo1, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably a variant of SEQ ID NO: 52 or 53 (corresponding to variant #028 or variant #029 disclosed in the examples), or a variant of SEQ ID NO: 54 (corresponding to variant #030, further comprising the N137Q mutation to suppress N-glycosylation).

[0062] Another example of such a chimeric variant is a variant comprising the FU1 and FU2 domains of Rspo2, as well as the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably the variant of SEQ ID NO: 55 (corresponding to variant #031 disclosed in the Examples).

[0063] Another example of such a chimeric variant is a variant comprising the FU2 domain of Rspo2 and the remaining part of the R-spondin protein from Rspo1, preferably the variant of SEQ ID NO: 56 (corresponding to variant #032 disclosed in the Examples).

[0064] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo1 and the FU2 domain of Rspo2, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably the variant of SEQ ID NO: 57 (corresponding to variant #033 disclosed in the Examples).

[0065] Another example of such a chimeric variant is a variant comprising the FU1 and FU2 domains of Rspo4, as well as the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably the variant of SEQ ID NO: 58 (corresponding to variant #036 disclosed in the Examples).

[0066] Another example of such a chimeric variant is a variant comprising the FU2 domain of Rspo4 and the remaining part of the R-spondin protein from Rspo1, preferably the variant of SEQ ID NO: 59 (corresponding to variant #037 disclosed in the Examples).

[0067] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo1 and the FU2 domain of Rspo4, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably the variant of SEQ ID NO: 60 (corresponding to variant #038 disclosed in the Examples).

[0068] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo4 and the FU2 domain of Rspo1, and optionally the TSP domain of Rspo1 of SEQ ID NO:7 and the BR domain of Rspo1 of SEQ ID NO:8, preferably a variant of SEQ ID NO:61 or 62 (corresponding to variant #039 or variant #040 disclosed in the Examples).

[0069] Another example of such a chimeric variant is a variant comprising the FU1 domain of Rspo2 and the FU2 domain of Rspo4, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8, preferably the variant of SEQ ID NO: 63 (corresponding to variant #042 disclosed in the Examples).

[0070] In addition to the chimeric proteins described above, it is also possible to optimize the ZNRF3-binding activity of the chimeric proteins by further replacing one or more amino acid residues in the FU1 domain of Rspo2 with one or more corresponding amino acid residues of Rspo1 at positions known to be important for Rspo1 / ZNRF3 binding affinity or to enhance binding affinity to ZNRF3.

[0071] Typically, the variants of the present disclosure comprise a hybrid FU1 domain having the same sequence as SEQ ID NO:5, except that it comprises one or more amino acid substitutions at positions E49, V50, D68, D85 to enhance binding affinity to ZNRF3 compared to the chimeric variants of SEQ ID NO:25 or SEQ ID NO:26: Preferably, the variants of the present disclosure comprise a hybrid FU1 domain having the same sequence as SEQ ID NO:5, except that it comprises one or more amino acid substitutions at positions E49, V50, D68, D85 to enhance binding affinity to ZNRF3 compared to the chimeric variants of SEQ ID NO:25 or SEQ ID NO:26: Preferably, the variants of the present disclosure are selected from among the FU2 domains of these functional variants comprising one or more of the following amino acid substitutions: E49K, V50D, D68G, D85G (corresponding to residues in the FU1 domain of Rspo1), and further comprising amino acid substitutions that maintain at least the same binding affinity to Rspo1, Rspo2, Rspo3, Rspo4, or LGR4, and preferably, said FU2 domain is 100% identical to the FU2 domain of Rspo1 of SEQ ID NO:6.

[0072] In specific embodiments, a variant of the present disclosure comprises a hybrid FU1 domain having a sequence identical to SEQ ID NO:5 (i.e., the FU1 domain of Rspo1), except for one or more amino acid substitutions at positions E49, V50, D68, and D85, preferably the following amino acid substitutions: 49K, 50D, 68G, 85G (corresponding to residues in the FU1 domain of Rspo2), and further, the FU2 domain is 100% identical to the FU2 domain of Rspo1 of SEQ ID NO: 6. For example, a variant of the present disclosure comprises or consists essentially of SEQ ID NO:27 (corresponding to variant #047 disclosed in the Examples).

[0073] In a specific embodiment, the variants of the present disclosure described above further comprise a hybrid Rspo1 FU1 and Rspo1 FU2 domains having one or more amino acid substitutions selected from among E45L, E49K, V50D, K55R, D68G, D85G, N88A, H108K, and N109D. For example, the variants of the present disclosure comprise or consist essentially of SEQ ID NO:28 (corresponding to variant #051 disclosed in the Examples, having E45L; E49K; V50D; K55R; D68G; D85G; N88A; H108K; and N109D mutations).

[0074] In specific embodiments, the variants of the present disclosure comprise the corresponding FU2 domain of human Rspo2, Rspo3 or Rspo4 of SEQ ID NO: 10, 14 or 18, combined with the FU1 domain of Rspo1 or a hybrid Rspo1 FU1 domain having one or more amino acid substitutions at positions E49, V50, D68 and D85 to increase binding to ZNRF3, such as, for example, E49K, V50D, D68G, D85G.

[0075] Amino acid substitutions in the BR domain to improve FO-glycosylation Variants of the present disclosure may further comprise one or more amino acid substitutions in the BR domain to modulate, optimize or improve O-glycosylation compared to Rspo1 of SEQ ID NO:41.

[0076] Amino acid residues relevant for improved O-glycosylation include, inter alia, amino acid residues T253, T258, and S259 of Rspo1.

[0077] In specific embodiments, the variants of the present disclosure comprise one or more amino acid substitutions at positions G252, T253, L257, T258, S259, A260, A263 in Rspo1, or the corresponding residues in Rspo2, Rspo3, or Rspo4 to improve O-glycosylation. More specifically, the variants of the present disclosure comprise the BR domain of Rspo1 with one or more of the following amino acid substitutions in Rspo1: G252T, T253E, L257S, T258E, S259E, A260T, or A263T, S268E, or the corresponding residues in Rspo2, Rspo3, or Rspo4.

[0078] In contrast, when the variant is fused to a C-terminal polypeptide, it may be advantageous to mutate the O-glycosylation site.Thus, in a specific embodiment, the variant of the present disclosure is a fusion protein and includes one or more of the following amino acid substitutions: T253A, T258A, S259A, and S268A.

[0079] In specific embodiments, the variants of the present disclosure comprise or consist essentially of the polypeptides of SEQ ID NOs:76-86.

[0080] G. Truncated R-spondin activity protein The inventors have surprisingly found that deletion of residues 21-31 and residues 245-263 of the Rspo1 protein results in similar activity in the Min6 proliferation assay compared to the native Rspo1 protein of SEQ ID NO: 41. This protein is the shortest native Rspo1 protein with activity. More specifically, the variants of the present disclosure comprise or consist of the protein of SEQ ID NO: 94, which corresponds to the human Rspo1 fragment comprising amino acid residues 32-224 of Rspo1 (see variant #116 disclosed in the Examples).

[0081] H. Rspo3 for the Treatment of Diabetes Moreover, the inventors have determined that in vivo, the native protein Rspo3 activates pancreatic β-cell proliferation to at least similar levels as Rspo1. Thus, in one embodiment, the present disclosure relates to an Rspo3 polypeptide (e.g., comprising SEQ ID NO:3), or a functional equivalent thereof, for use in treating diabetes (e.g., type I or type II diabetes) and / or for inducing proliferation of pancreatic β-cells either in vitro or in vivo.

[0082] Preferred variants of the present disclosure Table 1 shows preferred variants of the present disclosure, their amino acid sequences and the main changes compared to the native Rspondin protein.

[0083] [Table 1] TIFF2024526356000002.tif232169TIFF2024526356000003.tif231169TIFF20245263560 00004.tif232169TIFF2024526356000005.tif232169TIFF2024526356000006.tif194169

[0084] Conservative Modifications and Functional Equivalents Additional functional equivalents of variants as described in sections A-F hereinbefore, having similar advantageous properties of native R-spondin1 protein, or functional equivalents of Rspo3 polypeptides as described in section G hereinbefore, having similar advantageous properties of native human R-spondin3 protein, can be further identified by screening candidate molecules and testing whether such candidate molecules maintain the desired functional properties when compared to Rspo1, Rspo3, or any of the specific variants as disclosed herein (especially those preferred variants mentioned in Table 1 above).

[0085] In certain embodiments, the functional equivalents of the present variants bind to the LGR4 receptor with at least the same affinity as one of the preferred variants disclosed herein.

[0086] In certain embodiments, said functional equivalents of a particular variant as disclosed herein exhibit one or more of the following activities at least 90%, 100% or more relative to the Rspo1 protein of SEQ ID NO: 41: (i) binding affinity to the LGR4 receptor, for example as determined by an SPR assay; (ii) induction of functional β-cell proliferation, e.g., as determined by an in vitro Min6 β-cell proliferation assay; (iii) Induction of the proliferation of functional β-cells, for example as determined by an in vivo β-cell proliferation assay.

[0087] In certain embodiments, the functional equivalents of the Rspo3 polypeptides disclosed herein exhibit one or more of the following activities at least 90%, 100% or more relative to the Rspo3 protein of SEQ ID NO:3: (i) binding affinity to the LGR4 receptor, for example, as determined by SPR or ELISA assays; (ii) binding affinity to the ZNRF3 receptor, for example, as determined by SPR or ELISA assays; (iii) induction of functional β-cell proliferation, e.g., as determined by an in vitro Min6 β-cell proliferation assay; (iv) Induction of the proliferation of functional β-cells, for example as measured in an in vivo β-cell proliferation assay.

[0088] Further details of the assays and conditions for use in determining activity are disclosed in the experimental part below.

[0089] In certain embodiments, said functional equivalent of a particular variant exhibits at least 90%, more preferably 100% or more of the desired activity described above relative to one of the corresponding preferred variants as disclosed in Table 1.

[0090] Functional equivalents of variants as disclosed in the previous section can typically be obtained by amino acid substitutions, deletions or insertions compared to the corresponding variants at non-essential residues. In certain embodiments, said functional equivalents differ from the corresponding variants only by amino acid substitutions with natural or non-natural amino acids, preferably only by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with natural amino acids, in particular compared to one of the variants listed in Table 1.

[0091] In another embodiment, the functional equivalent is a polypeptide having 95% identity to at least one of SEQ ID NOs: 22-28, which polypeptide comprises a FU1 domain and a FU2 domain that are 100% identical to the FU1 domain and the FU2 domain of at least one of SEQ ID NOs: 22-28.

[0092] In more specific embodiments, the amino acid sequence of said functionally equivalent may differ from a variant as disclosed herein, in particular in Table 1, by mostly conservative amino acid substitutions; for example, at least 10 (at least 9, 8, 7, 6, 5, 4, 3, 2 or 1, etc.) of the substitutions in the variant are conservative amino acid residue substitutions.

[0093] In the context of the present disclosure, conservative substitutions may be defined by substitutions within classes of amino acids reflected as follows: Aliphatic residues I, L, V, and M Cycloalkenyl-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T and V Very small residues A, G, S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and R More conservative substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation of hydrophilicity / hydrophilic properties and residue weight / size may also be substantially retained in the variant mutant polypeptide compared to the parent variant of Table 1.

[0094] In specific embodiments, functional equivalents of the variants include polypeptides that are identical to any one of SEQ ID NOs: 22-28 (variants of Table 1), except for one, two or three amino acid residues that are replaced by another naturally occurring amino acid, preferably by a conservative amino acid substitution as defined above.

[0095] Additionally, one of skill in the art will appreciate that conserved residues among various species or different members of the Rspo family may be important for maintaining proper structure, and thus one of skill in the art may refrain from mutating such amino acid positions. For example, conserved residues among Rspo1, Rspo2, Rspo3 and Rspo4 are specifically shown in Figure 1 and preferably may not be mutated unless otherwise specified in the present disclosure.

[0096] Alternatively, at many sites, one or more amino acid positions show conservative variations between species variants and / or among other members of the Rspo family (Rspo2, Rpo3 and Rspo4, etc.). One skilled in the art will understand that some of such conservative substitutions do not adversely affect the function of Rspo1 and are therefore mutated compared to native R-spondin1 with such conservative variations.

[0097] In certain embodiments, the variant of the present disclosure comprises a FU2 domain having the following amino acid residues that are not mutated compared to the native R-spondin protein: F106, H108, F110, N109, E116, L118, P127, A128, S133, A136, G138, S143 in human Rspo1 of SEQ ID NO:1.

[0098] In certain embodiments, variants of the disclosure comprise a BR domain that has no amino acid changes compared to the native BR domain at one or more of the following positions: T253, L257, T258, S259, A260 or A263 in human Rspo1 of SEQ ID NO:1, or the corresponding residues in human Rspo2 of SEQ ID NO:2, human Rspo3 of SEQ ID NO:3 or human Rspo4 of SEQ ID NO:4; typically, the BR domain is 100% identical to SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:20.

[0099] Fusion Proteins of the Present Disclosure Various polypeptides other than the R-spondin1 polypeptide can be fused to the variants of the present disclosure or their functional equivalents described above (particularly the variants in Table 1) for various purposes (e.g., to increase the in vivo half-life of the protein, to facilitate the identification, isolation and / or purification of the protein, to enhance the activity of the protein, and to promote the oligomerization of the protein, etc.).

[0100] Many polypeptides can facilitate the identification and / or purification of the recombinant fusion protein of which they are a part. For example, polyarginine, polyhistidine are included. Polypeptides that contain polyarginine allow for effective purification by ion exchange chromatography. For example, in certain embodiments, the variant of the present disclosure (more specifically, any of the preferred variants as disclosed in Table 1) comprises a polyhistidine C-terminal tag, and optionally a peptide linker, such as the polypeptide of SEQ ID NO: 90 (GGGGSEPEAHHHHHH).

[0101] In a specific embodiment, a polypeptide comprising the Fc region of an antibody, optionally an IgG antibody, or a substantially similar protein, can be fused directly, or optionally via a peptide linker, to a variant of the present disclosure (typically one of the variants disclosed in Table 1) or a functional equivalent thereof, thereby forming an Fc fusion protein of the present disclosure. An example of such an Fc region is an IgG4 Fc fragment or a derivative thereof, typically a polypeptide of SEQ ID NO:29.

[0102] In a specific embodiment, the present disclosure also relates to an Fc fusion protein in which an Fc fragment is fused (at either the C-terminus or N-terminus, optionally via a peptide linker or a variant thereof) to the Rspo1 protein of SEQ ID NO: 66 (more specifically, the Fc fragment comprises SEQ ID NO: 29).

[0103] In another specific embodiment, the present disclosure also relates to an Fc fusion protein in which an Fc fragment is fused (at either the C-terminus or N-terminus, optionally via a peptide linker or a variant thereof) to the Rspo1 protein of SEQ ID NO: 24 (more specifically, the Fc fragment comprises SEQ ID NO: 29).

[0104] In another specific embodiment, the present disclosure also relates to an Fc fusion protein in which an Fc fragment is fused (at either the C-terminus or N-terminus, optionally via a peptide linker or a variant thereof) to the Rspo1 protein of SEQ ID NO: 94 (more specifically, the Fc fragment comprises SEQ ID NO: 29).

[0105] The fusion protein may also include one or more peptide linkers. Generally, a peptide linker is a stretch of amino acids that serves to link multiple polypeptides to form multimers, providing the flexibility or rigidity required for the desired function of the linked portions of the proteins. Typically, peptide linkers are about 1-30 amino acids in length. Examples of peptide linkers include, but are not limited to, -Gly-Gly-, GGGGS (SEQ ID NO: 46), (GGGGS)n (n is 1-8, typically 3 or 4). Linking moieties are described, for example, in Huston, JS, et al., Proc. Natl. Acad. Sci. 85: 5879-83 (1988), Whitlow, M., et al., Protein Engineering 6: 989-95 (1993), Newton, DL, et al., Biochemistry 35: 545-53 (1996), and U.S. Patent Nos. 4,751,180 and 4,935,233.

[0106] In certain embodiments, the Fc fusion is indirectly fused via a peptide linker at the C-terminus of the variant of the present disclosure or its functional equivalent. In other embodiments, the Fc fusion is indirectly fused via a peptide linker at the N-terminus of the variant of the present disclosure or its functional equivalent.

[0107] In certain embodiments, the inventors have shown that linker optimization is necessary to generate active Fc-fusion proteins, and indeed, the inventors have shown that extending the linker length improves the biological activity of Fc-fusion proteins.

[0108] Preferred peptide linkers that may be used between the Fc portion and the R-spondin portion of the Fc fusion protein include, for example, the linker (GGGGGGSGGGGSGGGGSA) (SEQ ID NO: 44) or (GGGGSGGGGSGGGGGG) (SEQ ID NO: 45).

[0109] In a specific embodiment, the Fc polypeptide of SEQ ID NO:29 is fused directly or indirectly via a peptide linker at the C-terminus of one of the preferred variants of Table 1.

[0110] The inventors have surprisingly shown that fusing an Fc polypeptide directly or indirectly via a peptide linker at the N-terminus of a variant of the present disclosure increases the production yield of the Fc fusion protein. Thus, in a preferred embodiment, an Fc polypeptide of SEQ ID NO: 29 is fused directly or indirectly via a peptide linker at the N-terminus of one of the preferred variants of Table 1, preferably the variant of SEQ ID NO: 24, 66, or 94.

[0111] The present disclosure also relates to Fc fusion proteins in which an Fc fragment is fused (either at the C-terminus or N-terminus, optionally via a peptide linker) to the Rspo1 protein of SEQ ID NO: 1 (more specifically, the Fc fragment comprises SEQ ID NO: 29). In specific embodiments, the variants of the present disclosure are Fc fusion proteins of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 92, SEQ ID NO: 93 or SEQ ID NO: 95 (corresponding to variant #063, variant #064, variant #121, variant #155 or variant #150, respectively).

[0112] Another modification of variants or their functional equivalents contemplated by the present disclosure is the conjugation or protein fusion of at least variants or equivalents to serum proteins (such as human serum albumin or fragments thereof) to increase the half-life of the resulting molecule. Such an approach is described, for example, in Ballance et al. (EP 0322094).

[0113] Another possibility is the fusion protein of the present disclosure, which comprises a protein capable of binding to serum proteins (proteins that bind to human serum albumin (i.e., anti-HSA fusion proteins)) to increase the half-life of the resulting molecule, for example, comprising an anti-HSA binding moiety derived from a Fab or nanobody that binds to HSA or any other domain type structure, such as DARPIN, nanophytin, finomer, etc. Such an approach is described, for example, in Journal of Controlled Release 301 (2019) 176-189; BioDrugs (2015) 29:215-239; J Pharmacol Exp Ther 370:703-714, September 2019; Biodrugs 2009; 23 (2): 93-109. Examples of such fusion proteins include the polypeptides of SEQ ID NO:64, SEQ ID NO:87 or SEQ ID NO:88.

[0114] In other embodiments, the fusion protein of the present disclosure comprises one of the albumin-binding peptides disclosed in Dennis et al 2002 (Journal of Biological Chemistry, 2002, Vol 277, No. 38, pp 35035-35043), in particular a peptide comprising the core sequence of SEQ ID NO:91 (DICLPRWGCLW).

[0115] Examples of such variants are the polypeptide of SEQ ID NO: 64 (variant #046), or the polypeptide of SEQ ID NO: 87 (variant #094), or the polypeptide of SEQ ID NO: 88 (variant #095).

[0116] The recombinant fusion protein of the present disclosure may include a polypeptide that includes a leucine zipper or other multimerization motif. Among the known leucine zipper sequences, there are sequences that promote dimerization and sequences that promote trimerization. See, for example, Landschulz et al. (1988), Science 240: 1759-64). Leucine zippers contain repeated heptad repeats, often with four or five leucine residues interspersed with other amino acids. The use and preparation of leucine zippers are well known in the art.

[0117] Another modification of the variants or functional equivalents disclosed herein that is contemplated by this disclosure is related techniques such as pegylation or hexylation or PASylation.

[0118] More generally, the variants or functional equivalents thereof may be conjugated to biodegradable bulking agents including natural and semi-synthetic polysaccharides, including O-linked and N-linked oligosaccharides, dextran, hydroxyethyl starch (HES), polysialic acid and hyaluronic acid, as well as unstructured protein polymers such as homoamino acid polymers, elastin-like polypeptides, XTEN and PAS.

[0119] For example, variants of the present disclosure or their functional equivalents can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate, the recombinant protein is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups are attached to the recombinant protein. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analog reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. Methods for pegylation of proteins are known in the art and can be applied to the proteins of the present disclosure. See, for example, Jevsevar et al 2010 Biotechnol J. 5(1): 113-28, or Turecek et al 2016 J Pharm Sci 2016 105(2): 460-375. Thus, in certain embodiments, the Rspo1 proteins of the present disclosure are pegylated.

[0120] Another modification of variants or their functional equivalents contemplated by the present disclosure is PASylation.See, for example, Protein Engineering, Design & Selection vol. 26 no. 8 pp. 489-501, 2013.Thus, in certain embodiments, the Rspo1 protein of the present disclosure is PASylation.

[0121] Xten technology is described and reviewed, for example, in Nature Biotechnology volume 27 number 12 2009: 1186-1192.

[0122] Nucleic acid molecules encoding proteins of the present disclosure Nucleic acid molecules encoding the disclosed variants or their functional equivalents are also disclosed herein.

[0123] The Examples in Tables 10 and 11 below provide specific examples of nucleotide sequences that encode particular amino acid sequences of the variants disclosed herein.

[0124] Exemplary nucleotide sequences are those that encode the amino acid sequence of any one of the examples set forth in Table 1, in particular any one of SEQ ID NOs:22-28, 42-43, 50-89, and 92-95, where the nucleic acid sequence is readily derived from Table 9, using the genetic code and optionally taking into account codon bias depending on the host cell type.

[0125] The present disclosure also relates to nucleic acid molecules derived from the latter sequence that have been optimized for protein expression in mammalian cells (eg, mammalian Chinese Hamster Ovary (CHO) cell lines, or human HEK293 cell lines).

[0126] The nucleic acid may be present in a whole cell, a cell lysate, or may be in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acid of the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In one embodiment, the nucleic acid may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[0127] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. Once DNA fragments encoding variants of the present disclosure are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques. In these manipulations, the DNA fragments encoding the variants may be operably linked to another DNA molecule, or to a fragment encoding another protein (e.g., an antibody constant region (Fc region) or a flexible linker). Exemplary nucleotide sequences include recombinant fusion proteins, particularly Fc fusion proteins comprising the coding sequence of the amino acid sequence SEQ ID NO: 1, or any one of their variants (e.g., SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NOs: 92-95) operably linked to the coding sequence of the Fc region.

[0128] As used in the present context, the term "operably linked" is intended to mean that two DNA fragments are linked in a functional manner, e.g., such that the amino acid sequences encoded by the two DNA fragments remain in frame or that a protein is expressed under the control of a desired promoter.

[0129] [Generation of transfectomas producing variants or fusion proteins of the present disclosure] The disclosed variants or their functional equivalents, and / or related proteins can be produced, for example, in host cell transfectomas using a combination of recombinant DNA technology and gene transfection methods that are well known in the art.

[0130] For example, to express a variant or related fusion protein of the present disclosure, or a corresponding functional equivalent thereof, DNA encoding a partial or full-length recombinant protein can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning) and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences.

[0131] In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the recombinant protein. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The gene encoding the protein is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0132] The recombinant expression vector can encode a signal peptide that facilitates secretion of the recombinant protein from the host cell. The gene encoding the variant can be cloned into the vector so that the signal peptide is linked in frame to the amino terminus of the recombinant protein. The signal peptide can be the native signal peptide of Rspo1, Rspo2, Rspo3, or Rspo4 or a heterologous signal peptide (i.e., a signal peptide from a non-Rspondin protein).

[0133] In addition to the gene encoding the variant, the recombinant expression vector disclosed herein carries a regulatory sequence that controls the expression of the recombinant protein in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the protein encoding the gene. It will be understood by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired protein, and the like. Regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements, such as cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma, that direct high levels of protein expression in mammalian cells. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or P-globin promoter, may be used. Additionally, there are also regulatory elements composed of sequences from different sources, such as the SRa promoter system, including the SV40 early promoter and sequences derived from the long terminal repeat of human T-cell leukemia virus type 1.

[0134] In addition to the gene and regulatory sequence encoding the variant, the recombinant expression vector of the present disclosure may carry additional sequences such as sequences that regulate the replication of the vector in a host cell (e.g., origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of a host cell into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically, the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0135] For expression of variants or related fusion proteins of the present disclosure, expression vectors encoding recombinant proteins are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells (e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc.). In theory, it is possible to express the proteins of the present disclosure in either prokaryotic or eukaryotic host cells. Expression of proteins in eukaryotic cells (e.g., mammalian host cells, yeast or filamentous fungi) is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and functional recombinant proteins than prokaryotic cells.

[0136] In a specific embodiment, a cloning or expression vector according to the present disclosure comprises one of the coding sequences for a variant set forth in Table 1 (typically SEQ ID NOs: 22-28 or 50-89), or a fusion protein (typically SEQ ID NOs: 42, 43, or 92-95), operably linked to a suitable promoter sequence.

[0137] Mammalian host cells for expressing the recombinant proteins of the present disclosure include Chinese Hamster Ovary (CHO cells), including dhfr-CHO cells (described in Urlaub and Chasin, 1980), CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, HEK293 cells and SP2 cells, used with a DHFR selection marker (described in Kaufman and Sharp, 1982). When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the recombinant protein is produced by culturing the host cell for a period of time sufficient to express the recombinant protein in the host cell, and, optionally, secrete the protein into the culture medium in which the host cell is grown.

[0138] The variant or related fusion proteins of the present disclosure can be recovered and purified from the culture medium following secretion, for example, using standard protein purification methods.

[0139] In a specific embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector having a coding sequence suitable for expression of one of the variants comprising any one of SEQ ID NOs: 22-28, 42-43, 50-89 and 92-95, each operably linked to a suitable promoter sequence.

[0140] The latter host cells may then be further cultured under conditions suitable for the expression and production of the recombinant variants or related fusion proteins of the present disclosure.

[0141] [Pharmaceutical composition] In another aspect, the disclosure provides compositions (e.g., pharmaceutical compositions) comprising the variants or related fusion proteins disclosed herein, or functional equivalents. Such compositions may contain one or a combination of (e.g., two or more different) recombinant variants or related fusion proteins, as described above.

[0142] For example, the pharmaceutical composition comprises a recombinant protein comprising any one of the suitable variants set forth in Table 1 (typically comprising any one of the polypeptides of SEQ ID NOs: 22-28 and SEQ ID NOs: 50-89), or a fusion protein (e.g., SEQ ID NOs: 42-43 or SEQ ID NOs: 92-95), or functional equivalents thereof, formulated together with a pharma- ceutically acceptable carrier.

[0143] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy may include a variant of the present disclosure, e.g., a recombinant protein comprising any one of the polypeptides of SEQ ID NOs: 22-28, 42-43, 50-89, and 92-95, or functional equivalents thereof, in combination with at least one anti-inflammatory agent or another anti-diabetic agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the recombinant variants or related fusion proteins of the present disclosure.

[0144] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The carrier should be suitable for parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration (e.g., by injection or infusion).

[0145] In one embodiment, the carrier should be suitable for subcutaneous or intravenous injection. Depending on the route of administration, the active compound, i.e., the variant of the present disclosure, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0146] Sterile phosphate-buffered saline is an example of a pharma- ceutically acceptable carrier. Other suitable carriers are known to those skilled in the art (Remington and Gennaro, 1995). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface.

[0147] The form of the pharmaceutical composition, the route of administration, the dose and the regimen will of course depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0148] The pharmaceutical compositions of the present disclosure can be formulated for oral, intranasal, sublingual, subcutaneous, intramuscular, intravenous, transdermal, parenteral, topical, or rectal administration, etc. The variants of the present disclosure as active ingredients can be administered to animals and humans alone or in combination with another active ingredient, in unit dosage form, in admixture with conventional pharmaceutical carriers.

[0149] Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0150] Preferably, the pharmaceutical compositions comprise a pharma- ceutically acceptable vehicle for injectable formulations, which may in particular be an isotonic, sterile, saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of these salts), or a dry, in particular lyophilized composition, which allows the constitution of an injectable solution, upon addition of sterile water or saline, as the case may be.

[0151] The dose used for administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, the pathology involved, or alternatively the desired duration of treatment.

[0152] To prepare a pharmaceutical composition, a therapeutically effective amount of the variants of the present disclosure may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0153] Pharmaceutical forms suitable for injection use may include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0154] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0155] The variant or related fusion protein of the present disclosure can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0156] The carrier may also be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include an isotonic agent (for example, sugar or sodium chloride). Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate and gelatin.

[0157] Sterile injection solution is prepared by mixing the required amount of active compound, i.e., Rspo1 protein, in a suitable solvent with various other ingredients as listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by mixing various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from the ingredients listed above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0158] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like can also be used.

[0159] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous dissolution or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some dosage variation will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

[0160] The variant or related fusion proteins of the present disclosure, or their functional equivalents, may be formulated into a therapeutic mixture to contain from about 0.01 mg to 1000 mg / kg, or from 1 mg to 100 mg / kg. Multiple doses are also possible.

[0161] Suitable formulations for solutions for infusion or subcutaneous injection of recombinant proteins are described in the art and are reviewed, for example, in Advances in Protein Chemistry and Structural Biology Volume 112, 2018, Pages 1-59 Therapeutic Proteins and Peptides Chapter One - Rational Design of Liquid Formulations of Proteins: Mark C. Manning, Jun Liu, Tiansheng Li, Ryan E. Holcomb.

[0162] Uses and Methods of the Disclosed Variant or Related Proteins The variant or related proteins of the present disclosure, or their functional equivalents, have in vitro and in vivo utilities. For example, these recombinant proteins can be administered to cells in culture, e.g., in vitro, ex vivo or in vivo, or administered to a subject (e.g., in vivo) to treat or prevent various disorders.

[0163] As used herein, the terms "treat," "treating," or "treatment" refer to one or more of: (1) inhibiting the disease; e.g., inhibiting the disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder (i.e., halting further progression of the pathology and / or symptomology); and (2) ameliorating the disease; e.g., reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease, thereby improving the disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomology).

[0164] In particular, with respect to the treatment of diabetes, and more particularly with respect to type 1 diabetes, the term "treatment" may refer to inhibiting the loss of pancreatic beta cells and / or increasing the amount of pancreatic beta cells (especially functional insulin-secreting beta cells in such subjects) and / or improving glycemic control, particularly in patients who have lost pancreatic beta cells and / or islets of Langerhans due to disease (e.g., type 1 diabetes).

[0165] The disclosed variants or related fusion proteins, or functional equivalents thereof, can induce proliferation of pancreatic beta cells in vivo and reconstitute functional insulin-secreting islets of Langerhans, and may thereby be used to treat diabetic patients or patients in need of functional insulin-secreting beta cells or patients with disorders related to hyperglycemia or patients with deficient glucose-stimulated insulin secretion.

[0166] As used herein, the term "diabetes" generally refers to any condition or disorder that results in a deficiency of insulin or resistance to its effects.

[0167] Examples of diabetes include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, and latent autoimmune diabetes in adults (LADA).

[0168] Thus, the present disclosure relates to a method for treating one of the above-disclosed disorders in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a variant or related protein of the present disclosure disclosed above, or a functional equivalent (typically a recombinant protein comprising any of the polypeptides of SEQ ID NOs: 22-28, 42-43, 50-89, and 92-95, or a functional equivalent thereof).

[0169] In certain embodiments, the subject is selected from among patients with low Rspo1 gene expression.

[0170] The variants or related fusion proteins for use as disclosed above, or functional equivalents thereof, may be administered as the sole active ingredient, e.g., for the treatment or prevention of the above-mentioned diseases, or may be administered in combination with other agents, e.g., as adjuvants or in combination with cytokines, antiviral agents, anti-inflammatory agents, antidiabetic or hypoglycemic agents, cell therapy products (e.g., beta cell compositions) and immunomodulatory agents.

[0171] For example, the variants or related fusion proteins for use as disclosed above, or functional equivalents thereof, may be used in combination with cell therapy, in particular beta cell therapy.

[0172] As used herein, the term "cell therapy" refers to a therapy that includes in vivo administration of at least a therapeutically effective amount of a cell composition to a subject in need thereof. The cells administered to the patient may be allogeneic or autologous. The term "β-cell therapy" refers to a cell therapy in which the cell composition includes β-cells, particularly insulin-secreting β-cells, as an active ingredient. The β-cells may be produced by in vitro methods described below using Rspo1 protein.

[0173] In certain embodiments, the Rspo1 protein, its variants or related fusion proteins, or functional equivalents thereof, for use in treating diabetic patients, or patients in need of functional insulin-secreting beta cells, or patients with disorders related to hyperglycemia, or patients with defective glucose-stimulated insulin secretion, are administered in combination with, before, simultaneously with, or after a beta cell composition, particularly a stem cell-derived beta cell composition.

[0174] In a particular embodiment, the beta cells are isolated from a living donor, a cadaveric donor.

[0175] In another particular embodiment, the beta cells are stem cell-derived beta cells. Stem cell-derived beta cells refer to insulin-secreting beta cells obtained by differentiation of pluripotent stem cells, particularly induced pluripotent stem cells or human embryonic stem cells. In one embodiment, when the stem cells are human stem cells, the human stem cells are not human embryonic stem cells. Stem cell-derived beta cells exhibit at least one marker indicative of pancreatic beta cells (e.g., PDX-1 or NKX6.1), express insulin, and exhibit a glucose-stimulated insulin secretion response (GSIS) characteristic of endogenous mature beta cells in vitro or in vivo.

[0176] As used herein, the term "insulin-secreting β-cells" refers to endocrine precursor cells that secrete insulin or cells differentiated from their precursor cells. Insulin-secreting β-cells include pancreatic β-cells and pancreatic β-like cells that express insulin.

[0177] As used herein, a "pluripotent stem cell" is an undifferentiated cell that has both the ability to self-renew (by mitosis) and to differentiate into specialized cell types derived from the three germ cell layers (ectoderm, endoderm, mesoderm) to give rise to cells of all tissues of the body. In certain embodiments, the pluripotent stem cell is an induced pluripotent stem cell or a human embryonic stem cell.

[0178] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., by induction or complete reversal) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0179] "Human embryonic stem cells" or "hESCs" as used herein refer to human stem cells derived from the inner cell mass (ICM) of a human embryo at the blastocyst stage. A human embryo reaches the blastocyst stage 4-5 days after fertilization, at which point it is composed of 50-150 cells. Embryonic stem cells are pluripotent stem cells. According to the present invention, human embryonic stem cells may be obtained from established cell lines or may be isolated from an embryo by different techniques known to those skilled in the art. In some embodiments, human embryos were not destroyed as a source of stem cells for use in the methods and compositions as disclosed herein.

[0180] Methods for generating beta cells from stem cells are well known in the art and are illustrated by, but not limited to, protocols described in D'Amour, KA et al. (2006); Jiang, J. et al. (2007); and Kroon, E. et al. (2008), Rezania et al. (2012, 2014), Felicia W. Pagliuca et al. (2014), and PCT International Application No. PCT / US2014 / 041992, the relevant portions of which are incorporated herein by reference. These protocols for inducing differentiation of pluripotent stem cells into insulin-secreting beta cells include inducing differentiation of stem cells into progenitor cells, such as pancreatic progenitor cells or endocrine progenitor cells, which can be induced to differentiate into insulin-secreting beta cells.

[0181] A cell therapy product refers to a cell composition administered to the patient for treatment purposes. The cell therapy product includes a therapeutically effective dose of cells, and optionally additional excipients, adjuvants, or other pharma- ceutically acceptable carriers.

[0182] Suitable antidiabetic or hypoglycemic agents may include, but are not limited to, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, cholesterol lowering drugs, biguanides, metformin, thiazolidinediones, hypoglycemic sulfamides, DPP-4 inhibitors, α-glucosidase inhibitors, insulin or derivatives thereof (including short-acting, rapid-acting or long-acting insulin), GLP1 analogues, derivatives of carbamoylmethylbenzoic acid; typically insulin receptors, SLGT2 inhibitors, GABR targeting molecules, and IL2R targeting molecules.

[0183] In accordance with the above, the present disclosure provides yet another aspect: The method as defined above, comprising the step of co-administering (e.g., simultaneously or sequentially) a therapeutically effective amount of a variant or related fusion protein of the present disclosure, or a functional equivalent thereof, and at least one second drug substance, wherein the second drug substance is, for example, a cytokine, an antiviral agent, an anti-inflammatory agent, an antidiabetic agent, a cell therapy product (e.g., a beta cell composition) as described above.

[0184] In certain embodiments of the methods for treating one of the disorders disclosed above, in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of an Rspo1 protein, or a variant or related fusion protein as disclosed herein, or a functional equivalent, in combination with a therapeutically effective amount of a beta cell composition (particularly a stem cell derived beta cell composition as disclosed herein), e.g., before, simultaneously with, or after the beta cell composition.

[0185] In certain embodiments, said beta cells are isolated from a living or cadaveric donor.

[0186] In another particular embodiment, said beta cells are stem cell-derived beta cells. Stem cell-derived beta cells refer to insulin-secreting beta cells obtained by differentiation of stem cells, in particular induced pluripotent stem cells, human embryonic stem cells or mesenchymal precursor cells. In one embodiment, when the stem cells are human stem cells, the human stem cells are not human embryonic stem cells.

[0187] Methods for transplanting beta cells or islets of Langerhans into patients are disclosed, for example, in Shapiro, et al (2000) The New England Journal of Medicine. 343 (4): 230-238, and Shapiro et al (2017) Nature Reviews Vol 13: 268-277.

[0188] In some embodiments, the β-cells described herein are administered to the patient as dispersed cells or in clusters. The β-cells can be transplanted into a suitable site in the subject, including, but not limited to, the liver, native pancreas, renal subcapsular space, fetal membrane, peritoneum, subserous space, intestine, stomach, or subcutaneous pocket.

[0189] In a specific embodiment, the beta cell composition is autologous to the subject in need of treatment, preferably derived from iPS obtained from somatic cells of the subject in need of treatment.

[0190] In a specific embodiment, said subject in need of such treatment is a subject suffering from diabetes, preferably type 1 diabetes.

[0191] In another embodiment, the disclosed variant or related proteins, or functional equivalents thereof, can be used in in vitro methods to induce proliferation of pancreatic beta cells and / or islets of Langerhans.

[0192] Thus, in one aspect, the present disclosure further provides a method for producing beta cells in vitro, comprising the steps of: (i) providing β cells; (ii) culturing said beta cells in the presence of a therapeutically effective amount of said variant or related protein, or a functional equivalent thereof, under conditions that induce proliferation of said beta cells.

[0193] In a specific embodiment of said production method, said beta cells are primary cells, preferably primary cells derived from a subject in need of beta cell therapy or transplantation of islets of Langerhans.

[0194] In other specific embodiments, said beta cells provided in step (i) are obtained from iPS cells after differentiating said iPS cells into beta cells.

[0195] Thus, in certain embodiments, the present disclosure relates to a method for in vitro production of beta cells from induced pluripotent stem cells, comprising the steps of: (i) providing induced pluripotent stem cells (iPSCs); (ii) differentiating the iPSCs in vitro into β cells of the islets of Langerhans; and (iii) culturing the differentiated β-cells under proliferation conditions; A sufficient amount of the variant or related fusion protein, or functional equivalent, is added in step (ii) and / or step (iii) to differentiate the iPS cells and / or induce proliferation of the beta cells.

[0196] Methods for differentiating iPSCs into islet β cells have been described in the art, e.g., Pagliuca, et al. Cell 159, 428-439 (2014) and Rezania et al. Nat Biotechnol. 2014 Nov;32(11):1121-33), the relevant portions of which are incorporated herein by reference.

[0197] The disclosure further includes compositions comprising the β-cells obtainable or obtained by the above method, and their use as cell therapy products, for example, in subjects for treating diabetes, preferably type 1 diabetes. Methods for transplanting β-cells or islets of Langerhans into patients are disclosed, for example, in Shapiro, et al (2000) The New England Journal of Medicine. 343 (4): 230-238, and Shapiro et al (2017) Nature Reviews Vol 13: 268-277.

[0198] A kit consisting of the compositions disclosed herein (including, for example, variants of the present disclosure) and instructions for use is also within the scope of the present disclosure. The kit can further include at least one additional reagent, or one or more additional antibodies or proteins. The kit typically includes a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded material that is provided on or with the kit, or that otherwise accompanies the kit. The kit may further include a tool for diagnosing whether a patient belongs to a group that responds to Rspo treatment, as defined above.

[0199] Another treatment strategy is based on the use of variants or related fusion proteins, or functional equivalents thereof, as agents to proliferate β-cells isolated from samples of human subjects.

[0200] The present disclosure relates to a method for treating a subject in need thereof, comprising the steps of: (a) isolating cells from a subject; (b) optionally expanding and / or reprogramming the cells into induced pluripotent stem cells; (c) differentiating the iPS cells into β cells; (d) expanding the beta cells in vitro, optionally with other cells, in the presence of a variant or related fusion protein of the present disclosure, or a functional equivalent thereof (e.g., a recombinant protein comprising any of SEQ ID NOs: 22-28, 42-43, 50-89, and 92-95, or a functional equivalent thereof); (e) optionally, harvesting the expanded beta cells and / or formulating the expanded beta cells and administering a therapeutically effective amount of said expanded beta cells to the subject.

[0201] The present disclosure further relates to the use of the above variants or related fusion proteins of the present disclosure, or functional equivalents thereof (e.g., a recombinant protein comprising any one of SEQ ID NOs:22-28, 42-43, 50-89, and 92-95, or functional equivalents thereof) as an agent for expanding beta cells in vitro.

[0202] The present disclosure also relates to variants or related fusion proteins of the present disclosure, or functional equivalents thereof (such as recombinant proteins comprising any one of SEQ ID NOs: 22-28, 42-43, 50-89, and 92-95, or functional equivalents thereof) for use in vivo as an agent for inducing proliferation of beta cells in humans, particularly in subjects having a loss of functional beta cells, typically subjects suffering from diabetes.

[0203] Thus, the present disclosure relates to a method of treating a subject suffering from diabetes, e.g., type 1 diabetes or another disorder involving loss of functional beta cells, comprising the steps of: (i) administering to the subject an effective amount of a variant or related fusion protein of the present disclosure, or a functional equivalent (typically a recombinant protein comprising any one of SEQ ID NOs: 22-28, 42-43, 92-95, and 50-89, or a functional equivalent thereof); and (ii) administering in the subject an effective amount of a beta cell composition; The effective amount of the variant or related fusion protein is capable of increasing proliferation of the beta cell composition. Steps (i) and (ii) can be performed simultaneously or sequentially, in particular, either step (i) or step (ii) is administered to the subject first.

[0204] The invention now being fully described, is further illustrated by the following examples, which are illustrative only and are not intended to be further limiting.

[0205] [Embodiment] E1. A recombinant variant of R-spondin protein, comprising the following domains: FU1, FU2, TSP and BR: a. FU1 is a domain having at least 80% identity to any of the FU1 domains of human Rspo1, Rspo2, Rspo3 or Rspo4 as set forth in SEQ ID NOs: 5, 9, 13 and 17, respectively; b. FU2 is a domain having at least 80% identity to any of the FU2 domains of human Rspo1, Rspo2, Rspo3 or Rspo4 as set forth in SEQ ID NOs: 6, 10, 14, 18, respectively; c. the TSP is a domain having at least 80% identity to any of the TSP domains of human Rspo1, Rspo2, Rspo3, or Rspo4 set forth in SEQ ID NOs: 7, 11, 15, 19, and d. BR is a domain having at least 80% identity to any of the BR domains of human Rspo1, Rspo2, Rspo3, or Rspo4 as set forth in SEQ ID NOs: 8, 12, 16, 20; Recombinant variants.

[0206] E2. A recombinant variant as described in E1, wherein each of the domains FU1, FU2, TSP and BR has at least 80% identity with the FU1, FU2, TSP and BR domains, respectively, of the human Rspo1 domain of SEQ ID NO:1.

[0207] E3. A recombinant variant according to E1 or E2, wherein the TSP domain and BR domain have 100% identity to the corresponding TSP domain and BR domain of human Rspo1, respectively, and the amino acid sequences of the FU1 domain and FU2 domain are at least 80% identical to the corresponding amino acid sequences of FU1 and FU2 of human Rspo1, with the differences being due to amino acid substitutions.

[0208] E4. A recombinant variant according to any one of E1 to E3, which has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in each of the FU1 domain or the FU2 domain of human Rspo1 of SEQ ID NO: 5 and the FU2 domain of Rspo1 of SEQ ID NO: 6, when aligned in a corresponding manner.

[0209] E5. A recombinant variant of any one of claims E1-E4, comprising a deletion of the first 10-14 N-terminal amino acids within the region 21-33 of Rspo1, or the equivalent region in Rspo2, Rspo3, or Rspo4, typically a deletion of residues 21-31 of Rspo1.

[0210] E6. A recombinant variant according to E5, comprising or consisting of the protein of SEQ ID NO:24.

[0211] E7. A recombinant variant according to any one of E1 to E6, comprising at least an amino acid substitution of R66 in the FU1 domain of human Rspo1 of SEQ ID NO:5, or an amino acid substitution of an equivalent arginine residue in the FU1 domain sequence of human Rspo2, Rspo3, or Rspo4, wherein said amino acid substitution reduces or eliminates ZNRF3 binding.

[0212] E8. A recombinant variant of claim E7, comprising the FU1 domain of Rspo1 of SEQ ID NO:5, with a single amino acid substitution at residue R66, typically R66A, that reduces or eliminates ZNRF3 binding.

[0213] E9. A recombinant variant according to E7 or E8, wherein the FU2, TSP and BR domains are 95%, preferably 100%, identical to Rspo1, e.g., the recombinant variant comprises or essentially consists of SEQ ID NO:23.

[0214] E10. A recombinant variant of any one of claims E1 to E9, wherein the variant of R-spondin binds to LGR4 with higher affinity than human Rspo1 of SEQ ID NO: 41, as measured in an Rspo1 / LGR4 binding affinity in vitro assay.

[0215] E11. The recombinant variant according to E10, comprising one or more amino acid substitutions at positions H108, N109, E116, L118, P127, A128, S133, A136, G138, or S143 of Rspo1, or the corresponding residues of Rspo2, Rspo3 or Rspo4, more specifically one or more of the following amino acid substitutions: H108K, N109D, E116V, L118F, P127D, A128F, S133F, A136L, G138E, or S143V, to enhance binding affinity to LGR4 compared to the chimeric Rspo1 of SEQ ID NO: 41.

[0216] E12. A recombinant variant as described in E10, comprising one or more amino acid substitutions at positions E45, L46, E49, V50, N51, K55, S57, 162, L63, D68, P77, F84, D85, N88, or 195 of Rspo1, or the corresponding residues of Rspo2, Rspo3, or Rspo4, which enhances binding affinity to ZNRF3 compared to Rspo1 of SEQ ID NO:41.

[0217] E13. The recombinant variant according to any one of E1 to E12, wherein the variant does not contain an N-glycosylation site between the FU2 domain and the TSP domain.

[0218] E14. A recombinant variant as described in E13, comprising a mutation at residue N137 of Rspo1, or the corresponding residue in Rspo2, Rspo3, or Rspo4, preventing N-glycosylation at this site.

[0219] E15. The recombinant variant of E14, comprising or consisting essentially of SEQ ID NO:22.

[0220] E16. A recombinant variant according to any one of E1 to E15, wherein the FU1 domain is 100% identical to the FU1 domain of Rspo2 of SEQ ID NO: 9, and the FU2 is selected from the FU2 domain of Rspo1, Rspo3, Rspo4, or a functional variant thereof having amino acid substitutions that maintain at least the same binding affinity to LGR4.

[0221] E17. A recombinant variant according to E16, comprising the FU1 domain of Rspo2 of SEQ ID NO: 9 and the FU2 domain of Rspo1 of SEQ ID NO: 6, and optionally the TSP domain of Rspo1 of SEQ ID NO: 7 and the BR domain of Rspo1 of SEQ ID NO: 8.

[0222] E18. The recombinant variant according to E16, comprising or consisting essentially of SEQ ID NO:25 or SEQ ID NO:26.

[0223] E19. A recombinant variant according to any one of E1 to E15, having the same sequence as SEQ ID NO: 9, except that it contains one or more amino acid substitutions at positions E49, V50, D68, and D85, and which has enhanced binding affinity to ZNRF3 compared to the protein of SEQ ID NO: 25.

[0224] E20. A recombinant variant according to E19, comprising the Rspo1 FU1 domain of SEQ ID NO:5, except that it comprises one or more amino acid substitutions at positions E49, V50, D68 and D85, which enhances the binding affinity to ZNRF3 compared to the protein of SEQ ID NO:25, preferably the amino acid substitutions E49K, V50D, D68G and D85G, and further wherein said FU2 domain is selected from among the FU2 domains of Rspo1, Rspo3, Rspo4 or functional variants thereof having amino acid substitutions which maintain at least the same binding affinity to ZNRF3.

[0225] E21. The recombinant variant according to E19 or E20, wherein the FU2 domain is 100% identical to the Rspo1 FU2 domain of SEQ ID NO:6.

[0226] E22. A recombinant variant according to E19 or E20, comprising the FU1 domain of Rspo1 and the FU2 domain of Rspo1 having one or more amino acid substitutions selected from among E45L, E49K, V50D, K55R, D68G, D85G, N88A, and H108K, N109D.

[0227] E23. The recombinant variant according to any one of E19 to E22, comprising or consisting essentially of SEQ ID NO:27.

[0228] E24. The recombinant variant according to any one of E19 to E22, comprising or consisting essentially of SEQ ID NO:28.

[0229] E25. A recombinant variant according to any one of E19 to E22, comprising the corresponding FU2 domain of human Rspo3 or Rspo4 of SEQ ID NO: 10, 14 or 18 in combination with the FU1 domain of Rspo2 or the FU1 domain of a hybrid Rspo1 (e.g., containing one or more amino acid substitutions at positions E49K, V50D, D68G, D85G), which enhances binding to ZNRF3, such as E49K, V50D, D68G, D85G.

[0230] E26. A recombinant variant according to E1, which is a variant of any one of SEQ ID NOs: 22 to 28 having not more than 10 amino acid substitutions compared to any one of SEQ ID NOs: 22 to 28.

[0231] E27. A recombinant variant according to E26, having at least 95% identity to at least one of SEQ ID NOs: 22 to 28, and wherein the FU1 domain and FU2 domain are 100% identical to at least one of SEQ ID NOs: 22 to 28.

[0232] E28. A recombinant variant according to E26, comprising an amino acid sequence identical to one of SEQ ID NOs: 22 to 28, but in which one, two or three amino acid residues have been replaced by another naturally occurring amino acid, preferably by a conservative amino acid substitution.

[0233] E29. A recombinant variant according to any one of E1 to E28, wherein the FU2 domain comprises amino acid residues F106, H108, F110, N109, E116, L118, P127, A128, S133, A136, G138, S143 in human Rspo1 of SEQ ID NO: 1, or the corresponding residues in human Rspo2 of SEQ ID NO: 2, human Rspo3 of SEQ ID NO: 3, or human Rspo4 of SEQ ID NO: 4.

[0234] E30. A recombinant variant according to any one of E1 to E29, wherein the BR domain comprises at least one or more of amino acid residues T253, L257, T258, S259, A260 or A263, and typically, the BR domain is 100% identical to SEQ ID NO: 8, 12, 16, 20.

[0235] E31. The recombinant variant of any one of E1-E30, comprising an amino acid substitution in the BR domain to improve O-glycosylation, e.g., the variant comprises one or more amino acid substitutions at positions G252, T253, L257, T258, S259, A260, A263 of Rspo1, or corresponding residues in Rspo2, Rspo3 or Rspo4, e.g., the variant comprises a BR domain of Rspo1 having one or more of the amino acid substitutions G252T, L257S, A260T or A263T.

[0236] E32. The recombinant variant according to any one of E1 to E31, wherein the recombinant variant exhibits one or more of the following characteristics at least at a level comparable to that of the Rspo1 protein of SEQ ID NO: 41: (i) binds to the LGR4 receptor with at least the same affinity as the reference human Rspo1 of SEQ ID NO:41, as measured in an Rspo1 / LGR4 binding affinity in vitro assay, e.g., as determined by an SPR assay; (ii) binds to the ZNRF3 receptor with at least the same affinity as the reference human Rspo1 of SEQ ID NO:41, as measured in an Rspo1 / ZNRF3 binding affinity in vitro assay, e.g., as determined by an SPR or ELISA assay; (iii) activates the wnt / β-catenin pathway to a level at least comparable to that of human Rspo1 of reference SEQ ID NO: 41, as determined, for example, by a topflash assay as described in the Examples below; (iv) induces the proliferation of functional beta cells to a level at least comparable to that of the reference human Rspo1 of SEQ ID NO: 41, e.g., as determined in an in vitro Min6 beta cell proliferation assay; and / or (v) induces the proliferation of functional beta cells to a level at least comparable to that of the reference human Rspo1 of SEQ ID NO: 41, eg, as determined in an in vivo beta cell proliferation assay.

[0237] E33. An Fc fusion protein comprising a recombinant variant according to any one of E1 to E32 and an Fc fragment fused to the recombinant variant directly or indirectly via a peptide linker.

[0238] E34. The Fc fusion protein of E33, further comprising an Fc fragment directly or indirectly fused to the N-terminus of the R-spondin protein.

[0239] E35. The Fc fusion protein of E33, further comprising an Fc fragment directly or indirectly fused to the C-terminus of the R-spondin protein.

[0240] E36. The Fc fusion protein according to any one of E33 to E35, wherein the peptide linker comprises, for example, a linker of (GGGGGGSGGGGSGGGGSA) (SEQ ID NO: 44) or (GGGGSGGGGSGGGGGG) (SEQ ID NO: 45).

[0241] E37. The Fc fusion protein according to any one of E33 to E36, wherein the Fc fragment comprises or consists essentially of SEQ ID NO:29.

[0242] E38. The Fc fusion protein according to any one of E33 to E37, comprising or consisting essentially of a sequence selected from the group consisting of SEQ ID NO: 42, 43, or SEQ ID NO: 92, 93, or 95.

[0243] E39. A recombinant variant according to any one of E1 to E38, for use as a medicament, in particular as a medicament for treating diabetes, more preferably type I or type II diabetes, in a patient in need thereof.

[0244] E40. The fusion protein according to any one of E33 to E39, for use as a medicament, in particular as a medicament for treating diabetes, more preferably type I or type II diabetes, in a patient in need thereof.

[0245] E41. A recombinant variant or fusion protein for use as described in claim E39 or for use as described in E40, wherein the recombinant variant or fusion protein is administered to the patient in combination with beta cells, preferably stem cell derived beta cells.

[0246] E42. A nucleic acid encoding a recombinant variant according to any one of E1 to E32.

[0247] E43. A vector containing the nucleic acid of E42.

[0248] E44. A host cell comprising the nucleic acid of E42.

[0249] E45. A method for producing a recombinant variant according to any one of E1 to E32, comprising: (i) culturing a host cell according to E44 under conditions for expression of the recombinant variant; (ii) recovering the recombinant variant; and (iii) optionally purifying the recombinant variant.

[0250] E46. A nucleic acid encoding the fusion protein according to any one of E33 to E38.

[0251] E47. A vector comprising the nucleic acid according to E46.

[0252] E48. A host cell comprising the nucleic acid according to E46.

[0253] E49. A method for producing a fusion protein according to any one of E33 to E38, comprising the steps of: (i) culturing a host cell according to E44 under conditions for expression of the fusion protein; (ii) recovering the recombinant variant; and (iii) optionally purifying the recombinant variant.

[0254] Description of the drawings FIG. 1 provides an alignment of the Rspo1, Rspo2, Rspo3 and Rspo4 amino acid sequences along with a schematic diagram of the different domains FU1, FU2, TSP and BR.

[0255] [Figure 2] Quantification of Min6 cells incubated with PBS (black control) or different doses of recombinant hRspo1.

[0256] [Figure 3] Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #009 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0257] FIG. 4: Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #008 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0258] FIG. 5: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #005 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0259] FIG. 6: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #034 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0260] FIG. 7: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #047 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0261] FIG. 8: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #051 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0262] FIG. 9: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #063 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0263] FIG. 10: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #064 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0264] FIG. 11: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #049 containing a histidine tag (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0265] FIG. 12: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #054 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0266] FIG. 13. Quantification of Min6 cells incubated for 24 hours with PBS (control) or untagged variant #049 at 200 nM and 400 nM.

[0267] FIG. 14. Quantification of Min6 cells incubated for 48 hours with PBS (control) or untagged variant #049 at 200 nM and 400 nM. Fresh protein was added after the first 24 hour incubation.

[0268] FIG. 15. Quantification of Min6 cells incubated with PBS (control) or untagged variant #049 at 200 nM and 400 nM for 72 hours. Fresh protein was added after the first 24 hour incubation and again after 48 hours.

[0269] FIG. 16: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #056 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0270] FIG. 17: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #082 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1.

[0271] FIG. 18: Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #0116 (gray). The left bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1 (#014).

[0272] [Figure 19] Percentage of BrdU-positive beta cells in islets of Langerhans from 129 / Sv mice after 4 days of culture and 72 hours of treatment with variants #014 (A), #49 (B), #008 (C), #064 (D), #051 (E), #121 (F) and GLP1 (1 nM).

[0273] FIG. 20. Blood glucose tracing and diabetes incidence in female NOD mice treated daily for 16 weeks by intraperitoneal injection of variant #014 (A, B) and variant #064 (C, D).

[0274] FIG. 21. Diabetes incidence in female NOD mice treated daily with 0.2 mg / Kg, 0.4 mg / Kg and 0.8 mg / Kg of variant #049.

[0275] FIG. 22. Blood glucose tracing of NOD mice treated daily with 0.2 mg / Kg, 0.4 mg / Kg and 0.8 mg / Kg of variant #049.

[0276] [Figure 23] Pancreatic islet area (μm2) in pancreatic sections of mice intraperitoneally injected with variant #014 for 5 consecutive days. 2 Quantification of Ki67 / insulin double positive cells normalized to .

[0277] [Figure 24] Pancreatic islet area (μm2) in pancreatic sections of mice intraperitoneally injected with variant #014 for 5 consecutive days. 2 Quantification of BrdU / insulin double positive cells normalized to .

[0278] FIG. 25. Blood glucose tracing of STZ-induced hyperglycemic mice treated with daily intraperitoneal injections of variant #014 starting 15 days before STZ treatment.

[0279] FIG. 26 Water intake in STZ-induced hyperglycemic mice treated with daily intraperitoneal injections of variant #014 starting 15 days before STZ treatment.

[0280] [Figure 27] Intraperitoneal glucose tolerance test performed on STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 every day starting 15 days before STZ treatment. This test was performed 74 days after the start of daily administration of variant #014.

[0281] FIG. 28. Blood glucose tracing of STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily starting 7 days after STZ treatment.

[0282] FIG. 29. Water intake in STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily starting 7 days after STZ treatment.

[0283] Figure 30: Intraperitoneal glucose tolerance test performed on STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily from 7 days after STZ treatment. This test was performed 63 days after the start of daily administration of variant #014.

[0284] FIG. 31. Fasting human c-peptide measured in the blood of animals transplanted with human islets and injected daily with either PBS, or 0.4 mg / Kg and 0.8 mg / Kg of variant #014.

[0285] FIG. 32. Intraperitoneal glucose tolerance test performed on mice transplanted with human islets after treatment for 28 days with either PBS (control) or 0.4 mg / Kg and 0.8 mg / Kg of variant #014.

[0286] FIG. 33. Basal and glucose-stimulated blood human c-peptide concentrations in mice transplanted with human islets after treatment for 28 days with either PBS (control), or 0.4 mg / Kg and 0.8 mg / Kg of variant #014.

[0287] FIG. 34. Quantification of total insulin capacity in transplanted human islets after treatment for 60 days with either PBS (control) or 0.4 mg / Kg and 0.8 mg / Kg of variant #014.

[0288] Figure 35: Comparison of 168-hour kinetics following SC administration of #63-1, #63-2, and #64 Fc fusion proteins at 0.8 mg / kg in male 129 / Sv mice (n=3 / time point) shows extended half-life relative to the reference non-Fc protein #14. For comparison, the single-dose PK profile of protein #14 is shown (dashed line).

[0289] [Example] 1.Functional assays for characterization of R-spondin proteins (ZNRF3 Binding Assay: ELISA (Enzyme-Linked Immunosorbent Assay) Protocol) ELISA is a plate-based assay method for detecting and quantitating soluble proteins (ligands) in liquid samples using a ligand binding molecule (antibody, fusion molecule, etc.) and a detection antibody.

[0290] procedure: An ELISA module (Nunc Maxisorp, Thermo Scientific) is treated with antigen (100 μl / well, 1 μg / ml in PBS pH 7.4, overnight at 4° C.). After antigen capture, the plate is washed 4 times with PBS pH 7.4 containing 0.05% Tween 20 (PBS-Tween) and blocked for 60 min with PBS-Tween containing 2% BSA (Merck). Then, serial 2-fold dilutions of the samples starting from 4000 ng / ml (for the second layer see molecules used) are made in PBS-Tween containing 1% BSA, applied to the plate (100 μl / well) and incubated for 60 min at room temperature on a shaker (300 rpm).

[0291] If a third layer is required (LGR4-His binding): Wash the plate 4 times with PBS-Tween, then add 100 μl of antibody (see antibody for use in the third layer) diluted in PBS-Tween with 1% BSA. Incubate the plate for 30 minutes at room temperature on a shaker (300 rpm).

[0292] The plate is then washed four times with PBS-Tween, and 100 μl of peroxidase-labeled goat anti-mouse IgG polyclonal antibody (Goat anti-mouse-IgG-HRP, 1:10000, 0.1 μg / mL, LabAs Estonia) diluted in PBS-Tween containing 1% BSA is added. The plate is incubated for 30 minutes at room temperature on a shaker (300 rpm).

[0293] After washing the plate four times with PBS-Tween, 100 μl of TMB substrate solution VII (Biopanda Diagnostics) is added. The reaction is carried out at room temperature on a shaker (300 rpm) for 10 minutes, and 50 μl of 0.5 M sulfuric acid is added to terminate the reaction.

[0294] Absorbance is measured at 450 nm using an ELISA plate reader (Thermo Scientific).

[0295] Antigen used: RSPO-1 (#120-38, Peprotech) ·RSPO-2(#3266-RS, R&D Biosystems) RSPO-3 (#120-44, Peprotech) Proteins #007, #008, #014, #059 (produced at Icosagen Cell Factory) Molecules used in the second layer: · LGR4-His (#LG4-H52H3, Acro) ·LGR4-Fc(#7750-GP, R&D Biosystems) ·ZNRF3-Fc(#7994-RF, R&D Biosystems) Antibodies used for the third layer (for LGR4-His binding): Mouse monoclonal anti-His antibody (1:2500, 0.2 μg / mL, GenScript, Cat. No. A00186) (TOP-FLASH-ASSSAY for measuring Wnt / β-catenin signaling pathway activity) The Super-TOP-Flash luciferase reporter assay can be used to monitor the concentration and activity of both Wnt and R-spondin proteins in conditioned medium. First, prepare Super-TOP-Flash reporter-expressing HEK 293 STF cells and plate them in a 96-well plate with serum-free DMEM (1–2 × 10 4 cells / well). After 24 h, serum-free reporter cells are exposed to different amounts of culture medium containing either Wnt protein or R-spondin protein. After 18-24 h of induction, luciferase activity is measured and the amount of growth factor present in the conditioned medium is compared to a known protein source (recombinant standard hRSPO1). This cell-based reporter assay can test the activity of both Wnt and R-spondin ligands.

[0296] Required reagents and cell lines: a. HEK-293 STF cells (ATCC). b. R-spondin proteins produced at Icosagen Cell Factory, and RSPO-1 (#120-38, Peprotech), RSPO-2 (#3266-RS, R&D Biosystems), and RSPO-3 (#120-44, Peprotech). d. Steady-Glo® Luciferase Assay System (Promega, cat#E2510) e. Complete growth medium: DMEM (Gibco) cell culture medium containing 10% FCS (Gibco) + 1x Pen / Strep f. Serum-free growth medium: DMEM (Gibco) culture medium + 1x Pen / Strep g. CELLSTAR® 96-well plates: flat-bottom white polystyrene wells (Greiner) Preparation of HEK-293 STF cells for Super-Top-Flash luciferase reporter assay: Day 0: Use 2 ml of trypsin solution to detach HEK-293-STF cells from the bottom of a 10 cm culture plate, and add 2 ml of complete growth medium after 5 min. Spin down at 200g for 5 min and suspend the cell pellet in serum-free DMEM. Count the cells and suspend the HEK-293-STF reporter cells (2 ml) in serum-free medium in a total volume of 100 ul. * 10 5 Seed 1000 cells / ml into a CELLSTAR 96-well plate. Culture the cells at 37°C in a humidified cell culture incubator for 22-26 hours. Day 1: Dilute R-spondin protein in DMEM medium and apply serial dilutions to cultured HEK-293 STF cells (3-fold dilutions were used in this study). Culture the cells with the inducer for 18-24 hours at 37°C. Day 2: Measure STF firefly activity by adding 50 μl of Steady-Glo® Luciferase Assay Substrate directly to the culture wells, wait at least 5 minutes, and then measure bioluminescence signal intensity using a Glomax Explorer luminometer (Promega). Export results to a PC for analysis.

[0297] (Min6 cell proliferation assay) Mouse insulinoma (Min6) cells are cultured in DMEM supplemented with 4.5 g / l glucose, 12.5% ​​FCS (fetal calf serum), 0.005% sodium bicarbonate, 100 U / mL penicillin, and 100 mg / mL streptomycin and maintained in a humidified atmosphere (37 °C; 95% air / 5% CO2). Low excess (up to P30) Min6 cells are plated in 12-well plates at a seeding density of 80000 cells / ml. Adherent cells are incubated for 24 h with target molecules diluted at different concentrations in low FCS medium (5%). For longer incubations, fresh proteins were added to the medium every 24 h. Cells are finally detached and quantified manually using a Thoma Chamber.

[0298] A variant is considered to be more effective than native hRspo1 if it exhibits an overall stronger ability to induce proliferation compared to Rspo1 at 400 nM, or if it exhibits a lower concentration that allows for a significant increase in proliferation compared to a comparable concentration of hRspo1.

[0299] (Wnt / β-catenin assay) Min6 cells are cultured as described in the previous paragraph. Low passage cells are plated in 6-well plates at a seeding density of 250000 cells / ml. Adherent cells are treated with different doses of hRspo1 analogues in low FCS medium (5%) for multiple time points. Cells are then detached and resuspended in PBS. Total protein content is isolated by sonication and β-catenin concentration is assessed by ELISA assay according to the manufacturer's instructions. BCA content is used for normalization of each protein sample.

[0300] In vivo proliferation assay In vivo proliferation studies are performed with 2-month-old wild-type 129SV mice. After an acclimatization period of at least 3 days, animals are injected intraperitoneally or subcutaneously daily with different concentrations of the variant of interest for several consecutive days. Mice injected daily with 150 μl of sterile PBS served as controls. Finally, mice are sacrificed by cervical dislocation 30 min after the last injection. The pancreas is harvested, fixed with Antigenfix (paraformaldehyde solution at pH 7.2-7.4; Microm Microtech France), washed with cold PBS and incubated for 1 h in 0.86% saline. After dehydration through a series of increasing ethanol dilutions (50%, 70%, 80%, 90%, and 100%), the pancreas is treated with isopropanol and toluene and embedded in paraffin. Paraffin blocks are cut into 6 μm slides and analyzed by immunofluorescence using antibodies against insulin, PC1 / 3, Ki67, and BrdU.

[0301] A variant is considered to be more active than the original hRspo1 if it induces more proliferative β-cells, a greater increase in β-cell mass, better glucose handling, or if the onset of action / required concentration is lower than that of native hRspo1.

[0302] Obviously, such an approach can also be used in different diabetic mouse models, including in particular streptozotocin-treated mice, NOD animals or Rip-B7 animals (King, Br J Pharmacol. 2012 Jun; 166(3): 877-894 and Karges et al., Diabetes 2002 Nov; 51(11): 3237-3244).

[0303] 〔result〕 A. Production of Recombinant Variants Table 2 below provides a list of recombinant Rspo proteins that were produced according to the following protocol.

[0304] [Table 2] TIFF2024526356000008.tif219169TIFF2024526356000009.tif219169TIFF2024526356000010.tif220169TIFF2024526356000011.tif154169

[0305] All of the above recombinant proteins were expressed using QMCF technology developed by Icosagen (US 7,790,446, see also US 8,377,653), involving CHO or HEK293-based QMCF cell lines.

[0306] Most of the proteins listed in Table 2 contain at their C-terminus, after the BR domain, a short linker and a C-His tag to facilitate purification, with the following exceptions: ·#014 (natural hRspo1), #46, whose C-terminus is functionalized with a short linker, resulting in a sequence with high affinity for albumin; ·Fc conjugated proteins.

[0307] One skilled in the art would be able to produce similar recombinant proteins using alternative linkers and / or tags for purification, or without such linkers and tags.

[0308] B. Determining β-catenin pathway stimulation in the TOP Flash assay All generated variants were tested for their ability to stimulate the β-catenin pathway and compared with native hRspo1.

[0309] Table 3 below shows the results of some of the variants produced:

[0310] [Table 3]

[0311] C. Measurement of Min proliferation activity using the in vitro Min6 proliferation assay The proliferation activity of each variant was evaluated in vitro using a mouse insulinoma (Min6) cell line. Recombinant hRspo1 stimulated Min6 proliferation with a bell-shaped dose-response curve that peaked at 400 nM (Figure 2).

[0312] Similar to native hRspo1, variant #009 showed a bell-shaped dose-response mitogenic effect on Min6 cells, peaking at 400 nM (Figure 3). However, at this dose, the proliferation rate of Min6 was significantly higher compared to original hRspo1 (Figure 3).

[0313] The number of Min6 cells was significantly increased by incubation with variant #008 at all concentrations tested (Figure 4). Interestingly, this number was also significantly higher at doses of 200 nM and 1 μM when compared to Min6 cells treated with hRspo1 (Figure 4).

[0314] Variant #005 showed the same bell-shaped dose-response curve as standard hRspo1, with a peak proliferative response at 400 nM (Figure 5). Nevertheless, Min6 cells incubated with variant #005 were significantly fewer in number than cells incubated with native hRspo1 (Figure 5).

[0315] Interestingly, variant #034 significantly stimulated proliferation of Min6 cells at all concentrations tested, but there was no significant difference compared to wild-type hRspo1 at 400 nM (Figure 6).

[0316] The proliferation dose-response curve for variant #047 was bell-shaped, with an onset of activity at 200 nM and a peak at 1 μM (Figure 7). At this latter concentration, the mitotic rate of Min6 cells was found to be significantly increased compared to cells incubated with original hRspo1 at 400 nM (Figure 7).

[0317] Importantly, stimulation of Min6 cells with variant #051 produced a dose-response curve with a similar shape to that observed upon treatment with variant #047 (Figure 8). However, variant #051 began to act earlier (100 nM) compared to native hRspo1 at 400 nM, and induced significantly stronger proliferation at both 400 nM and 1 μM (Figure 8).

[0318] Interestingly, we found that the Fc-binding hRspo1 variant (#063) was as efficient as the original protein when incubated at a concentration of 400 nM (Figure 9). However, this proliferative effect did not follow a bell-shaped dose-response curve, but rather remained stable at higher doses (Figure 9).

[0319] Conversely, the N-terminal Fc binding variant #064 had no significant proliferative effect on Min6 cells at 400 nM, and the protein only became effective when diluted above 1 μM (FIG. 10).

[0320] The mitotic effects of variants #049 and #056 containing His tags gave rise to the same bell-shaped dose-response curve and showed the same efficacy as native recombinant hRspo1 (Figures 11-12).

[0321] The efficacy of untagged variant #049 was also tested in Min6 cells. Min6 cell numbers were significantly increased after 24 hours of incubation with 400 nM variant #049 (Figure 13). Importantly, this increase was shown to be stronger and more pronounced when the same protein was incubated for 48 or 72 hours (Figures 14 and 15).

[0322] Similarly, variant #054 was observed to be increasingly effective up to a dose of 400 nM, with this efficiency gradually decreasing with increasing concentrations (Figure 16). However, this analog was found to be significantly more potent than the original hRspo1 in stimulating Min6 proliferation at the peak of its dose-response curve (Figure 16).

[0323] Surprisingly, variant #082 strongly stimulated proliferation of Min6 cells at all concentrations tested, and even at low doses it displayed significantly more potent mitogenic activity than hRspo1 (FIG. 17).

[0324] Variant #116 increases Min6 cell numbers in a bell-shaped manner with a peak in activity at 400 nM (FIG. 18).

[0325] Native human Rspo3 was also tested for its ability to induce β-cell proliferation in vivo, and additional analyses showed that short-term exposure (5–30 min) of wild-type mice to human Rspo3 also induced β-cell proliferation in vivo.

[0326] D. Determination of ZNRF3 binding We generated variant #009, variant #047 and variant #051 and tested them for their ability to improve binding to ZNRF3 compared to the original native Rspo1 using the ZNRF3-Fc receptor binding assay described above.

[0327] Variant #009 behaves similarly to the control Rspo1 (no mutations), whereas variants #047 and #051 show superior binding affinity for ZNRF3 compared to the control Rspo1.

[0328] 2. Beta Cell Proliferation in Isolated Islets 2.1 Method 2.1.1 Animals Male 129S2 / SvPasCrl mice were obtained from Charles River Laboratories (69210 Saint-Germain Nuelles, France): 60 mice aged 10–12 weeks were used. All animal experiments were performed as part of the certified project N2796, in accordance with the European guidelines for animal care (2010 / 63 / UE). Animals were acclimatized 1 week before the start of the experiments and kept in a temperature-controlled (22 ± 2 °C) area with a 12-h light / dark cycle (lights on at 7:00 am). All mice were fed with normal growth diet A04 from SAFE (Scientific Animal Food and Engineering-Route de Saint Bris-89290AUGY-France) and drank ad libitum. Bedding (sterile sawdust) was changed every other day. Mice were divided into groups of 6 mice per cage. Cage dimensions were 42.5 × 26.6 × 15.5 cm. Systemic signs were observed and only animals without abnormal signs were included in the study.

[0329] Mice were anesthetized with intraperitoneal injection of pentobarbital, and the pancreas was perfused with collagenase for further isolation of islets. After overnight stabilization in RPMI1640, the islets were isolated and treated with compounds and reference substances for 72 h and further proceeded to cell proliferation evaluation.

[0330] 2.1.2 Isolation and processing of islets Pancreatic islets from 129 / Sv mice were isolated by collagenase digestion of the pancreas, washed with Hank's Balanced Salt Solution (HBSS), and then purified on a density gradient using Histopaque 1077 and HBSS. Islets were hand-picked into Petri dishes at a density of 30 and cultured in RPMI 1640 supplemented with 10 mM Hepes, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, and SVF 10% at 37 °C in a humidified atmosphere of 90% air / 5% CO2. After overnight stabilization, the medium was removed and fresh medium without either the test compound or the reference substance (control) or fresh medium containing the test compound or the reference substance was added. The medium and treatments were renewed after 24 and 48 h. In the last 24 h of treatment, BrdU (10 μM) was added to the medium.

[0331] The islets were divided into 8 groups with 6 petri dishes per group, as described in Table 4 below:

[0332] [Table 4]

[0333] 2.1.3 Preparation of isolated islets for proliferation measurements After 72 hours of treatment, 30 islets per condition were collected from the Petri dish and transferred to a 1.5 ml Eppendorf tube. The islets were then washed three times with DPBS by centrifugation (1000 rpm, 30 seconds, 4°C) and digested with Trypsin-EDTA 0.25%. The reaction was stopped by adding RPMI1640, and the digested islets were then cell-aliquoted on a cytoslide.

[0334] The islets were then fixed with 3.7% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X100 in 5% BSA, and antigen retrieval was performed for 20 min at 100°C using citrate buffer, pH 6. To prevent nonspecific binding of antibodies, a blocking step was performed with PBS-5% BSA before immunostaining.

[0335] 2.1.4 Determination of β-cell proliferation Cell proliferation was estimated by measurement of BrdU positive cells in sections after double immunostaining with rat anti-BrdU antibody (Abcam, Ref. ab6326) coupled with goat anti-rat IgG Alexa Fluor 647 (ThermoFisher Scientific, Ref. A-21247) and mouse anti-insulin antibody (Sigma, Ref. I2018) coupled with goat anti-mouse IgG DyLight® 488 antibody (Diagomix - Ref. GtxMu-003-D488NHSX). Cell nuclei were stained with ProLong™ Gold Antifade Mountant with DAPI (Life Technologies - Ref. P36935). The number of cells stained with BrdU immunostaining was determined after scanning the slides and analyzing the slides using NDP view or case viewer imaging software. Analysis was performed on 5-6 samples per batch.

[0336] 2.2 Results This study aimed to evaluate the effect of 72 h exposure to six variants (#008, #014, #049, #051, #064 and #121) on β-cell proliferation in islets of 129 / Sv mice. #008, #014, #051 and #064 proteins were tested at three concentrations (0.2 μM, 1 μM and 3 μM) and #049 and #121 at five concentrations (0.1 μM, 0.2 μM, 0.4 μM, 1 μM, and 2 μM (#049) or 3 μM (#121)). Cell proliferation was calculated by dividing the number of BrdU-positive cells by the total number of cells and expressed as % (Figure 19). Results are expressed as mean ± SEM. The number of observations was 5–6 for each treatment. To analyze combined effects, statistical analysis was performed using Anova followed by Dunnett's test, or Kruskal-Wallis followed by Dunn's test if variances were significantly different (GraphPad PRISM® 8). A p-value <0.05 was considered significant.

[0337] 3. Changes in blood glucose levels and β-cell mass in female NOD mice 3.1 Method 3.1.1 Animals Female NOD / Mrk Tac mice were provided by Taconic Biosciences (4623 Ejby, Lille Skensved, Denmark): 55 mice were 6–8 weeks old when transferred to the CNRS-Universite de Paris-UMR 7592 animal facility. All animal experiments were performed as part of the certified projects #20173 and #31876, in accordance with the European guidelines for animal care (2010 / 63 / UE). Animals were acclimatized for 1 week prior to the experiments and kept in a temperature-controlled (22 ± 2 °C) area with a 12-h light / dark cycle (lights on at 7 am). All mice were fed NIH-31M diet from ALTROMIN (Altromin Spezialfutter GmbH & Co.KG- Im Seelenkamp 20- D-32791 Lage - Germany) and allowed to drink ad libitum. Bedding (sterile sawdust) was changed every other day. Mice were divided into groups of 5 per cage. The cage dimensions were 37.3 × 23.4 × 14.0 cm. Systemic signs were observed, and only animals without abnormal signs were included in the study.

[0338] 3.1.2 Study design During the 16 week period of the study, the proteins were administered to the mice by intraperitoneal route at a dose of 10 ml / kg, as provided by the sponsor, once daily for variant #014 and once weekly in the morning for variant #064, as described in Table 5 below:

[0339] [Table 5]

[0340] The drug solutions were prepared as follows: - #064: The appropriate amount of protein at 1 mg / ml in PBS 1X pH 7.4 was diluted with PBS 1X pH 7.4 to obtain a 240 μg / ml solution (2400 μg / kg / 10 ml).

[0341] - #014: An appropriate amount of protein at 1 mg / ml in PBS 1X pH 7.4 was diluted with PBS 1X pH 7.4 to obtain a 80 μg / ml solution (800 μg / kg / 10 ml).

[0342] This solution was further diluted 1:2 with PBS 1X, pH 7.4 to obtain a 40 μg / ml solution (400 μg / kg / 10 ml). The amount prepared was adjusted according to the number of animals to be injected.

[0343] All animals were dosed with vehicle or compound intraperitoneally once daily in the morning (#64 once weekly) from week 1 to week 18. Blood glucose levels were monitored weekly up to week 16 using an Accu-Check reader after tail vein blood sampling.

[0344] Ten-week-old female NOD mice were intraperitoneally injected daily for 13 weeks with untagged variant #049 at concentrations of 0.2 mg / Kg, 0.4 mg / Kg, and 0.8 mg / Kg. Blood glucose levels and body weights of mice were monitored weekly. Mice with blood glucose levels above 250 mg / dl were considered diabetic.

[0345] Analysis method Glucose concentrations are determined using a commercial kit from Horiba Medical (reference number: A11A01667). The procedure is based on a two-phase enzymatic reaction:

[0346]

number

[0347] The reaction is monitored kinetically by measuring the absorbance caused by the NADH produced in the second phase, and the glucose content of the sample can be calculated from the measured change in absorbance.

[0348] Insulin concentration is determined using a commercially available kit from Alpco (Reference Number: 80-INSMSU-E01 / E10). The ALPCO Mouse Ultrasensitive Insulin ELISA is a sandwich-type immunoassay. A 96-well microplate is coated with a monoclonal antibody specific for insulin. Standards, controls, and samples are added to the microplate wells along with the conjugate. The microplate is incubated on a microplate shaker at 700-900 rpm. After the first incubation is complete, the wells are washed with wash buffer and blotted dry. TMB substrate is added and a second incubation is performed on a microplate shaker at 700-900 rpm. After the second incubation is complete, Stop Solution is added and the optical density (OD) is measured at 450 nm in a spectrophotometer. The intensity of the color developed is directly proportional to the amount of insulin in the sample. The measurement range is 0.025-6.9 ng / mL.

[0349] 3.2 Results This study aimed to evaluate the effect of 18 weeks of intraperitoneal treatment with #014 protein at two doses (400 and 800 μg / kg) and #64 protein at one dose (2400 μg / kg) on ​​blood glucose course, diabetic phenotype and β-cell mass in female NOD mice (four groups: vehicle (n=15), #014 protein at two concentrations (n=13), #64 protein at one concentration (n=14) administered compounds for 18 weeks from 10 weeks of age).

[0350] Results in Figure 20 are expressed as mean ± SEM with number of individual observations (n). Statistical analysis was performed using Anova followed by Dunnett's t-test to compare treated groups with the STZ control group. If variances between groups calculated by Bartlett's test (GraphPad PRISM®8) were significantly different, Kruskal-Wallis test followed by Dunn's test was used. A p-value of 0.05 was considered significant.

[0351] To evaluate the ability of variant #049 to counteract β-cell loss in diabetic conditions, we used NOD mice as a type 1 diabetes model. Interestingly, daily intraperitoneal administration of variant #049 dose-dependently reduced diabetes onset (Figure 21). Importantly, rodents treated with variant #049 at a concentration of 0.8 mg / Kg showed negligible increase in basal blood glucose compared to controls (Figure 22).

[0352] 4. In Vivo Testing 4.1 Method 4.1.1 In vivo metaphase proliferation assay In vivo proliferation studies are performed using 2-month-old wild-type male 129SV mice. After a minimum 3-day acclimatization period, animals are injected intraperitoneally daily for 5 consecutive days with 0.4 mg / Kg #014 and 0.8 mg / kg #014. Mice injected daily with 150 ul of sterile PBS served as controls. In addition, bromodeoxyuridine (BrdU), a thymidine analogue, was diluted in drinking water at a concentration of 1 mg / ml and given to all mice for 72 hours before sacrifice. Finally, mice are sacrificed by cervical dislocation 30 minutes after the last injection. Pancreases are harvested, fixed in Antigenfix (paraformaldehyde solution at pH 7.2–7.4; Microm Microtech France), washed in cold PBS, and incubated in 0.86% saline for 1 hour. After dehydration through a series of increasing ethanol dilutions (50%, 70%, 80%, 90%, and 100%), the pancreas is treated with isopropanol and toluene and embedded in paraffin. Paraffin blocks are cut into 6 μm slides and analyzed by immunofluorescence using antibodies against insulin, Ki67, and BrdU.

[0353] 4.4.2 In vitro efficacy studies on streptozotocin-induced mouse hyperglycemia model These tests are performed on adult 2-month-old wild-type male 129SV mice. To induce hyperglycemia, 129SV male mice are intraperitoneally injected with streptozotocin (STZ), a glucosamine-nitrosourea compound that exhibits cytotoxic effects through DNA and chromosomal damage. Briefly, STZ was dissolved in 0.1 M sodium citrate buffer (pH 4.5) and administered at a dose of 50 mg / Kg for three consecutive days after 5 hours of starvation. The onset of hyperglycemia was assessed by monitoring blood glucose levels using an ONETOUCH glucometer (Life Scan, Inc. CA). Water intake was measured manually once a week. For the evaluation of glucose tolerance, an intraperitoneal glucose tolerance test (ip-GTT) was performed. In these tests, mice were fasted for 5 hours and intraperitoneally injected with D-(+)-glucose at 2 g / kg body weight. Blood glucose levels were measured at the indicated time points after injection using an ONETOUCH glucometer. PBS or variant #014 (SEQ ID NO: 41) was administered daily via intraperitoneal injection at a dose of 0.8 mg / Kg starting either 15 days before STZ treatment or 7 days after STZ treatment.

[0354] 4.4.3 In vivo efficacy studies on transplanted human islets For this experiment, 12 Rag2N12 immunodeficient mice were transplanted with human islets (500 islets equivalent / mouse). After gas anesthesia (isoflurane), a lumbar laparotomy was performed to access the left kidney. Islet pellets (500 islets equivalent) were injected through a catheter into the subcapsular space of the kidney of each animal. After transplantation, the capsule was cauterized to avoid bleeding and cell leakage. Both muscle and skin layers were sutured. Morphine (buprenorphine-0.05mg / kg) was administered subcutaneously to relieve perioperative pain. Each mouse was kept in a sterile cage in the SOPF animal facility. After 2 weeks of recovery, mice were treated daily for 60 days with PBS, or with variant #14 at doses of 0.4mg / Kg and 0.8mg / Kg. Every 2 weeks, blood c-peptide concentrations after 6 hours of starvation were measured. After 28 days of variant #014 administration, glucose handling was evaluated with an ip-GTT. Finally, BrdU was diluted in drinking water at a concentration of 1 mg / ml and given to the animals for 1 week before sacrifice. At the end of the experiment, the mice were sacrificed and the islet grafts were isolated, fixed, and analyzed by immunofluorescence using antibodies against insulin and BrdU.

[0355] 4.2 Results 4.2.1 Assessment of in vivo hRspo1 proliferative activity To evaluate the ability of hRspo1 to induce adult mouse β-cell neogenesis, we administered variant #014 intraperitoneally for 5 consecutive days. Importantly, an increase in cells co-expressing insulin and the endogenous nuclear proliferation marker Ki67 was observed in pancreatic samples from mice treated with variant #014 at all doses tested (Figure 23). Notably, a significant 6-fold increase was assessed in tissues isolated from animals treated with 0.8 mg / Kg variant #014. Thus, variant #014 administration was shown to enhance BrdU accumulation in pancreatic β-cells (Figure 24). Furthermore, BrdU / insulin co-expressing cells were significantly more abundant in pancreatic sections from mice injected with 0.8 mg / Kg variant #014.

[0356] 4.2.2 Effect of variant #014 on glycemic control in STZ-induced hyperglycemic mouse model This study aimed to evaluate the effect of chronic treatment with #014 protein (0.8mg / kg) on ​​glycemic control in a mouse model of hyperglycemia induced by multiple low doses of streptozotocin (STZ). Hyperglycemia was induced by three doses of STZ (50mg / Kg) administered over three consecutive days. Variant #014 or vehicle was administered daily by intraperitoneal injection, starting either 15 days before STZ treatment or 7 days after STZ treatment.

[0357] In both experimental settings, STZ effectively induced hyperglycemia in all animals. However, blood glucose levels in mice injected with variant #014 remained steadily lower compared to vehicle-injected controls throughout the entire 2-month experimental period (Figures 25 and 28). Importantly, this difference was more pronounced and significant when variant #014 was inoculated prior to the onset of STZ-mediated hyperglycemia. Accordingly, 24-hour water intake was consistently decreased in mice treated with variant #014, indicating that chronic administration of hRspo1 alleviates hyperglycemia-associated polydipsia (Figures 26 and 29).

[0358] After 2 months of blood glucose monitoring, ip-GTT revealed that mice IP treated with #14 protein had significantly improved glucose tolerance compared to controls in both experiments (Figures 27 and 30). These data indicate that variant #014 can improve glycemic control in a chronic hyperglycemic mouse model.

[0359] 4.2.3 Effect of variant #014 on transplanted human islets To test the proliferation activity of variant #014 on human β-cells, immunodeficient mice transplanted with human islets under the kidney capsule were injected daily with the molecule. Interestingly, administration of 0.4mg / Kg and 0.8mg / Kg of variant #014 to mice resulted in a gradual increase in fasting blood human peptide levels compared to PBS-injected controls (Figure 31). Notably, this increase became significant at day 45 in mice treated with both doses of variant #014 and remained significant at day 60 in the experimental animal group injected with 0.4mg / Kg (Figure 31). Furthermore, glucose tolerance at day 28, assessed by ip-GTT, was shown to be improved in variant #014-treated animals compared to controls (Figure 32). Accordingly, plasma human c-peptide was found to be significantly increased not only during fasting but also 15 minutes after stimulation with a glucose bolus (Figure 33).

[0360] Finally, for pancreatic tissue analysis, mice were given BrdU in drinking water for 1 week and sacrificed on day 60. These studies revealed a strong and significant increase in BrdU immunolabeled β-cells and β-cell mass in pancreatic sections from mice injected with variant #014 compared to controls (Figure 34). Taken together, these data suggest that chronic administration of variant #014 effectively stimulates human β-cell proliferation and hyperplasia.

[0361] 5. Pharmacokinetics 5.1 Method The pharmacokinetic profiles of Fc fusion variants #63-1, #63-2 and #64 were investigated following a single subcutaneous injection at 0.8 mg / kg in 129 / Sv mice.

[0362] Proteins were administered to mice via subcutaneous route (SC, skin along the back) at a dose of 10 ml / kg as shown in Table 6 below:

[0363] [Table 6]

[0364] Blood samples (3 timepoints + 1 terminal bleed per animal) were taken at 1, 3, 6, 24, 48, 72, 96 and 168 hours post-dose as detailed in the table above and plasma was separated for bioanalytical testing.

[0365] The concentrations of proteins #63-1, #63-2 and #64 in plasma were quantified using an optimized bioanalytical method based on the commercially available Human R-Spondin 1 DuoSet ELISA kit (R&D Systems).

[0366] Concentration versus time plots were generated in Excel where PK data were evaluated using the PK Solver add-in for Excel (Zhang Y, t al. Comput Methods Programs Biomed. 2010 Sep;99(3):306-14).

[0367] 5.2 Results The pharmacokinetic profiles of Fc fusion variants #64, #63-1 and #63-2 are shown in Figure 35 and Table 7. The profiles were overlaid with the single dose profile of reference protein #14 generated in a previous study (Study No: DX001-PHA-21-017) for comparison.

[0368] [Table 7]

[0369] After subcutaneous administration of 800 μg / kg to 129 / Sv mice, the 168 h-kinetics of the three proteins were compared. 最大 Between 1 and 3 hours after administration (T 最大) after this point. Plasma concentrations of the three proteins gradually declined until the final time point of the study (168 hours). Protein #14 was quantifiable up to 24 hours after SC injection, while all three Fc-fusion proteins were detectable in plasma even 168 hours after administration, demonstrating that the Fc portion extended their half-lives from approximately 6.5 hours (estimated for #14) to more than 30 hours (Figure 35).

[0370] 6. Production of two types of Rspo1-Fc fusion proteins The #063 (hRspo1 21-263-linker-Fc) variant and the #064 (Fc-linker-hRspo1-21-263) variant were transiently expressed in CHOEBNALT-85-E9 cells. The amounts of the two proteins produced were determined. The production yield of variant #064 is higher than that of variant #063 (11.25 mg vs. 4.5 mg). Thus, the fusion of an Fc polypeptide at the N-terminus of the variants of the present disclosure makes it possible to increase the production yield of the Fc fusion protein.

[0371] 7. Linker optimization of Fc fusion proteins We have shown that linker optimization is necessary to generate active Fc fusion proteins. Indeed, a commercially available Fc fusion protein without a linker (Fc-hRspo1; Creative Biomart 053H) showed no activity in the TopFlash assay. Unlike the previously tested variants #064 or #063, the Fc-hRspo1 protein (Creative Biomart 053H) does not appear to stimulate pancreatic β-cell proliferation.

[0372] Sequences useful in the practice of the invention

[0373] [Table 8] TIFF2024526356000019.tif227169TIFF2024526356000020.tif232169TIFF2024526356000021.tif233169TIFF2024526356000022.tif122169

[0374] [Table 9] TIFF2024526356000024.tif232169TIFF2024526356000025.tif231169TIFF2024526356000026.tif231169TIFF2024526356000027.tif231169 TIFF2024526356000028.tif231169TIFF2024526356000029.tif227169TIFF2024526356000030.tif231169TIFF2024526356000031.tif214169

[0375] [Table 10]

[0376] [Table 11] TIFF2024526356000034.tif230169TIFF2024526356000035.tif230169TIFF2024526356000036.tif224169 [Brief description of the drawings]

[0377] [Figure 1] An alignment of the Rspo1, Rspo2, Rspo3 and Rspo4 amino acid sequences is provided along with a schematic diagram of the different domains FU1, FU2, TSP and BR. [Diagram 2] Quantification of Min6 cells incubated with PBS (black control) or different doses of recombinant hRspo1. [Diagram 3] Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #009 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 4] Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #008 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Diagram 5] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #005 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 6] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #034 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1. [Figure 7] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #047 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 8] Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #051 (grey). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 9] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #063 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 10]Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #064 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1. [Figure 11] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #049 containing a histidine tag (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1. [Figure 12] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #054 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 13] Quantification of Min6 cells incubated for 24 hours with PBS (control) or untagged variant #049 at 200 nM and 400 nM. [Figure 14] Quantification of Min6 cells incubated for 48 hours with PBS (control) or untagged variant #049 at 200 nM and 400 nM. Fresh protein was added after the first 24 hour incubation. [Figure 15] Quantification of Min6 cells incubated with PBS (control) or untagged variant #049 at 200 nM and 400 nM for 72 h. Fresh protein was added after the first 24 h incubation and again after 48 h. [Figure 16] Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #056 (grey). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 17]Quantification of Min6 cells incubated with PBS (control, left black bar) or different doses of variant #082 (gray). The right bar at 400 nM corresponds to the quantification of Min6 with wild type hRspo1. [Figure 18] Quantification of Min6 cells incubated with PBS (control, black bar on the left) or different doses of variant #0116 (gray). The left bar at 400 nM corresponds to the quantification of Min6 with wild-type hRspo1 (#014). [Figure 19] Percentage of BrdU-positive β-cells in islets of Langerhans from 129 / Sv mice after 4 days of culture and 72 hours of treatment with variants #014 (A), #49 (B), #008 (C), #064 (D), #051 (E), #121 (F) and GLP1 (1 nM). [Figure 20] Blood glucose tracing and diabetes incidence in female NOD mice treated daily for 16 weeks by intraperitoneal injection of variant #014 (A, B) and variant #064 (C, D). [Figure 21] Diabetes incidence in female NOD mice treated daily with 0.2 mg / Kg, 0.4 mg / Kg and 0.8 mg / Kg of variant #049. [Figure 22] Blood glucose tracing of NOD mice treated daily with 0.2mg / Kg, 0.4mg / Kg and 0.8mg / Kg of variant #049. [Figure 23] Quantification of Ki67 / insulin double positive cells normalized by islet area (μm2) in pancreatic sections from mice intraperitoneally injected with variant #014 for 5 consecutive days. [Figure 24] Quantification of BrdU / insulin double positive cells normalized by islet area (μm2) in pancreatic sections from mice intraperitoneally injected with variant #014 for 5 consecutive days. [Diagram 25] Blood glucose tracing of STZ-induced hyperglycemic mice treated with daily intraperitoneal injections of variant #014 starting 15 days before STZ treatment. [Figure 26]Water intake in STZ-induced hyperglycemic mice treated with daily intraperitoneal injections of variant #014 starting 15 days before STZ treatment. [Figure 27] Intraperitoneal glucose tolerance test performed on STZ-induced hyperglycemic mice treated with daily intraperitoneal injections of variant #014 starting 15 days before STZ treatment. This test was performed 74 days after the start of daily treatment with variant #014. [Figure 28] Blood glucose tracing of STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily starting 7 days after STZ treatment. [Figure 29] Water intake in STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily starting 7 days after STZ treatment. [Diagram 30] Intraperitoneal glucose tolerance test performed on STZ-induced hyperglycemic mice treated with intraperitoneal injections of variant #014 daily from 7 days after STZ treatment. This test was performed 63 days after the start of daily administration of variant #014. [Diagram 31] Fasting human c-peptide measured in the blood of animals transplanted with human islets and injected daily with either PBS, or 0.4mg / Kg and 0.8mg / Kg of variant #014. [Diagram 32] Intraperitoneal glucose tolerance test performed on mice transplanted with human islets after treatment for 28 days with either PBS (control) or 0.4mg / Kg and 0.8mg / Kg of variant #014. [Diagram 33] Basal and glucose-stimulated blood human c-peptide concentrations in mice transplanted with human islets after treatment for 28 days with either PBS (control), or 0.4mg / Kg and 0.8mg / Kg of variant #014. [Diagram 34] Quantification of total insulin capacity in transplanted human islets after treatment for 60 days with either PBS (control) or 0.4mg / Kg and 0.8mg / Kg of variant #014. [Diagram 35]Comparison of 168-hour kinetics following SC administration of #63-1, #63-2, and #64 Fc fusion proteins at 0.8 mg / kg in male 129 / Sv mice (n=3 / time point) shows extended half-life relative to the reference non-Fc protein #14. For comparison, the single-dose PK profile of protein #14 is shown (dashed line).

Claims

1. A recombinant variant of an R-spondin protein comprising the following FU1 domain, FU2 domain, TSP domain, and BR domain: wherein a. FU1 is a domain having at least 80% identity with any one of the FU1 domains of human Rspo1, Rspo2, Rspo3, or Rspo4 shown in SEQ ID NOs: 5, 9, 13, and 17, respectively, b. FU2 is a domain having at least 80% identity with any one of the FU2 domains of human Rspo1, Rspo2, Rspo3, or Rspo4 shown in SEQ ID NOs: 6, 10, 14, and 18, respectively, c. TSP is a domain having at least 80% identity with any one of the TSP domains of human Rspo1, Rspo2, Rspo3, or Rspo4 shown in SEQ ID NOs: 7, 11, 15, and 19, respectively, and d. BR is a domain having at least 80% identity with any one of the BR domains of human Rspo1, Rspo2, Rspo3, or Rspo4 shown in SEQ ID NOs: 8, 12, 16, and 20, respectively, wherein the recombinant variant is a recombinant variant of an R-spondin protein comprising at least one amino acid substitution at position H108 or N109 of Rspo1, or the corresponding residue of Rspo2, Rspo3, or Rspo4.

2. The recombinant variant according to claim 1, comprising at least one amino acid substitution: H108K or N109D, at Rspo1, or the corresponding residue of Rspo2, Rspo3, or Rspo4.

3. When the FU1 domain of human Rspo1 of SEQ ID NO: 5 and the FU2 domain of Rspo1 of SEQ ID NO: 6 are aligned correspondingly and respectively, having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in each of the FU1 domain or the FU2 domain, the recombinant variant according to claim 1 or 2.

4. The recombinant variant according to claim 1 or 2, comprising a deletion of the first 10 - 14 N-terminal amino acids within the region of 21 - 33 of Rspo1, or within the equivalent region in Rspo2, Rspo3, or Rspo4, typically a deletion of residues 21 - 31 of Rspo1. **Claim 5**: The recombinant variant according to claim 1 or 2, comprising the following amino acid substitutions: H108K and N109D at the corresponding residues of Rspo1, or Rspo2, Rspo3 or Rspo4. **Claim 6**: The recombinant variant of the R-spondin protein according to claim 1 or 2, comprising SEQ ID NO: 66 or consisting essentially of SEQ ID NO:

66. **Claim 7** The recombinant variant according to claim 1 or 2, comprising at least an amino acid substitution of R66 in the FU1 domain of human Rspo1 of SEQ ID NO: 5, or an amino acid substitution of an equivalent arginine residue in the FU1 domain sequence of human Rspo2, Rspo3, or Rspo4, wherein the amino acid substitution reduces or abolishes ZNRf3 binding. **Claim 8**: The recombinant variant according to claim 1 or 2, which does not contain an N-glycosylation site between the FU2 domain and the TSP domain. **Claim 9**: The recombinant variant according to claim 1 or 2, comprising a mutation at residue N137 of Rspo1, or an equivalent residue of Rspo2, Rspo3, or Rspo4, which suppresses N-glycosylation at this site. **Claim 10** The recombinant variant according to claim 1 or 2, exhibiting one or more of the following characteristics at least to the same extent as the Rspo1 protein of SEQ ID NO: 41: (i) binds to the LGR4 receptor with at least the same affinity as human Rspo1 of the reference SEQ ID NO: 41, as measured in an in vitro assay of Rspo1 / LGR4 binding affinity; (ii) induces the proliferation of functional β-cells to at least the same level as human Rspo1 of the reference SEQ ID NO: 41; and / or (iii) induces the proliferation of functional β-cells to at least the same level as human Rspo1 of the reference SEQ ID NO:

41. **Claim 11** An Fc fusion protein comprising the recombinant variant according to claim 1 and an Fc fragment fused directly or indirectly via a peptide linker to the recombinant variant. **Claim 12**: The Fc fusion protein according to claim 11, wherein the Fc fragment comprises SEQ ID NO:

29. **Claim 13**: The Fc fusion protein according to claim 11 or 12, wherein the Fc fragment is fused directly or indirectly via a peptide linker to the Rspo1 protein of SEQ ID NO: 66 or a variant thereof at either the C-terminus or the N-terminus.

14. The Fc fusion protein according to claim 13, wherein the Fc fragment is fused to the Rspo1 protein of SEQ ID NO: 66 or a variant thereof directly or indirectly via a peptide linker at the C-terminus.

15. The Fc fusion protein according to claim 14, wherein the Fc fragment comprises SEQ ID NO: 29 and is fused to the Rspo1 protein of SEQ ID NO: 66 or a variant thereof via a peptide linker at the C-terminus.

16. A pharmaceutical composition for treating diabetes, comprising the recombinant variant according to claim 1 or the fusion protein according to claim 11.

17. The pharmaceutical composition according to claim 16, for treating type I or type II diabetes.

18. A nucleic acid encoding the recombinant variant according to claim 1 or the fusion protein according to claim 11.

19. A vector comprising the nucleic acid according to claim 18.

20. A host cell comprising the nucleic acid according to claim 18.

21. A host cell comprising the vector according to claim 19.

22. A method for producing the recombinant variant according to claim 1 or the fusion protein according to claim 11, comprising: (i) culturing a host cell comprising a nucleic acid encoding the recombinant variant or the fusion protein under conditions for expression of the recombinant variant or the fusion protein; (ii) recovering the recombinant variant or the fusion protein; and (iii) optionally, purifying the recombinant variant or the fusion protein.

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