Rspo1 proteins and uses thereof

Abstract

To provide a medicine for treating diabetes such as type 1 or type 2 diabetes.SOLUTION: Treatment with recombinant Rspo1 proteins was shown to induce in vivo proliferation of functional pancreatic β - cells, improve glucose tolerance and increase glucose-stimulated insulin secretion (GSIS) in a diabetic mouse model. In addition, nearly complete beta-cell ablation was performed and it was found that Rspo1 protein administration can induce proliferation and reconstitution of a functional beta-cell mass that allows the remaining beta-cells to maintain normoglycemia. Finally, the inventors have shown that Rspo1 can also induce human β - cell proliferation opening new and unexpected avenues for the treatment and prevention of human diabetes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to Rspo1 proteins for use as pharmaceutical agents, particularly for the treatment of diabetes. [Background technology]

[0002] Over the past few decades, diabetes has become one of the most widespread metabolic disorders with an epidemic dimension, affecting nearly 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 caused by 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) results from resistance to insulin action and eventual beta cell failure / loss over time.

[0003] Current treatments for diabetes fail to restore euglycemia, and in the case of type 1 diabetes, substituting exogenous insulin injections for defective insulin secretion may even appear to be symptomatic. Therefore, replenishing the pancreas with newly functioning β-cells and / or maintaining the health of remaining β-cells represents an important strategy for treating both conditions. However, to date, no treatments are available to prevent pancreatic β-cell loss or induce proliferation, particularly in human patients with 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 containing four structurally and functionally distinct domains. At the N-terminus, a signal peptide sequence ensures 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), which is involved in interactions with specific components of the extracellular matrix, followed by a carboxy-terminal basic amino acid-rich domain, the function of which is yet to be elucidated. R-spondin proteins are 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 G R-spondin has been reported to play an important role in processes such as WNT signaling (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 largely unexplored 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, V.S., Yeung, A., Schultz, W. & Brubaker, P.L. 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 findings: Rspo1 deficiency in mice is associated with increased beta 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, we surprisingly showed that treatment with recombinant Rspo1 protein induced the in vivo proliferation of functional pancreatic β-cells, improved glucose tolerance, and increased glucose-stimulated insulin secretion (GSIS) in a mouse model of diabetes. Furthermore, we performed near-complete β-cell ablation and found that Rspo1 protein administration could induce the remaining β-cells to proliferate and reconstitute functional β-cell mass capable of maintaining normoglycemia. Finally, we showed that Rspo1 can also induce human β-cell proliferation, opening new and unexpected avenues for the treatment and prevention of human diabetes. Summary of the Invention

[0008] The present disclosure relates to an isolated Rspo1 protein for use as a pharmaceutical, preferably in the treatment of diabetes in a subject in need thereof.

[0009] In specific embodiments, the Rspo1 protein of the present disclosure is any of the following: (i) a protein comprising an Rspondin-1 polypeptide; (ii) a protein comprising a functional fragment of an Rspondin-1 polypeptide, or (iii) a protein comprising a functional variant of an Rspondin-1 polypeptide.

[0010] In specific embodiments, the Rspo1 protein of the present disclosure is any of the following: (i) a protein comprising a human Rspondin 1 polypeptide of any one of SEQ ID NOs: 2 to 4; (ii) a protein comprising a functional fragment of a human Rspondin-1 polypeptide of any one of SEQ ID NOs: 2 to 4, or (iii) A protein comprising a functional variant of the Rspondin-1 polypeptide of any one of SEQ ID NOs: 2 to 4.

[0011] In a specific embodiment, the Rspo1 protein of the present disclosure is a protein comprising a functional fragment of an Rspondin-1 polypeptide, and the functional fragment preferably comprises or consists of a polypeptide having at least 40 to 100 consecutive amino acid residues in the FU1 and / or FU2 domain of the Rspondin-1 protein, typically at least 40 to 100 consecutive amino acid residues in the FU1 and / or FU2 domain of any of the polypeptides of SEQ ID NOs: 1 to 4 and 8 to 24.

[0012] In specific embodiments, the Rspo1 protein of the present disclosure is a recombinant protein comprising any of the following: (i) any one of SEQ ID NOs: 1 to 4 and 8 to 24, or (ii) A combination of fragments of the Rspo1 protein of SEQ ID NO: 1, typically comprising the functional domains FU1 and FU2, and optionally the functional domain TSP.

[0013] In a specific embodiment, the Rspo1 protein of the present disclosure binds to the LGR4 receptor.

[0014] In specific embodiments, the Rspo1 proteins of the present disclosure induce the proliferation of functional beta cells as determined in an in vitro beta cell proliferation assay and / or an in vivo beta cell proliferation assay.

[0015] In a specific embodiment, the Rspo1 protein of the present disclosure is a protein comprising a functional fragment or a functional variant of a natural R-spondin1 polypeptide, preferably human R-spondin-1 of SEQ ID NO: 3 or 4; The Rspo1 protein exhibits at least 50%, 60%, 70%, 80%, 90%, 100% or more of the following activity compared to the native Rspondin-1: (i) binding affinity to the LGR4 receptor, as determined, for example, by an SPR assay; (ii) induction of functional β-cell proliferation, e.g., as determined by an in vitro β-cell proliferation assay; (iii) induction of functional β-cell proliferation, e.g., as determined by an in vivo β-cell proliferation assay; (iv) increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vitro beta cell proliferation assay; or (v) Increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vivo β-cell proliferation assay.

[0016] The above functional assays are described in more detail, for example, in the Examples below.

[0017] In a specific embodiment, the Rspo1 protein of the present disclosure is a protein comprising a functional variant of R-spondin1, wherein the functional variant comprises or essentially consists of a polypeptide having at least 70%, 80%, 90%, or at least 95% identity to the parent R-spondin1 polypeptide sequence, preferably at least 70%, 80%, 90%, or at least 95% identity to one of the polypeptides of SEQ ID NOs: 1-4 and SEQ ID NOs: 8-24.

[0018] In a specific embodiment, said functional variant of R-spondin1 differs from the corresponding native R-spondin1 sequence only by amino acid substitutions.

[0019] In a specific embodiment, the Rspo1 protein of the present disclosure is a fusion protein, for example, a fusion protein that includes the Fc region of an antibody.

[0020] In a specific embodiment, the Rspo1 protein of the present disclosure is a pegylated protein or a PASylated protein.

[0021] According to the present disclosure, the Rspo1 protein is particularly useful in treating type 1 or type 2 diabetes and / or in inducing beta cell proliferation and increased islet mass in vivo. In a specific embodiment, a therapeutically effective amount of the Rspo1 protein is administered subcutaneously or intravenously to a subject in need thereof.

[0022] In a specific embodiment of such in vivo uses of Rspo1 protein, the subject is a human subject.

[0023] The present disclosure also relates to a pharmaceutical composition comprising an Rspo1 protein as defined above, and one or more pharmaceutically acceptable excipients.

[0024] In specific embodiments, the pharmaceutical composition further comprises one or more additional pharmaceutical ingredients for treating or preventing diabetes, typically selected from the group consisting of cytokines, antiviral agents, anti-inflammatory agents, antidiabetic or hypoglycemic agents, cell therapy products (e.g., beta cell compositions), and immunomodulatory agents.

[0025] The present disclosure also relates to the use of an Rspo1 protein or analogue as defined herein in an in vitro method for inducing proliferation of beta cells, typically human beta cells.

[0026] Typically, the in vitro method comprises the following steps: (i) providing induced pluripotent stem cells (iPSCs), preferably iPSCs derived from human cells; (ii) differentiating the iPSCs into beta cells of the islets of Langerhans in vitro; (iii) culturing the differentiated β-cells under growth conditions; (iv) A sufficient amount of the Rspo1 protein or analog is added in step (ii) and / or (iii) to induce differentiation of the iPS cells and / or proliferation of the β cells.

[0027] Detailed Description [Definition] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0028] The term "amino acid" refers to naturally occurring amino acids 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 later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, e.g., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a compound that has a structure that differs from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.

[0029] Substitution refers to the replacement of a naturally occurring amino acid with another naturally occurring or non-natural amino acid. For example, during the chemical synthesis of a synthetic peptide, a natural amino acid can be easily substituted with another naturally occurring or non-natural amino acid.

[0030] As used herein, the term "protein" refers to any organic compound composed 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 composed primarily of two or more amino acid chains. It also includes, but is not limited to, glycoproteins or other known post-translational modifications. It also includes, but is not limited to, known natural or artificial chemical modifications of native proteins, such as glycoengineering, pegylation, hesylation, PASylation, etc., incorporation of unnatural amino acids, amino acid modifications for chemical conjugates or other molecules, etc.

[0031] As used herein, the term "recombinant protein" includes proteins that are prepared, expressed, made, 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.).

[0032] 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 Rspondin-1 polypeptide or its fragment or variant, as described below, and at least one other moiety. The other moiety is a polypeptide other than the Rspondin-1 polypeptide or its functional variant or fragment. 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). The second moiety may be the Fc region of an antibody, and therefore, such a fusion protein is referred to as an "Fc fusion protein".

[0033] 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 comprising an insertion, deletion, or substitution of 5, 10, 15, or 20 or fewer amino acids relative to a native human Fc region. Native human Fc regions can be of any of the IgG1, IgG2, IgG3, IgG4, IgA, IgA, IgD, IgE, or IgM isotypes. The human IgG heavy chain Fc region is generally defined as comprising amino acid residues from position C226 or P230 of an IgG antibody to the carboxyl terminus. The numbering of residues in the Fc region is the EU numbering system of Kabat (EU index). The C-terminal lysine of the Fc region (residue K447) may be removed, for example, during production or purification of the Fc fusion protein.

[0034] As used herein, the percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that 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, as described below.

[0035] The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm.

[0036] The percent identity between two nucleotide or amino acid sequences may be determined using an algorithm 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, and a "gap extend penalty" of 0.5. A "zero" "end gap 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. Generally, "percent identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, 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 with the same primary amino acid or nucleic acid sequence are identical, regardless of any chemical and / or biological modifications.

[0037] 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.).

[0038] [Rspo1 protein] The present disclosure relates to certain Rspo1 proteins or analogs thereof and their use as pharmaceuticals, particularly in the treatment of diabetes in subjects in need thereof, or to induce the in vivo or in vitro production of pancreatic beta cells (preferably human beta cells) in the islets of Langerhans.

[0039] As used herein, the term "Rspo1 protein" refers to either the native R-spondin1 protein encoded by the corresponding Rspo1 gene, or a functional equivalent thereof.

[0040] As used herein, the term "analog" refers to a non-protein compound that has the same or substantially the same properties as R-spondin1 protein, particularly with respect to at least one or more of the desired properties described in the following section.Such analog includes small molecules or synthetic organic molecules up to 2000 Da, preferably 800 Da or less, as well as peptidomimetics, aptamers and structural or functional mimetics of R-spondin1 protein.Analog also includes antibodies (hereinafter referred to as "agonist antibodies") that have binding specificity for LGR4 and have the same or substantially the same properties as R-spondin1 protein.

[0041] As used herein, the term "aptamer" refers to a strand of oligonucleotide (DNA or RNA) that can adopt a highly specific three-dimensional conformation.

[0042] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, and antibody variants (including derivatives) and antibody fragments.

[0043] In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. Light chains contain two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and heavy chain (VH) determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding.

[0044] The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-binding site and an antigenic determinant. Antibody-binding sites are composed primarily of residues derived from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody-binding site or influence the overall domain structure and thus the binding site. Complementarity-determining regions or CDRs represent amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each contain three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. The framework region (FR) represents the amino acid sequence intervening between the CDRs. Thus, the variable regions of the light and heavy chains typically comprise four framework regions and three CDRs, each of which has the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0045] Therefore, from among anti-LGR4 antibodies obtained by conventional techniques such as hybridoma technology and / or phage display technology, those skilled in the art may further select anti-LGR4 antibodies that exhibit at least one of the desired properties described in the next section, and screen for agonist antibodies by using functional assays further described in this example.

[0046] The term "Rspo1 protein" includes in particular any protein, including a functional fragment of the native R-spondin1 protein, a functional variant of the native R-spondin-1 protein, or a recombinant protein, in particular a fusion protein comprising said fragment or functional variant of the native R-spondin1 protein, all generally denoted "functional equivalents".

[0047] Native R-spondin1 protein typically comprises, from its N-terminus to C-terminus, a signal peptide (SP), two cysteine-rich furin-like domains (FU1 and FU2), a thrombospondin (TSP1) motif (TSP) and a basic amino acid-rich (BR) domain. Figure 13 provides a schematic diagram of the various domains of human R-spondin-1. R-spondin1 protein is known to bind to the LGR4 receptor.

[0048] Examples of the furin-like 1 domain (FU1) of human R-spondin1 include any of SEQ ID NOs: 13-15.

[0049] Examples of the furin-like 2 domain (FU2) of human R-spondin1 include any of SEQ ID NOs: 16-18.

[0050] In a specific embodiment, the Rspo1 protein is a protein comprising a human R-spondin1 polypeptide (preferably the polypeptide of any one of SEQ ID NOs: 2 to 4) or a functional fragment thereof.

[0051] Another example of an Rspo1 protein is a protein comprising the mouse R-spondin1 polypeptide of SEQ ID NO: 6 or a functional fragment thereof.

[0052] Examples of R-spondin1 polypeptides or functional fragments thereof for use in the Rspo1 proteins of the present disclosure are listed in Table 1 below.

[0053] [Table 1]

[0054] The following commercially available R-spondin1 proteins or functional fragments thereof may also be used in accordance with the present disclosure: -Full-length mouse Rspo1 recombinant protein with His tag (SinoBiological Ref. 50316-M08S); - Short length mouse Rspo1 recombinant protein (aa 21-135) (CliniScience Ref. LS-G16201-100); -Human Rspo1 recombinant protein produced in CHO cells (Peprotech Ref. 120-38); -Human Rspo1 recombinant protein with His tag produced in E. coli (Creative Biomart Reference RSPO1-1942H); -Human Rspo1 recombinant protein Fc tag produced in HEK293 cells: (Creative Biomart Ref. RSPO1-053H).

[0055] In a more specific embodiment, the Rspo1 protein is an isolated recombinant protein comprising any one of the polypeptides set forth in SEQ ID NOs: 1 to 4 and 8 to 24. In a more specific embodiment, the recombinant protein of the present invention is a fusion protein (e.g., an Fc fusion protein), typically comprising any one of SEQ ID NOs: 1 to 4 and 8 to 24.

[0056] [Functional equivalent of R-spondin 1] Further functional equivalents of R-spondin1 protein having similar advantageous properties as the native R-spondin1 protein can also be identified by screening candidate molecules and testing whether the candidate molecules maintain the desired functional properties of a reference native R-spondin1 protein (typically the human R-spondin1 protein of SEQ ID NO: 3 or 4).

[0057] In one embodiment, the functional equivalent of R-spondin1 binds to the LGR4 receptor.

[0058] For example, the above-mentioned functional equivalents of R-spondin1 bind to the LGR4 receptor with at least the same affinity as the corresponding native R-spondin1 (typically human R-spondin1 of SEQ ID NO: 3 or 4), which affinity is determined, for example, by an SPR assay.

[0059] In another embodiment, the functional equivalent of R-spondin1 inhibits the binding of native R-spondin1 (eg, human R-spondin1) to the LGR4 receptor as determined by a competitive binding assay.

[0060] In specific embodiments, the functional equivalent of R-spondin1 exhibits at least 50%, 60%, 70%, 80%, 90%, 100% or more of the following activity compared to the corresponding native R-spondin-1 (preferably human native R-spondin1): (i) binding affinity to the LGR4 receptor, as determined, for example, by an SPR assay; (ii) induction of functional β-cell proliferation, e.g., as determined by an in vitro β-cell proliferation assay; (iii) induction of functional β-cell proliferation, e.g., as determined by an in vivo β-cell proliferation assay; (iv) increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vitro beta cell proliferation assay; or (v) Increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vivo β-cell proliferation assay.

[0061] Further details of the assays and conditions for use in determining activity are disclosed in the experimental part below.

[0062] In various embodiments, a functional equivalent is a recombinant protein that exhibits one, two, three, four, five, or all of the above desired activities. In specific embodiments, a functional equivalent is a recombinant protein that exhibits at least the above desired activities (ii) through (v).

[0063] In a specific embodiment, the functional equivalent is a recombinant protein that exhibits at least 50%, 60%, 70%, 80%, 90%, and more preferably 100% or more of the desired activity compared to the corresponding native human R-spondin1 of SEQ ID NO: 3.

[0064] [Functional fragment] In a specific embodiment, a functional equivalent of R-spondin1 is a protein comprising a fragment of a native R-spondin1 polypeptide.

[0065] In specific embodiments, a "fragment of an R-spondin1 polypeptide" refers to a polypeptide having at least 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 consecutive amino acid residues of any of the polypeptides of SEQ ID NOs: 1-4 and SEQ ID NOs: 8-24.

[0066] A fragment of R-spondin1 is by definition at least one amino acid shorter than the full-length wild-type R-spondin1. In a specific embodiment, said fragment of R-spondin1 lacks the thrombospondin-1 domains (TSP1 and TSP2) and / or the basic amino acid-rich domain (BR).

[0067] In a specific embodiment, the fragment of R-spondin1 comprises at least 40 to 52 consecutive amino acids of the FU2 domain (e.g., residues 91 to 143 of human R-spondin1 of SEQ ID NO: 4) and / or at least 40 to 61 consecutive amino acids of the FU1 domain of the R-spondin1 protein (e.g., residues 34 to 95 of human R-spondin1 of SEQ ID NO: 4).

[0068] In a more specific embodiment, a "fragment of R-spondin1" refers to a polypeptide having: (i) any one of SEQ ID NOs: 1 to 4 and 8 to 24, or (ii) A combination of fragments of the R-spondin1 polypeptide of SEQ ID NO: 1 (typically comprising the functional domains FU1 and FU2, and optionally the functional domain TSP).

[0069] Thus, in a particular embodiment, a functional equivalent of R-spondin1 is a protein comprising: (i) any one of SEQ ID NOs: 1 to 4 and 8 to 24, or (ii) a combination of fragments of the R-spondin-1 polypeptide of SEQ ID NO: 1 (typically comprising the functional domains FU1 and FU2, and optionally the functional domain TSP); The proteins exhibit at least 50%, 60%, 70%, 80%, 90%, 100% or more of the following activities compared to the corresponding native R-spondin1: (i) binding affinity to the LGR4 receptor, as determined, for example, by an SPR assay; (ii) induction of functional β-cell proliferation, e.g., as determined by an in vitro β-cell proliferation assay; (iii) induction of functional β-cell proliferation, e.g., as determined by an in vivo β-cell proliferation assay; (iv) increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vitro beta cell proliferation assay; or (v) Increased glucose-stimulated insulin secretion (GSIS), for example, as determined by an in vivo β-cell proliferation assay.

[0070] [Functional mutant variants] In a specific embodiment, said functional equivalent is a protein comprising a functional variant of the functional domains FU1 and / or FU2 of R-spondin1 (typically human R-spondin1).

[0071] In a specific embodiment, said "functional variant" comprises or essentially consists of a polypeptide having at least 50%, 60%, 70%, 80%, 90% or at least 95% identity to a parent (natural) R-spondin1 protein or a functional fragment of a parent (natural) R-spondin1. In a specific embodiment, said functional variant has at least 50%, 60%, 70%, 80%, 90% or at least 95% identity to one of the parent polypeptides of any one of SEQ ID NOs: 1-4 and 8-24.

[0072] A functional variant may typically be a mutant variant obtained by amino acid substitution, deletion or insertion compared to the corresponding native polypeptide or a functional fragment thereof. In certain embodiments, a functional variant may have a combination of amino acid deletion, insertion or substitution throughout its sequence compared to the parent polypeptide. In certain embodiments, the functional variant differs from the corresponding native R-spondin1 sequence or a functional fragment thereof only through amino acid substitution with natural or non-natural amino acids, preferably through substitution of only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids with natural amino acids, in particular compared to one of the native R-spondin1 polypeptides of SEQ ID NOs: 1-4 and 8-24. In a specific embodiment, the functional variant is a mutant variant having 1, 2 or 3 amino acid substitutions compared to the human R-spondin1 of SEQ ID NO: 4.

[0073] In another embodiment, the functional mutant variant is a polypeptide having at least 50%, 60%, 70%, 80%, 90% or at least 95% identity to a parent (natural) R-spondin1 protein or a functional fragment thereof, for example to any of the polypeptides of SEQ ID NOs: 1 to 4 and 8 to 24. The polypeptide comprises an FU1 domain that is 100% identical to the FU1 domain of the corresponding natural R-spondin1 protein (typically, a human R-spondin1 protein).

[0074] In another embodiment, the functional mutant variant is a polypeptide having at least 50%, 60%, 70%, 80%, 90% or at least 95% identity to a parent (natural) R-spondin1 protein or a functional fragment thereof, for example to any of the polypeptides of SEQ ID NOs: 1 to 4 and 8 to 24. The polypeptide comprises a FU2 domain that is 100% identical to the FU2 domain of the corresponding natural R-spondin1 protein (typically, human R-spondin1 protein).

[0075] In another embodiment, the functional mutant variant is a polypeptide having at least 50%, 60%, 70%, 80%, 90% or at least 95% identity to a parent (natural) R-spondin1 protein, for example to any of the polypeptides of SEQ ID NOs: 1 to 4 and 8 to 24. The polypeptide comprises FU1 and FU2 domains that are 100% identical to the corresponding FU1 and FU2 domains, respectively, of a natural R-spondin1 protein (typically, a human R-spondin1 protein).

[0076] In more specific embodiments, the amino acid sequence of said functional variant may differ from the native R-spondin1 sequence or functional fragment thereof mostly through 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.

[0077] In the context of the present disclosure, conservative substitutions may be defined by substitutions within a class of amino acids as 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 the turn are A, C, D, E, G, H, K, N, Q, R, S, P, and forming 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 hydrophobicity / hydrophilicity and residue weight / size may also be substantially retained in the variant polypeptides compared to the parent polypeptides of any one of SEQ ID NOS: 1-4 or 8-24.

[0078] In specific embodiments, a functional variant comprises a polypeptide that is identical to any one of SEQ ID NOs: 1-4 or 8-24, except for one, two or three amino acid residues that are substituted by another naturally occurring amino acid, preferably by a conservative amino acid substitution as defined above.

[0079] Xu et al. (Journal of Biological Chemistry, 2015, Vol. 290, No. 4, pp. 2455-2465) reported the crystal structure of the LGR4-Rspo1 complex. They reported that the two central tandem FU1 / FU2 domains of human Rspo1 are required for binding to the LGR receptor, particularly residues 34-135. More specifically, they reported that the connecting loops of the β4-β3, β5-β6, and β8-β7 hairpins of human Rspo1, located on the same side of Rspo1, are involved in binding to LGR4.

[0080] Therefore, in other specific embodiments that may be combined with the above embodiments, the functional mutant variant of human R-spondin1 comprises at least the following amino acid residues of human R-spondin1 protein: Asp-85, Arg-87, Phe-107, Asn-109, Phe-110 and Lys-122. In other specific embodiments that may be combined with the above embodiments, the conserved cysteines at amino acid residues 53, 56, 94, 97, 102, 106, 111, 114, 125 and 129 may also not be mutated (see also Figure 3 in Xu et al., 2015).

[0081] Furthermore, those skilled in the art will understand that residues that are conserved among various species are important for maintaining the proper structure and therefore refrain from mutating such amino acid positions. Alternatively, at many sites, one or more amino acid positions exhibit conservative variations among species variants and / or among other members of the Rspo family (e.g., Rspo2, Rpo3, and Rspo4); those 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-spondin1s with such conservative variations.

[0082] In particular, in other specific embodiments, the functional variant therefore comprises a polypeptide sequence substantially identical to human R-spondin1, except that it contains one or more of the following amino acid substitutions or deletions: K115Q, S134T, G138S, S143G, Q163R, Q164K, R170K, V184G, A188T, A189T, R198K, V204T, N226H, L227P, E231N, A235P, A237S, G238N, R242H, ΔQ248, Q251P, V254T, A260V, A263T.

[0083] These amino acid substitutions correspond to the amino acid substitutions from human R-spondin1 to mouse R-spondin1 when the two sequences are aligned as shown in FIG.

[0084] Other sites within R-spondin1 may tolerate further variation without affecting function. For example, one skilled in the art may also identify other possible amino acid substitutions or insertions to identify functional variants by comparing alignments of human Rspondin1 and other mammalian Rspondin1 proteins (primate, rat, dog, cat, etc.).

[0085] Also, the functional assays described above and in the experimental part below may be used to screen for the ability of any functional variant of R-spondin1 to maintain the advantageous desired properties of the native R-spondin1 polypeptide.

[0086] Recombinant proteins of the present disclosure In certain embodiments, the Rspo1 protein of the present disclosure is a soluble and / or recombinant protein.

[0087] In a more specific embodiment, the recombinant Rspo1 protein is a fusion protein, more specifically an Fc fusion protein.

[0088] Various polypeptides other than the R-spondin1 polypeptide can be fused to the R-spondin1 polypeptide or the above-mentioned functional equivalents thereof (particularly fragments or mutant variants) 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 increase the activity of the protein, and to promote the oligomerization of the protein, etc.).

[0089] Many polypeptides can facilitate the identification and / or purification of the recombinant fusion protein of which they are a part. For example, polyarginine and polyhistidine are included. Polypeptides containing polyarginine allow for efficient purification by ion exchange chromatography.

[0090] 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 an Rspondin-1 polypeptide, thereby forming an Fc fusion protein of the present disclosure.

[0091] Another modification of the antibody contemplated by the present disclosure is the conjugation or protein fusion of at least Rspondin-1 polypeptide (or its functional fragment or variant) with serum protein (for example, human serum albumin or its fragment) to increase the half-life of the resulting molecule. Such an approach is described, for example, in Ballance et al. (EP 0322094).

[0092] Another possibility is a fusion protein of the present disclosure that includes a protein capable of binding to a serum protein, e.g., human serum albumin (i.e., an anti-HSA fusion protein), to increase the half-life of the resulting molecule, and includes an anti-HSA binding moiety derived from, for example, a Fab or nanobody that binds to HSA or any other domain-type structure, such as a DARPin, nanophytin, phynomers, etc. Such an approach is described, for example, in Nygren et al. (EP 0486525).

[0093] The recombinant fusion proteins of the present disclosure can include polypeptides containing leucine zippers or other multimerization motifs. Among 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.

[0094] Fusion proteins may also contain one or more peptide linkers. Generally, peptide linkers are stretches of amino acids that serve 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 to 30 amino acids in length. Examples of peptide linkers include, but are not limited to, -Gly-Gly-, GGGGS (SEQ ID NO: 25), and (GGGGS)n (where n is 1 to 8, typically 4). Linking moieties are described, for example, in Huston, J.S., et al., Proc. Natl. Acad. Sci. 85: 5879-83 (1988), Whitlow, M., et al., Protein Engineering 6: 989-95 (1993), Newton, D.L., et al., Biochemistry 35: 545-53 (1996), and U.S. Patent Nos. 4,751,180 and 4,935,233.

[0095] The recombinant Rspo1 protein of the present disclosure can include an R-spondin1 protein or a functional equivalent thereof that lacks its normal signal sequence and instead has a different signal sequence replacing it. The choice of signal sequence depends on the type of host cell in which the recombinant protein is produced, and the different signal sequence can replace the native signal sequence.

[0096] Another modification of the R-spondin1 protein herein or related recombinant Rspo1 protein contemplated by the present disclosure is pegylation or related techniques such as hexylation or PASylation.

[0097] More generally, Rspo1 proteins 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.

[0098] The Rspo1 proteins of the present disclosure can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate the Rspo1 protein, it is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the Rspo1 protein. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).

[0099] 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)alkoxypolyethylene glycol or aryloxypolyethylene 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 protein of the present disclosure is pegylated.

[0100] Another modification of Rspo1 proteins or related recombinant proteins 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 proteins of the present disclosure are PASylated.

[0101] Xten technology is reviewed, for example, in Nature Biotechnology volume 27 number 12 2009: 1186-1192.

[0102] Nucleic acid molecules encoding proteins of the present disclosure Also disclosed herein are nucleic acid molecules encoding the Rspo1 proteins of the present disclosure.

[0103] Exemplary nucleotide sequences are those that encode any one of the amino acid sequences of Examples #1-#21 defined in Table 1, particularly those that encode any one of SEQ ID NOs: 1-4 and SEQ ID NOs: 8-24, where nucleic acid sequences are readily derived from Table 1, using the genetic code and optionally taking into account codon bias depending on the host cell type.

[0104] 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).

[0105] Nucleic acids may be present in whole cells, cell lysates, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants, e.g., 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. Nucleic acids of the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In one embodiment, the nucleic acids may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[0106] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. Once encoding DNA fragments (e.g., fragments encoding Rspo1) are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques. In these manipulations, the Rspo1-encoding DNA fragment 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 further include nucleotide sequences encoding recombinant fusion proteins, particularly Fc fusion proteins comprising the coding sequence of any one of amino acid SEQ ID NOS: 1-4 and 8-24 operably linked to the coding sequence of an Fc region.

[0107] 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 such that a protein is expressed under the control of a desired promoter.

[0108] [Generation of transfectomas producing the Rspo1 protein of the present disclosure] The Rspo1 proteins of the present disclosure can be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods known in the art.

[0109] For example, to express an Rspo1 protein or its corresponding fragment, 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 so that the gene is operably linked to transcriptional and translational control sequences.

[0110] 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 protein-encoding gene 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).

[0111] The recombinant expression vector can encode a signal peptide that facilitates secretion of the recombinant protein from the host cell. The Rspo1-encoding gene 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 or a heterologous signal peptide (i.e., a signal peptide from a non-Rspo1 protein).

[0112] In addition to the gene encoding the Rspo1 protein, the recombinant expression vectors disclosed herein carry regulatory sequences that control the expression of the recombinant protein in host cells. 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. Those skilled in the art will understand that the design of an expression vector, including the selection of regulatory sequences, depends on factors such as the choice of host cell to be transformed and the desired protein expression level. Regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high-level protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or P-globin promoter, may be used. Furthermore, regulatory elements composed of sequences from different sources also exist, such as the SV40 early promoter and the SRa promoter system, which includes sequences derived from the long terminal repeat of human T-cell leukemia virus type 1.

[0113] In addition to the gene encoding the Rspo1 protein and regulatory sequences, the recombinant expression vector of the present disclosure may carry additional sequences, such as sequences regulating replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells 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).

[0114] For expression of the Rspo1 protein, an expression vector encoding the recombinant protein is transfected into a host cell 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 the protein in eukaryotic cells (e.g., mammalian host cells, yeast, or filamentous fungi) is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, functional Rspo1 protein.

[0115] In a specific embodiment, a cloning or expression vector of the present disclosure comprises one of the coding sequences for the Rspo1 protein of any one of SEQ ID NOs: 1-4 and 8-24, operably linked to a suitable promoter sequence.

[0116] 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), the CHOK1 dhfr+ cell line, NSO myeloma cells, COS 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 the mammalian host cells, the recombinant protein is produced by culturing the host cells for a period of time sufficient to express the recombinant protein in the host cells, and, optionally, secrete the protein into the culture medium in which the host cells are grown.

[0117] Recombinant proteins of the present disclosure can be recovered and purified from the culture medium after secretion, for example, using standard protein purification methods.

[0118] 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 expressing an Rspo1 protein comprising any one of SEQ ID NOs: 1 to 4 and 8 to 24, each operably linked to a suitable promoter sequence.

[0119] For example, the present disclosure relates to a host cell comprising at least the nucleic acid of SEQ ID NO: 5 encoding the human R-spondin1 protein.

[0120] The latter host cells may then be further cultured under conditions suitable for the expression and production of the recombinant proteins of the present disclosure.

[0121] [Pharmaceutical composition] In another aspect, the present disclosure provides compositions (e.g., pharmaceutical compositions) comprising one or a combination of the Rspo1 proteins or analogs thereof disclosed herein, which may include one or a combination (e.g., two or more different) of the Rspo1 proteins, as described above.

[0122] For example, the pharmaceutical composition includes a recombinant protein comprising a polypeptide of any one of SEQ ID NOs: 1 to 4 and SEQ ID NOs: 8 to 24, or a functional variant thereof, formulated together with a pharmaceutically acceptable carrier.

[0123] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, i.e., in combination with other drugs. For example, the combination therapy may include a recombinant protein comprising a polypeptide of any one of SEQ ID NOS: 1-4 and 8-24, or a functional variant thereof, in combination with at least one anti-inflammatory agent or another antidiabetic 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 Rspo1 proteins of the present disclosure.

[0124] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The carrier should be suitable for parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration (e.g., by injection or infusion).

[0125] 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 Rspo1 protein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0126] Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. (Remington and Gennaro, 1995). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, and albumin to prevent protein loss on the vial surface.

[0127] 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.

[0128] The pharmaceutical compositions of the present disclosure can be formulated for oral, intranasal, sublingual, subcutaneous, intramuscular, intravenous, transdermal, parenteral, topical, ocular, or rectal administration, etc. The Rspo1 protein as an active ingredient can be administered to animals and humans alone or in combination with other active ingredients, in unit dosage form, in admixture with conventional pharmaceutical carriers.

[0129] 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.

[0130] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle for injectable formulations, which may in particular be an isotonic, sterile, saline solution (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), or a dry, in particular lyophilized, composition, which, on addition of sterile water or saline, as the case may be, allows the constitution of an injectable solution.

[0131] 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.

[0132] To prepare pharmaceutical compositions, an effective amount of Rspo1 protein may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0133] Pharmaceutical forms suitable for injection use may include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders or lyophilized materials for the extemporaneous preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and 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.

[0134] 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 normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0135] The Rspo1 proteins of the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) that are formed with inorganic acids (e.g., hydrochloric or phosphoric acid) or organic acids (e.g., acetic, oxalic, tartaric, mandelic, etc.). Salts formed with the 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.).

[0136] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., 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 (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferable to include an isotonic agent (e.g., sugar or sodium chloride). Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0137] Sterile injectable solutions are prepared by mixing the required amount of the active compound, i.e., Rspo1 protein, in an appropriate solvent with various other ingredients as listed above, as needed, followed by filtration sterilization. Generally, dispersions are prepared by mixing various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0138] 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 employed.

[0139] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first 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 vehicles that can be used will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous solution or injected at the proposed injection site (see, e.g., "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 dose for the individual subject.

[0140] The Rspo1 proteins or analogs thereof of the present disclosure may be formulated into a therapeutic mixture to contain about 0.01 mg to 1000 mg / kg or 1 mg to 100 mg / kg. Multiple doses are also possible.

[0141] Suitable formulations for infusion or subcutaneous injection of recombinant proteins have been 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.

[0142] Uses and Methods of the Proteins of the Present Disclosure The Rspo1 proteins of the present disclosure have in vitro and in vivo utilities. For example, these molecules can be administered to cells in culture, e.g., in vitro, ex vivo, or in vivo, or can be administered to a subject (e.g., in vivo) to treat or prevent various disorders.

[0143] 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 displaying the symptoms or symptomology of the disease, condition, or disorder (i.e., halting further progression of the symptoms and / or symptomology); and (2) ameliorating the disease; e.g., improving the disease, condition, or disorder in an individual experiencing or displaying the symptoms or symptomology of the disease, condition, or disorder (i.e., reversing the symptoms and / or symptomology), such as reducing the severity of the disease or alleviating or alleviating one or more symptoms of the disease.

[0144] 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).

[0145] The disclosed Rspo1 proteins or analogs thereof can induce proliferation of pancreatic β-cells in vivo and reconstitute functional insulin-secreting islets of Langerhans, and may therefore be used to treat diabetic patients or patients in need of functional insulin-secreting β-cells or patients with disorders related to hyperglycemia or patients with deficient glucose-stimulated insulin secretion.

[0146] 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.

[0147] Examples of diabetes include, but are not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, and latent autoimmune diabetes in adults (LADA).

[0148] Accordingly, the present disclosure relates to a method for treating one of the above-disclosed disorders in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the above-disclosed Rspo1 protein or analog (typically a recombinant protein comprising a polypeptide of any one of SEQ ID NOS: 1-4 and 8-24, or a functional variant thereof).

[0149] In certain embodiments, the subject is selected from among patients with low Rspo1 gene expression.

[0150] The Rspo1 proteins or analogs for use as disclosed above may be administered as the sole active ingredient, for example, for the treatment or prevention of the above-mentioned diseases, or may be administered in combination with, or as adjuvants to, other agents such as cytokines, antiviral agents, anti-inflammatory agents, antidiabetic or hypoglycemic agents, cell therapy products (e.g., beta cell compositions), and immunomodulatory agents.

[0151] For example, the Rspo1 proteins or analogs for use disclosed above may be used in combination with cell therapy, particularly beta cell therapy.

[0152] As used herein, the term "cell therapy" refers to a therapy involving the in vivo administration of at least a therapeutically effective amount of a cell composition to a subject in need thereof. The cells administered to a 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. Such β cells may be produced using Rspo1 protein by in vitro methods described below.

[0153] A cell therapy product refers to a cell composition administered to the patient for therapeutic purposes, comprising a therapeutically effective dose of cells, and optionally additional excipients, adjuvants, or other pharmaceutically acceptable carriers.

[0154] 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 analogs, derivatives of carbamoylmethylbenzoic acid; typically insulin receptors, SLGT2 inhibitors, GABR targeting molecules, and IL2R targeting molecules.

[0155] In accordance with the above, the present disclosure provides yet another aspect: The method defined above, comprising the step of co-administering (e.g., simultaneously or sequentially) a therapeutically effective amount of an Rspo1 protein or analog of the present invention 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, or a cell therapy product (e.g., a beta cell composition) as described above.

[0156] In another embodiment, the Rspo1 proteins or analogs of the present disclosure can be used in in vitro methods to induce proliferation of pancreatic beta cells and / or islets of Langerhans.

[0157] Thus, in one aspect, the present disclosure further provides a method for producing beta cells in vitro, comprising the steps of: (i) providing beta cells; (ii) culturing the beta cells in the presence of an effective amount of the Rspo1 protein or analog of the present disclosure under conditions that induce proliferation of the beta cells.

[0158] In a specific embodiment of the above-mentioned production method, the beta cells are primary cells, preferably primary cells derived from a subject in need of beta cell therapy or transplantation of islets of Langerhans.

[0159] In another specific embodiment, the beta cells provided in step (i) are obtained from iPS cells after differentiating the iPS cells into beta cells.

[0160] Thus, in certain embodiments, the present disclosure relates to a method for producing beta cells in vitro from induced pluripotent stem cells, comprising the steps of: (i) providing induced pluripotent stem cells (iPSCs); (ii) differentiating the iPSCs in vitro into beta cells in the islets of Langerhans; and (iii) culturing the differentiated β cells under growth conditions; A sufficient amount of the Rspo1 protein or analog is added in step (ii) and / or step (iii) to differentiate the iPS cells and / or induce proliferation of the β cells.

[0161] Methods for differentiating iPSCs into beta cells of the islets of Langerhans have been previously described in the art, for example, in 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.

[0162] The disclosure further includes compositions comprising the beta cells obtainable or obtained by the above method, and their use as cell therapy products, e.g., in a subject for treating diabetes, preferably type 1 diabetes. Methods for transplanting beta cells or islets of Langerhans into a patient are described, 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.

[0163] Kits consisting of the compositions disclosed herein (e.g., the Rspo1 proteins of the present disclosure) and instructions for use are also within the scope of the present disclosure. The kits can further include at least one additional reagent, or one or more additional antibodies or proteins. The kits typically include a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded material supplied on or with the kit, or which otherwise accompanies the kit. The kit may further include a tool for diagnosing whether a patient belongs to the group that will respond to Rspo1 treatment, as defined above.

[0164] Another therapeutic strategy is based on the use of the Rspo1 protein disclosed herein as an agent to proliferate β-cells isolated from samples of human subjects.

[0165] Accordingly, 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 to induced pluripotent stem cells; (c) differentiating the iPS cells into β cells; (d) growing the β cells in vitro, optionally with other cells, in the presence of an Rspo1 protein or analog, such as a recombinant protein comprising any one of SEQ ID NOS: 1 to 4 and 8 to 24, or a functional variant 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.

[0166] The present disclosure further relates to the use of the Rspo1 protein disclosed in this specification (e.g., a recombinant protein comprising any one of SEQ ID NOs: 1 to 4 and SEQ ID NOs: 8 to 24, or a functional variant thereof) as an agent for proliferating beta cells in vitro.

[0167] The present disclosure also relates to the Rspo1 proteins disclosed herein (such as recombinant proteins comprising any one of SEQ ID NOs: 1-4 and SEQ ID NOs: 8-24, or functional variants thereof) for in vivo use as agents for inducing beta cell proliferation in humans, particularly in subjects who have lost functional beta cells, typically subjects suffering from diabetes.

[0168] Accordingly, 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 an Rspo1 protein or analog disclosed herein, typically an Rspo1 protein; and (ii) administering in the subject an effective amount of a beta cell composition; The effective amount of Rspo1 protein or analog is capable of increasing proliferation of the β-cell composition. Steps (i) and (ii) can be performed simultaneously or sequentially, and in particular, either step (i) or step (ii) is administered to the subject first.

[0169] 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. [Brief explanation of the drawings]

[0170] [Figure 1] RT-qPCR analysis of R-spondin gene expression in the pancreas of WT mice. Rspo1 is expressed in the mouse pancreas, whereas Rspo2 and Rspo4 are undetectable. (n=5, age=3 months, p<0.0001 (****), p<0.001 (***), p<0.01 (**), and p<0.05, respectively.) [Figure 2] RT-qPCR analysis of Rspo1 expression in mouse pancreas from embryonic day 15.5 (E15.5) to 9 months of age (n=5, p<0.0001(****), p<0.001(***), p<0.01(**), and p<0.05(*)). [Figure 3] RNAscope of adult pancreas labeled with Rspo1 probe. Expression of both RNAs is restricted to acinar cells (intracellular puncta) within the exocrine compartment. [Figure 4] IPGTT in Rspo1KO mice. Loss of Rspo1 leads to improved glucose tolerance with a significant reduction in blood glucose peaks. (n=5, age=2.5 months, p<0.0001 (****), p<0.001 (***), p<0.01 (**), and p<0.05 (*)) [Figure 5] Quantitative analysis of the pancreas of Rspo1KO mice. Rspo1 loss does not induce structural changes in the islets of Langerhans, and the total islet surface remains unchanged upon Rspo1 loss (A). Indeed, no changes were observed in the number of insulin-producing (B), glucagon-producing (C), or somatostatin-producing cells (D) in Rspo1KO mice. (n=5; age=3 months, p<0.0001 (****), p<0.001 (***), p<0.01 (**), p<0.05 (*)) [Figure 6]Body weight and basal blood glucose of wild-type mice treated with Rspo1 recombinant protein. Rspo1 recombinant protein treatment did not induce any changes in body weight or basal blood glucose (n=6; age at start of treatment=2 months, p<0.0001 (****), p<0.001 (***), p<0.01 (**), and p<0.05 (*)). [Figure 7] IPGTT and insulinemia measurements upon Rspo1 treatment. Treated animals have better glucose tolerance, with a stronger reduction in blood glucose peaks and a faster return to normoglycemia compared to age-matched control mice (A). The improvement in glucose tolerance is caused by an increase in glucose-stimulated insulin secretion upon Rspo1 administration (B) (n = 6, age = 3 months, p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*)). [Figure 8] Immunofluorescence analysis of paraffin pancreatic sections upon Rspo1 administration. Mice treated with Rspo1 recombinant protein show significant islet hypertrophy (light gray) and an increased number of proliferating β-cells, labeled with BrdU (white). [Figure 9] Quantitative analysis of WT pancreas upon injection of Rspo1 recombinant protein. Mice receiving daily injections of Rspo1 showed a significant increase in β-cell proliferation. As a result, pancreatic islet area was increased in mice treated with recombinant Rspo1 compared with age-matched controls injected with saline alone. Rspo1 recombinant protein administration significantly increased β-cell mass (C) but had no effect on α-cell number (D). (n = 6, age = 3 months, p < 0.0001 (****), p < 0.001 (***), p < 0.01 (**), and p < 0.05 (*)). [Figure 10]Rspo1 treatment induces functional β-cell neogenesis upon β-cell ablation. WT mice were treated with high doses of streptozotocin (STZ) to ablate β-cells and then treated with Rspo1 (or saline) while overtly diabetic (blood glucose ≥ 300 mg / dL). Saline-treated animals developed severe hyperglycemia, whereas Rspo1-treated counterparts experienced a glycemic peak followed by gradual normalization of blood glucose levels. Quantitative immunohistochemical analysis (percentages in the colored rectangle) over the course of these experiments clearly demonstrated β-cell loss after STZ. Interestingly, upon Rspo1 treatment, a progressive increase in the number of insulin+ cells was observed, and this continued increase ultimately led to replenishment of the total β-cell mass. [Figure 11] Rspo1 treatment induces human β-cell proliferation. Human pancreatic islets were cultured for 5 days in the presence or absence of Rspo1 and in the presence of BrdU. Immunohistochemical analysis showed very little proliferation of insulin-producing cells (white dots) in the control (left). Interestingly, Rspo1 treatment (right) clearly demonstrated a significant increase in the number of proliferating human β-cells, demonstrating that Rspo1 can also induce human β-cell proliferation. [Figure 12] Alignment of human and mouse sequences of Rspondin1 obtained online using Clustal Omega using default settings ( https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). [Figure 13] 1 provides a schematic representation of the different domains for human Rspondin-1. [Figure 14] Min6 cells were treated with various concentrations of human recombinant Rspo1 (hR1) for 24 hours. Quantitation of Min6 revealed that hR1 could significantly stimulate immortalized mouse β cells at concentrations of 200 nM and 400 nM. [Figure 15] Recombinant hR1 was purified from endotoxin and incubated with Min6 cells at various concentrations. After 24 hours, the number of Min6 cells was significantly higher with 400 nM and 1 μM hR1 exposure compared to the control. [Figure 16] Quantitative analysis demonstrated that a single dose of hR1 was able to significantly increase the number of proliferating β-cells in WT mice. [Figure 17] Quantitative immunostained β-cell studies demonstrated that long-term treatment with hR1 significantly increased the number of proliferating β-cells and the overall size of the islets compared with PBS-injected controls. [Example]

[0171] [Detailed protocol for determining the activity of functional equivalents of the R-spondin 1 native protein] 1. Binding affinity to the LGR4 receptor as determined by SPR assay Surface plasmon resonance (SPR) was performed using a Biacore 3000 instrument (Biacore, Uppsala, Sweden). Ligand (mouse Lgr4) immobilization was achieved by activation of a dextran-coated CM5 chip followed by covalent binding of the ligand to the chip surface. After ligand stabilization, purified Rspo1 recombinant protein (100 mM) was flowed over the immobilized ligand surface, and the binding response of the analyte to the ligand was recorded. The level of interaction was expressed in response units (RU), with the maximum corresponding to the maximum level of affinity / interaction.

[0172] 2. Induction of Functional β-Cell Proliferation as Determined by an In Vitro β-Cell Proliferation Assay For proliferation assays with Rspo1 recombinant protein treatment, cells were seeded at a density of 150,000 cells / well in 6-well plates on glass and coverslips and maintained in serum-free standard culture medium (supplemented with 1% penicillin / streptomycin) 12 hours before treatment. Cells were further cultured for 5 minutes, 1 hour, 6 hours, and 24 hours using serum-free standard culture medium containing 67 ng / ml R1 or medium alone (control). After treatment, coverslips were first washed with PBS, then fixed in 4% PFA for 5 minutes, permeabilized in 0.1% Triton for 10 minutes, and stored in PBS at 4°C with agitation. Prior to immunolabeling, cells were blocked for 45 minutes in blocking solution (PBS, 10% FCS) and then incubated overnight at 4°C with primary antibody (Ki67 1:50, Dako, M7249). Subsequently, cells were washed with PBS (3 × 5 min) and incubated with secondary antibody (e.g., donkey anti-rat IgG secondary antibody, Alexa Fluor 488 conjugate, 1:1000) for 45 min. Coverslips were mounted with mounting medium containing DAPI (Vectashield, H-1200) and processed using a ZEISS Axiomanager Z1 Imaging System. Proliferation was quantified by counting the number of Ki67+ cells per section and normalizing to the total cell number per section.

[0173] 3. Induction of Functional β-Cell Proliferation as Determined by In Vivo β-Cell Proliferation Assay Transgenic mouse strains and 129-SV wild-type animals (Charles River) were housed and used according to the guidelines of the Belgian regulations for animal care and with the approval of the local ethical committee.

[0174] Rspo1 recombinant protein (SinoBiologic, 50316-M08S) was dissolved in PBS and administered intraperitoneally daily at a concentration of 400 μg / kg.

[0175] To assess cell proliferation upon Rspo1 addition, WT mice were treated with Rspo1 followed by BrdU (1 mg / ml via drinking water) for 7 days and then assayed. Cells that incorporated BrdU during DNA replication were detected using immunohistochemistry.

[0176] For immunohistochemistry, tissues are isolated, fixed in 4% PFA for 30 minutes at 4°C, dehydrated, embedded in paraffin, and sectioned onto 6 μm slides. Sections are rehydrated with decreasing concentrations of alcohol (xylene, 100% ethanol, 80% ethanol, 60% ethanol, 30% ethanol, and water) and then resuspended in blocking buffer (PBS). The slides were treated with 10% fetal calf serum (FCS) and incubated with primary antibodies overnight at 4°C. For experiments with mouse Rspo1, the primary antibodies used were guinea pig polyclonal anti-insulin (1 / 500), mouse monoclonal anti-glucagon (1 / 500), and mouse fluorescein-conjugated anti-bromodeoxyuridine (BrdU) (1 / 50). Slides were then incubated with secondary antibodies (1 / 1000) for 45 minutes at room temperature and processed using a ZEISS Axiomanager Z1 Imaging System. BrdU counts were assessed by manually counting proliferating cells within the islets of Langerhans and normalizing the final count to the total islet surface. All values ​​are reported as the mean ± SEM of datasets from at least five animals. Data were analyzed using Prism software (GraphPad) by first determining whether they followed a normal distribution using the D'Agostino-Parson Omnibus normality test. If this was not the case, an unpaired / nonparametric Mann-Whitney test was used. Conversely, an unpaired t-test (comparing two groups) or an unpaired Anova test (comparing more than two groups) was used, assuming a Gaussian distribution. p<0.0001 ( **** ), p<0.001( *** ), p<0.01( ** ), p<0.05( * ), the results are considered significant.

[0177] 4. Increased glucose-stimulated insulin secretion (GSIS) as determined by an in vitro beta cell proliferation assay To evaluate GSIS induced by Rspo1 recombinant protein supplementation, MIN6 cells were incubated with low-glucose (2 mM) or high-glucose (25 mM) serum-free standard culture medium for 2 hours, then treated with serum-free standard culture medium containing Rspo1 (67 ng / ml) or medium alone (control) for an additional 2 hours. The medium was then collected and centrifuged at 2000 × g for 3 minutes at 4 °C to collect the supernatant, which was then stored at -20 °C.

[0178] Insulin concentrations in MIN6 supernatants were assessed by ELISA immunoassay (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions. All reagents and samples were warmed to room temperature before use. Absorbance was read at 450 nm using a spectrophotometer (Sunrise Basic Tecan, Crailsheim, Germany) and supplemented with Tecan Magellan data analysis software. Insulin concentrations were calculated using a quadratic equation in Microsoft Excel. A calibration curve was calculated by plotting the known absorbance values ​​of each calibrator (except calibrator 0) against the average of the corresponding insulin concentration values.

[0179] 5. Increased glucose-stimulated insulin secretion (GSIS) as determined by an in vivo beta-cell proliferation assay Transgenic mouse strains and 129-SV wild-type animals (Charles River) were housed and used in accordance with the guidelines of the Belgian regulations for animal care and with the approval of the local ethical committee. Mouse Rspo1 recombinant protein was obtained from SinoBiological (50316-M08S).

[0180] Rspo1 recombinant protein was dissolved in PBS and administered intraperitoneally daily at a concentration of 400 μg / kg for 5 weeks. For insulinemia measurements, mice were anesthetized using isoflurane delivered in oxygen at a flow rate of 1 L / min. Whole blood samples were collected from the retroorbital sinus using glass capillaries into K3EDTA blood collection tubes. To measure basal insulinemia, blood samples were collected after 6 hours of fasting. To assess glucose-stimulated insulin secretion, an additional blood sample was collected 2 minutes after intraperitoneal injection of 2 g / kg body weight of D-(+)-glucose. The whole blood samples were immediately cooled in ice water. Plasma was separated by centrifugation at 2000 × g for 7 minutes at 4 °C. The resulting plasma was transferred to pre-cooled tubes, quickly frozen in liquid nitrogen, and finally stored at -80 °C.

[0181] Insulin concentrations in mouse plasma samples were measured using an ELISA immunoassay (Mercodia, Uppsala, The assay is performed by a lab at 1000 rpm in a 2000 rpm, ...

[0182] [material and method] (cell culture) Min6 cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 25 mmol / L glucose, supplemented with 15% heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 100 μg / ml L-glutamine, in a humidified 5% CO2, 95% air atmosphere. For proliferation assays with R1 treatment, cells were seeded into 6-well plates at a density of 150,000 cells / well and incubated with various concentrations of the tested molecules for 24 hours. Subsequently, cells were washed with phosphate-buffered saline (PBS), lifted from the plate with trypsin-EDTA, and manually quantified in a TOMA chamber.

[0183] (Animal Care and Operation) The mouse protocol was reviewed and approved by the Institutional Ethics Committee of the University of Nice (Ciepal-Azur), and all colonies were maintained in accordance with European animal research guidelines. Transgenic mouse strains and 129-SV wild-type animals (Charles River) were housed and used according to the guidelines of the Belgian Regulation for Animal Care and with the approval of the local ethical committee.

[0184] Rspo1 recombinant protein (SinoBiological, 50316-M08S; Peprotech, 120-38) was dissolved in PBS and administered intraperitoneally. To assess cell proliferation in response to Rspo1, WT mice were treated with Rspo1 followed by BrdU (1 mg / ml in drinking water) for 7 days before testing. Cells that incorporated BrdU during DNA replication were detected using immunohistochemistry.

[0185] (immunohistochemistry) Tissues were isolated, fixed in 4% PFA for 30 minutes at 4°C, dehydrated, embedded in paraffin, and sectioned onto 6-μm slides. Sections were rehydrated in decreasing alcohol concentrations (xylene, 100% ethanol, 80% ethanol, 60% ethanol, 30% ethanol, and water), then treated with blocking buffer (PBS 10% fetal calf serum-FCS) and incubated with primary antibodies overnight at 4°C. The primary antibodies used were: guinea pig polyclonal anti-insulin (1 / 500), mouse monoclonal anti-glucagon (1 / 500), goat monoclonal anti-somatostatin (1 / 250), rabbit monoclonal anti-amylase (1 / 100), rat Ki67 (1 / 50), and mouse fluorescein-conjugated anti-bromodeoxyuridine (BrdU) (1 / 50). Slides were then incubated with secondary antibodies (used at 1 / 1000) for 45 minutes at room temperature and processed using a ZEISS Axiomanager Z1 and Vectra Polaris Automated Quantitative Pathology Imaging System.

[0186] In situ RNA detection of Rspo1 (probe 401991) and Rspo3 (probe 402011) transcripts was performed using RNAscope (Advanced Cell Diagnostic). Tissues were rapidly isolated, fixed in 10% buffered formalin for 16 hours, dehydrated, embedded in paraffin, and sectioned onto 6-μm slides. Sample preparation, probe hybridization, and signal amplification and detection were performed according to the manufacturer's protocols.

[0187] (Intraperitoneal glucose tolerance test (IPGTT) and blood glucose measurement) For IPGTT, mice were fasted for 6 h and then intraperitoneally injected with a body weight dose of D-(+)-glucose (2 g / kg). Blood glucose levels were measured using an ONETOUCH Verio glucometer (LifeScan) at the indicated time points after glucose administration.

[0188] (Blood insulin level measurement) For insulinemia measurements, mice were anesthetized with isoflurane delivered in oxygen at a flow rate of 1 L / min. Whole blood samples were collected from the retroorbital sinus using glass capillaries into K3EDTA blood collection tubes. To measure basal insulinemia, blood samples were collected after 6 h of fasting. To assess glucose-stimulated insulin secretion, an additional blood sample was collected 2 min after intraperitoneal injection of 2 g / kg body weight of D-(+)-glucose. The whole blood samples were immediately cooled in ice water. Plasma was separated by centrifugation at 2000 g for 7 min at 4°C. The resulting plasma was transferred to pre-chilled tubes, quickly frozen in liquid nitrogen, and finally stored at -80°C.

[0189] (ELISA immunoassay) Plasma insulin concentrations were assessed by ELISA immunoassay (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions. All reagents and samples were warmed to room temperature before use. Absorbance was read at 450 nm using a spectrophotometer (Sunrise Basic Tecan, Crailsheim, Germany) and supplemented with Tecan Magellan data analysis software. Insulin concentrations were calculated using Microsoft Excel. A calibration curve was calculated by plotting the known absorbance values ​​of each calibrator (except calibrator 0) against the average of the corresponding insulin concentration values.

[0190] (Quantitative and Data Analysis) Quantitative analysis was performed on whole pancreases from at least five mice / genotypes / conditions using the HALO-Indica Labs module. BrdU counts were assessed by manually counting proliferating cells within the islets of Langerhans and normalizing the final counts on the total islet surface. All values ​​are reported as the mean ± SEM of datasets from at least five animals. Data were analyzed using Prism software (GraphPad) by first determining whether they followed a normal distribution using the D'Agostino-Person omnibus normality test. If not, an unpaired / nonparametric Mann-Whitney test was used. Conversely, an unpaired t-test (comparing two groups) or an unpaired Anova test (comparing more than two groups) was used, assuming a Gaussian distribution. p<0.0001 ( **** ), p<0.001( *** ), p<0.01( ** ), p<0.05( * ), the results are considered significant.

[0191] (Streptozotocin-mediated induction of diabetes) To induce hyperglycemia, STZ (Sigma) was dissolved in 0.1 M sodium citrate buffer (pH 4.5) and administered intraperitoneally at a single dose (115 mg / kg) within 10 minutes after dissolution. The progression of diabetes was assessed by monitoring blood glucose levels.

[0192] [result] Extensive quantitative analysis using RT-qPCR revealed that Rspo1 is expressed in the pancreas, whereas Rspo2 and Rspo4 mRNAs are completely undetectable (Figure 1). More precisely, Rspo1 is already detectable during embryonic development, starting at embryonic day 15.5 (E15.5), then peaks postnatally (around P6) and returns to embryonic levels during adulthood (Figure 2).

[0193] Due to the lack of antibodies specifically recognizing Rspo1, we used a relatively new in situ hybridization technique called RNAscope to assess its localization within the pancreas. The results indicated that Rspo1 is localized within the exocrine compartment, with its expression restricted to acinar cells (Figure 3). Interestingly, further analysis clearly demonstrated the expression of the Rspo1 receptor, Lgr4, exclusively in the islets of Langerhans, and specifically in β cells, a finding also seen in human islets (not shown).

[0194] To assess whether Rspo1 activity is essential for pancreatic development and function, we first analyzed the Rspo1-knockout (Rspo1KO) mouse line, in which targeted disruption of the Rspo1 transcript was achieved by inserting a LacZ reporter followed by a neomycin resistance cassette into the third exon of the Rspo1 gene (Chassot, AA et al. Hum Mol Genet 17, 1264–1277, doi:10.1093 / hmg / ddn016(2008)).

[0195] To determine whether Rspo1 expression, despite being restricted to acinar cells, could play a role in pancreatic physiology, we performed an intraperitoneal glucose tolerance test (IPGTT) to assess the body's ability to restore glucose-stimulated normoglycemia. After a 6-h starvation period, both Rspo1- / - mutant mice and Rspo1+ / + age-matched controls received a weight-dependent administration of glucose.

[0196] Importantly, mice lacking Rspo1 showed a significantly improved response, with a strongly reduced blood glucose peak. Furthermore, a faster return to normoglycemia was observed in Rspo1-deficient animals (Fig. 4).

[0197] To gain further insight into the mechanisms underlying the improved glucose disposal in Rspo1 loss-of-function animals, quantitative analysis was used. Therefore, pancreatic sections were immunostained with antibodies recognizing the hormones insulin, glucagon, and somatostatin, and the stained area was quantified. Initial analysis of the total islet surface area revealed no differences between the two groups tested (Figure 5A). Furthermore, more detailed quantification did not clearly reveal any differences in the number of cells expressing insulin (Figure 5B), glucagon (Figure 5C), or somatostatin (Figure 5D). Finally, the total number of islets per pancreatic section was also assessed, again demonstrating no discrepancy between Rspo1 − / − mice and their age-matched controls (not shown).

[0198] Given the results obtained, we hypothesized that the improvement in glucose tolerance upon Rspo1 loss may not be due to significant changes in pancreatic cell number and function, but rather to peripheral changes in insulin sensitivity caused by systemic genetic ablation of Rspo1.

[0199] Next, we investigated the consequences of Rspo1 overexpression. To this end, wild-type adult mice were intraperitoneally injected daily with recombinant Rspo1 protein for 4 weeks. Treated mice showed no differences in body weight or basal blood glucose compared with age-matched controls (injected with an equal volume of saline alone) throughout the entire treatment period (Figure 6).

[0200] Interestingly, IPGTT revealed that mice treated with recombinant Rspo1 protein had significantly improved glucose tolerance, as evidenced by a reduced blood glucose peak and a more rapid recovery to normal blood glucose compared with the control group (Fig. 7A). Furthermore, ELISA assays measuring blood insulin levels demonstrated enhanced glucose-stimulated insulin secretion (GSIS) in mice injected with recombinant Rspo1 compared with age-matched control animals (Fig. 7B).

[0201] Finally, to address the possible causes of these phenotypes, we relied on immunofluorescence techniques and stained paraffin pancreatic sections with insulin and BrdU, markers of cell proliferation. Surprisingly, we not only observed a higher number of proliferating β cells within the islets of mice treated with Rspo1 recombinant protein, but also noted an increased islet size in these mice (Figure 8). Using quantitative analysis, we were able to confirm the increased β cell proliferation with Rspo1 recombinant protein injection (Figure 9A). As a result, the islets of Langerhans in mice treated with recombinant Rspo1 were found to be significantly larger than those of age-matched control counterparts treated with saline alone (Figure 9B). This increased size was primarily driven by an increase in insulin-producing β cell mass (Figure 9C), whereas α cell mass remained unchanged after Rspo1 administration (Figure 9D).

[0202] To determine whether the supplementary insulin-producing cells were functional, WT animals were injected with a high dose of streptozotocin (STZ) to ablate pancreatic β-cell mass. Once these animals were overtly diabetic and had blood glucose levels of approximately 300 mg / dL, they were treated daily with Rspo1 or saline (control). Saline-treated control mice showed a further increase in blood glucose, whereas Rspo1-treated control mice showed a steady recovery (following a transient peak in blood glucose) (Figure 10). Quantitative immunohistochemistry was performed on sections of isolated saline- and Rspo1-treated pancreata. STZ treatment induced a loss of insulin-producing cells in all conditions, whereas Rspo1-treated animals showed a progressive regeneration of their β-cell mass, resulting in reconstituted islets after β-cell ablation (Figure 10). It is noteworthy that body weight (not shown) and blood glucose were normal in surviving animals, which had a longer lifespan compared to controls.

[0203] Finally, to determine whether these results could be translated to humans, we cultured human islets in RPMI in the presence of BrdU in the presence or absence of Rspo1 (75 mM for 5 days) and labeled proliferating cells. Immunohistochemical analysis revealed very few proliferating insulin-producing cells in controls (Fig. 11). Interestingly, Rspo1 treatment clearly demonstrated a significant increase in the number of proliferating human β-cells, demonstrating that Rspo1 can also induce human β-cell proliferation.

[0204] (human Rspo1) To assess whether human Rspo1 (hR1) stimulates mouse β-cell proliferation, hR1 was incubated with mouse insulinoma (Min6) cells at various concentrations for 24 hours. Notably, hR1 induced a significant 26% increase in Min6 cell numbers at concentrations of 200 nM and 400 nM compared with control cells incubated with PBS (Figure 14). To exclude the contribution of endotoxin to hR1 mitogenicity, the experiment was repeated using an endotoxin-purified preparation of hR1. Interestingly, this form of hR1 led to a 35% increase in β-cell numbers when incubated at concentrations of 400 nM or higher (Figure 15).

[0205] These data clearly demonstrate that hR1 exerts a proliferative effect on mouse β cells in vitro. To translate these experimental results to in vivo conditions, we performed short-term treatments on wild-type rodents. Specifically, mouse pancreases were harvested 30 minutes after injection of hR1 at concentrations of 100 μg / kg, 400 μg / kg, and 1350 μg / kg. Immunohistochemical and quantitative analysis of Ki67-labeled β cells demonstrated that hR1 can potently induce β cell proliferation when administered in vivo (Figure 16).

[0206] Encouraged by these experimental results, we performed long-term treatment of adult WT animals with daily injections of hR1 at different doses ranging from 30 μg / kg to 400 μg / kg. For this experiment, mice were administered an endotoxin-purified preparation of recombinant hR1 for 28 days. Subsequently, the animals were sacrificed, and pancreatic tissue was analyzed by immunofluorescence using antibodies recognizing the β-cell markers prohormone convertase 1 / 3 (PC1 / 3) and BrdU to identify proliferating cells. Interestingly, a significant increase in BrdU- and PC1 / 3-double-positive cells was observed in mice treated with recombinant hR1 at concentrations of 200 μg / kg and 400 μg / kg (Figure 17). Notably, β-cell hyperproliferation was associated with a significant increase in islet size (Figure 17).

[0207] [Conclusion] The obtained data demonstrate the essential role of Rspo1 in the mouse pancreas. Overexpression of Rspo1 alone, achieved by daily injections of recombinant full-length protein, not only significantly improved glucose tolerance in treated mice but also increased β-cell mass. Interestingly, these newly proliferated β-cells also appeared fully functional, allowing treated mice to produce greater amounts of insulin upon glucose stimulation. Importantly, nearly complete β-cell ablation enabled the remaining β-cells to proliferate and reconstitute functional β-cell mass capable of maintaining normoglycemia. These data also strongly demonstrate that human recombinant Rspo1 can stimulate mouse β-cell proliferation and increase the size of the islets of Langerhans. Finally, the demonstration that Rspo1 can also induce human β-cell proliferation opens new and unexpected avenues.

[0208] Taken together, these results suggest that Rspo1 plays an important paracrine role in the pancreas and that strategies aimed at increasing Rspo1 expression, for example, by in vivo administration of Rspo1 protein, may be beneficial in the treatment and / or prevention of diabetes in humans.

[0209] [Table 2-1]

[0210] Table 2-2

[0211] Table 2-3

[0212] Table 2-4

[0213] Table 2-5

Claims

[Claim 1] An isolated Rspo1 protein for use as a medicament, preferably in the treatment of diabetes in a subject in need thereof.

Images