CN116723867A - Liver-specific Wnt signal enhancement molecules and uses thereof - Google Patents

Abstract

The present disclosure provides liver-specific Wnt signaling enhancing molecules, and related methods of using these molecules to increase Wnt signaling in liver tissue and treat liver diseases and disorders.

Description

Liver-specific Wnt signal enhancement molecules and uses thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application Ser. No. 63/114,457, filed 11/16/2020, U.S. provisional application Ser. No. 63/182,106, filed 2021/4/30, and U.S. provisional application Ser. No. 63/248,157, filed 2021/9/24, each of which is incorporated herein by reference in its entirety for all purposes.

Statement regarding sequence listing

The sequence listing relevant to the present application is provided in text format in place of paper copies and is incorporated herein by reference. The name of the text file containing the sequence listing is srzn_019_03wo_st25.Txt. The text file is 100KB in size, created 11 at 2021, 11 at 11, and submitted electronically via the EFS-Web.

Technical Field

The present disclosure relates to liver-specific Wnt signaling enhancing molecules, e.g., fusion proteins, comprising a domain that binds to E3 ubiquitin ligase, ZNRF3 or RNF43 and a liver-specific cell surface receptor binding domain; and related methods of using liver-specific Wnt signaling-enhancing molecules to mediate liver-specific internalization of E3 ligase ZNRF3/RNF43 or sequestration (sequencing) of E3 ligase ZNRF3/RNF43, thereby stabilizing Wnt receptors and enhancing Wnt signaling in a liver-specific manner and treating and preventing a variety of diseases and disorders.

Background

Wnt ("wingless-related integration sites" or "wingless and Int-1" or "wingless-Int") ligands and their signals play a key role in the control of development, homeostasis and regeneration of many essential organs and tissues (including bone, liver, skin, stomach, intestine, kidneys, central nervous system, breast, oral mucosa, taste buds, ovaries, cochlea and many other tissues) (for example, reviewed by Clevers, loh and Nusse,2014; 346:1248012). Modulation of Wnt signaling pathways has the potential to treat degenerative diseases and tissue injury. To achieve this goal, strategies need to be developed to modulate Wnt signaling activity in a liver-specific or cell type-specific manner to avoid undesirable effects. One of the challenges in modulating Wnt signaling as a therapeutic agent is the presence of multiple Wnt ligands and Wnt receptors, frizzled 1-10 (Fzd 1-10), with many tissues expressing multiple and overlapping Fzds. Canonical Wnt signaling also relates to Low Density Lipoprotein (LDL) receptor-related protein 5 (LRP 5) or Low Density Lipoprotein (LDL) receptor-related protein 6 (LRP 6) as co-receptors, which are widely expressed in various tissues in addition to Fzds.

R-rachis 1-4 are a family of ligands that amplify Wnt signaling. Each R-spinal protein acts through a receptor complex containing zinc and ring finger 3 (ZNRF 3) or ring finger protein 43 (RNF 43) at one end and a leucine-rich repeat-rich G-protein coupled receptor 4-6 (LGR 4-6) at the other end (for example, reviewed by Knight and Hankenson2014, matrix Biology; 37:157-161). The R-vertebrate proteins can also act by additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target the Wnt receptors (Fzd 1-10 and LRP5 or LRP 6) for degradation. Binding of R-spondin to ZNRF3/RNF43 and LGR4-6 results in clearance or sequestration of the ternary complex, which removes the E3 ligase from the Wnt receptor and stabilizes the Wnt receptor, resulting in enhanced Wnt signaling. Each R-vertebrate protein contains two furin domains (1 and 2), where furin domain 1 binds ZNRF3/RNF43 and furin domain 2 binds LGR 4-6. Fragments of R-vertebrate proteins containing furin domains 1 and 2 are sufficient to amplify Wnt signaling. Although the R-spondin effect is dependent on Wnt signaling, the R-spondin effect is not tissue specific due to the broad expression of LGR4-6 and ZNRF3/RNF43 in various tissues.

There is clearly a need in the art for liver-specific Wnt signaling enhancing molecules for use in the treatment and prevention of specific diseases and disorders. The present invention addresses this need by providing compositions and methods for enhancing Wnt activity in a liver-specific manner.

Disclosure of Invention

The present invention relates to liver-specific Wnt signaling enhancement molecules and their use, for example, in increasing Wnt signaling in target tissues and in treating diseases and conditions that would benefit from increased Wnt signaling. In a particular embodiment, the tissue is liver.

In one embodiment, the invention provides a liver-specific Wnt signaling enhancement molecule or a pharmaceutically acceptable salt thereof, comprising a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from the group consisting of ZNRF3 and RNF43 and a second domain that specifically binds to a liver-specific cell surface molecule, wherein the molecule increases Wnt signaling in the tissue. In certain embodiments, the second domain specifically binds to a liver-specific cell surface molecule and increases Wnt signaling in the liver or liver cells. In various embodiments, one or both of the first domain and the second domain is a polypeptide, an antibody, a small molecule, a natural ligand, a non-natural ligand, or a variant thereof.

In a particular embodiment of the Wnt signaling enhancing molecule, the first domain comprises a first polypeptide sequence and/or the second domain comprises a second polypeptide sequence. In a particular embodiment, the molecule comprises a fusion protein comprising a first polypeptide sequence and a second polypeptide sequence.

In certain embodiments, the first polypeptide sequence comprises an R-vertebrate protein sequence, or a fragment or variant thereof. In particular embodiments, the R-spinal protein is R-spinal protein-1, R-spinal protein-2, R-spinal protein-3, or R-spinal protein-4, such as human R-spinal protein-1-4. In certain embodiments, the first polypeptide sequence comprises R-vertebrate furin domain 1 or a fragment or variant thereof. In a particular embodiment, the first polypeptide sequence is a wild-type R-spondin-derived sequence or a modified sequence. Furthermore, the first polypeptide sequence may have increased, similar or decreased binding to LGR4-6 compared to the corresponding native full-length R-spinal protein. In some embodiments, R-vertebrate protein or R-vertebrate furin 1 domain has at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% identity to any R-vertebrate protein or R-vertebrate furin 1 domain present in SEQ ID NOS 29-32 or 47-50. In certain embodiments, the first polypeptide is an antibody or antigen-binding fragment thereof that specifically binds ZNRF3 and/or RNF 43. In a particular embodiment, the first polypeptide is an antibody or antigen-binding fragment thereof comprising: a) CDRH1, CDRH2 and CDRH3 sequences shown herein; and/or b) CDRL1, CDRL2 and CDRL3 sequences as set forth herein, or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises fewer than 8 amino acid substitutions in said CDR sequence. In a particular embodiment, the first polypeptide is an antibody or antigen-binding fragment thereof comprising a nanobody, a VH or VL sequence as shown herein, or a fragment or variant thereof.

In certain embodiments, the second polypeptide sequence is a polypeptide, an antibody or fragment or variant thereof, or a ligand or fragment or variant thereof. In certain embodiments, the second polypeptide is an antibody or antigen-binding fragment thereof that specifically binds ASGR1 and/or ASGR 2. In a particular embodiment, the second polypeptide is an antibody or antigen binding fragment thereof comprising: a) CDRH1, CDRH2 and CDRH3 sequences shown herein; and/or b) CDRL1, CDRL2 and CDRL3 sequences as set forth herein, or a variant of said antibody, or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises fewer than 8 amino acid substitutions in said CDR sequence. In a particular embodiment, the first polypeptide is an antibody or antigen-binding fragment thereof comprising a nanobody, a VH or VL sequence as shown herein, or a fragment or variant thereof.

In certain illustrative embodiments of the liver-specific Wnt signaling enhancing molecules disclosed herein, the tissue is liver tissue, and the cell surface receptor is asialoglycoprotein receptor 1 (ASGR 1), asialoglycoprotein receptor 2 (ASGR 2), transferrin receptor 2 (TfR 2), or solute carrier family 10 member 1 (SLC 10 A1).

In certain embodiments of the liver-specific Wnt signaling enhancing molecules described herein, the first domain and the second domain are connected by a linker moiety. In certain embodiments, the linker moiety is a peptidyl linker sequence. In particular embodiments, the peptidyl linker sequence comprises one or more amino acids selected from glycine, asparagine, serine, threonine and alanine.

In certain embodiments, the liver-specific Wnt signaling enhancing molecules described herein consist of a single polypeptide, such as a fusion protein comprising a first domain and a second domain. In certain embodiments, the liver-specific Wnt signaling enhancing molecules described herein comprise two or more polypeptides, such as dimers or multimers comprising two or more fusion proteins, each comprising a first domain and a second domain, wherein the two or more polypeptides are linked, for example, by a linker moiety or via an intermolecular disulfide bond between amino acid residues in each of the two or more polypeptides, for example, cysteine residues. In certain embodiments, a liver-specific Wnt signaling enhancing molecule described herein comprises two or more polypeptide sequences. For example, a liver-specific Wnt signaling enhancing molecule may comprise an antibody heavy and light chain (or antigen-binding fragment thereof) that constitutes a first domain or a second domain, wherein the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy or light chain as a fusion protein (e.g., directly or through a peptide linker) or via a linker moiety. In a particular embodiment, the other domain is linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain or the C-terminus of the light chain. Such a structure may be referred to herein as an additional IgG scaffold or form.

In related embodiments, the present disclosure provides a liver-specific Wnt ("wingless-related integration site" or "wingless and Int-1" or "wingless-Int") signaling enhancement molecule, or a pharmaceutically acceptable salt thereof, comprising a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43), and a second domain that specifically binds to asialoglycoprotein receptor 1 (ASGR 1), wherein: (a) The first domain comprises a modified R-vertebrate polypeptide or fragment or variant thereof; and (b) the second domain comprises a modified antibody or antigen binding fragment thereof comprising: CDRH1, CDRH2 and CDRH3 sequences; CDRL1, CDRL2 and CDRL3 sequences. In certain embodiments, the R-spondin polypeptide, or fragment or variant thereof, comprises a furin domain 1 sequence and optionally a wild-type or mutant furin domain 2 sequence, or fragment or variant thereof, wherein the R-spondin polypeptide, or fragment or variant thereof, has reduced binding to G protein-coupled receptor 4-6 (LGR 4-6) enriched in leucine repeat sequences compared to the full-length wild-type R-spondin polypeptide. In certain embodiments, the R-vertebrate polypeptide, or fragment or variant thereof, comprises amino acid substitutions at positions corresponding to amino acids 105 and 109 of human R-vertebrate protein 2. In certain embodiments, the amino acid substitutions are: (a) F105R, F105A or F105E; and (b) F109A or F109E. In a particular embodiment, the two amino acid substitutions are: (a) F105R and F109A; (b) F105A and F109A; (c) F105E and F109A; or (d) F105E and F109E. In particular embodiments, the combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences is selected from the following: (a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; (b) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; or (c) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42), QHFWGTPFT (SEQ ID NO: 43), AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46), respectively. In certain embodiments, the second domain comprises an antibody light chain polypeptide and an antibody heavy chain polypeptide, and wherein the first domain is fused to the N-terminus of the antibody heavy chain polypeptide, optionally via a linker moiety. In a particular embodiment, the linker moiety is a peptidyl linker sequence. In certain embodiments, the linker sequence comprises one or more amino acids selected from the group consisting of: glycine, asparagine, serine, threonine and alanine. In certain embodiments, the Wnt signaling enhancing molecule comprises two antibody light chain polypeptides and two fusion polypeptides, wherein each fusion polypeptide comprises a modified R-spondin polypeptide, or fragment or variant thereof, fused to the N-terminus of an antibody heavy chain polypeptide via a linker moiety, optionally a peptidyl linker sequence, wherein the two fusion polypeptides are linked to each other, and the two antibody light chain polypeptides are each linked to a different heavy chain polypeptide of the fusion polypeptides. In a particular embodiment, the molecule comprises: (i) the two antibody light chain polypeptides comprise a sequence identical to SEQ ID NO: 1. 3, 5, 7, 9, 11, 14, 17, 18, 19, 21, 23, 25, or 27, or a variable region sequence thereof having at least 95% identity; and/or (ii) the two antibody heavy chain polypeptides comprise a variable region sequence having at least 95% identity to the variable region sequence of any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 15, 16, 20, 22, 24, 26, 28, 33 or 51, or a variable region thereof. In certain embodiments, each of the two fusion polypeptides comprises a sequence having at least 95% identity to any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 20, 22, 24, 26, 28, 33 or 51, or a variable region thereof. In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 7, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 8, or a variable region thereof. In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID NO. 25, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 26, or a variable region thereof.

In another related embodiment, the present disclosure provides a polypeptide comprising a sequence having at least 95% identity to any one of SEQ ID NOs 1-28, 33 or 51, or a variable region thereof, optionally wherein said polypeptide comprises one of the following sets of CDRs: (a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35) and DFSSRRWYLEY (SEQ ID NO: 36); (b) QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39); (c) QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39); (d) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42) and QHFWGTPFT (SEQ ID NO: 43); or (e) AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46). In certain embodiments, the polypeptide is a fusion protein comprising a modified R-vertebrate polypeptide, or fragment or variant thereof, fused to the N-terminus of an antibody heavy chain polypeptide via a linker moiety, optionally a peptidyl linker sequence, wherein the R-vertebrate polypeptide, or fragment or variant thereof, comprises two amino acid substitutions at positions corresponding to amino acids 105 and 109 of human R-vertebrate 2. In certain embodiments, the two amino acid substitutions are: (a) F105R, F105A or F105E; and (b) F109A or F109E. In certain embodiments, the two amino acid substitutions are: (a) F105R and F109A; (b) F105A and F109A; (c) F105E and F109A; or (d) F105E and F109E. In certain embodiments, the antibody heavy chain polypeptide comprises a combination of CDRH1, CDRH2, and CDRH3 sequences selected from the group consisting of: (a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), respectively; or (b) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42), QHFWGTPFT (SEQ ID NO: 43), respectively. In particular embodiments, the polypeptide comprises a modified antibody light chain polypeptide, in certain embodiments, the modified antibody light chain polypeptide comprises a combination of CDRL1, CDRL2, and CDRL3 sequences selected from the group consisting of: (a) QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; (b) QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; or (c) AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46), respectively.

In another related embodiment, the present disclosure provides a nucleic acid sequence encoding any of the polypeptides disclosed herein, such as an antibody light chain polypeptide or fusion polypeptide disclosed herein, or a polypeptide having at least 95% identity to any one of SEQ ID NOS: 1-33 or 51, or a variable region thereof. In particular embodiments, the nucleic acid sequence is DNA or mRNA.

In another embodiment, the present disclosure provides a vector comprising a nucleic acid sequence disclosed herein. In certain embodiments, the vector is an expression vector comprising a promoter sequence operably linked to a nucleic acid sequence. In certain embodiments, the vector is a virus comprising a promoter sequence operably linked to a nucleic acid sequence.

In related embodiments, the present disclosure provides host cells comprising the vectors disclosed herein. In another related embodiment, the present disclosure provides a method for producing an antibody light chain polypeptide or fusion polypeptide disclosed herein comprising culturing a host cell under conditions wherein the polypeptide is expressed by an expression vector. In certain embodiments, the method comprises the step of isolating the fusion polypeptide produced.

In related embodiments, the present disclosure provides a pharmaceutical composition comprising: a) A molecule disclosed herein, a nucleic acid sequence disclosed herein, a vector disclosed herein, or a host cell disclosed herein; and b) a pharmaceutically acceptable diluent, adjuvant or carrier.

In another embodiment, the invention provides a pharmaceutical composition comprising a liver-specific Wnt signaling enhancing molecule described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, adjuvant, or carrier.

In another embodiment, the invention provides a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, adjuvant, or carrier. In particular embodiments, the nucleic acid sequence comprises DNA or mRNA, optionally modified mRNA.

In another embodiment, the invention provides a pharmaceutical composition comprising a vector comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, adjuvant or carrier. In a particular embodiment, the vector comprises a promoter operably linked to the nucleic acid sequence that drives expression of the liver-specific Wnt signaling enhancing molecule. In certain embodiments, the vector is an expression vector or a viral vector.

In another embodiment, the present disclosure provides a method for increasing Wnt ("wingless-related integration site" or "wingless and Int-1" or "wingless-Int") signaling in liver tissue, comprising contacting the liver tissue with: a) Wnt signaling enhancing molecules disclosed herein; b) Nucleic acids disclosed herein; c) The vectors disclosed herein; d) A host cell as disclosed herein; or E) a pharmaceutical composition as disclosed herein, wherein the molecule binds to and sequesters in liver tissue one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43) or increases endocytosis of one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43) in liver tissue.

In related embodiments, the present disclosure provides methods for treating or preventing a liver disease or liver disorder in a subject in need thereof, wherein the liver disease or liver disorder is associated with reduced Wnt ("wingless-related integration sites" or "wingless and Int-1" or "wingless-Int") signaling or would benefit from increased Wnt signaling, the method comprising administering to the subject an effective amount of: a) Molecules disclosed herein; b) Nucleic acids disclosed herein; c) The vectors disclosed herein; d) A host cell as disclosed herein; or e) the disclosed pharmaceutical composition. In particular embodiments, the liver disease or liver disorder is selected from: acute liver failure of various causes, drug-induced acute liver failure, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in patients with cirrhosis, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, alcoholic liver disease, chronic liver failure of various causes, decompensated liver failure, advanced decompensated liver failure, cirrhosis, liver fibrosis of various causes, portal hypertension, chronic liver insufficiency of various causes, end-stage liver disease (ESLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, acute Alcoholic Hepatitis (AAH) or severe alcoholic hepatitis, chronic alcoholic hepatitis Alcoholic Liver Disease (ALD) (also known as alcohol-related liver disease (ARLD)), hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis B virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, surgery with symptoms of liver disease, liver injury, venous Occlusive Disease (VOD), sinus Obstructive Syndrome (SOS), primary cholangitis (PBC), primary Sclerosing Cholangitis (PSC), liver transplantation, liver surgery and "small volume" syndrome in transplantation, congenital liver diseases and disorders, liver failure due to APAP (acetaminophen) overdose, acetaminophen-induced liver injury and any other liver disease or condition caused by genetic disease, degeneration, aging, drugs or injury.

In certain embodiments, the liver disease or liver condition is selected from acute alcoholic hepatitis, acute liver failure including, but not limited to, acute Liver Failure (ALF) due to acetaminophen (APAP) overdose, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in cirrhosis patients, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, or cirrhosis. In certain embodiments, the liver disease is alcoholic hepatitis, such as acute or severe alcoholic hepatitis. In particular embodiments, the molecule, nucleic acid, vector, host cell or pharmaceutical composition is administered parenterally, orally, intramuscularly or topically to the liver. In a particular embodiment, the subject is a mammal, optionally a human.

In related embodiments, the present disclosure provides methods of producing, culturing, or maintaining a liver cell, liver tissue, or liver organoid comprising contacting the cell, tissue, or organoid with: a) Molecules disclosed herein; b) Nucleic acids disclosed herein; c) The vectors disclosed herein; d) A host cell as disclosed herein; or e) a pharmaceutical composition as disclosed herein. In some embodiments, the method is for maintaining viability of ex vivo liver tissue, comprising optionally contacting liver tissue obtained from a donor by perfusing ex vivo liver tissue with a composition comprising the molecule. In some embodiments, the method is for maintaining viability of liver tissue, comprising contacting donor liver tissue in vivo with a composition comprising the molecule. In some embodiments, the methods are for producing or maintaining a liver organoid culture, comprising optionally contacting the liver organoid culture by culturing the liver organoid culture in a medium comprising the molecule.

In another embodiment, the invention includes a method for increasing Wnt signaling in a target tissue comprising contacting the target tissue with a liver-specific Wnt signaling enhancement molecule described herein, wherein the second domain specifically binds to a cell-specific surface molecule on the target tissue, and wherein the liver-specific Wnt signaling enhancement molecule binds to the target tissue and sequesters one or more transmembrane E3 ubiquitin ligases in the target tissue selected from ZNRF3 and RNF43 or increases endocytosis of one or more transmembrane E3 ubiquitin ligases in the target tissue selected from ZNRF3 and RNF 43.

In certain embodiments, the target tissue or cell is contacted with a polynucleotide comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule, or a vector, such as an expression vector or a viral vector, comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule.

In another related embodiment, the invention includes a method for treating or preventing a disease or condition in a subject in need thereof, wherein the disease or condition is associated with reduced Wnt signaling or would benefit from increased Wnt signaling, the method comprising providing to the subject an effective amount of a pharmaceutical composition comprising a liver-specific Wnt signaling enhancing molecule or a pharmaceutically acceptable salt thereof, alone or in combination with Wnt, norrin, or Wnt activation/mimetic molecules. In certain embodiments, the method is performed using a pharmaceutical composition comprising a polynucleotide comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule (e.g., DNA or mRNA) or a vector (e.g., an expression vector or a viral vector) comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule.

In a particular embodiment of any of the methods of treatment described herein, the disease or disorder is a liver disease or disorder of a tissue selected from the group consisting of: acute liver failure of various causes, drug-induced acute liver failure, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in patients with cirrhosis, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, alcoholic liver disease, chronic liver failure of various causes, decompensated liver failure, late decompensated liver failure, cirrhosis, liver fibrosis of various causes, portal hypertension, chronic liver insufficiency of various causes, end-stage liver disease (ESLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, acute Alcoholic Hepatitis (AAH), chronic alcoholic hepatitis Alcoholic Liver Disease (ALD) (also known as alcohol-related liver disease (ARLD)), hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis B virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, surgery with symptoms of liver disease, liver injury, venous Occlusive Disease (VOD), sinus Obstructive Syndrome (SOS), primary cholangitis (PBC), primary Sclerosing Cholangitis (PSC), liver transplantation, liver surgery and "small volume" syndrome in transplantation, congenital liver diseases and disorders, liver failure due to APAP (acetaminophen) overdose, and any other liver disease or disorder caused by genetic disease, degeneration, aging, drugs or injury. In certain embodiments, the liver disease is alcoholic hepatitis, such as acute or severe alcoholic hepatitis. In particular embodiments of any of the therapeutic or prophylactic methods described herein, the pharmaceutical composition is provided systemically, parenterally, orally, intramuscularly, topically or topically. In a particular embodiment, the subject is a mammal, optionally a human.

In particular embodiments, the antibody or antigen binding fragment thereof comprises a heavy or light chain variable region or heavy or light chain comprising an amino acid sequence having at least 90% identity to an amino acid sequence disclosed herein (e.g., SEQ ID NOS: 1-28, 33 or 51) or a fragment or variant thereof (e.g., the variable domain of the sequence).

Brief description of the drawings

The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

FIG. 1 shows the light chain variable domain sequence of the initial αASGR1-RSPO2 Wnt signaling enhancer molecule and the fusion polypeptide heavy chain variable domain-RSPO 2 sequence. CDRs are underlined and deamidation or isomerization risks are bolded. Various amino acid substitutions are shown for each position to eliminate risk.

FIG. 2 provides SEC profiles for variants of the initial αASGR1-RSPO2 Wnt signaling enhancing molecule having various amino acid substitutions at the D62 position of CDR H2.

FIG. 3 provides SEC profiles for variants of the initial αASGR1-RSPO2 Wnt signaling enhancing molecule having various amino acid substitutions at the D25 position of CDR L1.

FIG. 4 provides SEC profiles for variants of the initial αASGR1-RSPO2 Wnt signaling enhancing molecule having various amino acid substitutions at the N51 position of CDRL 2.

FIG. 5 provides SEC profiles for variants of the initial αASGR1-RSPO2 Wnt signaling enhancing molecule having various amino acid substitutions at the N88 position of CDR L3.

FIG. 6 shows SDS-PAGE gel analysis of expression and folding of Wnt signaling enhancers comprising the indicated point mutations compared to the original αASGR1-RSPO2 Wnt signaling enhancers under non-reducing (left panel) or reducing (right panel) conditions.

Figure 7 shows SDS-PAGE gel analysis of expression and folding of Wnt signaling enhancement molecules comprising the combination of the indicated point mutations compared to the original aasgr 1-RSPO2 Wnt signaling enhancement molecule (left panel: lane 1 = marker, lanes 2-8 respectively = EESY, EEAL, EEAE, EEAH, EEAT, EEAY, EEAR, non-reduced, and lanes 9-15 respectively = EESY, EEAL, EEAE, EEAH, EEAT, EEAY, EEAR, reduced; right panel: lane 1 = marker, lanes 2-9 = EESN, EEAN, EESL, EESE, EESH, EEST, EESR, EESK).

FIG. 8 is a table summarizing the results of a given assay for Wnt signaling enhancement molecules comprising a combination of the indicated point mutations compared to the initial αASGR1-RSPO2 Wnt signaling enhancement molecule (wild-type).

FIG. 9 is a table summarizing ASGR1 binding assay results for Wnt signaling enhancers comprising the combination of point mutations shown compared to the initial αASGR1-RSPO2 Wnt signaling enhancer (wild-type).

FIG. 10 provides a graph showing the results of STF assays of Wnt signaling enhancing molecules comprising the combination of point mutations shown in Huh-7 (left panel) and Hek-203 (right panel) cells.

FIG. 11 provides a graph showing Axin2/ActB expression in animals treated with vehicle or the indicated Wnt signaling enhancing molecules. The data are presented as values for injured animals treated with delphinidin daily (left panel) or not (right panel).

FIG. 12 is a graph showing the increase in Axin2 expression in various tissues isolated from animals treated with anti-bgal, anti-GFP-mutSPO, EEST-EE Wnt signaling enhancement molecule (1R 34-EEST-EE) or Rspo2 (each tissue from left to right).

FIG. 13 provides a graph showing increased expression of the Wnt target gene in liver in animals treated with αGFP-IgG, αASGR1-RSPO 2-EEST-EEEEGR or αASGR1-RSPO2-EEST-RA Wnt signaling enhancing molecules (left to right) for a specified period of time.

FIG. 14A shows the increased expression of proliferation marker Ki67 in the livers of animals treated with αGFP-IgG, αASGR1-RSPO 2-EEST-EEE or αASGR1-RSPO2-EEST-RA (left to right) at the indicated time points.

FIG. 14B shows the increased expression of the proliferation marker Ki67 in the livers of animals treated with the indicated doses of αASGR1-RSPO2-EEST-EE or control.

FIG. 15 provides pharmacokinetic profiles following administration of EEST-EE Wnt signaling enhancing molecules in mice at 3, 10, 30, 100mg/kg Intravenous (IV) or at 10 or 30mg/kg intraperitoneal (i.p.).

Figure 16 is a graph of various Wnt signaling enhancing molecules tested and model of chronic thioacetamide induced mouse liver fibrosis.

FIGS. 17A-17C show INR (FIG. 17A), axin2 (FIG. 17B) and CYP2e1mRNA (FIG. 17C) after administration of indicated amounts of Wnt signaling enhancing molecules EEST-EE or EEST-RA.

FIG. 18 shows Ki67 expression in the livers of animals treated with control (anti-bgal), EEST-EE or Rspo2 in the presence or absence of TAA.

FIG. 19 shows INR (left), axin2 (middle) and CYP2e1mRNA (right) after administration of indicated amounts of Wnt signaling enhancing molecules, EEST-EE or EEST-RA.

FIG. 20 shows the presence or absence of CCl ₄ Expression of Ki67 in the liver of animals treated with control (anti bgal), EEAT-EE (aasgr 1-RSPO2-EEAT-EE or 1R 34-EEAT/EE) or RSPO 2.

FIG. 21 is a graph showing expression of the proliferation marker Ki67 in the liver of animals treated with control or αASGR1-RSPO 2-EEST-EE.

FIG. 22 is a graph showing the expression of the proliferation marker Ki67 in the small intestine of animals treated with control or αASGR1-RSPO 2-EEST-EE.

FIG. 23 shows the CCl in use ₄ Graph of the fibrotic area of animals treated with control (anti-GFP), rspo2 or αASGR1-RSPO2-EEAT-EE after treatment.

FIG. 24 is a graph showing ALP expression after treatment with vehicle, EEST-RA, EEST-EE, 8M24-EASE-RA or 8M 24-EASE-EE.

FIG. 25 provides the sequences of the variable domains of the heavy and light chains of the 8M24 antibodies and their various humanized versions.

Fig. 26A provides the sequences of VH and VL domains of the 8M24 antibody. CDRs are underlined and modified amino acids are shown in bold.

FIG. 26B shows various amino acid substitutions at each of the indicated positions of the 8M24 CDRs.

FIG. 27 is a graph showing the results of an STF assay for an 8M24 Wnt signaling enhancer molecule comprising the point mutation shown at N57.

FIG. 28 is a table summarizing the characteristics of various 8M24 Wnt signaling enhancing molecules comprising the indicated combinations of point mutations.

FIG. 29 provides a graph showing Axin2, ccnd and Ki67 expression in animals treated with different doses of the initial 8M24-RA Wnt signaling enhancing molecule or the Wnt signaling enhancing molecule in the form of EEST-EE IgG2 (1R 34-EEST/EE IgG 2).

FIG. 30 provides a graph showing Axin2, ccnd and Ki67 expression in animals treated with different doses of the 8M24-EASE-RA Wnt signaling enhancer molecule or a Wnt signaling enhancer molecule in the form of EEST-EE IgG2 (1R 34-EEST/EE IgG 2).

FIG. 31 provides pharmacokinetic profiles of EEST-RA (1R 34-EEST/RA), EEST-EE (1R 34-EEST/EE), 8M24-EASE-RA, or 8M24-EASE-EE Wnt signal enhancing molecules in mice.

Figure 32 shows the percentage of liver to body weight at termination. Statistical analysis: unidirectional ANOVA (GraphPad Prism) p <0.05, p <0.01, p <0.001, p <0.0001, error bars: mean value of SEM.

Fig. 33A-B show serum ALP (fig. 33A) and albumin (fig. 33B) at day 7 and day 14 after the start of test article dosing.

FIG. 34 shows expression analysis of liver RNA of Axin2, ccnd1 and Mki67 genes measured by qPCR.

FIG. 35 shows the position in CCl ₄ Immunofluorescent staining of liver sections of mice treated with SZN-043.v2, RSPO2, anti-GFP or control sections from mice injected with olive oil only. anti-Ki 67 nuclear antigen (green), hepatocyte-specific markers were stained with proliferation markers, and anti-hnf4α (red) DNA was stained with DAPI (blue).

Fig. 36A-B show the percentage of fibrotic area measured by staining with Sirius red (fig. 36A) followed by quantification using image J (fig. 36B).

FIG. 37A shows the general structure of the HuASGR1-CBD:8M24 complex. The molecular surface of HuASGR1-CBD is shown as a light gray transparent surface. The heavy and light chains of 8M24 were colored in the shades of deep black and light black, respectively. Three structural calcium ions are shown as dark spheres.

FIG. 37B shows a close-up view of the HuASGR1-CBD:8M24 interface, marking the positions of the CDR loops H1, H2, H3 of the heavy chain and L1, L2 and L3 of the light chain.

FIG. 38 shows an alignment of all four human R-spinal proteins (Rspo 1 (SEQ ID NO: 47), rspo2 (SEQ ID NO: 48), rspo3 (SEQ ID NO: 49), and Rspo4 (SEQ ID NO: 50), wherein furin domains 1 (Fu 1) and 2 (Fu 2) are colored in light and dark shades, respectively, fu1 domains generally correspond to about amino acid residues 38-94 of SEQ ID NO:47, about amino acid residues 37-93 of SEQ ID NO:48, about amino acid residues 39-95 of SEQ ID NO:49, and about amino acid residues 32-88.Fu2 domains of SEQ ID NO:50 generally correspond to about amino acid residues 97-144 of SEQ ID NO:47, about amino acid residues 96-143 of SEQ ID NO:48, about amino acid residues 98-144 of SEQ ID NO:49, and about amino acid residues 91-137 of SEQ ID NO: 50.

Figures 39A-C show binding of various Wnt signaling enhancing molecules comprising a combination of the indicated point mutations compared to the initial αasgr1-RSPO2 Wnt signaling enhancing molecule (NG or "wild-type"). Fig. 39A is a table showing kinetic fig. parameters from the data in fig. 39B and 39C. The data were fitted using a divalent analyte model in ForteBIO data analysis software. Figures 39B-39C provide data from serial dilutions of various Wnt signaling enhancing molecules bound using an Octet Red96e, using biotinylated ASGR1 assay captured on a streptavidin sensor.

FIGS. 40A-40D show STF activity of various combinations of mutations in Huh-7 (FIGS. 40A and 40C) and Hek-293 (FIGS. 40B and 40D) cells.

FIG. 41 shows the stability of constructs with the indicated amino acid substitution combinations.

Figure 42 shows STF activity of constructs with the indicated amino acid substitution combinations.

FIG. 43 provides a graph showing expression levels of Wnt target genes and cytochrome P450 (CYP) metabolizing enzymes shown in the liver of intact animals treated with αGFP-IgG, nac or αASGR1-RSPO 2-EEAT-EEWnt signaling enhancing molecules (left to right) or in the liver of APAP-injured animals at the indicated times.

FIG. 44 provides photomicrographs showing Ki67 and HNF4a expression in hepatocytes of livers treated with the indicated agents following acetaminophen-induced liver injury.

FIG. 45 provides photomicrographs showing Ki67 and CYP2F2 expression in hepatocytes of livers treated with the indicated agents following acetaminophen-induced liver injury.

Fig. 46 provides a photomicrograph showing the histology of the liver treated as shown. The necrotic areas after anti-GFP or Nac treatment are shown.

FIG. 47 provides a graph showing ALT, AAST, ALP and AMMN levels in the liver or APAP-injured liver of intact animals treated with αGFP-IgG, nac or αASGR1-RSPO 2-EEAT-EEWnt signaling enhancing molecules (left to right) at the indicated times.

FIG. 48 is a graph of the effect of 1R34-EEST-EE on liver function and tissue repair in an animal model of chronic alcoholism-induced liver injury.

FIG. 49 provides a graph showing expression of Wnt target genes indicated on day 0, day 3 or day 7 after treatment. On day 0, left to right bars correspond to paired feeding (pair fed) and EtOH, and on day 3 and on days 3 and 7, left to right bars correspond to treatment with anti-GFP or 1R 43-EEST-EE.

FIG. 50 provides a graph showing expression of liver proliferation markers shown on day 0, day 3 or day 7 after treatment. On day 0, left to right bars correspond to paired feeding and EtOH, and on day 3 and on days 3 and 7, left to right bars correspond to treatment with anti-GFP or 1R 43-EEST-EE.

FIG. 51 provides a micrograph showing immunofluorescent staining of Ki67 or HNF 4A.

FIG. 52 provides a graph showing expression of the molecules shown on day 0, day 3 or day 7 after treatment. On day 0, left to right bars correspond to paired feeding and EtOH, and on day 3 and on days 3 and 7, left to right bars correspond to treatment with anti-GFP or 1R 43-EEST-EE.

FIG. 53 is a graph showing the expression of Lect2 and angiogenin on day 0, day 3 or day 7 post-treatment. On day 0, left to right bars correspond to paired feeding and EtOH, and on day 3 and on days 3 and 7, left to right bars correspond to treatment with anti-GFP or 1R 43-EEST-EE.

FIG. 54 provides a graph showing the expression of inflammatory markers, interleukins IL1b and IL6 on day 0, day 3 or day 7 after treatment. On day 0, left to right bars correspond to paired feeding and EtOH, and on day 3 and on days 3 and 7, left to right bars correspond to treatment with anti-GFP or 1R 43-EEST-EE.

Detailed Description

The present disclosure provides liver-specific Wnt signaling enhancement molecules, wherein in certain embodiments the molecules: 1) Selectively binding to liver-specific cell surface receptors; 2) Mediate internalization or sequestration of ZNRF3/RNF43 in targeted liver tissues or cells; and/or 3) enhance Wnt signaling in a liver-specific manner. In certain embodiments, the molecule is a fusion protein. In certain embodiments, the molecule is an antibody having an additional binding domain. Also provided are pharmaceutical compositions and methods for using any of the molecules and compositions disclosed herein to enhance (i.e., increase) Wnt signaling in liver tissue or liver cells, e.g., for treating or preventing a liver disease or disorder. These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods as described more fully below.

Definition of the definition

As used herein, "vector" refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and that can be used to mediate delivery of the polynucleotide to a cell. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles.

The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. The nucleotide structure, if present, may be modified before or after assembly of the polymer. The term polynucleotide as used herein interchangeably refers to double-stranded and single-stranded molecules. Unless otherwise indicated or required, any embodiment of the invention described herein as a polynucleotide encompasses both the double stranded form and each of the two complementary single stranded forms known or predicted to constitute the double stranded form.

A polynucleotide or polypeptide (sequence of interest) has a certain percentage of "sequence identity" with another polynucleotide or polypeptide (reference sequence), meaning that when aligned, the percentage of bases or amino acids is the same when comparing the two sequences. As understood in the art, sequence identity refers to the percentage of identity obtained when sequences are aligned for maximum correspondence over a comparison window (e.g., a designated region of each sequence), which can be calculated by any algorithm described herein using default parameters that, when applied to similar sequences, are expected to yield the same alignment in most cases. Unless otherwise indicated, identity is calculated over the entire length of the reference sequence. Thus, a sequence of interest "shares at least x% identity" with a reference sequence if at least x% (rounded down) of the residues in the sequence of interest are aligned to exactly match the corresponding residues in the reference sequence when the sequence of interest is aligned with the reference sequence. Gaps may be introduced in the sequence of interest and/or the reference sequence to maximize correspondence over the comparison window.

Sequence similarity (i.e., identity) can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including those publicly available through the National Center for Biotechnology Information (NCBI), e.g., BLAST obtained through the global network in ncbi.nlm.nih.gov/BLAST/BLAST disclosures (e.g., BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1990) J.mol. Biol.215:403-410 and Altschul et al (1997) Nucleic acids Res. 25:3389-3402), respectively, such as BLASTP or BLASTN. For example, sequence identity may be determined by using the independently executable BLAST engine program (b 12 seq) for both BLAST sequences, which may be retrieved from NCBI ftp sites using default parameters (Tatusova and Madden, FEMS Microbiol Lett.1999,174, 247-250). Another alignment algorithm is FASTA, which is available from the genetics computing group (Genetics Computing Group, GCG) package of Madison, wis, USA (Oxford Molecular Group, inc fully owned subsidiaries). Other techniques for alignment are described in Methods in Enzymology, vol.266: computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, academic Press, inc., a division of Harcourt Brace & Co., san Diego, calif., USA. Of particular interest are alignment programs that allow gaps in the sequences. Smith-Waterman is a type of algorithm that allows gaps in sequence alignments. See Meth.mol.biol.70:173-187 (1997). Furthermore, the GAP program using Needleman and Wunsch alignment methods can be used to align sequences. See J.mol. Biol.48:443-453 (1970). Of interest is the BestFit program which uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. The gap creation penalty typically ranges from 1 to 5, typically from 2 to 4, and in many embodiments will be 3. The gap extension penalty typically ranges from about 0.01 to 0.20, and in many cases will be 0.10. The program has default parameters determined by the entered sequences to be compared. Sequence identity may be determined using default parameters determined by the program. The program is also available from the Genetics Computing Group (GCG) package from Madison, wis. Another destination program is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, macromolecule Sequencing and Synthesis, selected Methods and Applications, pp.127-149,1988,Alan R.Liss,Inc. Percent sequence identity was calculated by FastDB based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; connection penalty: 30.0. another objective program is CLUSTAL from uniprot. Org, and is available at https:// www.ebi.ac.uk/Tools/msa/clustalo. Unless indicated to the contrary, sequence identity is determined using the BLAST algorithm (e.g., bl2 seq) and default parameters.

"recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, as well as other methods that result in a different construct than the polynucleotide found in nature.

A "control element" or "control sequence" is a nucleotide sequence that is involved in molecular interactions that facilitates functional regulation of a polynucleotide, including replication (duplication), multiplication (transcription), splicing, translation, or degradation of a polynucleotide. Modulation may affect the frequency, speed, or specificity of the process, and may be enhanced or inhibited in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. Promoters are regions of DNA that are capable of binding RNA polymerase under certain conditions and promoting transcription of a coding region that is typically downstream (3' direction) of the promoter.

"operably linked (operatively linked)" or "operably linked" refers to the juxtaposition of genetic elements wherein the elements are in a relationship permitting them to operate in their intended manner. For example, a promoter is operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. Insertion residues may be present between the promoter and coding region, provided that this functional relationship is maintained.

An "expression vector" is a vector that comprises a region encoding a gene product of interest and is used to effect expression of the gene product in a desired target cell. The expression vector further comprises a control element operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements and their operably linked one or more genes for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

As used herein, the terms "polypeptide," "peptide," and "protein" refer to a polymer of amino acids of any length. The term also encompasses amino acid polymers that have been modified, e.g., to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation to a labeling component.

As used herein, the term "antibody" means an isolated or recombinant binding agent comprising the requisite variable region sequence that specifically binds an epitope of an antigen. Thus, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binds to a specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including but not limited to scFv, fab, and Fab ₂ Provided that they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody; linear antibodies (e.g., zapata et al, protein Eng.8 (10): 1057-1062 (1995)); single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. Pepsin treatment to produce F (ab') ₂ Fragments which have two antigen binding sites and which are still capable of cross-linking antigens。

By "comprising" is meant that the element is, for example, necessary in a composition, method, kit, etc., but that other elements may be included within the scope of the claims to form, for example, a composition, method, kit, etc. For example, an expression cassette "comprising" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to the gene and promoter, such as polyadenylation sequences, enhancer elements, other genes, linker domains, and the like.

By "consisting essentially of … …" is meant that the scope of the described, e.g., compositions, methods, kits, etc., is limited to the specified materials or steps that do not materially affect the basic and novel characteristics of, e.g., the compositions, methods, kits, etc. For example, an expression cassette consisting essentially of a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may comprise additional sequences, such as linker sequences, so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant, polypeptide fragment consisting essentially of the sequence has the amino acid sequence of the sequence plus or minus about 10 amino acid residues at the boundary of the sequence based on the full length native polypeptide from which it is derived, e.g., less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues of the boundary amino acid residues.

By "consisting of … …" is meant a composition, method or kit that excludes any element, step or component not specified in the claims. For example, a polypeptide or polypeptide domain consisting of the sequence comprises only the sequence.

As used herein, an "expression vector" encompasses vectors as discussed herein or known in the art, e.g., plasmids, microcircles, viral vectors, liposomes, and the like, comprising a polynucleotide encoding a gene product of interest, and for effecting expression of the gene product in a desired target cell. The expression vector further comprises a control element operably linked to the coding region to facilitate expression of the gene product in the target. The combination of control elements (e.g., promoters, enhancers, UTRs, miRNA targeting sequences, etc.) with one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette". Many such control elements are known and available in the art or may be readily constructed from components available in the art.

As used herein, "promoter" encompasses DNA sequences that direct RNA polymerase binding to promote RNA synthesis, i.e., minimal sequences sufficient to direct transcription. Promoter and corresponding protein or polypeptide expression may be ubiquitous, meaning having strong activity in a broad range of cell, tissue and species or cell type specificities, liver specificities or species specificities. Promoters may be "constitutive," meaning continuously active or "inducible," meaning that the promoter may be activated or deactivated by the presence or absence of an biological or non-biological agent. The nucleic acid constructs or vectors of the invention also include enhancer sequences, which may or may not be contiguous with the promoter sequence. Enhancer sequences affect promoter-dependent gene expression and may be located in the 5 'or 3' region of the native gene.

The term "native" or "wild-type" as used herein refers to a nucleotide sequence, such as a gene or gene product, such as RNA or protein, that is present in a wild-type cell, tissue, organ or organism. The term "variant" as used herein refers to a reference polynucleotide or polypeptide sequence, e.g., a mutant of a native polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity to the reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to a reference polynucleotide sequence (e.g., a native polynucleotide or polypeptide sequence). For example, a variant may be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity to a full-length native polynucleotide sequence, e.g., a polynucleotide having 75% or 80% or more, such as 85%, 90% or 95% or more, e.g., 98% or 99% identity to a full-length native polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence, e.g., a polypeptide having 75% or 80% or more, such as 85%, 90% or 95% or more, e.g., 98% or 99% identity to a full-length native polypeptide sequence. Variants may also include variant fragments of a reference sequence (e.g., a native sequence) that share 70% or more sequence identity with a fragment of a reference sequence (e.g., a native sequence), such as 75% or 80% or more, such as 85%, 90% or 95% or more, e.g., 98% or 99% identity with a native sequence.

As used herein, the terms "biological activity" and "bioactive" refer to activity that is attributed to a particular biological element in a cell. For example, "biological activity" of an R-spinal protein or fragment or variant thereof refers to the ability to enhance Wnt signaling. As another example, the biological activity of a polypeptide or functional fragment or variant thereof refers to the ability of the polypeptide or functional fragment or variant thereof to perform its natural function, e.g., binding, enzymatic activity, etc. As a third example, the biological activity of a gene regulatory element, e.g., a promoter, enhancer, kozak sequence, etc., refers to the ability of the regulatory element or a functional fragment or variant thereof, respectively, to modulate, i.e., promote, enhance, or activate translation of its operably linked gene expression.

The term "administering" or "introducing" or "providing" as used herein refers to delivering a composition to a cell, tissue and/or organ of a subject or a subject. Such administration or introduction may be performed in vivo, in vitro, or ex vivo.

The terms "treatment", "treatment" and the like as used herein generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, e.g., reducing the likelihood of the disease or symptoms thereof occurring in the subject; and/or may be therapeutic in terms of partial or complete cure of the disease and/or side effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, and includes: (a) inhibiting the disease, i.e., partially or completely arresting its development; or (b) alleviating the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of the disease or injury. Treatment of an ongoing disease in which the treatment stabilizes or alleviates undesirable clinical symptoms in a patient is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissue. During and in some cases after the symptomatic phase of the disease, it is desirable to administer the subject therapy.

The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including apes and humans; mammalian sports animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.) and rodents (e.g., mice, rats, etc.).

Various compositions and methods of the present invention are described below. Although specific compositions and methods are illustrated herein, it should be understood that any of a number of alternative compositions and methods are suitable and suitable for use in the practice of the present invention. It will also be appreciated that the evaluation of the expression constructs and methods of the invention can be performed using program standards in the art.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (M.J.Gait, 1984); "Animal Cell Culture" (r.i. freshney, 1987); "Methods in Enzymology" (Academic Press, inc.); "Handbook of Experimental Immunology" (D.M. Weir & C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller & M.P.Calos. Ed., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al, 1987); "PCR: the Polymerase Chain Reaction" (Mullis et al, 1994); and "Current Protocols in Immunology" (J.E. Coligan et al, 1991), each of which is expressly incorporated herein by reference.

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art, however, will readily recognize that the invention may be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "includes," including, "" has, "" having, "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.

The term "about" or "approximately" means within an acceptable error range for the particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, such as within a factor of 5 or within a factor of 2. When a particular value is described in the application and claims, unless otherwise specified, the term "about" shall be assumed to mean within an acceptable error range for the particular value.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that this disclosure supersedes any disclosure of the incorporated publications to the extent that there is a conflict.

It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

Unless otherwise indicated, all terms used herein have the same meaning as understood by those skilled in the art, and practice of the present invention will employ conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of those skilled in the art.

Sequence(s)

The following table (table a) is a list of representative sequences and related sequence identifier numbers. CDRs are underlined in plain font. For each heavy and light chain sequence, the CDRs are CDR1, CDR2, and CDR3 in succession. The linker sequence is shown in bold and the Rspo2 sequence is shown in italics. Thus, the heavy and light chain sequences and their variable domains can be readily determined based on the sequences provided. Heavy chain constant domains are shaded in gray. In a particular embodiment, the sequence is a polypeptide sequence present within the liver-specific Wnt signaling enhancing molecule. In a particular embodiment, the Wnt signaling enhancing molecule comprises two of the heavy chain fusion protein sequences and two of the light chain sequences, e.g., in the form of antibodies, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In a particular embodiment, the Wnt signaling enhancing molecule comprises two heavy chain fusion protein sequences, e.g., in the form of antibodies, comprising CDRs present in any of these sequences and two light chain sequences present in any of these sequences, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In a particular embodiment, the Wnt signaling enhancing molecule comprises two heavy chain fusion protein sequences, e.g., in the form of antibodies, each heavy chain fusion protein sequence comprising CDRs present in any of these sequences, and two light chain sequences, each light chain sequence comprising CDRs present in any of these sequences, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In particular embodiments, each of the two heavy chain fusion protein sequences and/or each of the two light chain sequences has at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the disclosed sequences, and in particular embodiments, amino acid modifications, such as insertions, deletions, or substitutions, are not present in the CDRs. In certain embodiments, the amino acid modification does not occur in (a) F105R, F105A or F105E; and/or (b) at either F109A or F109E.

Representative liver-specific Wnt signaling enhancing molecules 1R34-DDNN/RA, 8M24-v1, 1R34-EEST/EE, 1R34-EEST/RA, 1R34-EEAT/EE, 8M24 humanized 1, 8M24 humanized 2, 8M 24-eae-RA, 8M 24-eae-EE, 1R34-DDNN/RA sequences comprising two of the indicated light chain sequences and two of the indicated heavy chain fusion sequences in antibody form, e.g., bi-specific molecules that are bi-symmetrical: (a) Comprising or consisting of an IgG fused to 2 Rspo2 domains, wherein (i) one (1) Rspo2 domain is fused to each heavy chain of IgG, (ii) each arm of IgG binds to a liver-specific cell surface receptor binding domain (e.g., ASGR 1); and (b) wherein binding of such IgG arms activates downstream signaling.

/>

/>

/>

/>

/>

/>

In a particular embodiment, the sequence is a polypeptide sequence present within the liver-specific Wnt signaling enhancing molecule shown, comprising one or more of any polypeptides shown in table a or a variant thereof, or any combination thereof. In a particular embodiment, the Wnt signaling enhancing molecule comprises two of the heavy chain fusion protein sequences, e.g., in the form of antibodies, and two of the light chain sequences, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In a particular embodiment, the Wnt signaling enhancing molecule comprises two heavy chain fusion protein sequences, e.g., in the form of antibodies, comprising CDRs present in any of these sequences and two light chain sequences present in any of these sequences, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In a particular embodiment, the Wnt signaling enhancing molecule comprises two heavy chain fusion protein sequences, e.g., in the form of antibodies, each heavy chain fusion protein sequence comprising CDRs present in any of these sequences, and two light chain sequences, each light chain sequence comprising CDRs present in any of these sequences, wherein the two heavy chain fusion protein sequences bind to each other and each of the two light chain sequences binds to a different heavy chain fusion protein sequence. In particular embodiments, each of the two heavy chain fusion protein sequences and/or each of the two light chain sequences is a variant of any of the disclosed sequences and has at least 90%, at least 95%, at least 98% or at least 99% identity to any of the disclosed sequences, and in particular embodiments, any amino acid modification, such as an insertion, deletion or substitution, is not present within a CDR. In certain embodiments, the amino acid modification does not occur in (a) F105R, F105A or F105E; and/or (b) at either F109A or F109E. In a particular embodiment, the variant of the heavy chain comprises N297G. In particular embodiments, the RPOS2 sequence present in the variant comprises F105R and F109A substitutions. In particular embodiments, the RPOS2 sequences present in the variants comprise F105E and F109E substitutions. In particular embodiments, the molecule comprises amino acid substitutions compared to the parent or wild type of any of the following constructs: EEST/EE, EEST/RA, EEAT/EE, EESN/RA, EEAN/RA, 8M24-EAASE-0RA or 8M24-EASE-EE.

Liver-specific Wnt signaling enhancement molecules

In certain aspects, the present disclosure provides liver-specific Wnt signaling enhancement molecules that are capable of enhancing Wnt activity in a liver-specific manner. In certain embodiments, the liver-specific Wnt signaling enhancing molecule is a bifunctional molecule comprising a first domain that binds to one or more ZNRF3 and/or RNF43 ligases and a second domain that binds to liver tissue and/or liver cells. Each of the first domain and the second domain may be any moiety capable of binding to a ligase complex or a target tissue or cell, respectively. For example, each of the first domain and the second domain may be, but is not limited to, a moiety selected from the group consisting of: polypeptides (e.g., antibodies or antigen-binding fragments thereof or peptides or polypeptides other than antibodies), small molecules, and natural ligands or variants, fragments, or derivatives thereof. In certain embodiments, the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. The first domain and the second domain may be part of the same type as each other, or they may be part of different types. In certain embodiments, the liver-specific Wnt signaling enhancing molecule binds to a liver-specific cell surface receptor. In certain embodiments, for example, the liver-specific Wnt signaling-enhancing molecule increases or enhances Wnt signaling in liver tissue or liver cells by at least 50%, at least two-fold, at least three-fold, at least five-fold, at least ten-fold, at least twenty-fold, at least thirty-fold, at least forty-fold, or at least fifty-fold as compared to a negative control.

Liver-specific Wnt signaling enhancing molecules may have different forms. In a particular embodiment, the liver-specific Wnt signaling enhancing molecule is a fusion protein comprising a first polypeptide sequence that binds ZNRF3/RNF43 and a second polypeptide sequence that binds liver tissue or liver cells. In certain embodiments, the two polypeptide sequences may be fused directly or via a linker. In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises two or more polypeptides, such as dimers or multimers comprising two or more fusion proteins, each fusion protein comprising a first domain and a second domain, wherein the two or more polypeptides are linked, for example, by a linker moiety or via an intermolecular disulfide bond between amino acid residues in each of the two or more polypeptides, for example, cysteine residues.

In a particular embodiment, the liver-specific Wnt signaling enhancing molecule is an antibody comprising an antibody heavy chain and light chain (or antigen binding fragments thereof) that constitute a first domain or a second domain, wherein the other domain (i.e., the second domain or the first domain) is linked to the antibody heavy chain or light chain as a fusion protein or via a linker moiety. In a particular embodiment, the other domain is linked to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain or the C-terminus of the light chain. Such a structure may be referred to herein as an additional IgG scaffold. For example, the liver-specific Wnt signaling enhancing molecule may be an antibody that binds to a liver-specific cell surface receptor, wherein the binding domain that binds to ZNRF3/RNF43 is fused to or attached to the heavy or light chain of an antibody that binds to a tissue or cell specific receptor. In a particular embodiment, the liver-specific Wnt signaling enhancing molecule is an antibody or antigen-binding fragment thereof that binds ASGR1 or ASGR2, wherein the binding domain that binds ZNRF3/RNF43 is fused or appended to the heavy or light chain of the antibody or antigen-binding fragment thereof. In particular embodiments, the binding domain that binds ZNRF3/RNF43 is derived from an Rspo polypeptide, and in some embodiments, comprises Fu1 and Fu2 domains, wherein the Fu1 and Fu2 domains optionally comprise one or more amino acid modifications, including any of those disclosed herein, e.g., F105R, F105E, F a or F109E.

In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises a first domain that binds ZNRF3/RNF43 ("action module") and a second domain that binds, for example, a liver-specific receptor with high affinity ("targeting module"). In certain embodiments, each of the two domains has significantly reduced activity or no activity in enhancing Wnt signaling itself. However, when liver-specific Wnt signaling enhancing molecules bind to liver tissue or cells expressing liver-specific receptors, the E3 ligase ZNRF3/RNF43 is recruited into the ternary complex with liver-specific receptors, causing them to sequester and/or to clear from the cell surface via receptor-mediated endocytosis. The end result is an enhancement of Wnt signaling in a liver-specific manner.

In certain embodiments, the module of action is a binding agent for ZNRF3/RNF 43E 3 ligase and it may be designed based on R-spinal proteins, such as R-spinal protein-1-4, including but not limited to human R-spinal protein-1-4. In certain embodiments, the module of action is an R-spinal protein, such as wild-type R-spinal protein-1-4, optionally human R-spinal protein-1-4, or a variant or fragment thereof. In particular embodiments, it is a variant of any R-vertebrate protein-1-4 that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the corresponding wild-type R-vertebrate protein-1-4 sequence. In certain embodiments, the action module comprises or consists of furin domain 1 that binds to an R-vertebrate protein of ZNRF3/RNF43, e.g., any one of R-vertebrate proteins 1-4. Extended forms of furin domain 1 (including but not limited to those having mutated furin domain 2 that no longer binds to LGR4-6 or has reduced binding to LGR 4-6) or engineered antibodies or any other derivative or any engineered polypeptide other than antibodies capable of specifically binding ZNRF3/RNF43 may also be used. In certain embodiments, the action module comprises one or more furin domains 1 of R-spinal protein. In certain embodiments, it does not comprise furin domain 2 of R-spinal protein, or it comprises modified or variant furin domain 2 of R-spinal protein, e.g., furin domain 2 having reduced activity compared to wild-type furin domain 2. In certain embodiments, the action module comprises furin domain 1 of R-vertebrate protein but does not comprise furin domain 2. In certain embodiments, the action module comprises two or more furin domain 1 or a multimer of furin domain 1. The action domain may comprise one or more wild-type furin domains 1 of R-spinal protein. In particular embodiments, the action module comprises a modified or variant furin domain 1 having increased activity compared to wild-type furin domain 1, e.g., R-spinal protein binding to ZNRF3/RNF 43. Variants with increased binding to ZNRF3/RNF43 can be identified, for example, by screening phage or yeast display libraries comprising variants of R-vertebrate furin domain 1. Peptides or polypeptides that are not related to R-vertebrate furin domain 1 but that bind ZNRF3/RNF43 can also be identified by screening. The acting module may also comprise additional moieties or polypeptide sequences, e.g., additional amino acid residues, to stabilize the structure of the acting module or liver-specific Wnt signaling enhancing molecule present therein.

The R-spondin is capable of amplifying Wnt signaling. The minimal functional unit of R-vertebrate protein consists of two furin domains, furin domain 1 binding to ZNRF3/RNF 43E 3 ligase and furin domain 2 binding to LGR4-6 binding the ternary complexes of R-vertebrate protein, LGR and E3 ligase together. This results in internalization of the entire complex and removal of ZNRF3/RNF43 away from their destructive targets. Furin domain 1 alone is not fully functional, but it is able to bind ZNRF3 and RNF 43.

The functional module of the liver-specific Wnt signaling enhancing molecules described herein may be, but is not limited to, any functional moiety, such as a polypeptide or an organic chemical, capable of binding to the ZNRF3/RNF43 ligase. In particular embodiments, the targeting module alone or together with the targeting module, such as a furin domain 1 polypeptide comprising R-vertebrate protein, is substantially inactive in non-target tissues in order to minimize potential off-target effects. In the context of liver-specific Wnt signaling enhancement molecules, the effector moiety is fused to or bound to the targeting moiety, and when the liver-specific Wnt signaling enhancement molecule binds to target tissue expressing liver-specific receptors, the E3 ligase ZNRF3/RNF43 is recruited to the ternary complex with liver-specific receptors, causing them to be repositioned on, sequestered and/or cleared from the cell surface.

In certain embodiments, the action module comprises a fragment or variant of an R-vertebrate polypeptide (e.g., any of R-vertebrate proteins 1-4) or a functional fragment or variant thereof. In certain embodiments, the action module comprises a fragment of wild-type R-spinal protein, while in other embodiments, the action module comprises a fragment of R-spinal protein comprising one or more amino acid modifications. The R-vertebrate protein can be any R-vertebrate protein known in the art or a homologue thereof, including R-vertebrate proteins from any animal species, including but not limited to mammalian species, such as human R-vertebrate protein. R-vertebrate proteins have been identified and described, and their polypeptides and encoding polynucleotide sequences are known and available in the art. In particular embodiments, the R-vertebrate polypeptide is a human R-vertebrate or homolog found in other vertebrates or non-vertebrates, such as a mouse R-vertebrate. The amino acid sequences of human R-spondin 1, human R-spondin 2, human R-spondin 3, and human R-spondin 4 and their furin domain 1 are provided in FIGS. 38 and SEQ ID NOS.47-50, respectively. Their homologs and variants are available from general database searches, e.g.https:// www.dot.ncbi.dot.nlm.dot.nih.dot.gov/protein/. The invention includes, but is not limited to, a targeting module comprising or consisting of fragments and variants of any of these or other R-spinal proteins. In various embodiments, variants of any R-vertebrate polypeptide, and fragments thereof, comprise one or more amino acid modifications, e.g., deletions, additions, or substitutions, as compared to the wild-type R-vertebrate polypeptide. Modifications may be present in any region of the R-vertebrate protein variant or fragment thereof, including but not limited to furin domain 1 and/or furin domain 2. It is understood that amino acid modifications outside of furin domain 1 or furin domain 2 may alter the resulting variant such that the resulting variant has reduced LGR4-6 binding activity as compared to wild-type R-spinal protein or a fragment thereof.

In certain embodiments, the action module comprises or consists of an R-spinal protein sequence, such as full-length or wild-type R-spinal protein-1, -2, -3, or-4, optionally human R-spinal protein-1, -2, -3, or-4, or a variant or fragment thereof. In particular embodiments, it is a variant of any R-vertebrate protein-1-4 that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the corresponding wild-type R-vertebrate protein-1-4 sequence. In certain embodiments, the action module comprises or consists of a full length R-spinal protein (e.g., any of R-spinal protein-1-4) comprising one or more amino acid modifications, including but not limited to any of those disclosed herein. In certain embodiments, the action module comprises or consists of a fragment of a wild-type or modified R-spinal protein (e.g., any of R-spinal protein-1-4). In particular embodiments, the fragment is capable of binding ZNRF3 and/or RNF43. In certain embodiments, the action module comprises furin domain 1 of an R-spinal protein, or a fragment or variant of an R-spinal protein. In certain embodiments, the action module comprises, or consists of, one or more (e.g., one, two, or three or more) furin domain 1 of an R-vertebrate protein (e.g., R-vertebrate protein-1-4), or a variant thereof having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to R-vertebrate protein furin domain 1. In certain embodiments, the action module comprises an R-vertebrate furin 1 domain or variant or fragment thereof and an R-vertebrate furin 2 domain or variant or fragment thereof. In certain embodiments, the action module comprises an antibody or antigen-binding fragment thereof that binds ZNRF3/RNF 43. In certain embodiments, the action module specifically binds ZNRF3 or RNF43.

In certain embodiments, the action module comprises one or more furin domains 1 of an R-spinal protein, e.g., human R-spinal protein 1 or human R-spinal protein 2, or a variant thereof. In certain embodiments, the action module comprises one or more furin domains 1 of R-vertebrate protein, but it does not comprise furin domain 2 of R-vertebrate protein. In certain embodiments, the action module comprises one or more furin domains 1 of R-spinal protein, and it comprises a modified or variant furin domain 2 of R-spinal protein, e.g., furin domain 2 having reduced activity compared to wild-type furin domain 2. In certain embodiments, the action module comprises a modified or variant furin domain 2 having R-vertebrate protein, e.g., an R-vertebrate protein having a furin domain 2 with reduced activity compared to wild-type furin domain 2. In certain embodiments, the action module comprises two or more furin domain 1 or variants thereof or multimers of furin domain 1 or variants thereof. In certain embodiments, the action module comprises a variant R-vertebrate furin 1 domain comprising one or more point mutations at amino acid residues corresponding to K58, H76, S77, R86 and/or N91 of human R-vertebrate protein 2, for example. In certain embodiments, the action module comprises a variant R-vertebrate furin 2 domain comprising one or more point mutations at amino acid residues corresponding to F105, F109, and/or K121 of human R-vertebrate protein 2, for example. In particular embodiments, the action module comprises a modified or variant furin domain 1 having increased activity compared to wild-type furin domain 1, e.g., R-spinal protein binding to ZNRF3/RNF 43. The acting module may also comprise additional moieties or polypeptide sequences, e.g., additional amino acid residues, to stabilize the structure of the acting module or liver-specific Wnt signaling enhancing molecule present therein. In certain embodiments, the action module comprises a peptide or polypeptide that has no significant/strong sequence homology to the R-vertebrate protein but has a binding affinity to ZNRF3/RNF43 comparable to or greater than the binding affinity of the R-vertebrate protein to ZNRF3/RNF 43.

In certain embodiments, the action module comprises a furin domain 1 of an R-spinal protein polypeptide (e.g., a human R-spinal protein) or a functional fragment or variant thereof and a modified or variant furin domain 2 of an R-spinal protein polypeptide (e.g., a human R-spinal protein), wherein the modified furin domain 2 has a reduced binding affinity for LGR4-6 as compared to the corresponding wild-type furin domain 2. In certain embodiments, furin domain 2 comprises one or more point mutations, for example, at amino acid residues corresponding to F105 and/or F109 of human R-vertebrate protein 2. The corresponding amino acid residues in other R-spinal protein polypeptides can be readily determined by one skilled in the art by comparing their amino acid sequences with human R-spinal protein 2. In certain embodiments, the action module comprises furin domain 1 or a variant thereof and furin domain 2 or a variant thereof, wherein furin domain 1 and/or furin domain 2 comprises one or more point mutations. One or more point mutations within the action module (as compared to the corresponding wild-type R-spondin sequence) may occur at any amino acid residue within furin domain 1 and/or furin domain 2, including, but not limited to, amino acid residues K58, H76, S77, R86, N91, F105, F109, or K121 and other residues that may be modified to reduce binding affinity to LGR4-6, for example. The regions of furin domain 1 and furin domain 2 of human R-spinal protein 1 that are important for their functional activity have been identified, including conserved hydrophilic residues S48, N51, R66, R70 and Q71, and less conserved hydrophobic residues L46, L54, I62 and L64 that are important for binding to E3 ligase. Furthermore, in human R-vertebrate protein 1 furin domain 1, amino acid residues K59, S78, D85, R87, N88 and N92 form a hydrophilic interaction surface with LGR5, and FSHNF amino acid sequence has been identified as a loop important for a hydrophobic surface. In particular embodiments, the action module comprising R-vertebrate furin domain 1 and/or furin domain 2 may comprise one or more mutations within any of these regions, surfaces or amino acid residues.

In particular embodiments, the action modules comprising R-vertebrate furin domain 1 and/or furin domain 2 may comprise one or more mutations or other substitutions in addition to these regions, surfaces or amino acid residues that indirectly impair LGR4-6 binding by affecting the structure and/or stability of the binding surface.

In certain embodiments, the action module comprising R-vertebrate furin domain 1 and/or furin domain 2 may comprise one or more mutations at any amino acid residue, including but not limited to any of those depicted in the appended examples. In a particular embodiment, the action module comprises a modified furin domain 2 comprising an amino acid substitution at amino acid residues F105 and/or F109. In a particular embodiment, the action module comprises a furin 1 domain and a modified furin domain 2 comprising amino acid substitutions at amino acid residues F105 and/or F109. In certain embodiments, the action module comprises a modified furin 1 domain and a modified furin 2 domain, wherein in certain embodiments the modified furin 1 domain comprises one or more amino acid modifications at amino acids R65, R69, and/or Q70, and the modified furin domain comprises one or more amino acid modifications at amino acids F105 and/or F109. In certain embodiments, the modified R-vertebrate polypeptide, or fragment or variant thereof, comprises amino acid substitutions at positions corresponding to amino acids F105 and F109 of human R-vertebrate protein 2. In certain embodiments, the two amino acid substitutions comprise: (a) F105R, F105A or F105E; and (b) F109A or F109E. In a particular embodiment, the two amino acid substitutions are: (a) F105R and F109A; (b) F105A and F109A; (c) F105E and F109A; or (d) F105E and F109E. In certain embodiments, the modified R-vertebrate polypeptide, or fragment or variant thereof, has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOS.29-32. In certain embodiments, the modified R-vertebrate polypeptide, or fragment or variant thereof, hybridizes to SEQ ID NO:29-32, and comprises one of the following combinations of amino acid substitutions: (a) F105R and F109A; (b) F105A and F109A; (c) F105E and F109A; or (d) F105E and F109E. In particular embodiments, for example, in the context of full-length R-vertebrate proteins, modified furin domain 2 binds to LGR4-6 with less than 80%, less than 50%, less than 20%, or less than 10% of the binding of the corresponding wild-type furin domain 2.

In certain embodiments, the action module comprises furin domain 1 of an R-spinal protein polypeptide (e.g., human R-spinal protein) or a functional fragment or variant thereof and unmodified furin domain 2 of an R-spinal protein polypeptide (e.g., human R-spinal protein). Although in certain embodiments modified furin domain 2 having reduced binding affinity for LGR4-6 as compared to the corresponding wild-type furin domain 2 is more desirable to increase the specificity of tissue targeting, in certain embodiments unmodified furin domain 2 in combination with a targeting module has improved tissue targeting as compared to wild-type R-spinal protein without the targeting module and has utility in certain circumstances.

In certain embodiments, the action module comprises wild-type or modified R-vertebrate furin domain 1, e.g., from any of R-vertebrate protein-1, -2, -3, -4, optionally human R-vertebrate protein-1, -2, -3, or-4. In particular embodiments, the action module comprises an R-spinal protein furin 1 domain and a wild-type or modified R-spinal protein furin 2 domain, e.g., from any of R-spinal protein-1, -2, -3, -4, optionally human R-spinal protein-1, -2, -3, or-4. In a particular embodiment, the action module comprises a first R-vertebrate furin 1 domain and a second wild-type or modified R-vertebrate furin 1 domain, e.g., from any of R-vertebrate proteins-1, -2, -3, -4, optionally human R-vertebrate proteins-1, -2, -3 or-4. In particular embodiments, for example, in the context of full-length R-vertebrate proteins, the binding affinity of modified furin domain 2 to LGR4-6 is comparable to the binding affinity of the corresponding wild-type furin domain 2, or the binding affinity to LGR4-6 is less than 80%, less than 50%, less than 20% or less than 10% of the binding affinity of the corresponding wild-type furin domain 2.

In certain embodiments, the action module comprises an antibody or antigen-binding fragment thereof that specifically binds ZNRF3 and/or RNF 43. In particular embodiments, the action module comprises an antibody or antigen binding fragment thereof that binds to human RNF43 (hnnf 43, NCBI reference sequence xp_011523257.1, residues 44-198) or human ZNRF3 (hZNRF 3, NCBI reference sequence np_001193927.1, residues 56-219). In particular embodiments, the acting moiety is an antibody or antigen-binding fragment thereof comprising a nanobody, VH or VL sequence, or fragment or variant thereof.

In certain embodiments, the targeting moiety specifically binds to a liver-specific surface molecule, such as a liver-specific surface receptor, and may be, for example, a natural ligand, an antibody, or a synthetic chemical. In certain embodiments, the liver-specific surface molecules are preferentially expressed on liver organs, liver tissue, and/or liver cells. In certain embodiments, the liver-specific surface molecule has increased or enhanced expression on liver tissue or liver cells as compared to one or more other non-targeted organs, tissues or cell types. In certain embodiments, the liver-specific surface molecule is preferentially expressed on the surface of a liver organ, liver tissue, or liver cell, respectively, as compared to one or more other organs, tissues, or cell types. For example, in a particular embodiment, a cell surface receptor is considered to be a liver-specific or liver-specific cell surface molecule if its level expression in a liver organ, tissue or cell is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold or at least 1000-fold higher than its expression in one or more, five or more, all other organs, tissues or cells, or an average of all other organs, tissues or cells, respectively. In certain embodiments, the liver-specific or liver-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor comprising a region located within a cell surface membrane and an extracellular region to which a targeting moiety can bind. In various embodiments, the methods described herein can be practiced by specifically targeting cell surface molecules that are expressed only on liver tissue or a subset of tissues including liver tissue, or by specifically targeting cell surface molecules that have higher expression levels on liver tissue than all, most, or a substantial number of other tissues, e.g., expression on liver tissue is higher than at least two, at least five, at least ten, or at least twenty other tissues.

Liver-specific cell surface receptors are known in the art. Examples of liver-specific surface receptors include, but are not limited to, ASGR1, ASGR2, TFR2, and SLC10A1. Additional receptors for liver delivery are described, for example, in Yan et al, tuner Biology,2015;36:55-67.

In certain embodiments, the targeting module comprises an antibody or antigen binding fragment thereof that specifically binds ASGR1 and/or ASGR 2. In a particular embodiment, the targeting moiety comprises an antibody or antigen binding fragment thereof comprising: a) CDRH1, CDRH2 and CDRH3 sequences shown herein; and/or b) CDRL1, CDRL2 and CDRL3 sequences as set forth herein; or a variant of the antibody or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein the variant comprises less than 8 amino acid substitutions (e.g., less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2) in the CDR sequences. In certain embodiments, the targeting module comprises an antibody heavy chain variable domain comprising the CDRs of SEQ ID NOS: 34, 35 and 36 and an antibody light chain variable domain comprising the CDRs of SEQ ID NOS: 37, 38 and 39. In certain embodiments, the targeting module comprises an antibody heavy chain variable domain comprising the CDRs of SEQ ID NOS: 34, 35 and 36 and an antibody light chain variable domain comprising the CDRs of SEQ ID NOS: 37, 40 and 39. In certain embodiments, the targeting module comprises an antibody light chain variable domain comprising the CDRs of SEQ ID NOS 41, 42 and 43 and an antibody heavy chain variable domain comprising the CDRs of SEQ ID NOS 44, 45 and 46.

As used herein, a cell surface molecule is considered liver-specific if there is a greater amount of the molecule on a liver cell or liver tissue as compared to one or more other cell or tissue types or any other cell or tissue type. In certain embodiments, the greater amount is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to the amount in one or more other cell or tissue types or any other cell or tissue type. For example, in a particular embodiment, a cell surface receptor is considered to be a liver-specific or liver-specific cell surface molecule if its level expression in a target organ, tissue or cell is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold or at least 1000-fold higher than its expression in one or more, five or more, all other organs, tissues or cells, or an average of all other organs, tissues or cells, respectively. In certain embodiments, the liver-specific or cell-specific cell surface molecule is a cell surface receptor, e.g., a polypeptide receptor comprising a region located within a cell surface membrane and an extracellular region to which a targeting moiety can bind.

In certain embodiments, the targeting moiety binds to a liver-specific surface molecule. The targeting moiety that binds to each liver-specific surface molecule may be, but is not limited to, an antibody or antigen binding fragment thereof, a peptide, a natural ligand for a tissue or cell specific receptor or derivatives and synthetic small molecules thereof, and the like.

In certain embodiments, the liver-specific Wnt signaling enhancing molecule binds to a particular liver cell type, e.g., a particular cell type associated with a target tissue. For example, in liver tissue, the targeting module may bind hepatocytes, precursors of hepatocytes, and stem cells, biliary cells, and/or endothelial or other vascular cells.

The asialoglycoprotein receptor (ASGPR) consists of ASGR1 and ASGR2 (reviewed, for example, by stock, morell and Ashwell,1991,Targeted Diagnostics and Therapy4:41-64). The receptor is a transmembrane protein that plays a key role in serum glycoprotein homeostasis by mediating endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues. Thus, natural and synthetic ligands for AGPR include, but are not limited to, galactosylated cholinesterase, galactose (Gal) and N-acetylgalactosamine (GalNAc), galNAc containing molecules such as GalNAc terminating glycoproteins and mono-, oligo-or polysaccharides containing molecules or nanoparticles (reviewed, for example, by D' Souza and Devarajan 2015,Journal of Controlled Release,203:126-139).

In various embodiments, the liver-specific surface molecule is a liver-specific cell surface receptor. For the liver, these include, but are not limited to ASGR1 and ASGR2. In particular embodiments, the targeting module binds to human ASGR1 (hASGR 1; NCBI reference sequence NP-001662.1, residues 62-291), human ASGR2 (hASGR 2; NCBI reference sequence NP-550436.1, residues 66-292), cynomolgus ASGR1 (cynoaGR 1, sequence ID XP-005582755.1, residues 62-291), or cynomolgus ASGR2 (cynoaGR 2).

In certain embodiments, the targeting module comprises an antibody or antigen binding fragment thereof comprising the CDRH1, CDRH2, and CDRH3 sequences shown herein; and/or CDRL1, CDRL2 and CDRL3 sequences shown herein; or a variant of the antibody or antigen binding fragment thereof, comprising one or more amino acid modifications, wherein the variant comprises fewer than 8 amino acid substitutions in the CDR sequences. In particular embodiments, an isolated antibody or antigen binding fragment thereof comprises a heavy chain variable region, a light chain variable region, a nanobody, or an scFv sequence comprising an amino acid sequence that is at least 90% or at least 95% identical to a sequence disclosed herein (e.g., a sequence disclosed in any one of SEQ ID NOs: 1-24 or 29). In particular embodiments, the targeting module comprises a variable heavy chain region having at least 90% or at least 95% identity to the variable heavy chain domain shown in any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 15, 16, 20, 22, 24, 26, 28, 33 or 51. In particular embodiments, the targeting module comprises a variable light chain region having at least 90% or at least 95% identity to a variable light chain domain as set forth in any one of SEQ ID NOs 1, 3, 5, 7, 9, 11, 14, 17, 18, 19, 21, 23, 25 or 27. In particular embodiments, the targeting module comprises a heavy chain having at least 90% or at least 95% identity to the heavy chain shown in any one of SEQ ID NOs 2, 6, 8, 10, 12, 22, 26, 33 or 51. In particular embodiments, the targeting module comprises a light chain having at least 90% or at least 95% identity to the light chain shown in any one of SEQ ID NOs 1, 5, 7, 9, 11, 21 or 25. In particular embodiments, the constant region of the light chain and/or heavy chain is IgG1 or IgG2.

In certain embodiments, the Wnt signaling enhancing molecule comprises a fusion protein, e.g., a fusion protein comprising an antibody heavy or light chain that comprises a targeting domain fused to a scope. In certain embodiments, two regions of the fusion protein (e.g., the targeting domain region and the action domain) are fused via a linker moiety. In certain embodiments, the linker consists of amino acids linked together by peptide bonds. In particular embodiments, the linker comprises 1 up to about 40 amino acid residues, 1 up to about 20 amino acid residues, or 1 to about 10 amino acid residues in length. In certain embodiments, the amino acid residues in the linker are selected from twenty standard amino acids, and in certain embodiments, from cysteine, glycine, alanine, proline, asparagine, glutamine, and/or serine. In certain embodiments, the linker comprises one or more unnatural amino acids. In some embodiments, the peptidyl linker consists of most amino acids that are sterically unimpeded, such as glycine, serine, and alanine, linked by peptide bonds. Some linkers include poly glycine, poly serine, and poly alanine or a combination of any of these. Some exemplary peptidyl linkers are poly (Gly) 1-8, particularly (Gly) 3, (Gly) 4 (SEQ ID NO: 55), (Gly) 5 (SEQ ID NO: 56) and (Gly) 7 (SEQ ID NO: 57), and poly (Gly) 4Ser (SEQ ID NO: 58), poly (Gly-Ala) 2-4 and poly (Ala) 1-8. Other specific examples of peptidyl linkers include (Gly) 5Lys (SEQ ID NO: 59) and (Gly) 5LysArg (SEQ ID NO: 60). For the purposes of explaining the above nomenclature, for example, (Gly) 3Lys (Gly) 4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly (SEQ ID NO: 61). Other combinations of Gly and Ala are also useful. In addition, the peptidyl linker may also comprise a non-peptidyl moiety, such as a 6 carbon aliphatic molecule of the formula- -CH2- -CH2- -CH2- -CH2- -CH2- -. The peptidyl linker may be altered to form a derivative as described herein. In a particular embodiment, the linker is any of those identified in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 20, 22, 24, 26 or 28.

Exemplary non-peptidyl linkers include, for example, alkyl linkers, such as- -NH- - (CH 2) s- -C (O) - -, where s = 2-20. These alkyl linkers may also be substituted with any non-sterically hindered group, such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., cl, br), CN, NH2, phenyl, and the like. The non-peptide moiety, such as a non-peptidyl linker or a non-peptide half-life extending moiety, of the compositions of matter of the invention can be synthesized by conventional organic chemical reactions. Chemical groups that can be used to attach the binding domain include carbamates; amides (amine plus carboxylic acid); esters (alcohol plus carboxylic acid), thioethers (haloalkane plus mercapto; maleimide plus mercapto), schiff bases (amine plus aldehyde), ureas (amine plus isocyanate), thioureas (amine plus isothiocyanate), sulfonamides (amine plus sulfonyl chloride), disulfides; hydrazones, lipids, and the like, as known in the art.

The linkage between the domains may comprise a spacer, such as an alkyl spacer, which may be linear or branched, is typically linear, and may include one or more unsaturated bonds; typically having from 1 to about 300 carbon atoms; more typically about 1 to 25 carbon atoms; and may be about 3 to 12 carbon atoms. This type of spacer may also contain heteroatoms or functional groups, including amines, ethers, phosphodiesters, and the like. Specific structures of interest include: (CH) ₂ CH ₂ O) n, wherein n is 1 to about 12; (CH) ₂ CH ₂ NH) n, wherein n is 1 to about 12; [ (CH) ₂ )n(C＝O)NH(CH ₂ ) _m ]z, wherein n and m are 1 to about 6, and z is 1 to about 10; [ (CH) ₂ )nOPO ₃ (CH ₂ ) _m ] _z Wherein n and m are from 1 to about 6 and z is from 1 to about 10. Such linkers may include polyethylene glycol, which may be linear or branched.

In a specific embodiment, the liver-specific Wnt signaling enhancing molecule or pharmaceutically acceptable salt thereof comprises a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from the group consisting of zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43) and a second domain that specifically binds to asialoglycoprotein receptor 1 (ASGR 1) and/or asialoglycoprotein receptor 2 (ASGR 2), wherein: (a) The first domain comprises an Rspo sequence or a fragment or variant thereof; and/or (b) the second domain comprises an antibody or antigen binding fragment thereof comprising: (i) CDRH1, CDRH2 and CDRH3 sequences shown herein; and/or (ii) CDRL1, CDRL2 and CDRL3 sequences as set forth herein, or a variant of said antibody or antigen-binding fragment thereof, comprising one or more amino acid modifications, wherein said variant comprises fewer than 8 amino acid substitutions in said CDR sequence. In a particular embodiment, it comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to any one of SEQ ID NOs 1 to 29. In a particular embodiment, the polypeptide comprises the CDR sequences identified in any one of SEQ ID NOS 1-46 or 51 or any other sequences disclosed herein.

An "action or targeting moiety, such as an antibody or antigen binding fragment thereof, that" specifically binds "or" is specific for "a particular cell surface polypeptide or receptor is a moiety that binds the particular polypeptide or receptor without substantially binding any other polypeptide or polypeptide epitope. In some embodiments, the action and targeting modules of the present disclosure specifically bind to ZNRF3/RNF43 or liver-specific cell surface molecules (e.g., receptors), respectively, dissociation constants (K _d ) Equal to or lower thanAt 1000nM, at or below 100nM, at or below 10nM, at or below 1nM, at or below 0.5nM, at or below 0.1nM, at or below 0.01nM, at or below 0.005nM, at or below 0.001nM or at or below 0.0005nM. The affinity of an adhesive (e.g., an antibody) can be readily determined using conventional techniques, such as those described by Scatchard et al (Ann.N.Y. Acad. Sci. USA 51:660 (1949), ELISA assays, biological Layer Interference (BLI) assays, and Surface Plasmon Resonance (SPR) assays.

In certain embodiments, the acting moiety and/or targeting moiety of the liver-specific Wnt signaling molecule is a polypeptide, while in other embodiments, the acting moiety and/or targeting moiety of the liver-specific Wnt signaling molecule is a small organic molecule. In certain embodiments, both the targeting moiety and the targeting moiety are polypeptides, such as antibodies or antigen binding fragments thereof. In certain embodiments, the targeting moiety and the action moiety of the liver-specific Wnt signaling enhancement molecule are covalently bound to each other. In certain embodiments, the action module and the targeting module of the liver-specific Wnt signaling-enhancing fusion molecule are non-covalently bound to each other. In certain embodiments, the action module and the targeting module of the liver-specific Wnt signaling enhancement molecule are present within the same fusion protein. In other embodiments, the targeting moiety is present in a first polypeptide further comprising a first binding domain and the targeting moiety is present in a second polypeptide further comprising a second binding domain, wherein the first and second binding domains bind to each other. In some embodiments, the first and second binding domains are the same or variants thereof, such as, for example, an Fc polypeptide. In some embodiments, the first and second binding domains are different from each other. In particular embodiments, the invention includes the use of fragments or variants of any of the targeting or targeting modules described herein (including functional fragments or variants of reference molecules).

In certain embodiments, the liver-specific Wnt signaling enhancing molecule (e.g., fusion protein) has a formula selected from the group consisting of: r is R ₁ -L-R ₂ And R is ₂ -L-R ₁ Wherein R is ₁ Is a module for combining ZNRF3/RNF43, R ₂ Is a targeting module that binds liver-specific cell surface receptors, and L is a linker, and wherein L may be absent or present. R is R ₁ And R is ₂ Each of which may be any of the various functional and targeting modules described herein, respectively. R is R ₁ And R is ₂ May be any moiety capable of binding to one or more E3 ligases (ZNRF 3 or RNF 43) or target tissues or cells, respectively. For example, R ₁ And R is ₂ May be, but is not limited to, a moiety selected from the group consisting of: a polypeptide (e.g., an antibody or antigen-binding fragment thereof or a peptide or polypeptide other than an antibody), a small molecule, and a natural ligand or variant, fragment, or derivative thereof. In certain embodiments, the natural ligand is a polypeptide, a small molecule, an ion, an amino acid, a lipid, or a sugar molecule. Action and targeting modules (i.e. R ₁ And R is ₂ ) May be the same type of portion as each other, or they may be different types of portions. In particular embodiments, R ₂ Is an antibody or antigen binding fragment thereof, and in certain embodiments, R ₂ Comprising an Fc protein or an analog thereof.

In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises a single molecule (e.g., a polypeptide), while in other embodiments, the Wnt signaling enhancing fusion molecule comprises two or more molecules (e.g., polypeptides) that bind to each other, e.g., are non-covalently bound to each other. For example, in one embodiment, the liver-specific Wnt signaling enhancing fusion comprises Wnt signaling enhancing fusion having the formula R, respectively ₃ -L ₁ And R is ₄ -L ₂ Wherein R is ₃ Is an action module, R ₄ Is a targeting module, and wherein L ₁ And L ₂ The groups combine with each other, for example, to form dimers. In various embodiments, L ₁ And L ₂ The radicals are identical to or different from one another. L (L) ₁ Or L ₂ One example of a group is an Fc sequence, such as a murine Fc2b or a human Fc1, each of which is known in the art. R is R ₃ And R is ₄ Each of which may be any of the various functional and targeting modules described herein, respectively. R is R ₃ And R is ₄ May be any moiety capable of binding to one or more E3 ligases (ZNRF 3 and/or RNF 43) or target tissues or cells, respectively.

In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises an antibody or binding fragment thereof that binds to one or more E3 ligases (ZNRF 3 and/or RNF 43), wherein the antibody heavy chain and/or antibody light chain comprises an additional binding domain that binds to a target tissue or cell.

In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises an antibody or one or more binding fragments thereof that binds to a target tissue or cell, wherein the antibody heavy chain and/or antibody light chain comprises an additional binding domain that binds to one or more E3 ligases (ZNRF 3 and/or RNF 43). The additional binding domain may be fused directly to the N-terminus or C-terminus of the antibody, for example as a heavy chain or light chain fusion protein, or it may be attached to the heavy chain or light chain via a linker moiety, for example to the N-terminus, C-terminus, or an internal amino acid of the heavy chain or light chain. In certain embodiments, the antibody is an IgG, such as IgG1 or IgG2. In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises four polypeptides comprising two antibody light chains and two antibody heavy chains, wherein one or more of the antibody heavy chains and/or antibody light chains further comprises an additional binding domain that binds one or more E3 ligases (ZNRF 3 and/or RNF 43), such as Rspo2 variants disclosed herein. In certain embodiments, the additional binding domain is linked to one or both of the heavy and/or light chains (or binding fragments thereof) of the antibody via a linker (as any of the disclosure herein). In certain embodiments, two antibody heavy chains are linked via one or more disulfide bonds, and each antibody light chain is linked to a different antibody heavy chain via one or more disulfide bonds. In certain embodiments, each light chain in the antibody-like Wnt signaling enhancing molecule is identical. In certain embodiments, each heavy chain in the antibody-like Wnt signaling enhancing molecule is the same. In certain embodiments, each light chain in the antibody-like Wnt signaling enhancing molecule is different. In certain embodiments, each heavy chain in the antibody-like Wnt signaling enhancing molecule is different. In a particular embodiment, the Wnt signaling enhancing molecule comprises two different light chains and/or two different heavy chains, each of which binds to a different liver-specific cell surface molecule. In a particular embodiment, the Wnt signaling enhancing molecule comprises two different light chains and/or two different heavy chains, each of which binds to a different E3 ligase. In various embodiments, the binding domain is attached to a heavy chain or a light chain, and in particular embodiments, the liver-specific Wnt signaling enhancement molecule comprises two antibody light chains and two antibody heavy chains of an antibody that specifically bind to a liver-specific cell surface molecule (e.g., ASGR1 or ASGR 2), wherein the binding domain that binds to one or more E3 ligases (ZNRF 3 and/or RNF 43) is attached to the N-terminus of the two antibody heavy chains. In certain embodiments, the binding domain that binds to one or more E3 ligases is Rspo or a fragment or variant thereof. In certain embodiments, rspo has at least 90%, at least 95%, at least 98% or at least 99% sequence identity with any one of SEQ ID NOS: 29-32 or SEQ ID NOS: 47-50, or fragments thereof. In a specific embodiment, the liver-specific Wnt signaling enhancement molecule comprises two heavy chain fusion proteins and two light chain fusion proteins as disclosed in table a, e.g., the liver-specific Wnt signaling enhancement molecule is 1R34-DDNN/RA, 8M24-v1, 1R34-EEST/EE, 1R34-EEST/RA, 1R34-EEAT/EE, 8M24 humanized 1, 8M24 humanized 2, 8M24-EASE-RA, 8M24-EASE-EE, or 1R34-DDNN/RA.

In certain embodiments, the liver-specific Wnt signaling enhancement molecule or pharmaceutically acceptable salt thereof comprises a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from the group consisting of zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43), and a second domain that specifically binds to asialoglycoprotein receptor 1 (ASGR 1), wherein:

(a) The first domain comprises a modified R-vertebrate polypeptide or fragment or variant thereof; and

(b) The second domain comprises a modified antibody or antigen-binding fragment thereof comprising: CDRH1, CDRH2 and CDRH3 sequences; CDRL1, CDRL2 and CDRL3 sequences.

In a particular embodiment, the second domain comprises two modified antibody light chains and two modified antibody heavy chains, wherein the first domain is attached to the N-terminus of each antibody heavy chain. Thus, in a particular embodiment, the Wnt signaling enhancing molecule comprises two modified antibody light chains derived from an antibody that binds to ASGR1 and two fusion proteins, each comprising a modified antibody heavy chain derived from an antibody that binds to ASGR1 and an R-vertebrate polypeptide (or fragment thereof) fused to the N-terminus thereof, optionally via a linker moiety. In particular embodiments, the R-vertebrate polypeptide is a modified R-vertebrate polypeptide, e.g., a modified Rspo-2 polypeptide, comprising one or more amino acid modifications as compared to wild-type human Rspo-2.

In certain embodiments, the modified R-vertebrate polypeptide, or fragment or variant thereof, comprises amino acid substitutions at positions corresponding to amino acids F105 and F109 of human R-vertebrate protein 2. In certain embodiments, the two amino acid substitutions comprise: (a) F105R, F105A or F105E; and (b) F109A or F109E. In a particular embodiment, the two amino acid substitutions are: (a) F105R and F109A; (b) F105A and F109A; (c) F105E and F109A; or (d) F105E and F109E. In certain embodiments, the modified R-vertebrate polypeptide, or fragment or variant thereof, has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of the following:

in certain embodiments, a modified antibody heavy chain derived from an antibody that binds ASGR1 is fused to a modified R-vertebrate polypeptide via a linker moiety (including any of those disclosed herein). In particular embodiments, the linker moiety is a peptidyl linker comprising or having the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 62).

In particular embodiments, the modified heavy chain variable region comprises a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 20, 22, 24, 26, 28, 33 or 51. In particular embodiments, the modified light chain variable region comprises a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs 1, 3, 5, 7, 9, 11, 14, 17, 18, 19, 21, 23, 25 or 27. In certain embodiments, amino acid modifications, such as insertions, deletions, or substitutions, are not present in the CDRs. In certain embodiments, the amino acid modification does not occur in (a) F105R, F105A or F105E; and/or (b) at either F109A or F109E.

In particular embodiments, the modified heavy chain comprises a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs 2, 6, 8, 10, 12, 22, 26 or 33, or a fragment thereof comprising a heavy chain sequence or a heavy chain variable domain sequence (e.g., in the absence of RSPO2 and linker sequences). In particular embodiments, the modified light chain comprises a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs 1, 5, 7, 9, 11, 21 or 25. In certain embodiments, amino acid modifications, such as insertions, deletions, or substitutions, are not present in the CDRs. In certain embodiments, the amino acid modification does not occur in (a) F105R, F105A or F105E; and/or (b) at either F109A or F109E. In a particular embodiment, the variant of the heavy chain comprises N297G. In particular embodiments, the RPOS2 sequence present in the variant comprises F105R and F109A substitutions. In particular embodiments, the RPOS2 sequences present in the variants comprise F105E and F109E substitutions. In particular embodiments, the molecule comprises amino acid substitutions of any of the following constructs as compared to the parent or wild type: EEST/EE, EEST/RA, EEAT/EE, EESN/RA, EEAN/RA, 8M24-EAASE-RA or 8M24-EASE-EE.

In a particular embodiment, the two fusion polypeptides each comprise a sequence having at least 95% identity to any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 20, 22, 24, 26, 28, 33 or 51.

In certain embodiments, the modified heavy chain and modified light chain sequences are derived from or have at least 90%, at least 95%, at least 98% or at least 99% identity (CDR underlined) to an anti-ASGR 1 antibody comprising the following heavy chain and light chain sequences:

light chain:

heavy chain:

in particular embodiments, the light chain comprises amino acid substitutions at one or more amino acid positions selected from the group consisting of D25, N51, and N88 (shown above Wen Cuti). In certain embodiments, all three positions are substituted. In certain embodiments, D25 is substituted with E. In certain embodiments, N51 is substituted with S or a. In certain embodiments, N88 is substituted with T. In one embodiment, D25 is substituted with E, N51 is substituted with S, and N88 is substituted with T (EST). In one embodiment, D25 is substituted with E, N51 is substituted with a, and N88 is substituted with T (EAT). In particular embodiments, any Wnt signaling enhancing molecule disclosed herein comprises a light chain variable domain that has at least 90%, at least 95%, at least 98% or at least 99% identity to the variable domain of SEQ ID NO:1, optionally further comprising an amino acid substitution at one or more amino acid positions selected from the group consisting of D25, N51 and N88, including any of the substitutions disclosed above.

In a particular embodiment, the heavy chain comprises an amino acid substitution at D62 (shown above Wen Cuti). In certain embodiments, D62 is substituted with E (E). In particular embodiments, any Wnt signal enhancing molecule disclosed herein comprises a heavy chain variable domain having at least 90%, at least 95%, at least 98% or at least 99% identity to the variable domain of SEQ ID NO. 33, optionally further comprising an amino acid substitution at D62, including any of the substitutions disclosed above.

In a particular embodiment, the modified antibody portion of the molecule (or the variable domain thereof) comprises a combination of the following amino acid substitutions: (a) in the heavy chain, D62 is substituted with E; and (b) in the light chain, D25 is substituted with E, N51 is substituted with S, and N88 is substituted with T (EEST). In a particular embodiment, the modified antibody portion of the molecule (or variable domain thereof) comprises a combination of the following amino acid substituents: (a) in the heavy chain, D62 is substituted with E; and (b) in the light chain, D25 is substituted with E, N51 is substituted with a, and N88 is substituted with T (EEAT).

Thus, in certain embodiments, the modified anti-ASGR 1 antibody portion of the molecule comprises any one of the following combinations of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences:

(a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; or (b)

(b) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively.

In certain embodiments, the modified anti-ASGR 1 antibody portion of the molecule comprises VH and VL domains comprising any one of these combinations of CDRs in the context of the parent sequence, or variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the VH or VL domain. In certain embodiments, any of the variants disclosed herein do not comprise any additional amino acid modifications in their CDR sequences (other than those described herein).

In a particular embodiment, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to any one of SEQ ID NOs 1, 5, 7, 9, 11, 21, 25, or a variable region thereof.

In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 1, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 2, or a variable region thereof.

In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 7, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 8, or a variable region thereof.

In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 7, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 10, or a variable region thereof.

In certain embodiments, the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 11, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 12, or a variable region thereof.

In a particular embodiment, the two fusion polypeptides each comprise a sequence having at least 95% identity to any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 20, 22, 24, 26, 28, 33 or 51, or a variable region thereof.

In certain embodiments, the modified heavy chain and modified light chain sequences are derived from or have at least 90%, at least 95%, at least 98% or at least 99% identity (CDR underlined) to an 8M24 anti-ASGR 1 antibody comprising the following heavy chain and light chain sequences:

Light chain:

heavy chain:

in a particular embodiment, the light chain comprises an amino acid substitution at amino acid position D56 (shown above Wen Cuti). In certain embodiments, D56 is substituted with E, S or a. In certain embodiments, D56 is substituted with E (E). In particular embodiments, any Wnt signal enhancing molecule disclosed herein comprises a light chain variable domain having at least 90%, at least 95%, at least 98% or at least 99% identity to the variable domain of SEQ ID NO. 5, optionally further comprising an amino acid substitution at D56, including any of the substitutions disclosed above.

In particular embodiments, the heavy chain comprises amino acid substitutions at one or more amino acid positions selected from N31, N57, or D102 (shown above Wen Cuti). In certain embodiments, all three positions are substituted. In certain embodiments, N31 is substituted with a or Q. In certain embodiments, N57 is substituted with S, A or N. In certain embodiments, D102 is substituted with E, S or a. In one embodiment, N31 is substituted with a, N57 is substituted with S, and D102 is substituted with E (ASE). In certain embodiments, N31 is substituted with a, N57 is substituted with S, D102 is substituted with E, and D110 is unsubstituted (ASED). In particular embodiments, any Wnt signal enhancing molecule disclosed herein comprises a heavy chain variable domain having at least 90%, at least 95%, at least 98% or at least 99% identity to the variable domain of any of SEQ ID NOs 20, 22 or 51, optionally further comprising an amino acid substitution at one or more amino acid positions selected from N31, N57 or D102, including any of the substitutions disclosed above.

In a particular embodiment, the modified antibody portion of the molecule comprises a combination of the following amino acid substitutions: (a) in the light chain, D56 is substituted with E; and (b) in the heavy chain, N31 is substituted with a, N57 is substituted with S, D102 is substituted with E, and D110 is unsubstituted (EASE).

Thus, in certain embodiments, the modified anti-ASGR 1 antibody portion of the molecule comprises the following combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences:

(c) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42), QHFWGTPFT (SEQ ID NO: 43), AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46), respectively.

In certain embodiments, the modified anti-ASGR 1 antibody portion of the molecule comprises VH and VL domains comprising any one of these combinations of CDRs in the context of the parent sequence, or variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the VH or VL domain. In certain embodiments, the variant does not comprise any additional amino acid modifications (other than those described herein) in its CDR sequence.

In certain embodiments, the two antibody light chain polypeptides, or variable domains thereof, comprise sequences having at least 90% or at least 95% identity to any one of SEQ ID NOs 3, 5, 14, 17, 18, 19, 21 or 25, or variable regions thereof. In certain embodiments, each of the two fusion polypeptides or antibody heavy chain polypeptides or variable domains thereof comprises a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs 4, 6, 13, 15, 16, 20, 22 or 26, or a variable region thereof. In a particular embodiment, the two antibody light chain polypeptides each comprise a sequence having at least 95% identity to SEQ ID No. 25, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 26, or a variable region thereof.

In certain embodiments, the light chain polypeptide comprises the sequence: DIQMTQSPSSLSASVGDRVTITCRISENIYSNLAWYQQKPGKAPKLLIYAAINLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPFTFGQGTKLEIK (SEQ ID NO: 25), wherein the CDR sequences are underlined; and in certain embodiments, the heavy chain fusion polypeptide comprises the sequence:

wherein the Rspo2 domain is shown in italics, the linker is shown in bold, and the CDR sequences are underlined. In a related embodiment, R and A shown in bold are replaced with E and E.

It is understood that Wnt signaling enhancing molecules may comprise various combinations of acting and targeting modules. Thus, any variant anti-ASGR 1 antibody sequence (or fragment thereof) can be combined with various other functional modules, including but not limited to any of those disclosed herein.

The disclosure also provides polypeptides comprising any of the light chain or fusion polypeptides disclosed herein and polypeptides comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the light chain or fusion polypeptides disclosed herein, as well as functional or binding fragments thereof, e.g., VH or VL domains. One of skill in the art can readily determine the light chain region of any of the polypeptides disclosed herein based on the information provided in the sequence listing and by comparing these sequences to other sequences disclosed herein.

In certain embodiments, a liver-specific Wnt signaling enhancing molecule (e.g., fusion protein) increases Wnt signaling in liver tissue or liver cells contacted with the fusion protein. In certain embodiments, wnt signaling in liver tissue or liver cells is increased by at least 50%, at least two times, at least three times, at least four times, at least five times, or at least ten times.

Liver-specific Wnt signaling enhancing molecules may be produced by standard methods of organic synthesis and molecular biology known and available in the art. For example, a liver-specific Wnt signaling-enhancing fusion protein may be produced by fusing a targeting moiety (e.g., an antibody or antigen-binding fragment thereof that binds ASGR1 or ASGR 2) to an action moiety (e.g., human R-vertebral protein 2 furin domain 1 alone, corresponding to amino acid residues N37-R95; or human R-vertebral protein 2 furin domain 1 followed by furin domain 2, wherein the interaction of furin domain 2 with LGR protein is eliminated or compromised by a point mutation, e.g., F105A and F109A, alone or in combination). In certain embodiments, the targeting module and the targeting module are fused to any domain located at the N-terminus of the liver-specific Wnt signaling enhancement molecule via a linker (e.g., glycine-serine linker). In certain embodiments, the targeting module and the targeting module are fused via a protein linker (e.g., albumin). Additional ways of "fusing" the targeting module to the targeting module include, but are not limited to, for example, "knob-in-hole" or leucine zipper-mediated dimerization. The DNA sequences encoding the targeting module, the targeting module (and optionally the linker) can be genetically engineered to encode a desired fusion protein.

For liver-specific Wnt signaling enhancing molecules and domains thereof (e.g., fusion molecules, antibody heavy and light chains), DNA sequences encoding the different portions of the fusion protein may be inserted into bacterial or eukaryotic expression vectors and expressed in appropriate host cells using standard molecular cloning techniques. Expressed proteins can be purified to homogeneity using standard techniques in protein science, such as affinity, ion exchange and size exclusion chromatography. The present disclosure also includes functional fragments and variants of any of the polypeptide action modules, targeting modules, and fusion proteins described herein, including variants having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% polypeptide sequence identity to the action modules, targeting modules, or fusion proteins described herein. Such variants may comprise one or more amino acid modifications, such as one or more amino acid deletions, insertions, or substitutions, as compared to any of the sequences disclosed herein. In certain embodiments, functional fragments and variants of liver-specific Wnt signaling enhancement fusion proteins have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more Wnt signaling enhancement activity compared to the liver-specific Wnt signaling enhancement fusion protein from which they are derived. In certain embodiments, functional fragments and variants of the polypeptide action module have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more Wnt signaling enhancement activity as compared to the action module from which they are derived (as measured in the context of the whole liver-specific Wnt signaling enhancement molecule). In certain embodiments, functional fragments and variants of the targeting module have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more binding activity compared to the targeting module from which they are derived.

The present disclosure also includes polynucleotides or nucleic acid sequences encoding one or more liver-specific Wnt signaling enhancing molecules or components thereof, e.g., the proteins, fusion proteins, or variants thereof described herein, vectors (including expression vectors) comprising these polynucleotides, and cells comprising these vectors. In certain embodiments, the polynucleotide or nucleic acid sequence is DNA or RNA. In a particular embodiment, the RNA is messenger RNA (mRNA). In certain embodiments, the RNA is a modified mRNA comprising one or more modified nucleosides. Modified mrnas comprising one or more modified nucleosides have been described as having advantages over unmodified mrnas, including increased stability, higher expression levels, and reduced immunogenicity. Non-limiting examples of modified mRNAs that can be used according to the invention are described in, for example, PCT patent application publication Nos. WO2011/130624, WO2012/138453, WO2013052523, WO2013151666, WO2013/071047, WO2013/078199, WO2012045075, WO2014081507, WO2014093924, WO2014164253, U.S. Pat. No. 8,278,036 (describing modified mRNAs comprising pseudouridine), US 8,691,966 (describing modified mRNAs comprising pseudouridine and/or N1-methyl pseudouridine), US 8,835,108 (describing modified mRNAs comprising 5-methyl cytidine), US 8,748,089 (describing modified mRNAs comprising pseudouridine or 1-methyl pseudouridine). In certain embodiments, the modified mRNA sequence encoding the liver-specific Wnt signaling enhancement polypeptide comprises at least one modification compared to an unmodified A, G, U or C ribonucleoside. In particular embodiments, the at least one modified nucleoside comprises N1-methyl pseudouridine and/or 5-methylcytidine. In a particular embodiment, the modified mRNA comprises a 5 'terminal cap sequence, followed by a sequence encoding a liver-specific Wnt signaling enhancement polypeptide, followed by a 3' tailing sequence, such as a polyA or polyA-G sequence.

In certain embodiments, the polynucleotide is a vector, e.g., an expression vector, and the expression vector comprises a polynucleotide sequence encoding a liver-specific Wnt signaling enhancing fusion molecule described herein (e.g., a fusion protein or one or both strands of an additional antibody) operably linked to a promoter sequence, e.g., a promoter sequence that drives expression of the polynucleotide in a cell. In certain embodiments, the vector is a viral vector, e.g., a virus comprising a polynucleotide comprising an expression cassette comprising a promoter operably linked to a DNA or RNA sequence encoding a liver-specific Wnt signaling enhancement polypeptide. In particular embodiments, the expression cassette comprises 5 'and/or 3' cells or viral UTRs or derivatives thereof.

The disclosure also includes functional fragments and variants of the polynucleotides described herein, including variants having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% polynucleotide sequence identity to the polynucleotides described herein. Such variants may comprise one or more nucleotide or nucleoside modifications, such as one or more nucleotide deletions, insertions or substitutions, as compared to any of the sequences disclosed herein. In certain embodiments, the polynucleotides described herein are codon optimized, e.g., to enhance expression of the encoded polypeptide in a host cell. In particular embodiments, the polynucleotide variant comprises one or more modified nucleotides or nucleosides.

The present disclosure also includes cells comprising a polynucleotide or vector encoding a liver-specific Wnt signaling enhancement molecule described herein, such as a fusion protein or portion or domain thereof. In certain embodiments, the cell is a host cell, such as, for example, a HEK293 cell, which can be used to produce liver-specific Wnt signaling enhanced fusion proteins. In preparing the subject compositions, any host cell may be used, including, but not limited to, for example, mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In certain embodiments, the cell is heterologous or autologous to a subject treated with a liver-specific Wnt signaling enhancing polypeptide described herein. In certain embodiments, cells are obtained from a subject and transduced with a viral vector described herein. In certain embodiments, the transduced cells are delivered to a subject for treatment.

The present disclosure also includes pharmaceutical compositions comprising one or more liver-specific Wnt signaling enhancement molecules (e.g., fusion proteins or antibody-based constructs), or one or more polynucleotides or vectors comprising sequences encoding liver-specific Wnt signaling enhancement molecules or portions thereof.

Wnt signaling may be measured using techniques and assays known and available in the art. In certain embodiments, an increase in Wnt signaling is determined using a cell line corresponding to the target tissue or cell type. In a particular embodiment, the cell line contains a reporter plasmid bearing a marker gene (e.g., a luciferase gene) under the control of a Wnt signaling responsive promoter. Enhanced reporter activity of cells in response to Wnt3a, wnt3a conditioned medium, recombinant sources of Wnt3a, or Wnt mimetic agonists can be determined by adding furin domain 1 alone (or with furin domain 2, having the F105A and/or F109A point mutation) as a negative control or functional R-vertebral protein (full-length or furin domains 1 and 2) as a positive control. The reporter activity in response to the liver-specific Wnt signaling enhancement molecule may also be determined by contacting the reporter cell line with a tissue-specific Wnt signaling enhancement molecule. The negative control may be substantially, significantly or completely negative for the reporter activity, and the liver-specific Wnt signaling enhancement molecule and the positive control should exhibit an increase in Wnt signaling response with an increase in the reporter activity. Additional controls may include anti-ASGR 1 antibody alone (negative), where anti-GFP antibody is used in place of the fusion protein of anti-ASGR 1 antibody (negative) and the intact furin domain 1-furin domain 2 protein (positive). Tissue specificity of liver-specific Wnt signaling enhancement molecules may be determined by similarly measuring the reporter activity in response to treatment with liver-specific Wnt signaling enhancement molecules in cell types or tissues other than those targeted. In certain embodiments, the reporter activity is higher in the target tissue bound by the liver-specific Wnt signaling enhancement molecule, e.g., at least 50%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold higher, as compared to non-targeted tissue.

In certain embodiments, the liver-specific Wnt signaling enhancing polypeptide comprises any combination of an action module and a targeting module, including any combination of an action module and a targeting module described herein. In particular embodiments, they are linked by a linker, such as albumin (e.g., human serum albumin), a peptide-based linker or a non-peptide-based linker, wherein the targeting moiety and the targeting moiety are at the N-and C-terminus of the linker, such as Fc or albumin, a peptide-based linker or a non-peptide-based linker.

Liver-specific Wnt signaling enhancing molecules may also be linked to moieties known in the art such as polyethylene glycol (PEG), fc, albumin, etc. to enhance in vivo stability.

One example of a liver-specific Wnt signaling enhancing molecule is a Wnt signaling enhancing polypeptide that comprises an action module comprising a variant or fragment of an R-spinal protein (e.g., human R-spinal protein 2) having reduced ability to enhance Wnt signaling and a targeting module that specifically binds ASGR1, ASGR2, TFR2, or SLC10A1, wherein the tissue-specific Wnt signaling enhancing polypeptide increases Wnt signaling in liver tissue and is useful for treating a disease or condition of liver tissue.

Illustrative, non-limiting examples of liver-specific Wnt signaling enhancing molecules include those described in the accompanying examples and sequences. In certain embodiments, the liver-specific Wnt signaling enhancing molecule comprises two or more polypeptide sequences disclosed herein, e.g., in the form of additional IgG or antibodies. Polypeptides disclosed herein include, but are not limited to, polypeptides comprising or consisting of sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to any of the sequences set forth herein, and fragments and variants thereof. In particular embodiments, the polypeptides comprise a targeting or targeting moiety present in any of the sequences set forth herein, as well as fragments and variants thereof. In certain embodiments, the polypeptide has activity as an action module and/or a targeting module.

Illustrative, non-limiting examples of polynucleotides disclosed herein include any polynucleotide encoding any of the polypeptides, variants, and fragments described herein (including those described above). In certain embodiments, the polynucleotide encodes a polypeptide having activity as a functional domain and/or targeting module.

Pharmaceutical composition

Also disclosed are pharmaceutical compositions comprising a liver-specific Wnt signaling enhancing molecule or antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable diluents, carriers or excipients. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA.

In other embodiments, also disclosed are pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule or antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable diluents, carriers or excipients. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In particular embodiments, the polynucleotide is a modified mRNA that further comprises a 5 'cap sequence and/or a 3' tail sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence.

In other embodiments, also disclosed are pharmaceutical compositions comprising an expression vector (e.g., a viral vector) comprising a polynucleotide comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule or antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable diluents, carriers or excipients.

The invention also contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a liver-specific Wnt signaling enhancing molecule or antibody or antigen-binding fragment thereof described herein, and one or more pharmaceutically acceptable diluents, carriers or excipients. In particular embodiments, the cells are heterologous or autologous cells obtained from the subject to be treated. In particular embodiments, the cells are stem cells, such as adipose derived stem cells or hematopoietic stem cells.

The subject molecules may be combined with pharmaceutically acceptable carriers, diluents and agents for preparing generally safe, non-toxic and desirable formulations, and include acceptable excipients for mammalian (e.g., human or primate) use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds may also be incorporated into the formulation. Solutions or suspensions for use in the formulations may include sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediamine tetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; detergents, such as tween 20, to prevent aggregation; and compounds for modulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. In certain embodiments, the pharmaceutical composition is sterile.

The pharmaceutical composition may also comprise a sterile aqueous solution or dispersion and a sterile powder for extemporaneous preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic or Phosphate Buffered Saline (PBS). In some cases, the composition is sterile and should be fluid to the extent that easy injection is possible. In certain embodiments, they are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. 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 achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, in the compositions; polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the internal composition may be achieved by including agents in the composition that delay absorption, such as aluminum monostearate and gelatin.

Sterile solutions may be prepared by incorporating the liver-specific Wnt signaling enhancing molecule in the required amount in an appropriate solvent with one or a combination of the components listed above, followed by filter sterilization, if desired. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the pharmaceutical composition is prepared with a carrier that will protect the fusion protein from rapid clearance from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods of preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

For ease of administration and uniformity of dosage, it may be advantageous to formulate pharmaceutical compositions in dosage unit form. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of the active compound associated with the desired pharmaceutical carrier calculated to produce the desired therapeutic effect. The specifications of the dosage unit forms of the invention are determined by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of formulating such active compounds for use in the treatment of individuals.

The pharmaceutical composition may be included with the instructions for administration in a container, package or dispenser, such as a syringe, e.g., a prefilled syringe.

The pharmaceutical compositions of the invention encompass any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other compound that is capable of providing (directly or indirectly) a biologically active liver-specific Wnt signaling enhancing molecule when administered to an animal, including a human.

The invention includes pharmaceutically acceptable salts of liver-specific Wnt signaling enhancing molecules described herein. The term "pharmaceutically acceptable salt" refers to the physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects thereto. Various pharmaceutically acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17 th edition, alfonso r.gennaro (ed.), mark Publishing Company, easton, PA, USA,1985 (and newer versions thereof); "Encyclopaedia of Pharmaceutical Technology", 3 rd edition, james Swarbrick (eds.), informa Healthcare USA (Inc.), NY, USA,2007 and J.Pharm. Sci.66:2 (1977). For a review of suitable salts, see also "Handbook of Pharmaceutical Salts: properties, selection, and Use", stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., berge et al, "Pharmaceutical Salts," j.pharma sci.,1977,66,119). The base addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to produce the salt. The free acid form may be regenerated by contacting the salt form with an acid and separating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention, salts are equivalent to their respective free acids.

In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a liver-specific Wnt signaling enhancing molecule described herein in admixture with pharmaceutically acceptable carriers, diluents, and/or excipients, such as saline, phosphate buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars are octylphenoxy polyethoxylate ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. Preferably, the formulation is stable for at least 6 months at 4 ℃.

In some embodiments, the pharmaceutical compositions provided herein comprise buffers such as Phosphate Buffered Saline (PBS) or sodium phosphate/sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill, such as those described in Good et al (1966) Biochemistry 5:467. The pH of the buffer may range from 6.5 to 7.75, preferably from 7 to 7.5, and most preferably from 7.2 to 7.4.

Methods for increasing Wnt activity or Wnt receptor cell surface expression

The liver-specific Wnt signaling enhancing molecules exemplified herein with respect to fusion proteins may be used to increase Wnt signaling in liver tissue or liver cells. In certain embodiments, wnt signaling is canonical Wnt signaling. Thus, in some aspects, the invention provides methods for increasing or enhancing Wnt signaling in a liver tissue or liver cell comprising contacting the liver tissue or cell with an effective amount of a liver-specific Wnt signaling-enhancing molecule disclosed herein, wherein the molecule comprises a targeting module that binds to a cell surface receptor on the target tissue or cell in a tissue or cell specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In certain embodiments, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo, e.g., a subject liver-specific Wnt signaling enhancing molecule is administered or provided to the subject. In a particular embodiment, the cells are cultured cells and the contacting occurs in vitro.

In a related aspect, the invention provides a method for increasing Wnt signaling in a liver tissue or cell comprising contacting a target tissue or cell with an effective amount of one or more polynucleotides comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule disclosed herein, wherein the molecule comprises a targeting module that binds to a cell surface receptor on the target tissue or cell in a tissue or cell specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In certain embodiments, the polynucleotide is DNA or mRNA, e.g., modified mRNA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In certain embodiments, the polynucleotide is a modified mRNA that further comprises a 5 'cap sequence and/or a 3' tail sequence, such as a polyA tail. In other embodiments, the polynucleotide is an expression cassette comprising a promoter operably linked to a coding sequence.

In a related aspect, the invention provides a method for increasing Wnt signaling in a liver tissue or cell comprising contacting a target tissue or cell with an effective amount of one or more vectors comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on the liver tissue or cell in a liver-specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA. In certain embodiments, the vector is an expression vector and may comprise a promoter operably linked to a nucleic acid sequence. In a particular embodiment, the vector is a viral vector.

In a related aspect, the invention provides a method for increasing Wnt signaling in a liver tissue or cell comprising contacting a target tissue with an effective amount of a cell comprising one or more nucleic acid sequences encoding a liver-specific Wnt signaling enhancement molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on the liver tissue or cell in a liver-specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA. In particular embodiments, the cells are heterologous or autologous cells obtained from the subject to be treated. In certain embodiments, the cells are transduced with a vector comprising an expression cassette encoding a liver-specific Wnt signaling enhancement molecule. In particular embodiments, the cells are stem cells, such as adipose derived stem cells or hematopoietic stem cells.

Any of the methods described herein for increasing Wnt signaling may also be used to increase the number of Frizzled (Fz) receptors on the surface of a target cell (e.g., a liver tissue cell). In certain embodiments, the number of Fz receptors on the surface of the target cell is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 5-fold, or at least 10-fold. In particular embodiments, the Fz receptor comprises one or more human frizzled proteins Fz1, fz2, fz3, fz4, fz5, fz6, fz7, fz8, fz9, and Fz10. For example, the present disclosure provides a method for increasing Fz receptors on the surface of a liver cell comprising contacting the liver cell with an effective amount of a liver-specific Wnt signaling enhancing molecule disclosed herein, wherein the molecule comprises a targeting module that binds to a cell surface receptor on liver tissue or cells in a liver-specific manner. In certain embodiments, the targeting module binds ASGR1 or ASGR2. In certain embodiments, the targeting moiety comprises an antibody or antigen binding fragment thereof disclosed herein. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In some embodiments, the contacting occurs in vitro, ex vivo, or in vivo, e.g., the liver-specific Wnt signaling enhancing molecule is administered or provided to the subject. In a particular embodiment, the cells are cultured cells and the contacting occurs in vitro. In certain embodiments, the liver cell or tissue is initially contacted directly with the Wnt signaling enhancing molecule, whereas in other related embodiments, the liver tissue or cell is initially contacted with a polynucleotide (e.g., an expression vector) encoding the Wnt signaling enhancing molecule, whereby the cell ingests the polynucleotide and expresses the Wnt signaling enhancing molecule.

Any of the methods described herein for increasing Wnt signaling may also be used to increase Ki-67 on liver tissue or liver cells.

Methods for treating diseases and disorders

The liver-specific Wnt signaling enhancing molecules exemplified herein with respect to the fusion proteins may be used to treat a disease, disorder, or condition, for example, by increasing Wnt signaling in a target liver cell, tissue, or organ. Thus, in some aspects, the invention provides methods for treating a disease or condition in a subject in need thereof, such as a disease or condition associated with reduced Wnt signaling, or for which increased Wnt signaling would provide therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the disclosure. In a particular embodiment, the composition is a pharmaceutical composition comprising any one of the following: liver-specific Wnt signaling enhancing molecules, e.g., small molecules or polypeptides; one or more polynucleotides comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule (e.g., DNA or mRNA, optionally modified mRNA); one or more vectors comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancing molecule, e.g., an expression vector or a viral vector; or a cell comprising one or more nucleic acid sequences encoding a liver-specific Wnt signaling enhancement molecule, e.g., a cell transduced with an expression vector or viral vector encoding a liver-specific Wnt signaling enhancement molecule. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In particular embodiments, the disease or condition is a pathological disease or disorder, or injury, such as that caused by a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition includes, or would benefit from, increased tissue repair, healing, or regeneration. In some embodiments, the contacting occurs in vivo, i.e., the subject composition is administered to the subject.

In a related aspect, the invention provides a method for treating a disease or condition, such as a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising administering to or contacting a subject in need thereof a pharmaceutical composition comprising an effective amount of a liver-specific Wnt signaling enhancing molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on a target tissue or cell in a tissue or cell specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA.

In a related aspect, the invention provides a method for treating a disease or condition, such as a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising administering to or contacting a subject in need thereof a pharmaceutical composition comprising an effective amount of one or more polynucleotides comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on a target tissue or cell in a tissue or cell-specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA.

In a related aspect, the invention provides a method for treating a disease or condition, such as a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of one or more vectors comprising a nucleic acid sequence encoding a liver-specific Wnt signaling enhancement molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on a target tissue or cell in a tissue or cell specific manner. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA.

In a related aspect, the invention provides a method for treating a disease or condition, such as a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a cell comprising one or more nucleic acid sequences encoding a liver-specific Wnt signaling enhancement molecule of the invention, wherein the molecule comprises a targeting module that binds to a cell surface receptor on a target tissue or cell in a tissue or cell specific manner. In a particular embodiment, the cells are stem cells, such as adipose tissue-derived stem cells or hematopoietic stem cells. In a specific embodiment, the Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA.

Wnt signaling plays a key role in the developmental process and maintenance of stem cells. Reactivation of Wnt signaling is associated with regeneration and repair of most tissues following injury and disease. Liver-specific Wnt signaling enhancing molecules may provide benefits in response to healing and tissue repair of liver injury and disease. Causes of liver tissue damage and loss include, but are not limited to, aging, degeneration, genetic conditions, infection and inflammation, traumatic injury, toxin/metabolism-induced toxicity or other pathological conditions. Wnt signaling and enhancers of Wnt signaling have been shown to activate adult tissue resident stem cells. In some embodiments, the compounds of the invention are administered for treating diseased or damaged liver tissue, for liver tissue regeneration and for liver cell growth and proliferation, and/or for liver tissue engineering.

Human diseases associated with mutations in the Wnt pathway provide strong evidence for enhancement of Wnt signaling in disease treatment and prevention. Preclinical in vivo and in vitro studies provide additional evidence that Wnt signaling is involved in many disease states, and further support the use of liver-specific Wnt signaling enhancers in a variety of human diseases. For example, the compositions of the invention may also be used to enhance liver cell regeneration, e.g., liver regeneration, to treat liver cirrhosis, to enhance liver transplantation, to treat acute liver failure, to treat chronic liver disease accompanied by hepatitis (A, B or C) viral infection or post antiviral drug therapy, alcoholic liver disease, including alcoholic hepatitis, e.g., acute or severe alcoholic hepatitis, non-alcoholic liver disease accompanied by steatosis or steatohepatitis, and the like. The compositions of the invention may treat diseases and disorders, including but not limited to conditions requiring regenerative liver tissue or cell growth. In certain embodiments, the composition is used to treat, for example, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in cirrhosis patients, hepatorenal syndrome-acute kidney injury (HRS-AKI), or hepatic encephalopathy. In a particular embodiment, the composition comprises a Wnt signaling enhancing molecule selected from any one of those disclosed herein, or comprising any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA. In certain embodiments, the method is for treating alcoholic hepatitis, e.g., acute or severe alcoholic hepatitis, the Wnt signaling-enhancing molecule is EEST-EE, and in particular embodiments, the Wnt signaling-enhancing molecule is administered intravenously. In certain embodiments, the method is for treating alcoholic hepatitis, e.g., acute or severe alcoholic hepatitis, the Wnt signaling-enhancing molecule is 8m24EASE-EE or 8m2 EASE-RA, and in particular embodiments, the Wnt signaling-enhancing molecule is administered intravenously.

Specific populations of proliferating cells for hepatocyte homeostasis renewal have been identified by lineage-tracking studies, such as Axin2 positive cells in the pericentral region. Lineage follow-up studies also identified additional potential hepatic progenitors, including but not limited to Lgr positive cells. Self-renewing liver cells and other potential progenitor cell populations (including Lgr5 positive and Axin2 positive cells) were identified as capable of regenerating in response to Wnt signaling and/or R-spinal protein following injury. Many preclinical models of acute liver injury and failure and chronic liver disease show that recovery and regeneration of hepatocytes benefit from enhancement of Wnt signaling. In some embodiments of the present invention, in some embodiments, the composition of the present invention can be used for treating, for example, acute liver failure of various causes, drug-induced acute liver failure, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in patients with cirrhosis, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, alcoholic liver disease, chronic liver failure of various causes, decompensated liver failure, late decompensated liver failure, cirrhosis, hepatic fibrosis of various causes, portal hypertension, chronic liver insufficiency of various causes, end-stage liver disease (ESLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis Acute Alcoholic Hepatitis (AAH), chronic alcoholic hepatitis, alcoholic Liver Disease (ALD) (also known as alcohol-related liver disease (ARLD)), hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis D virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, symptomatic operation of liver disease, liver injury, venous Occlusive Disease (VOD), sinus Occlusion Syndrome (SOS), primary cholangitis (PBC), primary Sclerosing Cholangitis (PSC), liver transplantation, "small volume" syndrome in liver surgery and transplantation, congenital liver diseases and conditions, liver failure due to excess APAP (acetaminophen), and any other liver disease or condition caused by genetic disease, degeneration, aging, drugs, or injury. They may also be used to enhance regeneration of liver cells in vivo or in vitro. In certain embodiments, the methods result in increased hepatocyte regeneration, improved liver function, and/or reduced fibrosis. Methods for liver tissue regeneration benefit from administration of the compounds of the present invention, which may be systemic or local. These include, but are not limited to, methods of systemic administration and methods of topical administration, such as by injection into liver tissue, introduction into the liver by injection into veins or blood vessels, implantation of sustained release formulations, and the like. In a particular embodiment, the composition comprises a Wnt signaling enhancing molecule selected from any one of those disclosed herein, or comprising any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In a particular embodiment, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24EASE-EE or 8M2 EASE-RA.

In a particular embodiment, the liver-specific Wnt signaling enhancing molecules disclosed herein are used to treat, inhibit, or prevent Alcoholic Hepatitis (AH), such as Acute Alcoholic Hepatitis (AAH), also known as severe alcoholic hepatitis (severe AH). AAH (or severe AH) is a severe form of alcohol-related liver disease associated with significant short-term mortality. Alcoholic hepatitis usually occurs after a number of frequent drinking of over 10 years; the average consumption in one study was 100 g/day (equivalent to 10 drinking per day). Typical patients manifest recent jaundice, ascites and proximal muscle loss. Fever and leukocytosis are also common, but should suggest an assessment of infection, especially spontaneous bacterial peritonitis. Liver biopsies of these patients showed steatosis, swelling of hepatocytes with eosinophilic inclusion bodies (Mallory) and significant neutrophil infiltration. Because of the accuracy of clinical diagnosis, biopsies are rarely required, but rather rely on clinical and laboratory features for diagnosis. Acute Alcoholic Hepatitis (AH) is an acute decompensation of a severe form of Alcoholic Liver Disease (ALD), which develops in alcoholics and is characterized by jaundice, malaise, anorexia, tender hepatomegaly and rapid onset of the features of Systemic Inflammatory Response Syndrome (SIRS). Severe or acute Alcoholic Hepatitis (AH) is a catastrophic disease, with a very high mortality rate of 180 days and often requires hospitalization. It may be manifested as acute or chronic liver failure, with poorer prognosis in the presence of infection and higher levels of liver disease severity. Patients may have a recent history of high alcohol consumption within three months of exhibiting jaundice and characteristic patterns of elevated liver enzymes, as well as coagulopathies, hepatic encephalopathy, varicose bleeding, and sepsis leading to extrahepatic organ failure, as well as other potential symptoms such as itching and/or fever. In a particular embodiment, the liver-specific Wnt signaling enhancing molecule is selected from any one of those disclosed herein, or comprises any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8m24 EASE-RA. In certain embodiments, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In particular embodiments, it is administered intravenously. In certain embodiments, the methods result in increased hepatocyte regeneration, improved liver function, and/or reduced fibrosis.

The compositions of the invention may be used to treat end-stage liver disease (ESLD). ESLD or chronic liver failure is often the result of severe cirrhosis and resultant liver fibrosis. ESLD manifests itself in the development of ascites, variceal bleeding, hepatic encephalopathy, and/or impairment of liver function (e.g., decompensated liver disease). Common diseases or conditions associated with ESLD include: alcoholic hepatitis, chronic hepatitis C infection, chronic hepatitis B infection, chronic hepatitis C infection, nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH) and hereditary diseases such as cystic fibrosis, alpha-1 antitrypsin deficiency, hemochromatosis, wilson disease, galactosylation and glycogen storage disease. Prolonged exposure to drugs, toxic chemicals, parasitic infections, and recurrent heart failure and liver congestion can also lead to ESLD. In a particular embodiment, the composition comprises a Wnt signaling enhancing molecule selected from any one of those disclosed herein, or comprising any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In certain embodiments, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In particular embodiments, it is administered intravenously. In certain embodiments, the methods result in increased hepatocyte regeneration, improved liver function, and/or reduced fibrosis.

In particular embodiments, the composition is administered parenterally, for example, intravenously, orally, rectally, or by injection. In some embodiments, it is administered topically, e.g., topically or intramuscularly. In some embodiments, the composition is administered to a target tissue, such as the liver. The method of the invention may be carried out in vivo or ex vivo. In some embodiments, the contacting of the target cell or tissue with the liver-specific Wnt signaling enhancement molecule is performed ex vivo, followed by implantation of the cell or tissue (e.g., activated stem or progenitor cells) into the subject. One skilled in the art can determine the appropriate site and route of administration based on the disease or condition being treated. Methods of administration include, but are not limited to, methods of systemic administration and methods of topical administration, such as by injection into liver tissue, introduction into the liver by injection into veins or blood vessels, implantation of sustained release formulations, and the like.

The administration and dosage regimen may depend on a variety of factors readily determinable by the physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's medical history. In particular embodiments, the amount of liver-specific Wnt signaling enhancing molecule (e.g., fusion protein) administered or provided to the subject ranges from about 0.01mg/kg to about 50mg/kg, from 0.1mg/kg to about 500mg/kg, or from about 0.1mg/kg to about 50mg/kg of the body weight of the subject.

In certain embodiments, the subject may be any mammal, such as a human, rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cow, or primate.

In some embodiments, the subject methods produce therapeutic benefits, such as inhibiting or preventing the development of a liver disease or disorder, stopping the progression of a liver disease or disorder, reversing the progression of a liver disease or disorder, and the like. In some embodiments, the methods increase hepatocyte regeneration, increase liver function, and/or reduce liver fibrosis. In some embodiments, the subject methods include the step of detecting that a therapeutic benefit has been achieved. One of ordinary skill in the art will recognize that such measurement of therapeutic efficacy will be applicable to the particular disease or condition being alleviated, and will recognize appropriate detection methods for measuring therapeutic efficacy.

In certain embodiments, the present disclosure provides methods for treating or preventing a disease or disorder associated with reduced Wnt signaling or that would benefit from increased Wnt signaling activity in liver tissue, such as, for example, any disease or disorder disclosed herein that would benefit from liver regeneration, comprising providing a pharmaceutical composition comprising a Wnt signaling enhancing molecule comprising a targeting module that binds to liver tissue, e.g., a targeting module that specifically binds to ASGR1, to a subject in need thereof, wherein the Wnt signaling enhancing molecule increases or enhances Wnt signaling in liver tissue of the subject. In a particular embodiment, the composition comprises a Wnt signaling enhancing molecule selected from any one of those disclosed herein, or comprising any one of the polypeptide sequences disclosed herein, e.g., a Wnt signaling enhancing molecule known as EEST-EE, EEST-RA, EEAT-EE, or 8M24 EASE-RA. In certain embodiments, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In certain embodiments, the pharmaceutical composition is administered orally or systemically, e.g., parenterally. In a specific embodiment, the Wnt signaling enhancing molecule comprises an action module comprising R-vertebrate furin domain 1 or a fragment or variant thereof, and optionally, a mutated furin domain 2 or a fragment or variant thereof.

Methods for producing or maintaining liver cells, tissues and organoids

Other embodiments are directed in part to the use of a molecule disclosed herein for promoting or enhancing the growth or proliferation of liver cells, liver tissue, and organoids, e.g., by contacting a liver cell, liver tissue, or liver organoid with one or more Wnt signaling enhancing molecules disclosed herein, e.g., 1R34-EEST-EE, 8M24-EASE-EE, or 8M 24-EASE-RA. In certain embodiments, the Wnt signaling enhancing molecule is EEST-EE. In certain embodiments, the Wnt signal enhancing molecule is 8M24 EASE-EE or 8M2 EASE-RA. In certain embodiments, the methods may be used to enhance the growth or proliferation of liver cells, liver tissue, or liver organoids, or to maintain or increase the viability of liver cells, liver tissue, or liver organoids. In certain embodiments, the liver cells, liver tissue, or liver organoids are contacted ex vivo, in vitro, or in vivo. The methods disclosed herein may be used to generate and/or maintain liver cells, tissues or organoids for therapeutic use, e.g., for transplantation or implantation into a subject. They may also be used to produce and/or maintain liver cells, tissues or organoids for research purposes. Wnt signaling enhancing molecules find wide application in non-therapeutic approaches, such as in vitro research approaches.

In certain embodiments, liver tissue is contacted with a Wnt signaling enhancing molecule to maintain viability of the liver tissue. In certain embodiments, the liver tissue is donor liver tissue to be transplanted to a recipient in need thereof. In certain embodiments, the donor liver tissue is perfused in vivo with a solution comprising the Wnt signaling enhancing molecules disclosed herein, e.g., prior to removal of the liver tissue from the donor. In certain embodiments, donor liver tissue is perfused ex vivo with a solution comprising a Wnt signaling enhancing molecule disclosed herein, e.g., during storage or during transport from a donor to an acceptor. In certain embodiments, the time that liver tissue contacted with the Wnt signaling enhancing molecule remains transplant viable is prolonged by at least 10%, at least 20%, at least 50%, or at least 100% as compared to liver tissue not contacted with the Wnt signaling enhancing molecule.

In certain embodiments, the liver organoid culture is produced, grown, or maintained by contacting the liver organoid culture with one or more Wnt signaling molecules disclosed herein. In certain embodiments, the Wnt signaling enhancing molecule is present in a medium used to grow or maintain liver organoid tissue.

All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and/or listed in the application data sheet, are incorporated herein by reference, in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees celsius, and pressure is at or near atmospheric pressure.

General methods in molecular biology, cell biology and biochemistry can be found in standard textbooks, such as "Molecular Cloning: A Laboratory Manual, 3 rd edition" (Sambrook et al Harbor Laboratory Press 2001); "Short Protocols in Molecular Biology, 4 th edition" (Ausubel et al, john Wiley & Sons 1999); "Protein Methods" (Bollag et al, john Wiley & Sons 1996); "Nonviral Vectors for Gene Therapy" (Wagner et al, academic Press 1999); "Viral Vectors" (Kaplift & Loewy, academic Press 1995); "Immunology Methods Manual" (incorporated by reference, academic Press 1997); and "Cell and Tissue Culture: laboratory Procedures in Biotechnology" (Doyle & Griffiths, john Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors and kits for genetic manipulation mentioned in this disclosure are available from commercial suppliers such as BioRad, stratagene, invitrogen, sigma-Aldrich and ClonTech.

Materials and methods employed in the following examples include the following.

Protein production: unless otherwise indicated, all recombinant proteins were produced by transient transfection in Expi293F cells (Thermo Fisher Scientific). All IgG-based and Fc-containing constructs were purified with CaptivA protein a affinity (Repligen) and eluted with 0.1M glycine (ph 3.3). All proteins were further purified (polih) using Superdex 200Increase 10/300GL (GE Healthcare Life Sciences) Size Exclusion Chromatography (SEC) using either 1 XHBS buffer (20mM HEPES pH 7.4,150mM NaCl) or 2 XHBS buffer (40mM HEPES pH 7.4,300mM NaCl). The protein was supplemented with glycerol (to 10%) for long term storage at-80 ℃. All proteins tested were checked by SDS-polyacrylamide electrophoresis and the purity was estimated to be at least 90%.

superTop Flash (STF) assay: as reported (Janda et al, 2017;Nature 545:234), wnt signaling activity was measured using a cell line containing a luciferase gene controlled by a Wnt responsive promoterSuper Top FAnd (3) measuring a flash report and STF). Briefly, cells were seeded in 96-well plates at a density of 10,000 per well 24 hours prior to treatment and then treated with RSPO or mimetic proteins alone or with 100pm wnt3a surrogate R2M3-26 overnight. Cells were lysed with luciferase cell culture lysis reagent (Luciferase Cell Culture Lysis Reagent) (Promega) and activity was measured with a luciferase assay system (Promega) using methods suggested by the supplier. Data were plotted as mean of triplicate-/+ standard deviation and fitted by nonlinear regression using Prism (GraphPad software).

Semi-quantitative PCR analysis of gene expression: using MagMAX ^TM mirVana ^TM Total RNA isolation kit (ThermoFish)er, a 27828) extracts RNA from mouse tissue (liver and small intestine samples). cDNA was produced using high capacity cDNA reverse transcription kits (ThermoFisher, 43-688-14) or SuperScriptIV VILOMaster Mix (ThermoFisher, catalog number 11756050). By usingFast Advanced Master Mix (thermo fisher, 4444963) and Mm00443610 _m1axin2, mm01278617_m1Ki67, mm01300555 _g1wnt1, mm00470018 _m1wnt2, mm00437336_m1wnt3, mm01194003 _m1wnt4, mm00437347 _m1wn5a, mm01183986 _m1wnt5b, mm00437353 _m1wnt6, mm00437356_m1wnt7a, mm01301717 _m1wnt7b, mm01157914 _g1wn8a, mm00457102 _m1wn9b, mm00442104 _m1wn10b, M00437327 _g1wn1t11, M00446420 _m1wnt16, M00507077 _m1po1, M00555790 _m1po2, mrs83_m1po1Mrs3 and Mrs3_M676_m676 and Md1m672 (thermo probe) and Md6m327m6342. Values were normalized to the expression of the constitutive actin B gene using the Mm02619580_g1 probe (thermo fisher, 4351368).

Multispecific assay: ELISA methods were used to examine binding to non-target antigens including human insulin (Sigma 91077C-100 MG), keyhole Limpet Hemocyanin (KLH) (Sigma H7017-50 MG), lipopolysaccharide (LPS) from E.coli (Sigma L3012-10 MG), double-stranded DNA (dsDNA) (Sigma D1626-5G), and heparin. Before use, dsDNA was sheared to 200-200 bp with ultrasound. Will be96-well EIA/RIA Easy Wash ^TM Transparent flat bottom polystyrene high binding microplates (Corning 3369) were coated overnight at 4℃with 50. Mu.l KLH, LPS and dsDNA (10 mg/ml) in PBS. Insulin was coated at 5 mg/ml. Heparin coated plates (Thermo Scientific C995X 60) were purchased. The coated plates were blocked with 300. Mu.l 300SuperBlock (Thermo 37516) for 1 hour at room temperature, then probed with 100. Mu.l of the protein of interest (antibody or fusion) at 1000mg/ml, 250mg/ml, 125mg/ml, 62.5mg/ml for 1 hour at room temperature (or overnight at 4 ℃). anti-hFc-HRP (Jackson IR 109-035-098) was used for detection and chemiluminescent quantification.

Protein thermostability assay: protein thermostability was measured using a Uncle instrument (Unchained Labs). The reaction was performed by adding a protein sample in 1×hbs buffer to the units, then sealing and sealing in a frame by silicone seals. Fluorescence readings were measured at UV266nm and Blue 473nm at 1 ℃/min increments over a temperature range of 15℃to 95 ℃. Tm/Tagg was obtained using the un data analysis software.

Mouse study: male 6-week-old C57B1/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred in groups. All animal experiments were in accordance with the "guidelines for laboratory animal care and use" set by national academy of sciences (National Academy of Sciences). Animal protocol was approved by the Surrozen institutional animal care and use committee. Mice were acclimatized for a minimum of two days before starting the experiment. Mice can obtain purified laboratory grade acidified water ad libitum and be fed ad libitum (2018 Teklad global 18% protein rodent diet). Mice were kept in a 12/12 hour light/dark cycle at room temperature of 30% -70% humidity and 20 ℃ -26 ℃.

In the case of mice humanized for human ASGR gene expression, each mouse was treated with 1X10 on day 0 ¹¹ The ssAAV8-CAG-hASGR1 genomic copies (Vector Biolabs, malvern, pa.) were administered intravenously. On day 7, mice were injected intraperitoneally (i.p.) with αGFP, fc-RSPO2-WT, αGFP-RSPO2-RA, or αASGR1-RSPO2-RA. At the indicated time following protein administration, mice were anesthetized with isoflurane and blood was removed by cardiac puncture. A portion of the left lobe and duodenum were collected for analysis.

CCl ₄ Semi-quantitative PCR analysis of gene expression in study: using MagMAX ^TM mirVana ^TM Total RNA isolation kit (ThermoFisher, A27828) extracts RNA from mouse tissue (liver samples). Using high-capacity cDNA reverse transcription kit (ThermoFisher, 43-688-14) or SuperScript ^TM IV VILO ^TM Master Mix (ThermoFisher, catalog number 11756050) produced cDNA. By usingFast AdvancedMaster Mix (ThermoFisher, 4444963) and Mm00443610_ml axin2, mm00432359_m1Ccnd1, mm01278617_ml Mki67 measured mouse mRNA expression. Values were normalized to the expression of the constitutive actin B gene using the Mm02619580_gl probe (thermo fisher, 4351368).

Serum chemistry: blood was collected from the caudal tip on day 7 and terminated via cardiac puncture on day 14. Serum was isolated by centrifuging blood at 10,000rpm for 7 minutes in a serum separation tube with gel (Fisher, 22030401). The supernatant was transferred to a fresh tube and kept at-20 ℃ until analysis. Serum samples were analyzed using a VetAxcel clinical analyzer, alkaline phosphatase, and albumin assay kit (404200-3, SA2002 and SA2001, alfa-Wasserman Diagnostic Technologies, respectively).

Histological analysis and immunofluorescence: formalin-fixed and paraffin-embedded liver samples were sectioned and treated with anti-Ki-67 rabbit antibodies (Fisher, 50245564), anti-HNF 4a antibodies (Abcam, ab 199431), goat anti-rat IgG H &L(Alexa488 (ab 150157) and donkey anti-rabbit IgG H&L IgG H&L(Alexa/>647 (ab 150075) staining. The whole histological liver section was stained with picrosides Red (PSR) using standard procedures and scanned into digital images. Image J was used to quantify the percentage of sections stained with PSR.

Example 1

Development and characterization of liver-specific WNT signaling enhancement molecules

ASGR is a hetero-oligomer consisting of two polypeptides ASGR1 and ASGR2, which are expressed predominantly on hepatocytes and undergo rapid endocytosis. To generate liver-specific RSPO-like Wnt signaling enhancing molecules, asialoglycoprotein receptors (ASGR) were targeted.

A number of IgG-like liver-specific Wnt signaling-enhancing molecules were generated, each comprising two anti-ASGR 1 antibody light chains and two anti-ASGR 1 antibody heavy chains with modified RSPO2 polypeptides fused to their N-termini via a linker sequence (aagr 1-RSPO2 construct). In this design, the anti-ASGR 1 antibody portion of the molecule is a "targeting module" that provides liver specificity, while the RSPO2 portion of the molecule functions as an "action module" that interacts with the E3 ligase. Unless otherwise indicated, wnt signaling enhancing molecules comprise an IgG1 backbone.

The initial αasgr1-RSPO2 Wnt signaling-enhancing molecule prepared comprises an αasgr1 binding domain that binds to the stem region of ASGR1 and is referred to as 1R 34-DDNN/RA. The light chain sequence of the 1R34-DDNA/RA molecule is provided below, with the CDRs underlined:

SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSLERIGYLSYVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:1)。

the heavy chain sequence of the 1R34-DDNA/RA molecule with fused RSPO2 sequences is shown below, with the CDRs underlined, the RSPO2 sequences shown in italics, and the linker sequences shown in bold:

two amino acid substitutions compared to the wild-type human RSPO2 sequence are shown in bold, italics and underlined.

The subsequent αasgr1-RSPO2 Wnt signaling-enhancing molecule 8M24-v1 comprises an αasgr1 binding domain that binds to the carbohydrate binding domain of ASGR1 and is derived from an 8M24 antibody. The sequence of the variable domain of the light chain of the 8M24-v1 molecule is provided below, with the CDRs underlined:

DIQMTQSPSSLSASVGDRVTITCRISENIYSNLAWYQQKPGKAPKLLIYAAINLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPFTFGQGTKLEIK(SEQ ID NO:3)。

the light chain sequence of the 8M24-v1 molecule is provided below, with the CDRs underlined:

DIQMTQSPSSLSASVGDRVTITCRISENIYSNLAWYQQKPGKAPKLLIYAAINLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:5)。

the sequence of the variable domain of the heavy chain of the 8M24v1 molecule with the fused RSPO2 sequence is shown below, with the CDRs underlined, the RSPO2 sequence shown in italics, and the linker sequence shown in bold:

two amino acid substitutions compared to the wild-type human RSPO2 sequence are shown in bold, italics and underlined.

Each of these two starting molecules was modified for testing to identify modified forms with excellent properties.

1R34-DDNA/RA molecular modification

The initial αasgr1-RSPO2 Wnt signaling-enhancing molecule 1R34-DDNA/RA was subjected to various amino acid modifications and tested to identify molecules with improved properties. FIG. 1 provides the amino acid sequences of the light chain polypeptide and heavy chain-Rspo 2 fusion polypeptide of the 1R34-DDNA/RA initiation molecule, and indicates in bold the amino acid residues that are modified in the variant. All molecules included an N297G substitution in the IgG1 backbone (NG). In some molecules, to eliminate binding of RSPO2 polypeptides to LGR, point mutations were introduced into two highly conserved hydrophobic residues within the Fu2 domain of RSPO2, which are reported to be critical for binding to LGR proteins, F105 and F109. In comparison to the wild-type RSPO2 sequence, the 1R34-DDNA/RA heavy chain sequence shown in fig. 1 comprises F105R and F109A substitutions, which are shown in italics and underlined.

Four Asn and Asp sites with potential for deamidation or Asp isomerization are present in the CDRs of the ASGR1 binding IgG moiety of the 1R34-DDNA/RA Wnt signaling enhancing molecule (shown in bold in fig. 1). Various amino acid substitutions are made at each of these positions. Molecules were engineered using standard molecular biology techniques and expressed in Expi293 cells by transient transfection, followed by 2 column purifications (protein a followed by SEC). The resulting molecules were tested for the potential to introduce Wnt signaling enhancing molecules to eliminate deamidation or Asp isomerization capability.

Surprisingly, the D62 position in CDR2 of the heavy chain shows limited flexibility for substitution by other amino acids. As shown in fig. 2, D62 mutated to Ser or Ala resulted in a significant change in SEC spectrum of the molecule, indicating that these mutations disrupt folding of the molecule, whereas mutation to Glu remained folded. The latter mutation also retained STF activity (data not shown).

Similarly, the D25 position in CDR1 of the light chain cannot be replaced by serine because it disrupts protein folding, as shown in fig. 3. However, substitution of D25 with Glu or Ala maintained protein folding and STF activity (FIG. 3 and data not shown).

The N51 position in CDR2 of the light chain has the flexibility to be replaced by Gln, ser or Ala (fig. 4). Each of these substitutions maintained STF activity, although STF activity was slightly decreased with the Gln mutation at position 51 (data not shown).

As shown in fig. 5, modification of the N88 position in CDR3 of the light chain did not affect the protein SEC spectrum when replaced by Gln, ser or Ala. Each of these substitutions maintained STF activity, although STF activity was slightly decreased with the Gln mutation at position 88 (data not shown). However, SDS-PAGE analysis revealed that although the N88S and N88A mutants were well expressed, mutations to S or A resulted in incorrectly folded proteins, as shown in FIG. 6. In combination with other mutants, LC N88Q compromised activity and L88A compromised integrity. The N88 position in CD3 was further modified to His (EESH), thr (EEST), arg (EESR) or Lys (EESK) (and other amino acid residues) in the context of the heavy chain CDR 2D 62E, light chain CDR 1D 25E and light chain CDR 2N 51S substitutions and tested in Huh-7 and Hek-293 cells (FIGS. 50A-D). Mutants EESH and EESR have reduced STF activity, probably due to disruption of binding sites on the cell surface. Mutants EEST, EESK and EEAT have STF activity comparable to WT, however the following combination of mutations has reduced activity: EESL, EESE, EESH, EESR, EESY, EEAL, EEAE, EEAH, EEAY and EEAR. Protein folding was also examined for various combinations of mutants with different amino acid substitutions for N88 (fig. 41). Combinations of sequences with similar activities were compared, EEST with highest Emax and lowest EC50 (fig. 42). Surprisingly, the mutation of N88 to Thr maintained STF activity and proper protein folding (fig. 41).

Preferred substitutions at various modification positions were selected based on activity and are shown in table 1, wherein preferred amino acid substitutions are highlighted in bold (WT represents wild type).

TABLE 1

Wnt signaling enhancing molecules comprising various combinations of preferred amino acid substitutions identified above were prepared and tested by STF assay, oct binding and multispecific assay. These molecules comprise the following amino acids at positions 1-4 of table 1: EESY, EEAL, EEAE, EEAH, EEAT, EEAY, EEAR, EESN, EEAN, EESL, EESE, EESH, EEST, EESR and EESK. SDS-PAGE analysis was performed under non-reducing and reducing conditions to determine protein folding (FIG. 7: left panel: lane 1 = marker, lanes 2-8 = EESY, EEAL, EEAE, EEAH, EEAT, EEAY, EEAR, non-reducing, and lanes 9-15 = EESY, EEAL, EEAE, EEAH, EEAT, EEAY, EEAR, reduced; right panel: lane 1 = marker, lanes 2-9 = EESN, EEAN, EESL, EESE, EESH, EEST, EESR, EESK).

STF assays were performed to assess the ability of these molecules to modulate Wnt signaling in Huh-7 STF Wnt responsive reporter cells in the presence of a supplied Wnt source. Only mutants 1R34-EEST/RA ("EEST"), 1R34-EESA/RA ("EESA"), 1R34-EESN/RA ("EESN"), 1R34-EEAN/RA ("EEAN"), 1R34-EEAT/RA ("EEAT") and 1R34-EESK/RA ("EESK") have STF activity comparable to 1R34-DDNN/RA starter molecule ("NG") (FIGS. 8 and 39A-C).

As summarized in fig. 8, both EEST, EESN, EEAN and EEAT mutants were monodisperse and stable to freeze-thawing, including three rounds of freeze-thawing, between freezing with liquid nitrogen to room temperature. Freeze-thaw stability was determined by STF and SEC (data not shown). Binding to ASGR1 antigen was also determined. As shown in fig. 8 and 9, these four mutants and parent molecules have similar binding affinities for ASGR1 antigen. Multispecific binding to insulin, heparin, dsDNA, KLH and LPS was also examined. ELISA showed interactions in the mutants comparable to heparin. At high concentrations, the mutants also showed weak binding comparable to dsDNA, KLH and LPS, but not insulin. Thus, these constructs showed comparable activity and stability.

To eliminate LGR binding of RSPO2 polypeptides, different point mutations were made at two highly conserved hydrophobic residues within the Fu2 domains of huRSPO2, F105 and F109. In particular, these residues are replaced by F105R and F109A or F105E and F109E. Table 2 shows the specific combinations of substitutions present in each of these variants.

TABLE 2

The light and heavy chains present in each of these molecules are shown below: sequence of RSPO2 fusion protein.

The light chain sequence of the αASGR1-RSPO 2-EEST-EEE (1R 34-EEST/EE) molecule is provided below, with the CDRs underlined:

the heavy chain sequence of the αasgr1-RSPO2-EEST-EE molecule with fused RSPO2 sequences is shown below, with CDRs underlined, RSPO2 sequences shown in italics, and linker sequences shown in bold:

/>

two amino acid substitutions compared to the wild-type human RSPO2 sequence are shown in bold, italics and underlined.

The light chain sequence of the αASGR1-RSPO2-EEST-RA molecule is provided below, with the CDRs underlined:

the heavy chain sequence of the αasgr1-RSPO2-EEST-RA molecule with fused RSPO2 sequences is shown below, wherein the CDRs are underlined, the RSPO2 sequences are shown in italics, and the linker sequences are shown in bold:

two amino acid substitutions compared to the wild-type human RSPO2 sequence are shown in bold, italics and underlined.

The light chain sequence of the αASGR1-RSPO 2-EEAT-EEE molecule is provided below, with the CDRs underlined:

the heavy chain sequence of the αasgr1-RSPO2-EEAT-EE molecule with fused RSPO2 sequences is provided below, wherein the CDRs are underlined, the RSPO2 sequences are shown in italics, and the linker sequences are shown in bold:

two amino acid substitutions compared to the wild-type human RSPO2 sequence are shown in bold, italics and underlined.

As shown in FIG. 10, the combination of F105E and F109E mutations (SWEETS-1_RSPO 2EE_NG_EEST_G4S;1R 34-EEST/EE) had lower in vitro activity in the STF assay than the combination of F105R and F109A mutations (SWEETS-1_NG_EEST_G4S; 1R 34-EEST/RA), especially in Huh-7 cells, where the latter was almost 6-fold more potent. Surprisingly, however, this greater in vitro activity did not translate into an increase in vivo activity of the combination of F105R and F109A mutations. In contrast, when Axin mRNA expression levels were analyzed in non-human primates treated with either swets-1_ng_eest_g4 s (1R 34-EEST/RA) or swets-1_rspo2ee_ng_eest_g4 s (1R 34-EEST/EE), the combination of F105E and F109E mutations showed equal Axin2 expression or a greater increase in Axin2 expression compared to the combination of F105R and F109A mutations (fig. 11). This is in contrast to in vitro efficacy.

8M24-v1 molecular modification

Various amino acid modifications were made to the 8M 24-based αasgr1-RSPO2 Wnt signaling-enhancing molecule (8M 24-v 1) and tested to identify molecules with improved properties.

Initially, VH and VL domains of the starting 8M24 antibody sequence were each two different ways of humanization (H1, H2, V1 and V2). The original and humanized VH and VL sequences are shown in FIG. 25 (SEQ ID NOS: 13-18). Various combinations of humanized VH and VL sequences were combined in the context of the parent 8m24 iggl 1 constant region and tested for binding affinity to human ASGR1 as compared to the parent 8m24 VH and VL sequences. As shown in table 3, their combinations were selected based on the lowest-impact dynamic binding of L1 and H1 humanized VL and VH chains to human ASGR 1.

TABLE 3 Table 3

VH and VL	KD	kon	koff
				Parent strain	<1E-12	5.89E5	<1E-7
L1H1	5.73E-12	4.6E5	2.64E-6
				L1H2	<1E-12	5.11E5	<1E-7
L2H1	<1E-12	4.89E5	<1E-7
				L2H2	1.10E-10	5.73E5	6.29E-5

Potential deamidation or Asp isomerization capacity was identified in the CDRs of the 8m24 ASGR1 binding IgG moiety of the Wnt signaling enhancing molecule (shown in bold in fig. 26). Each of these amino acids was substituted with the various amino acids shown in fig. 26 and tested in the STF assay in the context of an aasgr 1-RSPO2 Wnt signaling enhancing molecule based on L1H1 humanized 8M24, as described above. RSPO2 sequences include the F105R and F109A substitutions described above. When tested in the context of a single mutation, the light chain D56E mutant was selected, and the heavy chain N31A mutant was the most effective mutation at that position and was selected. Both heavy chain N57Q, S and a mutants were almost equally effective. D102E mutants were also selected. All three mutations at D110 had poor potency, so wild-type D110 residues were selected (data not shown). Light chains comprising D56E mutations were tested in combination with the various heavy chain mutations identified above (which are shown in fig. 27). As shown in FIG. 27, the mutants containing N31A, N57S and D102E (EASE) heavy chain mutations had the best activity. Various combinatorial mutants were tested in various other assays, including protein folding, HIC, multispecific, tm/Tagg, stability and otte Kd. The results of these assays are summarized in fig. 28. The EASE mutant has an optimal combination of activity and otte Kd. The sequence of its light chain variable domain polypeptide is shown below, with CDRs underlined:

DIQMTQSPSSLSASVGDRVTITCRISENIYSNLAWYQQKPGKAPKLLIYAAINLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPFTFGQGTKLEIK(SEQ ID NO:19)。

The sequence whose heavy chain variable region is fused to the RSPO2 variant sequence is shown below, with RSPO2 sequences shown in italics, linkers shown in bold, and CDRs underlined:

the complete heavy chain also comprises, for example, the amino acid sequence of ID NO: the constant region sequences shown.

Based on the above studies Wnt signaling enhancing molecules were identified with specific combinations of mutations that could only be determined empirically to provide superior properties.

Example 2

In vivo liver action of liver-specific WNT signaling enhancing molecules

To demonstrate that the αasgr1-RSPO2 construct described in example 1 was able to activate the Wnt signaling pathway in vivo in a tissue-specific manner, mice were treated with various constructs.

To analyze tissue-specific activation of Wnt signaling pathway by Wnt signaling enhancement molecules, natural micemice) received a single i.p. dose of 10mg/kg of αasgr1-RSPO2-EEST-EE (1R 34-EEST/EE), R-spo2 or control (anti- βgal or anti-GFP-mutRSPO) (n=5 mice/group). EEST represents the substitutions present at positions 1-4 of fig. 1, and EE below represents the N105E and N109E substitutions in the RSPO2 region. 48 hours after treatment, various organs and tissues were collected for analysis. Axin2/ActB gene expression was determined and normalized to control. Axin2mRNA levels were significantly increased in most tissues after Rspo2 treatment. However, αASGR1-RSPO2-EEST-EE only resulted in an increase in Axin2mRNA in the liver (FIG. 12). For each tissue, anti-bgal, anti-GFP-mutSPO, αASGR1-RSPO 2-EEST-EEE and RSPO2 are shown from left to right. The resulting data shows that Wnt signaling enhancing molecules selectively activate the Wnt pathway in the liver (fig. 12). / >

mRNA and protein expression of Ki67 in liver and small intestine were determined by RT-PCR and immunofluorescence, and protein expression of HNF4α was determined by immunofluorescence. The antigen Ki-67 is a nuclear protein associated with proliferation and used as a cellular marker of proliferation, and hepatocyte nuclear factor 4a (HNF 4 a) is an orphan nuclear receptor that plays a major role in hepatic differentiation. As shown in fig. 21 and 22, αasgr1-RSPO2-EEST-EE (EE) stimulated proliferation of hepatocytes (fig. 21), but not small intestine cells (fig. 22). Furthermore, livers of animals treated with αASGR1-RSPO2-EEST-EE showed expression of Ki67 and HNF4α by immunofluorescence.

To analyze gene expression that was up-regulated upon Wnt-signaling pathway activation, native mice received a single i.p. dose of either 10mg/kg of αasgr1-RSPO2-EEST-EE (1R 34-EEST/EE), αasgr1-RSPO2-EEST-RA (1R 34-EEST/RA), or αgfp-IgG (n=20 mice/group). Serum and liver samples were collected 1 hour, 4 hours, 24 hours, and 72 hours after protein administration for expression analysis (n=5 per group at each time point). mRNA expression was analyzed by qPCR and samples were normalized to ActB. The relative fold was calculated by setting the average value of the anti-GFP group at 1 hour to a value of 1.

Liver Axin2, ccnd1 and Notum expression was significantly induced by treatment with αasgr1-RSPO2-EEST-EE or αasgr1-RSPO2-EEST-RA compared to expression in mice treated with αgfp-IgG negative control (fig. 13; αgfp-IgG, αasgr1-RSPO2-EEST-EE and αasgr1-RSPO2-EEST-RA; two-way ANOVA, holm-Sidak multiplex comparison (for anti-GFP), < p <0.05, < p <0.01, < p <0.001, < p < 0.0001) at each time point from left to right). These results indicate that the αasgr1-RSPO2 variant activates the Wnt pathway in the liver in vivo.

In another study, mice received a single i.p. dose of 10mg/kg of αASGR1-RSPO 2-EEST-EEE, αASGR1-RSPO2-EEST-RA, or αGFP-IgG (n=5 mice/group). Liver samples were collected after 1 hour, 4 hours, 24 hours and 72 hours for expression analysis and histoimmunochemistry. Treatment with αasgr1-RSPO2-EEST-EE or αasgr1-RSPO2-EEST-RA induced expression of the cell proliferation marker gene Mki67 when compared to mice treated with αasgr1-RSPO2-EEST-RA (fig. 14A; at each time point from left to right: αgfp-IgG, αasgr1-RSPO2-EEST-EE, and αasgr1-RSPO2-EEST-RA; two-way ANOVA, holm-Sidak multiple comparisons (for anti-GFP), <0.05, <0.01, <0.001, < 0.0001). These results indicate that the αasgr1-RSPO2 variants are able to stimulate proliferation of liver parenchymal cells. Additional studies showed that this effect was dose dependent (fig. 14B).

By IV injection of ssaV 8-CAG-hASGR1 (1X 10 per mouse) 7 days prior to treatment with the. Alpha. ASGR1-RSPO2 construct ¹¹ Individual genomic particles), which is shown to reach transgene expression levels equivalent to endogenous liver ASGR1 mRNA (data not shown).

The ability of the original 8M24-RA molecule (8M 24-v 1) and 8M24-RA EASE mutant (8M 24-EASE) to induce gene expression regulated by the Wnt signaling pathway was demonstrated. Mice received a single i.p. dose of 8M24-RA (8M 24-v 1) or 8M24-RA EASE (8M 24-EASE-RA;1, 3, 10 or 30 mg/kg), anti-GFP (10 mg/kg) or αASGR1-RSPO2-EEST-EE (1R 34-EEST/EE), with the IgG2 form replacing its normal IgG1 form. Serum and liver samples were collected 24h, 48h or 72h after treatment (n=4 at each time point). mRNA expression by qPCR was normalized to Actb. The relative fold was calculated by setting the average value of the anti-GFP group at each time point to 1. As shown in FIG. 29, 8M24-RA induced expression of Wnt signal target genes Axin2 and Ccnd1 and proliferation marker Mki 67. As shown in FIG. 30, 8M-24-EASE-RA also induced expression of Wnt signaling target genes Axin2 and Ccnd1 and proliferation marker Mki 67. Both 8M24-RA and 8M24-EASE-RA also induced a small but significant dose-dependent increase in ALP, consistent with the role of ASGR in eliminating serum ALP (data not shown).

Example 3

Pharmacokinetic profile of liver-specific WNT signaling-enhancing molecules

Pharmacokinetic profiles of Wnt signaling-enhancing molecules were examined in mice. Mice were divided into 6 groups (n=25/group) and received single doses of EEST-EE construct injected at 3, 10, 30, 100mg/kg IV or at 10 or 30mg/kg i.p.. Serum samples (rare) were collected at each time point 5 and 30 minutes (IV), 30 minutes and 1 hour (i.p.) and 2h, 6h, 24h, day 4, day 7, day 10 or day 14 (IV and i.p.) after protein dosing (n=5/group). Serum levels of EEST-EE were quantified by ELISA with ASGR1 or RNF43 capture ligands. Clearance, terminal half-life, cmax and MRT are shown in the table of figure 15. Liver samples were collected at the termination of 30 minutes (IV), 1h (i.p.), 6h, 24h, day 7 or day 14 (IV and i.p.) after protein dosing (n=5 for each time point). The relative fold was calculated by setting the average value of the anti-GFP group at each time point to 1. As shown in FIG. 15, 1R34-EEST/EE showed a nonlinear PK response to increased doses. Comparison of AUC obtained between IV and i.p. administration showed high bioavailability.

Similar studies were performed to compare 1R34-EEST/RA, 1R34-EEST/EE, 8M24-EASE-RA and 8M24-EASE-EE constructs. The results are provided in fig. 31.

Example 4

Liver targeting WNT signaling enhancement molecules improve liver function in mouse liver fibrosis models

The effect of Wnt signaling-enhancing molecules on liver function was examined in two mouse liver fibrosis models.

A chronic thioacetamide was used to induce a mouse liver fibrosis model. 6 week old C57B1/6J male mice were treated with Thioacetamide (TAA). TAA was added to drinking water at a concentration of 200mg/L for 13 weeks to induce liver fibrosis. Furthermore, during the last 8 weeks of TAA modulation, mice were i.p. administered TAA 3 times per week. TAA treatment was stopped 2 days prior to administration of the αasgr1-RSPO2 protein and mice were returned to purified laboratory grade acidified drinking water. Mice were injected daily with recombinant αASGR1-RSPO 2-EEST-EEEEE (1R 34-EEST/EE) or αASGR1-RSPO2-EEST-RA (1R 34-EEST/RA) or twice weekly intraperitoneally (i.p.) with αGFP-IgG or Rspo2 for one week as shown in FIG. 16.

At the time shown in FIG. 16, INR was measured using Roche CoaguChek-XS Plus. On day 3, day 10 and/or day 30 after the start of dosing, mice were anesthetized with isoflurane and blood was removed by cardiac puncture. A portion of the left lobe and duodenum were collected for analysis. Formalin fixed and paraffin embedded liver samples were sectioned and stained with anti-Ki-67 rabbit antibodies (Abcam, ab 15580). Calculation of Ki-67 in each randomly selected field of view using Image J ^- Number of positive nuclei (100 x magnification using 10x objective). Treatment with αASGR1-RSPO 2-EEST-EEEEEE (1R 34-EESD/EE) resulted in a significant decrease in INR, whereas treatment with αASGR1-RSPO2-EEST-RA (1R 34-EEST/RA) or Rspo2 was absent (FIG. 17A; one-way ANOVA compared to anti-GFP; (;) p)<.05,(**)p<.01,(***)p<.001,(****)p<.0001). Treatment with αasgr1-RSPO2-EEST-EE or RSPO2 also resulted in a significant increase in axin2 and CYP2E1mRNA expression, whereas treatment with αasgr1-RSPO2-EEST-RA showed a smaller but significant increase in axin2 and CYP2E1mRNA expression (fig. 17B and 17C; (:) p<.05,(**)p<.01,(***)p<.001,(****)p<.0001). Ki67 immunofluorescence staining also demonstrated hepatocyte specificity in TAA-induced injury modelsSex (fig. 18).

In CCl ₄ The effect of αASGR1-RSPO2-EEST-EE was also examined in the induced injury model. CCl (CCl) ₄ Male mice of p.C57BL/6J received CCl ₄ injection was performed twice weekly for 11 weeks. Control mice received only olive oil i.p. injection (n=8). Stopping CCl ₄ Mice were treated, divided into 10 groups (n=8), and dosed with either daily or every other day (q.o.d.) with either different doses of αasgr1-RSPO2-EEST-EE or αasgr1-RSPO2-EEST-RA (mg/kg), or twice weekly with anti-GFP (10 mg/kg) or RSPO2 (4.6 mg/kg). Blood and liver samples were collected at the end of day 7 or day 14.

As shown in FIG. 19, αASGR1-RSPO2-EEST-EE significantly induced Axin2 and MKi67mRNA levels on day 7. A larger increase in Axin2, ccnd1 and Mki67mRNA was observed with αASGR1-RSPO2-EEST-EE compared to αASGR1-RSPO 2-EEST-RA. Furthermore, immunofluorescence confirmed that this increase was hepatocyte-specific (fig. 20).

Example 5

WNT signal-enhancing molecules improve liver synthesis and reduce fibrosis

6 week old C57B1/6J male mice were treated with Thioacetamide (TAA). TAA was added to drinking water at a concentration of 200mg/L for 18 weeks to induce liver fibrosis. Furthermore, during the last 7 weeks of TAA modulation, TAA was administered i.p. to mice 3 times per week, resulting in liver fibrosis. TAA treatment was stopped 2 days prior to administration of αASGR1-RSPO 2-EEST-EEE (1R 34-EEST/EE) and mice were returned to purified laboratory grade acidified drinking water. Mice were injected intraperitoneally (i.p.) at 10mg/kg daily with recombinant αasgr1-RSPO2-EEST-EE or negative control for 14 days.

At the time shown in FIG. 16A, INR was measured using Roche CoaguChek-XS Plus. INR (internalization normalization ratio) measures the rate of clot formation. High INR levels reflect liver disease or cirrhosis and indicate related inability to produce normal amounts of protein and less desirable blood clotting. On days 3, 7 and 14 after the start of dosing, mice were anesthetized with isoflurane and blood was removed by cardiac puncture. Treatment with αasgr1-RSPO2-EEST-EE resulted in a significant decrease in INR compared to the control (fig. 16B). In this mouse fibrosis model, short-term treatment with Rspo and αasgr1-Rspo2-EEST-EE resulted in moderately variable fibrosis reduction (data not shown).

Example 6

Tissue-targeting RSPO mimics versus chronic CCl ₄ Effects of liver function in induced mouse liver fibrosis model

Also use chronic CCl ₄ The induced mouse liver fibrosis model examines liver fibrosis. In particular, 1R23-EEAT/EE vs CCl was compared in immunodeficient and immunocompetent mice ₄ Induced liver fibrosis.

To 32C 57BL/6J males (Jackson Laboratories) of 6 weeks of age and 32 NOD.CB17-Prkdc ^scid Intraperitoneal injection of CCl/J (SCID) ₄ (0.5 mL/kg, twice/week) for 10 weeks. 16 mice of each strain were injected intraperitoneally with olive oil vehicle (0.5 mL/kg). CCl (CCl) ₄ Following treatment, 8 mice of each strain were sacrificed (with or without cci ₄ Process), and blood and tissue are collected for baseline measurements. The rest CCl ₄ The treated mice were randomly divided into treatment groups based on body weight (table 4) and protein was administered for 2 weeks. The treatment groups for each strain were as follows: 20mg/kg of 1R34-EEAT/EE, n=8 (daily administration); 4.6mg/kg Fc-RSPO2-WT, n=8 (twice weekly); 10mg/kg of anti-GFP, n=8 (twice weekly administration). An additional control group, previously injected with oil only, was included, n=8. Blood was drawn from mice on day 7 (the first day of protein administration is denoted as day 0) for serum chemistry testing. Mice were sacrificed on day 14. Total body weight and liver weight were measured and blood, liver tissue were collected and saved for testing.

Table 4 provides CCl ₄ Description of treatment groups of induced mouse fibrosis model. Immunodeficient NOD.CB17-Prkdc ^scid J or immunocompetent C57BL/6J males with CCl diluted in olive oil carrier ₄ Or pretreated with olive oil alone. Mice were divided into groups a to L as shown. Mice were then injected with test articles at the doses and frequency shown and then terminated at day 0 at baseline or at day 14 after the start of test article dosing.

Table 4: CCl (CCl) ₄ Induced fibrosis treatment group

The ratio of liver to body weight was significantly increased in the group treated with RSPO2 positive control and the group treated with RSPO mimetic 1R34-EEAT/EE (fig. 32).

Serum alkaline phosphatase (ALP) and albumin were measured at time points one week and two weeks after protein treatment. Mouse serum was collected from tail blood collection on day 7, terminated on day 14, and analyzed using VetAxcel (Alfa-wasterman). The data are presented in fig. 33A and 33B. At both the week and two week time points, serum ALP was statistically higher, consistent with upregulation of alkaline phosphatase protein levels due to elimination of the ASGR receptor as described in the ASGR1-KO mouse model (see, e.g., cell Host microbe.2018Oct10;24 (4): 500-513). Serum albumin levels were significantly reduced in response to RSPO2 and 1R 34-EEAT/EE. The results indicate an expected temporary decrease in periportal hepatocyte function due to increased pericentral hepatocyte expansion induced by increased Wnt signaling.

Quantitative PCR was used to measure changes in liver gene expression. Treatment of mice with RSPO2 or 1R34-EEAT/EE resulted in increased expression of Wnt-inducible Axin2 gene in SCID mice, but not in C57BL/6J mice on day 14. These results indicate that after two weeks, the activity of the test article was sustained in immunodeficient mice, but not in immunocompetent mice.

Treatment with RSPO2 and 1R34-EEAT/EE also showed a trend towards an increase in the proliferation markers of Mki67 and cyclin D1 (Ccnd 1) in the liver following 1R34-EEAT/EE treatment, as well as a significant increase following RSPO2 treatment (fig. 34). Immunofluorescent staining of liver sections showed an increase in the number of Ki67-HNF4α biscationic cells in RSPO2 and 1R34-EEAT/EE liver samples from SCID mice (FIG. 35). Similar results were obtained using liver samples from C57BL/6J mice (data not shown). These results indicate that RSPO2 and 1R34-EEAT/EE promote hepatocyte proliferation.

Histological liver sections were stained with sirius scarlet to measure fibrosis levels (fig. 36A). The area stained with sirius red was quantified by Image J (NIH) (fig. 36B). These results show that 1R34-EEAT/EE significantly reduced the area percentage of collagen fibers stained with sirius scarlet in immunocompetent and immunodeficient mice. RSPO2 also reduced the percentage of area stained with sirius scarlet, although not significant in SCID mice.

Together, these data demonstrate that RSPO mimetic 1R34-EEAT/EE has a significant effect on the rate at which mouse livers can resolve (resolve) fibrosis and regenerate functional hepatocytes.

Example 7

Liver-targeted RSPO WNT signaling enhancement molecules activate WNT signaling and bind to targets in non-human primates

Non-human female primate were treated by intravenous bolus injection with 10mg/kg of αASGR1-RSPO 2-EEST-EEE (1R 34-EEST/EE), αASGR1-RSPO2-EEST-RA (1R 34-EEST/RA), 8M24 αASGR1-RSPO2-EASE-RA (M24-EASE/RA), 8M24-EASE-RSPO2-EASE-EE (8M 24/EASE/EE) or vehicle control on day 1 and 15 and then terminated 24 hours after the second administration on day 16. Liver samples were obtained and subjected to hematology analysis and histopathological examination. Liver samples were analyzed for AXIN2 expression by qPCR and normalized to ACTB expression. The relative fold was calculated by setting the mean value of the vehicle group to 1. Mean +/-SEM. One-way ANOVA, holm-Sidak multiple comparisons (for vehicle), p <0.05.

Treatment with αasgr1-RSPO2-EEST-EE, αasgr1-RSPO2-EEST-RA, 8M24 αasgr1-RSPO2-EASE-RA, or 8M24-EASE-RSPO2-EASE-EE resulted in unplanned death, no abnormal clinical observations, and no changes in body weight or food consumption. Axin2 mRNA levels were significantly increased in the liver and slightly higher after treatment with αasgr1-RSPO2-EEST-EE, 8M24 αasgr1-RSPO2-EASE-RA, or 8M24-EASE-RSPO2-EASE-EE than after treatment with αasgr1-RSPO2-EEST-RA (fig. 11). For clinical pathology, there was no substantial change in hematology, but there was a substantial transient increase in ALP, consistent with binding of the molecule to ASGR1, inhibiting the receptor from scavenging ALP (fig. 24).

These studies demonstrate that Wnt signaling enhancing molecules are well tolerated and active in non-human primates.

Example 8

Crystal structure of human ASGR1-CBD 8M24 complex

The asialoglycoprotein receptor (ASGR; ASGPR) is a C-type lectin expressed primarily in mammalian liver cells (hepatocytes), mediating clearance of desialylated galactose-or N-acetylgalactosamine-terminated plasma glycoproteins via receptor-mediated endocytosis. The assembly of ASGR is considered a heterotrimer consisting of two ASGR1 and one ASGR2 polypeptides, called H1 and H2, respectively. Structurally, both ASGR1 (H1) and ASGR2 (H2) polypeptides are type II membrane proteins with a short N-terminal cytoplasmic domain, followed by a single transmembrane helix and an cytoplasmic region comprising a helical stalk region that mediates oligomerization via a coiled-coil structure and a Carbohydrate Binding Domain (CBD) at their C-terminus. Human ASGR1 and ASGR2 share 54% sequence identity.

The crystal structure of the human ASGR1-CBD (HuASGR 1-CBD) domain (residues 154 to 291 of uniprot entry P07306; https:// www.dot.uniprot.org/uniprot/P07306) complexed with the Fab domain of the antibody designated 8M24 was determined. The sequence of the HuASGR1-CBD construct comprising an octahistidine motif and a biotin receptor peptide (BAP) at its N-terminus for structural studies was as follows:

HuASGR1-CBD_P07306_154-291

HHHHHHHHGSGSGLNDIFEAQKIEWHESGSGCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL(SEQ ID NO:52)。

Expression and purification of HuASGR1-CBD for structural studies

Plasmids expressing HuASGR1-CBD were transfected for expression in Expi293 cells (thermo fisher USA), typically at a scale of 1000mL, using a FectoPro transfection reagent according to the standard protocol of manufacturer (Polyplus Transfection NY USA). After 4 days of continuous cell growth, the medium was collected by centrifugation and HuASGR1-CBD was purified from the medium by incubation with HisComplite resin (1 mL/L culture; roche) pre-equilibrated in 50mM sodium dihydrogen phosphate, pH8.0, 300mM NaCl, washing with 10mM imidazole and elution with 250mM imidazole in equilibration buffer. The eluate was concentrated to 5mL and further purified on a HiLoad 16/600Superdex 200pg column (GE Life Sciences) pre-equilibrated with HBS (20 mM HEPES pH7.4 and 150mM sodium chloride). The contents were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; tris-HCl 4-15% gel from Bio-Rad, hercules, calif.). Samples were prepared in Laemmli sample buffer and heated at 100 ℃ for 5 minutes. The HuASGR1-CBD containing fractions were concentrated to 1.78mg/mL and frozen in the presence of 10% glycerol for storage at-80 ℃ until further use. Protein concentration was determined by direct UV a280 method using a NanoDrop spectrophotometer (Thermo Scientific). Based on the Beer-Lamber equation, the relationship between absorbance and protein concentration is linear, a=ε1c; a is the absorbance value, epsilon is the wavelength dependent extinction coefficient, l is the path length in cm, and c is the protein concentration. The extinction coefficients of all the produced proteins were estimated by their amino acid sequences.

Expression and purification of 8M24 Fab

Plasmids expressing 8m24 Fab (version L1H1, corresponding to SZP19057+ 19056) light and heavy chains (with a hexahistidine tag at their C-terminus) were transfected for expression in Expi293 cells (ThermoFisher USA) at a scale of typically 1000mL, using FectoPro transfection reagent according to the standard protocol of the manufacturer (Polyplus Transfection NY USA). After 4 days of continuous cell growth, the medium was collected by centrifugation and bound to Complete-His resin pre-equilibrated in PBS (2.5 mL/1L culture; roche) and eluted under gravity flow using 250mM imidazole in PBS. The eluate containing the Fab conjugate was concentrated to-5 mL and further purified on a hilload 16/600Superdex 200pg column (GE Life Sciences) pre-equilibrated with HBS. The contents were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; tris-HCl 4-15% gel from Bio-Rad, hercules, calif.). Samples were prepared in Laemmli sample buffer and heated at 100 ℃ for 5 minutes. The fraction containing 8m24 Fab was concentrated to 7.12mg/mL and frozen in the presence of 10% glycerol for storage at-80 ℃ until further use. Protein concentration was determined by direct UV a280 method using a NanoDrop spectrophotometer (Thermo Scientific). Based on the Beer-Lamber equation, the relationship between absorbance and protein concentration is linear, a=ε1c; a is the absorbance value, epsilon is the wavelength dependent extinction coefficient, l is the path length in cm, and c is the protein concentration. The extinction coefficients of all the produced proteins were estimated by their amino acid sequences.

HuASGR1-CBD 8M24 Complex formation, crystallization and Structure determination

Purified HuASGR2-CBD and 8m24 Fab were mixed at a molar ratio of 1.1:1 (slight excess HuASGR 1-CBD) and incubated with carboxypeptidase a and B at a w/w ratio of 100:1 overnight at 4 ℃. Complex formation was confirmed by observing a single main peak on a SuperdexS200Increase (10/300 GL) column pre-equilibrated in HBS. The fraction containing the complexes was further checked by SDS-PAGE and concentrated to 20mg/mL for crystallization screening. Crystallization screening using commercially available MCSG1, MCSG2, MCSG3, MCSG4, PACT (Molecular Dimensions USA), PEGs I and PEGs II (Qiagen USA) was performed using a Mosquito (TTP LabTech) liquid processor and equilibrated in an echo incubator (Torrey Pines Scientific USA) at 18 ℃. The 96-well plate crystal screening experiments were periodically monitored manually via a discover v20 stereo microscope (Zeiss USA) and the crystals were frozen by plunging into liquid nitrogen in the presence of various cryoprotectants (typically 15-30% v/v glycerol or ethylene glycol) to collect the data. The X-ray diffraction dataset was collected at the Bocley structural biology center (Berkeley Center for Structural Biology) of Advanced Light Source (ALS) of Bocley, calif., and treated with XDS [ XDS.Kabsch W. (2010) actaCryst.D 66,125-132] and XDSME [ Legrand P. (2017) XDSME: XDS Made Easier GitHub repository, https:// gitub.com/legandp/XDSME DOI 10.5281/zenoduo.837885 ] procedures. Structure of the HuASGR1-CBD 8M24 Complex by using Phaser [ [ Phasecrystallization software. McCoy AJ, grosse-Kunstleve RW, adams PD, win MD, storoni LC and Read RJ. (2007) J Appl Crystallogr, 658-674] and the polyalanine model of the published structure of ASGR1-CBD [ PDB code:5JPV; efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor, sanhueza, baksh MM, thumb B, roy MD, dutta S, preville C, chunyk BA, beaumont K, dullea R, ammirati M, liu S, gebhard D, finley JE, salatto CT, king-Ahmad A, stock I, atkinson K, reidich B, linW, kumar R, tu M, menhaji-Klotz E, price DA, liras S, finn MG, masciitti V. (2017) J.am.chem.Soc.139:3528-3536] and Surrozen Inc. previously determined variable and constant domains of the crystal structure of the unrelated Fab are then passed through Phenix [ PHENIX: python comprehensive systems for macromolecular structure resolution (acomprehensive Python-acomprehensive Python) and (35-858, kumart, 221, Z.35.213, etc.; the structure of the HuASGR1-CBD:8M24 complex was determined by molecular replacement methods by modification and validation of MolProbit:all-atom structure validation for macromolecular crystallograph, chen VB, arendall WB, headJJ, keedy DA, immorcino RM, kapral GJ, murray LW, richardson JS and Richardson DC (2010) Acta Cryst.D66,12-21 ]. The crystallographic model was manually checked and built using COOT [ Features and development of coot.emsley P, lohkamp B, scott WG and Cowtan k. (2010) Acta cryst.d66,486-501]. Analysis of fine crystal structure and image creation were performed using MOE (CCG) and PyMol (Schrodinger).

Structure of HuASGR2-CBD 8M24 Complex

The sequences of the light and heavy chains of 8m24 Fab used for structural studies are shown below.

8M24L1 light chain:

DIQMTQSPSSLSASVGDRVTITCRISENIYSNLAWYQQKP GKAPKLLIYAAINLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHFWGTPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:53)。

8M24H1 heavy chain:

EVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYGINWVR QAPGQGLEWMGEIFPRSDNTFYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARKGRDYGTSHYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGSGHHHHHH(SEQ ID NO:54)。

diffraction-quality crystals of HuASGR1-CBD:8M24 complex (concentration = 20 mg/mL) were grown under PACT-A6 crystallization conditions with 0.1M SPG (succinic acid, sodium dihydrogen phosphate monohydrate, glycine buffer) ph9.0 and 25% w/v PEG 1500. The crystals were cryogenically protected with 20% glycerol in the pore solution. HuASGR1-CBD 8M24 Complex at P2 ₁ 2 ₁ 2 ₁ Space groupAnd each asymmetric unit has a complex molecule. The structure of the HuASGR1-CBD 8M24 complex is +.>Is determined at a resolution of (2) and is accurate to 17.3% and 20.5% R, respectively _cryst And R is _free Factors.

FIG. 37A shows the general structure of the HuASGR1-CBD:8M24 complex. Structural analysis of HuASGR1-CBD showed three Ca2 important for the structural stability of CBD and its overall folding ⁺ The ion is similar to its previously disclosed structure [ PDB Code:1DV8; crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein collector. Meier M, bider MD, malshkevich VN, spiess M and Burkhard P. (2012) J Mol Biol 300,857-865 ]. The structure also shows that the 8M24 epitope on ASGR1 is distant from Ca ²⁺ Ion localization, the Ca ²⁺ The ions are part of the natural ligand binding site (fig. 37B).

The structure of the complex was used to identify an epitope of 8M24 on human ASGR1, where the following residues define the core interaction site [ ]Cut-off value):

gly163, pro165, val166, asn167, cys175, trp177, ser181, lys183, ala184, ala186, asp187, asn190, tyr191, arg193, leu194, glu195, asp196, trp285, thr289, glu290 and Leu291.

Furthermore, the following residues on human ASGR1 were identified as direct interaction sites for 8M24 (interatomic distanceAnd->)：

Cys164, trp168, arg173, ser174, phe178, ser179, arg180, gly182, trp185, ala188, asp189, cys192, ala197, gin 227, cys279, gin 280, arg281, cys287 and Glu288.

The structure of the HuASGR1-CBD 8M24 complex is used to identify any atom from human ASGR1 as being less than or equal toThe following residues of 8M 24:

8M24 heavy chain: asn31, phe52, arg54, ser55, asn57, phe59, lys99, arg101, asp102, tyr103, gly104, thr105, ser106, and His107.

8M24 light chain: tyr30, ser31, asn32, phe91, trp92, gly93 and Phe96.

Furthermore, huASGR1-CBD: the structure of the 8M24 complex shows that the following residues of 8G8 are interatomic distancesAnd->Direct interaction site of human ASGR 1:

8M24 heavy chain: thr28, thr30, tyr32, gly33, asn35, glu50, ile51, pro53, asp56, thr58, gly100, tyr108 and Phe109.

8M24 light chain: ile2, asn28, ile29, ala50, his90 and Thr94.

Example 9

Effects of hepatocyte-targeted RSPO mimics on liver function and tissue repair following acetaminophen-induced liver injury

The acetaminophen (APAP) hepatotoxicity mouse model is a well-established model of acute liver injury, the mechanism and severity of which is similar between mice and humans. The ability of 1R34-EEAT-EE to repair the hepatotoxic effects of APAP-induced liver injury was examined.

Based on body weight, 100 male C57BL/6J mice (9 weeks old) were randomized into 10 study groups (groups A-J) and then fasted overnight. To induce liver injury, mice were administered a single Intraperitoneal (IP) dose of APAP 300mg/kg (groups B-J, n=90); saline was administered IP to the control group (group a, n=10). The mice were then returned to the cages and food was obtained ad libitum. At 2 hours post APAP administration, mice in group B-J received a single IP injection of one of the following treatments according to the random cohort: 10mg/kg of anti-GFP (negative control), 1200mg/kg of N-acetylcysteine (NAC) (positive control) or 10mg/kg of 1R34-EEAT-EE. Group a mice continue to be tracked without additional injections of vehicle administered. Mice were then tracked for up to 72 hours and terminated 24 hours, 48 hours or 72 hours after treatment.

At various termination time points, blood and liver were collected for clinical chemistry and immunohistochemical analysis. Quantitative polymerase chain reaction (qPCR) analysis of liver mRNA measures expression levels of Wnt target genes and key cytochrome P450 (CYP) metabolizing enzymes.

Table 5: APAP-induced liver injury treatment group

1R-34 EEAT-EEAT activates the Wnt/β -catenin signaling pathway as shown by the increased hepatic mRNA expression of the marker Axin2 of Wnt/β -catenin activation and induction of the proliferation marker cyclin D1 (Ccnd 1) of the G1/S phase transition of the Wnt target gene and cell cycle (FIG. 43). In addition, notum, a physiological negative regulator of Wnt target genes and Wnt ligands, was induced by 1R 34-EEAT-EE.

After APAP-induced liver injury, the expression of Cyp1a2 and Cyp2e1 genes was significantly reduced in all groups (fig. 43). At 48 hours and 72 hours post-dose, 1R 34-EEAT-EEE treatment resulted in significantly higher levels of Cyp1a2 and Cyp2e1 expression.

Spontaneous repair was observed by immunofluorescence in the pericentral region of the liver treated with anti-GFP 72 hours after treatment, as evidenced by the presence of immunoreactive ki67+ nuclei. The ability of 1R34-EEAT-EE to regenerate was demonstrated by an increase in Ki67+/HNF4α+ double positive hepatocytes (FIG. 44, white squares) over the ability to spontaneously occur, evenly distributed in all liver regions, including the periportal region, as shown by CYP2F2 staining with Ki-67 double immunofluorescence and periportal expression (FIG. 45). In contrast, a significant proportion of non-hepatocytes appear to proliferate in the anti-GFP and N-acetylcysteine groups based on the presence of ki67+ nuclei (yellow squares) with undetectable hnf4α.

Histological analysis showed a large area of diffuse necrosis of the area around the center of the liver in the control group, but decreased necrosis in 1R34-EEAT-EE treated liver (fig. 46).

Serum alkaline phosphatase (ALP) increased significantly at 24 and 48 hours post-treatment, consistent with the occupancy of ASGR1 by 1R34-EEAT-EE and the known role of ASGR in ALP clearance (FIG. 47). At all time points measured, 1R34-EEAT-EE significantly reduced blood ammonia levels compared to anti-GFP. At any of the time points measured, there was no significant difference in serum AST or ALT levels between 1R34-EEAT-EE and anti-GFP.

This study in a murine model of APAP-induced liver injury suggests that 1R34-EEAT-EE alleviates APAP-induced hepatotoxicity by targeted activation of Wnt/β -catenin signaling in the liver and stimulation of hepatocyte-specific regeneration.

Example 10

Effects of hepatocyte-targeted RSPO mimics on liver function and tissue repair following chronic alcohol-induced liver injury

Animal models of alcoholic liver injury reproduce to varying degrees many features of AH in patients, such as steatosis and elevated liver expression of inflammatory body components (e.g., IL-1β). Chronic drinking models (chronic-binge model) are a common model consisting of ad libitum feeding using diets containing Lieber-decelli liquid ethanol (EtOH) and ethanol delivered by oral gavage (Bertola et al 2013). The present study examined the effect of 1R34-EEST-EE on hepatocyte expansion and function in a long-term chronic, multiple binge eating, acute Alcoholic Hepatitis (AAH) model in aged mice, where spontaneous regeneration ability of the mouse liver was significantly impaired.

The 11 month-old C57BL/6J female mice were fed a control Lieber-DeCarli diet ad libitum for 5 days to accommodate fluid diet and gavage (FIG. 48). All mice were then randomized (fig. 55). Mice in the EtOH fed group were left free to acquire a Lieber-DeCarli (L-D) diet containing 5% (vol/vol) EtOH for 7 weeks, and the pair fed (pair-fed) group received an L-D control diet with an equicaloric amount of maltodextrin in place of ethanol. From week 2 of the feeding period and for up to week 7, etOH fed and pair fed mice received twice weekly gavage doses of EtOH 20% (5.23 g/kg body weight) or equicaloric maltodextrin, respectively. After the end of the EtOH feeding period, mice were returned to the control L-D fluid diet and were treated with 1R34-EEST-EE (30 mg/kg) or anti-GFP (10 mg/kg) at random; treatments were administered once daily via intraperitoneal injection. Mice were then sacrificed 3 or 7 days after treatment.

Table 6: ethanol-induced injury treatment group

At various termination time points, blood and tissue samples were collected for clinical chemistry analysis. Wnt pathway activation, proliferation and inflammatory markers were examined by quantitative polymerase chain reaction (qPCR) analysis of liver mRNA.

1R34-EEST-EE activates the Wnt/β -catenin signaling pathway as shown by the increased hepatic mRNA expression of the two Wnt target genes Axin2 and Ccnd1 (FIG. 49) and the induction of liver proliferation markers such as Rrm, cdk1, ccnb1, cdc20 and Mki67 (FIG. 50). The hepatocyte-specific regenerative activity of 1R34-EEST-EE was confirmed by double immunofluorescent staining with proliferation marker Ki67 and hepatocyte-specific marker HNF4A (FIG. 51).

Furthermore, 1R34-EEST-EE significantly increased both serum biomarkers of Wnt activation, namely leukocyte-derived chemokine-2 (Lect 2) and angiogenin, as measured by enzyme-linked immunosorbent assay (ELISA) (fig. 53). Compared to anti-GFP (control group), 1R34-EEST-EE also reduced mRNA expression of inflammatory markers (interleukins Il1b and Il 6) (fig. 54), ALT was slightly elevated, and AST responded to 1T34-EEST-EE significantly decreased, with significant changes observed after 3 days of treatment with 1R34-EEST-EE resulting in a significant decrease in AST/ALT ratio (fig. 52). Furthermore, on day 3, 1R34-EEST-EE significantly reduced circulating ammonia compared to anti-GFP (FIG. 52).

In summary, 1R34-EEST-EE induced liver-specific Wnt/β catenin signaling in the AAH model of aged mice. In addition, 1R34-EEST-EE stimulates hepatocyte-specific cell regeneration. These data provide proof of concept as follows: 1R34-EEST-EE stimulates hepatocyte expansion under conditions of impaired proliferation due to age and alcohol use.

Example 11

Biomarkers of drug-induced liver failure regeneration

Acute Liver Failure (ALF) due to acetaminophen (APAP) overdose or other drug-induced liver injury (DILI) has limited therapeutic options. Although 65% of patients with APAP-induced ALF spontaneously survive, approximately 500 cases die annually in the united states due to APAP hepatotoxicity. In DILI, the survival rate without transplantation is about 25% and there are estimated 300-500 deaths in the united states. For liver transplantation, patients are listed who are not expected to recover after first line treatment (e.g., intravenous N-acetylcysteine). As the demand for liver transplantation increases and the supply is limited, a reliable test for predicting liver recovery is needed.

Wnt signaling plays an important role in hepatocyte expansion during development and tissue repair. Downstream canonical Wnt signaling mediated by β -catenin stabilization was associated with increased regeneration in ALF patients (Apte 2009,Bhushan 2014).

Herein, the difference in serum levels of liver injury biomarkers (alpha fetoprotein and cholinesterase) and Wnt signaling markers (angiogenin and leukocyte-derived chemokine 2) between ALF patients not receiving liver transplantation and ALF patients undergoing liver transplantation or death was determined.

Alpha Fetoprotein (AFP) is secreted by immature hepatocytes proliferating and is elevated in liver injury. In ALF, the ratio of AFP on day 3 to day 1 of spontaneous survivors was higher than in dead or transplanted patients, indicating that the prognostic value of AFP level changes [ ]2006)。

Butyrylcholinesterase (BChE) is a nonspecific esterase produced by the liver and is reduced in many liver dysfunctions including acute and chronic liver injury, inflammation and infection.

Angiogenin is a direct Wnt target secreted primarily by hepatocytes. Angiogenin induces angiogenesis and plays a role in cell growth and survival. Whether there is a link between angiogenin and ALF results has not been reported.

Leukocyte-derived chemokine 2 (LECT 2) is a liver factor secreted almost exclusively by hepatocytes and is a direct Wnt target. LECT2 plays a key role in liver regeneration: specific for ALF, LECT2 levels increased when liver function was restored (Sato 2004), but low LECT2 levels during the first 3 days after injury correlated with higher survival probability (Slowik 2019).

Serum samples were selected from the American adult acute liver failure study group accession (NCT 00518440). The etiology of liver failure, as specified by ALFSG, is divided into two groups: APAP or DILI/other/indeterminate. Spontaneous Survivors (SS) are defined as patients recovered without transplantation and compared to patients undergoing transplantation or death (LT/D). Samples of 10 APAP overdose patients and 5 DILI patients in SS groups withdrawn on day 1, day 3 and day 7 after enrollment, and samples of 10 APAP and 10 DILI patients in LT/D groups withdrawn on day 1 and day 3 (see table 7).

Table 7: survival of liver failure patients

AFP, angiogenin and LECT2 were measured by enzyme-linked immunosorbent assay (ELISA), and BChE was measured by enzyme activity assay.

The time since study registration represents different times during the patient's clinical course. To illustrate this, biomarker levels are explained in the time context since admission. Log scale of biomarker levels was simulated by performing a random mixing effect model, where covariates included time terms since admission and status sets indicating SS or LT/D. Assume that unstructured correlations illustrate the correlation of repeated measurements over time for the same patient. A 95% Confidence Interval (CI) of the point estimate and least squares mean for each state group is estimated, as well as the difference between the two states.

The distribution of AFP, angiogenin, LECT2 and BChE was plotted against admission time for SS and LT/D, respectively. The point estimate between SS and LT/D is compared to 95% CI of the least squares mean. The fold difference between SS and LT/D is summarized in Table 8.

Table 8: AFP, angiogenin, LECT2 and BChE distribution

The fold difference in the point estimates of angiogenin and LECT2 between SS and LT/D was significant, whereas the fold difference of AFP and BChE was not.

For the etiology of liver failure (APAP and DILI/other), the fold difference in point estimates between SS and LT/D, respectively, is compared. There was a significant fold difference between angiogenin and LECT2 in APAP patients, but only angiogenin was a significant fold difference in DILI/other patients (table 9).

Table 9: AFP, angiogenin, LECT2 and BChE distribution

In ALF, a > 2.5-fold increase in circulating angiogenin and a > 6-fold increase in circulating LECT2 was observed early after admission in patients recovered without liver transplantation. These results indicate that serum markers can function as prognostic indicators of regeneration and as biomarkers of Wnt signaling activation. Furthermore, since 1R34-EEST-EE was shown in example 10 to have significantly increased LECT2 and angiogenin, these data indicate that treatment with 1R34-EEST correlates with increased Wnt signaling and regeneration.

Reference is made to:

Acute liver failure induced by idiosyncratic reaction to drugs:challenges in diagnosis and therapy.Authors:Tujios,S；Lee,W.Journal Title:Liver International.Publisher:Wiley.Publication Date:01/2018.Volume:38.Issue:1.Pages:6-14.DOI:10.1111/liv.13535.PMID:28771932.

Beta-catenin activation promotes liver regeneration after acetaminophen-induced injury.Authors:Apte,U；Singh,S；Zeng,G；Cieply,B；Virji,M；Wu,T；Monga,S.Journal Title:The American Journal of Pathology.Publisher:Elsevier.Publication Date:09/2009.Volume:175.Issue:3.Pages:1056-1065.DOI:10.2353/ajpath.2009.080976.PMID:19679878.

Pro-regenerative signaling after acetaminophen-induced acute liver injury in mice identified using a novel incremental dose model.Authors:Bhushan,B；Walesky,C；Manley,M；Gallagher,T；Borude,P；Edwards,G；Monga,S；Apte,U.Journal Title:The American Journal of Pathology.Publisher:Elsevier.Publication Date:11/2014.Volume:184.Issue:11.Pages:3013-3025.DOI:10.1016/j.ajpath.2014.07.019.PMID:25193591.

Alpha-fetoproitein and prognosis in acute liver failure.Authors:F；Ostapowicz,G；Murray,N；Satyanarana,R；Zaman,A；Munoz,S；Lee,W.Journal Title:Liver Transplantation.Publisher:Wiley.Publication Date:12/2006.Volume:12.Issue:12.Pages:1776-1781.DOI:10.1002/lt.20886.PMID:17133565.

Serum LECT2 level as a prognostic indicator in acute liver failure.Authors:Sato,Y.；Watanabe,H.；Kameyama,H.；Kobayashi,T.；Yamamoto,S.；Takeishi,T.；Hirano,K.；Oya,H.；Nakatsuka,H.；Watanabe,T.；Kokai,H.；Yamagoe,S.；Suzuki,K.；Oya,K.；Kojima,K.；Hatakeyama,K.Journal Title:Transplantation Proceedings.Publisher:Elsevier.Publication Date:10/2004.Volume:36.Issue:8.Pages:2359-2361.DOI:10.1016/j.transproceed.2004.07.007.PMID:15561249.

Leukocyte cell derived chemotaxin-2(Lect2)as a predictor of survival in adult acute liver failure.Authors:Slowik,V；Borude,P；Jaeschke,H；Woolbright,B；Lee,W；Apte,U,the Acute Liver Failure Study Group.Journal Title:Translational Gastroenterology and Hepatology.Publisher:AME Publishing Company.Publication Date:03/2019.Volume:4.Issue:17.Pages:2359-2361.DOI:10.21037/tgh.2019.03.03.PMID:30976720.

the various embodiments described above may be combined to provide further embodiments.

Aspects of the embodiments can be modified to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and/or listed in the application data sheet are incorporated herein by reference, in their entirety.

Sequence listing

<110> Surrozen, Inc.

<120> liver-specific Wnt signaling enhancement molecules and uses thereof

<130> SRZN-019/03WO 328202-2142

<150> US 63/114,457

<151> 2020-11-16

<150> US 63/182,106

<151> 2021-04-30

<150> US 63/248,157

<151> 2021-09-24

<160> 62

<170> PatentIn version 3.5

<210> 1

<211> 215

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 2

<211> 572

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of fusion construct 1R34-DDNN/RA heavy chain fused to RSPO2

<400> 2

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

245 250 255

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

340 345 350

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

355 360 365

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

370 375 380

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

385 390 395 400

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

405 410 415

Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr

420 425 430

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

435 440 445

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

450 455 460

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

465 470 475 480

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

485 490 495

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

500 505 510

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

515 520 525

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

530 535 540

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

545 550 555 560

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-humanized VL Domain engineered from 8M24 antibody

<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 4

<211> 244

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-fusion construct heavy chain variable Domain fused to RSPO2

<400> 4

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Asn Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Asp

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser

<210> 5

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-humanized VL chain engineered with 8M24 antibody

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 574

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-fusion construct heavy chain fused with RSPO2

<400> 6

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Asn Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Asp

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

465 470 475 480

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 7

<211> 215

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of mutated light chain sequences

<400> 7

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Glu Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Ser Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 8

<211> 572

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of mutated EEST/EE heavy chain of fusion construct fused to RSPO2

<400> 8

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Glu Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

245 250 255

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

340 345 350

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

355 360 365

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

370 375 380

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

385 390 395 400

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

405 410 415

Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr

420 425 430

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

435 440 445

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

450 455 460

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

465 470 475 480

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

485 490 495

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

500 505 510

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

515 520 525

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

530 535 540

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

545 550 555 560

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 9

<211> 215

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of a mutant 1R34-EEST/RA light chain

<400> 9

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Glu Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Ser Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 10

<211> 572

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-fusion construct mutant 1R34-EEST/RA heavy chain fused to RSPO2

<400> 10

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

245 250 255

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

340 345 350

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

355 360 365

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

370 375 380

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

385 390 395 400

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

405 410 415

Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr

420 425 430

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

435 440 445

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

450 455 460

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

465 470 475 480

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

485 490 495

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

500 505 510

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

515 520 525

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

530 535 540

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

545 550 555 560

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 11

<211> 215

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of 1R34-EEAT/EE light chain

<400> 11

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Glu Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Ala Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 12

<211> 572

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of 1R34-EEAT/EE heavy chain mutated with fusion construct fused to RSPO2

<400> 12

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Glu Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

245 250 255

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

340 345 350

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

355 360 365

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

370 375 380

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

385 390 395 400

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

405 410 415

Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr

420 425 430

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

435 440 445

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

450 455 460

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

465 470 475 480

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

485 490 495

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

500 505 510

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

515 520 525

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

530 535 540

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

545 550 555 560

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 13

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the heavy chain variable Domain of the 8M24 antibody in the laboratory

<400> 13

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Ser Asp Asn Thr Phe Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Gly Arg Asp Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 14

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-8M 24 antibody light chain variable Domain in the laboratory

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro His Leu Leu Val

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 15

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the heavy chain variable Domain of the 8M24 antibody in the laboratory (humanization 1)

<400> 15

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Phe Pro Arg Ser Asp Asn Thr Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Arg Asp Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 122

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the heavy chain variable Domain of the 8M24 antibody in the laboratory (humanization 2)

<400> 16

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Ser Asp Asn Thr Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Arg Asp Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the light chain variable Domain of the-8M 24 antibody in the laboratory (humanized 1)

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the light chain variable Domain of the-8M 24 antibody in the laboratory (humanized 2)

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-8M 24-EASE-RA light chain variable Domain in the laboratory

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 20

<211> 244

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of a fusion construct 8M24-EASE-RA heavy chain variable Domain fused to RSPO2

<400> 20

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Ser Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Glu

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser

<210> 21

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of 8M24-EASE-EE light chain in laboratory

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 22

<211> 574

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of fusion construct 8M24-EASE-EE heavy chain fused to RSPO2

<400> 22

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Glu Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Ser Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Glu

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

465 470 475 480

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 23

<211> 109

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-1R 34-DDNN/RA light chain variable Domain in the laboratory

<400> 23

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 24

<211> 242

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-fusion construct 1R34-DDNN/RA heavy chain variable Domain fused to RSPO2

<400> 24

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 25

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of 8M24-EASE light chain in laboratory

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 26

<211> 574

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of fusion construct 8M24-EASE heavy chain fused to RSPO2 (human IgG 1-N297G)

<400> 26

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Ser Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Glu

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

465 470 475 480

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 27

<211> 109

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-1R 34-EEST/EE light chain variable Domain in the laboratory

<400> 27

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Glu Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Lys Ser Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Thr Ser Leu Glu Arg Ile Gly Tyr Leu

85 90 95

Ser Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 28

<211> 242

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-fusion construct 1R34-EEST/EE heavy chain variable region fused to RSPO2

<400> 28

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Glu Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

165 170 175

Gly Gly Ser Thr Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Phe Ser Ser

210 215 220

Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 29

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of-modified R-vertebrate protein-2

<400> 29

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

100 105

<210> 30

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of-modified R-vertebrate protein-2

<400> 30

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Ala Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

100 105

<210> 31

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of-modified R-vertebrate protein-2

<400> 31

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

100 105

<210> 32

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of-modified R-vertebrate protein-2

<400> 32

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Glu Ser Lys Asp Glu Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu

100 105

<210> 33

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of 1R34-DDNN/RA heavy chain in laboratory

<400> 33

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Phe Ser Ser Arg Arg Trp Tyr Leu Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 34

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRH 1 in laboratory

<400> 34

Ser Tyr Ala Met Ser

1 5

<210> 35

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRH 2 in laboratory

<400> 35

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Glu Asp Ser Val Lys

1 5 10 15

Gly

<210> 36

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRH 3 in laboratory

<400> 36

Asp Phe Ser Ser Arg Arg Trp Tyr Leu Glu Tyr

1 5 10

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRL 1 in laboratory

<400> 37

Gln Gly Glu Ser Leu Arg Ser Tyr Tyr Ala Ser

1 5 10

<210> 38

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRL 2 in laboratory

<400> 38

Tyr Gly Lys Ser Asn Arg Pro Ser

1 5

<210> 39

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEST/EECDRL 3 in laboratory

<400> 39

Cys Thr Ser Leu Glu Arg Ile Gly Tyr Leu Ser Tyr Val

1 5 10

<210> 40

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-1R 34-EEAT/EECDRL 2 in laboratory

<400> 40

Tyr Gly Lys Ala Asn Arg Pro Ser

1 5

<210> 41

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRL1 in laboratory

<400> 41

Arg Ile Ser Glu Asn Ile Tyr Ser Asn Leu Ala

1 5 10

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRL2 in laboratory

<400> 42

Ala Ala Ile Asn Leu Ala Glu

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRL3 in laboratory

<400> 43

Gln His Phe Trp Gly Thr Pro Phe Thr

1 5

<210> 44

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRH1 in laboratory

<400> 44

Ala Tyr Gly Ile Asn

1 5

<210> 45

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRH2 in laboratory

<400> 45

Glu Ile Phe Pro Arg Ser Asp Ser Thr Phe Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 46

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of-8M 24-EASE CDRH3 in laboratory

<400> 46

Lys Gly Arg Glu Tyr Gly Thr Ser His Tyr Phe Asp Tyr

1 5 10

<210> 47

<211> 263

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 47

Met Arg Leu Gly Leu Cys Val Val Ala Leu Val Leu Ser Trp Thr His

1 5 10 15

Leu Thr Ile Ser Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile

20 25 30

Ser Ala Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser

35 40 45

Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu

50 55 60

Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro

65 70 75 80

Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Lys

85 90 95

Cys Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr

100 105 110

Lys Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr Pro Ala

115 120 125

Cys Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met Glu Cys Ser Ser

130 135 140

Pro Ala Gln Cys Glu Met Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser

145 150 155 160

Lys Lys Gln Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr

165 170 175

Arg Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp

180 185 190

Thr Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu

195 200 205

Gly Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn

210 215 220

Arg Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg

225 230 235 240

Arg Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr Val Gly Pro

245 250 255

Leu Thr Ser Ala Gly Pro Ala

260

<210> 48

<211> 243

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 48

Met Gln Phe Arg Leu Phe Ser Phe Ala Leu Ile Ile Leu Asn Cys Met

1 5 10 15

Asp Tyr Ser His Cys Gln Gly Asn Arg Trp Arg Arg Ser Lys Arg Ala

20 25 30

Ser Tyr Val Ser Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys

35 40 45

Asp Asn Gly Cys Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg

50 55 60

Arg Glu Gly Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser

65 70 75 80

Gly Tyr Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys

85 90 95

Arg Ile Glu Asn Cys Asp Ser Cys Phe Ser Lys Asp Phe Cys Thr Lys

100 105 110

Cys Lys Val Gly Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys

115 120 125

Pro Asp Gly Phe Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly

130 135 140

Cys Glu Val Gly His Trp Ser Glu Trp Gly Thr Cys Ser Arg Asn Asn

145 150 155 160

Arg Thr Cys Gly Phe Lys Trp Gly Leu Glu Thr Arg Thr Arg Gln Ile

165 170 175

Val Lys Lys Pro Val Lys Asp Thr Ile Leu Cys Pro Thr Ile Ala Glu

180 185 190

Ser Arg Arg Cys Lys Met Thr Met Arg His Cys Pro Gly Gly Lys Arg

195 200 205

Thr Pro Lys Ala Lys Glu Lys Arg Asn Lys Lys Lys Lys Arg Lys Leu

210 215 220

Ile Glu Arg Ala Gln Glu Gln His Ser Val Phe Leu Ala Thr Asp Arg

225 230 235 240

Ala Asn Gln

<210> 49

<211> 272

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 49

Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met

1 5 10 15

Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg

20 25 30

Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys

35 40 45

Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala

50 55 60

Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys

65 70 75 80

Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr

85 90 95

Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr

100 105 110

Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn

115 120 125

Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser

130 135 140

Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr

145 150 155 160

Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val

165 170 175

Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro

180 185 190

Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys

195 200 205

Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn

210 215 220

Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser

225 230 235 240

Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys

245 250 255

Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His

260 265 270

<210> 50

<211> 234

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 50

Met Arg Ala Pro Leu Cys Leu Leu Leu Leu Val Ala His Ala Val Asp

1 5 10 15

Met Leu Ala Leu Asn Arg Arg Lys Lys Gln Val Gly Thr Gly Leu Gly

20 25 30

Gly Asn Cys Thr Gly Cys Ile Ile Cys Ser Glu Glu Asn Gly Cys Ser

35 40 45

Thr Cys Gln Gln Arg Leu Phe Leu Phe Ile Arg Arg Glu Gly Ile Arg

50 55 60

Gln Tyr Gly Lys Cys Leu His Asp Cys Pro Pro Gly Tyr Phe Gly Ile

65 70 75 80

Arg Gly Gln Glu Val Asn Arg Cys Lys Lys Cys Gly Ala Thr Cys Glu

85 90 95

Ser Cys Phe Ser Gln Asp Phe Cys Ile Arg Cys Lys Arg Gln Phe Tyr

100 105 110

Leu Tyr Lys Gly Lys Cys Leu Pro Thr Cys Pro Pro Gly Thr Leu Ala

115 120 125

His Gln Asn Thr Arg Glu Cys Gln Gly Glu Cys Glu Leu Gly Pro Trp

130 135 140

Gly Gly Trp Ser Pro Cys Thr His Asn Gly Lys Thr Cys Gly Ser Ala

145 150 155 160

Trp Gly Leu Glu Ser Arg Val Arg Glu Ala Gly Arg Ala Gly His Glu

165 170 175

Glu Ala Ala Thr Cys Gln Val Leu Ser Glu Ser Arg Lys Cys Pro Ile

180 185 190

Gln Arg Pro Cys Pro Gly Glu Arg Ser Pro Gly Gln Lys Lys Gly Arg

195 200 205

Lys Asp Arg Arg Pro Arg Lys Asp Arg Lys Leu Asp Arg Arg Leu Asp

210 215 220

Val Arg Pro Arg Gln Pro Gly Leu Gln Pro

225 230

<210> 51

<211> 574

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory of fusion construct 8M24-EASE-RA heavy chain fused to RSPO2

<400> 51

Asn Pro Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys Asp Asn Gly Cys

1 5 10 15

Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg Arg Glu Gly Met

20 25 30

Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser Gly Tyr Tyr Gly

35 40 45

His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys Arg Ile Glu Asn

50 55 60

Cys Asp Ser Cys Arg Ser Lys Asp Ala Cys Thr Lys Cys Lys Val Gly

65 70 75 80

Phe Tyr Leu His Arg Gly Arg Cys Phe Asp Glu Cys Pro Asp Gly Phe

85 90 95

Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln

115 120 125

Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr Gly Ile Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile Phe Pro Arg

165 170 175

Ser Asp Ser Thr Phe Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile

180 185 190

Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu

195 200 205

Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Lys Gly Arg Glu

210 215 220

Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

245 250 255

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

290 295 300

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

305 310 315 320

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

325 330 335

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

340 345 350

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

355 360 365

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

370 375 380

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

385 390 395 400

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

405 410 415

Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val

420 425 430

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

435 440 445

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

450 455 460

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

465 470 475 480

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

485 490 495

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

500 505 510

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

515 520 525

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

530 535 540

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

545 550 555 560

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

565 570

<210> 52

<211> 169

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation in laboratory-modified human ASGR1

<400> 52

His His His His His His His His Gly Ser Gly Ser Gly Leu Asn Asp

1 5 10 15

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Ser Gly Ser Gly Cys

20 25 30

Pro Val Asn Trp Val Glu His Glu Arg Ser Cys Tyr Trp Phe Ser Arg

35 40 45

Ser Gly Lys Ala Trp Ala Asp Ala Asp Asn Tyr Cys Arg Leu Glu Asp

50 55 60

Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gln Lys Phe Val Gln

65 70 75 80

His His Ile Gly Pro Val Asn Thr Trp Met Gly Leu His Asp Gln Asn

85 90 95

Gly Pro Trp Lys Trp Val Asp Gly Thr Asp Tyr Glu Thr Gly Phe Lys

100 105 110

Asn Trp Arg Pro Glu Gln Pro Asp Asp Trp Tyr Gly His Gly Leu Gly

115 120 125

Gly Gly Glu Asp Cys Ala His Phe Thr Asp Asp Gly Arg Trp Asn Asp

130 135 140

Asp Val Cys Gln Arg Pro Tyr Arg Trp Val Cys Glu Thr Glu Leu Asp

145 150 155 160

Lys Ala Ser Gln Glu Pro Pro Leu Leu

165

<210> 53

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-8M 24L1 light chain in laboratory

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ile Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ile Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 54

<211> 236

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of the-8M 24H1 heavy chain in laboratory

<400> 54

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Phe Pro Arg Ser Asp Asn Thr Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Lys Gly Arg Asp Tyr Gly Thr Ser His Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Gly Ser Gly Ser Gly His His His His His His

225 230 235

<210> 55

<211> 4

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 55

Gly Gly Gly Gly

1

<210> 56

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 56

Gly Gly Gly Gly Gly

1 5

<210> 57

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 57

Gly Gly Gly Gly Gly Gly Gly

1 5

<210> 58

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 58

Gly Gly Gly Gly Ser

1 5

<210> 59

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 59

Gly Gly Gly Gly Gly Lys

1 5

<210> 60

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 60

Gly Gly Gly Gly Gly Lys Arg

1 5

<210> 61

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 61

Gly Gly Gly Lys Gly Gly Gly Gly

1 5

<210> 62

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> preparation of peptide linker in laboratory

<400> 62

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims (51)

1. A liver-specific Wnt ("wingless-related integration site" or "wingless and Int-1" or "wingless-Int") signaling enhancer molecule or a pharmaceutically acceptable salt thereof, comprising a first domain that specifically binds to one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43), and a second domain that specifically binds to asialoglycoprotein receptor 1 (ASGR 1), wherein:

(a) The first domain comprises a modified R-vertebrate polypeptide or fragment or variant thereof; and

(b) The second domain comprises a modified antibody or antigen-binding fragment thereof comprising: CDRH1, CDRH2 and CDRH3 sequences; CDRL1, CDRL2 and CDRL3 sequences.

2. The molecule of claim 1, wherein the R-spondin polypeptide or fragment or variant thereof comprises a furin domain 1 sequence and optionally a wild-type or mutant furin domain 2 sequence or fragment or variant thereof, wherein the R-spondin polypeptide or fragment or variant thereof has reduced binding to G protein-coupled receptor 4-6 (LGR 4-6) that is rich in leucine repeat sequences compared to a full-length wild-type R-spondin polypeptide.

3. The molecule of claim 1 or claim 2, wherein the R-vertebrate protein polypeptide or fragment or variant thereof comprises amino acid substitutions at positions corresponding to amino acids 105 and 109 of human R-vertebrate protein 2.

4. A molecule according to claim 3, wherein the amino acid substitutions are:

(a) F105R, F a or F105E; and

(b) F109A or F109E.

5. The molecule of claim 4, wherein the two amino acid substitutions are:

(a) f105R and F109A;

(b) f105A and F109A;

(c) f105E and F109A; or (b)

(d) f105E and F109E.

6. The molecule of any one of claims 1-5, wherein the combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences is selected from the group consisting of:

(a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively;

(b) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; or (b)

(c) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42), QHFWGTPFT (SEQ ID NO: 43), AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46), respectively.

7. The molecule of any one of claims 1-6, wherein the second domain comprises an antibody light chain polypeptide and an antibody heavy chain polypeptide, and wherein the first domain is fused to the N-terminus of the antibody heavy chain polypeptide, optionally via a linker.

8. The molecule of claim 7, wherein the linker moiety is a peptidyl linker sequence.

9. The molecule of claim 8, wherein the linker sequence comprises one or more amino acids selected from the group consisting of: glycine, asparagine, serine, threonine and alanine.

10. The molecule of any one of claims 1-9, comprising two antibody light chain polypeptides and two fusion polypeptides, wherein each fusion polypeptide comprises a modified R-spondin polypeptide or fragment or variant thereof fused to the N-terminus of the antibody heavy chain polypeptide via a linker moiety, optionally a peptidyl linker sequence, wherein the two fusion polypeptides are linked to each other and the two antibody light chain polypeptides are each linked to a different heavy chain polypeptide of the fusion polypeptides.

11. The molecule of claim 10, wherein:

(i) The two antibody light chain polypeptides comprise a sequence identical to SEQ ID NO: 1. 3, 5, 7, 9, 11, 14, 17, 18, 19, 21, 23, 25, or 27, or a variable region sequence thereof having at least 95% identity; and/or

(ii) The two antibody heavy chain polypeptides comprise a variable region sequence having at least 95% identity to the variable region sequence of any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 15, 16, 20, 22, 24, 26, 28, 33 or 51, or a variable region thereof.

12. The molecule of claim 10 or claim 11, wherein the two fusion polypeptides each comprise a sequence having at least 95% identity to any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 20, 22, 24, 26, 28, 33 or 51, or a variable region thereof.

13. The molecule of any one of claims 10-12, wherein the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 7, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID No. 8, or a variable region thereof.

14. The molecule of any one of claims 10-12, wherein the two antibody light chain polypeptides comprise a sequence having at least 95% identity to SEQ ID No. 25, or a variable region thereof; and each of the two fusion polypeptides comprises a sequence having at least 95% identity to SEQ ID NO. 26, or a variable region thereof.

15. A nucleic acid sequence encoding the antibody light chain polypeptide or fusion polypeptide of any one of claims 10-14, or a polypeptide having at least 95% identity to any one of SEQ ID NOs 1-33 or 51, or a variable region thereof.

16. The nucleic acid sequence of claim 15, wherein the nucleic acid sequence is DNA or mRNA.

17. A vector comprising the nucleic acid sequence of claim 16.

18. The vector of claim 17, wherein the vector is an expression vector comprising a promoter sequence operably linked to the nucleic acid sequence.

19. The vector of claim 17, wherein the vector is a virus comprising a promoter sequence operably linked to the nucleic acid sequence.

20. A host cell comprising the vector of any one of claims 17-19.

21. A method of producing the antibody light chain polypeptide or fusion polypeptide of any one of claims 10-14, comprising culturing the host cell of claim 20 under conditions wherein the polypeptide is expressed by the expression vector.

22. The method of claim 21, further comprising the step of isolating the resulting fusion polypeptide.

23. A pharmaceutical composition comprising:

a) The molecule of any one of claims 1-14, the nucleic acid sequence of any one of claims 15-16, the vector of any one of claims 17-19, or the host cell of claim 20; and

b) A pharmaceutically acceptable diluent, adjuvant or carrier.

24. A method for increasing Wnt ("wingless-related integration sites" or "wingless and Int-1" or "wingless-Int") signaling in liver tissue, comprising contacting the liver tissue with:

a) The molecule of any one of claims 1-14;

b) The nucleic acid of any one of claims 15-16;

c) The vector of any one of claims 17-19;

d) The host cell of claim 20; or (b)

e) The pharmaceutical composition of claim 23,

wherein the molecule binds to and sequesters one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43) in the liver tissue or increases endocytosis of one or more transmembrane E3 ubiquitin ligases selected from zinc and ring finger 3 (ZNRF 3) and ring finger protein 43 (RNF 43) in the liver tissue.

25. A method of treating or preventing a liver disease or liver disorder in a subject in need thereof, wherein the liver disease or liver disorder is associated with reduced Wnt ("wingless-related integration site" or "wingless and Int-1" or "wingless-Int") signaling, or would benefit from increased Wnt signaling, the method comprising administering to the subject an effective amount of

a) The molecule of any one of claims 1-14;

b) The nucleic acid of any one of claims 15-16;

c) The vector of any one of claims 17-19;

d) The host cell of claim 20; or (b)

e) The pharmaceutical composition of claim 23.

26. The method of claim 25, comprising administering to the subject an effective amount of the molecule of any one of claims 1-14.

27. The method of claim 25, comprising administering to the subject an effective amount of the nucleic acid of any one of claims 15-16.

28. The method of claim 25, comprising administering to the subject an effective amount of the vector of any one of claims 17-19.

29. The method of claim 25, comprising administering to the subject an effective amount of the host cell of claim 20.

30. The method of claim 25, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 23.

31. The method of any one of claims 25-30, wherein the liver disease or disorder is selected from the group consisting of: acute liver failure of various causes, drug-induced acute liver failure, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in patients with cirrhosis, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, alcoholic liver disease, chronic liver failure of various causes, decompensated liver failure, late decompensated liver failure, cirrhosis, liver fibrosis of various causes, portal hypertension, chronic liver insufficiency of various causes, end-stage liver disease (ESLD), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, acute Alcoholic Hepatitis (AAH), chronic alcoholic hepatitis, chronic alcoholism-induced liver injury Alcoholic Liver Disease (ALD) (also known as alcohol-related liver disease (ARLD)), hepatitis C virus-induced liver disease (HCV), hepatitis B virus-induced liver disease (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver disease (HAV) and hepatitis B virus-induced liver disease (HDV)), primary biliary cirrhosis, autoimmune hepatitis, surgery with symptoms of liver disease, liver injury, venous Occlusive Disease (VOD), sinus Obstructive Syndrome (SOS), primary cholangitis (PBC), primary Sclerosing Cholangitis (PSC), liver transplantation, liver surgery and "small volume" syndrome in transplantation, congenital liver diseases and disorders, liver failure due to APAP (acetaminophen) overdose, and any other liver disease or disorder caused by genetic disease, degeneration, aging, drugs or injury.

32. The method of claim 31, wherein the liver disease or liver condition is selected from acute alcoholic hepatitis, acute liver failure, chronic acute liver failure (ACLF), acute liver decompensation, ascites due to cirrhosis, hyponatremia in cirrhosis patients, hepatorenal syndrome-acute kidney injury (HRS-AKI), hepatic encephalopathy, or cirrhosis.

33. The method of any one of claims 25-32, wherein the molecule, nucleic acid, vector, host cell, or pharmaceutical composition is administered parenterally, orally, intramuscularly, or topically to the liver, optionally intravenously to the liver.

34. The method of any one of claims 25-33, wherein the subject is a mammal, optionally a human.

35. A method of producing, culturing or maintaining a liver cell, liver tissue or liver organoid comprising contacting the cell, tissue or organoid with:

a) The molecule of any one of claims 1-14;

b) The nucleic acid of any one of claims 15-16;

c) The vector of any one of claims 17-19;

d) The host cell of claim 20; or (b)

e) The pharmaceutical composition of claim 23.

36. The method of claim 35 for maintaining viability of ex vivo liver tissue comprising optionally contacting liver tissue obtained from a donor by perfusing the ex vivo liver tissue with a composition comprising a molecule of any of claims 1-14.

37. The method of claim 35 for maintaining viability of liver tissue comprising contacting donor liver tissue in vivo with a composition comprising the molecule of any one of claims 1-14.

38. A method according to claim 35 for producing or maintaining a liver organoid culture, comprising optionally contacting the liver organoid culture by culturing the liver organoid culture in a medium comprising a molecule according to any of claims 1-14.

39. A polypeptide comprising a sequence having at least 95% identity to any one of SEQ ID NOs 1-28, 33 or 51, or a variable region thereof, optionally wherein said polypeptide comprises one of the following sets of CDRs:

(a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35) and DFSSRRWYLEY (SEQ ID NO: 36);

(b) QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39);

(c) QGESLRSYYAS (SEQ ID NO: 37), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39)

(d) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42) and QHFWGTPFT (SEQ ID NO: 43); or (b)

(e) AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46).

40. The polypeptide of claim 39, wherein the polypeptide is a fusion protein comprising a modified R-vertebrate polypeptide, or fragment or variant thereof, fused to the N-terminus of an antibody heavy chain polypeptide via a linker moiety, optionally a peptidyl linker sequence, wherein the R-vertebrate polypeptide, or fragment or variant thereof, comprises two amino acid substitutions at positions corresponding to amino acids 105 and 109 of human R-vertebrate 2.

41. The polypeptide of claim 40, wherein the two amino acid substitutions are:

(a) F105R, F a or F105E; and

(b) F109A or F109E.

42. The polypeptide of claim 41, wherein the two amino acid substitutions are:

(a) f105R and F109A;

(b) f105A and F109A;

(c) f105E and F109A; or (b)

(d) f105E and F109E.

43. The polypeptide of any one of claims 40-42, wherein the antibody heavy chain polypeptide comprises a combination of CDRH1, CDRH2, and CDRH3 sequences selected from:

(a) SYAMS (SEQ ID NO: 34), AISGSGGSTYYEDSVKG (SEQ ID NO: 35), DFSSRRWYLEY (SEQ ID NO: 36), respectively; or (b)

(b) RISENIYSNLA (SEQ ID NO: 41), AAINLAE (SEQ ID NO: 42), QHFWGTPFT (SEQ ID NO: 43), respectively.

44. The polypeptide of claim 39, wherein the polypeptide comprises a modified antibody light chain polypeptide.

45. The polypeptide of claim 44, wherein the modified antibody light chain polypeptide comprises a combination of CDRL1, CDRL2, and CDRL3 sequences selected from the group consisting of:

(a) QGESLRSYYAS (SEQ ID NO: 37), YGKSNRPS (SEQ ID NO: 38) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively;

(b) QGESLRSYYAS (SEQ ID NO: 38), YGKANRPS (SEQ ID NO: 40) and CTSLERIGYLSYV (SEQ ID NO: 39), respectively; or (b)

(c) AYGIN (SEQ ID NO: 44), EIFPRSDSTFYNEKFKG (SEQ ID NO: 45) and KGREYGTSHYFDY (SEQ ID NO: 46), respectively.

46. The molecule of any one of claims 1-14, the nucleic acid sequence of claim 15 or claim 16, the vector of any one of claims 17-19, the host cell of claim 20, the method of claim 21 or claim 22, the pharmaceutical composition of claim 23, or the method of any one of claims 24-38, wherein the molecule is selected from Wnt signaling enhancing molecules known as EEST-EE, EEST-RA, EEAT-EE, 8m24 EASE-EE, or 8m24 EASE-RA.

47. The molecule, nucleic acid sequence, vector, host cell, method, pharmaceutical composition or method of claim 46, wherein said molecule is EEST-EE.

48. The molecule, nucleic acid sequence, vector, host cell, method, pharmaceutical composition or method of claim 46, wherein said molecule is EEST-RA.

49. The molecule, nucleic acid sequence, vector, host cell, method, pharmaceutical composition or method of claim 46, wherein said molecule is EEAT-EE.

50. The molecule, nucleic acid sequence, vector, host cell, method, pharmaceutical composition or method of claim 46, wherein said molecule is 8m24 EASE-EE.

51. The molecule, nucleic acid sequence, vector, host cell, method, pharmaceutical composition or method of claim 46, wherein said molecule is 8m24 EASE-RA.