FGF21 mutant polypeptide

Abstract

The present disclosure provides a complex comprising: (a) a first monomer comprising an Fc domain fused to two FGF21 domains, which may be the same or different, comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; and (b) a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region.

Description

[Technical Field]

[0001] The present disclosure relates to polypeptides and complexes comprising multiple mutant FGF21 domains.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 405,758, filed September 12, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing, created on September 11, 2023, filed concurrently herewith and hereinafter identified as a 100,787 byte XML file named "58224_Seqlisting.xml", is incorporated by reference in its entirety. [Background technology]

[0004] Fibroblast growth factor 21 (FGF21) is an endocrine hormone that acts in the liver, pancreas, muscle, and adipose tissue to regulate lipid, carbohydrate, and protein metabolism. Human FGF21 also functions as a paracrine hormone, playing an important role in protecting cells from stress. There is increasing evidence that metabolic disease states such as type 2 diabetes (T2D) and NASH are associated with resistance to endogenous FGF21, i.e., FGF21 is less effective in maintaining insulin sensitivity and protecting hepatocytes from multiple cellular stresses associated with high intracellular lipids. Although FGF21 levels appear to be higher in patients with metabolic diseases, this does not appear to be associated with improved FGF21 signaling. This may be related to lower expression levels of FGF21 receptors. Genetic loss of function mutations in one or both of the FGF21 receptors, beta-Klotho (KLB) and FGFR1c, are associated with resistance to endogenous FGF21, including increased liver fibrogenesis. Extremely rare cases with heterozygous LOF mutations in both KLB and FGFR1c are associated with severe resistance to FGF21 and insulin, early-onset T2D, and hepatic steatosis. Stone et al. Digenic variants in the FGF21 signaling pathway associated with severe insulin resistance and pseudoacromegaly. J Endocr Soc 4, bvaa 138 (2020).

[0005] While the properties of FGF21 make FGF21 agonism a compelling therapeutic mechanism, native FGF21 is limited by its short half-life in the bloodstream, and several studies have shown that the beneficial effects associated with FGF21 administration diminish with longer treatment periods. For example, evidence using the single-chain FGF21 analog pegbelfermin shows that pharmacological responses (adiponectin induction) and markers of efficacy decline with longer treatment periods (e.g., 24 weeks) compared with biological responses achieved early in the treatment period (e.g., 8–12 weeks). (Sanyal et al., AASLD The Liver Meeting 2021, Poster LP-40) Summary of the Invention

[0006] The present disclosure provides a complex comprising: (a) a first monomer comprising an Fc domain fused to two FGF21 domains, which may be the same or different, comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; and (b) a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region. Optionally, the (a) first monomer comprises the amino acid sequence of SEQ ID NO:4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 fused to the C-terminus of the Fc domain. In other aspects, the (a) first monomer comprises one or more FGF21 domains of the amino acid sequence of SEQ ID NO:2 fused to the N-terminus of the Fc domain (optionally via a linker), followed by another FGF21 domain comprising the amino acid sequence of SEQ ID NO:2. In various aspects, the (b) second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain. Alternatively, the (b) second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 4 fused to the C-terminus of the Fc domain. In some aspects, the (a) first monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain, and optionally, the (b) second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain. The present disclosure also provides a conjugate comprising two monomers comprising the amino acid sequence of SEQ ID NO: 18, two monomers comprising the amino acid sequence of SEQ ID NO: 19, or two monomers comprising the amino acid sequence of SEQ ID NO: 20.

[0007] Also provided is a complex comprising: (a) a first monomer comprising an Fc domain fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:4; and (b) a second monomer comprising an Fc domain fused at its N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2.

[0008] The present disclosure also provides peptides comprising an Fc domain fused at its N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:4 and fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2, as well as peptides comprising the amino acid sequence of SEQ ID NO:4 fused to the amino acid sequence of SEQ ID NO:2 via a linker (e.g., a linker comprising the amino acid sequence of SEQ ID NO:9 and / or the amino acid sequence of SEQ ID NO:10).

[0009] The foregoing summary is not intended to define all aspects of the invention; additional aspects are described in other sections, such as the detailed description. It should be understood that the entire document is intended to relate as a unified disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. Additionally, the present invention includes, as additional aspects, all aspects of the invention that are in no way narrower in scope than the variations specifically mentioned above.

[0010] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms unless expressly stated otherwise. When an embodiment of the invention is described as "comprising" a feature, it is also contemplated that the embodiment "consisting of" or "consisting essentially of" the feature. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the disclosure and do not limit the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Other than in the operational examples, or where otherwise indicated, all numbers expressing quantities should be understood to be modified in all instances by the term "about" as that term would be interpreted by one of ordinary skill in the relevant art. With respect to aspects of the invention described or claimed with "a," "the," or "an," these terms should be understood to mean "one or more," unless the context clearly dictates a more limited meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated.

[0011] Also, when describing a range of values, it should be understood that the present disclosure contemplates each individual value found within the range. In any of the ranges described herein, the endpoints of the range are included. However, the description also contemplates the same range but excluding the lower and / or higher endpoints. When the term "about" is used, it means plus or minus 5%, 10%, or more of the recited number. The actual variation intended can be determined from the context.

[0012] Additional features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are contemplated as aspects of the present invention. Similarly, features of the present invention described herein may be recombined into additional embodiments that are also contemplated as aspects of the present invention, regardless of whether a combination of features is identified as an aspect of the present invention. It should be understood that the entire document is intended to relate as a unified disclosure, and that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. Furthermore, only those limitations described herein as essential to the present invention should be considered as such; variations of the present invention lacking limitations not described herein as essential are contemplated as aspects of the present invention. The use of section headings, if any, is merely for convenience of reading, and it should be understood that all combinations of features described herein are contemplated. [Brief explanation of the drawings]

[0013] [Figures 1A-1O] 1 is a schematic diagram of representative mutant FGF21-based complexes and peptides of the present disclosure. The lines and rectangles represent the Fc domain, and the ovals represent FGF21 domains. The oval with the elongated end represents the FGF21 domain containing the free beta-Klotho binding region. [Figure 2] Various amino acid sequences are provided according to the present disclosure. Amino acid sequences are indicated N-to-C. Plain text corresponds to the Fc domain region. Underlined text corresponds to the linker sequence. Italicized text represents the FGF21 domain. [Figure 3]1 is a graph showing the results of a cell-based potency assay performed in HEK293 cells overexpressing human KLB and FGFR1c, measuring Elk1 in response to FGF21 analog signaling via a luciferase reporter system. EFX are shown as circles, FGF21 containing the L98R, P171G, and A180E modifications relative to mature human FGF21 are shown as squares, and the 3 / 4EFX described herein are shown as triangles. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure provides polypeptide complexes and peptides comprising at least two mutant FGF21 domains, and in some instances at least three (e.g., four) mutant FGF21 domains. The mutant FGF21 domains contain three modifications introduced into the native FGF21 sequence: L125R, P198G, and A207E (corresponding to L98R, P171G, and A180E in mature human FGF21). These modifications 1) reduce susceptibility to proteolysis in vivo, 2) increase affinity for beta-klotho, and 3) reduce the tendency to aggregate (Hecht et al., PLoS One 2012;7(11):e49345; Stanislaus et al., Endocrinology. 2017;158(5):1314-1327). For clarity, the invention described herein does not encompass effluxifermin (EFX). EFX is a long-acting fibroblast growth factor 21 (FGF21) analogue, containing a monomer that is a fusion protein containing a human immunoglobulin IgG1 Fc fragment fused to a mutant FGF21 domain containing modifications at positions L98R, P171G, and A180E (relative to mature human FGF21) at 92.1 kDa. Each EFX molecule is a homodimer containing a dimerized Fc domain, with each Fc domain fused to a single chain of modified FGF21 polypeptide. EFX has eight disulfide bonds, six intrachain bonds, and two interchain bonds. Two of the intrachain disulfide bonds are in the FGF21 polypeptide between Cys318 and Cys336, one for each monomer. EFX contains the amino acid sequence set forth in SEQ ID NO: 1. EFX (and the various components that make up the molecule) are further described in U.S. Pat. Nos. 8,034,770, 8,410,051, 8,642,546, 8,361,963, 9,273,106, 10,011,642, 8,188,040, 8,835,385, 8,795,985, 8,618,053, and 11,072,640, or International Patent Publication Nos. WO 2009 / 149171 and WO 2010 / 129503, the disclosures of which are incorporated herein by reference in their entireties.

[0015] In various aspects, the present disclosure provides a complex comprising: (a) a first monomer comprising an Fc domain fused to two or more FGF21 domains (e.g., two FGF21 domains), which may be the same or different, comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66); and (b) a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66), wherein at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region. The first and second monomers associate via the Fc domains to form the complex. The configuration of the first monomer is such that an FGF21 domain is fused to the N-terminus of the Fc domain and another FGF21 domain is fused to the C-terminus of the Fc domain (i.e., the Fc domain is adjacent to two FGF21 domains, which may be the same or different, and is selected from FGF21 domains comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NOs: 63-66). The configuration of the complex is such that at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region. The "beta-Klotho binding region" of an FGF21 domain refers to the portion of the domain that binds to beta-Klotho, which portion is located at the end of the domain comprising the amino acid sequence QGRSPSYES (SEQ ID NO: 3) found in SEQ ID NO: 2 (or SEQ ID NO: 63 or 65) or the amino acid sequence SEYSPSRGQ (SEQ ID NO: 6) found in SEQ ID NO: 4 (or SEQ ID NO: 64 or 66). A "free beta-Klotho binding region" refers to a configuration in which the beta-Klotho binding region of an FGF21 domain is exposed and available for binding to beta-Klotho, i.e., the end of the FGF21 domain containing the beta-Klotho region is not fused to an Fc domain or another FGF21 domain. In various embodiments, the first monomer comprises one or more FGF21 domains comprising the amino acid sequence of SEQ ID NO:2 fused (optionally via a linker) to the N-terminus of an Fc domain, followed by another FGF21 domain comprising the amino acid sequence of SEQ ID NO:2.Thus, the present disclosure contemplates a structure in which two, three, or four FGF21 domains of the amino acid sequence of SEQ ID NO: 2 are present in tandem at the N-terminus and fused to the N-terminus of an Fc domain, followed by one or more (e.g., one) FGF21 domains comprising SEQ ID NO: 2. Fusion to the Fc domain can be via a linker, although this is optional.

[0016] The present disclosure contemplates embodiments in which an FGF21 domain comprises an amino acid sequence at least about 90% identical (e.g., at least 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) to SEQ ID NO: 2 or 4. Indeed, at least one naturally occurring isoform of FGF21 has been reported, in which proline at amino acid position 146 in the mature FGF21 protein is substituted with leucine. The two isoforms are often referred to as the "P-type" (having proline at position 146) and the "L-type" (having leucine at position 146). In this regard, the present disclosure includes an FGF21 domain comprising the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64, where X is proline (e.g., SEQ ID NO: 2 or 4, respectively) or leucine (e.g., SEQ ID NOs: 65 and 66, respectively). In cases where an FGF21 domain is less than 100% identical to SEQ ID NO: 2, 4, or 63-66, the FGF21 domain retains at least a glutamic acid at position 180 relative to mature human FGF21, and optionally retains an arginine at position 98 and a glycine at position 171 as set forth in SEQ ID NOs: 2, 4, and 63-66. The disclosure herein regarding SEQ ID NOs: 2 and 4 also applies to an FGF21 domain comprising the amino acid sequence of any one of SEQ ID NOs: 63-66. For example, the disclosure provides a complex comprising: (a) a first monomer comprising an Fc domain fused to two FGF21 domains, which may be the same or different, comprising the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64; and (b) a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64, wherein at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region.

[0017] For example, the first monomer optionally comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain. In various aspects, the second monomer comprises (i) the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain, (ii) the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the N-terminus of the Fc domain, (iii) the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the C-terminus of the Fc domain, or (iv) the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain. In various aspects, the second monomer comprises the amino acid sequence of SEQ ID NO: 4 (or SEQ ID NO: 64 or 66) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 63 or 65) fused to the C-terminus of the Fc domain.

[0018] One or more FGF21 domains (two or more, three or more, etc.) may be fused to the N-terminus and / or C-terminus of the Fc domain (optionally via a linker), and the FGF21 domains may comprise the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 (or any other FGF21 domain sequence mentioned herein). For example, the present disclosure contemplates complexes in which the first monomer and / or the second monomer comprise two or more FGF21 domains comprising SEQ ID NO: 2 and / or SEQ ID NO: 4 fused to the N-terminus of the Fc domain (e.g., a domain comprising SEQ ID NO: 2 fused in tandem with a domain comprising SEQ ID NO: 2, a domain comprising SEQ ID NO: 4 fused in tandem with a domain comprising SEQ ID NO: 4, or a combination thereof).

[0019] Alternatively, the first monomer comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the C-terminus of the Fc domain. Optionally, (i) the second monomer comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain, or (ii) the second monomer comprises the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain. In various aspects, the second monomer comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the C-terminus of the Fc domain.

[0020] In various aspects, the first monomer comprises the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain. Optionally, (i) the second monomer comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain, or (ii) the second monomer comprises the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain. In some embodiments, the second monomer comprises the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain.

[0021] The present disclosure provides a conjugate comprising two monomers comprising the amino acid sequence of SEQ ID NO: 18, a conjugate comprising two monomers comprising the amino acid sequence of SEQ ID NO: 19, and a conjugate comprising two monomers comprising the amino acid sequence of SEQ ID NO: 20. In various aspects of the present disclosure, the monomers comprise an amino acid sequence at least about 90% identical (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) to SEQ ID NO: 18, 19, or 20. For example, the present disclosure contemplates a monomer in which one or more of the FGF21 domains is the L-isoform of an FGF domain within SEQ ID NOs: 18-20.

[0022] The FGF21 domain is fused to the Fc domain. An "Fc region" or "Fc domain" is a polypeptide comprising an antibody constant region, e.g., at least the second and third constant region domains (CH2 and CH3 regions) of an antibody, and all or part of the hinge. Thus, in various aspects of the present disclosure, the Fc domain can refer to the last two antibody constant region domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, and the last three antibody constant region domains of IgE and IgM, and the hinge N-terminal to these domains. In IgA and IgM, the Fc domain can include the J chain. In IgG, the Fc domain can include immunoglobulin domains Cy2 and Cy3 and the lower hinge region between Cγ1 and Cγ2. In some aspects of the present disclosure, the Fc domain comprises the CH2 and CH3 domains with a truncated CH1 domain. For illustrative purposes only, the human IgG Fc region is typically defined as including residues E216, C226, or P230 and extending to the C-terminus (numbering is according to the EU index in Kabat), with CH1 generally corresponding to positions 118-220 according to the EU index in Kabat, CH2 generally corresponding to positions 237-340 according to the EU index in Kabat, and CH3 generally corresponding to positions 341-447 according to the EU index in Kabat, all in the context of IgG. The Fc domain is preferably derived from an IgG Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain. In exemplary embodiments of the present disclosure, the Fc domain is an IgG1 Fc domain. A representative Fc domain of the present disclosure is provided as SEQ ID NO: 7. The Fc domain optionally comprises an amino acid sequence at least about 90% identical (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) identical to SEQ ID NO: 7. In any of the embodiments described herein, one or both of the Fc domains may comprise the amino acid sequence of SEQ ID NO:7.The Fc domain may be a variant of SEQ ID NO: 7, optionally including modifications that further extend the half-life of the molecule. The Fc domain of the present disclosure may alternatively comprise the amino acid sequences of SEQ ID NOs: 76-83. The complex may be a dimer or multimer. The present disclosure contemplates complexes in which monomers of the present disclosure associate to form an antibody-like dimeric Fc domain structure. To illustrate, the present disclosure contemplates two FGF21 domains, each fused to an Fc domain, and the Fc domains of two monomers associate to form an antibody-like structure (or complex), with an FGF21 binding domain fused, for example, to each CH2-CH3 arm, providing four FGF21 domains available for binding to beta-Klotho or FGFR1c.

[0023] In any of the embodiments of the disclosure described herein, the FGF21 domain and the Fc domain are optionally fused via a linker. A "linker" is an amino acid sequence that connects multiple peptide domains, such as an FGF21 domain and an Fc domain, or two FGF21 domains. Any suitable linker can be used so long as the FGF21 domain retains biological activity (e.g., retains the ability to bind to beta-Klotho and / or FGFR1c). The linker can be of any length, for example, 1 to 60 amino acids, such as 1 to 5, 1 to 10, 2 to 18, 4 to 30, 5 to 15, or 10 to 25 amino acids. Suitable linkers include, but are not limited to, for example, (GS)n, (GGGGS)n (also referred to as "G4S"; SEQ ID NO: 67), (SGGGG)n (SEQ ID NO: 68), (GSSSS)n (SEQ ID NO: 69), and (GGGS)n (also referred to as "G3S"; SEQ ID NO: 70), where n is an integer of at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Examples of linkers include, but are not limited to, GGGGSGGGGS (SEQ ID NO: 71), GGGGSGGGGSGGGGS (SEQ ID NO: 72), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 73), and GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 74). In any of the complexes or peptides described herein, one or more FGF21 domains may be fused to the Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or both. Although the above description relates to the use of a linker flanking the FGF21 domain, or the FGF21 domain and the Fc domain, the linker sequence can also be added to the N-terminus and / or C-terminus of the monomer.

[0024] Also provided herein are peptides comprising an Fc domain (e.g., an Fc domain comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NOs:76-83) fused at the N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) and fused at the C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2. In various embodiments, the FGF21 domain comprising SEQ ID NO:4 is fused to the Fc domain via a linker such as any of the linkers described herein (e.g., a linker comprising the amino acid sequence of SEQ ID NO:10), and the FGF21 domain comprising SEQ ID NO:2 (or SEQ ID NO:63 or 65) is fused to the Fc domain via a linker such as any of the linkers described herein (e.g., a linker comprising the amino acid sequence of SEQ ID NO:9). For example, the present disclosure provides a peptide comprising the amino acid sequence of SEQ ID NO:21. In various aspects of the disclosure, the disclosure provides a peptide comprising an amino acid sequence at least about 90% identical (e.g., at least 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) to SEQ ID NO: 21. For example, the disclosure contemplates a monomer in which one or more of the FGF21 domains is the L-isoform of the FGF domain in SEQ ID NO: 21. The disclosure further provides a complex comprising the peptide and a second Fc domain.

[0025] The present disclosure further provides a complex comprising: (a) a first monomer comprising an Fc domain (e.g., an Fc domain comprising the amino acid sequence of SEQ ID NO: 7 or any of SEQ ID NOs: 76-83) fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO: 4 (or SEQ ID NO: 64 or 66); and (b) a second monomer comprising an Fc domain (e.g., an Fc domain comprising the amino acid sequence of SEQ ID NO: 7 or any of SEQ ID NOs: 76-83) fused at its N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 63 or 65). In this regard, the FGF21 domain comprising SEQ ID NO: 4 is optionally fused to the Fc domain of the first monomer via a linker, such as any of the linkers described herein (e.g., a linker comprising the amino acid sequence of SEQ ID NO: 10). The FGF21 domain comprising SEQ ID NO: 2 (or SEQ ID NO: 63 or 65) is optionally fused to the Fc domain of the second monomer via a linker, such as any of the linkers described herein (e.g., a linker comprising the amino acid sequence of SEQ ID NO: 9). The present disclosure provides a conjugate wherein a first monomer comprises the amino acid sequence of SEQ ID NO: 27 and a second monomer comprises the amino acid sequence of SEQ ID NO: 28. In various aspects of the present disclosure, the present disclosure provides a monomer comprising an amino acid sequence at least about 90% identical (e.g., at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) identical to SEQ ID NO: 27 or 28. For example, the present disclosure contemplates a monomer wherein one or more of the FGF21 domains is the L-isoform of the FGF domain within SEQ ID NO: 21.

[0026] Also provided herein are peptides comprising the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) via a linker such as any one or more of the linkers described herein (e.g., a linker comprising the amino acid sequence of SEQ ID NO:9 and / or a linker comprising the amino acid sequence of SEQ ID NO:10).

[0027] The present disclosure further provides nucleic acids (i.e., polynucleotides) comprising a nucleic acid sequence encoding any of the domains, monomers, and peptides described herein. The present disclosure further provides expression vectors comprising the nucleic acids described herein. For example, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a first monomer of any of the complexes described above, as well as an expression vector comprising the nucleic acid. The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding a second monomer of any of the complexes described herein, as well as an expression vector comprising the nucleic acid. Additionally, the present disclosure provides a system or composition comprising an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a first monomer of any of the complexes described herein, and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a second monomer of any of the complexes described herein.In this regard, the present disclosure provides a method for producing a FGF21 antibody comprising: (a) a first monomer comprising an Fc domain fused to two FGF21 domains, which may be the same or different, comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66) (e.g., the first monomer comprises the amino acid sequence of SEQ ID NO:4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain; or the first monomer comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:64 or 66) fused to the C-terminus of the Fc domain; or the first monomer comprises the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) fused to the C-terminus of the Fc domain). ), or a first monomer comprises an Fc domain fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65), or a first monomer comprises an Fc domain fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66); and (b) an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66) (e.g., fused at the N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 (or SEQ ID NO:63 or 65) or SEQ ID NO:4 (or SEQ ID NO:64 or 66)).

[0028] The expression vector provides expression in vitro and / or in vivo (e.g., in a suitable host cell or organism). Many expression vectors are commercially available. The nucleic acid molecule can be provided, for example, in a plasmid, cosmid, YAC, or viral vector. Suitable viral vectors include, for example, retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), picornaviruses, alphaviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), lentiviruses, and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Vector components may include an origin of replication, one or more marker genes, a multiple cloning site containing recognition sequences for restriction endonucleases, an enhancer element, a promoter, a transcription termination sequence, etc. Indeed, the nucleic acids of the present disclosure may be operably linked to one or more regulatory elements, such as a promoter, an enhancer, and / or a terminator.

[0029] The present disclosure further provides a host cell comprising a nucleic acid or expression vector described herein. A "host cell" refers to a cell (e.g., a prokaryotic or eukaryotic cell) into which exogenous nucleic acid has been introduced, including the progeny of such a cell. A host cell can be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In various aspects, the cell is a eukaryotic cell such as a mammalian cell (e.g., a human cell; or a cell from a non-human primate such as an ape, chimpanzee, monkey, or orangutan; or a cell from a companion animal such as a dog or cat; or a cell from a livestock animal such as a horse, cow, pig, sheep, or goat; or a cell from another mammalian species, including, but not limited to, a mouse, rat, guinea pig, rabbit, hamster, bird (e.g., chicken, duck, goose, quail, or pheasant), etc.). An exemplary bacterial host cell is E. coli. Examples of human cells include, but are not limited to, PER.C6 cells (e.g., as described in International Patent Publication No. WO 01 / 38362), MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HEK-293 cells (ATCC CRL-1573), HeLa cells (ATCC CCL2), and fetal rhesus lung cells (ATCC CL-160). Non-human primate cells include Vero cells (ATCC CCL81), COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). An example of a canine cell is MDCK cells (ATCC CCL-34). An example of a rodent cell is a hamster cell, such as BHK21-F, HKCC, or Chinese hamster ovary (CHO) cells. Examples of insect cells include, but are not limited to, SF9 cells (ATCC CRL-1711), Sf21 cells (IPLB-Sf21), MG1 cells (BTI-TN-MG1), and High Five™ cells (BTI-TN-5B1-4).

[0030] Also contemplated are methods for producing the FGF21 domain-containing complexes described herein, comprising culturing host cells containing the expression vectors or systems described herein and harvesting the complexes. Similarly, the present disclosure provides methods for producing the FGF21 domain-containing peptides described herein, comprising culturing host cells containing the expression vectors or systems described herein and harvesting the peptides. Culture conditions and methods for producing recombinant proteins are known in the art. Similarly, protein purification methods are known in the art and are utilized herein for recovering recombinant proteins from cell culture media. In some embodiments, methods for protein purification include filtration, affinity column chromatography, cation exchange chromatography, anion exchange chromatography, and concentration. Optionally, the methods include formulating the recovered product.

[0031] The present disclosure further provides pharmaceutical compositions comprising the conjugates or peptides described herein. Such pharmaceutical compositions can include a therapeutically effective amount of the conjugates or peptides described herein in admixture with a pharmaceutically or physiologically acceptable formulation selected for compatibility with the mode of administration. Also provided are pharmaceutical compositions comprising nucleic acids (e.g., mRNA) encoding any one or more of the monomers or peptides described herein.

[0032] Pharmaceutical compositions optionally include ingredients to improve, sustain, or maintain, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition.Suitable formulation ingredients include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, trehalose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium carbonate), and the like. sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thiomersal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 or polysorbate 80, triton, tromethamine, lecithin, cholesterol, or tyloxapearl), stability enhancers (such as sucrose or sorbitol), tonicity enhancers (such as alkali metal halides including sodium or potassium chloride or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (e.g., Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions thereof, which are incorporated herein by reference for any purpose.

[0033] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, or supplemented with other materials common to compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer (e.g., about pH 7.0-8.5) or acetate buffer (e.g., about pH 4.0-5.5). In various aspects of the present disclosure, FGF21 domain-containing complexes or peptides can be prepared for storage by mixing a selected composition having the desired purity with optional compounding agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Furthermore, FGF21 polypeptide variant products can be formulated as lyophilizates.

[0034] The conjugates or peptides disclosed herein can be used to treat, ameliorate, prevent, or reverse several diseases, disorders, or conditions, including, but not limited to, genetic disorders, metabolic disorders, and fibrotic disorders. In various aspects, the present disclosure provides methods of treating a disease or disorder, the methods comprising administering a pharmaceutical composition comprising a conjugate or peptide described herein to a subject (e.g., a human) in need thereof. The disease or disorder can be non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated fatty liver disease (MASLD), metabolic dysfunction-associated alcoholic liver disease (MetALD), fatty liver disease of unknown etiology (SLD), fatty liver, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), or alcoholic fatty liver disease (AFLD), diabetes (e.g., type 2 diabetes), obesity, hypertriglyceridemia, dyslipidemia, cardiovascular disease (such as atherosclerosis), or aging. In various aspects, the present disclosure provides methods for reversing liver cirrhosis or reducing fibrosis, such as liver fibrosis associated with NASH, NAFLD, MASH, MASLD, MetALD, ASH, ALD, or AFLD, as well as liver fibrosis associated with non-metabolic diseases. Optionally, after treatment, a subject's fibrosis score based on the NASH Clinical Research Network (CRN) histological scoring system (Kleiner D et al., 2005 Hepatology 41, 1313) may regress from F4 (cirrhosis) to F3 (advanced fibrosis) or below. Fibrosis in other organs or tissues (e.g., lung, pancreas, or heart) is also contemplated. The present disclosure also contemplates methods for reducing or minimizing cravings or addictions (such as alcohol-related or other food-related) by administering a pharmaceutical composition comprising a conjugate or peptide described herein. In exemplary embodiments, addiction encompasses persistent, compulsive dependence on a behavior or substance, such as alcohol, drugs, or nicotine. In an exemplary embodiment, craving encompasses a strong, urgent, or abnormal desire for a particular substance or activity, such as sugar.

[0035] The present disclosure also provides methods for normalizing liver fat content, lowering blood glucose levels, increasing insulin sensitivity, and / or lowering uric acid levels by administering a pharmaceutical composition comprising a conjugate or peptide disclosed herein to a subject in need thereof. In exemplary embodiments, normalizing liver fat content refers to lowering liver fat content (e.g., absolute liver fat content), preferably to that of a typical, healthy, disease-free subject (i.e., a subject not suffering from one or more of the diseases / disorders described herein). In various embodiments, the liver fat content is reduced to an absolute liver fat of less than 5%. An absolute liver fat content of greater than 5% is associated with fatty liver (fatty liver disease), and an absolute liver fat content of less than 5% is considered within the clinically normal range in non-diseased subjects (see, e.g., Chalasani et al., 2018 Hepatology; 67(1):328-357).

[0036] "Subject in need thereof" refers to a subject, such as a human, who will benefit from the administration of pharmaceutical compositions, and may be diagnosed with or suffer from any of the symptoms of the disorders described herein.For example, the subject who needs to reduce uric acid levels may be a subject suffering from gout.The subject who needs to reverse liver cirrhosis or reduce the fibrosis associated with NASH, MASH, ASH, ALD, or AFLD may be suffering from NASH, MASH, ASH, ALD, or AFLD, or may be recovering from NASH, ASH, ALD, or ALFD.

[0037] The term "treat," as well as related terms, does not necessarily imply 100% or complete cure or remission. Rather, the degree of treatment that one skilled in the art recognizes as having potential benefit or therapeutic effect varies. In this regard, methods of treating a disease or disorder can provide any amount or level of treatment. Furthermore, the treatment provided by the methods can include treating one or more conditions, symptoms, or signs of the disease being treated and / or improving the quality of life of a subject with the condition or disease. The treatment methods of the present disclosure can inhibit one or more symptoms of the disease. The treatment provided by the methods of the present disclosure can also include slowing or reversing the progression of the disease.

[0038] Improvement in a subject's quality of life can be measured by determining one or more quality of life parameters, for example, using the European Quality of Life 5 Questionnaire (EQ-5D) to determine mobility, mood, and overall impact on the patient's quality of life, as reported by the patient. The EQ-5D questionnaire also includes a visual analog scale (VAS) that allows respondents to report their self-perceived health status. See, for example, Balestroni et al., Monaldi Arch Chest Dis. 2012 Sep; 78(3): 155-9, incorporated by reference in its entirety. Treatment can also be monitored using a liver disease questionnaire. See, for example, Younossi et al., Clin Gastroenterol Hepatol. 2019 Sep; 17(10): 2093-2100.e3, incorporated by reference in its entirety. Liver treatment can also be monitored by measuring histological data (e.g., regression of fibrosis, resolution of NASH, etc.) and objective parameters such as non-invasive biomarkers of liver fibrosis (Pro-C3, ELF), injury (e.g., alanine aminotransferase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT), and / or alkaline phosphatase (ALP)), stiffness (e.g., VCTE, FibroScan), and inflammation and fibrosis (e.g., T1-corrected MRI). Exemplary methods for liver histopathology scoring in NASH patients are disclosed, for example, in Kleiner et al., 2005 Hepatology 41, 1313, incorporated by reference in its entirety.

[0039] With respect to the aforementioned methods, the composition containing the conjugate or peptide can be administered by any suitable route of administration, including intravenous, intraperitoneal, intracerebral (intraparenchymal), intramuscular, intraocular, intraarterial, intraportal, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intranasal, inhalation (e.g., upper and / or lower respiratory tract), enteral, epidural, urethral, ​​vaginal, or rectal. In various examples, the composition is administered to the subject intravenously, intramuscularly, or subcutaneously. For example, in some embodiments, the composition is administered subcutaneously. The amount or dose (i.e., "effective amount") of the conjugate or peptide in the administered composition should be sufficient to achieve the desired biological effect in the subject over a clinically reasonable time frame (e.g., about 0.1 mg to about 100 mg).

[0040] In jurisdictions that prohibit the patenting of methods performed on the human body, the meaning of "administering" a composition to a human subject may be limited to prescribing a controlled substance that the human subject can self-administer by any technique (e.g., injection, insertion, etc.). The present disclosure contemplates the use of the pharmaceutical composition for treating any of the diseases or disorders described herein. The present disclosure further contemplates the use of the composition in the preparation of a medicament for treating any of the diseases or disorders described herein. The present disclosure further provides the compositions described herein for use in treating any of the diseases or disorders referred to herein. In jurisdictions that do not prohibit the patenting of methods performed on the human body, "administering" a composition includes both methods performed on the human body and the aforementioned activities.

[0041] In a further aspect, a kit is provided that includes a pharmaceutical composition comprising a conjugate or peptide described herein, packaged in a manner that facilitates administration to a subject. In one aspect, the kit includes a pharmaceutical composition / formulation comprising a conjugate or peptide described herein, packaged in a container such as a sealed bottle, vessel, single-use or multi-use vial, pre-filled device (e.g., a single- or dual-chamber syringe or dual-chamber cartridge for use in an automatic injection device), or pre-filled injection device, optionally with a label attached to the container or included in the packaging that explains the use of the pharmaceutical composition in performing a method. In one aspect, the composition comprising a conjugate or peptide described herein is packaged in a unit dosage form. The kit may include a device suitable for administering the pharmaceutical composition according to a specific administration route, but this is not required.

[0042] The various sequences referred to in this application are provided in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Example]

[0043] A study using the single-chain FGF21 analog pegbelfermin was reportedly discontinued after more than 24 weeks of treatment due to insufficient efficacy, including a decline in pharmacodynamic effects between weeks 4 and 24 of treatment. (Sanyal, AASLD Poster 2021 LP-40) The studies underlying this disclosure demonstrate that having at least two, and potentially more, covalently linked FGF21 moieties within the molecule better preserves signaling through FGF21's receptors (KLB and FGFR1c), including when lower levels of receptors are present on the cell surface or when the receptors have mutations that result in loss of FGF21 signaling. As illustrated in Figure 3, the addition of an Fc domain to a single-chain FGF21 domain containing the RGE modification described above results in a 3 / 4EFX (Figure 1A), which reduces potency (shift to the right) and the degree of agonism (shift down). However, the addition of a second FGF21 domain to 3 / 4EFX more than doubled the potency and restored the degree of agonism, suggesting that two FGF21 domains linked via a single Fc domain have significantly greater potency than twice the concentration of the 3 / 4EFX molecule, which has only a single FGF21 domain linked to an Fc dimer.

[0044] Furthermore, the addition of an Fc domain to a single FGF21(RGE) domain (Figure 1A, 3 / 4EFX) reduces the association rate of the FGF21 analog to the surface of live cells compared to "free" FGF21(RGE), whereas the addition of a second FGF21 domain to the Fc scaffold enhances the association rate and slows the dissociation rate, suggesting a significantly more stable interaction (>100-fold) between the bivalent FGF21 analog and the surface of cells compared to either free FGF21 or a single FGF21 domain bound to an Fc domain.

[0045] All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. 1. A complex comprising: (a) a first monomer comprising an Fc domain fused to two FGF21 domains, which may be the same or different, comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; and (b) a second monomer comprising an Fc domain fused to at least one FGF21 domain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, wherein at least two of the FGF21 domains of the complex comprise a free beta-Klotho binding region.

2. 2. The conjugate of claim 1, wherein (a) the first monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

3. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

4. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain.

5. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the N-terminus of the Fc domain.

6. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the C-terminus of the Fc domain.

7. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

8. 2. The conjugate of claim 1, wherein (a) the first monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 4 fused to the C-terminus of the Fc domain.

9. 9. The conjugate of claim 8, wherein (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 4 fused to the C-terminus of the Fc domain.

10. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain.

11. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

12. 2. The conjugate of claim 1, wherein (a) the first monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

13. 13. The conjugate of claim 12, wherein (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the N-terminus of the Fc domain and the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

14. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 4 fused to the N-terminus of the Fc domain.

15. (b) the second monomer comprises the amino acid sequence of SEQ ID NO: 2 fused to the C-terminus of the Fc domain.

16. The conjugate of any one of claims 1 to 15, wherein one or both Fc domains comprise the amino acid sequence of SEQ ID NO:

7.

17. The conjugate of any one of claims 1 to 16, wherein one or more FGF21 domains are fused to the Fc domain via a linker comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

18. The complex of claim 1, comprising two monomers comprising the amino acid sequence of SEQ ID NO:

18.

19. The complex of claim 1, comprising two monomers comprising the amino acid sequence of SEQ ID NO:

19.

20. 2. The complex of claim 1, comprising two monomers comprising the amino acid sequence of SEQ ID NO:

20.

21. A peptide comprising an Fc domain fused at its N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:4 and at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:

2.

22. 22. The peptide of claim 21, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

7.

23. 23. The peptide of claim 21 or 22, wherein the FGF21 domain comprising SEQ ID NO: 4 is fused to the Fc domain via a linker comprising the amino acid sequence of SEQ ID NO: 10, and the FGF21 domain comprising SEQ ID NO: 2 is fused to the Fc domain via a linker comprising the amino acid sequence of SEQ ID NO:

9.

24. 22. The peptide of claim 21, comprising the amino acid sequence of SEQ ID NO:

21.

25. A complex comprising: (a) a peptide according to any one of claims 21 to 24; and (b) a second Fc domain.

26. A complex comprising: (a) a first monomer comprising an Fc domain fused at its C-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO: 4; and (b) a second monomer comprising an Fc domain fused at its N-terminus to an FGF21 domain comprising the amino acid sequence of SEQ ID NO:

2.

27. 27. The conjugate of claim 26, wherein at least one Fc domain comprises the amino acid sequence of SEQ ID NO:

7.

28. 28. The conjugate of claim 26 or 27, wherein the FGF21 domain comprising SEQ ID NO: 4 is fused to the Fc domain of the first monomer via a linker comprising the amino acid sequence of SEQ ID NO: 10, and the FGF21 domain comprising SEQ ID NO: 2 is fused to the Fc domain of the second monomer via a linker comprising the amino acid sequence of SEQ ID NO:

9.

29. 27. The complex of claim 26, wherein the first monomer comprises the amino acid sequence of SEQ ID NO:27 and the second monomer comprises the amino acid sequence of SEQ ID NO:

28.

30. A peptide comprising the amino acid sequence of SEQ ID NO: 4 fused to the amino acid sequence of SEQ ID NO: 2 via a linker comprising the amino acid sequence of SEQ ID NO: 9 and / or the amino acid sequence of SEQ ID NO:

10.

31. The complex or peptide according to any one of claims 1 to 30, wherein the Fc domain is an IgG1 Fc domain.

32. A nucleic acid comprising a nucleotide sequence encoding a first monomer according to any one of claims 1 to 20.

33. A nucleic acid comprising a nucleotide sequence encoding a second monomer according to any one of claims 1 to 20.

34. 33. An expression vector comprising the nucleic acid of claim 32.

35. 34. An expression vector comprising the nucleic acid of claim 33.

36. 21. A system comprising: an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the first monomer of any one of claims 1 to 20; and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the second monomer of any one of claims 1 to 20.

37. (a) an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the first monomer according to any one of claims 1 to 7, and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the second monomer according to any one of claims 1 to 7; (b) an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the first monomer according to any one of claims 8 to 11, and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the second monomer according to any one of claims 8 to 11; or (c) an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the first monomer of any one of claims 12 to 15, and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the second monomer of any one of claims 12 to 15.

38. A host cell comprising an expression vector according to claim 34 and / or 35.

39. 38. A method of producing a complex, comprising culturing a host cell comprising the system of claim 37 and harvesting the complex.

40. A nucleic acid comprising a nucleotide sequence encoding the peptide of any one of claims 21 to 24 and 30.

41. A nucleic acid comprising a nucleotide sequence encoding the first monomer of any one of claims 26 to 29 or the second monomer of any one of claims 26 to 29.

42. 42. An expression vector comprising the nucleic acid of claim 40 or 41.

43. 43. A host cell comprising the expression vector of claim 42.

44. 30. A method of producing a complex, the method comprising culturing a host cell comprising an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the first monomer of any one of claims 26 to 29 and an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding the second monomer of any one of claims 26 to 29; and harvesting the complex.

45. A pharmaceutical composition comprising the complex or peptide of any one of claims 1 to 30.

46. 46. ​​A method for treating nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

47. 46. ​​A method of reversing NASH associated with cirrhosis, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

48. 46. ​​A method for treating alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), or alcoholic fatty liver disease (AFLD), comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

49. 46. ​​A method for normalizing liver fat content in a subject in need thereof, comprising administering to a subject in need thereof the pharmaceutical composition of claim 45.

50. 46. ​​A method for reversing cirrhosis or reducing fibrosis associated with NASH, NAFLD, ASH, ALD, or AFLD, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

51. 46. ​​A method of treating type 2 diabetes, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

52. 46. ​​A method of treating obesity, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

53. 46. ​​A method for treating dyslipidemia, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

54. 46. ​​A method of lowering blood glucose, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

55. 46. ​​A method of increasing insulin sensitivity, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

56. 46. ​​A method for reducing uric acid, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

57. 46. ​​A method of treating craving or addiction, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

58. The method of any one of claims 46 to 57, wherein the pharmaceutical composition comprises a conjugate of any one of claims 18 to 20.

59. 46. ​​A method for treating metabolic dysfunction-associated steatohepatitis (MASH) or metabolic dysfunction-associated fatty liver disease (MASLD), comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

60. 46. ​​A method of reversing MASH associated cirrhosis, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

61. 46. ​​A method for treating metabolic dysfunction-related alcoholic liver disease (MetALD), comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

62. A method for reversing cirrhosis or reducing fibrosis associated with MASH, MAFLD, or MetALD, comprising administering the pharmaceutical composition of claim 45 to a subject in need thereof.

63. 18. The conjugate of any one of claims 1 to 17, wherein the first monomer and / or the second monomer comprises two or more tandem FGF21 domains comprising SEQ ID NO:2 and / or SEQ ID NO:4 fused to the N-terminus of the Fc domain.

64. The complex or peptide of any one of claims 1 to 15, 17 to 21, 23 to 26, and 28 to 30, wherein the Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 76 to 83.

Images