Engineered α-Klotho peptides and their applications

Abstract

This disclosure provides engineered α-klotho polypeptides and methods for their production and use. The soluble α-klotho polypeptides disclosed herein comprise an α-klotho moiety having one or more mutations relative to human α-klotho for enhanced activity, productivity, and / or stability. This disclosure further provides pharmaceutical compositions comprising the engineered α-klotho polypeptide, and methods for using the engineered α-klotho polypeptide in treatments, including the treatment of age-related conditions and kidney disease. Also disclosed are nucleic acids encoding the engineered α-klotho polypeptide, recombinant cells expressing the engineered α-klotho polypeptide, and methods for producing the engineered α-klotho polypeptide.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 597,872, filed November 10, 2023, and U.S. Provisional Application No. 63 / 657,247, filed June 7, 2024, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence List

[0004] This application contains a list of electronically submitted sequences, which are hereby incorporated by reference in their entirety. The copy created on November 5, 2024, is named RGN-019WO_SL.xml and is 231,499 bytes in size. Background Technology

[0005] α-Klotho (commonly referred to as Klotho (KL)) is a single-transmembrane protein located in the plasma membrane and primarily expressed in the kidneys, parathyroid glands, and choroid plexus. Human KL (hKL) has a relatively short intracellular region lacking any functional domains, while its extracellular region contains two major domains, KL1 and KL2, which share amino acid sequence homology with family 1 glycosidases.

[0006] KL can be cleaved by proteolysis to produce a 130 kDa soluble form containing the KL extracellular region, which is then released into circulation. Despite its sequence homology with glycosidases, the KL extracellular region does not exhibit glycosidase-mediated activity. Instead, the extracellular portion of KL acts as an FGF23 co-receptor via the FGF receptor and participates in P... i And the regulation of vitamin D metabolism, ion homeostasis and oxidative stress.

[0007] KL has been associated with maintaining kidney health. For example, KL deficiency is associated with chronic kidney disease in humans (Barker et al., 2014, Nephrol Dial Transplant 30(2):223-233), affecting more than 700 million individuals worldwide and associated with more than 1 million deaths globally. KL has also shown protective effects against age-related symptoms, and circulating levels of soluble KL decrease with age (Kim et al., 2015, J Lifestyle Med.5(1):1-6).

[0008] Therefore, there is a need to develop KL peptides that exhibit effective biological activity and can be produced efficiently on a large scale. Summary of the Invention

[0009] This disclosure relates to engineered α-klotho peptides.

[0010] Typically, the engineered α-klotho peptides of this disclosure are soluble, for example, lacking transmembrane domains.

[0011] The engineered α-klotho polypeptide disclosed herein comprises the α-klotho moiety. Exemplary α-klotho moieties are described in Section 6.3.

[0012] In some embodiments, the engineered klotho peptide of this disclosure comprises a KL2 domain having an amino acid substitution at position 521 and a C-terminal tail region (relative to human KL number; SEQ ID NO:1) that does not contain a cysteine ​​residue at position 970 (if present). As described herein, these modifications produce proteins with surprisingly enhanced activity, productivity, and stability relative to human KL.

[0013] The engineered α-klotho polypeptide may further include a stabilizing portion, which is optionally linked to the α-klotho portion via a linker. Exemplary stabilizing portions are described in Section 6.4. Exemplary linkers are described in Section 6.5.

[0014] Exemplary engineered α-klotho peptides of this disclosure are described in Section 6.2 and in Examples 1 through 181.

[0015] This disclosure further provides nucleic acids encoding the engineered α-klotho peptide of this disclosure. This disclosure further provides host cells and cell lines engineered to express the nucleic acids and engineered α-klotho peptide of this disclosure. This disclosure further provides methods for generating the engineered α-klotho peptide of this disclosure. Exemplary nucleic acids, host cells and cell lines, and methods for generating engineered α-klotho peptides are described in Section 6.6 and in Examples 182 through 188.

[0016] This disclosure further provides pharmaceutical compositions comprising the engineered α-klotho polypeptide of this disclosure. Exemplary pharmaceutical compositions are described in Section 6.7 and in Example 189.

[0017] This document further provides methods for using the engineered α-klotho peptides and pharmaceutical compositions of this disclosure, for example, for activating FGFR signaling, for treating age-related conditions, for treating kidney disease, or for supplementing endogenous α-klotho protein. Exemplary methods are described in Section 6.8 and in Examples 190 through 205. Attached Figure Description

[0018] Figure 1This is a schematic representation of the extracellular region of α klotho, which, from the N-terminus to the C-terminus, comprises: the α klotho-1 (KL1) domain (AA 34-506), the interdomain region (AA 507-514), the α klotho-2 (KL2) domain (AA 515-950), and the C-terminal tail region (950-981). The amino acid numbers shown are relative to the amino acid sequences of full-length human α klotho (Genbank BAA23382.1; SEQ ID NO:1 and UniProtKB Q9UEF7-1; SEQ ID NO:2).

[0019] Figure 2A-2J This is a schematic representation of an exemplary engineered α-klotho polypeptide.

[0020] Figure 2A An engineered α-klotho polypeptide chain is shown, comprising a KL1 domain, an interdomain region, a KL2 domain with a C521S mutation, and a C-terminal tail region oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2A The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0021] Figure 2B An engineered α-klotho polypeptide is shown, comprising a KL1 domain, an interdomain region, a KL2 domain with a C521S mutation, a C-terminal tail region, an optional linker (L), and a stabilizing moiety oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2B The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0022] Figure 2C An engineered α-klotho polypeptide is shown, comprising a KL1 domain with F353V and C370S mutations, an interdomain region, a KL2 domain with a C521S mutation, and a C-terminal tail region oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2C The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0023] Figure 2DAn engineered α-klotho polypeptide is shown, comprising, in an N-terminal to C-terminal orientation, a KL1 domain with F353V and C370S mutations, an interdomain region, a KL2 domain with a C521S mutation, a C-terminal tail region, an optional linker (L), and a stabilizing moiety. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2D The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0024] Figure 2E An engineered α-klotho polypeptide is shown, comprising a KL1 domain with an F353V mutation, an interdomain region, a KL2 domain with a C521S mutation, and a C-terminal tail region oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2E The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0025] Figure 2F An engineered α-klotho polypeptide is shown, comprising, in an N-terminal to C-terminal orientation, a KL1 domain with an F353V mutation, an interdomain region, a KL2 domain with a C521S mutation, a C-terminal tail region, an optional linker (L), and a stabilizing moiety. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2F The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0026] Figure 2G An engineered α-klotho polypeptide is shown, comprising a KL1 domain with a C370S mutation, an interdomain region, a KL2 domain with a C521S mutation, and a C-terminal tail region oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2G The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0027] Figure 2H An engineered α-klotho polypeptide is shown, comprising, in an N-terminal to C-terminal orientation, a KL1 domain with a C370S mutation, an interdomain region, a KL2 domain with a C521S mutation, a C-terminal tail region, an optional linker (L), and a stabilizing moiety. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2H The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0028] Figure 2I An engineered α-klotho polypeptide is shown, comprising a KL1 domain with an L111S mutation, an interdomain region, a KL2 domain with a C521S mutation, and a C-terminal tail region oriented from the N-terminus to the C-terminus. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2I The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0029] Figure 2J An engineered α-klotho polypeptide is shown, comprising, in an N-terminal to C-terminal orientation, a KL1 domain with an L111S mutation, an interdomain region, a KL2 domain with a C521S mutation, a C-terminal tail region, an optional linker (L), and a stabilizing moiety. In some embodiments, the engineered α-klotho polypeptide of this disclosure (e.g., as shown in the image) Figure 2J The one shown is a monomer, consisting of only a single polypeptide chain, and may further contain a signal sequence.

[0030] for Figure 2A-2J In each of the depicted constructs, the C-terminal tail region can be a full-length C-terminal tail (optionally having one or more amino acid substitutions) or a C-terminal tail region truncated relative to human KL. In some embodiments, Figure 2A-2J The C-terminal tail region of the depicted construct contains a C-terminal deletion between 12 and 31 amino acids relative to human KL. All amino acid numbers are relative to the amino acid sequences of full-length human α klotho (Genbank BAA23382.1; SEQ ID NO:1 and UniProtKB Q9UEF7-1; SEQ ID NO:2).

[0031] Figure 3 The amino acid sequence of full-length human klotho (UniProt KB Q9UEF7-1; SEQ ID NO:2) is shown. The first 33 underlined amino acids with dashed lines correspond to the native hKL signal sequence. Italicized amino acid residues correspond to the KL1 domain, bold amino acid residues correspond to the KL2 domain, and underlined amino acid residues with solid lines indicate interdomain regions (the regions located between the KL1 and KL2 domains). Amino acid residues presented within solid boxes show the C-terminal ADAM10 / ADAM17 cleavage target sequence, while the vertical dashed line between two bold and underlined amino acid residues indicates the cleavage site. Amino acid residues presented within dashed boxes correspond to the transmembrane region, and underlined amino acid residues with double lines correspond to the intracellular region of hKL. Amino acids between the KL2 domain and the transmembrane region correspond to the C-terminal tail region.

[0032] Figures 4A-4C The effects of C-terminal truncation and surface Cys residue engineering on the generation and activity of engineered KL peptide constructs are shown. Figure 4A The graphs showing the post-purification yields and aggregations of the His-tagged constructs REGN14416 (KL981 His), REGN14225 (KL958 His), REGN 14226 (KL958 C521S His), and REGN14227 (KL958 C521S C910S His) are presented, where the bar graphs represent the yields and the line graphs show the percentage (HMW%) of high molecular weight species after size exclusion chromatography (SEC), which is used as a measure of aggregation. Figure 4B This is a bar graph depicting the fold change in phosphorylated extracellular signal-regulated kinase (pERK) levels in NIH3T3 cells relative to treatment with engineered KL peptide alone, as a result of co-treatment with engineered KL peptide and FGF23. Figure 4C It is a bar graph depicting the fold change in pERK levels in NHDF cells relative to treatment with engineered KL peptide alone, as a result of co-treatment with engineered KL peptide and FGF23.

[0033] Figures 5A-5C The effect of pH on the yield and activity of His and / or HSA-labeled KL958 constructs was demonstrated. Figure 5A Images of non-reducing (NR) and reducing (R) polyacrylamide gel electrophoresis (PAGE) are shown, depicting the differences in yield and aggregation when His or HSA-labeled constructs are eluted with a His column at pH 7 or an HSA column at pH 3. The presence of smears with values ​​higher than 169 kDa under NR conditions indicates aggregate formation. Figure 5B This is a bar graph showing the results of pERK homogeneous time-resolved fluorescence (HTRF) assays, depicting pERK-related fluorescence in untreated cells and in cells treated with FGF23, positive control human α-klotho protein (haKL), or FGF23 with haKL. Figure 5C This is a bar graph showing pERK HTRF in cells treated with the construct KL958 SS HSA His in the presence or absence of FGF23, eluted with a His column at pH 7 or an HSA column at pH 3. Background levels correspond to... Figure 5B pERK HTRF levels in untreated cells.

[0034] Figures 6A-6D It is a sketch depiction of engineered KL peptides containing different stable components. Figure 6AThis is a sketch illustration of an engineered KL peptide with a C-terminal HSA-stabilized moiety. Figure 6B This is a sketch illustration of an engineered KL polypeptide dimer with a C-terminal Fc stabilizing moiety. Figure 6C This is a sketch illustration of an engineered KL peptide with a C-terminal Fc1.5 stable moiety. Figure 6D This is a sketch illustration of an engineered KL polypeptide with a stable C-terminal monomeric Fc (moFc) moiety, where moFc is a non-dimerized Fc containing one or more mutations on the hydrophobic surface of its CH3 region. Figures 6A-6D The small ellipse at the C-terminus of the illustrated construct represents an optional additional tag (OAT) that can be used to facilitate construct purification. Non-limiting examples of such additional tags include the His tag (HHHHHH; SEQ ID NO:73) and the Twin-Strep tag. ® (WSHPQFEKGGGSGGGSGGSAWSHPQFEK; SEQ ID NO:74).

[0035] Figures 7A-7E The production yield and purity of four different constructs were demonstrated, each containing a different C-terminal tag linked to REGN14226 (KL958 C521S). Figure 7A These are polyacrylamide gel electrophoresis images showing the yields of reduced and non-reduced constructs containing Fc, moFc, Fc1.5, and HSA-stabilized moieties, respectively. All constructs also contain Twin-Strep tags. The scissor icons above the lanes indicate that the samples in that lane were treated with 3C protease prior to loading to remove the Twin-Strep tags. Figure 7B The size exclusion chromatography (SEC) chromatogram of the Fc-labeled KL958 C521S peptide construct KL958(C521S)-Fc is shown. Figure 7C The size exclusion chromatography (SEC) chromatogram of the moFc-labeled KL958 C521S peptide construct KL958(C521S)-MoFc is shown. Figure 7D The size exclusion chromatography (SEC) purification chromatogram of the Fc1.5-labeled KL958 C521S peptide construct KL958(C521S)-Fc1.5 is shown. Figure 7E The size exclusion chromatography (SEC) chromatogram of the HSA-labeled KL958 C521S peptide construct KL958(C521S)-HSA is shown. The striped peaks represent high molecular weight (HMW) proteins and aggregates. The checkerboard patterned peaks correspond to the monomeric KL958 C521S peptide construct.

[0036] Figures 8A-8BThe results of improved high-salt purification of the KL peptide construct KL958(C521S)-HSA at neutral pH are shown. Figure 8A These are polyacrylamide gel electrophoresis images showing the yields of reduced and non-reduced KL958(C521S)-HSA. Lane 1 was loaded with molecular weight markers. Lanes 2 and 5 were loaded with non-reduced and reduced samples containing aggregates and HMW proteins, respectively. Lanes 3 and 6 were loaded with non-reduced and reduced SEC eluents containing monomeric KL958(C521S)-HSA constructs. Figure 8B The SEC purification spectrum of KL958(C521S)-HSA is shown. Peak 1 corresponds to aggregated HMW protein, peak 2 corresponds to monomeric KL958C521S peptide construct, and peak 3 corresponds to low molecular weight peptide.

[0037] Figures 9A-9F A step-by-step improvement to the purification of KL958(C521S)-HSA using a three-step modified ion exchange chromatography (IEX) method is shown. Figures 9A-9B The images shown are polyacrylamide gel electrophoresis images of different fractions of protein obtained by the Q-agarose ion exchange step. Figure 9A ) and SEC chromatogram of the eluent ( Figure 9B ). Figure 9C-9D The images shown are polyacrylamide gel electrophoresis images of different fractions of protein obtained by HSA affinity column purification. Figure 9C ) and the SEC chromatogram of the eluent ( Figure 9D ). Figure 9E-9F The image shown is a polyacrylamide gel electrophoresis image of the final eluent obtained by the SD-200 purification step. Figure 9E ) and the SEC chromatogram of the final eluent ( Figure 9F ).

[0038] Figures 10A-10B The effects of His and HSA tags on the pharmacokinetic (PK) profiles of engineered KL peptide constructs are shown. Figure 10A This is a graph showing the average serum concentration of the KL peptide construct over time. Figure 10B This is a graph showing the dose-normalized PK plot of the KL peptide construct.

[0039] Figure 11A-11C The effects of His and HSA tags on the engineered KL peptide-mediated proliferation of cultured 3T3 cells were demonstrated. Figure 11A This is a graph showing the dose-dependent proliferation of cells treated with KL958(C521S)-His in the presence or absence of FGF23. Figure 11B This is a graph showing the dose-dependent proliferation of cells treated with KL958(C521S)-HSA in the presence or absence of FGF23. Figure 11C This is a graph showing the growth differences between cells treated with KL958(C521S)-His and FGF23, KL958(C521S)-HSA and FGF23, bFGF alone or FGF23 alone, and untreated cells.

[0040] Figure 12 This is a graph showing the effects of different C-terminal truncations on the activity of certain engineered KL peptide constructs.

[0041] Figures 13A-13F An exemplary engineered α-klotho peptide construct with an N-terminal to C-terminal configuration is shown: [mROR1]-[KL1 domain]-[KL2 domain]-[G4S linker]-[HSA domain]. The first 33 amino acids of human KL (corresponding to the native KL signal sequence) are replaced by the mROR1 signal sequence, and the KL2 domain is truncated at residue 958. Figure 13A The polypeptide construct containing the C521S mutation in its KL2 domain is shown. Figure 13B The polypeptide construct is shown, which contains F352V and C370S mutations in its KL1 domain and C521S mutation in its KL2 domain. Figure 13C The polypeptide construct is shown, which contains the F353V mutation in its KL1 domain and the C521S mutation in its KL2 domain. Figure 13D The polypeptide construct is shown, which contains a C370S mutation in its KL1 domain and a C521S mutation in its KL2 domain. Figure 13E The polypeptide construct is shown, which contains an L111S mutation in its KL1 domain and a C521S mutation in its KL2 domain. Figure 13F The polypeptide construct is shown, which contains a C521S mutation in its KL2 domain and a C34S mutation in its HSA domain. Detailed Implementation

[0042] 6.1. Definition

[0043] Approximately: Throughout the specification, the terms “approximately,” “about,” etc., are used before numbers to indicate that the number is not necessarily precise (e.g., considering variations in fractions, measurement accuracy and / or precision, timing, etc.). It should be understood that disclosures of “approximately X” or “about X”, where X is a number, are also disclosures of “X.” Thus, for example, a disclosure of an embodiment in which one sequence has “approximately X% sequence identity” with another sequence is also a disclosure of an embodiment in which that sequence has “X% sequence identity” with another sequence.

[0044] And / or: Unless otherwise stated, the conjunction “or” should be used in its proper sense as a Boolean logical operator, encompassing both the selection of features in alternatives (A or B, where the selection of A and B are mutually exclusive) and the selection of joint features (A or B, where both A and B are selected). In some places in the text, the term “and / or” is used for the same purpose, which should not be interpreted as implying that “or” is used to refer to mutually exclusive alternatives.

[0045] EC50: The term "EC50" refers to the half-maximal effective concentration of a molecule (such as a polypeptide of this disclosure) that elicits half the response between baseline and maximum after a specific exposure time. EC50 essentially represents the concentration of the polypeptide at which 50% of its maximum effect is observed. In some embodiments, the EC50 value is equal to the concentration of the polypeptide that gives half-maximal activation in a luciferase reporter assay, as described in Section 9.1.5.

[0046] Fc domains and Fc regions: The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain on a separate polypeptide chain. In some embodiments, an Fc domain comprises a CH2 domain followed by a CH3 domain, having or not having a hinge region at the N-terminus of the CH2 domain. In some embodiments, the Fc domain is non-dimerizing. Optionally, in addition to the CH2 domain followed by a CH3 domain, the non-dimerizing Fc domain further comprises an additional CH3 domain (e.g., "Fc1.5 domain") linked to the CH3 domain via a linker. In some embodiments, an Fc domain comprises a CH3 domain that cannot dimerize with another Fc domain (e.g., "monomer Fc"). The term "Fc region" refers to a region formed by the association of two heavy chain Fc domains on a separate polypeptide chain. The two Fc domains within an Fc region may be identical or different from each other.

[0047] Fibroblast growth factor receptor or FGFR: Unless otherwise stated, as used herein, the terms “fibroblast growth factor receptor” and “FGFR” mean any of the FGFRs 1-4 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats), and include naturally occurring variants of FGFRs, such as splice variants or allele variants (e.g., FGFR1c).

[0048] Host cell: As used herein, the term "host cell" refers to a cell in which the nucleic acids of this disclosure have been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer to a specific subject cell and to the progeny or potential progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the terminology used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, such as vertebrate cells, such as mouse, rat, monkey, or human cell lines, such as HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0049] KL1 domain or α Klotho KL1 domain: As used herein, the terms “KL1 domain” and “α Klotho KL1 domain” refer to the amino acid sequence corresponding to the KL1 domain of an α klotho protein (e.g., human α klotho or mouse α klotho), as well as its derivatives and variants. Therefore, a KL1 domain can be the amino acid sequence of the KL1 domain of an α klotho protein (e.g., human α klotho or mouse α klotho) or a sequence having one, two, three, four, five, or more amino acid substitutions relative to the wild-type sequence. For example, in some embodiments, the KL1 domain of the α klotho portion has L111S, F352V, and / or C370S substitutions relative to human α klotho (GenBank accession number BAA23382.1 - SEQ ID NO:1). In some embodiments, the KL1 domain is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the KL1 domain is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of SEQ ID NO:9 or SEQ ID NO:10 and having one, two, or all three substitutions for L111S, F352V, and / or C370S (e.g., only L111S, only F352V, only C370S, or both F352V and C370S).

[0050] KL2 domain or α Klotho KL2 domain: As used herein, the terms “KL2 domain” and “α Klotho KL2 domain” refer to the amino acid sequence corresponding to the KL2 domain of the α klotho protein (e.g., human α klotho or mouse α klotho), as well as its derivatives and variants. Therefore, the KL2 domain can be the amino acid sequence of the KL2 domain of the α klotho protein (e.g., human α klotho or mouse α klotho) or a sequence having one, two, three, four, five, or more amino acid substitutions relative to the wild-type sequence. For example, in some embodiments, the KL2 domain of the α klotho portion has the C521S mutation relative to the human α klotho (GenBank accession number BAA23382.1; SEQ ID NO:1). In some embodiments, the KL2 domain is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with SEQ ID NO:3 or SEQ ID NO:4. In specific embodiments, the KL2 domain is an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:3 and containing an amino acid sequence substituted with C521S relative to the human α klotho (GenBank accession number BAA23382.1). In some embodiments, the KL2 domain is the amino acid sequence of SEQ ID NO:4.

[0051] Klotho moiety, α-Klotho moiety, or KL moiety: As used herein, the terms "klotho moiety," "α-klotho moiety," and "KL moiety" refer to an amino acid sequence comprising at least one KL1 domain and / or at least one KL2 domain. In specific embodiments, the α-klotho moiety comprises both the KL1 and KL2 domains oriented from the N-terminus to the C-terminus. In some embodiments, the α-klotho moiety further comprises an interdomain region between the KL1 and KL2 domains. In some embodiments, the interdomain region comprises the amino acid sequence of SEQ ID NO:21 or a variant thereof having one, two, three, four, or five amino acid substitutions relative to SEQ ID NO:21. In some embodiments, the α-klotho moiety further comprises, at its C-terminus, a C-terminal tail of or a portion thereof of the KL extracellular domain. In some embodiments, the C-terminal tail comprises amino acids 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, or 1-8 of SEQ ID NO:7, or an amino acid thereof relative to SEQ ID NO:7. The amino acid variants of NO:7, consisting of 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, or 1-8, have one, two, three, four, or five amino acid substitutions. In a specific embodiment, the α-klotho moiety comprises, in the N-terminal to C-terminal orientation: (i) a KL1 domain; (ii) an interdomain region; (iii) a KL2 domain; and (iv) a C-terminal tail region.

[0052] In some embodiments, the α klotho portion comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with SEQ ID NO:13. In some embodiments, the α klotho portion comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity with SEQ ID NO:14. In some embodiments, the α klotho portion comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with SEQ ID NO:15. In some embodiments, the α klotho portion comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% sequence identity with SEQ ID NO:16.

[0053] Linker: As used herein, the term "linker" refers to a connecting peptide between two parts. For example, a linker can connect the α-klotho moiety to a stable domain.

[0054] Operable ligation: As used herein, the term “operable ligation” refers to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are ligated to produce a functional polypeptide, or two or more nucleic acid sequences are ligated to, for example, produce an in-frame fusion of two polypeptide components or to ligate a regulatory sequence to a coding sequence.

[0055] Polypeptides, peptides, and proteins: The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this document and refer to polymers of amino acid residues.

[0056] Subject: The term “subject” includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise stated, the terms “patient” or “subject” are used interchangeably in this document.

[0057] Treatment, Treatment, and Treating: As used herein, the terms “treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a condition as described herein; an improvement in one or more symptoms (preferably one or more identifiable symptoms) of a condition or symptom as described herein; or prevention of a condition or symptom as described herein (e.g., kidney disease or age-related condition or symptom caused by the administration of a molecule or composition (e.g., one or more α-klotho peptides of this disclosure). In specific embodiments, the terms “treat,” “treatment,” and “treating” refer to the improvement of at least one measurable physical parameter of a condition (e.g., kidney disease or age-related symptom) that may not necessarily be identifiable by the patient. In other embodiments, the terms “treat,” “treatment,” and “treating” refer to the suppression of the progression or onset of a condition physically by, for example, stabilizing identifiable symptoms, or physiologically by, for example, stabilizing physical parameters, or both.

[0058] 6.2. Soluble α-Klotho polypeptide

[0059] This disclosure relates to engineered α-klotho polypeptides (also known as "engineered KL polypeptides") comprising an α-klotho moiety. Typically, the α-klotho moiety has a KL2 domain having an amino acid substitution at position C521 corresponding to full-length human α-klotho (SEQ ID NO: 1), and, if present, also lacking cysteine ​​at position C970 corresponding to full-length human α-klotho (SEQ ID NO: 1).

[0060] Unbound by theory, amino acid residues C521 and C970 of full-length human α klotho (SEQ ID NO:1) are understood to be free cysteine ​​residues that do not pair with any other cysteine ​​residues in the native protein via disulfide bonds. Although many other free cysteine ​​residues are present in the α klotho protein, it has been surprisingly found that modifications, particularly to these two cysteine ​​residues (e.g., by substituting C521 and by deleting a portion of the α klotho protein containing C970), result in significantly enhanced activity, productivity, and stability of the α klotho protein relative to the wild type. Therefore, the polypeptides of this disclosure generally comprise the α klotho moiety comprising 1) a KL2 domain with an amino acid substitution at position 521, and 2) a C-terminal tail region containing no cysteine ​​residue at position 970 (if present) or a C-terminal deletion of at least 12 amino acids such that position 970 is absent. Exemplary α klotho moieties are described in Section 6.3.

[0061] In some embodiments, the soluble α-klotho polypeptide of this disclosure may further include a stabilizing portion optionally linked via a linker to the α-klotho portion (e.g., an N-terminus or a C-terminus). Exemplary stabilizing portions are described in Section 6.4 and include an Fc domain (as described in Section 6.4.1), an albumin portion (as described in Section 6.4.2), and other stabilizing portions (e.g., as described in Section 6.4.3). Exemplary linkers are described in Section 6.5.

[0062] An exemplary engineered α-klotho peptide is shown in Figure 2A-2J The amino acid sequences of example engineered α-klotho peptides are provided as SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, and 63. The sequences of example nucleic acids encoding the engineered α-klotho peptides are provided as SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.

[0063] 6.3.α Klotho section

[0064] Naturally occurring α-klotho (KL) is encoded by the α-klotho gene located on human chromosome 13. The product of the α-klotho gene is a single-transmembrane protein that comprises an extracellular region, a transmembrane region, and a cytoplasmic region from the N-terminus to the C-terminus. The extracellular region of KL has two domains (sometimes referred to as "daughter domains"), called KL1 and KL2. Furthermore, the amino acid sequence between the domains lies between the KL1 and KL2 domains, and the C-terminal tail region is located at the C-terminus of the KL2 domain.

[0065] The following is a reproduction of the amino acid sequence of human KL (GenBank accession number BAA23382.1), in which the signal sequence is shown in italics, the KL1 domain is shown in straight underline, the KL2 domain is shown in bold, and the transmembrane region is shown in dashed underline.

[0066]

[0067] The following reproduces the alternative amino acid sequence of human KL with a single amino acid substitution relative to SEQ ID NO:1 (UniProtKB accession number Q9UEF7-1), wherein the signal sequence is shown in italics, the KL1 domain is shown in straight underline, the KL2 domain is shown in bold, and the transmembrane region is shown in dashed underline.

[0068]

[0069] Extracellular region of human KL ( Figure 1 The α-klotho moiety is composed of amino acids 34 to 981 and, as discussed above, includes the KL1 domain (amino acids 34-506; SEQ ID NO: 9), the interdomain region (amino acids 507-514; SEQ ID NO: 21), the KL2 domain (amino acids 515-950; SEQ ID NO: 3), and the C-terminal tail region (amino acids 951-981; SEQ ID NO: 7). The α-klotho moiety, as discussed herein in the context of engineered α-klotho peptides, comprises at least a portion of the extracellular region of human KL or its variants or derivatives.

[0070] In some aspects, the α klotho portion includes the α klotho KL2 domain. In some embodiments, the α klotho KL2 domain has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, 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%, at least about 99.5%, or at least about 99.8% sequence identity with the KL2 domain of human KL. The α klotho KL2 domain may contain 1, 2, 3, 4, 5, or more amino acid mutations (e.g., deletions, additions, or substitutions) relative to the KL2 domain of human KL. In some embodiments, the α-klotho KL2 domain has an amino acid substitution at position C521 corresponding to the full-length human KL (SEQ ID NO:1). In some embodiments, this amino acid substitution is a substitution of cysteine ​​to serine. In other embodiments, this amino acid substitution is the substitution of cysteine ​​with a different amino acid that is not serine.

[0071] In some embodiments, the α klotho moiety lacks a cysteine ​​residue at the position corresponding to C970 of the full-length human KL (SEQ ID NO:1), if present. Therefore, in some aspects, the α klotho moiety includes the position corresponding to C970 of the full-length human KL (SEQ ID NO:1), where this position is not a cysteine ​​residue. For example, the α klotho moiety may include an amino acid substitution at the position corresponding to C970, such as a substitution from cysteine ​​to serine. Alternatively, in some aspects, the α klotho moiety completely lacks the position corresponding to C970 of the full-length human KL (SEQ ID NO:1). For example, the α klotho moiety may contain only a portion of the C-terminal tail region of the extracellular region of human KL (SEQ ID NO:7), which lacks the position corresponding to C970 of the full-length human KL. Therefore, in some embodiments, the α klotho moiety lacks 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids from the C-terminal tail of the full-length human KL (SEQ ID NO:7). In some embodiments, the α klotho moiety lacks 23 amino acids from the C-terminal tail of the full-length human KL (SEQ ID NO:7). Without being bound by theory, it is believed that, in the case of engineered α klotho peptides, α klotho moiety having a C521 mutation (e.g., C521S) and lacking cysteine ​​at position 970 (or completely lacking position 970) provides a significant increase in both productivity and biological activity compared to α klotho moiety containing a wild-type human KL2 domain and a C-terminal tail region. Therefore, in some embodiments, the α klotho portion (1) comprises a KL2 domain having a C521S mutation (relative to SEQ ID NO:1), and (2) lacks the C-terminal 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids of the C-terminal tail of the full-length human KL (SEQ ID NO:7). In some embodiments, the α klotho portion (1) comprises a KL2 domain having a C521S mutation (relative to SEQ ID NO:1), and (2) lacks an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0072] In some aspects, the α klotho portion further comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, 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% sequence identity with the KL1 domain of human KL. The α klotho KL1 domain may contain 1, 2, 3, 4, 5, or more amino acid mutations (e.g., deletions, additions, or substitutions) relative to the KL1 domain of human KL. In some embodiments, the α klotho KL1 domain has an amino acid substitution at the L111 position corresponding to the full-length human KL (SEQ ID NO:1). In some embodiments, this amino acid substitution is a leucine-to-serine substitution. In some embodiments, the α klotho KL1 domain has an amino acid substitution at position F352 corresponding to full-length human KL (SEQ ID NO:1). In some embodiments, this amino acid substitution is a substitution from phenylalanine to valine. In some embodiments, the α klotho KL1 domain has an amino acid substitution at position C370 corresponding to full-length human KL (SEQ ID NO:1). In some embodiments, this amino acid substitution is a substitution from cysteine ​​to serine.

[0073] Sequences of certain example α-klotho portions of this disclosure are provided in Table 1 below. In some embodiments, the α-klotho portions have at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, 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%, at least about 99.5%, or 100% sequence identity with the sequences in Table 1.

[0074]

[0075] 6.4. Stable Part

[0076] The engineered α-klotho peptide of this disclosure may include a stabilizing moiety that prolongs the serum half-life of the molecule in vivo. Serum half-life is typically divided into an α-phase and a β-phase. By adding an appropriate stabilizing moiety, either or both phases can be significantly improved. For example, relative to a corresponding stable α-klotho peptide without a stabilizing moiety, the stabilizing moiety can increase the serum half-life of the engineered α-klotho peptide by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 400%, 600%, 800%, 1000%, or more. For the purposes of this disclosure, serum half-life may refer to the half-life in humans or other mammals (e.g., mice or non-human primates).

[0077] The stabilizing portion includes, but is not limited to, a polyoxyethylene portion (e.g., polyethylene glycol), a sugar (e.g., sialic acid), and a well-tolerated protein portion (e.g., Fc and its fragments and variants, transferrin, and serum albumin). In some embodiments, the stabilizing portion is human serum albumin (or a variant thereof having 1, 2, 3, 4, 5, or more amino acid substitutions relative to human serum albumin). In some embodiments, the stabilizing portion is an Fc domain.

[0078] Other stable moieties that can be used for the engineered α-klotho peptides of this disclosure include those described in Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76. These stable moieties include, but are not limited to, human serum albumin, human serum albumin conjugates (e.g., Adnectin PKE, AlbudAb, ABD), and XTEN. ® PAS (a recombinant PEG mimic based on three amino acids proline, alanine, and serine), carbohydrates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid, and fatty acids.

[0079] In some embodiments, the engineered α-klotho peptide comprising the stabilizing moiety will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the bioactivity associated with the unmodified engineered α-klotho peptide. In some embodiments, bioactivity refers to its ability to bind to FGFR or both FGFR and FGF23, such as via K D k on or k off The assessment.

[0080] The stabilizing portion may be connected via a linker to one or more other components (e.g., the α-klotho portion) of the engineered α-klotho polypeptide of this disclosure, such as as described in Section 6.5. In some embodiments, the stabilizing portion is located at the C-terminus of the α-klotho portion. In some embodiments, the stabilizing portion is located at the N-terminus of the α-klotho portion.

[0081] In some embodiments, the stable portion is a variant of an Fc domain or a fragment thereof, as described in Section 6.4.1. In some embodiments, the stable portion is a non-dimerized Fc domain, as described in Section 6.4.1.1. In some embodiments, the stable portion is an Fc1.5 domain, as described in Section 6.4.1.1.1.

[0082] In other embodiments, the stable component is serum albumin or a variant or fragment thereof, as described in Section 6.4.2.

[0083] This document also considers additional stabilizing components, including polyethylene glycol portions or other polymers, as described in Section 6.4.3.

[0084] 6.4.1. Fc structural domain

[0085] In some embodiments, the engineered α-klotho polypeptide of this disclosure comprises one or more Fc domains as a stabilizing part.

[0086] The Fc domain can be derived from any suitable species. In one embodiment, the Fc domain is derived from the human Fc domain. In some embodiments, the α-klotho moiety is fused to the IgG Fc domain. The α-klotho moiety can be fused to either the N-terminus or the C-terminus of the IgG Fc domain.

[0087] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0088] Exemplary sequences of the Fc domains from IgG1, IgG2, IgG3 and IgG4 are provided in Table Y-1 below.

[0089]

[0090] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:23. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO:23 (e.g., sequence identity between 90% and 99% with SEQ ID NO:23), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions to prevent dimerization (e.g., as described in Section 6.4.1.1) and / or one or more substitutions to alter effector function (e.g., as described in Section 6.4.1.2).

[0091] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:24. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO:24 (e.g., sequence identity between 90% and 99% with SEQ ID NO:24), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions to prevent dimerization (e.g., as described in Section 6.4.1.1) and / or one or more substitutions to alter effector function (e.g., as described in Section 6.4.1.2).

[0092] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:25. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO:25 (e.g., sequence identity between 90% and 99% with SEQ ID NO:25), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions to prevent dimerization (e.g., as described in Section 6.4.1.1) and / or one or more substitutions to alter effector function (e.g., as described in Section 6.4.1.2).

[0093] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:26. Where the Fc domain has at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO:26 (e.g., sequence identity between 90% and 99% with SEQ ID NO:26), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions to prevent dimerization (e.g., as described in Section 6.4.1.1) and / or one or more substitutions to alter effector function (e.g., as described in Section 6.4.1.2).

[0094] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form the Fc region. The α-klotho polypeptide disclosed herein may contain an Fc domain comprising heavy chain constant domains from one or more different classes (e.g., one, two, or three different classes) of antibodies.

[0095] In some other embodiments, the stabilizing portion comprises two Fc domains that form a dimer. The two Fc domains may be identical or different from each other. In some embodiments, the two Fc domains are identical. However, Fc domains that allow heterodimerization can be used to manufacture engineered α-klotho peptides comprising different polypeptide components. Engineered α-klotho peptides with different polypeptide components may comprise, for example, an α-klotho moiety and another polypeptide, or a first α-klotho moiety fused to another polypeptide and a second α-klotho moiety not fused to another polypeptide.

[0096] 6.4.1.1. Non-dimerizing Fc domain

[0097] In some embodiments, the Fc domain is a non-dimerizing (or “monomerizing”) Fc domain, which is an Fc domain whose self-association ability is reduced or completely absent relative to the wild-type Fc domain, for example, as in Helm et al., 1996, J. Biol. Chem. 271: 7494-7500 or Ying et al., 2012, J Biol Chem. 287(23):19399–19408. Exemplary non-dimerizing Fc domains contain amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3 (according to Kabat EU index numbers), as described in U.S. Patent Publication No. 2019 / 0367611, which is incorporated herein by reference. Specific amino acid substitutions that may be included in the nondimerizing Fc domain include, for example, L351S, T366R, L368H, P395K, L242C, K334C, L351S, P343C, A431C, L351Y, T366Y, L368A, P395R, F405R, Y407M, K409A, F405E, Y407K, L351K, T366S, P395V, Y407A, and K409Y (according to Kabat EU index numbers). The nondimerizing Fc domain of this disclosure may contain any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of the above substitutions.

[0098] Exemplary sequences of the non-dimerized Fc domains are provided in Table Y-2 below. Bold residues indicate the positions of amino acid substitutions relative to the wild-type IgG sequence.

[0099]

[0100] In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:27. In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:28. In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:29. In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:30. In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:31. In some embodiments, the nondimerizing Fc domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:32.

[0101] 6.4.1.1.1.Fc1.5 Structural Domain

[0102] In some embodiments, the Fc domain, in addition to the CH2 and CH3 domains, further comprises an additional CH3 domain connected to the first CH3 domain via a linker (e.g., a linker as described in Section 6.5). Fc domains comprising this configuration (CH2 – CH3 – linker – CH3) are sometimes referred to herein as “Fc1.5 domains” or simply “Fc1.5”. The linker between the first and second CH3 domains of the Fc1.5 domain preferably has sufficient length and flexibility to allow dimerization of the first and second CH3 domains. Thus, in some embodiments, the stabilizing portion is the Fc1.5 domain, which comprises a linker at least 5, at least 10, at least 15, or at least 20 amino acid lengths connecting the first and second CH3 domains.

[0103] The following table Y-3 provides an exemplary sequence of Fc1.5 structural domains.

[0104]

[0105] In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:33. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:34. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:35. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:36. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:37. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:38. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:39. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:40.

[0106] 6.4.1.2. Fc domain with altered effector function

[0107] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce the binding of Fc receptor and / or effector functions.

[0108] In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is selected from one or more of the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0109] In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (according to Kabat EU index numbers). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such embodiment, the Fc domain is an Igd Fc domain, particularly a human Igd Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (according to Kabat EU index numbers). In one embodiment, the Fc domain contains an amino acid substitution at position P329 and additional amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to Kabat EU index numbers). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain contains amino acid substitutions at positions P329, L234, and L235 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain contains amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0110] In one embodiment, the Fc domain is the IgG1 Fc domain, particularly the human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU number) to reduce effector function.

[0111] In another embodiment, the Fc domain is an IgG4 Fc domain that reduces binding to the Fc receptor. Exemplary IgG4 Fc domains with reduced binding to the Fc receptor may include amino acid sequences selected from Table F below. In some embodiments, the Fc domain includes only the bolded portions of the sequences shown below:

[0112]

[0113] In a specific embodiment, the reduced effector function IgG4 comprises the bold portion of the amino acid sequence of SEQ ID NO:31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4.

[0114] 6.4.2. Albumin portion

[0115] Human serum albumin (HSA) has a relatively long average half-life of approximately 19 days. HSA is the most abundant protein in human plasma and provides a variety of functions, including maintaining plasma pH, transporting fatty acids and other metabolites, and maintaining blood pressure. The amino acid sequence of mature HSA (lacking the signal sequence and propeptide) is shown below.

[0116] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDMPADLPSLAADFVESKDVCNKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:19)

[0117] The albumin portion of the engineered α-klotho polypeptide disclosed herein can be an HSA, a portion thereof, or a variant thereof. Thus, in some embodiments, the albumin portion comprises a sequence of wild-type, mature HSA. In some embodiments, the albumin portion comprises an amino acid sequence having 1, 2, 3, 4, 5, or more amino acid substitutions relative to wild-type, mature HSA. For example, in some embodiments, the albumin portion comprises an amino acid sequence having a C34S mutation relative to wild-type, mature HSA. Without being bound by theory, it is believed that C34S in HSA improves stability by eliminating free cysteine. Additional HSA variants are well known in the art and are considered herein, including, for example, the K573P mutant HSA. In some embodiments, the albumin portion comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:19. In some embodiments, the albumin portion comprises an amino acid sequence having at least about 90%, 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% sequence identity with SEQ ID NO:20.

[0118] 6.4.3. Other stable components

[0119] In some embodiments, the engineered α-klotho polypeptide comprises polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing portion, such as a copolymer of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homogeneous or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer can have any molecular weight and can be branched or unbranched.

[0120] Serum albumin can participate in half-life extension through modules with the ability to interact non-covalently with albumin. Therefore, the engineered α-klotho polypeptide of this disclosure can include an albumin-binding protein as a stable component. The albumin-binding protein can be conjugated or genetically fused to one or more other components of the engineered α-klotho polypeptide of this disclosure. Proteins with albumin-binding activity are known to exist in certain bacteria. For example, Streptococcus protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Further examples of serum albumin-binding proteins are those described in U.S. Publications 2007 / 0178082 and 2007 / 0269422. Fusion of the albumin-binding domain to the protein results in a significantly extended half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0121] 6.5. Connector

[0122] In some respects, this disclosure provides engineered α-klotho polypeptides in which two or more components are interconnected via peptide linkers. By way of example and not limitation, linkers may be used to connect (a) the α-klotho moiety and a stable moiety and / or (b) the stable moiety and another moiety, such as a purified tag moiety.

[0123] The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in some respects, the length of the peptide linker ranges from 3 amino acids to 50 amino acids, 4 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, 10 amino acids to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids.

[0124] In certain respects, the length of the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids, and optionally, the length is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids.

[0125] In some of the foregoing embodiments, the length of the connector ranges from 5 to 50 amino acids, for example, from 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other foregoing embodiments, the length of the connector ranges from 6 to 50 amino acids, for example, from 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In some of the foregoing embodiments, the length of the connector ranges from 7 to 50 amino acids, for example, from 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0126] Electrically charged (e.g., electrically charged hydrophilic connectors) and / or flexible connectors are particularly preferred.

[0127] Examples of flexible linkers that can be used for the engineered α-klotho peptides of this disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or contain repetitive sequences of glycine and serine, such as G... n S or SG n The monomer or polymer, where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the connector is or contains a repeating sequence of G4S, such as (GGGGS). n (SEQ ID NO:75).

[0128] Polyglycine linkers can be suitably used in the engineered α-klotho peptides of this disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly), five consecutive glycines (5 Gly), six consecutive glycines (6 Gly), seven consecutive glycines (7 Gly), eight consecutive glycines (8 Gly), or nine consecutive glycines (9 Gly).

[0129] Exemplary linker sequences are listed in Table L below. The engineered α-klotho polypeptide of this disclosure may include one or more linkers from Table L. For example, in some embodiments, the engineered α-klotho polypeptide of this disclosure includes an α-klotho portion and a stable portion linked via linkers from Table L.

[0130]

[0131] In some embodiments, the engineered KL peptide includes a connector L1. In some embodiments, the engineered KL peptide includes a connector L2. In some embodiments, the engineered KL peptide includes a connector L3. In some embodiments, the engineered KL peptide includes a connector L4. In some embodiments, the engineered KL peptide includes a connector L5. In some embodiments, the engineered KL peptide includes a connector L6. In some embodiments, the engineered KL peptide includes a connector L7. In some embodiments, the engineered KL peptide includes a connector L8. In some embodiments, the engineered KL peptide includes a connector L9. In some embodiments, the engineered KL peptide includes a connector L10. In some embodiments, the engineered KL peptide includes a connector L11. In some embodiments, the engineered KL peptide includes a connector L12. In some embodiments, the engineered KL peptide includes a connector L13. In some embodiments, the engineered KL peptide includes a connector L14. In some embodiments, the engineered KL peptide includes a connector L15. In some embodiments, the engineered KL peptide includes a connector L16. In some embodiments, the engineered KL peptide includes a connector L17. In some embodiments, the engineered KL peptide includes a connector L18. In some embodiments, the engineered KL peptide includes a connector L19. In some embodiments, the engineered KL peptide includes a connector L20. In some embodiments, the engineered KL peptide includes a connector L21. In some embodiments, the engineered KL peptide includes a connector L22. In some embodiments, the engineered KL peptide includes a connector L23. In some embodiments, the engineered KL peptide includes a connector L24. In some embodiments, the engineered KL peptide includes a connector L25. In some embodiments, the engineered KL peptide includes a connector L26. In some embodiments, the engineered KL peptide includes a connector L27. In some embodiments, the engineered KL peptide includes a connector L28. In some embodiments, the engineered KL peptide includes a connector L29. In some embodiments, the engineered KL peptide includes a connector L30. In some embodiments, the engineered KL peptide includes a connector L31. In some embodiments, the engineered KL peptide includes a connector L32. In some embodiments, the engineered KL peptide includes a connector L33. In some embodiments, the engineered KL peptide includes a connector L34. In some embodiments, the engineered KL peptide includes a connector L35. In some embodiments, the engineered KL peptide includes a connector L36. In some embodiments, the engineered KL peptide includes a connector L37. In some embodiments, the engineered KL peptide includes a connector L38. In some embodiments, the engineered KL peptide includes a connector L39. In some embodiments, the engineered KL peptide includes a connector L40. In some embodiments, the engineered KL peptide includes a connector L41. In some embodiments, the engineered KL peptide includes a connector L42.In some embodiments, the engineered KL peptide includes a connector L43. In some embodiments, the engineered KL peptide includes a connector L44. In some embodiments, the engineered KL peptide includes a connector L45. In some embodiments, the engineered KL peptide includes a connector L46. In some embodiments, the engineered KL peptide includes a connector L47. In some embodiments, the engineered KL peptide includes a connector L48. In some embodiments, the engineered KL peptide includes a connector L49. In some embodiments, the engineered KL peptide includes a connector L50. In some embodiments, the engineered KL peptide includes a connector L51. In some embodiments, the engineered KL peptide includes a connector L52. In some embodiments, the engineered KL peptide includes a connector L53. In some embodiments, the engineered KL peptide includes a connector L54. In some embodiments, the engineered KL peptide includes a connector L55. In some embodiments, the engineered KL peptide includes a connector L56. In some embodiments, the engineered KL peptide includes a connector L57. In some embodiments, the engineered KL peptide includes a connector L58. In some embodiments, the engineered KL peptide includes a connector L59. In some embodiments, the engineered KL peptide includes a connector L60. In some embodiments, the engineered KL peptide includes a connector L61. In some embodiments, the engineered KL peptide includes a connector L62. In some embodiments, the engineered KL peptide includes a connector L63. In some embodiments, the engineered KL peptide includes a connector L64. In some embodiments, the engineered KL peptide includes a connector L65. In some embodiments, the engineered KL peptide includes a connector L66. In some embodiments, the engineered KL peptide includes a connector L67. In some embodiments, the engineered KL peptide includes a connector L68. In some embodiments, the engineered KL peptide includes a connector L69. In some embodiments, the engineered KL peptide includes a connector L70. In some embodiments, the engineered KL peptide includes a connector L71. In some embodiments, the engineered KL peptide includes a connector L72. In some embodiments, the engineered KL peptide includes a connector L73. In some embodiments, the engineered KL peptide includes a connector L74. In some embodiments, the engineered KL peptide includes a connector L75. In some embodiments, the engineered KL peptide includes a connector L76. In some embodiments, the engineered KL peptide includes a connector L77. In some embodiments, the engineered KL peptide includes a connector L78. In some embodiments, the engineered KL peptide includes a connector L79.

[0132] 6.6. Nucleic Acids and Host Cells

[0133] On the other hand, this disclosure provides a nucleic acid encoding the engineered α-klotho polypeptide of this disclosure. The nucleic acid of this disclosure may be DNA (e.g., plasmid) or RNA (e.g., mRNA).

[0134] In some embodiments, the nucleic acid of this disclosure comprises a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, 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%, at least about 99.5%, or 100% sequence identity with any of SEQ ID NO:41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.

[0135] In some respects, this disclosure provides host cells and vectors containing the nucleic acids of this disclosure. The nucleic acids may be present in a single vector or in a single vector within the same host cell or a separate host cell, as described in more detail below.

[0136] 6.6.1. Carrier

[0137] This disclosure provides vectors containing nucleotide sequences encoding the α-klotho polypeptide described herein or components thereof (e.g., a polypeptide chain of the α-klotho polypeptide). Vectors include, but are not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs).

[0138] Various vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rouse sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semleeki Forest virus, Eastern Equine Encephalitis Virus, and Flavivirosis.

[0139] Additionally, cells that have stably integrated their DNA into their chromosomes can be selected by introducing one or more markers that allow selective transfection of host cells. Markers can provide, for example, tropism against auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals (such as copper). The selection marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal mRNA synthesis may also require additional elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0140] Once the expression vector or DNA sequence containing the construct is prepared, it can be transfected or introduced into a suitable host cell. This can be achieved using various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used to culture the resulting transfected cells and to recover the expressed peptide are known to those skilled in the art and can be varied or optimized based on this specification, depending on the specific expression vector and mammalian host cell used.

[0141] 6.6.2. Host Cell

[0142] This disclosure also provides host cells containing the nucleic acids contained in this disclosure.

[0143] In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids as described herein.

[0144] In one embodiment, host cells are genetically engineered using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of influencing gene expression in a host compatible with such a sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in influencing expression, such as, for example, inducible promoters, may also be used.

[0145] This disclosure also provides host cells containing the vectors described herein. Cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0146] 6.7. Pharmaceutical Compositions

[0147] The engineered α-klotho peptide disclosed herein can be in the form of a composition comprising the engineered α-klotho peptide and one or more carriers, excipients, and / or diluents. The composition can be formulated for a specific purpose, such as for veterinary or human pharmaceutical use. The form of the composition used (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers will depend on the intended use of the engineered α-klotho peptide and, for therapeutic purposes, also on the mode of administration.

[0148] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient via a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topically, or locally. The most appropriate route of administration in any given situation will depend on the specific engineered α-klotho peptide, the subject, the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition is administered intravenously or subcutaneously.

[0149] The pharmaceutical composition can be conveniently available in unit dosage forms containing a predetermined amount of the engineered α-klotho peptide of this disclosure per dose. The amount of engineered α-klotho peptide included in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized powder containing a predetermined amount of engineered α-klotho peptide suitable for a single administration, or in liquid form. The powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components for administration. The liquid unit dose can be conveniently supplied in the form of a syringe pre-filled with a predetermined amount of engineered α-klotho peptide suitable for a single administration.

[0150] The pharmaceutical composition can also be supplied in bulk from a certain amount of α-klotho polypeptide suitable for multiple administrations.

[0151] Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing engineered α-klotho peptides of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives) commonly used in the art. See Remington’s Pharmaceutical Sciences, 16th edition (edited by Osol, 1980). Such additives should be non-toxic to the recipient at the dosage and concentration used.

[0152] Buffers help maintain pH values ​​within a range close to physiological conditions. They can be present in a variety of concentrations, but are typically found in concentrations ranging from about 2 mM to about 50 mM. Buffers suitable for use in this disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., mixtures of monosodium citrate and disodium citrate, mixtures of citrate and trisodium citrate, mixtures of citrate and monosodium citrate, etc.), succinate buffers (e.g., mixtures of succinate and monosodium succinate, mixtures of succinate and sodium hydroxide, mixtures of succinate and disodium succinate, etc.), tartrate buffers (e.g., mixtures of tartaric acid and sodium tartrate, mixtures of tartaric acid and potassium tartrate, mixtures of tartaric acid and sodium hydroxide, etc.), and fumarate buffers (e.g., mixtures of fumaric acid and monosodium fumarate, etc.). Fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can also be used.

[0153] Preservatives may be added to delay microbial growth, and may be added in amounts ranging from about 0.2% to 1% (w / v). Preservatives suitable for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride halides (e.g., chlorides, bromides, and iodides), hexamethyl chloride, and alkylparaben esters (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents (sometimes referred to as “stabilizers”) may be added to ensure the isotonicity of the liquid compositions of this disclosure, and isotonic agents include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a large class of excipients whose functional range includes fillers to additives, capable of dissolving therapeutic agents or helping to prevent denaturation or adhesion to container walls. Typical stabilizers can be polyols (listed above); amino acids (such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.); organic sugars or sugar alcohols (such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc.); including cyclic alcohols, such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as... Urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount of 0.5 to 10% by weight per weight of the α-klotho peptide.

[0154] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect them from agitation-induced aggregation. This also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and Pranic polyols. Nonionic surfactants can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0155] Other miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0156] The engineered α-klotho peptide of this disclosure can be formulated into pharmaceutical compositions comprising the engineered α-klotho peptide, for example, containing one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition comprising the engineered α-klotho peptide of this disclosure, the engineered α-klotho peptide formulation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0157] For example, formulations of engineered α-klotho peptides can be prepared by mixing engineered α-klotho peptides with physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NY; Avis et al. (ed.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman et al. (ed.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (ed.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker). NY; Weiner and Kotkoskie, 2000, ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0158] The effective dose for a particular subject can vary depending on a number of factors, such as the disease being treated, the subject’s overall health, the route and dosage of administration, and the severity of side effects (see, for example, Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Florida; Dent (2001) Good Laboratory and Good Clinical Practice, UrchPubl., London, United Kingdom).

[0159] The compositions of this disclosure can also be administered via one or more routes of administration using one or more of the various methods known in the art. As those skilled in the art will understand, the route of administration and / or mode will vary depending on the desired outcome. Selected routes of administration for the engineered α-klotho peptide include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other general routes of administration, such as by injection or infusion. General administration can represent modes of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of this disclosure can be administered via non-general routes, such as local, epidermal, or mucosal administration routes, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration. In one embodiment, the engineered α-klotho peptide is administered by infusion. In another embodiment, the engineered α-klotho peptide of this disclosure is administered subcutaneously.

[0160] 6.8. Indications and Methods of Use

[0161] This disclosure provides methods of use and applications of the engineered α-klotho polypeptide used in this disclosure.

[0162] The engineered α-klotho peptide disclosed herein can be used as a treatment for a variety of conditions, including age-related conditions, metabolic disorders, and kidney diseases.

[0163] In some aspects, this disclosure provides a method for treating age-related conditions, comprising administering the engineered α-klotho peptide described herein to a subject in need of treatment. Age-related conditions include, but are not limited to, hearing loss, cataracts and refractive errors, osteoarthritis, chronic obstructive pulmonary disease, diabetes, and dementia. The engineered α-klotho peptide of this disclosure can be administered to subjects who have or are diagnosed with age-related conditions. The engineered α-klotho peptide can also be administered to subjects at risk of developing age-related conditions. For example, the engineered α-klotho peptide can be administered prophylactically to subjects at increased risk of developing age-related conditions, thereby reducing the risk of developing age-related conditions.

[0164] In some respects, this disclosure provides a method for treating kidney disease, comprising administering the engineered α-klotho peptide described herein to a subject in need of treatment. Kidney disease includes both acute kidney injury and chronic kidney disease. Therefore, the engineered α-klotho peptide can be administered to a subject after acute kidney injury or after a diagnosis of chronic kidney disease. The engineered α-klotho peptide can also be administered to subjects at risk of developing kidney disease. For example, the engineered α-klotho peptide can be administered prophylactically to subjects at increased risk of developing chronic kidney disease, thereby reducing the risk of developing chronic kidney disease.

[0165] A method for replenishing endogenous α-klotho loss is also disclosed, comprising administering the engineered α-klotho peptide described herein to a subject in need of such replenishment. Endogenous levels of α-klotho protein decrease with age, and the engineered α-klotho peptide can be used to replenish this loss. Therefore, in some embodiments, the engineered α-klotho peptide of this disclosure is administered to a subject who has a reduced α-klotho protein level relative to previous levels in the same subject (e.g., a reduction of at least about 5%, 10%, 15%, 20%, 25%, or 30% relative to α-klotho protein levels at least 6 months prior in the same subject).

[0166] This document also discloses a method for activating FGFR signaling using an engineered α-klotho peptide. The exemplary engineered α-klotho peptide of this disclosure can interact with FGF23 and activate FGFR signaling in cells. Therefore, a method for activating FGFR signaling in cells is disclosed, comprising contacting cells with the engineered α-klotho peptide of this disclosure. The cells can be any cells expressing FGFR on their surface. In some embodiments, the cells are kidney cells.

[0167] 7. Sequence

[0168] Some sequences of this disclosure are provided in Table S below.

[0169] 8. Specific Implementation Examples

[0171] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by way of examples with reference to the numbers set forth below.

[0172] In the embodiments numbered below, the α-klotho portion is preferably derived from mammalian α-klotho, the albumin portion is preferably derived from mammalian albumin, the Fc domain is preferably derived from mammalian antibodies, and the subject is preferably a mammal. More preferably, the mammal is a human.

[0173] 1. A polypeptide, wherein:

[0174] (a) Contains an α klotho KL2 domain, said domain (i) having at least about 80% sequence identity with SEQ ID NO:3, and (ii) having an amino acid substitution at the position corresponding to amino acid C521 of SEQ ID NO:1; and

[0175] (b) At the amino acid position corresponding to amino acid C970 of SEQ ID NO:1, if said amino acid is present, cysteine ​​is lacking.

[0176] 2. The polypeptide according to Example 1 lacks the amino acid corresponding to amino acid C970 of SEQ ID NO:1.

[0177] 3. The polypeptide according to Example 1, if the amino acid corresponding to C973 of SEQ ID NO:1 is present, is deficient in cysteine.

[0178] 4. The polypeptide according to Example 3 lacks the amino acid corresponding to amino acid C973 of SEQ ID NO:1.

[0179] 5. The polypeptide according to Example 1, if the amino acid corresponding to C963 of SEQ ID NO:1 is present, is deficient in cysteine.

[0180] 6. The polypeptide according to Example 5 lacks the amino acid corresponding to amino acid C963 of SEQ ID NO:1.

[0181] 7. The polypeptide according to any one of Examples 1 to 6, wherein the amino acid at the position corresponding to amino acid C521 of SEQ ID NO:1 is substituted with a mutation from cysteine ​​to serine.

[0182] 8. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 12 amino acids.

[0183] 9. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 13 amino acids.

[0184] 10. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 14 amino acids.

[0185] 11. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 15 amino acids.

[0186] 12. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 16 amino acids.

[0187] 13. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 17 amino acids.

[0188] 14. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 18 amino acids.

[0189] 15. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 19 amino acids.

[0190] 16. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 20 amino acids.

[0191] 17. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 21 amino acids.

[0192] 18. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 22 amino acids.

[0193] 19. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 23 amino acids.

[0194] 20. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 24 amino acids.

[0195] 21. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 25 amino acids.

[0196] 22. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 26 amino acids.

[0197] 23. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 27 amino acids.

[0198] 24. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 28 amino acids.

[0199] 25. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 29 amino acids.

[0200] 26. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 30 amino acids.

[0201] 27. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion of at least 31 amino acids.

[0202] 28. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 12 and 31 amino acids.

[0203] 29. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 13 and 31 amino acids.

[0204] 30. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 14 and 31 amino acids.

[0205] 31. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 15 and 31 amino acids.

[0206] 32. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 16 and 31 amino acids.

[0207] 33. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 17 and 31 amino acids.

[0208] 34. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 18 and 31 amino acids.

[0209] 35. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 19 and 31 amino acids.

[0210] 36. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 20 and 31 amino acids.

[0211] 37. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 21 and 31 amino acids.

[0212] 38. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 22 and 31 amino acids.

[0213] 39. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 23 and 31 amino acids.

[0214] 40. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 12 and 23 amino acids.

[0215] 41. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 13 and 23 amino acids.

[0216] 42. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 14 and 23 amino acids.

[0217] 43. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 15 and 23 amino acids.

[0218] 44. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 16 and 23 amino acids.

[0219] 45. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 17 and 23 amino acids.

[0220] 46. ​​The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 18 and 23 amino acids.

[0221] 47. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 19 and 23 amino acids.

[0222] 48. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 20 and 23 amino acids.

[0223] 49. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 21 and 23 amino acids.

[0224] 50. The polypeptide according to any one of Examples 1 to 7, wherein the amino acid sequence of SEQ ID NO:13 contains a C-terminal deletion between 22 and 23 amino acids.

[0225] 51. The polypeptide according to any one of Examples 1 to 50, which lacks an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8.

[0226] 52. The polypeptide according to any one of Examples 1 to 51, which lacks an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:8.

[0227] 53. The polypeptide according to any one of Examples 1 to 52, which lacks the amino acid sequence of SEQ ID NO:8.

[0228] 54. The polypeptide according to any one of Examples 1 to 53, wherein the α klotho KL2 domain has at least about 85% sequence identity with SEQ ID NO:3.

[0229] 55. The polypeptide according to any one of Examples 1 to 54, wherein the α klotho KL2 domain has at least about 90% sequence identity with SEQ ID NO:3.

[0230] 56. The polypeptide according to any one of Examples 1 to 55, wherein the α klotho KL2 domain has at least about 95% sequence identity with SEQ ID NO:3.

[0231] 57. The polypeptide according to any one of Examples 1 to 56, wherein the α klotho KL2 domain has at least about 99% sequence identity with SEQ ID NO:3.

[0232] 58. The polypeptide according to any one of Examples 1 to 57, wherein the α klotho KL2 domain has at least about 99.5% sequence identity with SEQ ID NO:3.

[0233] 59. The polypeptide according to any one of Examples 1 to 53, wherein the α klotho KL2 domain has at least about 85% sequence identity with SEQ ID NO:4.

[0234] 60. The polypeptide according to any one of Examples 1 to 54, wherein the α klotho KL2 domain has at least about 90% sequence identity with SEQ ID NO:4.

[0235] 61. The polypeptide according to any one of Examples 1 to 55, wherein the α klotho KL2 domain has at least about 95% sequence identity with SEQ ID NO:4.

[0236] 62. The polypeptide according to any one of Examples 1 to 56, wherein the α klotho KL2 domain has at least about 99% sequence identity with SEQ ID NO:4.

[0237] 63. The polypeptide according to any one of Examples 1 to 57, wherein the α klotho KL2 domain has at least about 99.5% sequence identity with SEQ ID NO:4.

[0238] 64. The polypeptide according to any one of Examples 1 to 63, wherein the α klotho KL2 domain comprises the amino acid sequence of SEQ ID NO:4.

[0239] 65. The polypeptide according to any one of Examples 1 to 64, further comprising an α klotho KL1 domain having at least about 80% sequence identity with SEQ ID NO:9.

[0240] 66. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0241] 67. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 95% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0242] 68. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 99% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0243] 69. The polypeptide according to Example 65, wherein the α klotho KL1 domain comprises the amino acid sequence of SEQ ID NO:9.

[0244] 70. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0245] 71. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 95% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0246] 72. The polypeptide according to Example 65, wherein the α klotho KL1 domain has at least about 99% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0247] 73. The polypeptide according to Example 65, wherein the α klotho KL1 domain comprises the amino acid sequence of SEQ ID NO:10.

[0248] 74. The polypeptide according to any one of Examples 65 to 73, wherein the α klotho KL1 domain contains an amino acid substitution at position C370 corresponding to SEQ ID NO:1.

[0249] 75. The polypeptide according to Example 74, wherein the amino acid at position C370 corresponding to SEQ ID NO:1 is replaced by a substitution from cysteine ​​to serine.

[0250] 76. The polypeptide according to any one of Examples 65 to 75, wherein the α klotho KL1 domain comprises the amino acid sequence of SEQ ID NO:9.

[0251] 77. The polypeptide according to any one of Examples 1 to 76, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:11.

[0252] 78. The polypeptide according to any one of Examples 1 to 76, comprising an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:11.

[0253] 79. The polypeptide according to any one of Examples 1 to 76, comprising an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:11.

[0254] 80. The polypeptide according to any one of Examples 1 to 77, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:12.

[0255] 81. The polypeptide according to any one of Examples 1 to 77, comprising an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:12.

[0256] 82. The polypeptide according to any one of Examples 1 to 77, comprising an amino acid sequence having at least about 99% sequence identity with SEQ ID NO:12.

[0257] 83. The polypeptide according to any one of Examples 1 to 77, comprising an amino acid sequence having at least about 99.5% sequence identity with SEQ ID NO:12.

[0258] 84. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having an amino acid substitution relative to SEQ ID NO:12.

[0259] 85. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having two amino acid substitutions relative to SEQ ID NO:12.

[0260] 86. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having three amino acid substitutions relative to SEQ ID NO:12.

[0261] 87. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having four amino acid substitutions relative to SEQ ID NO:12.

[0262] 88. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having five amino acid substitutions relative to SEQ ID NO:12.

[0263] 89. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having substituted 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids relative to SEQ ID NO: 12.

[0264] 90. The polypeptide according to any one of Examples 80 to 83, comprising an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid variations (substitution, deletion or insertion) relative to SEQ ID NO:12.

[0265] 91. The polypeptide according to any one of Examples 1 to 77, comprising the amino acid sequence of SEQ ID NO:12.

[0266] 92. The polypeptide according to any one of Examples 1 to 91, comprising an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:15.

[0267] 93. The polypeptide according to any one of Examples 1 to 91, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:15.

[0268] 94. The polypeptide according to any one of Examples 1 to 91, comprising an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:16.

[0269] 95. The polypeptide according to any one of Examples 1 to 91, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:16.

[0270] 96. The polypeptide according to any one of Examples 92 to 95, wherein the length of the amino acid sequence is between 850 and 950 amino acids.

[0271] 97. The polypeptide according to any one of Examples 92 to 95, wherein the length of the amino acid sequence is between 900 and 925 amino acids.

[0272] 98. The polypeptide according to any one of Examples 92 to 95, wherein the length of the amino acid sequence is between 910 and 920 amino acids.

[0273] 99. The polypeptide according to any one of Examples 92 to 95, wherein the length of the amino acid sequence is between 915 and 920 amino acids.

[0274] 100. The polypeptide according to any one of Examples 1 to 99, comprising the amino acid sequence of SEQ ID NO:16.

[0275] 101. The polypeptide according to any one of Examples 1 to 100, which lacks an amino acid sequence having at least 95% sequence identity with SEQ ID NO:17.

[0276] 102. The polypeptide according to any one of Examples 1 to 100, which lacks an amino acid sequence having at least 98% sequence identity with SEQ ID NO:17.

[0277] 103. The polypeptide according to any one of Examples 1 to 100, which lacks the amino acid sequence of SEQ ID NO:17.

[0278] 104. The polypeptide according to any one of Examples 1 to 103, further comprising a signal peptide.

[0279] 105. The polypeptide according to Example 104, wherein the signal peptide is α-klotho signal peptide.

[0280] 106. The polypeptide according to Example 104, wherein the signal peptide is not the α-klotho signal peptide.

[0281] 107. The polypeptide according to Example 106, wherein the signal peptide is a serum albumin (SA) signal peptide.

[0282] 108. The polypeptide according to Example 106, wherein the signal peptide is astragalin (AZ) signal peptide.

[0283] 109. The polypeptide according to Example 106, wherein the signal peptide is the SP1 signal peptide.

[0284] 110. The polypeptide according to any one of Examples 1 to 103, which lacks a signal peptide.

[0285] 111. The polypeptide according to any one of Examples 1 to 110, further comprising a stable portion.

[0286] 112. The polypeptide according to Example 111, wherein the stable portion is located at the C-terminus of the α klotho KL2 domain.

[0287] 113. The polypeptide according to Example 111, wherein the stable portion is located at the N-terminus of the α klotho KL2 domain.

[0288] 114. The polypeptide according to any one of Examples 111 to 113, wherein the stable portion comprises an Fc domain.

[0289] 115. The polypeptide according to Example 114, wherein the Fc domain comprises a CH2 domain and a CH3 domain.

[0290] 116. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:23.

[0291] 117. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:23.

[0292] 118. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:23.

[0293] 119. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:23.

[0294] 120. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:23.

[0295] 121. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:23.

[0296] 122. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:23.

[0297] 123. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:26.

[0298] 124. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:26.

[0299] 125. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:26.

[0300] 126. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:26.

[0301] 127. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:26.

[0302] 128. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:26.

[0303] 129. The polypeptide according to Example 114 or 115, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:26.

[0304] 130. The polypeptide according to Example 114, wherein the Fc domain further comprises an additional CH3 domain.

[0305] 131. The polypeptide according to Example 130, wherein the additional CH3 domain is connected to the CH3 domain via a linker.

[0306] 132. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:33-40.

[0307] 133. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 85% sequence identity with any of SEQ ID NO:33-40.

[0308] 134. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 90% sequence identity with any of SEQ ID NO:33-40.

[0309] 135. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity with any of SEQ ID NO:33-40.

[0310] 136. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 98% sequence identity with any of SEQ ID NO:33-40.

[0311] 137. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises an amino acid sequence having at least 99% sequence identity with any of SEQ ID NO:33-40.

[0312] 138. The polypeptide according to Example 130 or 131, wherein the Fc domain comprises the amino acid sequence of any of SEQ ID NO:33-40.

[0313] 139. The polypeptide according to Example 114, wherein the Fc domain is a non-dimerized Fc domain.

[0314] 140. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO:27-32.

[0315] 141. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 85% sequence identity with any of SEQ ID NO:27-32.

[0316] 142. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 90% sequence identity with any of SEQ ID NO:27-32.

[0317] 143. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 95% sequence identity with any of SEQ ID NO:27-32.

[0318] 144. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 98% sequence identity with any of SEQ ID NO:27-32.

[0319] 145. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises an amino acid sequence having at least 99% sequence identity with any of SEQ ID NO:27-32.

[0320] 146. The polypeptide according to Example 139, wherein the nondimerized Fc domain comprises the amino acid sequence of any one of SEQ ID NO:27-32.

[0321] 147. The polypeptide according to any one of Examples 111 to 113, wherein the stable portion is an albumin portion.

[0322] 148. The polypeptide according to Example 147, wherein the albumin portion is human serum albumin.

[0323] 149. The polypeptide according to Example 147, wherein the albumin portion is a variant of human serum albumin.

[0324] 150. The polypeptide according to Example 149, wherein the human serum albumin variant has an amino acid substitution C34S.

[0325] 151. The polypeptide according to Example 149 or 150, wherein the albumin portion comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:20.

[0326] 152. The polypeptide according to Example 149 or 150, wherein the albumin portion comprises an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:20.

[0327] 153. The polypeptide according to Example 149 or 150, wherein the albumin portion comprises an amino acid sequence having at least about 98% sequence identity with SEQ ID NO:20.

[0328] 154. The polypeptide according to Example 149 or 150, wherein the albumin portion comprises the amino acid sequence of SEQ ID NO:20.

[0329] 155. The polypeptide according to any one of Examples 111 to 154, further comprising a linker.

[0330] 156. The polypeptide according to Example 155, wherein the polypeptide comprises, in order from N-terminus to C-terminus: an α klotho portion comprising a v KL2 domain, the linker, and the stabilizing portion.

[0331] 157. The polypeptide according to Example 155, wherein the polypeptide comprises, in order from N-terminus to C-terminus: the stabilizing portion, the linker, and the α klotho portion comprising the α klotho KL2 domain.

[0332] 158. The polypeptide according to any one of Examples 155 to 157, wherein the linker comprises one or more amino acid sequences shown in Table L.

[0333] 159. The polypeptide according to any one of Examples 1 to 158, wherein the polypeptide is a monomer.

[0334] 160. The polypeptide according to any one of Examples 1 to 158, wherein the polypeptide is a dimer.

[0335] 161. A polypeptide, optionally a polypeptide according to any one of Examples 1 to 160, comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16, wherein the position corresponding to amino acid 488 of SEQ ID NO:16 is not cysteine, and which lacks an amino acid sequence having at least 80% sequence identity with SEQ ID NO:8.

[0336] 162. The polypeptide according to Example 161, wherein the position of amino acid 488 corresponding to SEQ ID NO:16 is serine.

[0337] 163. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:16.

[0338] 164. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:16.

[0339] 165. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:16.

[0340] 166. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 96% sequence identity with SEQ ID NO:16.

[0341] 167. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:16.

[0342] 168. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO:16.

[0343] 169. The polypeptide according to Example 161 or 162, comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:16.

[0344] 170. The polypeptide according to Example 161 or 162, comprising the amino acid sequence of SEQ ID NO:16.

[0345] 171. The polypeptide according to any one of Examples 161 to 170, which lacks an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8.

[0346] 172. The polypeptide according to any one of Examples 161 to 170, which lacks an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8.

[0347] 173. The polypeptide according to any one of Examples 161 to 170, which lacks the amino acid sequence of SEQ ID NO:8.

[0348] 174. The polypeptide according to any one of Examples 1 to 173, comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:62.

[0349] 175. The polypeptide according to Example 174, comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO:62.

[0350] 176. The polypeptide according to Example 174, comprising an amino acid sequence having at least 96% sequence identity with SEQ ID NO:62.

[0351] 177. The polypeptide according to Example 174, comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO:62.

[0352] 178. The polypeptide according to Example 174, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO:62.

[0353] 179. The polypeptide according to Example 174, comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO:62.

[0354] 180. The polypeptide according to Example 174, comprising an amino acid sequence having at least 99.5% sequence identity with SEQ ID NO:62.

[0355] 181. The polypeptide according to Example 174, comprising the amino acid sequence of SEQ ID NO:62.

[0356] 182. A nucleic acid encoding a polypeptide according to any one of Examples 1 to 181.

[0357] 183. A host cell engineered to express a polypeptide according to any one of Examples 1 to 181 or a nucleic acid according to Example 182.

[0358] 184. A method for producing a polypeptide according to any one of Examples 1 to 181, the method comprising culturing a host cell according to Example 183, and recovering the polypeptide expressed therefrom.

[0359] 185. The method according to Example 184 further includes purifying the polypeptide.

[0360] 186. The method according to Example 185, wherein the polypeptide is purified at a pH between 6.5 and 7.5.

[0361] 187. The method according to Example 185 or 186, wherein the polypeptide is purified at a pH between 6.9 and 7.1.

[0362] 188. The method according to any one of Examples 185 or 187, wherein the polypeptide is purified at a pH of about 7.0.

[0363] 189. A pharmaceutical composition comprising a polypeptide according to any one of Examples 1 to 181 and an excipient.

[0364] 190. A method for activating FGFR signaling in a cell, the method comprising contacting the cell with a polypeptide according to any one of Examples 1 to 181.

[0365] 191. The method according to Example 190, wherein the cell is a kidney cell.

[0366] 192. The method according to Example 190 or 191, wherein the method is an in vitro method.

[0367] 193. The method according to Example 190 or 191, wherein the method is an in vivo method.

[0368] 194. The method according to Example 193, wherein the method includes administering the polypeptide to a subject who requires it.

[0369] 195. The method according to Example 194, wherein the subject is at risk of developing age-related conditions.

[0370] 196. The method according to Example 194, wherein the subject is a patient suffering from age-related symptoms.

[0371] 197. The method according to Example 194, wherein the subject is at risk of developing kidney disease.

[0372] 198. The method according to Example 197, wherein the kidney disease is acute kidney injury.

[0373] 199. The method according to Example 197, wherein the kidney disease is chronic kidney disease.

[0374] 200. A method of treating a subject suffering from age-related symptoms, the method comprising administering to the subject a polypeptide according to any one of Examples 1 to 181 or a pharmaceutical composition according to Example 189.

[0375] 201. A method for preventing age-related symptoms, the method comprising administering to a subject in need of the polypeptide according to any one of Examples 1 to 181 or the pharmaceutical composition according to Example 189.

[0376] 202. A method of treating a subject with kidney disease, the method comprising administering to the subject a polypeptide according to any one of Examples 1 to 181 or a pharmaceutical composition according to Example 189.

[0377] 203. A method for preventing kidney disease, the method comprising administering to a subject in need of the polypeptide according to any one of Examples 1 to 181 or the pharmaceutical composition according to Example 189.

[0378] 204. The method according to Example 202 or 203, wherein the kidney disease is acute kidney injury.

[0379] 205. The method according to Example 202 or 203, wherein the kidney disease is chronic kidney disease.

[0380] 9. Example

[0381] 9.1. Materials and Methods

[0382] 9.1.1. Design and generation of KL peptide constructs

[0383] The KL polypeptide construct was created using the wild-type human α-Klotho (hKL) amino acid sequence (UniProtKB accession number Q9UEF7-1); Figure 3 Designed to be _____. Where applicable, the native KL signaling sequence is replaced with a 29-amino acid signaling sequence from the murine inactive tyrosine protein kinase transmembrane receptor ROR1 (mROR1) to improve productivity; the hKL amino acid sequence is truncated at the C-terminus to remove the C-terminal protease cleavage site; one or more Cys amino acids are mutated to Ser to remove free surface Cys residues; and one or more stable moieties are added to the N-terminus or C-terminus of the construct via a linker. Some exemplary KL peptide constructs are shown in _____. Figure 2A-2J middle.

[0384] Constructs encoding KL peptides were generated in standard mammalian protein expression DNA vectors (pcDNA3.4 or similar vectors) suitable for high-yield protein production and containing standard elements such as promoter sequences, polyA sequences, regulatory elements, and resistance genes. Where applicable, the sequences were codon-optimized. The KL peptide constructs were expressed in suitable cells (e.g., Expi293 or CHO cells) via transient transfection. The protein in the cell supernatant was purified using one of the methods described in Section 9.1.2, neutralized, dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C. The samples were further analyzed by size exclusion chromatography (SEC) to determine the presence of high or low molecular weight species relative to the species of interest, as described in Section 9.1.2. Exemplary sequences are shown in Table E1 below.

[0385]

[0386] 9.1.2. Purification of KL peptide construct

[0387] 9.1.2.1. Affinity column purification

[0388] For some evaluations, His and HSA-labeled KL peptides were purified using Ni-NTA agarose (Qiagen) or CaptureSelect™ human albumin affinity matrix (ThermoFisher). First, the Ni-NTA and albumin affinity matrix columns were equilibrated with 5 column volumes (CV) of equilibration buffer. Next, sterile filtered supernatant containing the KL peptides was loaded onto the pre-equilibrated column at a flow rate of 2.0 mL / min. Any non-specifically bound material was washed away from the column using 5 CV of washing buffer at a flow rate of 2.0 mL / min. Finally, the affinity-bound KL protein was eluted from the column with 1 CV of elution buffer and further purified using SEC. The buffer composition used for His and HSA affinity columns is shown in Table E2 below.

[0389]

[0390] 9.1.2.2. Twin-Strep Purification

[0391] Twin-Strep-labeled KL peptides were purified using Strep-Tactin® XT resin (IBA). First, the column was equilibrated with 5 column volumes (CV) of PBS buffer (pH 7.4). Next, sterile filtered supernatant containing the KL peptides was loaded onto the pre-equilibrated column at a flow rate of 2.0 mL / min. Any non-specifically bound material was washed away from the column using 5 CV of PBS at a flow rate of 2.0 mL / min. 1 CV of PBS and HRV-3C protease (Sigma-Aldrich, 1:100) were added to the column, and the column was incubated at 4 °C for 24 h, with the flow-through collected. Next, the column was washed with 3 CV of PBS, and the resulting flow-through was collected. The two flow-throughs were combined and further purified using SEC.

[0392] 9.1.2.3. Improved High-Salt Purification

[0393] Collect 500 mL of CM from a CHO cell line expressing the KL peptide construct. Use CaptureSelect. TM HSA affinity resin (ThermoFisher) was bound to PBS overnight at 4°C. KL peptides were eluted from the resin using a high-salt elution buffer (Tris-HCl containing 2.0 M MgCl2, pH 7.4). Aggregated proteins, HMW proteins, and other contaminants were removed using SEC.

[0394] 9.1.2.4. Improved Ion Exchange Purification

[0395] A three-step improved ion exchange (IEX) purification process was used to capture KL peptides from large-volume samples. In the first step, sterile filtration supernatant containing KL peptides was loaded onto a Q-agarose column. The column was washed with 20 mM PBS pH 6.5. Proteins were eluted with a NaCl gradient (50 mM to 2 M). SDS-PAGE gels were used to guide fraction collection. In the next step, HSA-labeled KL peptides were specifically captured and contaminants were removed using CaptureSelect™ human albumin affinity matrix. Proteins were eluted with Pierce™ Gentle Ag / Ab elution buffer pH 6.6 (ThermoFisher). KL peptides were further purified using SEC.

[0396] 9.1.3. Pharmacokinetic Analysis of KL Peptide

[0397] To assess the pharmacokinetic properties of KL peptide in plasma, adult C57BL / 6J mice were intraperitoneally injected with a single dose of KL peptide or a control antibody diluted in saline. Blood samples were collected at 0, 2, 8, 24, 48, 72, and 96 hours post-administration, and on days 6 and 14. Plasma was separated by centrifugation of the samples.

[0398] 9.1.4. pERK HTRF Measurement

[0399] Phospho-ERK (pERK) homogeneous time-resolved fluorescence (HTRF) assays utilize ERK signaling as a reading of FGF receptor activity, which is triggered by the co-binding of FGF23 and α-Klotho.

[0400] Use 10 mL of Ca-free 2+ and Mg 2+ Wash the cultured NIH3T3 cells (ATCC) twice with PBS. Add 4 mL of TrypLE TM (ThermoFisher) was added to the washed cells, and the cells were incubated at 37°C and 5% CO2 for 5 minutes. 10 mL of PBS was added to the cells, and the cell clumps were broken up by repeated pipetting. The dissociated cell mixture was transferred to a 50 mL conical tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in 1 mL of assay medium (OptiMEM containing 0.5% BSA) by repeated pipetting. Next, 10 mL of assay medium was added to the resuspended cells, and the cells were counted using AutoT4. The cells were diluted to 2.0 x 10⁻⁶ cells in the assay medium. 5 Cells / mL were seeded at 20,000 cells / well in a 96-well plate and incubated overnight at 37°C and 5% CO2.

[0401] On the second day, replace the medium with 50 µL of assay medium per well and incubate the plate at 37°C, 5% CO2 for 2 hours. Dilute the KL peptide to 80 nM in the assay medium and further dilute 1:2. Dilute FGF23 and anti-pERK antibody to 40 nM and 400 nM, respectively, in the assay medium. Pretreat the wells with the antibody for 10 minutes. Add the KL peptide diluent and FGF23 to the corresponding wells and incubate the plate at 37°C, 5% CO2 for 15 minutes. Replace the supernatant in each well with 30 µL of NP40 lysis buffer containing a mixture of protease and phosphatase inhibitors. The HTRF assay procedure was performed according to the manufacturer's Advanced Phospho-ERK (Thr202 / Tyr204) Cell HTRF Kit Protocol (CiSBio).

[0402] 9.1.5. SRE / ERK luciferase assay

[0403] The HEK293.FGFR1KO.hFGFR1c.Sre-Luc report cell line was treated with the KL peptide of interest for 5 hours.

[0404] 9.1.6.3 T3 cell proliferation assay

[0405] 3T3 cells were maintained in culture according to the supplier's recommendations. For assays, cells were seeded in 96-well plates with culture medium and cultured at 37°C and 5% CO2 for 24–48 hours until use. Next, cells were treated with either KL peptides in the culture medium or a control substance. Cell proliferation was monitored every three hours for 66 hours and expressed as a percentage of confluence. Untreated cells served as a control.

[0406] 9.2. Example 1: Sequence Engineering of KL Peptides

[0407] To engineer the KL amino acid sequence for efficient production of bioactive KL peptides, one or more modifications are introduced into... Figure 3 The full-length hKL sequence is shown as described in Section 9.1.1. The resulting peptide was purified using a His affinity column as described in Section 9.1.2.1, followed by SEC, and the bioactivity of the purified KL peptide was assessed using pERK HTRF assay as described in Section 9.1.4.

[0408] Four distinct KL peptide sequences (i.e., KL981, KL958, KL958 C521S, and KL958 C521S C910S) were engineered as follows: KL981 was generated by replacing its native signal sequence corresponding to the first 33 amino acids with the mROR1 signal sequence (SS). KL958 was generated by truncating KL981 at the C-terminus to remove the C-terminal loop. KL958 C521S was generated by replacing the Cys residue at position 521 of KL958 with Ser. KL958 C521S C910S was generated by replacing the Cys residue at position 910 of KL958C521S with Ser. A C-terminal His tag was added to all four constructs.

[0409] First, the yield and purity of each construct were evaluated. The yield of KL958 C521S was approximately 5-7 times that of the other three constructs. Figure 4A Compared to KL981, the truncated construct at the C-terminus has a lower level of high molecular weight species (HMW). Figure 4A ).

[0410] To determine whether sequence engineering affects KL bioactivity, changes in phospho-ERK (pERK) levels in NIH3T3 and NHDF cells after co-treatment with KL and FGF23 were measured and quantified as fold changes in pERK relative to KL treatment alone. A positive control KL protein (hKL) was used for comparison. All four sequence-engineered constructs were associated with increased fold changes in pERK in NIH3T3 and NHDF cells (respectively). Figure 4B and 4C Of the four engineered constructs, KL958C521S showed the highest fold change in pERK across both cell lines, exceeding the fold change observed with the positive control hKL protein.

[0411] In summary, C-terminal truncation combined with the C521S mutation not only resulted in a relatively high yield of KL peptides and a low HMW level, but was also associated with enhanced biological activity.

[0412] 9.3. Example 2: The effect of purification pH on the yield and activity of KL peptides

[0413] To evaluate the effects of different affinity columns on the yield and activity of KL peptides, a novel dual-labeled construct, KL958 (C521S C910S)-HSA-His, was designed and generated as described in Section 9.1.1. The resulting peptides were purified using either a His or HSA affinity column as described in Section 9.1.2.1, followed by SEC assay, and the bioactivity of the purified KL peptides was evaluated using pERK HTRF as described in Section 9.1.4.

[0414] The yield and purity of the dual-labeled KL peptide KL958 (C521S C910S)-HSA-His were evaluated by SDS-PAGE under non-reducing (NR) and reducing (R) conditions using 10 µL of purified peptide sample per well. Although HSA affinity column purification achieved significantly higher yields (i.e., thicker bands at approximately 160 kDa under reducing conditions), it was associated with higher molecular weight aggregates (i.e., smears above approximately 160 kDa under non-reducing conditions). Figure 5A And it did not show activity because the pERK HTRF signal in KL+ FGF23 co-treatment was lower than the background signal of cells alone. Figure 5B and 5C Purification of the same construct using a His affinity column resulted in higher pERK HTRF signaling in cells co-treated with FGF23. Figure 5C Considering the different pH values ​​of the elution buffers, these results indicate that the acidic pH of the HSA elution buffer inactivates the purified KL peptides.

[0415] 9.4. Example 3: Effects of different C-terminal tags on the yield and purity of KL peptides

[0416] KL peptide constructs were designed by linking different C-terminal tags to REGN 14226 (KL958 C521S) and generated as described in Section 9.1.1. The resulting peptides were purified using the Twin-Strep purification method as described in Section 9.1.2.2.

[0417] This example uses a set of four KL peptide constructs. KL958(C521S)-HSA contains a C-terminal HSA and a Twin-Strep tag ( Figure 6A KL958(C521S)-Fc contains a C-terminal Fc and a Twin-Strep tag ( Figure 6B KL958(C521S)-Fc1.5 contains a C-terminal Fc1.5 and a Twin-Strep tag, where Fc1.5 contains one CH2 region and two CH3 regions ( Figure 6C); and KL958(C521S)-moFc contains C-terminal moFc and Twin-Strep tags ( Figure 6D ).

[0418] After purification of the twin-strep peptide, the samples were treated with 3C protease to remove the twin-strep tag. The yield and purity of the 3C-treated and untreated constructs were assessed by SDS-PAGE under both reducing and non-reducing conditions. The 3C-protease-treated and untreated KL peptide constructs resulted in relatively thick bands at slightly lower molecular weights in each group, indicating that protease treatment successfully removed the twin-strep tag from each peptide construct. Figure 7A KL958(C521S)-HSA and KL958(C521S)-Fc1.5 achieved the highest yields, while KL958(C521S)-Fc was associated with high molecular weight aggregation. Figure 7A ).

[0419] The yield and purity of KL peptide constructs containing Fc, Fc1.5, moFc, or HSA tags were also evaluated using SEC. Consistent with SDS-PAGE results, the Fc-tagged KL peptides exhibited the highest HMW peak, while the peak corresponding to the KL958 C521 peptide was relatively smaller. Figure 7B The yields of MoFc- and Fc1.5-labeled Kl peptides were higher than those of Fc-labeled peptide constructs; however, the highest yield and lowest HMW level were achieved using HSA-labeled KL peptide constructs. Figures 7C-7E ).

[0420] 9.5. Example 4: Enhanced purification of HSA-labeled KL peptides

[0421] The experiments in Example 3 showed that the yield of HSA-labeled KL peptides was higher than that of KL peptides with other tags. However, the results in Example 2 indicated that HSA affinity purification using a low-pH elution buffer rendered the HSA-labeled KL peptides biologically inactive. The aim of the experiments in this example was to enhance the purification of the biologically active KL958(C521)-HSA peptide.

[0422] First, as described in Section 9.1.2.3, a modified high-salt purification method was used, where HSA-labeled KL peptides in 500 mL of sample were bound to HSA affinity resin, and the protein was eluted from the resin with a high-salt buffer at pH 7.4. Next, HMW, which accounted for approximately 75% of the total yield, was removed using SEC. SDS-PAGE analysis was performed to determine the concentrations of reduced and non-reduced HMW (…). Figure 8B Peak 1) and eluted KL peptide ( Figure 8BPeak 2) fractions were observed. Under non-reducing conditions, thin bands corresponding to the monomeric KL peptide were observed in the HMW lane, while under reducing conditions, the amount of monomeric KL peptide in the HMW lane was comparable to the amount of eluted KL peptide in the lane. Figure 8A This indicates that most of the peptides in the HMW fraction are aggregates of KL peptides. The final yield of monomeric KL peptides purified by this method was 8 mg (16 mg / L).

[0423] To further enhance the large-scale purification of KL peptides, an improved ion exchange (IEX) purification method as described in Section 9.1.2.4 was used, which involves three steps: a Q-agarose step to concentrate the KL peptides ( Figure 9A and 9B ), and then captured the KL peptide with HSA affinity resin ( Figure 9C and 9D Then, the SD-200 step is performed to further remove any remaining contaminants. Figure 9E and 9F Using this method, KL958 (C521)-HSA was purified from a 25L sample to yield approximately 5 mg / L of monomeric KL peptide.

[0424] 9.6. Example 5: Pharmacokinetic profiles of His- and HSA-labeled KL peptides

[0425] As described in Section 9.1.3, the pharmacokinetic properties of His-labeled and HSA-labeled KL peptides were evaluated in mice. Briefly, mice received a single injection of one of three doses of KL958 (C521)-HSA (3, 10, or 30 mg / kg) or 20 mg / kg KL958 (C521)-His. Positive control mice received a single injection of 3 mg / kg control mAb.

[0426] Eight hours after administration, KL958 (C521)-His was undetectable, indicating that the peptide was cleared relatively rapidly. At each dose evaluated, KL958 (C521)-HSA exhibited a slower clearance rate than His-labeled KL peptides. Figure 10A Dosage normalization of the data revealed that although the HSA-labeled KL peptides exhibited similar clearance rates at all three doses, clearance was slightly faster at 3 and 10 mg / kg after 24 hours. Figure 10B ).

[0427] 9.7. Example 6: Effects of His- and HSA-labeled KL peptides on 3T3 cell proliferation

[0428] As described in Section 9.1.6, the proliferative effects of His-tagged and HSA-tagged KL peptides KL958 (C521)-His and KL958 (C521)-HSA were evaluated.

[0429] When co-incubated with 10 nM FGF23, both KL peptides promoted 3T3 cell proliferation. However, KL958(C521)-His was more potent than KL958(C521)-HSA. Figure 11A and 11B Next, KL958 (C521)-His and KL958 (C521)-HSA at the same concentration (20 nM) were added to cells with or without 10 nM FGF23. Both KL peptides promoted 3T3 cell proliferation in the presence of FGF23, but the confluence percentage of the KL958 (C521)-His construct was significantly higher. Figure 11C ).

[0430] 9.8. Example 7: Differences in C-terminal truncation affect the activity of KL peptides

[0431] The experiments in the previous examples evaluated the activity of the KL958 peptide, derived from full-length human KL981 by truncating 23 amino acids at the C-terminus. To determine whether a slightly different length construct would yield a construct with similar activity, the KL peptide KL961(C521S)-HSA, truncated with 20 amino acids at the C-terminus, was designed and generated, as described in Section 9.1.1. The activities of KL958(C521S)-HSA and KL961(C521S)-HSA were measured, as described in Section 9.1.5.

[0432] Both KL958 (C521S)-HSA and KL961 (C521S)-HSA showed activity; however, KL958 (C521S)-HSA had a higher signal amplitude. Figure 12 Similarly, KL958 (C521S)-HSA is more efficient, with an EC50 value of 2.8E-08 M, which is about an order of magnitude higher than the EC50 value obtained with KL961 (C521S)-HSA.

[0433] 9.9. Example 8: Targeted sequence engineering to enhance KL peptide yield and activity

[0434] A new set of KL-peptides was designed by introducing additional mutations into the HSA-tagged KL958 C521S. Figures 13A-13FIt was generated as described in Section 9.1.1 and purified as described in Section 9.1.2.2. The activity of the construct KL958 (C521S C370S)-G4S-HSA was evaluated as described in Section 9.1.5. Figure 13D And compared with the activity of KL958 (C521)-HSA ( Figure 13A ).

[0435] Compared to KL958 (C521)-G4S-HSA, KL958 (C521S C370S)-G4S-HSA has a lower EC50 value and a better yield (Table E3).

[0436]

Claims

1. A polypeptide, wherein: (a) Contains an α klotho KL2 domain, which (i) has at least about 80% sequence identity with SEQ ID NO:3, and (ii) has an amino acid substitution at the position of amino acid C521 corresponding to SEQ ID NO:1; as well as (b) At the amino acid position corresponding to amino acid C970 of SEQ ID NO:1, if said amino acid is present, cysteine ​​is lacking.

2. The polypeptide according to claim 1, wherein the amino acid corresponding to amino acid C970 of SEQ ID NO:1 is missing.

3. The polypeptide according to claim 1, wherein, if the amino acid corresponding to C973 of SEQ ID NO:1 is present, it lacks cysteine, and optionally, it lacks the amino acid corresponding to amino acid C973 of SEQ ID NO:

1.

4. The polypeptide according to claim 1, wherein, if the amino acid corresponding to C963 of SEQ ID NO:1 is present, it lacks cysteine, and optionally, it lacks the amino acid corresponding to amino acid C963 of SEQ ID NO:

1.

5. The polypeptide according to any one of claims 1 to 4, wherein the amino acid at the position corresponding to amino acid C521 of SEQ ID NO:1 is substituted with a mutation from cysteine ​​to serine.

6. The polypeptide according to any one of claims 1 to 5, wherein, compared with the amino acid sequence of SEQ ID NO:13, it comprises a C-terminal deletion of at least 21 amino acids or at least 23 amino acids.

7. The polypeptide according to any one of claims 1 to 6, wherein it lacks an amino acid sequence having at least 95%, at least 98%, or 100% sequence identity with SEQ ID NO:

8.

8. The polypeptide according to any one of claims 1 to 7, wherein the α klotho KL2 domain has at least about 95% sequence identity with SEQ ID NO:

3.

9. The polypeptide according to any one of claims 1 to 8, wherein the α klotho KL2 domain has at least about 99% or 100% sequence identity with SEQ ID NO:

4.

10. The polypeptide according to any one of claims 1 to 9, further comprising an α klotho KL1 domain having at least about 80%, at least about 95%, at least about 99%, or 100% sequence identity with SEQ ID NO:

9.

11. The polypeptide of claim 10, wherein the α klotho KL1 domain has at least about 80%, at least about 95%, at least about 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:

10.

12. The polypeptide according to claim 10 or 11, wherein the α klotho KL1 domain comprises an amino acid substitution at position C370 corresponding to SEQ ID NO: 1, wherein the amino acid substitution is optionally a substitution from cysteine ​​to serine.

13. The polypeptide according to any one of claims 1 to 12, comprising an amino acid sequence having at least about 99% or 100% sequence identity with SEQ ID NO:

12.

14. The polypeptide according to any one of claims 1 to 13, comprising an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:

15.

15. The polypeptide according to any one of claims 1 to 14, comprising an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO:

16.

16. The polypeptide according to any one of claims 1 to 15, wherein the length of the amino acid sequence is between 900 and 925 amino acids.

17. The polypeptide according to any one of claims 1 to 16, wherein the amino acid sequence of SEQ ID NO:17 is missing.

18. The polypeptide according to any one of claims 1 to 17, further comprising a signal peptide.

19. The polypeptide according to any one of claims 1 to 18, further comprising a stable portion located at the C-terminus of the α klotho KL2 domain.

20. The polypeptide of claim 18, further comprising a stable portion located at the N-terminus of the α klotho KL2 domain.

21. The polypeptide according to claim 19 or 20, wherein the stable portion comprises an Fc domain comprising a CH2 domain and a CH3 domain, and the Fc domain optionally comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:23 or SEQ ID NO:

26.

22. The polypeptide of claim 21, wherein the Fc domain further comprises an additional CH3 domain connected to the CH3 domain via a linker.

23. The polypeptide of claim 21, wherein the Fc domain is a non-dimerized Fc domain, optionally comprising an amino acid sequence having at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO:27-32.

24. The polypeptide according to claim 19 or 20, wherein the stable portion is an albumin portion.

25. The polypeptide of claim 24, wherein the albumin portion is human serum albumin.

26. The polypeptide of claim 24, wherein the albumin portion is a human serum albumin variant, optionally having an amino acid substitution C34S.

27. The polypeptide of claim 26, wherein the albumin portion comprises the amino acid sequence of SEQ ID NO:

20.

28. The polypeptide according to any one of claims 19 to 27, further comprising a linker, optionally a linker comprising the sequence listed in Table L.

29. The polypeptide of claim 28, wherein the polypeptide comprises, in order from N-terminus to C-terminus: an α klotho portion comprising the KL2 domain, the linker, and the stabilizing portion.

30. The polypeptide of claim 28, wherein the polypeptide comprises, in order from N-terminus to C-terminus: the stabilizing portion, the linker, and the α klotho portion comprising the α klotho KL2 domain.

31. The polypeptide according to any one of claims 1 to 30, wherein the polypeptide is a monomer.

32. The polypeptide according to any one of claims 1 to 30, wherein the polypeptide is a dimer.

33. A polypeptide, optionally a polypeptide according to any one of claims 1 to 32, comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:16, wherein the position corresponding to amino acid 488 of SEQ ID NO:16 is not cysteine, and which lacks an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

8.

34. The polypeptide according to claim 33, wherein the position of amino acid 488 corresponding to SEQ ID NO:16 is serine.

35. The polypeptide according to claim 33 or 34, comprising the amino acid sequence of SEQ ID NO:

16.

36. The polypeptide according to any one of claims 33 to 35, wherein the amino acid sequence of SEQ ID NO:8 is missing.

37. The polypeptide according to any one of claims 1 to 36, comprising the amino acid sequence of SEQ ID NO:

62.

38. A nucleic acid encoding a polypeptide according to any one of claims 1 to 37.

39. A host cell engineered to express a polypeptide according to any one of claims 1 to 37 or a nucleic acid according to claim 38.

40. A method for producing a polypeptide according to any one of claims 1 to 37, the method comprising culturing a host cell according to claim 39, and recovering the polypeptide expressed therefrom.

41. The method of claim 40, further comprising purifying the polypeptide.

42. The method of claim 41, wherein the polypeptide is purified at a pH between 6.9 and 7.

1.

43. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 37 and an excipient.

44. A method for activating FGFR signaling in a cell, the method comprising contacting the cell with a polypeptide according to any one of claims 1 to 37.

45. The method of claim 44, wherein the cell is a kidney cell.

46. ​​The method according to claim 44 or 45, wherein the method is an in vitro method.

47. The method according to claim 44 or 45, wherein the method is an in vivo method.

48. A method for preventing age-related conditions in a subject or treating a subject suffering from age-related conditions, the method comprising administering to the subject a polypeptide according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 43.

49. A method for preventing age-related symptoms, the method comprising administering to a subject in need of the polypeptide according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 43.

50. A method for preventing kidney disease in a subject or treating a subject suffering from kidney disease, the method comprising administering to the subject a polypeptide according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 43.

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