Stabilized FGF23 fusion proteins, compositions and a targeted therapy for hyperphosphatemia

Abstract

Provided are novel modified humanized bioactive FGF23 fusion proteins, compositions comprising such fusion proteins, and methods for making such fusion proteins for the treatment of an individual with hyperphosphatemia. Also contemplated are methods for treating hyperphosphatemia in an individual.

Description

STABILIZED FGF23 FUSION PROTEINS, COMPOSITIONS AND A TARGETED THERAPY FOR HYPERPHOSPHATEMIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 484,284 filed on February 10, 2023, which is incorporated by reference in its entirety.FIELD OF DISCLOSURE

[0002] The present disclosure relates to the field of fibroblast growth factor-23 (FGF23) analogs and compositions containing such analogs for treating a hyperphosphatemia.REFERENCE TO A SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML copy, created on February 6, 2024 is named "IU202109401W02" and is 56,773 bytes in size.BACKGROUND

[0004] Phosphate is essential for many cellular processes, including as a cornerstone molecule for nucleic acid synthesis, energy metabolism, and mineralized calcium-phosphate hydroxyapatite formation to form the skeleton. Control of blood phosphate occurs through endocrine regulation in the skeleton, kidneys, and intestine. Disturbance in blood phosphate levels in the feedback loops controlling phosphate levels can cause severe disorders.

[0005] The phosphaturic hormone, fibroblast growth factor-23 (FGF23), is a peptide hormone that is secreted in response to dietary phosphorus load. The main function of FGF23 is to promote urinary phosphate excretion and to suppress active vitamin D (1,25D) production in the kidney. FGF23 can be detected at low levels in many tissues, including heart, liver,thyroid / parathyroid, and small intestine; it is predominantly expressed in bone by osteoblasts, osteocytes, flattened bone lining cells, and osteoprogenitor cells (ADHR Consortium.,“Autosomal dominant hypophosphatemic rickets is associated with mutations in FGF23,” Nat. Genet., 2000, 26(3): 345-348).

[0006] Secreted FGF23 travels to the kidney via the bloodstream. FGF23 binds to a complex of an FGF receptor (FGFR) and its co-receptor, alpha-Klotho. Formation of the three-component complex triggers a signal cascade in the kidney to reduce phosphate reabsorption by downregulating kidney phosphate transporters.

[0007] FGF23 down regulates the vitamin D 1 alpha-hydroxylase (also known as cytochrome p450 27B1 (CYP27B1) and Cyp27bl) that produces 1,25(OH)2 vitamin D, the active form of vitamin D that increases phosphate absorption in the intestines. In parallel with regulating renal phosphate transport, FGF23 also stimulates the vitamin D 24-hydroxylase (encoded by gene Cyp24al), which inactivates 1,25(OH)2 vitamin D. Thus, FGF23 acts via the kidney to lower blood phosphate and 1,25(OH)2 vitamin D. Reduction of both blood phosphate and 1,25(OH)2 vitamin D level in turn inhibits bone production of FGF23. Excess FGF23 in disorders such as X-linked hypophosphatemia (XLH) and autosomal dominant hypophosphatemic rickets (ADHR) leads to hypophosphatemia, osteomalacia, and rickets.

[0008] The biochemical converse disease to hypophosphatemic rickets is hyperphosphatemic familial tumoral calcinosis (hfTC). Human fTC (hfTC) is a rare, genetically heterogeneous disorder due to mutations in the FGF23 gene or in genes that control FGF23 intracellular processing, including the O-glycosylating enzyme, GALNT3. Patients with hfTC have mutations in the FGF23 gene or in genes that effectively modify the FGF23 protein intracellularly. The FGF23 mutant proteins produce very low concentrations of full-length, bioactive FGF23 (alsoknown as intact bioactive FGF23). These patients show increased reabsorption of phosphate into the bloodstream and very high phosphate absorption in the intestines given high levels of 1,25(OH)2 vitamin D. The high phosphate in the bloodstream then binds calcium in these patients. The phosphate to calcium binding precipitates in blood vessels causing calcifications and enlarged tissues that may appear tumor like (tumoral), but are not tumors or cancerous.

[0009] hfTC is characterized by reduced circulating bioactive ‘intact’ FGF23, markedly elevated blood phosphate, and the development of large, often painful, ectopic and vascular calcifications. Patients develop large, extremely disabling calcified tumoral masses in soft tissues up to two to four pounds in size, and can undergo amputation of the digits due to severe vascular calcifications and loss of blood flow.

[0010] Currently, there are no FDA approved drugs for the treatment of hfTC in a subject. The only available treatments are anecdotal off-label therapies, such as oral phosphate binders that provide very limited relief from hyperphosphatemia, and do not ablate calcification formation or recurrence. Another problem with anecdotal off-label therapies is that they can induce idiopathic hyperphosphatemia. Surgery is not an effective method for the removal of the tumoral masses because many tumoral masses cannot be completely removed or accessed by surgical means. Moreover, surgeries fail to stop formation / recurrence of tumoral masses.

[0011] There is a need for an effective treatment for this genetic disorder, given the shortcomings of the existing treatments.SUMMARY

[0012] The present disclosure provides a fusion protein including a bioactive fibroblast growth factor-23 (FGF23) analog and an FcRn binding partner. The FGF23 protein includes a first mutation at residue 176 and a second mutation in the Fam20c kinase site. Further, the FcRnbinding partner extends the half-life of the bioactive FGF23 analog. In another embodiment of the fusion protein, the first mutation is R176Q and the second mutation is S180A. In an additional embodiment of the fusion protein, the FcRn binding partner is an Fc region of IgG. In an additional aspect of the fusion protein of the additional embodiment, the FGF23 analog is attached to the Fc region using at least one GGGS linker. In an additional embodiment of the fusion protein, the amino acid sequence has at least 98% sequence identity with SEQ ID NO: 9.

[0013] The present disclosure provides a composition including: a fusion protein including a bioactive fibroblast growth factor-23 (FGF23) analog and an FcRn binding partner; and a pharmaceutically acceptable excipient. The FGF23 protein includes a first mutation at residue 176 and a second mutation in the Fam20c kinase site. Further, the FcRn binding partner extends the half-life of the bioactive FGF23 analog.

[0014] The present disclosure further provides a composition including: a fusion protein including a bioactive fibroblast growth factor-23 (FGF23) analog and an FcRn binding partner; and a pharmaceutically acceptable excipient. The FGF23 protein includes a first mutation at residue 176 and a second mutation in the Fam20c kinase site. Further, the FcRn binding partner extends the half-life of the bioactive FGF23 analog and the first mutation is R176Q and the second mutation is S180A.

[0015] The present disclosure provides a composition including: a fusion protein including a bioactive fibroblast growth factor-23 (FGF23) analog and an FcRn binding partner; and a pharmaceutically acceptable excipient. The FGF23 protein includes a first mutation at residue 176 and a second mutation in the Fam20c kinase site. Further, the FcRn binding partner extends the half-life of the bioactive FGF23 analog and the FcRn binding partner is an Fc region of IgG. In a different embodiment of the composition, the FGF23 analog is derived from a human FGF23or a primate FGF23. In a further embodiment of the composition, the composition further includes a pharmaceutically-acceptable carrier and / or a stabilizer. In an embodiment of the composition, the composition is formulated for oral administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, implantation, intraarterial administration, or transdermal administration.

[0016] The present disclosure provides a medicament including any one of the previously described fusion proteins, or the composition of any one of previously described compositions for use in treating an individual with hyperphosphatemia.

[0017] The present disclosure provides a method of treating a subject with hyperphosphatemia including administering to the subject any one of the fusion proteins previously described or any one of the compositions previously described. In another embodiment of the method, the fusion protein comprises an FGF23 analog, wherein the FGF23 analog is attached at the N-terminus or the C-terminus to the Fc region of IgG by at least one GGGS linker. In a further embodiment of the method, the FGF23 analog is a human FGF23 analog. In an additional embodiment of the method, the subject is a human. In another embodiment of the method, the hyperphosphatemia is a hyperphosphatemic familial tumoral calcinosis (hfTC), an idiopathic hyperphosphatemia, or a condition associated with absolute or relative deficiency of a circulating biointact FGF23. In an additional embodiment of the method, the administering step is performed orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, transdermally, or by application to mucous membranes. In a further embodiment of the method, the fusion protein is formulated with apharmaceutically acceptable carrier. In an embodiment of the method, the fusion protein is a bioactive FGF23 analog including the amino acid sequence of SEQ ID NO: 9, which is administered to the subject in an amount sufficient to lower blood phosphate concentrations in the subject as compared to a blood phosphate concentration assessed in the subject prior to administration of the bioactive FGF23 analog.

[0018] The present disclosure provides an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises an FGF23 signal peptide domain and an FGF23 polypeptide. In an embodiment of the nucleic acid, the nucleic acid further includes an operably linked nucleic acid encoding an Fc region of IgG and a nucleic acid encoding at least one glycine serine (GS) linker. In a further embodiment of the nucleic acid, the nucleic acid encoding Fc is operably located between the nucleic acid encoding a FGF23 signal peptide and a FGF23 polypeptide. In a different embodiment of the nucleic acid, the nucleic acid comprises in 5’ to 3’ order an operably linked promoter, a nucleic acid encoding the FGF23 signal peptide, the FGF23 polypeptide, at least one GS linker, and an Fc nucleic acid.

[0019] The present disclosure provides a polynucleotide including a nucleic acid sequence that has at least 85% sequence identity to the entire length of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO:30, or the complement thereof.

[0020] The present disclosure provides a vector including the nucleic acid sequence that has at least 85% sequence identity to the entire length of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQID N0:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO: 30, or the complement thereof.

[0021] The present disclosure provides a host cell comprising a vector including the nucleic acid sequence that has at least 85% sequence identity to the entire length of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or the complement thereof. In an embodiment of the host cell, the host cell is an E. coli cell.

[0022] The present disclosure provides a host cell including a nucleic acid including an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein includes an FGF23 signal peptide domain and an FGF23 polypeptide.

[0023] The present disclosure provides a protein encoded by a nucleic acid including an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein includes an FGF23 signal peptide domain and an FGF23 polypeptide.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] FIG. 1. Depicts FGF23 structure and mutagenesis for the double mutant design;

[0026] FIG. 2. Depicts FGF23-Fc constructs for humanized analogs. The constructs for the both double mutant ADHR +Fam20c designs, R176Q + S180G, and R176Q + S180A, are linkedat the C-terminus to an Fc region using one GlyGlyGlySer (‘GGGS’ represented in SEQ ID NO: 15) linker sequence;

[0027] FIG. 3 - Immunoblot analysis of purification of double mutant FGF23-Fc (R176Q and S180G);

[0028] FIG. 4 - Immunoblot analysis of purification of double mutant FGF23-Fc (R176Q and SI 80 A);

[0029] FIG. 5 - Immunoblot analysis of purification of triple mutant having the S180A mutation as well as two ADHR mutations, R176Q, R179Q;

[0030] FIG. 6 - Bioactivity assay of triple mutant having the S180A mutation as well as twoADHR mutations, R176Q, R179Q (designated FGFFclOA);

[0031] FIG. 7 - Depicts FGF23-Fc constructs for humanized analogs. The constructs for the FGF23-RQ (having only the R176Q mutation), are linked at the C-terminus to an Fc region using one GlyGlyGlySer (‘GGGS’ represented in SEQ ID NO:15) linker sequence (FGF-Fcl) or two GlyGlyGlySer (‘GGGSGGGS’ represented in SEQ ID NO: 16) linker sequences (FGF-Fc2);

[0032] FIG. 8. - Immunoblot analysis after mammalian cell transfection of FGF23-RQ control protein and FGF23-Fc analogs is shown;

[0033] FIG. 9 - Human C-terminal assay after a 24h transfection;

[0034] FIG. 10 - In vitro bioactivity assay (p-ERKl / 2 activation) using conditioned media from FGF-Fc and control transfected cells;

[0035] FIG. 11 - Immunoblot analysis after mammalian cell transfection of FGF23-RQ Fc fusion protein is shown;

[0036] FIG. 12 - ELISA assay comparing ADHR FGF23-Fc as compared to wild type FGF23 unconjugated protein at 24 and 48 hours;

[0037] FTG. 13 A-D - Bioactivity of dual mutants. Analysis on FGF23 double mutants impact on EGR1 (FIG 13A and FIG 13B) and CYP24al (FIG 13C) and CYP27bl (FIG 13D).

[0038] FIG. 14. - Depicts FGF23-ABD constructs for humanized analogs. All FGF23-ABD constructs were designed to contain the ‘R176Q’ mutation. The construct for the “parent” is not linked to an ABD tag. It only contains the naturally-occurring human R176Q mutation. The Nl, N2, Cl, and C2 human FGF23 sequences are linked to an ABD sequence (‘Albumod’) at the N- or C-terminus using one or two GlyGlyGlySer (‘GGGS’) linker sequences, as represented by SEQ ID NO: 15 and SEQ ID NO: 16, respectively, to form FGF23-ABD ‘Nl’, ‘N2’, ‘Cl ’, and ‘C2’ analogs;

[0039] FIG. 15. Immunoblot analysis after mammalian cell transfection of untagged FGF23- RQ control protein and FGF23-ABD analogs is shown. The FGF23-ABD analogs expressing the ABD tag had a predictably ~5 kD higher (~34 kD total) molecular mass than the untagged FGF23 counterpart (~29 kD);

[0040] FIG. 16. Human C-terminal FGF23 ELISA assay results are shown. The ELISA assay compares the level of FGF23 in the media of all N- and C-terminal FGF23-ABD analogs to the FGF23-RQ control protein and the media of non-transfected (‘Non-Tf) cells;

[0041] FIG. 17. Human FGF23 ‘Intact’ ELISA assay results are shown. The human FGF23 ‘Intact’ ELISA (Kainos, Inc.) recognizes whole molecule, bioactive “intact” FGF23. The ELISA assay compares the level of FGF23 in the media of all N- and C-terminal FGF23-ABD analogs with the FGF23-RQ control protein and the media of non-transfected (‘Non-Tf) cells;

[0042] FIG. 18. Early growth response gene-1 (EGR1) mRNA Response as a Bioassay for media containing FGF23 constructs is shown. The assay compares the level of EGR1 mRNA inthe media of all N- and C-terminal FGF23-ABD analogs with FGF23-RQ control protein and media of non-transfected cells / non-treated cells;

[0043] FIG. 19A illustrates the initial SUMO-FGF23-RQ expression using an SDS-PAGE gel. FIG. 19B illustrates the digestion of SUMO-FGF23-RQ on an SDS-PAGE gel. FIG. 19C illustrates the purified FGF23-RQ protein on an SDS-PAGE gel;

[0044] FIG. 20A illustrates the initial SUMO-FGF23-ABD-C2 expression on an SDS-PAGE gel. FIG. 20B illustrates the digestion of SUMO-FGF23-ABD-C2 on an SDS-PAGE gel. FIG. 20C illustrates the purified FGF23-ABD-C2 protein on an SDS-PAGE gel;

[0045] FIG. 21. Immunoblot analysis with anti-human FGF23 antibodies is shown. The immunoblot compares the molecular masses and purity of the FGF23-RQ and FGF23-ABD-C2 proteins;

[0046] FIG. 22. Immunoblot analysis with p-ERKl / 2 & total-ERK is shown. The immunoblot compares the stimulation of pERKl / 2 by FGF23-ABD-C2 and untagged FGF23- RQ proteins;

[0047] FIG. 23. RT-qPCR analysis of FGF23-ABD-C2 and untagged FGF23-RQ protein is shown. RT-qPCR compares the stimulated production of EGR1 mRNA by FGF23-ABD-C2 with untagged FGF23-RQ protein in HEK-mKL cells;

[0048] FIG. 24A illustrates RT-qPCR analysis comparing the level of Egrl mRNA production. FIG. 24B illustrates RT-qPCR analysis comparing the level of catabolic renal vitamin D 24-hydroxylase (Cyp24al) production. FIG. 24C illustrates RT-qPCR analysis comparing the level of anabolic vitamin D 1 alpha-hydroxylase (Cyp27bl) production. RT-qPCR compares the RNA extract from mice injected with FGF23-RQ with RNA extracted from mice injected with FGF23-ABD-C2;

[0049] FIG. 25. Results of in vitro binding assays of human (HSA) and mouse (MSA) serum albumin to FGF23-ABD and FGF23-ABD-C2 are shown;

[0050] FIG. 26A. Results of in vitro binding assays of human (HSA) serum albumin to FGF23-ABD and FGF23-ABD-C2 are shown. FIG. 26B. Results of in vitro binding assays of mouse (MSA) serum albumin to FGF23-ABD and FGF23-ABD-C2 are shown. The assay tests whether the binding is dose dependent;

[0051] FIG. 27. Optimized DNA sequence and corresponding encoded polypeptide of Construct 1 (FGF23-ABD-N1): DNA sequences represented in SEQ ID NOs: 17 and 18; amino acid sequence represented in SEQ ID NO: 19;

[0052] FIG 28. Optimized DNA sequence and corresponding encoded polypeptide of Construct 2 (FGF23-ABD-N2): DNA sequences represented in SEQ ID NOs:20 and 21; amino acid sequence represented in SEQ ID NO:22;

[0053] FIG 29. Optimized DNA sequence and corresponding encoded polypeptide of Construct 3 (FGF23-ABD-C1): DNA sequences represented in SEQ ID NOs:23 and 24; amino acid sequence represented in SEQ ID NO:25;

[0054] FIG 30. Optimized DNA sequence and corresponding encoded polypeptide of Construct 4 (FGF23-ABD-C2): DNA sequences represented in SEQ ID NOs:26 and 27; amino acid sequence represented in SEQ ID NO:28;

[0055] FIG 31. Optimized DNA sequence and corresponding encoded polypeptide of Construct 5 (FGF23-RQ): DNA sequences represented in SEQ ID NOs:29 and 30; amino acid sequence represented in SEQ ID NO:31;

[0056] FIG. 32. Optimized DNA sequence (SEQ ID NO:8) and corresponding encoded polypeptide of Construct FGFFcl OA (Triple Mutant) represented by SEQ ID NO:7;

[0057] FIG 33. Optimized DNA sequence (SEQ ID NO: 10) and corresponding encoded polypeptide of Construct FGFFc2 OA (Double Mutant) represented by SEQ ID NO: 9;

[0058] FIG 34. Optimized DNA sequence (SEQ ID NO: 12) and corresponding encoded polypeptide of double mutant FGFCFc OG (Double Mutant) represented by SEQ ID NO: 11;

[0059] FIG 35. Optimized DNA (SEQ ID NO: 14) and corresponding encoded polypeptide of Construct FGFFcl represented by SEQ ID NO: 13;

[0060] FIG 36. FGF23-QA2 in vitro production by plasmid (immunoblot with anti-human FGF23 Ab): FGF23-QA2 shows proper molecular mass of 56 kD and high weight Fc-interacting species due to non-reducing conditions under this analysis. Human FGF23(RQ) with no Fc tag was used as the positive control (32 kD);

[0061] FIG 37. FGF23-QA2 from in vitro conditioned media is recognition by the human Intact FGF23 ELISA (control media = no transfection). These results are consistent with the concept that FGF23-QA2 retains native FGF23 structure;

[0062] FIG 38. In vitro FGF23-QA2: HEK-mKL cells treated with 50 ng / mL purified recombinant FGF23-QA2 demonstrate pERKl / 2 signaling similar to non-Fc tagged FGF23 (FGF23-RQ);

[0063] FIG 39A. Wild type mice injected with FGF23-QA2 (4h, 500 ng / g bw) demonstrate appropriate kidney activity on vitamin D metabolizing enzymes similar to non-Fc tagged FGF23 (FGF23-RQ). The 1,25D catabolic enzyme Cyp24al mRNA;

[0064] FIG 39B. Wild type mice injected with FGF23-QA2 (4h, 500 ng / g bw) demonstrate appropriate kidney activity on vitamin D metabolizing enzymes similar to non-Fc tagged FGF23(FGF23-RQ). The 1,25D anabolic enzyme Cyp27bl mRNA;

[0065] FIG 40. FGF23-QA2 double mutant versus human FGF23 (FGF23-RQ) with no Fc tag, one 0.5 ng / g bw i.p. injection for 24 hours. Two human FGF23 ELISAs detect stabilizedFGF23-QA2 in plasma supporting increased half life;

[0066] FIG 41 A. FGF23-QA2 (1 ng / g bw, 3 hours) rescues kidney enzyme mRNA in pre- clinical hfFC model FGF23-KO mice in vivo and stimulates Egrl mRNA short term;

[0067] FIG 41B. FGF23-QA2 (1 ng / g bw, 3 hours) rescues kidney enzyme mRNA in pre- clinical hfTC model FGF23-KO mice in vivo and appropriately reduces basally elevated 1,25D anabolic enzyme Cyp27bl mRNA;

[0068] FIG 41C. FGF23-QA2 (1 ng / g bw, 3 hours) rescues kidney enzyme mRNA in pre- clinical hfTC model FGF23-KO mice in vivo and increases 1,25D catabolic Cyp24al mRNA;

[0069] FIG 42. FGF23-QA2 reduces serum phosphate in FGF23-KO mice in vivo over an extended time course: (1 or 2 pg / injection 2x per day for 3 d);

[0070] FIG 43 A. FGF23-QA2 Rescues kidney enzymes in FGF23-KO mice in vivo over an extended time course: (1 or 2 pg / injection 2x per day for 3 d). Delivery of FGF23-QA2 with extended delivery time reduced kidney Egrl mRNA;

[0071] FIG 43B. FGF23-QA2 Rescues kidney enzymes in FGF23-KO mice in vivo over an extended time course: (1 or 2 pg / injection 2x per day for 3 d). Delivery of FGF23-QA2 with extended delivery time increased catabolic 1,25D enzyme Cyp24al;

[0072] FIG 43C. FGF23-QA2 Rescues kidney enzymes in FGF23-KO mice in vivo over an extended time course: (1 or 2 pg / injection 2x per day for 3 d). Delivery of FGF23-QA2 with extended delivery time appropriately reduces anabolic Cyp27bl mRNA to levels not different from wild type (WT) mice; and

[0073] FIG 44. Shows the performance of in vivo half life comparison for single mutation versus double mutation FGF23.DETAILED DESCRIPTION OF THE DISCLOSURE

[0074] This disclosure exploits the use of FGF23 analogs as fusion proteins with a compound, wherein the compound extends the half-life if the FGF23. The disclosure further exploits the use of FGF23 analogs with an FcRn binding partner, including the Fc region and albumin binding domain (ABD), for the treatment of a patient with hyperphosphatemia.

[0075] The present disclosure provides novel fusion proteins for the treatment of a subject suffering from hyperphosphatemia. The present disclosure also provides a novel composition for the treatment of a subject suffering from hyperphosphatemia.

[0076] The present disclosure relates to a method of treatment of a subject with hyperphosphatemia. Also provided is a medicament for use in treating a subject with hyperphosphatemia.

[0077] The present disclosure provides a novel fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and a compound, wherein the compound extends the half-life of the FGF23. The compound can be a polyethylene glycol (PEG), an albumin binding domain, an Fc (human antibody Fc region) binding domain, or any other compound that can extend the half-life of the bioactive FGF23 analogs.

[0078] FGF23 is a 32-kDa protein that is mainly secreted by osteocytes and osteoblasts in bone. Human FGF23 consists of 251 amino acids and includes a signal peptide composed of 24 amino acids in the N-terminal portion of the protein. Additional structural information of Homo sapiens FGF23 can be retrieved, e.g., from Uniprot (Q9GZV9) and other similar databases. A member of the FGF19 subfamily of FGFs, FGF23 plays a key role in balancing mineral ionhomeostasis and bone mineralisation. For example, FGF23 suppresses phosphate reabsorption and vitamin D hormone synthesis in the kidney. Its synthesis and physiological functions are described, e.g., by Noonan & White, Curr Mol Biol Rep. 2019 Mar;5(l): 18-25 and Erben, Front Endocrinol (Lausanne). 2018; 9: 267.

[0079] The present disclosure also provides a novel fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an albumin binding domain (ABD). The fusion protein can be a fusion protein, wherein the FGF23 analog is attached to the ABD at the N- terminus or the C-terminus of the FGF23 analog using at least one GGGS linker, e.g., as represented in SEQ ID NO: 15. Also provided is a novel fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an Fc binding region, such as the fragment, crystallizable domain of an antibody. In some embodiments, the antibody is an isotype IgG, IgA, or an IgD antibody. The fusion protein can be a fusion protein, wherein the FGF23 analog is attached to the Fc binding domain at the N-terminus or the C-terminus of the FGF23 analog using at least one GGGS linker.

[0080] In some embodiments, the fusion protein comprises an amino acid sequence which has at least 75%, 80%, 85%, 90%, 98%, 99%, or 100% to the entire length of any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NON, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:31.

[0081] In some embodiments, the fusion protein comprises an amino acid sequence which has about 75%, 80%, 85%, 90%, 98%, 99%, or 100% to the entire length of any one of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NON, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO 20, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:31.

[0082] In some embodiments, the fusion protein comprises or consists of the entire length of any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, or SEQ ID NO:31.

[0083] Provided here is a composition comprising a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and a compound, wherein the compound extends the half-life of the FGF23. Also provided here is a composition comprising a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an albumin binding domain (ABD). Also provided is a composition comprising a novel fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an Fc binding region. The composition can be a composition, wherein the FGF23 analog is attached to the ABD using at least one GGGS linker. The composition can be a composition, wherein the FGF23 analog is attached to the Fc binding domain using at least one GGGS linker. The composition can be a composition, wherein the FGF23 analog is humanized.

[0084] The composition can be formulated for oral administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, implantation, intraarterial administration, or transdermal administration. The composition can further comprise a pharmaceutically acceptable carrier, a stabilizer, and / or an excipient.

[0085] Provided herein is a medicament for use in treating a subject with hyperphosphatemia comprising administering to the subject a fusion protein comprising a bioactive fibroblast growthfactor-23 (FGF23) analog and a compound, wherein the compound extends the half-life of the FGF23. Also provided herein is a medicament for use in treating a subject with hyperphosphatemia comprising administering to the subject a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an albumin binding domain (ABD) in a clinically effective amount. Additionally provided herein is a medicament for use in treating a subject with hyperphosphatemia comprising administering to the subject a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an Fc binding domain in a clinically effective amount.

[0086] Described herein is a method of treatment of a subject with a hyperphosphatemia comprising: administering to the subject a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and a compound, wherein the compound extends the half-life of the FGF23. Also described herein is a method of treatment of a subject with a hyperphosphatemia comprising: administering to the subject a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an albumin binding domain (ABD) and / or administering to the subject a fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an Fc binding region. The method can further include an FGF23 analog that is attached to the ABD using one or more GGGS linkers.

[0087] The method can utilize a humanized FGF23 analog. The method can be a method of treatment of a human. The method can be for the treatment of a hyperphosphatemia, wherein the hyperphosphatemia can be a hyperphosphatemic familial tumoral calcinosis (hfTC), an idiopathic hyperphosphatemia, or any disorders caused by relative or absolute deficiency of circulating biointact FGF23.

[0088] The administrating step of the method could be performed orally, parenterally, subcutaneously (SC), intravenously (IV), intramuscularly (IM), intraperitoneally, by implantation, intraarterially, or transdermally.

[0089] The method can be a method, wherein the FGF23 analog is formulated with a pharmaceutically-acceptable carrier.

[0090] The method can be a method, wherein the bioactive FGF23 analog is administered to the subject in an amount sufficient to lower blood phosphate concentrations in the subject as compared to a blood phosphate concentration assessed prior to the administration of the bioactive FGF23 analog.

[0091] Also provided herein is an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide. In some embodiments, the nucleic acid sequence further comprises a Kozak sequence. In some embodiments, the nucleic acid sequence further comprises at least one transcriptional stop codon. In some embodiments, the nucleic acid sequence further comprises one, two, three, four, or five transcriptional stop codon(s).

[0092] The polynucleotides disclosed herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, restriction enzyme sites (e.g., any one or more of Bell, Pfml, AccI, and Xhol), internal ribosomal entry sites (IRES), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. A Kozak sequence is a nucleotide sequence thatfacilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. Exemplary consensus Kozak sequences are described by Kozak, 1986. Cell. 44(2):283-92 and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48.

[0093] The nucleic acid can further comprise an operably linked nucleic acid encoding an albumin binding domain (ABD) and a nucleic acid encoding at least one glycine serine linker. The nucleic acid encoding ABD can be located between the nucleic acid encoding a FGF23 signal peptide and a FGF23 polypeptide. The nucleic acid can be a nucleic acid, wherein the nucleic acid comprises in 5’ to 3’ order an operably linked promoter, a nucleic acid encoding the FGF23 signal peptide, the FGF23 polypeptide, at least one GS linker and an ABD nucleic acid.

[0094] In some embodiments, the nucleic acid is a polynucleotide which has at least 75%, 80%, 85%, 90%, 98%, 99%, or 100% to the entire length of any one of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or the complement thereof.

[0095] In some embodiments, the nucleic acid is a polynucleotide which has about 75%, 80%, 85%, 90%, 98%, 99%, or 100% to the entire length of any one of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or the complement thereof.

[0096] In some embodiments, the nucleic acid is a polynucleotide which comprises or consists of the entire length of any one of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO 27, SEQ ID NO: 29, SEQ ID NO:30, or the complement thereof.

[0097] Provided herein is a vector comprising an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide. The vector can further comprise an operably linked nucleic acid encoding an albumin binding domain (ABD) and a nucleic acid encoding at least one glycine serine linker.

[0098] The term “vector” is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, mammalian expression vectors, and viral vectors. An exemplary mammalian expression vector is a pcDNA vector comprising a CMV promotoer. Exemplary viral vectors include, e.g., replication defective retroviruses and lentiviruses.

[0099] Provided herein is a host cell comprising a vector which comprises an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide. Exemplary heterologous promoters which can be operably linked to a nucleic acid sequence encoding a FGF23 fusion protein include, e.g., viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g.,immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters.

[0100] Also provided herein is a host cell comprising an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide. The host cell can further comprise an operably linked nucleic acid encoding an albumin binding domain (ABD) and a nucleic acid encoding at least one glycine serine linker. In some embodiments, the host-cell is a mammalian cell. In other embodiments, the host cell is a microbial cell. In preferred embodiments, the microbial cell is an Escherichia coli (E. coll) cell.

[0101] Provided herein is a protein encoded by an isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide.

[0102] As envisioned in the present disclosure with respect to the disclosed compositions of matter and methods, in one aspect, the embodiments of the disclosure comprise the components and / or steps disclosed herein. In another aspect, the embodiments of the disclosure consist essentially of the components and / or steps disclosed herein. In yet another aspect, the embodiments of the disclosure consist of the components and / or steps disclosed herein. The therapeutic methods and compositions used in these methods as described herein can be alternatively considered as a use of FGF23 analogs for treating a patient with hyperphosphatemia, or for use in the preparation of a medicament for treating hyperphosphatemia. The use of thedisclosed FGF23 analog can be applied to any of the methods and combinations described above and infra.I. Definitions

[0103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. The following terminology will have the indicated meanings unless specifically indicated otherwise.

[0104] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0105] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, recitation of “a cell,” for example, includes a plurality of the cells of the same type.

[0106] " About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or + / - 10%, or + / - 5%, or + / - 1%, and + / - 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0107] As used herein, the term “bioactive FGF23” is used interchangeably with the term “intact FGF23.” The terms “bioactive peptides” and “bioactive proteins” are meant respectively bioactive polypeptide portions of a full protein, with or without any signal sequence or the full bioactive protein (mature form or immature form), wherein the bioactivity relates to the regulation of phosphate levels in a subject’s blood.

[0108] "Effective amount" refers to an amount that provides a therapeutic and / or prophylactic benefit to a subject given the effective amount. It is understood, however, that the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses or never fully occur to an extent that the subject would be considered to fall within a normal range for the effect being treated. Thus, an effective amount may be administered in one or more administrations. In the context of therapeutic or prophylactic applications, the amount of active agent administered to the subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs. It will also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

[0109] "Encoding" refers to the inherent property of a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for the synthesis of a peptide or polypeptide. For example, a gene encodes a protein if transcription and translation of an mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0110] The term "expression" as used herein is defined as the transcription and possibly translation of the produced mRNA of a particular nucleotide sequence driven by its promoter.

[0111] "Expression vector" refers to a vector comprising a recombinant polynucleotide of interest comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked orcontained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0112] ‘ ‘Fc binding partner,” “FcRn binding partner,” or “FcRn BP” as used herein, means functional neonatal Fc receptor (FcRn) binding partners. FcRn is a receptor that is responsible for the protection of IgG and albumin from catalytic degradation. Thus, the term FcRn BP includes any molecule, including any variants or fragments of IgG Fc or albumin, that can be specifically bound by the FcRn receptor with consequent active transport through the cell by the FcRn receptor. FcRn BP described herein include the Fc region of an IgG and an albumin binding domain (ABD) which binds to natural albumin in order to utilize natural albumin as the FcRn binding partner.

[0113] As used herein, the term “fusion protein” or “fusion polypeptide” is a polypeptide comprised of at least two polypeptides and optionally a linking sequence, and that are operatively linked into one continuous protein. The expression of such a fusion protein would be through a nucleic acid encoding the two or more polypeptides or peptide sequences. The two or more polypeptides / peptides linked in a fusion protein are typically derived from two independent sources (i.e., not from the same parental polypeptide), and therefore a fusion protein comprises two linked polypeptides not found linked in nature. Typically, the two polypeptides can be operably attached directly by a peptide bond, or may be connected by a linking group, such as a spacer domain. An example of a fusion polypeptide is a polypeptide that functions as a receptor for an antigen, wherein an antigen binding polypeptide forming an extracellular domain is fused to a different polypeptide, forming a “chimeric antigen receptor.”

[0114] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode thesame amino acid sequence. The phrase “nucleotide sequence that encodes a protein or an RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0115] The term “linker,” also referred to as a “spacer” or “spacer domain” as used herein, refers to an amino acid or sequence of amino acids that is optionally located between two amino acid sequences in a fusion protein. An example of a linker here is one or more Glycine-Serine (GS) linkers, e.g., GGGS (SEQ ID NO: 15) is such an amino acid sequence.

[0116] The term "operably linked" (or “under transcriptional control”) refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0117] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and can include a human being.

[0118] The term "polynucleotide" is a chain of nucleotides, also referred to as a “nucleic acid.”

[0119] “Analog” refers to a nucleic acid encoding a fusion protein or a fusion protein that has a similar structure to another nucleic acid encoding a fusion protein or fusion protein, but differs from it in respect to a certain component. For example, an analog can differ from another by the presence or absence of one or more linkers between the ABD and the FGF23. The analogcan include human FGF23 variants, such as the R176Q variant of GenBank Sequence or primate variants of FGF23 known in the art.

[0120] The terms "peptide," "polypeptide," and "protein" are used herein interchangeably and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein, polypeptide, or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. The terms "peptide," "polypeptide," and "protein" can encompass native or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.

[0121] The term "promoter” means a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0122] The term "transfected" or "transformed" or "transduced” means a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected," "transformed," or "transduced" cell includes an exogenous nucleic acid that has been introduced into the cell by some means. The transfected / transformed / transduced cell includes the primary subject cell and its progeny.

[0123] To "treat" a disease or condition as the term is used herein means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Contemplated is treatment of a condition associated with the absolute or relativedeficiency of circulating biointact FGF23. The absolute or relative deficiency of circulating biointact FGF23 can include, e.g., due to genetic mutations, hyperphosphatemia-hyperostosis syndrome (HHS), hypervitaminosis D, infection-associated hyperphosphatemia, muscle injury or cell injury (e.g. cell lysis) associated hyperphosphatemia, and hyperphosphatemia resulting from diabetic ketoacidosis.

[0124] As used herein, the term “pharmaceutically acceptable” refers to a formulation of a compound that does not significantly abrogate the biological activity, a pharmacological activity and / or other properties of an active agent when the formulated compound is administered to a patient. In certain embodiments, a pharmaceutically acceptable formulation does not cause significant irritation to a patient.

[0125] By " vector" is meant a nucleic acid that can have other nucleic acids of interest operably introduced and then delivered into cells. Examples of vectors include but are not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, retroviral vectors, and the like.

[0126] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. Forexample, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6 and 0.1 decimal points there between. This applies regardless of the breadth of the range.II. Method of Treating a Patient with Hyperphosphatemia

[0127] In some aspects, provided herein are methods of treating a subject with hyperphosphatemia, such as by administering a disclosed fusion protein or a nucleic acid encoding a fusion protein as described herein to the subject. While a native FGF23 can be used in a fusion protein as a therapeutic in treating a subject diagnosed with hfTC or a related hyperphosphatemia, it is known that many native FGF23 proteins possess short-circulating halflives. Thus, a means of identifying a bioactive protein having FGF23 capability and a longer halflife was needed and is newly described herein.

[0128] The present disclosure provides that using an albumin binding domain (ABD) technology, a humanized FGF23-ABD protein can be produced and has bioactivity not different from endogenous FGF23 but has a longer half-life in the blood stream. Albumin binding domain polypeptides can be derived from Gram-positive bacteria, such as a streptococcal G protein. An exemplary albumin binding domain polypeptide is ABDCon (an engineered albumin binding domain expressed in the soluble fraction of E. coli; S. Jacobs et al., “Fusion to a highly stable consensus albumin binding domain allows for tunable pharmacokinetics,” Protein Eng. Des. Selection, 2015, 28(10): 385-93). Any research grade albumin binding domain peptide or variant thereof can be used for the subject fusion proteins.

[0129] The half-life of serum albumin is 21 days. Other ABD-fusion proteins have been created and have been found to have half-lives greater than their native protein counterparts lacking ABD (therapies for stroke and clotting).

[0130] The present disclosure provides a FGF23 fusion to an ABD polypeptide. The disclosed FGF23-ABD fusion proteins can lower serum phosphate and 1,25(OH)2 vitamin D in a subject when administered to the subject. Further, idiopathic hyperphosphatemia is a common occurrence around the world. Thus, an FGF23-ABD fusion protein can also be used to reduce blood phosphate in a subject diagnosed as having idiopathic hyperphosphatemia.

[0131] The compound and compositions administered to the subject can be administered on a weekly, bimonthly, or monthly schedule for as long as the medical personnel caring for the patient deemed necessary.

[0132] The normal plasma inorganic phosphate (Pi ) concentration in an adult human is about 2.5 to 4.5 mg / dl. In children, the normal range is about 4 to 7 mg / dl. A plasma phosphate level higher than 4.5 mg / dL is hyperphosphatemia. The compounds and compositions disclosed herein would be administered to the individual in need thereof in an amount sufficient to lower and / or normalize their blood phosphate levels. Blood phosphate levels may be determined according to methods available to one of skill in the art. For example, clinical laboratory methods for evaluating plasma or serum phosphate are described, e.g., by Bazydlo et al., Calcium, Magnesium, and Phosphate, Laboratory Medicine, February 2014; 45(l):e44-e50. Renal function tests, vitamin D, serum calcium, and parathyroid levels may also be evauated to determine treatment efficacy.

[0133] The amount of compound or composition can include 0.1 pg / kg subject body weight to 100 pg / kg subject body and any 0.1 value and integer value in between those ranges.

[0134] The FGF23-ABD fusion proteins can be administered to a subject in need thereof for example intravenously on a weekly, bimonthly, or monthly periodicity. Other contemplated means of administration to a subject in need of an FGF23-ABD fusion protein would includeintramuscular administration, oral administration, parenteral administration, subcutaneous administration, intraperitoneal administration, implantation, intraarterial administration, or transdermal administration.

[0135] The FGF23-ABD can be lyophilized and then resuspended in a resuspension buffer. The resuspension buffer can be sodium chloride, saline, water, glucose infusions with or without stabilizing excipients, or other pharmaceutically acceptable carrier.III. Compounds and Compositions

[0136] FGF23-ABD fusion proteins are described herein. The described fusion proteins can be used to treat a patient with hyperphosphatemia alone or in combination with other medicines to ameliorate conditions affiliated with hyperphosphatemia. The fusion proteins can be used to prepare a medicament for use in treating a patient having hyperphosphatemia. The FGF23-ABD fusion proteins described herein stimulate the FGF23 receptor-Klotho receptor complex and activate FGF23 -responsive genes known to lower blood phosphate.

[0137] The ABD of the FGF23-ABD fusion protein binds to blood albumin, the most abundant protein in plasma. Albumin has a half-life of 21 days in humans (M.S. Dennis et al., “Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins,” Protein Struct. Folding, 2002, 227(38): P35035-43). Described herein are FGF23-ABD analogs as fusion proteins that act in vitro and in vivo in a fashion similar to endogenous FGF23 but having an extended half-life.

[0138] The practice of the disclosure is illustrated by the following non-limited examples.EXAMPLESEXAMPLE 1: FGF23 Double Mutant

[0139] As described in this and later examples, we first created a human FGF23 having a R176Q point mutation in the amino-acid sequence of the wild type FGF23 cDNA (FIG. 1 and 31. This is a naturally-occurring human mutation which is associated with the human disorder autosomal dominant hypophosphatemic rickets (ADHR). This R176Q residue change interrupts an FGF23 proteolysis site and is known to prevent intracellular FGF23 cleavage, thus stabilizing the secreted intact, bioactive form of FGF23 for subsequent protein purification.

[0140] To develop an FGF23 with further stability and increased half-life, we designed an FGF23 having a second mutation in addition to the R176Q mutation to create a 'double mutant' form of FGF23. In tandem with the R176Q mutation, the double mutant FGF23 also has a mutation that replaces SI 80 in the FAM20C kinase site. (FIG. 1) Serine 180 is known to be phosphorylated by the Golgi-localized kinase FAM20c, which blocks the glycosylation of T178. The glycosylation of Thrl78 inhibits the cleavage of FGF23 by intracellular furin proteases.

[0141] Two variations of the ADHR+Fam20 double mutant were created: R176Q + S180G (FIG. 34), and R176Q + S180A (FIG. 32 and FIG. 33). When these constructs were expressed in Expi293 mammalian cells as Fc fusion proteins, as described further in these Examples, the R176Q / S180G mutant produced less than half the total protein versus the R176A / S180A mutant (only 0.64mg total and 0.32 mg / mL) (FIG. 3 and 4). Thus, the R176A / S180A mutant is the preferred double mutant design.EXAMPLE 2: FGR23 Triple Mutant

[0142] In addition, we designed a triple mutant, having the S180A mutation as well as two ADHR mutations, R176Q, R179Q. However, when the triple mutant construct was expressed inExpi293 cells by Bon Opus, Inc., the protein was very unstable (only 0.6 mg total, and 0.2 mg / mL recovered). It is clear from the purification gels that very little mature protein was expressed and recovered (FIG. 5) when compared to previous FGF23-Fc proteins (such as FC1) described in these examples. This protein had little or no activity as well when tested in vitro for EGR1 increases in HEK-mKL cells versus single mutant (ADHR R176Q mutation only) FGF23-Fc and purified commercial rhFGF23 (FIG. 6).EXAMPLE 3: Human IgGl Antibody Fc Region Approach for Long-acting FGF23

[0143] The Fc region of antibodies has also been successfully used to extend the half-lives of clinical grade recombinant protein drugs through its binding to FcRn. To this end, the human Fc sequence was designed as a C-terminal fusion protein (See FIG. 1 and FIG. 7 for schematics) to the human R176Q-FGF23 isoform. The human Fc sequence was derived from the construct used in the article: Albright et al, Nature Communications, 2015.

[0144] As the fusion of an FcRn binding partner, such as the Fc region of an antibody, can cause steric or spatial interference with protein being used, we tested linkers of two different lengths between the FGF23 protein and the Fc. As shown schematically in FIG. 7, construct FGF-Fcl included one GlyGlyGlySer (‘GGGS’) linker sequence, as represented by SEQ ID NO: 15, between the two proteins and construct FGF-Fc2 included two GGGS linkers, as represented by SEQ ID NO: 16, between the FGF23 protein and the Fc.

[0145] Amino acid sequences: FGF23-FC1 and FGF23-FC2

[0146] SEQ ID NO: 5 (‘FGF-FC1’ (FGF23-RQ + one linker + human Fc region))

[0147] KOZAK+MLGARLRLWVCALCSVCSMSVLRAYPNASPLLGSSWGGLIHLYT ATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRQHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI GGGS DKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK + 3 Stop codons

[0148] SEQ ID NO: 6 (‘FGF23-FC2’ (FGF23-RQ + two linkers + human Fc region))

[0149] KOZAK+MLGARLRLWVCALCSVC SMSVLRAYPNASPLLGSSWGGLIHLYT ATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNI FGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSR RNEIPLIHFNTPIPRQHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMA SDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI GGGSGGGS DKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G K +3 Stop codonsEXAMPLE 4: FGF23-Fc Plasmid Constructs for Humanized Fusion Proteins

[0150] To develop plasmids for the production of humanized FGF-Fc and untagged control protein, GeneArt Gene Synthesis software (ThermoFisher Scientific) was used. The first plasmidcarries no Fc tags, only the naturally-occurring human R176Q mutation. The human FGF23 sequence was joined to the Fc sequence at the C-terminus using one or two linker sequences (‘GGGS’ as set forth in SEQ ID NO: 15 or ‘GGGSGGGS’ as set forth in SEQ ID NO: 16) to form ‘FGF-Fcl’ and ‘FGF-Fc2’ analogs, respectively (FIG. 7). The native FGF23 secretion signal peptide was used for all constructs, for predicted optimal secretion. All fusion proteins and the point mutation FGF23-RQ protein sequences were then back-translated and the nucleotide codon sequences were designated for mammalian expression. For all constructs, a Kozak consensus sequence and 3 transcriptional stop-codons were added at the N- or C-terminus, respectively. The plasmids were assembled from synthetic oligonucleotides and inserted into a pcDNA3.1(+) backbone vector. The plasmid DNA was purified from transformed E. coli (strain K12 DH10B™ T1R) according to standard protocols, and the DNA concentration determined by UV spectroscopy. Final constructs were verified by DNA sequencing to confirm 100% sequence identity within the insertion sites.

[0151] The FGF-Fc amino acid sequences were uploaded to Invitrogen, Inc, and the cDNA plasmids were optimized for human codon expression. The plasmids carrying the individual human FGF-Fc were then produced by Invitrogen for subsequent analysis.EXAMPLE 5: FGF-Fc Mammalian Cell Transfection: Secretion of Stable Protein

[0152] To test the effects of the C-terminal Fc tag on FGF23 secretion from mammalian cells, the FGF-Fcl and -2 plasmids were transfected into Human embryonic kidney 293 cells (HEK293T; ATCC) cells grown in EMEM media (ATCC) supplemented with 10% fetal bovine serum (FBS; R&D Systems) and 1% penicillin-streptomycin (Hyclone) at 37°C. For the transfection, 1X106 HEK293T cells were plated in a 6-well plate in 2 ml EMEM media supplemented with 10% FBS for overnight. At 70% confluency, cells were transfected with theFGF-Fcl and -2 plasmids in duplicate wells for 24 hours using a 3: 1 ratio of FuGENE HD Transfection Reagent:DNA (control-media wells were not subjected to treatment). Then, some cells were washed with pre-warmed PBS and incubated with a 2 mL pre-warmed serum-free media for an additional 48 hours before collecting cell lysates and media in serum free media.

[0153] The FGF-Fc conditioned media (37.5 pL each serum-free media) were electrophoresed on an ‘Any kD’ precast acrylamide gel (Bio-Rad), and electrotransferred to PVDF membranes (Bio-Rad). The immunoblots were incubated in blocking solution at room temperature for 1 hour (5% non-fat dry milk powder dissolved in TBS) then incubated with antihuman FGF23 (Quidel, Inc; 1 : 1000 dilution) overnight at 4°C, followed by 1 :2000 of rabbit-anti- goat-IgG-HRP (Invitrogen) for 1 hour at room temperature. The FGF-Fc immunoblot detection was performed using the ECL Prime Western Blotting Detection Reagents (Amersham-GE Healthcare, Pittsburgh, PA, USA) and a GE AB 1600 digital imager.

[0154] Analysis of the immunoblots showed that both FGF-Fc constructs were efficiently secreted into the culture media, similarly to the untagged FGF-RQ control protein (FIG 8). The FGF-Fc analogs expressing the Fc tag had a predictably ~24 kD higher (-53 kD total) molecular mass than the untagged FGF23 counterpart (-29 kD). All FGF3-ABD protein lanes showed very slight proteolysis breakdown products, which is consistent with the insertion of the R176Q change, known to stabilize recombinant FGF23 protein. The control media showed no recombinant FGF-Fc fusion proteins.

[0155] Each of the FGF-Fc analogs, regardless of the C-terminal tags were efficiently secreted from mammalian cells. These findings are consistent with the idea that human FGF-Fc can be produced in scale-up mammalian culture systems to derive properly folded and glycosylated recombinant protein for future toxicology and therapeutic clinical trials.EXAMPLE 6: FGF-Fcl and FGF-Fc2 tested by Human FGF23 ELISA

[0156] The human FGF23 ‘C-terminaF ELISA (Quidel, Inc) contains capture and detection antibodies that are C-terminal to the R176Q FGF23 ADHR mutation. Thus, this assay recognizes whole molecule FGF23 as well as FGF23 C-terminal protease fragments. The conditioned media from mammalian FGF-Fcl and -2 cell transfections shortly after transfection, and then 24h after transfection were assessed undiluted in this assay.

[0157] Both FGF-Fc analogs were detected in the human C-terminal assay after a 24h transfection (FIG. 9), confirming the immunoblot analyses in FIG. 8. Media from nontransfected (‘Control’) cells was negative for FGF23 protein. Further, the presence of the Fc tags did not interfere with FGF-Fc detection.

[0158] The human C-terminal FGF23 ELISA assay recognizes the human FGF-Fcl and -2 analogs regardless of the Fc tag. Thus these data support that this assay can be used to detect, and track the human FGF-Fc proteins (ELISA is specific for human FGF23), e.g. for pharmacokinetic assessments in preclinical toxicology and pharmacology experiments.EXAMPLE 7: Performance of in vitro Bioactivity Assay (p-ERKl / 2 activation) Using Conditioned Media from FGF-Fc and Control Transfected Cells

[0159] FGF23 is known to stimulate the MAPK signaling cascade downstream of binding to KL-FGFR complexes, and phosphorylated ERK1 / 2 (pERKl / 2) is a readout for this activity. To test conditioned media transfected with FGF-Fcl and -2 for signaling bioactivity, HEK-mKL cells were plated in a 12-well plate (5x105 cells / well, triplicate for each condition). Cells were treated with control media, or conditioned FGF-Fcl and -2 media for one hour. Recombinant FGF23 (500ng / mL; R&D Systems) or untreated cell lysates from a previous study were used as positive and negative controls (cells previously treated for 15 min). The FGF-Fc treated cells were collected with IX Lysis buffer (Cell Signaling, Inc) and 10 ug protein lysates were loadedonto ‘Any kD’ gels (Bio-Rad) for immunoblotting as described above. Antibodies used were: pERKl / 2 (1 :2000; from Cell Signaling, Inc); total ERK (1 :2000; from Cell Signaling, Inc). The blots were developed by ECL and imaged as described above for FIG. 10.

[0160] Immunoblotting showed that the FGF-Fcl and -2 both stimulated pERKl / 2 similarly to commercial, recombinant FGF23 protein (FIG. 10). The immunoblot loading controls, ‘total ERK’ and ‘Beta-actin’, were not different across groups, and showed that protein was present in the control groups, but only when FGF-Fcl, FGF-Fc2, or recombinant FGF23 were present was MAPK signaling and pERKl / 2 activation detected.

[0161] The FGF-Fcl and FGF-Fc2 conditioned media activated MAPK signaling as detected by pERKl / 2, a known readout of Klotho-dependent FGF23 bioactivity. FGF-Fcl and -2 signaling capabilities reflected those of recombinant FGF23, providing support for these polypeptides as potential therapeutics for hfTC.EXAMPLE 8: Scale up Expression of FGF23-Fcl in Mammalian Cells

[0162] The purified humanized FGF23-Fcl plasmid was shipped to Bon Opus, Inc. (New Jersey) for protein production and initial larger-scale purification. Bon Opus uses the Expi293 cell system following transient plasmid transfection to generate secreted recombinant mammalian protein. In this regard, FGF23-Fcl protein was obtained at an excellent yield of 1.44 mg from 50ml of conditioned media from Expi293 cells. The FGF23-Fcl protein was one-step purified with Protein A columns (binds the human Fc region). Consistent with the pilot-trial experiment in using plasmids in HEK-293 cells (see FIG. 8), the recombinant mammalian FGF23-Fcl protein was secreted and purified in the scale up system (1.44mg total protein and 0.48mg / ml) but was purified as 2-3 primary bands which likely represent the full-length protein and the N-terminus carrying the Fc tag, as the protein was purified using Protein A (binds Fcregions) (FIG. 11). To test for extended half-life in vivo, the FGF23-Fc (R176Q) single mutant protein was injected intraperitoneallyinto wild type mice (500 ng / g body weight). After 24 and 48 hrs the human FGF23 C-terminal ELISA was performed. FGF23 circulates in the pg / mL range normally, however the circulating concentration of FGF23-Fc was 200 ng / mL after 24 hr and 30 ng / mL after 48 hr (FIG. 12). The untagged rhFGF23 was undetectable at 24 and 48h (FIG. 12), thus the Fc tag dramatically extends FGF23 half-life.EXAMPLE 9: Activity of the Double Mutant FGF23-Fc Proteins

[0163] To test bioactivity of the double mutant proteins in vitro, HEK-mKL cells were treated with 50 ng / mL of the generated recombinant FGF23-Fc as well as purified, recombinant human FGF23 (rhFGF23) for 1 and 4 hours. The proteins FGF23-Fc with the following mutations were tested: 1 ADHR mutation (R176Q), 2 mutations (1 ADHR and 1 FAM20c): (R176Q / S180A and R176Q / S180G). The FGF23-Fc analogs stimulated the FGF23 marker gene EGR1 200-fold; purified, rhFGF23 increased EGR1 300-fold (FIG. 13A). In vivo, WT mice were injected intraperitoneally with lOug of rhFGF23 or the above mutants and renal FGF23 bioactivity was assessed. The FGF-Fc R176Q / S180A analog showed increased EGR1 (FIG. 13B) and CYP24al (FIG. 13C) when compared to the R176Q / S180G recombinant protein; and reduced Cyp27bl in the same manner (the R176Q / S180A protein was more efficacious; FIG 13D). Thus the double mutant R176Q / S180A had better activity than the R176Q / S180G protein, and the additional mutation did not compromise activity when compared to the single mutant (R176Q ADHR mutation only) protein.EXAMPLE 10: FGF23-QA2 Recombinant Protein Expression In Vitro

[0164] Humanized FGF23-QA2 and mammalian expression: Human codon optimized plasmids were designed containing the FGF23 ADHR R176Q mutation in tandem with a pointmutation (SI 80A) that replaced the destabilizing FAM20C target SI 80 residue. This is followed by an inert linker (gly-gly-gly-ser: ‘GGGS’ represented in SEQ ID NO: 15) and IgGl-Fc tail. The construct possesses a native FGF23 secretion signal, and is referred to as ‘FGF23-QA2’ (see FIG. 2).

[0165] To produce FGF23-QA2 protein, the cDNA was transfected into HEK-293 cells and after 24h conditioned media was harvested. Immunoblotting using an anti-human FGF23 antibody (Quidel, Inc) showed that FGF23-Fcl was efficiently secreted and detectable similar to FGF23-RQ control protein (Fig. 36). FGF23-QA2 had 2 higher weight bands (>150 kDa) that are known to occur in non-reduced forms of Fc-tagged proteins due to steric interactions of the Fc regions with each other, as well as a predicted ~24 kD higher (~56 kD total) molecular mass than its untagged FGF23-RQ counterpart (~32 kD), with very limited evidence of proteolysis to lower weight molecules. A non-transfected ‘Control’ cell lane had no bands (Fig. 36).Example 11: The human Intact FGF23 ELISA detects FGF23-QA2

[0166] The human ‘Intact’ FGF23 ELISA which only recognizes bioactive iFGF23, detected FGF23-QA2 in the conditioned media in a dilution-dependent manner with the assay having higher absorbances with decreasing dilutions of the conditioned media (1 :500-1 : 10), supporting FGF23-QA2 had proper secondary structure (Fig. 37).Example 12: Performance Of Scaled Up Humanized FGF23-QA2 Production And Testing Bioactivity

[0167] To scale up FGF23-QA2 protein for purification, a company with extensive production expertise, Bon-Opus, Inc (Milburn, NJ), was employed in a fee for service agreement. The enlisted strategy was to purify the secreted recombinant FGF23-QA2 via one-step protein- A binding from conditioned media following FGF23-QA2 cDNA transfection into the Expi293cell system. FGF23-QA2 protein was efficiently expressed and purified, yielding 1.14 mg total protein, and used in all subsequent experiments (see Fig. 4).

[0168] Performance of FGF23-QA2 bioactivity in vitro using a bioassay: FGF23 stimulates MAPK signaling after binding to KL-FGFR complexes, and phosphorylated ERK1 / 2 (p- ERK1 / 2) is a reliable readout for FGF23 activity To first test the purified FGF23-QA2, HEK- mKL cells (HEK293 cells stably expressing KL) were treated with vehicle (PBS; ‘Control’; lanes), and 100 ng / mL FGF23-QA2 or FGF23-RQ (R&D Systems) for 10 min. Immunoblots showed that FGF23-QA2 stimulated p-ERKl / 2 similar to untagged human commercial FGF23- RQ, and the controls total-ERKl / 2 and P-actin confirmed equal protein loading (Fig. 38). Thus, FGF23-QA2 retains FGF23-like MAPK stimulating bioactivity.Example 13: Performance Of In Vivo Renal FGF23 Target Gene Expression Analyses Using Scaled Up, Purified Recombinant FGF23-QA2 Protein In Wild Type Mice

[0169] To test FGF23-QA2 activity in vivo, seven-week-old C57BL / 6 wild type (WT) female mice were purchased (Jackson Labs), and acclimated prior to the study. Mice were randomly assigned to treatment groups (n=3-4 per group). The investigators were not blinded during allocation or animal handling, only for endpoint measurements where arbitrary numbers were assigned for each sample. The mice were each injected with vehicle, 500 ng / g body weight intraperitoneally (i.p.) of FGF23-RQ or FGF23-QA2 or vehicle control (IX PBS; ‘Veh’) for 3 hours, a previously established time point for FGF23 -mediated effects on kidney target genes controlling 1,25D metabolism. Mice were euthanized by approved CO2 inhalation / cervical dislocation, and blood was collected by cardiac puncture for serum and plasma (collected inEDTA tubes).

[0170] Vitamin D 24-hydroxylase production: FGF23 is known to activate production of mRNA encoding the catabolic renal vitamin D 24-hydroxylase (Cyp24al), which inactivatescirculating 1,25D hormone. Both FGF23-RQ and FGF23-QA2 stimulated Cyp24al mRNA at 3h in WT mice (Fig. 38A). The activity was not statistically different between the proteins. The Cyp24al mRNA expression was markedly increased in the mice by FGF23-QA2 (Fig. 39A).

[0171] Vitamin D 1 -alpha hydroxylase suppression: The anabolic vitamin D 1 alphahydroxylase (Cyp27bl) stimulates the production of active 1,25D hormone in kidney, andFGF23 is known to suppress the expression of this enzyme, consistent with its physiological role of reducing blood 1,25D concentrations. Administration of FGF23-RQ and FGF23-QA2 suppressed Cyp27bl mRNA in the mice (Fig. 39B). Administration of FGF23-QA2 resulted in a marked decrease of Cyp27bl mRNA, showing the expected down-regulation of this enzyme with FGF23 delivery (Fig. 39B).Example 14: Performance Of FGF23-QA2 In In Vivo Half-Life Experiments In Wild Type Mice

[0172] To test for extended half-life in vivo, the FGF23-QA2 mutant protein was injected intraperitoneal injection into wild type mice (500 ng / g bw). After 24 hrs the human FGF23 C- terminal and Intact ELISAs were performed. FGF23 circulates in the pg / mL range normally, however the circulating concentration of FGF23-Fc was 250 ng / mL after 24 hr and 17 ng / mL as detected by the human bioactive FGF23-specific Intact ELISA (Fig. 40). The untagged FGF23- RQ was undetectable in both assays (Fig. 40), thus the Fc tag dramatically extends FGF23 halflife.Example 15: Performance Of In Vivo Renal FGF23 Target Gene Expression Analyses In Acute Regulation Using Scaled Up Purified FGF23-QA2 Protein In The Preclinical hfTCFGF23-KO Mouse Model

[0173] FGF23-KO mice are a pre-clinical model of hfTC, demonstrating severe soft tissue calcifications, hyperphosphatemia and high active vitamin D (consistent with human patientphenotypes) due to lack of circulating FGF23. Building upon our parallel initial studies in WT mice, FGF23-KO mice were injected with recombinant FGF23-QA2 (n=4-6 / group). Following euthanasia, RNA was extracted from kidney homogenates. Early growth response gene-1 (Egrl) mRNA levels are a simple and sensitive marker for normal FGF23 -dependent MAPK activity. Compared to WT mice, Egrl mRNA was up-regulated in FGF3-KO kidneys at baseline (FGF23- KO, Vehicle; Fig. 41 A), potentially as a compensatory attempt to normalize renal function. The short term treatment with FGF23-QA2 increased Egrl expression above these levels (Fig. 41A;*p<0.05; **p<0.01; ***p<0.001). Our results show that there are no significant barriers to restoring renal FGF23-QA2 signaling in an hfTC preclinical model.

[0174] FGF23-QA2 reverses the molecular defects associated with dysregulated vitamin D in the FGF23-KO mouse: Under normal circumstances, FGF23 reduces circulating 1,25(OH)2 vitamin D (‘ 1,25D’, the active form of vitamin D) via its actions on the kidney. Due to loss of FGF23 bioactivity in hfTC, 1,25D is markedly elevated in these patients. In addition to the excessive renal phosphate reabsorption, this disease manifestation further increases serum phosphate via 1,25D actions to increase intestinal dietary phosphate absorption.

[0175] The kidney enzyme Cyp27bl (vitamin D la-OHase) increases 1,25D and FGF23 inhibits its expression, whereas the Cyp24al enzyme (24-OHase) reduces 1,25D, and FGF23 acts to increase its mRNA expression. Due to lack of FGF23, Cyp27bl is elevated in FGF23-KO mice at basal levels (Fig. 41B; FGF23-KO, ‘Veh”), and Cyp24al is inappropriately normal considering the level of circulating 1,25D (Fig. 41 C; FGF23-KO, ‘Veh’). In our studies, we showed that delivery of FGF23-QA2 suppressed kidney Cyp27bl mRNA (Fig. 41B), and conversely increased Cyp24al (Fig. 41C). Thus, FGF23-QA2 controlled enzymatic expressionof these critical enzymes in a pre-clinical model of hfTC towards restoring renal vitamin D metabolism.Example 16: Performance Of In Vivo Renal FGF23 Target Gene Expression Analyses In More Chronic Regulation Using Scaled Up Purified FGF23-QA2 Protein In The Preclinical Hftc FGF23-KO Mouse Model

[0176] FGF23-QA2 reduces serum phosphate in FGF23 KO-mice with hyperphosphatemia: To test FGF23-QA2 on serum biochemistries, FGF23-KO mice were given 2 daily injections of FGF23-QA2 6 hours apart (1 or 2 pg / mouse, n=4-10 / group) for 3 days. The mice were then euthanized 24 h later and serum biochemistries were tested. FGF23-QA2 reduced serum phosphate by 23% (Fig. 42) with a very slight effect on increasing serum calcium at the lower dose, with no effect on serum blood urea nitrogen (BUN; marker of renal function), or alkaline phosphatase (Aik Phos; marker of bone formation) over this time course (Fig. 42). Thus, FGF23- QA2 reduces serum phosphate in a pre-clinical model of hfTC.Example 17: Performance of FGF23-QA2 on renal 1,25D metabolizing enzymes in more chronic administration to FGF23-KO mice

[0177] The mRNA encoding the renal transcription factor EGR1 is basally elevated in the FGF23-KO mouse model of hfTC (Fig. 43 A) as described above, potentially as a compensatory mechanism due to lack of FG23 signaling. In the kidneys from the FGF23-KO mice described immediately above for biochemical analyses, more chronic delivery of FGF23-QA2 and return towards normal phosphate metabolism resulted in a significant reduction in kidney EGR1 mRNA. Due to lack of FGF23, Cyp27bl is elevated in FGF23-KO mice at basal levels (Fig. 43B; FGF23-KO, ‘Veh”), and Cyp24al is inappropriately normal considering the level of circulating 1,25D (Fig. 43C; FGF23-KO, ‘Veh’). We demonstrate that delivery of FGF23-QA2 suppressed inappropriate kidney elevation of EGR1 and Cyp27bl mRNAs (Fig. 43B), and converselyincreased Cyp24al (Fig. 43C). Thus, FGF23-QA2 chronically controlled expression of these critical enzymes in a pre-clinical model of hfTC towards restoring renal vitamin D metabolism.Example 18: Performance Of in vitro FGF23-QA2 Degradation Studies

[0178] To test whether the FGF23-QA2 double mutant (ADHR mutation plus Fam20c mutation) had increased stability over FGF23-Fcl (single ADHR mutation), normal, wild type mice (n=5, 8 weeks of age) were i.p. injected with Vehicle (Veh), or 5pg of either the FGF23- FC1 (carrying only the R176Q ADHR mutation) or the FGF23-QA2 (carrying the ADHR and FAM20C S180A mutations) one-step Protein A-purified recombinant scale up protein to compare in vivo concentrations with time. Plasma was harvested following euthanasia after 24 h and the Intact Human FGF23 ELISA performed. Samples were normalized by ELISA to control for any lot-to-lot differences in purified intact FGF23 protein in the starting injected protein preparations. As shown in FIG. 44, this experiment showed higher in vivo concentrations of intact, bioactive FGF23-QA2 versus FGF23-FC1.EXAMPLE 19: FGF23-ABD Constructs for Humanized Analogs

[0179] To develop plasmids for the production of humanized FGF23-ABD and untagged control protein, GeneArt Gene Synthesis software (ThermoFisher Scientific) was used to customize a human FGF23-‘RQ’ point mutation amino-acid sequence in the wild type human FGF23 cDNA (Gene ID: 8074 (FIG 14)). FGF23-RQ containing plasmid is not linked to an ABD tag; it only contains the naturally-occurring human R176Q mutation, which is associated with the human disorder of autosomal dominant hypophosphatemic rickets (ADHR). The R176Q mutation interrupts an FGF23 proteolysis site and is known to prevent intracellular FGF23 cleavage. The FGF23 possessing the R176Q mutation is stabilized allowing it to be secreted as an ‘intact,’ bioactive form of FGF23 for subsequent protein purification. The FGF23-ABDconstructs were designed to contain the R176Q mutation. The human FGF23 with R176Q mutation sequence was joined to the ABD sequence (‘Albumod’) from Affibody Labs either at the N- or C-terminus of the FGF23 - R176Q variant separated by one or two linker sequences. The linker sequence used in the examples is GlyGlyGlySer (‘GGGS’) linkers and form FGF23- ABD ‘Nl’, ‘N2’, ‘CL and ‘C2’ analogs as depicted in FIG. 1. The fusion proteins can use 0, 1 (SEQ ID NO: 15) or 2 linkers (SEQ ID NO:16) to be sufficient to provide spatial separation between FGF23 and the ABD to reduce the changes arising from steric interference. Additional linkers of various sizes can be used, for example, 3, 4, 5, 6, 7, 8, 9, and 10 linkers, e.g., as set forth in SEQ ID NOs: 15, 16, 32, 33, 34, 35, 36, 37, 38, and 39, respectively. The native FGF23 secretion signal peptide was used for all FGF23-R176Q constructs (the R176Q mutation occurs in position 176 in the GenBank sequence NM 020638.3 and related NP 065689.1 and a GGGS linker (SEQ ID NO: 15) was used to separate the FGF23 signal sequence from the ABD in the Nl and N2 constructs for predicted optimal secretion. All fusion proteins and the point mutation FGF23-RQ protein sequences were then back-translated, and the nucleotide codon sequences were optimized for mammalian expression. For all constructs, a Kozak consensus sequence and 3 transcriptional stop-codons were added at the N- or C-terminus, respectively. A pcDNA3.1(+) (ThermoFisher) plasmid backbone was used for incorporating the cDNA encoding the FGF23- ABD sequence. The plasmids were assembled from synthetic oligonucleotides and inserted into a pcDNA3. 1 (+) backbone vector (Invitrogen). The engineered pcDNA3.1 (+) now containing the FGF23 nucleic acid sequences was purified from transformed E. coli (strain KI 2 DH1 OB™ T1R) according to standard protocols; the DNA concentration of the amplified DNA was determined by UV spectroscopy. Final constructs were verified by DNA sequencing to confirm 100% sequence identity of the inserted nucleic acids within the insertion sites.

[0180] Results: The FGF23-ABD amino acid sequences containing cDNA plasmids were further optimized for human codon expression by Invitrogen, Inc.EXAMPLE 20: FGF23-ABD Mammalian Cell Transfection: Secretion of Stable Protein

[0181] Plasmid transfections: To test the effects of the N- and C-terminal ABD tags on FGF23 secretion from transfected mammalian cells, the FGF23-ABD-N1, N2, Cl, and C2, and FGF23-RQ control plasmids were transfected into human embryonic kidney 293 cells (HEK293T; ATCC) cells grown in EMEM media (ATCC) supplemented with 10% fetal bovine serum (FBS; R&D Systems) and 1% penicillin-streptomycin (Hyclone) at 37°C. For the transfection, 1X106HEK293T cells were plated in a 6-well plate in 2 ml EMEM media supplemented with 10% FBS overnight. When the cells achieved about 70% confluency, the cells were transfected with the FGF23-RQ, or FGF23-ABD-N1, -N2, -Cl and -C2 cDNA plasmids for 24 hours using a 3: 1 ratio of FuGENE HD Transfection Reagent:DNA (Promega). Afterwards, the cells were washed with pre-warmed phosphate buffered saline (PBS) and incubated with a 2 mL pre-warmed serum-free EMEM media (from ATCC) for an additional 48 hours before collecting cell lysates and media in serum free media. Cell lysates were collected in 250 pl of IX Cell Lysis Buffer (Cell Signaling) with 0.1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, Sigma) for each well. Cell media were stored in -80 °C for future tests.

[0182] Immunoblots: The FGF23-ABD conditioned media (37.5 pL each of the serum-free media) were electrophoresed on an ‘Any kD’ precast acrylamide gel (Bio-Rad) and electrotransferred to PVDF membranes (Bio-Rad). The immunoblots were incubated in blocking solution at room temperature for 1 hour with 5% non-fat dry milk powder dissolved in IX Tris- Buffered Saline (TBS) then incubated with anti-human FGF23 (Quidel, Inc.) using a 1: 1000 dilution overnight at 4°C. After the overnight incubation, the membranes were then incubatedwith a 1 :2000 dilution of rabbit-anti -goat-TgG-HRP (Invitrogen) for 1 hour at room temperature.The FGF23-ABD immunoblot detection was performed using the ECL Prime Western Blotting Detection Reagents (Amersham-GE Healthcare, Pittsburgh, PA, LISA) and a GE AB 1600 digital imager.

[0183] Results: Analysis of the immunoblots showed that all FGF23-ABD constructs were efficiently secreted into the culture media by the transfected cells as compared to the untagged FGF23-RQ control protein (FIG. 15). The FGF23-ABD analogs expressing the ABD tag had a predictably ~5 kD higher (~34 kD total) molecular mass than the untagged FGF23 counterpart (~29 kD). The FGF23 tagged and untagged proteins showed no detectable proteolysis breakdown products, which is consistent with the insertion of the R176Q mutation, known to stabilize recombinant FGF23 protein. Each of the FGF23-ABD analogs, regardless of the N- or C-terminal tag locations were efficiently secreted from mammalian cells.EXAMPLE 21: FGF23-ABD 'C-terminal' FGF23 Human ELISA

[0184] The human FGF23 ‘C-terminal’ constructed was analyzed via ELISA (Quidel, Inc.) using capture and detection antibodies that recognize domains on FGF23 that are C-terminal to the R176Q FGF23 ADHR mutation. Thus, the assay using this ELISA system recognizes whole molecule FGF23 as well as FGF23 C-terminal protease fragments. The conditioned media from mammalian cell transfections were assessed undiluted in this assay using manufacturer instructions.

[0185] Results: All N- and C-terminal FGF23-ABD analogs, as well as the FGF23-RQ control proteins, were detected in the human C-terminal assay (FIG. 16), confirming the immunoblot analyses data depicted in FIG. 2. Media from non-transfected (‘Non-Tf ) cells was negative for the presence of FGF23 protein. The data reflects that neither the C- or N-terminallocations of the ABD tags interfered with FGF23-ABD detection. The human C-terminal FGF23 ELISA assay recognizes the human FGF23-ABD analogs regardless of ABD tag location.

[0186] Thus, these data support that this assay can be used to detect and track the human FGF23-ABD proteins (ELISA is specific for human FGF23), e.g., for pharmacokinetic assessments in preclinical toxicology and pharmacology experiments.EXAMPLE 22: FGF23-ABD 'Intact', bioactive FGF23 Human ELISA

[0187] The human FGF23 ‘Intact’ form was analyzed via ELISA system (“Kainos human intact ELISA System,” Kainos, Inc.) using capture and detection antibodies that are conformation specific, binding to N- and C-terminal portions of FGF23. Using this ELISA assay, one can identify the full-length FGF23 molecule. The conditioned media from mammalian cell transfections were tested undiluted using this assay system.

[0188] Results: All N- and C-terminal FGF23-ABD analogs, as well as the control FGF23- RQ protein lacking an ABD tag, were detected using the Kainos human Intact ELISA system (FIG. 17). The data confirms the immunoblot analyses in FIG. 2 and C-terminal ELISA in FIG.3. Media from non-transfected (‘Non-Tf ) cells was negative for FGF23 protein. The assay demonstrates that the ABD tags did not interfere with intact FGF23-ABD detection. The human Intact FGF23 ELISA assay recognized the recombinant FGF23-ABD analogs regardless of the location of the ABD tag.EXAMPLE 23: FGF23-ABD Activity in a Mammalian Cell Bioassay: EGR1 mRNA Expression with FGF23-ABD Conditioned Media

[0189] To further test the bioactivity of the FGF23-ABD analogs, a reliable in vitro cell activity assay was developed. An HEK293 cell line that stably expresses the FGF23 co-receptor, membrane-bound Klotho (HEK-‘mKL’ cells) was designed. The cells also express FGFreceptors (FGFRs), which partner with Klotho (‘KL’) and FGF23 to initiate high affinity FGF23 intracellular signaling. The production of the transcription factor, early growth response gene-1 (EGR1), mRNA produces a reliable means of assessing FGF23 bioactivity in vitro and in vivo.

[0190] To perform the assay, IxlO5HEK293 cells were plated in 12-well plates and grown in EMEM media (ATCC) supplemented with 10% fetal bovine serum (FBS; R&D Systems) and 1% penicillin-streptomycin (Hyclone) and incubated at 37°C. The conditioned media obtained from the cells (0.7 mL of the serum-containing and serum free media as obtained from the data in FIG. 15, or using a commercial recombinant human FGF23 (500 ng / mL; R&D Systems, Inc.)) were then added to the HEK-mKL cells for 30 min.

[0191] The cells were lysed and total RNA was isolated using the Bioline RNA isolation kit as described above. RT-qPCR was then performed to detect the presence of EGR1 mRNA using exon spanning primers (AB I, Inc.). The TaqMan One-Step RT-PCR kit (ThermoFisher) was used to perform the qPCR reactions under cycling conditions: 30 min. at 48°C, 10 min. at 95°C, followed by 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. The data were collected using a StepOne Plus system (Applied Biosystems / ThermoFisher Scientific). The expression levels of mRNAs were calculated relative to vehicle-treated casein diet or osteogenic media controls, and data were analyzed by the 2-AACT method. The 2-AACT method is described in Livak et al., “Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method,” Methods, 2001, 25(4): 402-8.

[0192] Results: The conditioned media (with or without serum) containing the FGF23- ABDs, FGF23-RQ control, and the commercial recombinant FGF23 all produced markedly increased EGR1 mRNA in HEK-mKL cells (FIG. 18). The negative controls (i.e., nontransfected (Non-Tf) media and non-treated cell wells) showed no changes in EGR1 expression.The FGF23-ABD conditioned media, with or without serum, strongly stimulated EGR1 mRNA expression in HEK-mKL cells, similar to commercial FGF23, consistent with these analogs possessing FGF23-like activity. Thus, the ABD tags do not diminish the bioactivity of FGF23 to stimulate known FGF23 target genes.EXAMPLE 24: FGF23-'RQ' Control Protein Bacterial Protein Production

[0193] The amino acid sequences for FGF23-RQ and FGF23-ABD-C2 were uploaded to Bon-Opus (Milbum, NJ), and the nucleic acid sequences were optimized for E. coli expression. The overall strategy was to purify the recombinant proteins produced from E. coli via the SUMO- tag method, wherein the desired fusion FGF23s are produced with a SUMO tag. The SUMO tag is then proteolytically removed to produce recombinant FGF23-RQ and FGF23-ABD-C2 proteins.

[0194] SEQ ID NO: 1 (Sequence for FGF23-RQ in E. coliy.

[0195] ATGGGTCACCATCACCATCACCATATGTCGGACTCAGAAGTCAATCAAG AAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAA GGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAA GAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAG ATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACA TGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGCTACCCGAA TGCAAGTCCGCTGCTGGGCAGCAGTTGGGGCGGCTTAATTCATCTGTATACCGCCA CCGCACGTAATAGTTATCATCTGCAGATTCATAAAAACGGTCATGTTGATGGTGCC CCGCATCAGACCATCTATAGCGCCCTGATGATTCGTAGTGAAGATGCAGGTTTTGT GGTTATTACCGGCGTTATGAGTCGTCGTTATCTGTGCATGGATTTTCGCGGCAATATTTTTGGCAGTCATTATTTTGATCCGGAAAATTGTCGCTTTCAGCATCAGACCCTGGAAAATGGTTATGATGTTTATCATAGTCCGCAGTATCATTTTCTGGTGAGTCTGGGTCGCGCAAAACGCGCCTTTCTGCCGGGTATGAATCCGCCGCCGTATAGCCAGTTTCTGAGTCGTCGCAATGAAATTCCGCTGATTCATTTTAATACCCCGATTCCGCGTCAGCATACCCGCAGCGCAGAAGATGATAGCGAACGCGATCCGCTGAATGTGCTGAAACCGCGTGCCCGCATGACCCCGGCACCGGCAAGTTGTAGCCAGGAACTGCCGAGTGCCGAAGATAATAGTCCGATGGCCAGCGATCCGCTGGGTGTGGTTCGCGGTGGTCGTGTTAATACCCATGCAGGCGGCACCGGCCCGGAAGGTTGCAGACCTTTTGCAAAATTCATTTAA

[0196] SEQ ID NO: 2 (FGF23-RQ protein):

[0197] YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQYHFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRQHTRSAEDDSERDPLNVLKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKFI*

[0198] SEQ ID NO: 3 (Sequence for FGF23-ABD-C2 in E. coli)

[0199] AATGGGTCACCATCACCATCACCATATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGCTACCCGAATGCAAGTCCGCTGCTGGGCAGCAGTTGGGGCGGCTTAATTCATCTGTATACCGCCACCGCACGTAATAGTTATCATCTGCAGATTCATAAAAACGGTCATGTTGATGGTGCCCCGCATCAGACCATCTATAGCGCCCTGATGATTCGTAGTGAAGATGCAGGTTTTGTGGTTATTACCGGCGTTATGAGTCGTCGTTATCTGTGCATGGATTTTCGCGGCAATA TTTTTGGCAGTCATTATTTTGATCCGGAAAATTGTCGCTTTCAGCATCAGACCCTGG AAAATGGTTATGATGTTTATCATAGTCCGCAGTATCATTTTCTGGTGAGTCTGGGTC GCGCAAAACGCGCCTTTCTGCCGGGTATGAATCCGCCGCCGTATAGCCAGTTTCTGAGTCGTCGCAATGAAATTCCGCTGATTCATTTTAATACCCCGATTCCGCGTCAGCATACCCGCAGCGCAGAAGATGATAGCGAACGCGATCCGCTGAATGTGCTGAAACCGC GTGCCCGCATGACCCCGGCACCGGCAAGTTGTAGCCAGGAACTGCCGAGTGCCGA AGATAATAGTCCGATGGCCAGCGATCCGCTGGGTGTGGTTCGCGGTGGTCGTGTTA ATACCCATGCAGGCGGCACCGGCCCGGAAGGTTGCAGACCTTTTGCAAAATTCATTGGTGGTGGCAGTGGCGGCGGCAGTCTGGCTGAAGCAAAAGTTCTGGCAAATCGTG AACTGGATAAATATGGCGTTAGCGATTTTTATAAGCGTCTGATTAATAAGGCAAAG ACCGTGGAAGGCGTTGAAGCCCTGAAACTGCATATTCTGGCAGCACTGCCGTAA

[0200] SEQ ID NO: 4 (FGF23-ABD-C2 E. coli optimized Protein Sequence):

[0201] YPNASPLLGSSWGGLIHLYTATARNSYHLQIHKNGHVDGAPHQTIYSALMIRSEDAGFVVITGVMSRRYLCMDFRGNIFGSHYFDPENCRFQHQTLENGYDVYHSPQY HFLVSLGRAKRAFLPGMNPPPYSQFLSRRNEIPLIHFNTPIPRQHTRSAEDDSERDPLNV LKPRARMTPAPASCSQELPSAEDNSPMASDPLGVVRGGRVNTHAGGTGPEGCRPFAKF IGGGSGGGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP*7te lilts: Bon-Opus successfully purified both the FGF23-RQ analog (FIGS. 19A-19C) and FGF23-ABD-C2 (FIGS. 20A-20C) analog (via SUMO tag purification method) from E. coli transfected with vectors encoding these proteins. These proteins were expressed (2 mg total protein producedfrom 1 Liter cultures) at the correct molecular weights, and FGF23-ABD-C2 had the predictably higher molecular mass due to the ABD fusion tag.EXAMPLE 25: FGF23-ABD-'C2' and Non-Tagged, FGF23-RQ Immunoblot with AntiHuman FGF23 Antibodies

[0202] Immunoblots were used to analyze the molecular masses and purity of the FGF23- RQ and FGF23-ABD-C2 proteins (same materials and methods of antibody -FGF23 protein detection as used in FIG. 2 were used in this example). For analysis, 200 ng / gel well of each protein were loaded on an ‘Any kD’ precast acrylamide gel (Bio-Rad) and electrotransferred to PVDF membranes (Bio-Rad). The immunoblots were incubated in blocking solution (5% milk powder dissolved in IX TBS) at room temperature for 1 hour then incubated with anti-human FGF23 antibody (Quidel, Inc.) using a 1 : 1000 dilution (diluted in 5% milk in IX TBST (Tris- Buffered Saline, 0.1% TWEEN 20 Detergent (Affymetrix)) overnight at 4°C. After the overnight incubation, the membranes were then incubated with a 1 :2000 dilution (diluted in 5% milk in IX TBST buffer) of rabbit-anti-goat-IgG-HRP (Invitrogen) for 1 hour at room temperature. The immunoblot detection was performed using the ECL Prime Western Blotting Detection Reagents (Amersham-GE Healthcare), and a GE AB 1600 digital imager was used.

[0203] Results-. As shown in FIG. 21, the FGF23-RQ and FGF23-ABD-C2 were both recognized by anti-human FGF23 (Quidel, Inc.) on the immunoblots at appropriate molecular sizes.

[0204] The ABD tag again did not interfere with antibody recognition of the FGF23 proteins similar to detection of the FGF23 proteins expressed from mammalian cells. Both the FGF23- RQ and FGF23-ABD-C2 were expressed at the appropriate sizes and showed no detectable breakdown products.EXAMPLE 26: Purified FGF23-ABD In Vitro Bioactivity (p-ERKl / 2 activation)

[0205] FGF23 is known to stimulate the mitogen-activated protein kinase (MAPK) signaling cascade downstream of binding to KL-FGFR complexes. Detection of phosphorylated ERK1 / 2 (pERKl / 2) is a readout for MAPK signaling activity. To assay purified recombinant FGF23- ABD-C2 signaling bioactivity, HEK-mKL cells were plated in 12-well plates (5xlO5cells / well, in triplicate for each condition assayed). HEK-mKL cells were treated with 5 pl of PBS (Hyclone) vehicle, 500 ng / mL of FGF23-C2, or FGF23-RQ for 15 min. The cells were collected with IX Lysis buffer (Cell Signaling, Inc.), and the protein concentrations of cell lysates were measured using the Coomassie Plus (Bradford) Assay kit (ThermoFisher Scientific). Ten pg of protein lysates were loaded onto ‘Any kD’ gels (Bio-Rad) for immunoblotting and processed as described above. Antibodies used were: pERKl / 2 (1:2000 dilution; from Cell Signaling, Inc.) and total ERK (1:2000 dilution; from Cell Signaling, Inc.). The blots were developed by ECL and imaged as described above for the example associated with FIG. 2.

[0206] Results: Immunoblotting showed that the FGF23-ABD-C2 stimulated pERKl / 2 in amount similar to pERKl / 2 stimulation from produced by the untagged FGF23-RQ protein (FIG. 22). The control, total ERK, was not different across groups, and showed that protein was present in the vehicle group, but only when FGF23-ABD-C2 or FGF23-RQ were present was MAPK signaling and pERKl / 2 activation detected.

[0207] The FGF23-ABD-C2 activated MAPK signaling as detected by pERKl / 2, and representative of a known Klotho-dependent FGF23 bioactivity. FGF23-ABD-C2 signaling capabilities did not significantly differ from untagged FGF23-RQ, supporting that the FGF23- ABDs have comparable function to FGF23-RQ on known FGF23 targets.EXAMPLE 27: Purified FGF23-ABD Tn Vitro Bioactivity (EGR1 mRNA production)

[0208] FGF23 is known to stimulate the MAPK signaling cascade. EGR1 mRNA production is a positive indicator for FGF23 induced MAPK activity. To test purified recombinant FGF23- ABD-C2 signaling bioactivity, HEK-mKL cells were plated on a 12-well plate (5xl05cells / well in triplicate). Cells were treated with vehicle, or 500 ng / mL of FGF23-C2 or 500 ng / mL FGF23- RQ for 30 min. Control cells were treated with saline (vehicle). The cells were lysed in Lysis Buffer (Bioline), and total RNA were extracted using RNA Mini Kit (Bioline) exactly according to the manufacturer’s protocols. The extracted RNAs were analyzed using RT-qPCR for the presence of EGR1 mRNA as described in the example for FIG. 5. P-actin was used as the internal control mRNA. The RT-qPCR was performed as described in the Example associated with FIG. 5.

[0209] Results: The RT-qPCR analysis showed that after 30 minutes of incubation in the presence of an FGF23 protein, the FGF23-ABD-C2 stimulated the production of EGR1 mRNA in an amount similar to that stimulated by the untagged FGF23-RQ protein in HEK-mKL cells, with over 50-fold elevation over the control (FIG. 23). The FGF23-ABD-C2 activated rapid EGR1 mRNA production is demonstrated to not significantly differ in its ability to stimulate EGR1 mRNA than stimulation by the non-tagged FGF23-RQ. These data support that the purified FGF23-ABD has a comparable level of function to FGF23-RQ on known FGF23 target genes.EXAMPLE 28: In Vivo Bioactivity: Gene Targets in WT mice and Fgf23-KO Mouse Rescue

[0210] In vivo FGF23-ABD bioactivity: To assess FGF23-ABD activity in vivo, seven- week-old C57BL / 6 wild type (WT) female mice were purchased (Jackson Labs) and acclimated prior to the study. Fgf23 -knockout (Fgf23-KO) mice, which mimic the key aspects of humanhfTC and generalized hyperphosphatemia, were previously purchased. Mice were housed in cages with up to five (5) mice per cage in accord with IUSM standard protocols and were randomly assigned to treatment groups. The investigators were not blinded during allocation or animal handling, only for endpoint measurements where arbitrary numbers were assigned for each sample. The mice were each injected with vehicle, 500 ng / g body weight intraperitoneally (i.p.) of FGF23-RQ or FGF23-ABD-C2. Three hours post i.p. delivery of either the FGF23 protein or vehicle was the previously established time point for FGF23 -mediated effects on kidney target genes controlling 1,25D metabolism. At the 3 hour post i.p. administration, mice were euthanized by approved CO2 inhalation / cervical dislocation. Blood was collected by cardiac puncture for serum and plasma from each mouse (collected in EDTA tubes).

[0211] RNA preparation and quantitative RT-PCR (qPCR): To assess the effects of FGF23- RQ control and FGF23-ABD-C2 on known FGF23 targets in kidney, following i.p. injection, the kidneys of each of the mice were harvested and homogenized in 1 mb of Trizol reagent (Invitrogen / ThermoFisher Scientific) according to the manufacturer’s protocol. RNA from the homogenized kidneys was purified using the Isolate II RNA Mini Kit (Bioline, Inc.). RNA samples were tested by RT-qPCR using commercial intron-spanning primers specific for mouse early growth response gene-1 (Egr-1), the vitamin Dla-hydroxylase (Cyp27bl), and the vitamin D 24-hydroxylase (Cyp24al). -actin was used as an internal control for RT-qPCR (Applied Biosystems / ThermoFisher Scientific). The TaqMan One-Step RT-PCR kit (ThermoFisher) was used to perform the RT-qPCR reactions under cycling conditions: 30 min 48°C, 10 min 95°C, followed by 40 cycles of 15 sec 95°C and 1 min 60°C. The data were collected using a StepOne Plus system (Applied Biosystems / ThermoFisher Scientific). The expression levels of mRNAswere calculated relative to vehicle-treated casein diet or osteogenic media controls, and data were analyzed by the 2- A ACT method (Livak et al.).

[0212] Results: Egrl production: Egrl mRNA production is rapid in vivo marker for FGF23 bioactivity in kidney. In wild-type (WT) mice, Egrl mRNA is known to reach its maximal level in 30-60 min. At 3 hours, Egrl mRNA was significantly upregulated following FGF23-ABD-C2 injections as compared to the animals injected with vehicle. Animals administered FGF23-RQ showed a trend towards increased expression (FIGS. 24A-24C). The FGF23-ABD-C2 may have a longer half-life and was associated with increasing the observed Egrl mRNA levels at the 3- hour time point. This observation will be further explored.

[0213] In the Fgf23-KO mice, FGF23-ABD-C2 markedly stimulated Egrl mRNA versus animals administered only vehicle. Vitamin D 24-hydroxylase production: FGF23 is known to activate the production of mRNA encoding the catabolic renal vitamin D 24-hydroxylase (Cyp24al), which inactivates circulating 1,25D hormone. Both FGF23-RQ and FGF23-ABD-C2 stimulated Cyp24al mRNA at 3h in WT mice (FIGS. 24A-24C). The detectable activity was not different between the proteins. The Cyp24al mRNA expression was significantly increased in the Fgf23-KO mice by FGF23-ABD-C2 (FIGS. 24A-24C ).

[0214] Vitamin D 1 -alpha hydroxylase suppression: The anabolic vitamin D 1 alphahydroxylase (Cyp27bl) stimulates the production of active 1,25D hormone in kidney, andFGF23 is known to suppress the expression of this enzyme, consistent with its physiological role of reducing blood 1,25D hormone concentrations. Administration of FGF23-RQ and FGF23-ABD- C2 suppressed Cyp27bl mRNA transcription in WT mice (FIGS. 24A-24C). In Fgf23-KO mice, Cyp27bl mRNA expression is increased in the basal state due to loss of FGF23 suppression. Administration of FGF23-ABD-C2 resulted in a statistically significant decrease of Cyp27blmRNA transcription (>40-fold), demonstrating the expected down-regulation of this enzyme with FGF23 delivery in the form of FGF23-ABD-C2 (FIGS. 11 A-l 1C). FGF23-ABD-C2 did not act differently in controlling the vitamin D metabolizing enzymes (Cyp24al and Cyp27bl) in WT mice versus the control untagged FGF23-RQ. Further, the FGF23-ABD-C2 rescued key gene expression disturbances in Fgf23-KO mice that shares common pathologies with patients. These results demonstrate that FGF23-ABD analogs possess activity to the stabilized FGF23 mutant and can be provided in vivo to rescue key hyperphosphatemic / hfTC phenotypes and reducing medical conditions affiliated with the phenotype.EXAMPLE 29: FGF23-ABD Binding to Human and Mouse Albumin

[0215] Albumin binding assay: A 96-well Nunc MaxiSorp ELISA plate (BioLegend) was coated with 100 pL of 5 pg / ml HSA (Sigma) or MSA (Sigma) overnight at 4°C; 5% milk powder in phosphate buffered saline (PBS) was used to block the ELISA plate for non-specific binding at room temperature for 1 hour (h). After washing the blocked ELISA plate with IX PBS twice, 50 pL FGF23-ABD-C2 or FGF23-RQ diluted in 1% milk were added for a 2 h incubation at room temperature; the plate was washed 5 times with 350 pl washing buffer per well (Quidel, Inc.; from the C-terminal human FGF23 ELISA kit). Next, 20 pl HRP FGF23 antibody (Quidel, Inc.; from the C-terminal human FGF23 kit) was added for a 1 h incubation according to manufacturer instructions, followed by 5 washes using washing buffer as described above. After washing, the HRP substrate (Quidel, Inc.; C-terminal human FGF23 ELISA kit reagent) was added, and absorbance was read at 450 nm. Data was plotted in Excel.

[0216] Results: To identify the albumin binding capacity of FGF23-ABD-C2 versus nativeFGF23-RQ, a 96-well MaxiSorp plate was coated with either human serum albumin (HSA) or mouse serum albumin (MSA). A characterized anti-human FGF23 antibody (HRP-FGF23 Abfrom High Sensitivity FGF23 C-term Human ELISA Kit (Quidel) was used for detection in a custom sandwich ELISA assay. FGF23-ABD-C2 bound to both HSA and MSA, and showed dose-dependent binding capacity with increasing protein concentrations. In contrast, the untagged FGF23-RQ had minimal binding to MSA or HSA even at the highest protein concentrations. The assay absorbances were plotted as depicted in FIG. 25. FGF23-ABD-C2 showed similar affinity for HSA and MSA, and its binding capacity was > 18-fold greater than FGF23-RQ at the maximal concentration of 100 ng / ml. These data demonstrate that the FGF23- ABD-C2 can bind to both mouse and human albumin in vitro, and at levels markedly higher than the FGF23-RQ untagged control (>1800% greater). The data confirmed albumin binding properties of the FGF23-ABD analogs, supporting their feasibility for therapeutic use.EXAMPLE 30: Mammalian FGF23-ABD in Serum Free Media Binding to Human / Mouse Albumin

[0217] Albumin binding assay: Serum free conditioned media from HEK-293 cells transfected with FGF23-RQ or FGF23-ABD-C1, -C2, -Nl, -N2 was assayed to determine whether the FGF23 analog present in the conditioned media could bind human and / or mouse serum albumin in vitro (HSA and MSA, respectively). A 96-well Nunc MaxiSorp ELISA plate (BioLegend) was coated with 100 pL of 5 pg / mL HSA (Sigma) or MSA (Sigma) overnight at 4°C; 5% milk powder in PBS was used to block the plate for non-specific binding at room temperature for 1 h. After washing the ELISA plates with IX PBS twice, 50 pL FGF23-ABD in media diluted in 1% milk were added for a 2 h incubation at room temperature. The plate was then washed 5 times with 350 pl washing buffer per well (Quidel, Inc.; from the C-terminal human FGF23 ELISA kit). Next, 20 pl HRP FGF23 antibody was added to each well (Quidel,Inc.; from the C-terminal human FGF23 kit) for a 1 h incubation, followed by 5 washes using thesame wash buffer as previously. After washing, the HRP substrate (Quidel, Inc.; C-terminal human FGF23 ELISA kit reagent) was added, and absorbance was read at 620nm. Data was plotted in Excel.

[0218] Results: All of the FGF23-ABDs bound to both HSA and MSA, and showed dosedependent binding capacity with increasing protein concentrations as reflected in panels A and B of FIG. 26. In contrast, the untagged FGF23-RQ had minimal binding capacity to either MSA or HSA, even at the highest concentrations. FGF23-ABD-C1 / C2 showed similar affinity to bind to both HSA and MSA. FGF23-ABD-C1 / C2 had a binding capacity that is ~10-fold higher than the untagged FGF23-RQ control conditioned media. These data demonstrate that the FGF23- ABDs analogs produced in mammalian cells bind mouse and human albumin in vitro, and at levels markedly higher than the FGF23-RQ untagged control.

[0219] While the disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope used in the practice of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.EXEMPLARY EMBODIMENTS

[0220] Among the embodiments provided herein are:

[0221] Embodiment 1 . A fusion protein comprising a bioactive fibroblast growth factor- 23 (FGF23) analog and a compound, wherein the compound extends the half-life of the FGF23.

[0222] Embodiment 2. The fusion protein of embodiment 1, wherein the compound is an albumin binding domain (ABD).

[0223] Embodiment 3 The fusion protein of embodiment 2, wherein the FGF23 analog is attached to the ABD using at least one GGGS linker.

[0224] Embodiment 4. A composition comprising the fusion protein of embodiment 1 and a pharmaceutically acceptable excipient.

[0225] Embodiment 5. A composition comprising the fusion protein of embodiment 2 or embodiment 3 and a pharmaceutically acceptable excipient.

[0226] Embodiment 6. The composition of embodiment 5, wherein the FGF23 analog is derived from a human FGF23 or a primate FGF23.

[0227] Embodiment 7. The composition of embodiment 5, wherein the composition further comprises a pharmaceutically acceptable carrier, a stabilizer, and / or an excipient.

[0228] Embodiment 8. The composition of any one of embodiments 4 to 7, wherein the composition is formulated for oral administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, implantation, intraarterial administration, or transdermal administration.

[0229] Embodiment 9. A medicament comprising the fusion protein of any one of embodiments 1-3, or the composition of embodiment 4 or embodiment 5 for use in treating an individual with hyperphosphatemia.

[0230] Embodiment 10. A method of treating a subject with hyperphosphatemia comprising:

[0231] administering to the subject a fusion protein of any one of embodiments 1-3 or a composition of embodiments 4-8.

[0232] Embodiment 11. The method of treatment of embodiment 10, wherein the fusion protein is a FGF23 analog, wherein the FGF23 analog is attached to the ABD using at least one GGGS linker.

[0233] Embodiment 12. The method of treatment of embodiment 11, wherein the FGF23 analog is a human FGF23 analog.

[0234] Embodiment 13. The method of treatment of embodiment 10, wherein the subj ect is a human.

[0235] Embodiment 14. The method of treatment of embodiment 10, wherein the hyperphosphatemia is a hyperphosphatemic familial tumoral calcinosis (hfFC), an idiopathic hyperphosphatemia, or a condition associated with absolute or relative deficiency of circulating biointact FGF23.

[0236] Embodiment 15. The method of embodiment 10, wherein the administering step is performed orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, transdermally, or by application to mucous membranes.

[0237] Embodiment 16. The method of embodiment 10, wherein the fusion protein is formulated with a pharmaceutically acceptable carrier.

[0238] Embodiment 17. The method of embodiment 10, wherein the fusion protein is a bioactive FGF23 analog comprising the amino acid sequence of SEQ ID NO:9, which is administered to the subject in an amount sufficient to lower blood phosphate concentrations in the subject as compared to a blood phosphate concentration assessed in the subject prior to administration of the bioactive FGF23 analog.

[0239] Embodiment 18. An isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises a FGF23 signal peptide domain and a FGF23 polypeptide.

[0240] Embodiment 19. The nucleic acid of embodimentl8, further comprising an operably linked nucleic acid encoding an albumin binding domain (ABD) and a nucleic acid encoding at least one glycine serine (GS) linker.

[0241] Embodiment 20. The nucleic acid of embodiment 18, wherein the nucleic acid encoding ABD is operably located between the nucleic acid encoding a FGF23 signal peptide and a FGF23 polypeptide.

[0242] Embodiment 21. The nucleic acid of embodiment 18, wherein the nucleic acid comprises in 5’ to 3’ order an operably linked promoter, a nucleic acid encoding the FGF23 signal peptide, the FGF23 polypeptide, at least one GS linker, and an ABD nucleic acid.

[0243] Embodiment 22. A vector comprising the nucleic acid of embodiment 18 or embodiment 19.

[0244] Embodiment 23. A host cell comprising the vector of embodiment 22.

[0245] Embodiment 24. The host cell of embodiment 23, wherein the host cell is an E. coli cell.

[0246] Embodiment 25. A host cell comprising the nucleic acid of embodiment 18 or embodiment 19.

[0247] Embodiment 26. A protein encoded by the nucleic acid of embodiment 18 or embodiment 19.

[0248] Embodiment 27. A polynucleotide comprising a nucleic acid sequence that has at least 85% sequence identity to the entire length of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO: 30, or the complement thereof.

Claims

ClaimsWe claim:Claim 1. A fusion protein comprising a bioactive fibroblast growth factor-23 (FGF23) analog and an FcRn binding partner, wherein the FGF23 protein includes a first mutation at residue 176 and a second mutation in the Fam20c kinase site and wherein the FcRn binding partner extends the half-life of the bioactive FGF23 analog.Claim 2. The fusion protein of claim 1 wherein the first mutation is R176Q and the second mutation is S180A.Claim 3. The fusion protein of claim 1, wherein the FcRn binding partner is an Fc region of lgG.Claim 4. The fusion protein of claim 3, wherein the FGF23 analog is attached to the Fc region using at least one GGGS linker.Claim 5. The fusion protein of claim 1 wherein the amino acid sequence has at least 98% sequence identity with SEQ ID NO: 9.Claim 6. A composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable excipient.Claim 7. A composition comprising the fusion protein of claim 2 and a pharmaceutically acceptable excipient.Claim 8. A composition comprising the fusion protein of claim 3 and a pharmaceutically acceptable excipient.Claim 9. The composition of claim 7 or claim 8, wherein the FGF23 analog is derived from a human FGF23 or a primate FGF23.Claim 10. The composition of claim 7 or claim 8, wherein the composition further comprises a pharmaceutically-acceptable carrier and / or a stabilizer.Claim 11. The composition of any one of claims 6 to 8, wherein the composition is formulated for oral administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, implantation, intraarterial administration, or transdermal administration.Claim 12. A medicament comprising the fusion protein of any one of claims 1-5, or the composition of any one of claims 6-8 for use in treating an individual with hyperphosphatemia.Claim 13. A method of treating a subject with hyperphosphatemia comprising: administering to the subject a fusion protein of any one of claims 1-5 or a composition of claims 6-8.Claim 14. The method of treatment of claim 13, wherein the fusion protein comprises an FGF23 analog, wherein the FGF23 analog is attached at the N-terminus or the C-terminus to the Fc region of IgG by at least one GGGS linker.Claim 15. The method of treatment of claim 14, wherein the FGF23 analog is a human FGF23 analog.Claim 16. The method of treatment of claim 13, wherein the subject is a human.Claim 17. The method of treatment of claim 13, wherein the hyperphosphatemia is a hyperphosphatemic familial tumoral calcinosis (hfTC), an idiopathic hyperphosphatemia, or a condition associated with absolute or relative deficiency of a circulating biointact FGF23.Claim 18. The method of claim 13, wherein the administering step is performed orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, transdermally, or by application to mucous membranes.Claim 19. The method of claim 13, wherein the fusion protein is formulated with a pharmaceutically acceptable carrier.Claim 20. The method of claim 13, wherein the fusion protein is a bioactive FGF23 analog comprising the amino acid sequence of SEQ ID NO: 9, which is administered to the subject in an amount sufficient to lower blood phosphate concentrations in the subject as compared to a blood phosphate concentration assessed in the subject prior to administration of the bioactive FGF23 analog.Claim 21. An isolated recombinant nucleic acid comprising an operably linked promoter and an operably linked nucleic acid sequence encoding a FGF23 fusion protein, wherein the FGF23 fusion protein comprises an FGF23 signal peptide domain and an FGF23 polypeptide.Claim 22. The nucleic acid of claim 21, further comprising an operably linked nucleic acid encoding an Fc region of IgG and a nucleic acid encoding at least one glycine serine (GS) linker. Claim 23. The nucleic acid of claim 22, wherein the nucleic acid encoding Fc is operably located between the nucleic acid encoding a FGF23 signal peptide and a FGF23 polypeptide.Claim 24. The nucleic acid of claim 21, wherein the nucleic acid comprises in 5’ to 3’ order an operably linked promoter, a nucleic acid encoding the FGF23 signal peptide, the FGF23 polypeptide, at least one GS linker, and an Fc nucleic acid.Claim 25. A polynucleotide comprising a nucleic acid sequence that has at least 85% sequence identity to the entire length of SEQ ID NO:3, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO:21, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, or the complement thereof.Claim 26. A vector comprising the nucleic acid sequence of claim 25.Claim 27. A host cell comprising the vector of claim 26.Claim 28. The host cell of claim 27, wherein the host cell is an E. coli cell.Claim 29. A host cell comprising the nucleic acid of claim 21 or claim 22.Claim 30. A protein encoded by the nucleic acid of claim 21 or claim 22.

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