Human FGF23 antagonist

Abstract

The problem that this invention aims to solve is to improve hFGF23 antagonist compounds. [Solution] The present invention relates to a novel hFGF23 antagonist polypeptide, its long-acting derivatives, and its use in pharmaceuticals, and more particularly to inhibiting hFGF23-induced signaling in patients who require it, such as patients suffering from hypophosphatemic disorders like X-linked hypophosphatemic rickets (XLH), and pharmaceutical compositions containing such compounds.

Description

[Technical Field]

[0001] Human fibroblast growth factor 23 (hFGF23) antagonists, and compositions containing such compounds for use in pharmaceuticals.

[0002] Inclusion by referencing the sequence list This application is submitted together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference. [Background technology]

[0003] Human fibroblast growth factor 23 (hFGF23) is a human protein with 251 amino acid residues that acts by binding to its receptor complex, primarily the fibroblast growth factor receptor 1 isoform c (FGFR1c) and alpha-klotho. Activation of FGFR1c-alpha-klotho by hFGF23 in the kidney leads to inhibition of the sodium phosphate cotransporter in the proximal tubule, resulting in decreased phosphate reabsorption, thereby increasing urinary phosphate excretion and lowering blood phosphate levels. Therefore, inhibition of hFGF23 activity can be used to treat certain hypophosphatemic disorders. X-linked hypophosphatemic rickets (hereinafter referred to as XLH) is a disease characterized by elevated blood hFGF23 levels due to loss-of-function mutations in the PHEX (phosphate-regulating endopeptidase homolog, X-linked) gene. Until recently, the only treatment for chronic hypophosphatemia in XLH was high-dose oral supplementation of phosphate and active vitamin D (referred to as conventional therapy). However, conventional therapies are burdensome, poorly tolerated, have limited efficacy, and are associated with dangerous adverse events. In 2018, burosumab, a monoclonal IgG1 anti-hFGF23 antibody, was approved for the treatment of XLH. Inhibition of hFGF23 by burosumab increases 1,25-dihydroxyvitamin D levels and phosphate reabsorption, resulting in improved serum phosphate levels in XLH patients (WO2008 / 099969).

[0004] Despite burosumab's ability to improve blood phosphate levels, many patients still experience hypophosphatemia during treatment, making burosumab a burdensome treatment. In pediatric patients treated with burosumab, many do not achieve phosphate concentrations within the age-appropriate normal range. For adults with burosumab, in addition to a subset of patients not reaching the normal range, phosphate levels are not stable throughout the course of treatment, and therefore many patients experience hypophosphatemia during the latter period. The high doses and large injection volumes required for burosumab treatment make it unsuitable for self-administering devices such as injection pens. In most cases, burosumab is administered by healthcare professionals, placing a heavy burden on patients and caregivers. In addition, the injection itself can be burdensome and painful due to the large volume and the process of loading the drug from the vial into the syringe. Each dose requires up to 3 ml for adults and typically 1-2 ml for children. Therefore, there is a need in the art for improvements in hFGF23 antagonist compounds. [Overview of the project]

[0005] The present invention relates to compounds in the form of hFGF23 antagonist polypeptides and derivatives thereof, comprising an extended portion (i.e., a portion that extends the half-life in the blood) that binds to hFGF23 and can antagonize hFGF23-induced signaling activity. The hFGF23 antagonist polypeptides and derivatives thereof disclosed herein are designed to provide effective treatment for hypophosphatemic disorders such as X-linked hypophosphatemic rickets (XLH).

[0006] In one embodiment, the present invention relates to the hFGF23 on the hFGF23 determined by SEQ ID NO: 1 at 3.5 Å a) R76, T86, R91, and R114, b) M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170, c) R76, T86, R91, Y93, Y107, R114, F169, and N170, d) W36, R76, T86, R91, Y107, R114, I167, F169, and N170, and e) R76, T86, R91, R114, L166, and F169, relates to an hFGF23 antagonist polypeptide capable of binding to an epitope consisting of amino acid residues selected from the group consisting of.

[0007] In another aspect, the present invention is an hFGF23 antagonist polypeptide capable of binding to hFGF23 according to SEQ ID NO: 1, i) X according to SEQ ID NO: 2 , g , , e , , , k , i , , , h , , f , , <00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ii) It includes a binding motif (BM) consisting of an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in i).

[0008] In another aspect, the present invention relates to the above-described hFGF23 antagonist polypeptide comprising a binding motif (BM), wherein the polypeptide a) X1X2X3X4X5X6X7X8-(BM)-X according to sequence number 3 36 X 37 PSQX 41 X 42 X 43 LLX 46 EARX 50 LX 52 X 53 X 54 QX 56 X 57 X 58 , In the formula, (BM) consists of the amino acid residue sequence defined above, During the ceremony, they acted independently of each other. X1 is G, I, L, N, or V. X2 is D, E, or Q, X3 is D, E, G, N, or Q. X4 is D, E, H, N, R, or T. X5 is E, I, L, T, or V. X6 is F, W, or Y. X7 is A, G, H, I, L, N, P, Q, R, T, or V. X8 is A, L, or E. X 36 is A, D, or E, X 37 is D or E, X 41 is A, R, or W, X 42 is A, D, or T, X 43 is E, N, Q, or S, X 46 is A, E, or K, X 50 is Q, or R, X 52 is N or E, X 53 is D, E, K, or T, X 54 is A or I, X 56 is A or C, X 57 is K or P, X 58 This is an amino acid residue sequence that is C, K, or E. and b) Includes an amino acid residue sequence selected from amino acid residue sequences having at least 93% identity with the sequence defined in a).

[0009] One aspect of the present invention relates to hFGF23 antagonist polypeptides, such as those represented by chemical substances 89, 106, 107, 111, 197, 201, 204, 206, 207, and 208.

[0010] In another embodiment, the present invention relates to the above-mentioned hFGF23 antagonist polypeptide, which is bound to the polypeptide such that the extended portion reaches the hFGF23 antagonist polypeptide derivative.

[0011] In another embodiment, the present invention relates to compounds selected from the group consisting of: [ka] [ka] [ka] [ka]

[0012] In another aspect, the present invention relates to further compounds disclosed herein.

[0013] In another aspect, the present invention relates to the above-mentioned hFGF23 antagonist polypeptide or its derivatives, as well as polynucleotides, vectors, and host cells suitable for producing the compound.

[0014] In another embodiment, the present invention relates to a pharmaceutical composition comprising the above-described hFGF23 antagonist polypeptide, its derivatives, or compounds.

[0015] In another embodiment, the present invention relates to a kit comprising the above-described hFGF23 antagonist polypeptide, its derivatives, compounds, or compositions, and instructions for use.

[0016] In another aspect, the present invention relates to the pharmaceutical uses of the above-described hFGF23 antagonist polypeptide or its derivatives, compounds, compositions, or kits, particularly, but not limited to, use for the treatment of hypophosphatemic diseases such as X-linked hypophosphatemic rickets (XLH).

[0017] The present invention can also solve further problems that become apparent from the disclosure of exemplary embodiments. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows plasma phosphate levels after intravenous administration of compounds (chemical 197, chemical 201, and chemical 204) to miniature pigs. [Figure 2] Figure 2 shows plasma phosphate levels after subcutaneous administration of compounds (chemicals 204, 206, 207, and 208) to male beagle dogs. [Figure 3] Figure 3 shows plasma phosphate levels after intravenous administration of the compound (chemical substance 206) to miniature pigs.

[0019] A brief explanation of arrays Sequence ID 1 represents the amino acid sequence of mature human FGF23 (hFGF23) after removing a 24-amino acid signal peptide from full-length human FGF23.

[0020] Sequence ID 2 represents the amino acid sequence of the binding motif of the present invention, including positions with fixed or unfixed residues.

[0021] Sequence ID 3 represents the amino acid residue sequence of the hFGF23 antagonist polypeptide of the present invention, including positions with fixed or unfixed residues.

[0022] Sequence ID 4 represents the amino acid residue sequence of the hFGF23 fragment used in Example 8 of this specification.

[0023] Sequence ID 5 represents the amino acid residue sequence of the hFGF23 variant used in Example 2 of this specification.

[0024] Sequence ID 6 represents the amino acid residue sequence of the hFGF23 variant used in Example 3 of this specification.

[0025] Sequence ID 7 represents the amino acid sequence of the heavy chain of brosumab.

[0026] Sequence ID 8 represents the amino acid sequence of the heavy chain of brosumab.

[0027] Sequence ID 9 represents the amino acid sequence of the peptide extension factor.

[0028] Sequence ID No. 10 represents the amino acid residue sequence of the hFGF23 antagonist polypeptide used in Example 8 of this specification.

[0029] Sequence IDs 11-52 represent the amino acid residue sequences of the hFGF23 antagonist polypeptide binding motif (BM).

[0030] Sequence IDs 53-280 represent the amino acid residue sequences of the hFGF23 antagonist polypeptide. [Modes for carrying out the invention]

[0031] The present invention relates to compounds that can bind to human FGF23 (hFGF23) and antagonize its biological activity. hFGF23 (represented by Sequence ID No. 1 herein) is involved in the metabolism of phosphate and calcium in the body. Inhibition of hFGF23 activity can be used to treat certain hypophosphatemic disorders, such as X-linked hypophosphatemic rickets (XLH), in which blood hFGF23 levels are elevated due to loss-of-function mutations in the PHEX (phosphate-regulating endopeptidase homolog, X-linked) gene. Further diseases that can be prevented or treated by the compounds of the present invention include diseases characterized by excessive hFGF23 activity, such as neoplastic bone disease, autosomal dominant hypophosphatemic rickets (ADHR), fibrous dysplasia, and McCune-Albright syndrome.

[0032] The hFGF23 antagonist polypeptides disclosed herein contain a 3-helix vandal protein domain derived from the Z domain of Staphylococcus aureus protein A. By randomizing the amino acids two levels above the three helices, a large library was constructed from which potent binding agents were identified. Thus, the compounds of the present invention are designed to provide effective treatment of hypophosphatemia by binding to hFGF23, thereby antagonizing hFGF23-induced signaling activity at high potency, and are compatible with low-burden dosing devices suitable for self-administered or caregiver-administered drug delivery at home. In one embodiment, the hFGF23 antagonist polypeptides disclosed herein specifically bind to hFGF23, thereby reducing hFGF23-induced signaling mediated by both FGFR1c as the receptor and α-cloto(KL) as the co-receptor. This, for example, increases blood phosphate concentration.

[0033] The compounds disclosed herein preferably have a molecular weight of 5 to 10 kDa, for example, 7 to 8 kDa, which is substantially smaller than that of an antibody designated as burosumab (approximately 147 kDa), and have a therapeutically useful in vivo half-life in the blood that is expected to allow for a convenient administration frequency resulting in effective reduction of hFGF23-induced signaling. The presence of an extended portion plays a role in increasing the blood half-life of the compounds disclosed herein.

[0034] While not bound by theory, this suggests that lower doses and / or lower injection volumes at mg / kg may be used for near- or equivalent potency to burosumab at nmole / kg, which in turn means compatibility with self-administering devices such as injectable pens. Furthermore, improved phosphate upcontrol with less peak variability is expected.

[0035] Therefore, the molecular weight of the compound is expected to enable high concentrations in the pharmaceutical formulation. The pharmacokinetic properties of the compound disclosed herein enable a favorable drug exposure profile that is expected to minimize dose-to-dose phosphate fluctuations, which is expected to reduce dose-to-dose hypophosphatemia and consequently improve disease outcomes. In addition, the compound disclosed herein is expected to have significantly better efficacy than burosumab per mg when used in patients who need it, such as patients with XLH. The improved efficacy of the compound means that it can provide greater phosphate improvement than burosumab can achieve, and therefore the rate of phosphate normalization in such patients should be improved.

[0036] In one embodiment, the compounds of the present invention are characterized by referring to amino acid residues on hFGF23 that constitute their target binding sites, i.e., the major epitope residues or the complete set of epitope residues of the hFGF23 antagonist polypeptide. Accordingly, in some embodiments, the present invention relates to the amino acid residues on hFGF23 determined at 3.5 Å by Sequence ID No. 1 a) R76, T86, R91, and R114, b) M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170, c) R76, T86, R91, Y93, Y107, R114, F169, and N170, d) W36, R76, T86, R91, Y107, R114, I167, F169, and N170, and e) R76, T86, R91, R114, L166, and F169, This invention relates to an hFGF23 antagonist polypeptide capable of binding to an epitope consisting of amino acid residues selected from the group comprising the following.

[0037] In preferred embodiments, the compounds of the present invention are hFGF23 antagonist polypeptides that can bind to epitopes on hFGF23 including M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170 as determined by SEQ ID NO: 1 at 3.5 Å.

[0038] In one embodiment, the compounds of the present invention are characterized by reference to their binding motifs (BM). In such one embodiment, the compounds can be represented as hFGF23 antagonist polypeptides that can bind to hFGF23 according to SEQ ID NO: 1, and the polypeptide is i) X by Sequence ID 2 ab X c AWX d EIX e X f X g PX h LX i DX j QWPAFIEX k LH, During the ceremony, they acted independently of each other. X a is F or Y, X b is F, I, L, or V, X c is A or Q, X d is A, F, H, Y, or W, X e is F or Y, X f is N, Q, or T, X g is A or L, X h is C, H, N, Q, W, or Y, X i is D, N, Q, S, or T, X j is A, D, E, G, H, L, N, Q, S, T, or Y, X k This is an amino acid residue sequence that is A, Q, or S, ii) It includes a binding motif (BM) consisting of an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in i).

[0039] In a preferred embodiment, X a is Y. In a preferred embodiment, X b is L. In a preferred embodiment, X c Q is Q. In a preferred embodiment, X f is N. In a preferred embodiment, X a Y is X b L is X c Q is X f It is N.

[0040] In some embodiments, (BM) is YLQAWYEIFNLPNLNDNQWPAFIESLH (Sequence ID 11), YLQAWYEIFNLPWLNDNQWPAFIESLH (Sequence ID 12), YLQAWYEIFNLPYLNDNQWPAFIESLH (Sequence ID 13), YLQAWYEIFQLPNLNDNQWPAFIESLH (Sequence ID 14), YLQAWWEIFNLPNLNDNQWPAFIESLH (Sequence ID 15), YLQAWFEIFNLPNLNDNQWPAFIESLH (Sequence ID 16), YLQAWHEIFNLPNLNDNQWPAFIESLH (Sequence ID 17), YLAAWYEIFNLPNLNDNQWPAFIESLH (Sequence ID 18), YLQAWYEIFNLPHLNDNQWPAFIESLH (Sequence ID 19) YLQAWYEIFNLPNLNDNQWPAFIEALH (Sequence ID 20), YLQAWYEIFNLPNLNDNQWPAFIEQLH (Sequence ID 21), YLQAWYEIFNLPNLQDNQWPAFIESLH (Sequence ID 22), YLQAWYEIFNLPNLNDQQWPAFIESLH (Sequence ID 23), YLQAWYEIFNLPNLNDDQWPAFIESLH (Sequence ID 24), YLQAWYEIFNLPNLNDEQWPAFIESLH (Sequence ID 25), YLQAWYEIFNLPNLNDSQWPAFIESLH (Sequence ID 26), YLQAWYEIFNLPNLNDTQWPAFIESLH (Sequence ID 27), YLQAWYEIFNLPNLNDHQWPAFIESLH (Sequence ID 28), YLQAWYEIFNLPNLNDAQWPAFIESLH (Sequence ID 29), YLQAWYEIFNLPNLNDLQWPAFIESLH (Sequence ID 30), YLQAWYEIFNLPNLTDNQWPAFIESLH (Sequence ID 31), YLQAWYEIFNLPNLTDEQWPAFIESLH (Sequence ID 32), YLQAWYEIFNLPNLTDQQWPAFIESLH (Sequence ID 33), YLQAWYEIFNLPNLSDNQWPAFIESLH (Sequence ID 34), YLQAWYEIFNLPNLNDGQWPAFIESLH (Sequence ID 35), YVQAWYEIFNLPNLNDNQWPAFIESLH (Sequence ID 36), YIQAWYEIFNLPNLNDNQWPAFIESLH (Sequence ID 37) YFQAWYEIFNLPNLNDNQWPAFIESLH (Sequence ID 38) YLQAWYEIFNLPQLNDNQWPAFIESLH (Sequence ID 39), YLQAWYEIFNLPNLQDQQWPAFIESLH (Sequence ID 40), YLQAWYEIFNLPHLNDQQWPAFIESLH (Sequence ID 41), YLQAWYEIFNLPNLDDAQWPAFIESLH (Sequence ID 42), YLQAWYEIFNLPNLTDAQWPAFIESLH (Sequence ID 43), YLQAWYEIFNLPNLDDQQWPAFIESLH (Sequence ID 44), YLQAWYEIFNLPHLNDAQWPAFIESLH (Sequence ID 45) YLQAWYEIFNLPNLNDYQWPAFIESLH (Sequence ID 46), YLQAWYEIFNLPHLTDQQWPAFIESLH (Sequence ID 47), YLQAWYEIFNLPNLTDYQWPAFIESLH (Sequence ID 48), YLQAWYEIFTLPHLTDQQWPAFIESLH (Sequence ID 49), YLQAWYEIFNLPCLNDNQWPAFIESLH (Sequence ID 50), YLQAWYEIFNLPCLNDQQWPAFIESLH (Sequence ID 51), The selection is made from the group consisting of YLQAWYEIFNLPCLTDSQWPAFIESLH (Sequence ID 52).

[0041] In one aspect, the compounds of the invention are characterized by their binding motifs (BM), and the amino acid residues N and C terminally adjacent to the (BM), and thus can be represented as hFGF23 antagonist polypeptides comprising the binding motif (BM) as described above, and the polypeptide is a) X1X2X3X4X5X6X7X8-(BM)-X according to SEQ ID NO: 3 36 X 37 PSQX 41 X 42 X 43 LLX 46 EARX 50 LX 52 X 53 [[ID={20]]X 54 QX 56 X 57 X 58 , wherein, (BM) consists of the amino acid residue sequence defined in (i) above, wherein, independently of each other, X1 is G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 is D, E, H, N, R, or T; X5 is E, I, L, T, or V; X6 is F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T, or V; X8 is A, L, or E; X 36 is A, D, or E; [[ID=5#]]X 37 is D, or E; X 41 is A, R, or W; X 42 is A, D, or T; X 43 is E, N, Q, or S; X 46 [[ID=#2]]is A, E, or K; X 50 is Q, or R; X 52 is N or E, X 53 is D, E, K, or T, X 54 is A or I, X 56 is A or C, X 57 is K or P, X 58 This is an amino acid residue sequence that is C, K, or E. and b) Selected from amino acid residue sequences that have at least 93% identity with the sequence defined in a).

[0042] In a preferred embodiment, independently of each other, X1 is G, I, L, or V. X2 is either D or E, X3 is either D or E. X4 is either D or E. X5 is E, X6 is F, X7 is either I or Q. X8 is A or L, X a Y is, X b V is, X c Q is, X d Y is, X e is F or Y, X f is N or Q, X g L is, X h N is, X i is N or T, Xj is A, N, or Q. X k S is, X 36is D or E, X 37 is D or E, X 41 is W, X 42 A is, X 43 N is, X 46 is A or E, X 50 is Q, or R, X 52 N is, X 53 E is, X 54 A is, X 56 A is, X 57 P is, X 58 It is K.

[0043] As those skilled in the art will understand, it is possible to make minor changes to the amino acid sequence in a polypeptide without significantly affecting its function. Therefore, this disclosure includes modified variants of polypeptides that retain their antagonist properties. Thus, included in this disclosure are hFGF23 antagonist polypeptides containing amino acid sequences having 93% or more identity with the hFGF23 antagonist polypeptide or (BM) as defined above, such as 95%, 96%, 97%, 98%, or 99% or more identity. For example, it may be possible to replace amino acid residues belonging to a specific functional group of an amino acid residue (e.g., hydrophobic, hydrophilic, polar, etc.) with other amino acid residues from the same functional group.

[0044] In some embodiments, the hFGF23 antagonist polypeptide of the present invention may contain 50 to 70 amino acid residues. In preferred embodiments, the hFGF23 antagonist polypeptide may contain 56 to 60 amino acid residues. In more preferred embodiments, the hFGF23 antagonist polypeptide contains 58 amino acid residues.

[0045] In preferred embodiments, the hFGF23 antagonist polypeptide includes an extended portion. The extended portion or an hFGF23 antagonist polypeptide containing the extended portion may also be called an "hFGF23 antagonist polypeptide derivative," where the word derivative indicates the presence of the extended portion or the extended portion.

[0046] Preferred compounds of the present invention are chemical substances 89, 106, 107, 111, 197, 201, 204, 206, 207, and 208 (see Table 4 below). Non-limiting examples are provided below.

[0047] Chemical substance 204 is represented by the following formula: [ka]

[0048] Chemical substance 206 is represented by the following formula: [ka]

[0049] Chemical substance 207 is represented by the following formula: [ka]

[0050] Chemical substance 208 is represented by the following formula: [ka]

[0051] In one embodiment, the present invention relates to a pharmaceutical formulation comprising the hFGF23 antagonist polypeptide or an hFGF23 antagonist polypeptide derivative of the present invention, and one or more pharmaceutically acceptable excipients.

[0052] In one embodiment, the present invention relates to the pharmaceutical use of hFGF23 antagonist polypeptides, hFGF23 antagonist polypeptide derivatives, and pharmaceutical formulations comprising hFGF23 antagonist polypeptides or derivatives thereof. Such pharmaceutical uses include, but are not limited to, use in the treatment of hypophosphatemic disorders such as X-linked hypophosphatemic rickets (XLH).

[0053] In other embodiments, the present invention relates to an hFGF23 antagonist polypeptide or polypeptide derivative, or a pharmaceutical composition thereof. A polynucleotide encoding the hFGF23 antagonist polypeptide or polypeptide derivative, an expression vector encoding such polynucleotide, Host cells that produce the aforementioned hFGF23 antagonist polypeptide or polypeptide derivative, Furthermore, a method for producing the polypeptide or its derivatives, The present invention relates to a method comprising: a) culturing a host cell according to any one of the preceding claims under conditions that allow expression of the polypeptide encoded by the expression vector; b) isolating the polypeptide; and c) optionally attaching an extended portion to the isolated polypeptide.

[0054] The terms "a" or "an" are intended to mean "one or more."

[0055] In this specification, the term "approximately" means roughly, roughly, or around. When used in conjunction with a numerical range, the term "approximately" modifies the range by extending the boundary above and below the stated number. Generally, the term "approximately" can be used to modify a number within a range of 10% above or below the stated number (higher or lower).

[0056] When preceding an enumeration of processes or elements, the term "comprise," and its variations such as "comprises" and "comprising," are intended to indicate that the addition of further processes or elements is optional and not excluded.

[0057] As used throughout this disclosure, the terms “amino acid” or “amino acid residue” encompass both standard amino acids (genetically encoded) and non-natural amino acids. Non-exclusive examples of non-natural amino acids include Aib (α-aminoisobutyric acid), desaminohistidine (also known as 3-(imidazole-4-yl)propanoic acid, abbreviated as Imp (imidazolpropionyl)), and the d-isomers of standard amino acids. All amino acid residues in polypeptides for which optical isomers are not described should be understood herein to mean L-isomers unless otherwise specified.

[0058] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, for example, between a polypeptide and an antigen. The term "binding affinity" is used to describe monovalent interactions.

[0059] The binding affinity between two molecules due to monovalent interactions, for example, between an hFGF23 binder such as the hFGF23 antagonist polypeptide disclosed herein and hFGF23, can be quantified by determining the equilibrium dissociation constant (KD). D This can be determined by measuring the dynamics of complex formation and dissociation, for example, by surface plasmon resonance (SPR) spectroscopy.

[0060] The rate constants corresponding to the association and dissociation of monovalent complexes are, respectively, the association rate constant k. a (or k on ), and the dissociation rate constant k d (or k off ) is called K D is, formula K D =k d / k a Through, k a and k d This relates to the K of individual binder / antigen complexes (such as the interaction between the compounds disclosed herein and hFGF23), as defined above. D They can be compared by comparing their values.

[0061] The K of the hFGF23 antagonist peptide against its target D This may include less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 10 nm, less than 2 nm, less than 1 nm, and less than 100 μM. D It is preferable that the concentration is less than 0.1 μM, for example less than 10 nM, for example less than 2 nM, for example less than 1 nM. More preferably, K D It is less than 2 nM.

[0062] As used in this disclosure, the term binding motif (BM) refers to the hFGF23 binding region of an hFGF23 antagonist polypeptide. A (BM) comprises a key binding residue that enables tight binding to hFGF23 and forms part of a three-helix bundle protein domain. For example, a (BM) may constitute two alpha helices having interconnection loops within the three-helix bundle protein domain.

[0063] In some embodiments, (BM) contains 25 to 28 amino acid residues. In preferred embodiments, (BM) contains 27 amino acid residues.

[0064] In some embodiments, the (BM) sequence corresponds to a sequence from position 9 to 35 (both positions included) in a sequence selected from the group consisting of sequence numbers 11 to 52, or a sequence having 90% identity with sequence numbers 11 to 52, for example, sequence numbers 91, 92, 93%, or higher, for example, 94%, 95%, 96, 97, 98, or 99% identity with sequence numbers 11 to 52(BM).

[0065] In some embodiments, (BM) is characterized by comprising fixed or unfixed (variable) amino acid residues. Unfixed amino acid residues are referred to as X in this disclosure. x Identified by, the characters within the subscripts identify their position within (BM).

[0066] At the N-terminus and C-terminus, (BM) is adjacent to a non-(BM) amino acid residue, and together the (BM) residue and the non-(BM) residue form the hFGF23 antagonist polypeptide.

[0067] The “cross-species reactive” polypeptide binds to FGF23 from all indicated species (e.g., human, dog, pig, and cynomolgus monkey) with equivalent affinity, particularly within a KD range of 100, such as within a coefficient range of 50, within a coefficient range of 20, or within a coefficient range of 10. The KD of the defined coefficient X is... DWithin the range, it means that the highest affinity for a particular enumerated species does not exceed X times the lowest affinity measured for binding to a different enumerated species. Those skilled in the art can use any method for measuring affinity to determine if a heterospecific reactive polypeptide is K for all enumerated species under the same conditions. D To the extent applicable to the measurement, a given K as described herein D It will be understood that it is possible to verify that the target antigens from all listed species within the coefficient range bind. D The value is preferably measured using SPR, particularly at 25°C.

[0068] As used herein, the term “epitope” is defined in relation to molecular interactions between an “antigen-binding polypeptide” and its corresponding antigen (Ag), such as an hFGF23 binder, including the hFGF23 antagonist polypeptide and its derivatives disclosed herein. Generally, an “epitope” refers to a region or area on the antigen to which the binder binds, i.e., a region or area that is in physical contact with the binder. Physical contact can be defined using various criteria (e.g., a distance cutoff of 4-5 Å, such as 4 Å, 4.5 Å, or 5 Å, or solvent exposure) with respect to atoms in the binding molecule and the antigen molecule.

[0069] hFGF23 may contain a number of different epitopes, including, but not limited to, (1) linear peptide epitopes and (2) structural epitopes consisting of one or more discontinuous amino acids located in close proximity to each other in the hFGF23 configuration.

[0070] The epitopes of a given binder / antigen pair can be described and characterized with varying levels of detail using a variety of experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (HDX-MS), various competitive binding methods, and computational methods such as Alphafold 2, as well as methods known in the field.

[0071] For example, in the context of X-ray-derived crystal structures defined by the spatial coordinates of the complex of a binding molecule and its antigen, such as the hFGF23 antagonist polypeptide disclosed herein, the term epitope as used herein is specifically defined as an hFGF23 residue having a heavy atom (i.e., a non-hydrogen atom) within a distance of 3.5 Å from the heavy atom of the binding molecule, unless otherwise specifically specified or contradicted by the context.

[0072] For example, epitopes described at the amino acid level, as determined from X-ray structure, are said to be identical if they contain the same set of amino acid residues. Epitopes are said to be duplicated if at least one amino acid residue is shared by them. Epitopes are said to be separate (unique) if no amino acid residues are shared by them.

[0073] Epitopes may be identified by routine methods. For example, the general location of an epitope may be determined by evaluating the ability of hFGF23 antagonist polypeptides to bind to different fragments or variants of hFGF23. Specific amino acids within hFGF23 that come into contact with the binder (epitope) can also be determined using routine methods. For example, the binder and target molecules may be combined, and the resulting complex may be crystallized. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target. Epitope residues common to several different compounds may be considered key epitope residues.

[0074] As used herein, the term hFGF23 antagonist polypeptide refers to a polypeptide that can bind to hFGF23 according to Sequence ID No. 1 and can partially or completely block its biological activity, such as the hFGF23-induced signaling activity of the FGFRFGFR1c-alpha-klotho complex. The hFGF23 antagonist polypeptide contains three helix bundle protein domains, which are variants of protein Z derived from domain B of Staphylococcus protein A.

[0075] In some embodiments, the hFGF23 antagonist polypeptide is characterized by containing either fixed or unfixed amino acid residues. Unfixed (variable) amino acid residues are X x The number of subscripts / letters identified herein by this specification identify the position within the hFGF23 antagonist polypeptide.

[0076] When used in this disclosure, the terms “half-life” or “blood half-life” refer to the time required for half the amount of a substance administered to a patient to be metabolized or removed from the patient’s blood by normal biological processes.

[0077] The term “identity” as known in the art refers to the relationship between sequences of two or more polypeptides, determined by comparing their sequences. In the art, “identity” also means the degree of relevance of sequences between polypeptides, such as that determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percentage of perfect matches between the smaller of two or more sequences, with gap adjustments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithm”). The identity of relevant polypeptides can be readily calculated by known methods.

[0078] In this invention, identity was determined using Needleman from EMBOSS-6.6.0 (Needleman et al. J.Mol.Biol.1970;48:443-453), with parameters 10 and 0.5 for gap start and gap extend, respectively (gapopen=10, gapextend=0.5). The score for each paired sequence was calculated as the number of matching sequences in the aligned sequence, normalized by the sequence length, and multiplied by 100.

[0079] The term "pharmaceutically acceptable excipient" refers to excipients that are generally safe, non-toxic, and acceptable for human medicinal use, and are useful in preparing pharmaceutical compositions. Such excipients may be, for example, solids, liquids, or semi-solids.

[0080] As used herein, the term "polypeptide" refers to a single chain of amino acids linked by one or more peptide bonds, including oligopeptides.

[0081] A "substituted" variant preferably involves the substitution of one or more amino acids having the same number of amino acids. The substitution may be, but is not limited to, a conservative substitution. For example, amino acids may be substituted with amino acids having similar biochemical properties; for example, a basic amino acid may be substituted with another basic amino acid (e.g., lysine to arginine); an acidic amino acid may be substituted with another acidic amino acid (e.g., glutamic acid to aspartic acid); a neutral amino acid may be substituted with another neutral amino acid (e.g., threonine to serine); a charged amino acid may be substituted with another charged amino acid (e.g., glutamic acid to lysine); a hydrophilic amino acid may be substituted with another hydrophilic amino acid (e.g., asparagine to glutamine); a hydrophobic amino acid may be substituted with another hydrophobic amino acid (e.g., alanine to valine); a polar amino acid may be substituted with another polar amino acid (e.g., serine to threonine); an aromatic amino acid may be substituted with another aromatic amino acid (e.g., phenylalanine to tryptophan); and an aliphatic amino acid may be substituted with another aliphatic amino acid (e.g., leucine to isoleucine). These types of substitutions are called conservative substitutions.

[0082] The present invention encompasses variants of the hFGF23 antagonist polypeptide disclosed herein, which may include configurations comprising one, two, or three amino acid substitutions in the individual binding motifs and hFGF23 antagonist polypeptide sequences disclosed herein. In some embodiments, the substitutions are conservative substitutions.

[0083] As used herein, the term “treatment” or any variation thereof refers to a medical therapy for any human subject in need of treatment. This term includes administering a therapeutically effective amount of a polypeptide disclosed herein that is sufficient to reduce or eliminate at least one symptom of the disorder in question. However, “treatment” does not necessarily have to be curative. The timing and purpose of such treatment may vary from individual to individual, depending on the subject’s current health status. Thus, such treatment may be prophylactic, disease-modifying, palliative, symptomatic, and / or curative. With respect to the present invention, prophylactic, disease-modifying, palliative, symptomatic, and / or curative treatments may represent distinct embodiments of the present invention.

[0084] As used in this disclosure, the term extension portion (PM) refers to a portion that can extend the half-life in the blood of the compound to which it is bound. The extension portion consists of an extension factor "P" and an optional linker (L P ) includes. Therefore, the term “extension” refers to the extension of the half-life, and thus the extension factor or extension portion serves the purpose of extending the plasma half-life of the hFGF23 antagonist polypeptide disclosed herein. The terms “extension” and “half-life extension” are used interchangeably and generally refer to the extension of the plasma half-life of the compounds disclosed herein.

[0085] The extended portion is preferably covalently bonded to the side chain of a surface-exposed lysine or cysteine ​​residue in the polypeptide, preferably via an epsilon-amino group, after the polypeptide has been produced (e.g., by synthesis or recombinant expression). In this specification, the residue constituting the binding site of such a bond is generally referred to as "R1" (in the case of two or more extended portions "R2", "R3" asf.).

[0086] In view of the disclosures herein, those skilled in the art will be able to identify other surface-exposed residues suitable for binding.

[0087] The extension can consist of one extension factor (and without a "linker (Lp)").

[0088] The extension portion may be configured to include one linker (L P ) and one elongation factor (P).

[0089] The extension portion may be configured to include one linker and two or more elongation factors (P).

[0090] When the linker (L P ) is present, the extension portion binds to the hFGF23 antagonist polypeptide backbone via LP. When the linker (L P ) is absent, P binds directly to the polypeptide backbone.

[0091] As disclosed in the present disclosure, the hFGF23 antagonist polypeptide derivative, i.e., the hFGF23 antagonist polypeptide to which the extension portion is bound, may contain a single cysteine residue or lysine residue, and a single extension portion is bound.

[0092] The hFGF23 antagonist polypeptide derivative disclosed in the present disclosure may be configured to include two lysine residues and two extension portions.

[0093] The hFGF23 antagonist polypeptide derivative disclosed in the present disclosure may be configured to include two lysine residues and two identical extension portions.

[0094] The hFGF23 antagonist polypeptide derivative disclosed in the present disclosure may be configured to include two lysine residues and two non-identical extension portions.

[0095] The hFGF23 antagonist polypeptide derivative disclosed in the present disclosure may be configured to include three lysine residues and three extension portions.

[0096] The hFGF23 antagonist polypeptide derivative disclosed in the present disclosure may be configured to include three lysine residues and three identical extension portions.

[0097] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may contain three lysine residues and three non-identical extension portions (for example, the first and second extension portions may be the same, and the third extension portion is different from the first and second portions).

[0098] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing two cysteine residues and two extension portions.

[0099] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing two cysteine residues and two identical extension portions.

[0100] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing two cysteine residues and two non-identical extension portions.

[0101] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing three cysteine residues and three extension portions.

[0102] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing three cysteine residues and three identical extension portions.

[0103] The hFGF23 antagonist polypeptide derivatives disclosed in the present disclosure may have a configuration containing three cysteine residues and three identical extension portions.

[0104] When the hFGF23 antagonist polypeptide derivative contains two or three extension portions, the extension portions are preferably similar, more preferably substantially identical, or most preferably identical.

[0105] In this specification, an hFGF23 antagonist polypeptide comprising one or more extensions is optionally referred to as a polypeptide comprising extensions, or simply as a polypeptide derivative.

[0106] The extended portion may be capable of non-covalent binding to albumin, thereby promoting the circulation of hFGF23 antagonist polypeptide derivatives in the bloodstream and extending their half-life. Therefore, in one embodiment, the extended portion is an albumin-binding portion.

[0107] Growth factors may include acyl groups. Extension factors may include fatty acyl groups. Acyl groups may be branched or unbranched. Acyl groups may be saturated or unsaturated.

[0108] The extension factor may also have a configuration that includes a distal carboxylic acid group.

[0109] The extension factor may contain at least two acidic groups, one of which is attached to the terminal end.

[0110] The extension factor may also have a configuration that includes a fatty acid group.

[0111] The extension factor may also have a configuration that includes fatty acid groups and amide groups.

[0112] The extension factor may also have a configuration that includes a distal carboxylic acid group and an amide group.

[0113] The extension factor may also contain an alkyl group.

[0114] The extension factor may also contain an aryl group.

[0115] The extension factor may also contain a tetrazole group.

[0116] The extension factor may also have a configuration that includes a sulfonic acid group.

[0117] The extender may have a structure containing a phenoxy group.

[0118] The extender may have a structure containing a benzoic acid group.

[0119] The extender may have a structure containing 8 to 30 carbon atoms. The extender may have a structure containing 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

[0120] The extender may have a structure containing 6 to 30 consecutive -CH2- groups. The extender may have a structure containing a carbon chain with at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive -CH2- groups.

[0121] The extender may have a structure containing 12 to 26 carbon atoms. The "extender" may have a structure containing 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms.

[0122] The extender may have a structure containing 10 to 26 consecutive -CH2- groups. The extender may have a structure containing a carbon chain with 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive -CH2- groups.

[0123] The extender may have a structure containing 16 to 22 carbon atoms. The extender may have a structure containing 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

[0124] The extension factor may consist of 14 to 20 consecutive -CH2- groups. The extension factor may consist of a carbon chain containing 14, 15, 16, 17, 18, 19, or 20 consecutive -CH2- groups.

[0125] The extension factor may also consist of 16 to 22 consecutive carbon atoms and 14 to 20 consecutive -CH2- groups.

[0126] The extension factor may have a configuration containing 16 consecutive carbon atoms and 14 consecutive -CH2- groups.

[0127] The extension factor may have a configuration containing 18 consecutive carbon atoms and 16 consecutive -CH2- groups.

[0128] The extension factor may have a configuration containing 20 consecutive carbon atoms and 18 consecutive -CH2- groups.

[0129] The extension factor may also have a configuration containing 22 consecutive carbon atoms and 20 consecutive -CH2- groups.

[0130] The extension factor is, Chemical 1: HOOC-(CH2) n It contains a group defined by -CO-* (where n is an integer in the range of 8 to 30), which is a C(n+2) diacid, or Chemical 1: [ka] This is sometimes also called (where n is an integer between 8 and 30).

[0131] The extension factor may also consist of an oligopeptide. The extension factor oligopeptide may consist of 10 to 40 amino acids, for example 10 to 30 amino acids, for example 15 to 25 amino acids, and preferably 20 amino acids. The extension factor oligopeptide sequence composition may be, for example, GlnArgLeuMetGluAspIleCysLeuProArgTrpGlyCysLeuTrpGluAspAspPhe (single amino acid notation QRLMEDICLPRWGCLWEDDF (chemical substance 1f, SEQ ID NO: 9)) (see also WO01 / 45746 A2).

[0132] The extension factor is amino acid linker L P It may be conjugated to ISVD or an hFGF23 antagonist polypeptide via [a specific method].

[0133] In one embodiment, L P This allows for the attachment of an extension factor (P) to the side chain of a lysine or cysteine ​​residue in the hFGF23 antagonist polypeptide backbone.

[0134] In one embodiment, L P The growth factor may be bound to the N-terminal or C-terminal residue of the hFGF23 antagonist polypeptide backbone.

[0135] In one embodiment, L P The growth factor may be bound to residues 46K, 53K, or 57K in hFGF23.

[0136] The hFGF23 antagonist polypeptide derivative may also have a configuration that includes two extended portions, each containing 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

[0137] The hFGF23 antagonist polypeptide derivative may contain two extension factors, each extension factor comprising 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups, and such extension factors are also referred to as C12, C13, C14, C15, C16, C17, or C18 extension factors. In some embodiments, the extension factors include acidic groups, such as carboxylic acid groups (diacids) adjacent to the consecutive -CH2- groups at each terminal.

[0138] The hFGF23 antagonist polypeptide derivative may also have a configuration comprising two C14 diacides, two C16 diacides, or two C18 diacides.

[0139] The hFGF23 antagonist polypeptide derivative may contain three extensions, each containing an extension factor comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The hFGF23 antagonist polypeptide may contain three extensions, each containing 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups.

[0140] In some embodiments, the growth factor comprises C16, C17, C18, C19, C20, C21, or C22 fatty acids, such as C16, C17, C18, C19, or C20 fatty diacides.

[0141] In a preferred embodiment, the hFGF23 antagonist polypeptide derivative comprises a diacid extension factor (C20 diacid extension factor) containing 20 consecutive -CH2- groups.

[0142] In a preferred embodiment, the C20 diacid extension factor is chemical substance 1c shown in Table 1 below.

[0143] In some embodiments, the hFGF23 antagonist polypeptides disclosed herein may include an extension factor selected from any one of those shown in Table 1.

[0144] [Table 1] [Table 1-2]

[0145] Here, the asterisk (*) represents L P This indicates a linker element binding site or an amino acid residue within the hFGF23 antagonist polypeptide.

[0146] As used herein, the term “extension portion” means having half-life extension properties and comprising an “extension factor” ("P") and an optional “linker” ("L"). P」 This refers to the portion containing ), and if present, the linker links the extension factor to the hFGF23 antagonist polypeptide.

[0147] L P The linker, if present, may consist of Ado, Aeep, or Aeeep, sulfonamide, Trx, ε-Lys, Ahx, Glu, γGlu, Gly, Ser, Ala, and / or Thr.

[0148] L P The linker may include, for example, at least a portion that can be represented by the following chemicals, and asterisk(*) is a further L in the hFGF23 antagonist polypeptide. P This indicates a linker element, extension factor, or binding site to an amino acid residue. Those skilled in the art will understand that the first and second * in the individual chemicals below do not indicate binding sites on the same type of element; that is, linkers following the formula below are not intended to link two hFGF23 antagonist polypeptides or two extension factors.

[0149] Chemical 2a: *-NH-(CH2)2-(O-(CH2)) k -O-(CH2) n -CO-* or Chemical 2a: [ka] The configuration may include at least a part that can be expressed by (wherein k is an integer in the range of 1 to 5, and n is an integer in the range of 1 to 5).

[0150] When k=1 and n=1, the linker element may be designated as Ado, or 8-amino-3,6-dioxaoctanoyl, which is the following chemical: Chemical 2b: *NH-(CH2)2-O-(CH2)2-O-CH2-CO-* or Chemical 2b: [ka] It may also be represented by [another method].

[0151] For k=1 and n=2, the linker element may be designated as Aeep, which may be represented by the following chemical: Chemical substance 2c: *NH-(CH2)2-O-(CH2)2-O-(CH2)2-CO-* or Chemical 2c: [ka]

[0152] For k=2 and n=2, the linker element may be designated as Aeeep, which may be represented by the following chemical: Chemical 2d: *-NH-(CH2)2-O-(CH2)2O-(CH2)2-O-(CH2)2-CO-* or Chemical 2d: [ka] Optional linker (L P) may also have a configuration including a sulfonamide-C4 moiety. The sulfonamide-C4 group is a sulfonamide group bonded to a 4-butanoyl group and has the following chemical substances. Chemical 2e:*-NH-S(O)2-CH2-CH2-CH2-CO-* or Chemical 2e: [ka]

[0153] Optional linker L P This may also be a composition containing Trx. Trx is also known as tranexamic acid, trans-4-(aminomethyl)cyclohexanecarboxylic acid, and has the following chemical components: Chemical substance 2f:*-NH-CH2-(C6H 10 )-CO-* or Chemical 2f: [ka]

[0154] Linker L P This may also be a composition containing epsilon-lysine (ε-Lys).

[0155] Linker L P This may also include a lysine (Lys) component.

[0156] Linker L P This may also include Ahx. Ahx, also known as aminocaproic acid or 6-aminohexanoic acid, is defined as follows: Chemical substance 2g:*-NH-(CH2)5-CO-* or Chemistry 2g: [ka]

[0157] Linker LP teeth, Chemicals 2h: [ka] The Glu diradical may be present in p instances, where p is an integer between 1 and 3.

[0158] Chemical 2h is also the gamma-carboxyl group of the amino acid glutamic acid (E) used herein to link to the epsilon-amino group of lysine, and may therefore be referred to as gamma-Glu, or simply γGlu. As described above, other linker elements may be, for example, another Glu residue or an Ado molecule. The amino group of Glu is then linked with the carboxyl group of the extended portion, or if present, with, for example, the carboxyl group of an Ado molecule, or if present, with, for example, the gamma-carboxyl group of another Glu, or if present, [ka] or [ka] or [ka] And so on, forming an amide bond.

[0159] In preferred embodiments, the growth factors exemplified by chemical substances 1a to 1f are linkers (L) selected from Table 2 below. P The molecule is bound to the polypeptide skeleton using ), where "R1" represents the binding site in the hFGF23 antagonist polypeptide and "P" represents the growth factor.

[0160] In a preferred embodiment, the growth factor chemicals 1a to 1f are linked to the linker (L1 (chemical 2i)) as shown in Table 2 below. P It is attached to the polypeptide backbone using ).

[0161] In a preferred embodiment, the binding site R1 is located on the epsilon-amino group of the polypeptide backbone.

[0162] In one such embodiment, the binding site R1 is located on the epsilon-amino group of the lysine C-terminal residue of the polypeptide backbone.

[0163] In another such embodiment, the binding site R1 is located on the epsilon-amino group of lysine at position 46, 53, or 57 of the hFGF23 antagonist polypeptide.

[0164] Therefore, in a preferred embodiment, the residues at positions 46, 53, and / or 57 are lysine (K).

[0165] In a preferred embodiment, the residue at position 58 is lysine (K).

[0166] Based on the disclosure herein, those skilled in the art will be able to determine the optimal L for use with the specific hFGF23 antagonist polypeptide derivatives disclosed herein. P Determining the linker may be possible through arbitrary selection, after a few limited routine experiments.

[0167] [Table 2]

[0168] The compounds disclosed herein can be produced by solid-phase peptide synthesis as described in the Examples section below. The Fmoc-Lys(Mtt)-OH group placed at the extension subbinding site allows for the selective removal of the Mtt group during peptide synthesis, followed by the introduction of the extension subgroup. The resin-bound peptide was deprotected post-synthesis and then purified by reverse-phase HPLC.

[0169] The compounds disclosed herein can be generated by recombinant nucleic acid technology. Generally, the cloned wild-type compounds disclosed herein are modified to encode a desired protein. This modified sequence is then inserted into an expression vector, which is then transformed or transfected into host cells.

[0170] The nucleic acid constructs encoding the compounds disclosed herein may be of genomic origin, cDNA origin, or synthetic origin. Amino acid sequence modifications are achieved by modifying the genetic code using well-known techniques.

[0171] The DNA sequences encoding the compounds disclosed herein are typically inserted into a recombinant vector, which may be any vector conveniently available for recombinant DNA procedures, and the choice of vector often depends on the host cell into which the vector is introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, whose replication is independent of chromosomal replication and may be, for example, a plasmid. Alternatively, the vector may be integrated into the host cell genome upon introduction into the host cell and replicate together with the chromosome into which it is integrated.

[0172] The vector is preferably an expression vector in which the DNA sequence encoding the compound disclosed herein is operably linked to additional segments necessary for DNA transcription. The term “operably linked” means that the segments are positioned so that they function in coordination for their intended purpose, for example, so that transcription proceeds through the polypeptide-encoding DNA sequence from a promoter until said transcription terminates within a terminator.

[0173] Accordingly, an expression vector for use in the expression of the compounds disclosed herein comprises a promoter capable of initiating and directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence that exhibits transcriptional activity in a selected host cell, and may be derived from a gene encoding a protein that is either homologous or heterologous to the host cell.

[0174] Furthermore, the expression vectors used for the expression of the compounds disclosed herein also include a terminator sequence, which is a sequence recognized by the host cell for terminating transcription. The terminator sequence is operably ligated to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in a selected host cell may be used in the present invention.

[0175] The expression of the compounds disclosed herein can be directed either to intracellular expression in the cytosol of a host cell or to a secretory pathway for extracellular expression into a growth medium.

[0176] Intracellular expression is the default pathway and requires an expression vector comprising a promoter, followed by a DNA sequence encoding the compound disclosed herein, followed by a terminator.

[0177] To direct the compounds disclosed herein to the secretory pathway of a host cell, a secretory signal sequence (also known as a signal peptide or pre-sequence) is required as an N-terminal extension of the compounds disclosed herein. The DNA sequence encoding the signal peptide is ligated to the 5' end of the DNA sequence encoding the compound disclosed herein in the precise read frame. The signal peptide may be one that is typically associated with a protein, or it may be derived from a gene encoding another secretory protein.

[0178] Procedures used to ligate DNA sequences encoding the compounds, promoters, terminators, and / or secretion signal sequences disclosed herein, and to insert them into a suitable vector containing the information necessary for replication, are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

[0179] The host cells into which the DNA sequences encoding the compounds disclosed herein are introduced may be any cells capable of expressing the compounds disclosed herein, either intracellularly or extracellularly. If post-translational modification is required, suitable host cells include higher eukaryotic cells such as yeast, fungal, insect, and mammalian cells.

[0180] The extended portion can be attached to the polypeptide backbone as follows.

[0181] To a solution of polypeptide backbone (10-40 mg / mL, 0.25 M Na2CO3 (aqueous solution)), N-methylpyrrolidinone (0.25 × volume of peptide solution) was added while stirring. To obtain selective reaction conditions for the epsilon amino group of lysine, aqueous sodium hydroxide solution was gradually added to increase the pH until it reached 10.5-11.

[0182] Add the acylation reagent solution (2-4 molar equivalents in terms of polypeptide backbone) to the peptide solution under stirring for 2-60 minutes, while continuously adjusting the pH to 10.5-11 by adding aqueous sodium hydroxide solution during this time. Stir the final reaction mixture at a constant pH at room temperature until the acylation reagent is consumed (0-4 hours). Neutralize the reaction mixture by adding acetic acid dropwise and purify it by reverse-phase HPLC.

[0183] Therefore, the extended portion can be bound to the polypeptide described above. In other embodiments of the present invention, the extended portion may be a fusion polypeptide that depends on a part of the polypeptide backbone, i.e., a bond via a peptide amide bond. The fusion polypeptide can be obtained synthetically or by recombinant expression by methods known to those skilled in the art.

[0184] The hFGF23 antagonist polypeptide derivatives disclosed herein may be prepared in a pharmaceutical composition. In some embodiments, such a composition comprises at least one pharmaceutically acceptable excipient.

[0185] Excipients can serve a variety of purposes, for example, as carriers, vehicles, fillers, binders, lubricants, disintegrants, flow regulators, crystallization inhibitors, solubilizers, stabilizers, colorants, flavoring agents, surfactants, emulsifiers, delivery agents, hydrotropes, or combinations thereof, and / or to improve the administration and / or absorption of active pharmaceutical ingredients.

[0186] The amounts of each excipient used may be modified within the conventional range in the art. Possible techniques and excipients are, for example, Remington: The Science and Practice of Pharmacy, 22 nd This is described in edition, Remington and Allen, Eds., Pharmaceutical Press (2013).

[0187] Compositions containing the compounds disclosed herein may be administered for prophylactic treatment and / or, in some embodiments, for on-demand therapy.

[0188] The composition is typically administered to subjects already suffering from a disease, such as those listed below, in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications. An amount sufficient to achieve this is defined as the “therapeutic effective dose.” As will be understood by those skilled in the art, the effective dose for this purpose depends on the severity of the disease, as well as the subject’s weight and overall condition.

[0189] The compounds disclosed herein, such as hFGF23 antagonist polypeptide derivatives, may be administered parenterally in a suitable pharmaceutical composition, for example, intravenously, intramuscularly, or subcutaneously.

[0190] In some embodiments, the dose of the compound delivered by subcutaneous administration may be about 0.1 mg to 500 mg of the compound per day, preferably about 0.5 mg to 150 mg per day, depending on the severity of the condition.

[0191] The preferred dose may also be adjusted for a particular compound based on its properties, including its in vivo half-life or mean residence time in the blood and its biological activity. In one embodiment, the present invention relates to an injection device having the contents of the composition.

[0192] In one embodiment, the compound can be used in a low-load dosing device suitable for self-administration or caregiver-administered drug delivery at home.

[0193] In one embodiment, the hFGF23 antagonist polypeptide or polypeptide derivative is an interspecific reactive polypeptide or polypeptide derivative.

[0194] In one embodiment, an hFGF23 antagonist polypeptide or polypeptide derivative exhibits an IC50 of less than 1 nM in the inhibitory efficacy assay disclosed herein. 50 Provide a value.

[0195] In one embodiment, an hFGF23 antagonist polypeptide or polypeptide derivative provides less than 50% residual activity at 1 nM in the inhibitory efficacy assay disclosed herein.

[0196] In one embodiment, the compound of the present invention provides improved phosphate levels compared to prior art compounds, such as brosumab.

[0197] Further embodiments 1. On hFGF23 as determined by Sequence ID 1 at 3.5 Å: a) R76, T86, R91, and R114, b) M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170, c) R76, T86, R91, Y93, Y107, R114, F169, and N170, d) W36, R76, T86, R91, Y107, R114, I167, F169, and N170, and e) Bindable to an epitope containing an amino acid residue sequence selected from the group consisting of R76, T86, R91, R114, L166, and F169. hFGF23 antagonist polypeptide. 2. The hFGF23 antagonist polypeptide described in Embodiment 1, which can bind to hFGF23 according to Sequence ID No. 1, i) X by Sequence ID 2 a X b X c AWX d EIX e X f X g PX h LX i DX j QWPAFIEX k LH, During the ceremony, they acted independently of each other. X a is F or Y, X b is F, I, L, or V, Xc is A or Q, X d is A, F, H, Y, or W, X e is F or Y, X f is N, Q, or T, X g is A or L, X h is C, H, N, Q, W, or Y, X i is D, N, Q, S, or T, X j is A, D, E, G, H, L, N, Q, S, T, or Y, X k This is an amino acid residue sequence that is A, Q, or S, ii) An hFGF23 antagonist polypeptide comprising a binding motif (BM) consisting of an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in i). 3. An hFGF23 antagonist polypeptide that is capable of binding to hFGF23 according to Sequence ID No. 1, i) X by Sequence ID 2 a X b X c AWX d EIX e X f X g PX h LX i DX j QWPAFIEX k LH, During the ceremony, they acted independently of each other. X a is F or Y, X b is F, I, L, or V, X c is A or Q, X d is A, F, H, Y, or W, X e is F or Y, Xf is N, Q, or T, X g is A or L, X h is C, H, N, Q, W, or Y, X i is D, N, Q, S, or T, X j is A, D, E, G, H, L, N, Q, S, T, or Y, X k This is an amino acid residue sequence that is A, Q, or S, ii) An hFGF23 antagonist polypeptide comprising a binding motif (BM) consisting of an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in i). 4. An hFGF23 antagonist polypeptide according to Embodiment 2 or 3, wherein (BM) is Selected from a group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52, an hFGF23 antagonist polypeptide, optionally comprising one, two, or three substitutions, the substitutions of which may occur at any one of the positions 1, 2, 3, 6, 9, 10, 11, 13, 15, 17, and / or 25 of the selected (BM) sequence. 5. The hFGF23 antagonist polypeptide according to Embodiment 4, wherein the substitution is a conservative substitution. 6. An hFGF23 antagonist polypeptide as described in any one of the prior embodiments, The polypeptide comprises a binding motif (BM), a) X1X2X3X4X5X6X7X8-(BM)-X according to sequence number 3 36 X 37 PSQX 41 X 42 X 43 LLX 46 EARX 50 LX 52 X 53 X 54 QX 56 X 57 X 58 , In the formula, (BM) consists of the amino acid residue sequence defined in Embodiment 2(i), During the ceremony, they acted independently of each other. X1 is G, I, L, N, or V. X2 is D, E, or Q, X3 is D, E, G, N, or Q. X4 is D, E, H, N, R, or T. X5 is E, I, L, T, or V. X6 is F, W, or Y. X7 is A, G, H, I, L, N, P, Q, R, T, or V. X8 is A, L, or E. X 36 is A, D, or E, X 37 is D or E, X 41 is A, R, or W, X 42 is A, D, or T, X 43 is E, N, Q, or S, X 46 is A, E, or K, X 50 is Q, or R, X 52 is N or E, X 53 is D, E, K, or T, X54 is A or I, X 56 is A or C, X 57 is K or P, X 58 This is an amino acid residue sequence that is C, K, or E. and b) an hFGF23 antagonist polypeptide comprising an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in a). 7. An hFGF23 antagonist polypeptide that is capable of binding to hFGF23 according to SEQ ID NO: 1, and the polypeptide is a) X1X2X3X4X5X6X7X8X according to Sequence ID 3 a X b X c AWX d EIX e X f X g PX h LX i DX j QWPAFIEX k LHX 36 X 37 PSQX 41 X 42 X 43 LLX 46 EARX 50 LX 52 X 53 X 54 QX 56 X 57 X 58 During the ceremony, they acted independently of each other. X1 is G, I, L, N, or V. X2 is D, E, or Q, X3 is D, E, G, N, or Q. X4 is D, E, H, N, R, or T. X5 is E, I, L, T, or V. X6 is F, W, or Y. X7 is A, G, H, I, L, N, P, Q, R, T, or V. X8 is A, L, or E. X a is F or Y, X b is F, I, L, or V, X c is A or Q, X d is A, F, H, Y, or W, X e is F or Y, X f is N, Q, or T, X g is A or L, X h is C, H, N, Q, W, or Y, X i is D, N, Q, S, or T, X j is A, D, E, G, H, L, N, Q, S, T, or Y, X k is A, Q, or S, X 36 is A, D, or E, X 37 is D or E, X 41 is A, R, or W, X 42 is A, D, or T, X 43 is E, N, Q, or S, X 46 is A, E, or K, X 50 is Q, or R, X 52 is N or E, X 53 is D, E, K, or T, X 54 is A or I, X 56 is A or C, X 57 is K or P, X58 This is an amino acid residue sequence that is C, K, or E. and b) an hFGF23 antagonist polypeptide comprising an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in a). 8. hFGF23 antagonist polypeptide that is capable of binding to hFGF23 according to Sequence ID No. 1, X1X2X3X4X5X6X7X8X by Sequence Number 3 a X b X c AWX d EIX e X f X g PX h LX i DX j QWPAFIEX k LHX 36 X 37 PSQX 41 X 42 X 43 LLX 46 EARX 50 LX 52 X 53 X 54 QX 56 X 57 X 58 , During the ceremony, they acted independently of each other. X1 is G, I, L, N, or V. X2 is D, E, or Q, X3 is D, E, G, N, or Q. X4 is D, E, H, N, R, or T. X5 is E, I, L, T, or V. X6 is F, W, or Y. X7 is A, G, H, I, L, N, P, Q, R, T, or V. X8 is A, L, or E. X a is F or Y, X b is F, I, L, or V, X cis A or Q, X d is A, F, H, Y, or W, X e is F or Y, X f is N, Q, or T, X g is A or L, X h is C, H, N, Q, W, or Y, X i is D, N, Q, S, or T, X j is A, D, E, G, H, L, N, Q, S, T, or Y, X k is A, Q, or S, X 36 is A, D, or E, X 37 is D or E, X 41 is A, R, or W, X 42 is A, D, or T, X 43 is E, N, Q, or S, X 46 is A, E, or K, X 50 is Q, or R, X 52 is N or E, X 53 is D, E, K, or T, X 54 is A or I, X 56 is A or C, X 57 is K or P, X 58 This is an amino acid residue sequence that is C, K, or E. An FGF23 antagonist polypeptide comprising an amino acid residue sequence selected from, optionally comprising one, two, or three substitutions, wherein the substitutions may occur at any one of the above non-fixed positions. 9. The hFGF23 antagonist polypeptide according to Embodiment 8, wherein the substitution is a conservative substitution. 10. An hFGF23 antagonist polypeptide according to any one of Embodiments 6 to 9, wherein each is independent of the others. X1 is G, I, L, or V. X2 is either D or E, X3 is either D or E. X4 is either D or E. X5 is E, X6 is F, X7 is either I or Q. X8 is A or L, X a Y is, X b V is, X c Q is, X d Y is, X e is F or Y, X f is N or Q, X g L is, X h N is, X i is N or T, Xj is A, N, or Q. X k S is, X 36 is D or E, X 37 is D or E, X 41 is W, X 42 A is, X 43 N is, X 46 is A or E, X 50 is Q, or R, X 52 N is, X 53 E is, X 54 A is, X 56 A is, X 57 P is, X 58 This is the K, hFGF23 antagonist polypeptide. 11. hFGF23 antagonist polypeptide that is capable of binding to hFGF23 according to Sequence ID No. 1, Chemical substance 197 listed in Sequence ID No. 168, Chemical substance 201, as described in Sequence ID No. 176. Chemical substance 204 listed in Sequence ID No. 234, Chemical substance 206, as described in Sequence ID No. 236, Chemical substance 207 as described in Sequence ID No. 237, and An hFGF23 antagonist polypeptide having at least 93% identity to an hFGF23 antagonist polypeptide selected from the group consisting of chemical substance 208 described in Sequence ID No. 238. 12. The hFGF23 antagonist polypeptide described in Embodiment 11, Chemical substance 197 listed in Sequence ID No. 168, Chemical substance 201, as described in Sequence ID No. 176. Chemical substance 204 listed in Sequence ID No. 234, Chemical substance 206, as described in Sequence ID No. 236, Chemical substance 207 as described in Sequence ID No. 237, and An hFGF23 antagonist polypeptide having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an hFGF23 antagonist polypeptide selected from the group consisting of chemical substance 208 described in Sequence ID No. 238. 13. An hFGF23 antagonist polypeptide according to Embodiment 11 or 12, wherein the hFGF23 is determined at 3.5 Å according to Sequence ID No. 1. a) R76, T86, R91, and R114, b) M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170, c) R76, T86, R91, Y93, Y107, R114, F169, and N170, hFGF23 antagonist polypeptides capable of binding to epitopes containing amino acid residues selected from the group consisting of d) W36, R76, T86, R91, Y107, R114, I167, F169, and N170, and e) R76, T86, R91, R114, L166, and F169. 14. An hFGF23 antagonist polypeptide according to any one of Embodiments 2 to 6, wherein the binding motif (BM) forms part of a 3-helix vandal protein domain, and the 3-helix vandal protein domain is a variant of protein Z derived from domain B of Staphylococcus protein A. 15. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide comprises 58 amino acid residues. 16. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide is chemicals 89, chemicals 106, Chemical 107, chemicals 111, chemicals 197, Chemical substances 201, chemicals 204, chemicals 206, Chemical substances 207; and hFGF23 antagonist polypeptide selected from the group consisting of 208 chemical substances. 17. An hFGF23 antagonist polypeptide according to any one of the preceding embodiments, wherein the polypeptide may be less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 10 nM, less than 1 nM, or less than 100 μM, preferably less than 0.1 μM, less than 10 nM, less than 2 nM, less than 1 nM, or less than 0.1 μM. D hFGF23 antagonist polypeptide capable of binding to hFGF23 at a given value. 18. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide comprises three helix bundle protein domains which are variants of protein Z derived from domain B of Staphylococcus protein A. 19. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide is a 3-helix vandal protein domain which is a variant of protein Z derived from domain B of Staphylococcus protein A. 20. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide is Affibody® or derived from Affibody®. 21. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the polypeptide comprises an extended portion. 22. An hFGF23 antagonist polypeptide according to any one of the prior embodiments, wherein the extended portion is covalently bonded to an amino acid residue side chain group on the polypeptide. 23. An hFGF23 antagonist polypeptide according to any one of Embodiments 1 to 21, wherein the extended portion is covalently bonded to the polypeptide via a peptide amide bond. 24. An hFGF23 antagonist polypeptide according to Embodiment 21 or 22, wherein the extended portion comprises at least two acidic groups, with one acidic group located at the terminal end. 25. hFGF23 antagonist polypeptide according to Embodiment 21, 22, or 24, wherein the extended portion comprises a C16, C17, C18, C19, C20, C21, or C22 fatty acid, such as C16, C17, C18, C19, or C20 fatty acid. 26. An hFGF23 antagonist polypeptide according to Embodiments 21, 22, 24, or 25, wherein the extended portion is [ka] (chemical substance 1a); [ka] (Chemical 1b): [ka] (Chemical 1c): [ka] (Chemical 1d): [ka] (chemical substance 1e); QRLMEDICLPRWGCLWEDDF-*(chemical substance 1f); *-QRLMEDICLPRWGCLWEDDF (chemical substance 1f), In the formula, * represents the linker (L PAn hFGF23 antagonist polypeptide comprising an extension factor "P" selected from the group consisting of ) or representing a binding site to either an hFGF23 antagonist polypeptide. 27. hFGF23 antagonist polypeptide according to Embodiments 1-22, 24-26, wherein the polypeptide is a polypeptide derivative comprising an extended portion, and the extended portion is *‐NH-(CH2)2-(O-(CH2)) k -O-(CH2) n -CO-*(chemical substance 2a), (wherein k is an integer in the range of 1 to 5, and n is an integer in the range of 1 to 5); *NH-S(O)2-CH2-CH2-CH2-CO-*(chemical substance 2e); *-NH-CH2-(C6H 10 )-CO-*(chemical substance 2f); *-NH-(CH2)5-CO-*(chemical substance 2g); [ka] [ka] [ka] [ka] Linker L selected from the group consisting of the following p hFGF23 antagonist polypeptide, including 28. An hFGF23 antagonist polypeptide according to any one of Embodiments 21 to 27, wherein the polypeptide is a polypeptide derivative including an extended portion, and the extended portion is bound to the N-terminal amino acid residue of the polypeptide. 29. An hFGF23 antagonist polypeptide according to any one of Embodiments 21 to 27, wherein the polypeptide is a polypeptide derivative comprising an extended portion, and the extended portion is bound to a C-terminal amino acid residue in the polypeptide. 30. An hFGF23 antagonist polypeptide according to any one of Embodiments 21-22 or 24-29, wherein the polypeptide is a polypeptide derivative comprising an extended portion, and the extended portion is covalently bonded to a side chain of a cysteine ​​residue or a lysine residue in the polypeptide. 31. A compound, [ka] [ka] A compound selected from the group consisting of the following. 32. An hFGF23 antagonist polypeptide derivative that can bind to hFGF23 represented by the following structure. [ka] 33. hFGF23 antagonist polypeptide derivatives that can bind to hFGF23 represented by the following structure. [ka] 34. hFGF23 antagonist polypeptide derivatives that can bind to hFGF23 represented by the following structure. [ka] 35. hFGF23 antagonist polypeptide derivatives that can bind to hFGF23 represented by the following structure. [ka] 36. The compound according to Embodiment 31, wherein the compound is an hFGF23 antagonist polypeptide derivative that can bind to hFGF23. 37. An hFGF23 antagonist polypeptide or polypeptide derivative according to any one of the prior embodiments, wherein the polypeptide or polypeptide derivative is an interspecific reactive polypeptide or polypeptide derivative. 38. An hFGF23 antagonist polypeptide or polypeptide derivative described in a prior embodiment, wherein the polypeptide or polypeptide derivative is capable of binding to one or more cynomolgus monkey FGF23, canine FGF23, or porcine FGF23. 39 A pharmaceutical composition comprising an hFGF23 antagonist polypeptide, polypeptide derivative, described in any one of the prior embodiments, and one or more pharmaceutically acceptable excipients. 40. An hFGF23 antagonist polypeptide derivative, polypeptide derivative, compound, or composition described in any one of the prior embodiments, for use in pharmaceuticals. 41. An hFGF23 antagonist polypeptide derivative, polypeptide derivative, compound, or composition according to any one of the preceding embodiments, for use in the treatment of a disease characterized by elevated hFGF23 concentration compared to that in a healthy subject. 42. An hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of Embodiments 1 to 39, for use in the treatment of hypophosphatemia. 43. An hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of Embodiments 1 to 39, for use in the treatment of tumor-induced bone disease, fibrous dysplasia, McCune-Albright syndrome, autosomal dominant hypophosphatemic rickets (ADHR), or X-linked hypophosphatemic rickets (XLH). 44. An hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of Embodiments 1 to 39, for use in the treatment of X-linked hypophosphatemic rickets (XLH). 45. A method for inhibiting hFGF23-induced signaling in a patient in need thereof, comprising administering to the subject an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39. 46. ​​A method for treating a hypophosphatemic disorder characterized by elevated hFGF23 concentration compared to concentrations observed in a healthy subject, comprising administering to the subject an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39. 47. A method for increasing serum phosphate concentration in a subject suffering from a hypophosphatemic disorder characterized by elevated FGF23 concentration, compared to a concentration observed in a healthy subject, comprising administering to the subject an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39. 48. A method for treating a subject suffering from a hypophosphatemic disease, such as neoplastic osteomalacia, fibrous dysplasia, McCune-Albright syndrome, autosomal dominant hypophosphatemic rickets (ADHR), or X-linked hypophosphatemic rickets (XLH), comprising administering to the subject an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39. 49. A method for treating a subject suffering from X-linked hypophosphatemic rickets (XLH), comprising administering to the subject an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39. 50. Use of an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of Embodiments 1 to 39 for the manufacture of a drug for the treatment of a subject requiring treatment. 51. Use of an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of Embodiments 1 to 39 for the manufacture of a pharmaceutical product for the treatment of hypophosphatemic diseases such as X-linked hypophosphatemic rickets (XLH). 52. A kit comprising an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition described in any one of Embodiments 1 to 39, and instructions for use. 53. A polynucleotide encoding an hFGF23 antagonist polypeptide, polypeptide derivative, or compound as described in any one of Embodiments 1 to 38. 54. An expression vector encoding a polynucleotide according to a prior embodiment. 55. A host cell producing an hFGF23 antagonist polypeptide, polypeptide derivative, or compound according to any one of Embodiments 1 to 38. 56. A method for producing an hFGF23 antagonist polypeptide, polypeptide derivative, or compound according to any one of Embodiments 1 to 38, a) A step of culturing host cells according to a prior embodiment under conditions that allow expression of the polypeptide encoded by the expression vector of Embodiment 54, b) A step of isolating the polypeptide, c) A method comprising the optional step of attaching an extended portion to the isolated polypeptide. 57. hFGF23 antagonist polypeptide derivatives obtainable by the method described in Embodiment 56. [Examples]

[0198] The following experimental section begins with a list of abbreviations, followed by sections on general methods for preparing the compounds and methods for measuring exposure profiles and other properties related to the compounds. Several specific examples are incorporated into each section to illustrate the present invention. All example compounds were prepared according to the general methods described herein.

[0199] Abbreviation Ado: 8-amino-3,6-dioxaoctanoic acid Boc: tert-butyloxycarbonyl Collidine: 2,4,6-trimethylpyridine DCM: Dichloromethane DIC: N,N'-Diisopropylcarbodiimide DMF: N,N-dimethylformamide DTT: 1,4-Dithiothreitol FBS: Fetal Bovine Serum Fmoc: 9-Fluorenylmethyloxycarbonyl HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HFIP: 1,1,1,3,3,3-Hexafluoro-2-propanol or hexafluoroisopropanol HPLC: High-Performance Liquid Chromatography iv: intravenous LC: Reset Chromatography LCMS: Liquid Chromatography Mass Spectrometry MeCN: Acetonitrile MQ: Milli-Q MS: mass spectrometry MSA: Multiple Sequence Alignment Mtt: 4-methyltrityl NMP: N-methyl-2-pyrrolidone OtBu: tert-butoxy Oxyma Pure®: Cyano(hydroxyamino)ethyl acetate OVA: Ovalbumin Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS: Phosphate-buffered saline RP: Out of phase RP-HPLC: Reverse-phase high-performance liquid chromatography RT: room temperature RU: Response Unit sc: subcutaneous SPPS: Solid-phase peptide synthesis SPR: Surface Plasmon Resonance tBu: tert-butyl TFA: Trifluoroacetic acid TIPS Triisopropylsilane Trt: Triphenylmethyl(trityl) UPLC: Ultra-high-performance liquid chromatography UV: Ultraviolet light

[0200] Methods for peptide synthesis, purification, and analysis: The Fmoc-protected amino acid derivatives used are as follows: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH (unless otherwise specified, and sourced from Gyros Protein Technologies AB). Fmoc-Glu-OtBu and resins for solid-phase peptide synthesis were sourced from Novabiochem (Merck Millipore). Fmoc-Ado-OH was sourced from Bachem. Triisopropylsilane was sourced from Sigma. Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH) was sourced from Flamma Group in Italy. 16-(tert-butoxy)-16-oxohexadecanoic acid was obtained from ABCR, 18-(tert-butoxy)-18-oxooctadecanoic acid from Solvias, and 20-(tert-butoxy)-20-oxoeicosanoic acid was prepared as known in the art, for example, as described in WO 2010102886 A1. Oxima was obtained from Gyros Protein Technologies. N,N-dimethylformamide, piperidine, N,N'-diisopropylcarbodiimide, trifluoroacetic acid, and 1,1,1,3,3,3-hexafluoro-2-propanol were obtained from Biosolve. HPLC-grade acetonitrile was obtained from Merck.

[0201] Automatic Fmoc SPPS, Symphony TMThe procedure was performed on X (Gyros Protein Technologies AB). Fmoc deprotection was achieved by treating with 20% by volume piperidine in 0.1 M oxima in DMF for 2 × 10⁻⁶ minutes. Peptide coupling was performed using DIC / collidine. The amino acid / oxima solution (0.3 M / 0.3 M DMF solution in a 5-fold molar excess) was added to the resin, followed by the addition of the same molar equivalents of DIC (1.5 M DMF solution) and the same molar equivalents of colidine (1.5 M DMF solution). Unless otherwise specified, the coupling procedure was allowed to proceed for 60 minutes.

[0202] After the synthesis of the polypeptide skeleton, the extended portion was selectively placed on the resin-bound polypeptide at the desired position(s) via orthogonally protected amino acids such as Fmoc-Lys(Mtt)-OH. The Mtt group was selectively removed by treatment with HFIP / DCM / TIPS (75:22.5:2.5) (3 × 15 min). Subsequently, the resin-bound polypeptide was treated with 20 vol% piperidine in 0.1 M oxima in DMF (1 × 5 min). After deprotection, the resin was then washed with DCM and DMF, and the extended portion was introduced using the standard SPPS procedure described above.

[0203] The resin-bound peptide was deprotected post-synthesis, the resin was washed with DCM, and then treated with TFA / TIPS / DTT / water (95:2.5:2.5:2.5, v / v / v / v) for 2-3 hours. Following the treatment, the pellet was precipitated in ice-cold diethyl ether and collected by centrifugation. The pellet was resuspended in diethyl ether and centrifuged again. The washed pellet was dissolved in a suitable mixture of water, MeCN, and acetic acid, and then purified by reverse-phase preparative HPLC.

[0204] X h Position and X 56The crude peptide containing cysteine ​​at the 12th position was folded before purification, dissolved in DMSO (10 ml), and then 30 mM HEPES buffer (40 ml, pH 7.8, aqueous solution) was added and stirred overnight. Before purification by reverse-phase preparative HPLC, the pH was reduced to 3 using trifluoroacetic acid.

[0205] Purification of polypeptides and final analogs was performed using a reverse-phase HPLC column, Waters TM Preparative purification was performed on a Deltaprep 4000 preparative chromatography system. Elution was performed using a mobile phase consisting of 0.1 vol%. TFA in HPLC-grade acetonitrile (solvent B) using a linear gradient with Milli-Q® water (solvent A) and 0.1 vol% TFA.

[0206] Elution was performed using a linear gradient with a mobile phase consisting of 0.1 vol% TFA, 10 vol% acetonitrile in Milli-Q water (solvent A), and 0.1 vol% TFA in HPLC-grade acetonitrile (solvent B). The relevant fractions were evaluated by LC-MS, and the purity of the pooled fraction was evaluated by analytical HPLC or UPLC. The fractions containing the pure target peptide were pooled and lyophilized.

[0207] Analysis Procedure [Table 3]

[0208] Method: M111 LCMS111(M111) is performed using a Waters Acquity UPLC system setup with an adjustable UV detector (TUV) and a Waters Acquity QDa mass spectrometer (QDa). Eluents: A: 0.05% trifluoroacetic acid aqueous solution; B: Acetonitrile, 0.05% trifluoroacetic acid. Analysis was performed at 40°C by injecting an appropriate amount of sample (2-10 μl) into a column eluted with gradients A and B. The UPLC conditions, detector settings, and mass spectrometer settings are as follows: Column: Waters Acquity UPLC BEH, C18, 1.7 μm, 2.1 mm x 50 mm. Gradient: Linear 10%-90% acetonitrile for 1.6 minutes at 0.9 ml / min. UV detection: 214 nm using a TUV detector. MS detection: Atmospheric pressure ionized electrospray (API-ES) with a scan range of 100-1250 Da using a QDa detector operating in positive mode.

[0209] compound Table 4 lists the chemical numbers, sequence numbers, complete skeletal sequences, linker-bound residue R1, linkers, and growth factors. Selected examples are shown below.

[0210] Chemical substance 4, SEQ ID NO: 53, VDNREFNAYLQAWYEIFNLPNLNDNQWPAFIESLHEDPSQWANLLAEARRLNDAQAKE, R1: 57K, Linker: L1, Extension factor. The structure of chemical substance 1b is shown below, with the linker bonded to the epsilon-amino group of lysine at position 57. [ka]

[0211] Chemical substance 106, SEQ ID NO: 138, LDEEEFQAYLQAWYEIFNLPNLNDNQWPAFIESLHEDPSQWANLLAEARRLNDAQAPK, 58K, Linker: L1, Extension factor. The structure of chemical substance 1c is shown below, with the linker bonded to the epsilon-amino group of lysine at position 58. [ka]

[0212] Chemical Substance 246, Sequence ID No. 274, VDQEEFQAYLQAWYEIFNLPCLNDNQWPAFIESLHEDPSQWDNLLEEARRLNDAQCPK, 58K, Linker: L1, Extension Factor. The structure of Chemical Substance 1b is shown below, with the linker bonded to the epsilon-amino group of lysine at position 58. Furthermore, two cysteine ​​(C) are at position X h and X 56 When present in this arrangement, it will be understood by those skilled in the art that a disulfide bridge is formed between the two cysteine ​​molecules, as shown in the following structure. [ka]

[0213] Table 4 below discloses examples of compounds of the present invention. The table provides information on amino acid residue sequences and extensions, as well as their binding sites to the hFGF23 antagonist polypeptide. The linker and growth factor columns provide designations of the linkers and growth factors used. See also Tables 1 and 2 above.

[0214] Table 5 below provides technical information about the compounds, such as molecular weight and m / z value. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 [Table 4-22] [Table 4-23] [Table 4-24] [Table 4-25] [Table 4-26] [Table 4-27] [Table 4-28] [Table 4-29] [Table 4-30] [Table 4-31]

[0215] The combined motif (BM) is highlighted in bold in the sequence shown in Table 4. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14]

[0216] (Average) refers to the average molecular weight peak, and (Mono) refers to the monoisotopic peak in the MS spectrum.

[0217] Example 1: Design of Compounds and Binding Motifs (BMs) The hFGF23 antagonist polypeptides disclosed herein are designed molecules developed on a 3-helix vandal domain derived from the Z domain of Staphylococcus aureus protein A, which binds to the Fc portion of immunoglobulins. A large library was constructed by randomizing selected amino acids in the range of residues 9 to 35 on the two first helices constituting the Fc binding surface, from which potent hFGF23 conjugates were isolated by various methods. Randomization of the Fc binding surface residues in the range of residue 9 to residue 35, and subsequent selection of polypeptide variants, leads to replacing Fc binding ability with the ability to bind to hFGF23 (selection #1). Thus, the binding motif (BM) corresponds to residues 9-35. A new library was designed after analysis of the results of selection #1 (selection #2) by randomizing other residues within the binding motif (BM) to further improve the hFGF23 binding ability. Polypeptide variants from Option #2 were subsequently optimized for potency by amino acid substitutions within and outside the binding motif (BM) (Option #3) to yield the hFGF23 antagonist polypeptides disclosed herein. Polypeptide variants from Option #1 and Option #2 had their in vivo half-lives optimized by identifying positions within the polypeptide backbone where extensions could be added while maintaining binding ability to hFGF23.

[0218] The hFGF23 antagonist polypeptide of the present invention can be functionalized by gene fusion to a target protein module, or by covalent amino acid residue side chain addition to a functional portion such as a toxin, contrast agent, and / or one or more extensions.

[0219] Example 2: Inhibitory effect on hFGF23-induced signaling in a serum luciferase reporter gene assay. The objective of this assay is to demonstrate and characterize the extent to which hFGF23 antagonist polypeptide derivatives (polypeptides) inhibit hFGF23-induced signaling mediated by both FGFR1c as the receptor and alpha-cloto (KL) as the co-receptor. hFGF23 is known to specifically signal via this trimer complex of FGFR1c / KL / hFGF23 through the MAPK / ERK pathway. Treatment of cells with hFGF23 results in luciferase expression, which, after the addition of the detection reagent, ultimately leads to a luminescence signal. The compounds of the present invention are IC23. 50 <1nM inhibited hFGF23-induced signaling, or >50% reduced hFGF23-induced signaling at 1nM.

[0220] Assay principle A reporter gene assay was established to determine the ability of polypeptide compounds to inhibit signal transduction. The inventors measured standard hFGF23-induced signal transduction using luciferase under the control of serum response elements (SREs).

[0221] HEK293 cells were first stably transfected with a human KL expression plasmid encoding a zeosin resistance gene for mammalian selection. Monoclonal HEK293 / KL cell lines were generated from a single clone in the presence of 50 μg / ml zeosin (Invitrogen, #R25001), and then stably transfected with a reporter plasmid based on pGL4.33[luc2P / SRE / Hygro] (Promega, #E1340), which encodes luciferase, under the control of SRE. The result was a HEK293 / KL / SRE cell line derived from a single clone named 17-3, selected for 200 μg / ml hygromycin. HEK293 / KL / SRE cells were maintained in complete medium under standard cell culture conditions at 37°C, based on DMEM (Gibco #10687010) supplemented with 1% penicillin-streptomycin (Gibco #31966-021), 10% FBS (Thermo Fisher Scientific #15140-122), 50 μg / ml zeosin, and 200 μg / ml hygromycin (Invitrogen #10091148).

[0222] To evaluate polypeptide compound-mediated inhibition of hFGF23-induced signaling, HEK293 / KL / SRE cells were seeded at a density of 10,000 cells per well in starvation medium in poly-D-lysine coated black 384-well plates (Corning #354663). The starvation medium was based on DMEM supplemented with 0.5% human serum albumin and 1% penicillin-streptomycin. The following day, polypeptide compounds and recombinant hFGF23 (SEQ ID NO: 5) (RnD Systems #2604-FG-025) were diluted in starvation medium and co-incubated at 22°C for 1 hour before being added to the cells. Cells were incubated at 37°C for 4 hours, followed by detection of lysis and luminescence using an EnVision 2104 plate reader with supersensitive luminescence detection (PerkinElmer).

[0223] Polypeptide compounds were tested using either a single-dose or dose-response method. For single-dose experiments, the polypeptide compound was applied at a final concentration of 1 nM. For dose-response experiments, the polypeptide compound was serially diluted and applied to cells at concentrations ranging from 50 to 0.032 nM. For both assay settings, hFGF23's EC was measured. 80 A corresponding concentration of 45 pM hFGF23 was applied. In single-dose experiments, compound response data were normalized to the minimum and maximum responses in the absence and presence of hFGF23, respectively. Results were expressed as residual activity %. The following formula was applied.

number

[0224] Dose-response experiments are conducted to compare the in vitro potency of compounds by measuring the half-maximal inhibitory concentration (IC). 50 The objective is to determine the following. To that end, the four-parameter logistic equation (4PL fitted) was applied.

number

[0225] Both single-dose and dose-response analyses were performed in Datalab using JSON scripts.

[0226] material: Zeosin (Invitrogen #R2500) Hygromycin (Invitrogen #10687010) pGL4.33[luc2P / SRE / Hygro](Promega #E1340) DMEM (Gibco #31966-021) Penicillin-streptomycin (Gibco #15140-122) FBS (Thermo Fischer #10091148) Hygromycin (Invitrogen #10687010) Poly-D-lysine coated black 384-well plate (Corning #354663) Recombinant Human FGF23 (Amino Acids 25-251) (RnD Systems #2604-FG-025)

[0227] result The results are shown in Table 6, and the IC of the compound of the present invention. 50 The value, or the inhibitory efficacy in the form of residual hFGF23 activity at a compound concentration of 1 nM, is shown collectively. IC values ​​less than 1 nM 50 A value, or a residual activity percentage at 1 nM of less than 50%, means that the compound has surprisingly potent inhibitory activity under experimental conditions. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0228] Analysis of the data indicates that the compounds disclosed herein can inhibit hFGF23-induced signaling. Compounds with high percentages of sequence identity, such as at least 93%, have been demonstrated to retain inhibitory activity against hFGF23. Therefore, these compounds are suitable for use in the treatment of hypophosphatemic disorders, such as X-linked hypophosphatemic rickets (XLH).

[0229] Example 3: Surface plasmon resonance (SPR) characterization of the interaction between hFGF23 antagonist polypeptide and hFGF23 The objective of this assay is to demonstrate that the hFGF23 antagonist polypeptide (polypeptide) of the present invention binds to hFGF23 and to determine its binding affinity. The binding test is performed using a Biacore® T200, Biacore® 8K, or Biacore® 8K+ (all manufactured by Cytiva), instruments designed to quantify molecular interactions in real time using surface plasmon resonance (SPR). The experiment is conducted at either 10°C or 25°C, while the sample is maintained at 10°C within the sample compartment. The SPR instrument's reported signal, expressed in response units (RU), directly correlates with the mass on the sensor chip surface in eight parallel channels, each containing two serial flow cells.

[0230] The procedure begins with the covalent immobilization of the in-house anti-HPC4 monoclonal antibody onto both flow cells of the Series S sensor chip CM4 (Cytiva) using an amine coupling kit (Cytiva), in accordance with the manufacturer's guidelines. Subsequently, HPC4-tagged recombinant hFGF23 (SEQ ID NO: 6) is captured by injecting it onto flow cell 2. This creates an active target surface on flow cell 2 and a reference surface in flow cell 1, which present only the immobilized anti-HPC4 antibody.

[0231] The binding of polypeptides to the captured target is examined by injecting the analyte into both flow cells, allowing for comparative analysis of the binding of different compounds to the captured target relative to a reference surface. The compounds of the present invention are serially diluted in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20) and 50 μl is injected. -1 Inject at a rate of 240 seconds and allow to dissociate for 900 seconds. After each analyte injection cycle, the CM4 surface is regenerated via injection of 10 mM glycine pH 1.5. This step removes the captured target and any bound polypeptides from the immobilized antibody surface and prepares them for subsequent interaction sample pairs. In this regeneration procedure, the covalently immobilized anti-HPC4 capture antibody is not removed from the tip surface.

[0232] The polypeptide binding curve is processed with subtraction of the reference surface signal across the captured hFGF23 and the blank buffer injection. This process allows for correction of instrument noise, bulk shift, and drift during sample injection. The polypeptide association and dissociation rate constants, i.e., k, are also processed. a (Meeting velocity) and k d The dissociation rate is extracted by globally fitting a 1:1 Langmuir model to the data using Biacore Insight evaluation software (version 5.0.18.22102). The affinity between the compound and the target of the present invention is expressed by the equilibrium dissociation constant (K). D Quantified by formula K D =k d / k a It is calculated from the dynamics determined by [the specified method]. The results are shown in Table 7 below. [Table 7]

[0233] Data analysis indicates that the compound exhibits desirable binding to hFGF23. Compounds with high sequence identity have affinity (K) of 2 nM or less, especially 1.8 nM or less. DIt was further demonstrated that it binds to hFGF23.

[0234] Example 4: Surface plasmon resonance (SPR) characterization of the interaction between a non-human species hFGF23 antagonist polypeptide and FGF23. The purpose of this assay is to demonstrate that the hFGF23 antagonist polypeptide (polypeptide) of the present invention binds to FGF23 derived from a non-human species, and to determine the binding affinity. The assay was carried out in the same manner as in Example 3, except that hFGF23 was substituted with FGF23 from dogs, pigs, or monkeys. The results are shown in Table 8. [Table 8]

[0235] Analysis of the data indicates that this compound exhibits desirable interspecies reactivity binding to FGF23 from dogs, pigs, and monkeys.

[0236] Example 5: Pharmacokinetic studies in miniature pigs The objective of this study was to determine the half-life in the blood after intravenous administration of a specific compound of the present invention.

[0237] The study was conducted on female Goettingen minipigs (Ellegaard Minipigs A / S, Dalmos, Denmark) weighing approximately 25 kg. Polypeptides were administered intravenously to the minipigs, with n=3 per group and a dose of 10 nmol / kg. The vehicle for the formulation was 50 mM phosphate and 70 mM sodium chloride at pH 7.4.

[0238] Blood samples were collected via an endogenous permanent venous catheter for up to 312 hours and collected in EDTA tubes (Salstrett, Germany). Plasma was separated and analyzed for compound concentrations using liquid chromatography-mass spectrometry (LC-MS).

[0239] Non-compartmental (NCA) pharmacokinetic analysis was performed using Phoenix® WinNonlin® v.8.1 (Certara LPPrinceton, NJ, USA). Terminal phase elimination phases were fitted via linear regression with uniform weighting. Nominal sampling time and actual dose were used for the NCA.

[0240] Polypeptide concentrations in miniature pig plasma were analyzed by plasma protein precipitation followed by turboflow liquid chromatography-mass spectrometry (TF-LC-MS). Calibration standards and quality control samples were prepared by spiked compounds into blank plasma and then serially diluting them. All plasma samples were prepared by adding 4 volumes of ethanol to 1 volume of plasma sample. The precipitation solvent contained a 20 nM internal standard. After centrifugation at 4°C, the supernatant was diluted with 1 volume of purified water containing 1% formic acid. The precipitated plasma samples were first washed and concentrated on a TurboFlow Cyclone column (0.5 × 50 mm from Thermo Fisher Scientific) operated at 30°C, and then transferred to the analytical column. The analytical column was an XBridge Peptide BEH C18 column (2.1 × 50 mm, particle size 3.5 μm from Waters) operated at 60°C. Analytical separation was performed using a linear gradient. Mobile phase A was water containing 5% organic solvent (acetonitrile / methanol (1:1, by volume)) and 1% formic acid, and mobile phase B was water containing 95% organic solvent (acetonitrile / methanol (1:1, by volume)) and 1% formic acid. The mass spectrometer coupled to the TF-LC system was either a TSQ Altis or a Q Exactive (both manufactured by Thermo Fisher Scientific). The mass spectrometer was operated in cationization mode, reaction monitoring was selected on the TSQ Altis, and reaction monitoring was performed in parallel on the Q Exactive. The sample was subjected to 1 / X 2Quantification was performed using linear regression to the response from a weighted calibration standard. The response was defined as the analyte peak area divided by the internal standard peak area. The concentrations measured for the quality control samples and calibration standard were within ±15% of the nominal concentration, with the exception of one quality control sample which was -17% of the nominal concentration. The limit of quantification was 0.250 nM to 1.00 nM.

[0241] result The results are shown in Table 9 below, with the column T1 / 2 indicating the half-life (T1 / 2) in the blood. A higher half-life value is desirable. [Table 9]

[0242] No adverse clinical findings considered to be related to the test compound were observed during the study.

[0243] The results indicate that prolongation at position 58, where C18 diacid or C20 diacid is present, results in a clinically significant half-life in the blood in polypeptide compounds with high sequence identity.

[0244] A long half-life indicates that a given compound can provide its inhibitory effect for a longer period after administration compared to compounds with relatively short half-lives. A longer half-life allows for less frequent administration, which is desirable as it provides improved convenience for the patient.

[0245] Example 6: Pharmacokinetic and pharmacodynamic studies in dogs The purpose of this study was to evaluate the pharmacokinetics and pharmacodynamics of the compound of the present invention in male beagle dogs after a single subcutaneous (sc) injection.

[0246] Chemicals 206, 207, and 208 were administered to male beagle dogs (n=3 per compound, target dose 10 nmol / kg). The composition of the injectable formulations was as follows: 20 mM L-histidine, 150 mM L-arginine hydrochloride, pH=6.49–6.51.

[0247] Fifteen male Beagle dogs from China and the Marshall Islands, aged 2.60–9.10 years and weighing 11.3–15.0 kg, were included in the study. The dogs were housed in groups during the experiment and tattooed with unique numbers to identify each animal. The housing chamber was maintained at a temperature of 16–25°C and a humidity level of 40–70%. The photoperiod was set to 12 hours of light and 12 hours of dark. During the study period, dogs were fed commercially available dog food (Adult Canine Culture Medium Dog Food, Royal Canin®, Shanghai, China, 150-225g / dog / day) once a day at 13:00. The animals were fasted overnight for morning sampling. The animals had free access to high-quality drinking water.

[0248] On the first day of the experiment, dogs received a single bolus injection of the test compound at approximately 9:00 AM. Blood samples (1.2 mL at each time point) were collected in EDTA-coated tubes at the following time points for measurement of plasma concentrations of the test compound and plasma phosphate: before administration (0 min), 5, 15, 30, 45 min, 1, 1.5, 2, 4, 7, 10 hours, 24, 30, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, and 336 hours. Blood samples were kept on ice until centrifugation (4 minutes, 4°C, 4000 rpm). After centrifugation, two sets of 100 μl EDTA-coated plasma (one for quantification of the test compound and one for quantification of phosphate) from each sampling time point were immediately transferred to micronic tubes and stored at -20°C until analysis.

[0249] Test compound plasma analysis Plasma concentrations of chemicals 206, 207, and 208 were assayed by plasma protein precipitation and analyzed by turbo-flow liquid chromatography-mass spectrometry (TF-LC-MS). Calibrators were prepared by spiking blank dog plasma with chemicals 206, 207, and 208 in the range of 0.5–2000 nM. Standards, plasma blanks, or test samples were prepared for TF-LC-MS by protein precipitation by adding 3 volumes of ethanol containing a 50 nM internal standard (ISTD) (chemical 175) to 1 volume of the sample, followed by centrifugation at 4000 rpm at 4°C for 60 minutes. 1 volume of supernatant was mixed with 2 volumes of water (containing 0.1% formic acid). The mixture was analyzed by TF-LC-MS using a Cyclone turboflow column (0.5 × 50 mm, ThermoFisher Scientific) and an Agilent Poloshell 120 SB-C18 column (2.1 × 50 mm, 2.7 μm particle size). Gradient elution was performed using mobile phase A (consisting of milli-Q water containing 0.1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (consisting of methanol / acetonitrile (50 / 50) containing 0.1% formic acid and 5% milli-Q water). The mass spectrometer was operated in cationization mode. Chemical Substance 206: m / z; Chemical Substance 207: m / z 1912.4600@1748.5375; Chemical Substance 208: m / z 1905.4500@2211.0012; 1926.9600@1767.5369; Chemical Substance 175: m / z 1923.4500@1762.8643) was analyzed using the Labrap Exploris 240 mass spectrometer (ThermoFisher Scientific) as the detector in parallel reaction monitoring mode. A linear calibration curve (weighted 1 / x2) was used to calculate the concentration in the plasma sample. Quality control samples were included. The deviation between the nominal concentration and the calculated concentration in the standard substance and quality control samples was less than 15%. The limit of quantification (LLOQ) is 2.0 nM.

[0250] Pharmacokinetic analysis of test compounds Plasma concentrations of the test compound at each sampling time point were imported into Phoenix® WinNonlin® version 8.4.0.6172 for non-compartmental analysis (NCA) using a linear uplog-down method and weighting 1 / (Y*Y). Actual sampling times were used for NCA calculations. Individual concentration values ​​below LLOQ were treated as zero for time points before the first detectable exposure and as missing values ​​for time points after Tmax. Actual doses calculated using the actual concentrations of the formulation were used for NCA analysis.

[0251] The results are shown in Table 10 below. [Table 10]

[0252] At the test dose, the compound was well-tolerated, and no adverse effects were observed in the test animals. The results indicate that prolongation at position 58 using C20 diacid results in a clinically significant half-life in the blood for polypeptide compounds with high sequence identity.

[0253] Example 7: Plasma phosphate levels in miniature pigs and dogs To determine the in vivo effect of the compound of the present invention, plasma phosphate levels were measured in the PK studies described in Examples 5 and 6.

[0254] Method Serum Pi: Plasma phosphate levels were measured using the PHOS2 inorganic phosphate assay v2.0 (Roche) on a Cobas Pro c503 analyzer. Animals were administered a single intravenous dose of 10 nmol / kg of the compound (miniature pig, Example 5) or a single subcutaneous dose of 10 nmol / kg (beagle dog, Example 6). Plasma samples for analysis were collected at (0), 5, 15, and 30 minutes after administration, and at 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 30, 48, 72, 96, and 144 hours after administration. Data are reported in Figure 1 / Table 11 and Figure 2 / Table 12, with plasma phosphate levels expressed in mmol / L. [Table 11] [Table 12]

[0255] The results demonstrate that the compounds of the present invention can increase plasma phosphate levels in vivo.

[0256] Example 8: Chemical Substances Using X-ray Crystallography 3: Crystallization and Epitope Mapping To determine the binding site of the compound of the present invention to hFGF23, a crystallization test was performed to identify the epitope residue on hFGF23.

[0257] hFGF23 and test compounds for complex purification and initial crystallization The hFGF23 core domain (SEQ ID NO: 4) and chemical 3 (SEQ ID NO: 10) were purified by size exclusion chromatography (SEC) using a Superdex75 16 / 60 column running on 10 mM HEPES pH 7.5 and 500 mM NaCl. The mixture was incubated at 4°C for 2 hours using an electrochromic material containing a sequence of 3 (SEQ ID NO: 10) with a 1.5 × molar excess of chemical hFGF23 core domain (SEQ ID NO: 4). The purified complex was collected from the SEC peak fraction, concentrated to 3.7–7.4 mg / ml, and used for crystallization testing in 96-well sitting drops using a commercially available crystallization screen incubated at 18°C. Initial crystals were obtained in dropwise infusions containing 0.18 μl of storage solution (0.2 M potassium formate, 20% (w / v) PEG 3350) and 0.36 μl of protein solution. The initial crystals were used to prepare seed material for subsequent seed crystallization zones. The seed material was prepared as follows: 1) the initial crystals in the droplets were crushed; 2) the droplets containing the crushed crystals were transferred to a seed bead tube (HR2-320 Hampton Research) containing 50 μl of storage solution (0.2 M potassium formate, 20% (w / v) PEG 3350); and 3) the mixture was shaken on a vortex mixer for 2 minutes.

[0258] Crystallization and structural determination of the hFGF23 core domain (SEQ ID NO: 4) complexed with chemical substance 3. Crystals were obtained at 18°C ​​by sowing 35 nl of seeds in a dropper containing 0.36 μl of protein solution and 0.36 μl of storage solution (0.2 M calcium acetate hydrate, 20% w / v polyethylene glycol 3,350). The crystals were cryoprotected in glycerol and frozen in liquid nitrogen. Synchrotron X-ray diffraction data were collected with a Swiss Light Source and processed with XDS / XSCALE to a resolution of 1.8 Å in space group 4 (P21). The structure was determined by molecular substitution performed in Phenix using the following pdb entries, 2P39 and 5DJT (chain B only), as search models. The solution from molecular substitution was in space group 4 (P21) and had two complexes in the asymmetric unit (complex 1 and complex 2). The structure was refined in Phenix to a resolution of 1.8 Å.

[0259] A brief explanation and interpretation of the structure The two complexes of the asymmetric units showed high structural similarity to the RMSDs of 0.658 Å (backbone) and 1.058 Å (total atoms) calculated in PyMOL using “Align” without outlier rejection. The overall structure of the hFGF23 core domain (SEQ ID NO: 4) was similar to the previous crystal structure of hFGF23 (pdb entry 2P39), which has a backbone RMSD of 1.304 Å calculated in PyMOL using “Align” without outlier rejection.

[0260] The interaction between hFGF23 and its receptor (heterodimer of FGFR1c and alpha-cloto) has been previously characterized by X-ray crystallography (pdb entry 5W21). Structural comparison of the hFGF23 / receptor complex (5W21) and the hFGF23 core domain (SEQ ID NO: 4) complex of Chem.3 revealed that the binding sites of the hFGF23 receptor and chemical 3 largely overlap on hFGF23. This supports the idea that chemical 3 directly inhibits the interaction between hFGF23 and the hFGF23 receptor, thereby suppressing the function of hFGF23.

[0261] Determination of the epitope of chemical substance 3 The epitope residues (a 3.5 Å distance between any non-hydrogen atom of chemical 3 and any non-hydrogen atom of hFGF23) include the following residues in hFGF23 (SEQ ID NO: 1): M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170.

[0262] Example 9: In silicoepitope determination Using Alphafold v 2.3 (https: / / www.biorxiv.org / content / 10.1101 / 2021.10.04.463034v2), hFGF23 (sequence number 1) was folded in silico using chemicals 206, 207, and 208, respectively. For multiple sequence alignments, a reduction in database settings was applied, and the number of recycles was set to 5. All other model parameters were set to default.

[0263] The identified epitopes, determined by the 3.5 Å distance between any non-hydrogen atom in the tested compound and hFGF23, include the following residues in hFGF23: Chemical substances 206: R76, T86, R91, Y93, Y107, R114, F169, and N170.

[0264] Chemical substances 207: W36, R76, T86, R91, Y107, R114, I167, F169, and N170.

[0265] Chemicals 208: R76, T86, R91, R114, L166, and F169.

[0266] Along with Example 8, all tested compounds bind to epitopes on hFGF23, specifically the amino acid residues R76, T86, R91, and R114. Therefore, these residues (highlighted in bold above) are considered the primary epitope residues of the compounds of the present invention.

[0267] Example 10: Pharmacokinetic study in miniature pigs 2 The purpose of this study is to determine the blood half-life of a specific compound of the present invention after intravenous administration.

[0268] The study was conducted using female Göttingen minipigs (Ellegaard Minipigs A / S, Dalmose, Denmark) weighing approximately 25 kg. Polypeptides were administered intravenously to the minipigs, with n=3 in each group, at a dose of 14 nmol / kg. The vehicle for the formulation consisted of 50 mM phosphate, 70 mM sodium chloride, and 0.007% polysorbate 20 at pH 7.4.

[0269] Blood was sampled via an indwelling permanent venous catheter for up to 312 hours and collected in EDTA tubes (Sarstedt, Germany). Plasma was separated and compound concentrations were analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0270] Non-compartmental (NCA) pharmacokinetic analysis was performed using Phoenix® WinNonlin® v. 8.1 (Certara LP Princeton, NJ, USA). Terminal elimination phases were fitted using equally weighted linear regression. Nominal sampling time and actual dose were used for NCA.

[0271] Polypeptide concentrations in miniature pig plasma were evaluated using plasma protein precipitation followed by turboflow liquid chromatography-mass spectrometry (TF-LC-MS). Calibration standards and quality control samples were prepared by spiked blank plasma with compounds and serially diluting the mixture. All plasma samples were sampled by adding 4 volumes of ethanol to 1 volume of plasma sample. The precipitation solvent contained a 20 nM internal standard. After centrifugation at 4°C, the supernatant was diluted 1 volume with purified water containing 1% formic acid. The precipitated plasma samples were first purified and concentrated on a TurboFlow cyclone column (Thermo Fisher Scientific, 0.5 x 50 mm) operated at 30°C, and then transferred to an analytical column. An XBridge Peptide BEH C18 column (2.1 x 50 mm, particle size 3.5 μm, Waters) was used for analysis, operated at 60°C. Analytical separation was performed using a linear gradient. Mobile phase A consisted of water with 5% organic solvent (acetonitrile / methanol (volume ratio 1:1)) and 1% formic acid added, while mobile phase B consisted of water with 95% organic solvent (acetonitrile / methanol (volume ratio 1:1)) and 1% formic acid added. The mass spectrometer coupled to the TF-LC system was either a TSQ Altis or a Q Exactive (both manufactured by Thermo Fisher Scientific). The mass spectrometer was operated in cationization mode, with selective reaction monitoring (SRM) performed on the TSQ Altis and parallel reaction monitoring (PRM) performed on the Q Exactive. The sample was 1 / X 2 Quantification was performed using linear regression on the response from the calibration standard material with weighting. The response was defined as the analyte peak area divided by the internal standard peak area. The measured concentrations of the quality control samples and calibration standard material were within ±15% of the nominal concentration, except for one quality control sample which was -17% of the nominal concentration. The limit of quantification was between 0.250 nM and 1.00 nM. result The results are shown in Table 13. 1 / 2 The column shows the half-life (T) in the blood. 1 / 2 ) indicates a high half-life. [Table 13]

[0272] No adverse clinical findings thought to be related to the test compound were observed during this study.

[0273] The results indicate that the extension at position 58 by C20 diacitate results in a clinically significant half-life in the blood.

[0274] A long half-life indicates that the inhibitory effect can be exerted for a longer period after administration compared to compounds with relatively short half-lives. A longer half-life allows for a reduction in the frequency of administration, which is desirable for improving patient convenience.

[0275] Example 11 To evaluate the in vivo effect of the compound of the invention (referred to herein as chemical substance 206), plasma phosphate concentration was measured in the PK test described in Example 10.

[0276] Method for serum Pi: Plasma phosphate was measured using the PHOS2 inorganic phosphate assay v2.0 (Roche) on a Cobas pro c503 analyzer. Animals were administered a single intravenous dose of the compound at 14 nmol / kg. Plasma samples for analysis were collected at administration (0), and at 5, 15, 30 minutes, and 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 24 hours, 30 hours, 48 ​​hours, 72 hours, 96 hours, and 144 hours post-administration. The data are reported in Figure 3 / Table 14, showing plasma phosphate concentrations in mmol / L. [Table 14]

[0277] The results indicate that the compounds of the present invention can increase plasma phosphate levels in vivo.

[0278] While specific configurations of the present invention have been illustrated and described herein, many variations, substitutions, alterations, and equivalents will be conceivable to those skilled in the art. It should therefore be understood that the appended claims are intended to encompass all such variations and alterations that fall within the true spirit of the invention.

Claims

1. The location on hFGF23 determined at 3.5 Å by Sequence ID No. 1 a) R76, T86, R91, and R114, b) M74, R76, T86, R91, F108, P110, R114, L166, I167, and N170, c) R76, T86, R91, Y93, Y107, R114, F169, and N170, d) W36, R76, T86, R91, Y107, R114, I167, F169, and N170, and e) R76, T86, R91, R114, L166, and F169, hFGF23 antagonist polypeptide capable of binding to an epitope containing an amino acid residue selected from the group consisting of the following.

2. hFGF23 antagonist polypeptide, which is capable of binding to hFGF23 according to SEQ ID NO: 1, i) X according to SEQ ID NO: 2 ａ X ｂ X ｃ AWX ｄ EIX ｅ X ｆ X ｇ PX ｈ LX ｉ DX ｊ QWPAFIE X ｋ being LH During the ceremony, they acted independently of each other. X ａ is F or Y, X ｂ is F, I, L, or V, X ｃ is A or Q, X ｄ is A, F, H, Y, or W, X ｅ is F or Y, X ｆ is N, Q, or T, X ｇ is A or L, X ｈ is C, H, N, Q, W, or Y, X ｉ is D, N, Q, S, or T, X ｊ is A, D, E, G, H, L, N, Q, S, T, or Y, X ｋ This is an amino acid residue sequence that is A, Q, or S. and ii) An amino acid residue sequence having at least 93% identity with the sequence defined in i) hFGF23 antagonist polypeptide containing a binding motif (BM) consisting of a selected amino acid residue sequence.

3. The hFGF23 antagonist polypeptide according to claim 2, wherein (BM) is Selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52; an hFGF23 antagonist polypeptide, optionally comprising one, two, or three substitutions, the substitutions of which may occur at any one of the positions 1, 2, 3, 6, 9, 10, 11, 13, 15, 17, and / or 25 of the selected (BM) sequence.

4. The hFGF23 antagonist polypeptide according to claim 3, wherein the substitution is a conservative substitution.

5. An hFGF23 antagonist polypeptide according to any one of claims 1 to 4, The polypeptide contains a binding motif (BM), a) X according to Sequence ID 3 １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ -(BM)-X ３６ X ３７ PSQX ４１ X ４２ X ４３ LLX ４６ EARX ５０ LX ５２ X ５３ X ５４ QX ５６ X ５７ X ５８ And, In the formula, (BM) consists of the amino acid residue sequence defined in claim 2(i), During the ceremony, they acted independently of each other. X １ is G, I, L, N, or V, X ２ is D, E, or Q, X ３ is D, E, G, N, or Q, X ４ is D, E, H, N, R, or T, X ５ is E, I, L, T, or V, X ６ is F, W, or Y, X ７ is A, G, H, I, L, N, P, Q, R, T, or V, X ８ is A, L, or E, X ３６ is A, D, or E, X ３７ is D or E, X ４１ is A, R, or W, X ４２ is A, D, or T, X ４３ is E, N, Q, or S, X ４６ is A, E, or K, X ５０ is Q or R, X ５２ is N or E, X ５３ is D, E, K, or T, X ５４ is A or I, X ５６ is A or C, X ５７ is K or P, X ５８ This is an amino acid residue sequence that is C, K, or E. and b) An hFGF23 antagonist polypeptide comprising an amino acid residue sequence selected from an amino acid residue sequence having at least 93% identity with the sequence defined in a).

6. The hFGF23 antagonist polypeptide according to claim 5, wherein each is independently of the other X １ is G, I, L, or V, X ２ is D or E, X ３ is D or E, X ４ is D or E, X ５ E is, X ６ F is, X ７ is I or Q, X ８ is A or L, X ａ Y is, X ｂ V is, X ｃ Q is, X ｄ Y is, X ｅ is F or Y, X ｆ is N or Q, X ｇ L is, X ｈ N is, X ｉ These are N and T, X ｊ is A, N, or Q, X ｋ S is, X ３６ is D or E, X ３７ is D or E, X ４１ W is, X ４２ A is, X ４３ N is, X ４６ is A or E, X ５０ is Q, or R, and X ５２ N is, X ５３ E is, X ５４ However, A is, X ５６ However, A is, X ５７ However, it is P, X ５８ This is K, an hFGF23 antagonist polypeptide.

7. The hFGF23 antagonist polypeptide according to any one of claims 2 to 6, wherein the binding motif (BM) forms part of a 3-helix vandal protein domain, and the 3-helix vandal protein domain is a variant of protein Z derived from domain B of Staphylococcus protein A.

8. hFGF23 antagonist polypeptide, which is capable of binding to hFGF23 according to SEQ ID NO: 1, Chemical substance 197 according to Sequence ID No. 168; Chemical substance 201 according to Sequence ID No. 176; Chemical substance 204 according to Sequence ID No. 234; Chemical substance 206 according to Sequence ID No. 236; Chemical substance 207 according to Sequence ID No. 237; and An hFGF23 antagonist polypeptide having at least 93% identity with an hFGF23 antagonist polypeptide selected from the group consisting of chemical substance 208, as defined by Sequence ID No.

238.

9. The hFGF23 antagonist polypeptide according to any one of claims 1 to 8, wherein the hFGF23 polypeptide is Chemical substances 89, Chemical substance 106, Chemical substance 107, Chemical substance 111, Chemical substances 197, Chemical substance 201, Chemical substance 204, Chemical substance 206, Chemical substance 207; and hFGF23 antagonist polypeptide, selected from the group consisting of 208 chemical substances.

10. An hFGF23 antagonist polypeptide according to any one of claims 1 to 9, wherein the polypeptide is capable of binding to hFGF23 at a KD value of less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 10 nM, less than 1 nM, less than 100 μM, preferably less than 0.1 μM, such as less than 10 nM, less than 2 nM, or less than 1 nM.

11. An hFGF23 antagonist polypeptide according to any one of claims 1 to 10, including an extended portion.

12. The hFGF23 antagonist polypeptide according to claim 11, wherein the extended portion is covalently bonded to the side chain group of an amino acid residue on the polypeptide.

13. The hFGF23 antagonist polypeptide according to claim 11 or 12, wherein the extended portion comprises a C16, C17, C18, C19, C20, C21, or C22 fatty acid such as C18, C19, or C20 fatty acid.

14. The hFGF23C antagonist polypeptide according to any one of claims 11 to 13, wherein the extended portion is 【Chemistry 1】 (Chemical substance 1a); 【Chemistry 2】 (Chemical substance 1b): 【Transformation 3】 (Chemical substance 1c); 【Chemistry 4】 (Chemical substance 1d); 【Transformation 5】 (Chemical substance 1e); QRLMEDICLPRWGCLWEDDF-* (chemical substance 1f); and *-QRLMEDICLPRWGCLWEDDF (chemical substance 1f); (where * represents a binding site to either a linker (L Ｐ ) or an hFGF23 antagonist polypeptide) hFGF23 antagonist polypeptide containing extension factor P selected from the group consisting of the following.

15. An hFGF23 antagonist polypeptide according to any one of claims 1 to 14, wherein the polypeptide derivative includes an extended portion, and the extended portion is *-NH-(CH ２ ) ２ - (O - (CH ２ )) ｋ -O-(CH ２ ) ｎ -CO-* (chemical substance 2a) (wherein k is an integer in the range of 1 to 5, and n is an integer in the range of 1 to 5); *-NH-S(O) ２ -CH ２ -CH ２ -CH ２ -CO-*; (chemical substance 2e); *-NH-CH ２ - (C) ６ H １０ )-CO-*(chemical substance 2f); *-NH-(CH) ２ ) ５ -CO-* (2g of chemical substance); 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 Linker L selected from the group consisting of the following Ｐ hFGF23 antagonist polypeptide, including

16. The hFGF23 antagonist polypeptide according to claim 15, wherein the extended portion "P" is a chemical substance 1c and linker L Ｐ However, the chemical substance 2i (L1) is the hFGF23 antagonist polypeptide.

17. The hFGF23 antagonist polypeptide according to any one of claims 1 to 16, wherein the polypeptide derivative includes an extended portion, and the extended portion is covalently bonded to the side chain of a cysteine ​​or lysine residue in the polypeptide.

18. It is a compound, 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 A compound selected from the group consisting of the following.

19. A compound according to claim 18, which is an hFGF23 antagonist polypeptide derivative that can bind to hFGF23 according to SEQ ID NO:

1.

20. The hFGF23 antagonist polypeptide derivative is represented by the following formula. 【Chemistry 14】

21. A pharmaceutical composition comprising an hFGF23 antagonist polypeptide, polypeptide derivative, or compound according to any one of claims 1 to 20, and one or more pharmaceutically acceptable excipients.

22. An hFGF23 antagonist polypeptide derivative, polypeptide derivative, compound, or composition according to any one of claims 1 to 21, for use in pharmaceuticals.

23. An hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of claims 1 to 22, for use in the treatment of tumor-induced bone disease, fibrous dysplasia, McCune-Albright syndrome, autosomal dominant hypophosphatemic rickets (ADHR), or X-linked hypophosphatemic rickets (XLH).

24. A kit comprising an hFGF23 antagonist polypeptide, polypeptide derivative, compound, or composition according to any one of claims 1 to 21, and instructions for use.

Images