Antagonists of human fgf23, pharmaceutical compositions and medicines comprising the same, and uses thereof

Abstract

This invention relates to novel hFGF23 antagonistic peptides and their long-acting derivatives, and their use in medicine, specifically for inhibiting hFGF23-induced signaling in patients in need, such as those suffering from hypophosphatemia, such as X-linked hypophosphatemia rickets (XLH); and pharmaceutical compositions comprising such compounds.

Description

Technical Field

[0001] Human fibroblast growth factor 23 (hFGF23) antagonists and compositions containing such compounds are used in medicine. [Sequence lists are incorporated by reference] [ ]

[0002] This application is submitted electronically as a sequence list. The entire contents of the sequence list are hereby incorporated by reference. Prior Technology

[0003] Human fibroblast growth factor 23 (hFGF23) is a 251-amino acid protein that exerts its effects by binding to its receptor complex, primarily composed of fibroblast growth factor receptor 1 isoform c (FGFR1c) and alpha-klotho. In the kidneys, hFGF23 activates FGFR1c-alpha-klotho, inhibiting the sodium phosphate cotransporter in the proximal tubule, leading to reduced phosphate reabsorption, increased urinary phosphate excretion, and decreased blood phosphate concentration. Therefore, inhibition of hFGF23 activity can be used to treat certain hypophosphatemia. X-linked hypophosphatemic rickets (hereinafter referred to as XLH) is one such disease in which blood hFGF23 concentrations are elevated due to a loss-of-function mutation in the PHEX (phosphate-regulated X-linked endopeptidase homolog) gene. Until recently, the only treatment for chronic hypophosphatemia in XLH was high-dose oral phosphate and active vitamin D supplementation (known as conventional therapy). However, conventional therapy is cumbersome, poorly tolerated, has limited efficacy, and is associated with dangerous adverse events. In 2018, the monoclonal IgG1 anti-hFGF23 antibody burosumab was approved for the treatment of XLH. Burosumab inhibits hFGF23, increasing 1,25-dihydroxyvitamin D levels and renal reabsorption of phosphate, thereby increasing blood phosphate levels in XLH patients (WO2008 / 099969).

[0004] Although bromozumab can increase blood phosphate levels, many patients still experience hypophosphatemia during treatment, and the treatment burden of bromozumab is significant. In pediatric patients treated with bromozumab, many fail to achieve age-appropriate reference phosphate concentrations. In adults taking bromozumab, phosphate levels are unstable during treatment, except for a small group who do not reach normal ranges, and therefore many patients experience hypophosphatemia later in the dosing interval. The high doses and large injection volumes required for bromozumab treatment make it incompatible with self-administered devices such as injectable pens. In most cases, bromozumab is administered by healthcare professionals, placing a heavy burden on both patients and caregivers. Furthermore, the injection itself is cumbersome and painful due to the large volume and the process of loading the drug from a vial into a syringe. Each dose requires up to 3 ml for adults and typically 1-2 ml for children. Therefore, modified hFGF23 antagonist compounds are needed in this technology. Summary of the Invention

[0005] This invention relates to compounds in the form of hFGF23 antagonistic peptides and their derivatives, comprising an extended portion (i.e., a portion with an extended blood half-life), which are capable of binding to hFGF23 and antagonizing hFGF23-induced signal transduction activity. The hFGF23 antagonistic peptides and their derivatives disclosed herein are designed for the effective treatment of hypophosphatemia such as X-linked hypophosphatemia rickets (XLH).

[0006] In one embodiment, the present invention relates to an hFGF23 antagonistic peptide capable of binding to an antigenic determinant on hFGF23 according to SEQ ID NO:1, the antigenic determinant comprising amino acid residues selected from the group consisting of: 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; As measured at 3.5 Å.

[0007] In another embodiment, the present invention relates to hFGF23 antagonistic peptides capable of binding hFGF23 according to SEQ ID NO:1, wherein such antagonistic peptides comprise a binding motif (BM) consisting of an amino acid residue sequence selected from the following. i) According to SEQ ID NO:2, XaXbXcAWXdEIXeXfXgPXhLXiDXjQWPAFIEXkLH Among them, each independently, Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; and ii) The amino acid residue sequence has at least 93% similarity to the sequence defined in i).

[0008] In another embodiment, the present invention relates to hFGF23 antagonistic peptides comprising the binding motif (BM) as described above, wherein such peptides comprise an amino acid residue sequence selected from the following a) According to SEQ ID NO:3, X1X2X3X4X5X6X7X8-(BM)-X36X37PSQX41X42X43LLX46EARX50LX52X53X54QX56X57X58, Wherein (BM) is composed of an amino acid residue sequence as defined above, Among them, each independently, X1 can be G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 can be D, E, H, N, R, or T; X5 can be E, I, L, T, or V; X6 can be F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T or V; X8 can be A, L, or E; X36 can be A, D, or E; X37 is either D or E; X41 can be A, R, or W; X42 represents A, D, or T; X43 is E, N, Q, or S; X46 is A, E, or K; X50 is either Q or R; X52 is either N or E; X53 is D, E, K, or T; X54 is either A or I; X56 is either A or C; X57 is either K or P; X58 is C, K, or E; and b) An amino acid residue sequence that has at least 93% similarity to the sequence defined in a).

[0009] In one embodiment, the present invention relates to hFGF23 antagonistic peptides, such as those referred herein by the following antagonistic peptides: Chem. 89, Chem. 106, Chem. 107, Chem. 111, Chem. 197, Chem. 201, Chem. 204, Chem. 206, Chem. 207, and Chem. 208.

[0010] In another embodiment, the present invention relates to hFGF23 antagonistic peptides as described above, wherein such peptides are linked with an extended portion in order to obtain hFGF23 antagonistic peptide derivatives.

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

[0012] In another embodiment, the invention relates to other compounds disclosed herein.

[0013] In another embodiment, the present invention relates to polynucleotides, carriers, and host cells suitable for producing the hFGF23 antagonistic peptides, derivatives, and compounds described above. [ ]

[0014] In another embodiment, the present invention relates to pharmaceutical compositions comprising the hFGF23 antagonistic polypeptide or its derivatives or compounds as described above.

[0015] In another embodiment, the present invention relates to a kit comprising the hFGF23 antagonistic polypeptide or its derivatives, compounds or compositions as described above, and an instruction manual.

[0016] In another embodiment, the present invention relates to the medical use of the hFGF23 antagonistic polypeptide or its derivatives, compounds, compositions and kits as described above, specifically for the treatment of hypophosphatemia, such as, but not limited to, X-linked hypophosphatemia rickets (XLH).

[0017] The present invention can also solve further problems that are obvious from the disclosure of exemplary embodiments. Simple Explanation of the Diagram

[0018] [Figure 1] shows the plasma phosphate levels in miniature pigs after intravenous administration of compounds (Chem. 197, Chem. 201, Chem. 204). [Figure 2] shows the plasma phosphate levels after subcutaneous administration of compounds (Chem. 204, 206, 207, 208) to male Beagle dogs. [Figure 3] shows the plasma phosphate content in miniature pigs after intravenous administration of compound (Chem. 206). Implementation

[0019] [Brief Explanation of the Sequence] [ ] SEQ ID NO:1 represents the amino acid residue sequence of mature human FGF23 (hFGF23) after the removal of 24 amino acid signal peptides from the full-length human FGF23. SEQ ID NO:2 represents the amino acid residue sequence of the binding motif of the present invention, which includes positions with fixed residues or non-fixed residues. SEQ ID NO:3 represents the amino acid residue sequence of the hFGF23 antagonistic polypeptide of the present invention, which includes positions with fixed residues or non-fixed residues. SEQ ID NO:4 represents the amino acid residue sequence of the hFGF23 fragment used in Example 8 of this article. SEQ ID NO:5 represents the amino acid residue sequence of the hFGF23 variant used in Example 2 of this paper. SEQ ID NO:6 represents the amino acid residue sequence of the hFGF23 variant used in Example 3 of this article. SEQ ID NO:7 represents the amino acid residue sequence of the heavy chain of brosuumab. SEQ ID NO:8 represents the amino acid residue sequence of the light chain of brosuumab. SEQ ID NO:9 represents the amino acid residue sequence of the peptide elongator. SEQ ID NO:10 represents the amino acid residue sequence of the hFGF23 antagonistic peptide used in Example 8 of this article. SEQ ID NO:11-52 represents the amino acid residue sequence of the hFGF23 antagonistic peptide binding motif (BM). SEQ ID NO:53-280 represents the amino acid residue sequence of the hFGF23 antagonistic peptide.

[0020] This invention relates to compounds capable of binding to and antagonizing the biological activity of human FGF23 (hFGF23). hFGF23 (represented herein by SEQ ID NO:1) is involved in phosphate and calcium metabolism in the body. Inhibition of hFGF23 activity can be used to treat certain hypophosphatemia, such as X-linked hypophosphatemic rickets (XLH), in which the concentration of hFGF23 in the blood is elevated due to a loss-of-function mutation in the PHEX (phosphate-regulated X-linked endopeptidase homolog) gene. Other diseases that the compounds of this invention can prevent or treat include diseases characterized by excessive hFGF23 activity, such as tumor-induced osteomalacia, autosomal dominant hypophosphatemic rickets (ADHR), fibrous dysplasia, and McCune-Albright syndrome.

[0021] The hFGF23 antagonistic peptide disclosed herein comprises a triple-helical bundled protein domain derived from the Z domain of Staphylococcus aureus protein A. A large library was constructed by randomly grouping the amino acids on two of the three helices, and strong binders were identified. Therefore, the compounds of this invention are designed to bind to hFGF23 to efficiently antagonize hFGF23-induced signaling activity and achieve effective treatment of hypophosphatemia, while being compatible with low-burden delivery devices suitable for self- or caregiver home administration. In one embodiment, the hFGF23 antagonistic peptide disclosed herein specifically binds to hFGF23 to reduce hFGF23-induced signaling mediated by FGFR1c as a receptor and α-croxoprotein (KL) as a co-receptor, thereby, for example, increasing blood phosphate levels.

[0022] The compounds disclosed herein preferably have a molecular weight between 5 kDa and 10 kDa, such as 7 kDa and 8 kDa, which is significantly smaller than that of an antibody called bromosumab (approximately 147 kDa); and are expected to have a therapeutically suitable blood half-life in vivo to achieve an appropriate dosing frequency, thereby effectively reducing hFGF23-induced signaling. The presence of the extended moiety serves to increase the blood half-life of the compounds disclosed herein.

[0023] Not being bound by theory means that when the efficacy (nemerol / kg) is close to or equivalent to brososumab, a lower dose (mg / kg) and / or a lower injection volume can be used, thus making it compatible with self-administering devices such as injectable pens. Additionally, phosphate upregulation is expected to improve with smaller peak / trough variations.

[0024] Therefore, the molecular weight of the compound is expected to allow for high concentrations in pharmaceutical formulations. The pharmacokinetic properties of the compound, as disclosed herein, enable a favorable drug exposure profile, which is expected to minimize dosing-to-dosing changes in phosphate levels, thereby reducing hypophosphatemia and improving disease outcomes. Furthermore, the compound disclosed herein is expected to be significantly more effective than bromozumab ( / mg) in patients in need, such as those with XLH. This increased efficacy should allow for a greater phosphate elevation than bromozumab can achieve, thereby improving the rate of phosphate normalization in these patients.

[0025] In one embodiment, the compounds of the present invention are characterized by reference to their target binding sites, namely, amino acid residues on hFGF23, which constitute the key antigenic determinant residues or the complete set of antigenic determinant residues of the hFGF23 antagonistic peptide. Therefore, in some embodiments, the present invention relates to an hFGF23 antagonistic peptide capable of binding to an antigenic determinant on hFGF23 according to SEQ ID NO:1, the antigenic determinant comprising amino acid residues selected from the group consisting of: 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; As measured at 3.5 Å.

[0026] In a preferred embodiment, the compound of the present invention is an hFGF23 antagonistic polypeptide capable of binding to an antigenic determinant on hFGF23 according to SEQ ID NO:1, the antigenic determinant comprising amino acid residues M74, R76, T86, R91, F108, P110, R114, L166, I167, N170, as determined at 3.5 Å.

[0027] In one embodiment, the compounds of the present invention are characterized by reference to their binding motif (BM). In one such embodiment, the compounds may be described as hFGF23 antagonistic peptides capable of binding hFGF23 according to SEQ ID NO:1, the antagonistic peptides comprising a binding motif (BM) composed of an amino acid residue sequence selected from the following. i) According to SEQ ID NO:2, XaXbXcAWXdEIXeXfXgPXhLXiDXjQWPAFIEXkLH Among them, each independently, Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; and ii) The amino acid residue sequence has at least 93% similarity to the sequence defined in i).

[0028] In a preferred embodiment, Xa is Y. In a preferred embodiment, Xb is L. In a preferred embodiment, Xc is Q. In a preferred embodiment, Xf is N. In a preferred embodiment, Xa is Y, Xb is L, Xc is Q, and Xf is N.

[0029] In some embodiments, (BM) is selected from the group consisting of: YLQAWYEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:11); YLQAWYEIFNLPWLNDNQWPAFIESLH (SEQ ID NO:12); YLQAWYEIFNLPYLNDNQWPAFIESLH (SEQ ID NO:13); YLQAWYEIFQLPNLNDNQWPAFIESLH (SEQ ID NO:14); YLQAWWEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:15); YLQAWFEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:16); YLQAWHEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:17); YLAAWYEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:18); YLQAWYEIFNLPHLNDNQWPAFIESLH (SEQ ID NO:19); YLQAWYEIFNLPNLNDNQWPAFIEALH (SEQ ID NO:20); YLQAWYEIFNLPNLNDNQWPAFIEQLH (SEQ ID NO:21); YLQAWYEIFNLPNLQDNQWPAFIESLH (SEQ ID NO:22); YLQAWYEIFNLPNLNDQQWPAFIESLH (SEQ ID NO:23); YLQAWYEIFNLPNLNDDQWPAFIESLH (SEQ ID NO:24); YLQAWYEIFNLPNLNDEQWPAFIESLH (SEQ ID NO:25); YLQAWYEIFNLPNLNDSQWPAFIESLH (SEQ ID NO:26); YLQAWYEIFNLPNLNDTQWPAFIESLH (SEQ ID NO:27); YLQAWYEIFNLPNLNDHQWPAFIESLH (SEQ ID NO:28); YLQAWYEIFNLPNLNDAQWPAFIESLH (SEQ ID NO:29); YLQAWYEIFNLPNLNDLQWPAFIESLH (SEQ ID NO:30); YLQAWYEIFNLPNLTDNQWPAFIESLH (SEQ ID NO:31); YLQAWYEIFNLPNLTDEQWPAFIESLH (SEQ ID NO:32); YLQAWYEIFNLPNLTDQQWPAFIESLH (SEQ ID NO:33); YLQAWYEIFNLPNLSDNQWPAFIESLH (SEQ ID NO:34); YLQAWYEIFNLPNLNDGQWPAFIESLH (SEQ ID NO:35); YVQAWYEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:36); YIQAWYEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:37); YFQAWYEIFNLPNLNDNQWPAFIESLH (SEQ ID NO:38); YLQAWYEIFNLPQLNDNQWPAFIESLH (SEQ ID NO:39); YLQAWYEIFNLPNLQDQQWPAFIESLH (SEQ ID NO:40); YLQAWYEIFNLPHLNDQQWPAFIESLH (SEQ ID NO:41); YLQAWYEIFNLPNLDDAQWPAFIESLH (SEQ ID NO:42); YLQAWYEIFNLPNLTDAQWPAFIESLH (SEQ ID NO:43); YLQAWYEIFNLPNLDDQQWPAFIESLH (SEQ ID NO:44); YLQAWYEIFNLPHLNDAQWPAFIESLH (SEQ ID NO:45); YLQAWYEIFNLPNLNDYQWPAFIESLH (SEQ ID NO:46); YLQAWYEIFNLPHLTDQQWPAFIESLH (SEQ ID NO:47); YLQAWYEIFNLPNLTDYQWPAFIESLH (SEQ ID NO:48); YLQAWYEIFTLPHLTDQQWPAFIESLH (SEQ ID NO:49); YLQAWYEIFNLPCLNDNQWPAFIESLH (SEQ ID NO:50); YLQAWYEIFNLPCLNDQQWPAFIESLH (SEQ ID NO:51); YLQAWYEIFNLPCLTDSQWPAFIESLH (SEQ ID NO:52).

[0030] In one state, the compounds of the invention are characterized by reference to their binding motif (BM) and amino acid residues side-attached at the N-terminus and C-terminus (BM), and can therefore be described as hFGF23 antagonistic peptides comprising the binding motif (BM) as described above, wherein such peptides comprise amino acid residue sequences selected from the following a) According to SEQ ID NO:3, X1X2X3X4X5X6X7X8-(BM)-X36X37PSQX41X42X43LLX46EARX50LX52X53X54QX56X57X58, Wherein (BM) consists of an amino acid residue sequence as defined in (i) above, Among them, each independently, X1 can be G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 can be D, E, H, N, R, or T; X5 can be E, I, L, T, or V; X6 can be F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T or V; X8 can be A, L, or E; X36 can be A, D, or E; X37 is either D or E; X41 can be A, R, or W; X42 represents A, D, or T; X43 is E, N, Q, or S; X46 is A, E, or K; X50 is either Q or R; X52 is either N or E; X53 is D, E, K, or T; X54 is either A or I; X56 is either A or C; X57 is either K or P; X58 is C, K, or E; and b) has an amino acid residue sequence that is at least 93% identical to the sequence defined in a).

[0031] In a preferred embodiment, they are independent of each other. X1 can be 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 either A or L; Xa is Y; Xb is V; Xc is Q; Xd is Y; Xe is either F or Y; Xf is N or Q; Xg is L; Xh is N; Xi is N or T; Xj can be A, N, or Q; Xk is S; X36 is either D or E; X37 is either D or E; X41 is W; X42 is A; X43 is N; X46 is either A or E; X50 is either Q or R; X52 is N; X53 is E; X54 is A; X56 is A; X57 is P; X58 is K.

[0032] As those skilled in the art will understand, minor alterations can be made to the amino acid sequence of a polypeptide without significantly affecting its function. Therefore, this invention covers modified variants of polypeptides that retain their inhibitory characteristics. In this manner, this invention covers an hFGF23 antagonistic polypeptide whose amino acid sequence has 93% or greater similarity to the hFGF23 antagonistic polypeptide or (BM) as defined above, such as 95%, 96%, 97%, 98%, or 99% or greater similarity. For example, an amino acid residue belonging to a functional group of amino acid residues (e.g., hydrophobic, hydrophilic, polar, etc.) can be exchanged for another amino acid residue from the same functional group.

[0033] In some embodiments, the hFGF23 antagonistic peptide of the present invention may comprise 50 to 70 amino acid residues. In a preferred embodiment, the hFGF23 antagonistic peptide may comprise 56 to 60 amino acid residues. In a more preferred embodiment, the hFGF23 antagonistic peptide comprises 58 amino acid residues.

[0034] In a preferred embodiment, the hFGF23 antagonistic peptide includes an elongation portion. An hFGF23 antagonistic peptide containing an elongator or elongation portion may also be referred to as an "hFGF23 antagonistic peptide derivative," the term "derivative" indicating the presence of an elongator or elongation portion.

[0035] Preferred compounds of the present invention are Chem. 89, Chem. 106, Chem. 107, Chem. 111, Chem. 197, Chem. 201, Chem. 204, Chem. 206, Chem. 207 and Chem. 208 (see Table 4 below). Non-limiting examples are shown below.

[0036] Chem. 204 is expressed by the following formula:

[0037] Chem. 206 is expressed by the following formula:

[0038] Chem. 207 is expressed by the following formula:

[0039] Chem. 208 is expressed by the following formula:

[0040] In one embodiment, the present invention relates to a pharmaceutical formulation comprising the hFGF23 antagonistic polypeptide or an hFGF23 antagonistic polypeptide derivative of the present invention and one or more pharmaceutically acceptable excipients.

[0041] In one embodiment, the present invention relates to the medical use of hFGF23 antagonistic peptides, hFGF23 antagonistic peptide derivatives, and pharmaceutical formulations comprising hFGF23 antagonistic peptides or derivatives thereof. Such medical uses include the use for treating hypophosphatemia, such as, but not limited to, X-linked hypophosphatemia rickets (XLH).

[0042] In other embodiments, the present invention relates to a kit comprising an hFGF23 antagonistic polypeptide or a polypeptide derivative or the pharmaceutical composition thereof; Polynucleotides encoding the hFGF23 antagonistic peptide or peptide derivative, and expression vectors encoding such polynucleotides; Host cells that produce the hFGF23 antagonistic peptide or its derivative; And methods for producing the polypeptide or its derivatives, the methods comprising: a) Culturing host cells according to the aforementioned technical solution under conditions that allow for the expression of the polypeptide encoded by the expression vector; b) Isolating the polypeptide; and c) Linking the extended portion to the isolated polypeptide as needed.

[0043] The term "one" or "a" is intended to mean "one or more".

[0044] The term "about" is used herein to mean approximately, roughly, roughly, or about. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. Generally, the term "about" can modify a numerical value by increasing or decreasing (above or below) the value by 10%.

[0045] When describing steps or elements, the term "comprise" and its variations, such as "comprises" and "comprising," are intended to mean, as appropriate and without excluding, the addition of other steps or elements.

[0046] As used throughout, "amino acid" or "amino acid residue" includes both typical amino acids (which are encoded in genes) and non-natural amino acids. Non-limiting examples of non-natural amino acids include Aib (α-aminoisobutyric acid), deaminohistidine (also known as 3-(imidazol-4-yl)propionic acid, abbreviated as Imp (imidazolyl)), and d-isomers of typical amino acids. Unless otherwise stated, all amino acid residues within a polypeptide for which optical isomers are not stated should be understood herein to mean L-isomers.

[0047] The term "binding affinity" is used in this paper as a measure of the non-covalent interaction between two molecules, such as a peptide and an antigen. The term "binding affinity" is used to describe monovalent interactions.

[0048] The binding affinity between two molecules, such as the hFGF23 binder (e.g., the hFGF23 antagonistic peptide disclosed herein) and hFGF23, arising from monovalent interactions, can be quantified by measuring the equilibrium dissociation constant (KD). KD can be determined by measuring the complex formation and dissociation kinetics, for example, by surface plasma resonance (SPR) methods.

[0049] The rate constants corresponding to association and dissociation of monovalent complexes are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is related to ka and kd through the equation KD = kd / ka. According to the above definition, the binding affinity associated with different molecular interactions can be compared by comparing the KD values ​​of individual conjugate / antigen complexes, such as comparing the binding affinity of different conjugates for a specified antigen (such as the interaction between the compound disclosed in this paper and hFGF23).

[0050] The KD of the hFGF23 antagonistic peptide to its target can be less than 100 µM, such as less than 10 µM, such as less than 9 µM, such as less than 8 µM, such as less than 7 µM, such as less than 6 µM, such as less than 5 µM, such as less than 4 µM, such as less than 3 µM, such as less than 2 µM, such as less than 1 µM, such as less than 0.9 µM, such as less than 0.8 µM, such as less than 0.7 µM, such as less than 0.6 µM, such as less than 0.5 µM, such as less than 0.4 µM, such as less than 0.3 µM, such as less than 0.2 µM, such as less than 0.1 µM, such as less than 10 nM, such as less than 2 nM, such as less than 1 nM. More preferably, the KD is less than 2 nM.

[0051] As used herein, the term "binding motif" (BM) refers to the hFGF23 binding region of an hFGF23 antagonistic polypeptide. The BM contains key binding residues that bind tightly to hFGF23 and "forms part of a triple-helical bundle domain." For example, the BM may consist of two α-helices with interconnecting loops within the triple-helical bundle domain.

[0052] In some embodiments, (BM) comprises 25 to 28 amino acid residues. In a preferred embodiment, (BM) comprises 27 amino acid residues.

[0053] In some embodiments, the (BM) sequence corresponds to a sequence selected from the group consisting of positions 9 to 35 (inclusive) of the following: SEQ ID NO: 11-52, or a sequence that is 90% identical to it, such as a sequence that is 91, 92, 93% or greater identical to it, such as a sequence that is 94, 95, 96, 97, 98 or 99% identical to the (BM).

[0054] In some embodiments, (BM) is characterized by containing fixed or non-fixed (variable) amino acid residues. Non-fixed amino acid residues are denoted herein as Xx, where the subscript letters identify their positions in (BM).

[0055] (BM) has non-(BM) amino acid residues attached to its N-terminus and C-terminus, and (BM) and non-(BM) residues are combined to form hFGF23 antagonistic peptides.

[0056] "Cross-species reactive" peptides bind with similar affinity to FGF23 in all designated species (e.g., humans, dogs, pigs, and cynomolgus monkeys), specifically within a factor of 100, such as within a factor of 50, a factor of 20, or a factor of 10. A KD range with a given coefficient X means that the highest affinity for the specifically listed species is no more than X times higher than the lowest affinity for different listed species. Those skilled in this art will understand that any method used to measure affinity can be used to verify that cross-species reactive peptides bind to target antigens from all listed species within the specified KD factor range as described herein, provided that the KD measurements for all listed species are performed under the same conditions. Preferably, SPR is used, especially at 25°C, to measure KD values.

[0057] As used herein, the term "antigen determinant" is defined in the context of molecular interactions between an antigen-binding polypeptide (such as the hFGF23 binder, such as the hFGF23 antagonistic polypeptide and its derivatives disclosed herein) and its corresponding antigen. Generally, an "antigen determinant" refers to a region or area on the antigen for binding of the binder, i.e., a region or area in contact with the binder entity. Entity contact can be defined using various criteria for atoms in the binding molecule and the antigen molecule (e.g., a distance cutoff of 4–5 Å, such as 4, 4.5, or 5 Å; or solvent accessibility).

[0058] hFGF23 may contain multiple different antigenic determinants, which may include (but are not limited to) (1) linear peptide antigenic determinants; (2) conformational antigenic determinants, which consist of one or more discontinuous amino acids in the conformation of hFGF23 that are close to each other.

[0059] The antigenic determinants of a specified binder / antigen pair can be described and characterized at different levels of detail using a variety of experimental and computational methods for antigenic determinant localization. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, high-density ion exchange mass spectrometry (HDX-MS), various competitive binding methods, and computational methods such as Alphafold 2; methods known in this technique.

[0060] In the context of defining X-ray derived crystal structures by spatial coordination of the complex between a binding molecule (such as the hFGF23 antagonistic peptide disclosed herein) and its antigen, unless otherwise stated or contradicted by the context, the term antigenic determinant is specifically defined herein as an hFGF23 residue characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 3.5 Å from the heavy atom in the binding molecule.

[0061] Epitopes containing the same group of amino acid residues are called identical (e.g., determined by X-ray structure analysis). Epitopes sharing at least one amino acid residue are called overlapping. Epitopes not sharing amino acid residues are called separate (unique).

[0062] Antigenic determinants can be identified using conventional methods. For example, the approximate location of an antigenic determinant can be determined by assessing the ability of an hFGF23 antagonistic peptide to bind to different fragments or variants of hFGF23. Specific amino acids within hFGF23 that contact the conjugate (antigenic determinant) can also be determined using conventional methods. For instance, the conjugate can be combined with a target molecule, and the resulting complex can be crystallized. The crystalline structure of the complex can be determined and used to identify specific locations of the interaction between the antibody and its target. Antigenic determinant residues common to several different compounds can be considered "key" antigenic determinant residues.

[0063] As used herein, the term "hFGF23 antagonistic peptide" refers to a peptide that can bind to hFGF23 according to SEQ ID NO:1 and can partially or completely block its biological activities, such as hFGF23-induced signal transduction activities, such as hFGF23-induced signal transduction activities of the FGFR1c-α-croxo protein complex. The hFGF23 antagonistic peptide contains a triple-helical bundle protein domain, which is a protein Z variant derived from domain B of Staphylococcus protein A.

[0064] In some embodiments, the hFGF23 antagonistic peptide is characterized by containing fixed or non-fixed amino acid residues. Non-fixed (variable) amino acid residues are designated as Xx herein, where subscript numbers / letters indicate their positions in the hFGF23 antagonistic peptide.

[0065] As used herein, the term "half-life" or "blood half-life" refers to the time required for half of the amount of a substance administered to a patient to be metabolized or eliminated from the patient's blood through normal biological processes.

[0066] The term "identity" as known in this technique refers to the relationship between two or more polypeptide sequences as determined by comparing sequences. In this technique, "identity" also means the degree of sequence relevance between polypeptides as determined by the number of matches between two or more amino acid residue strings. "Identity" measures the percentage of identical matches among two or more sequences having the smaller of the vacancy alignments (if any) resolved by a specific mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be easily calculated using known methods.

[0067] In this invention, Needleman (Needleman et al., J. Mol. Biol. 1970; 48:443-453) from EMBOSS-6.6.0 was used to determine consistency using parameters 10 and 0.5 (vacancy open = 10, vacancy extended = 0.5), respectively, for vacancy opening and extension. The score of each sequence in the sequence pair was calculated based on the number of matches in the aligned sequence, normalized according to the sequence length, and multiplied by 100.

[0068] The term "pharmaceutical-acceptable excipient" means an excipient suitable for the preparation of pharmaceutical compositions that are generally safe and non-toxic, including excipients acceptable for human medical use. Such excipients may be, for example, solid, liquid, or semi-solid.

[0069] As used herein, the term "peptide" includes oligopeptides and refers to a single amino acid chain linked by one or more peptide bonds.

[0070] The "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids. Substitution can be, but is not limited to, conservative substitution. For example, an amino acid can be substituted with an amino acid having similar biochemical properties; for instance, a basic amino acid can be substituted with another basic amino acid (e.g., lysine replaced with arginine), an acidic amino acid can be substituted with another acidic amino acid (e.g., glutamic acid replaced with aspartate), a neutral amino acid can be substituted with another neutral amino acid (e.g., threonine replaced with serine), a charged amino acid can be substituted with another charged amino acid (e.g., glutamic acid replaced with lysine), and a hydrophilic amino acid... Amino acids can be substituted with another hydrophilic amino acid (e.g., asparagine substituted with glutamine), hydrophobic amino acids can be substituted with another hydrophobic amino acid (e.g., alanine substituted with valine), polar amino acids can be substituted with another polar amino acid (e.g., serine substituted with threonine), aromatic amino acids can be substituted with another aromatic amino acid (e.g., phenylalanine substituted with tryptophan), and aliphatic amino acids can be substituted with another aliphatic amino acid (e.g., leucine substituted with isoleucine). Such substitutions are called "conservative" substitutions.

[0071] This invention covers variants of the hFGF23 antagonistic peptide as disclosed herein, which may include individual binding motifs disclosed herein and substitutions of one, two, or three amino acids in the hFGF23 antagonistic peptide sequence. In some embodiments, the substitutions are conservative substitutions.

[0072] As used herein, the term "treatment" and its variations refer to medical treatment for any human individual in need. The term includes the administration of a therapeutically effective amount of a polypeptide as disclosed herein, sufficient to reduce or eliminate at least one symptom of the condition under discussion. However, "treatment" is not necessarily a cure. The timing and purpose of treatment may vary from individual to individual depending on their current health status. Therefore, treatment can be preventative, disease-modifying, palliative, symptomatic, and / or curative. For the purposes of this invention, preventative, disease-modifying, palliative, symptomatic, and / or curative treatments may represent various aspects of this invention.

[0073] As used herein, the term "extended fraction" (PM) refers to a fraction that extends the half-life of a compound to which it is linked in the blood. An extended fraction comprises an "elongator" (P) and, if desired, a "linker" (LP). The term "extended" therefore refers to an extension of half-life, and the elongator or extended fraction is thus used for the purpose of extending the half-life of the hFGF23 antagonistic peptide disclosed herein in the blood. The terms "extended" and "half-life extension" are used interchangeably and generally refer to an extension of the plasma half-life of the compound as disclosed herein.

[0074] After the polypeptide is generated (e.g., by synthesis or recombination), the elongated portion is preferably covalently linked to the side chain of the lysine exposed on the surface or to a cysteine ​​residue in the polypeptide, preferably via an ε-amino group. In this document, the residue constituting the linker for such a link is generally referred to as "R1" (and in the case of more than one elongated portion, as "R2", "R3", etc.).

[0075] Given the disclosures herein, those skilled in this technique will be able to identify other surface-exposed residues suitable for attachment.

[0076] The extension portion may consist of an extension (without a connector ("LP")).

[0077] The extension may include a connector ("LP") and an extension ("P").

[0078] The extension may include one connector and two or more extensions ("P").

[0079] When the linker ("LP") is present, the extended portion is linked to the hFGF23 antagonistic polypeptide backbone via LP. When the linker ("LP") is absent, "P" is directly linked to the polypeptide backbone.

[0080] The hFGF23 antagonistic peptide derivatives disclosed in this article, namely, hFGF23 antagonistic peptides linked to an elongated portion, may contain a single cysteine ​​or lysine residue linked to a single elongated portion.

[0081] The hFGF23 antagonistic peptide derivatives revealed in this article may contain two lysine residues and two elongated moieties.

[0082] The hFGF23 antagonistic peptide derivatives disclosed in this article may contain two lysine residues and two identical elongated moieties.

[0083] The hFGF23 antagonistic peptide derivatives revealed in this article may contain two lysine residues and two different elongated moieties.

[0084] The hFGF23 antagonistic peptide derivatives revealed in this article may contain three lysine residues and three elongated moieties.

[0085] The hFGF23 antagonistic peptide derivatives revealed in this article may contain three lysine residues and three identical elongated moieties.

[0086] The hFGF23 antagonistic peptide derivatives disclosed in this paper may contain three lysine residues and three different elongated portions (e.g., the first elongated portion and the second elongated portion may be the same and the third elongated portion may be different from the first and second portions).

[0087] The hFGF23 antagonistic peptide derivatives revealed in this article may contain two cysteine ​​residues and two elongated portions.

[0088] The hFGF23 antagonistic peptide derivatives disclosed in this article may contain two cysteine ​​residues and two identical elongated moieties.

[0089] The hFGF23 antagonistic peptide derivatives revealed in this article may contain two cysteine ​​residues and two different elongated moieties.

[0090] The hFGF23 antagonistic peptide derivatives revealed in this article may contain three cysteine ​​residues and three elongated portions.

[0091] The hFGF23 antagonistic peptide derivatives revealed in this article may contain three cysteine ​​residues and three identical elongated moieties.

[0092] The hFGF23 antagonistic peptide derivatives revealed in this article may contain three cysteine ​​residues and three different elongated moieties.

[0093] In the case where the hFGF23 antagonistic peptide derivative contains two or three elongated moieties, the elongated moieties are preferably similar, more preferably substantially the same, or best preferably identical.

[0094] In this article, hFGF23 antagonistic peptides containing elongated portions are referred to in some cases as peptides containing elongated portions or simply peptide "derivatives".

[0095] The extended portion can non-covalently bind to albumin, thereby promoting the circulation of the hFGF23 antagonistic peptide derivative in the bloodstream and prolonging its half-life. Therefore, in one embodiment, the extended portion is an albumin-binding portion.

[0096] The elongator P may contain a amide group. The elongator may contain an aliphatic amide group. The amide group may be branched or unbranched. The amide group may be saturated or unsaturated.

[0097] The elongator may contain a distal carboxylic acid group.

[0098] The elongator may contain at least two acidic groups, one of which is attached at the end.

[0099] The elongator may contain a fatty acid group.

[0100] The elongator may contain a fatty acid group and a amide group.

[0101] The elongator may contain a distal carboxylic acid group and a amide group.

[0102] The elongator may contain an alkyl group.

[0103] The elongator may contain an aryl group.

[0104] The elongator may contain a tetrazolium group.

[0105] The elongator may contain a sulfonic acid group.

[0106] The elongator may contain a phenoxy group.

[0107] The elongator may contain a benzoic acid group.

[0108] The elongator can contain 8 to 30 carbon atoms. The elongator can contain 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.

[0109] The elongator may contain 6 to 30 consecutive -CH2- groups. The elongator may contain a carbon chain containing 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.

[0110] The elongator may contain 12-26 carbon atoms. The elongator may contain 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbon atoms.

[0111] The elongator may include 10-26 consecutive -CH2- groups. The elongator may include a carbon chain containing at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive -CH2- groups.

[0112] The elongator can contain 16-22 carbon atoms. Specifically, it can contain 16, 17, 18, 19, 20, 21, or 22 carbon atoms.

[0113] The elongator may contain 14-20 consecutive -CH2- groups. The elongator may contain a carbon chain containing 14, 15, 16, 17, 18, 19 or 20 consecutive -CH2- groups.

[0114] The elongator may contain 16-22 consecutive carbon atoms and 14-20 consecutive -CH2- groups.

[0115] The elongator may contain 16 consecutive carbon atoms and 14 consecutive -CH2- groups.

[0116] The elongator may contain 18 consecutive carbon atoms and 16 consecutive -CH2- groups.

[0117] The elongator may contain 20 consecutive carbon atoms and 18 consecutive -CH2- groups.

[0118] The elongator may contain 22 consecutive carbon atoms and 20 consecutive -CH2- groups.

[0119] The elongator may contain groups defined as follows: Chem. 1: HOOC-(CH2)n-CO-*, where n is an integer in the range of 8-30, it can also be called C(n+2) diacid or... Chem. 1: , where n is an integer in the range of 8-30.

[0120] The elongator may contain an oligopeptide. The elongator oligopeptide may be 10 to 40 amino acids, such as 10 to 30 amino acids, such as 15 to 25 amino acids, and preferably 20 amino acids. The elongator oligopeptide sequence composition may be, for example, GlnArgLeuMetGluAspIleCysLeuProArgTrpGlyCys LeuTrpGluAspAspPhe (single amino acid notation QRLMEDICLPRWGCLWEDDF (chem. 1f, SEQ ID NO:9) (see also WO01 / 45746 A2).

[0121] The elongator can be linked to the hFGF23 antagonistic peptide via the amino acid linker LP.

[0122] In one embodiment, the LP may link the elongator to the side chain of a lysine or cysteine ​​residue in the backbone of the hFGF23 antagonistic polypeptide.

[0123] In one embodiment, LP can link the elongator to N-terminal or C-terminal residues in the backbone of the hFGF23 antagonistic polypeptide.

[0124] In one embodiment, the LP can link the elongator to residues 46K, 53K, or 57K in the hFGF23 antagonistic peptide.

[0125] hFGF23 antagonistic peptide derivatives may contain two elongated moieties, each containing 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

[0126] hFGF23 antagonistic peptide derivatives may comprise two elongated portions, each elongator containing 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups; such elongators are also referred to as C12, C13, C14, C15, C16, C17, or C18 elongators. In some embodiments, the elongator comprises an acidic group, such as a carboxylic acid group (diacid) with consecutive -CH2- groups attached to each end.

[0127] hFGF23 antagonistic peptide derivatives may contain two C14 diacids, two C16 diacids, or two C18 diacids.

[0128] hFGF23 antagonistic peptide derivatives may comprise three elongated moieties, each of which contains an elongator having 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. hFGF23 antagonistic peptide derivatives may comprise three elongated moieties, wherein each elongator contains 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive -CH2- groups.

[0129] In some embodiments, the elongator comprises a C16, C17, C18, C19, C20, C21, or C22 fatty acid, such as C16, C17, C18, C19, or C20 fatty acid diacid.

[0130] In a preferred embodiment, the hFGF23 antagonistic polypeptide derivative comprises a diacid elongator (C20 diacid elongator) containing 20 consecutive -CH2- groups.

[0131] In a preferred embodiment, the C20 diacid elongator is Chem. 1c in Table 1 below.

[0132] In some embodiments, the hFGF23 antagonistic peptide derivatives disclosed herein may include an elongator selected from any of the elongators described in Table 1. surface [1]. Examples of extensions (“P”) Extended Sub-ID structure C16 diacid (Chem. 1a) C18 diacid (Chem. 1b) C20 diacid (Chem. 1c) C17 tetrazolium (Chem. 1d) C18 tetrazolium (Chem. 1e) Albumin-binding oligopeptide (Chem. 1f, SEQ ID NO:9) An asterisk (*) indicates a connection point to an LP linker element or an amino acid residue in an hFGF23 antagonistic peptide.

[0133] As used herein, the term "extended fraction" refers to a fraction with extended half-life characteristics, which includes an "elongator" ("P") and, if desired, a "linker" ("LP") that, when present, links the elongator to an hFGF23 antagonistic peptide.

[0134] The linker LP, when present, may contain Ado, Aeep or Aeeep, sulfadiazine, Trx, ε-Lys, Ahx, Glu, γGlu, Gly, Ser, Ala and / or Thr.

[0135] An LP linker may, for example, contain at least one portion that can be represented by the following chemical formula, wherein an asterisk (*) indicates a linking site to an amino acid residue in another LP linker element, elongator, or hFGF23 antagonist polypeptide. Those skilled in the art will understand that the first and second * in the individual chemical formulas below do not represent linking sites on elements of the same type; that is, the linker according to the following formula is not intended to link two hFGF23 antagonist polypeptides, nor is it intended to link two elongators. Chem. 2a: *‑NH-(CH2)2-(O-(CH2))kO-(CH2)n-CO-* or Chem. 2a: , Where k is an integer in the range of 1 to 5, and n is an integer in the range of 1 to 5. When k=1 and n=1, this linker element can be called Ado, or 8-amino-3,6-dioxaoctyl, which can be represented by the following chemical formula: Chem. 2b: *‑NH-(CH2)2-O-(CH2)2-O-CH2-CO-* or Chem. 2b: When k=1 and n=2, this connecting element can be called Aeep, which can be represented by the following chemical formula: Chem. 2c: *‑NH-(CH2)2-O-(CH2)2-O-(CH2)2-CO-* or Chem. 2c: When k=2 and n=2, this connector element can be called Aeeep, which can be represented by the following chemical formula: Chem. 2d: *-NH-(CH2)2-O-(CH2)2O-(CH2)2-O-(CH2)2-CO-* or Chem. 2d: The optional linker (LP) may contain a sulfonylurea-C4 moiety. The sulfonylurea-C4 group is a sulfonylurea group attached to a 4-butyryl group and has the following chemical formula: Chem. 2e: *-NH-S(O)2-CH2-CH2-CH2-CO-* or Chem. 2e: The linker LP, if desired, may include Trx. Trx is also known as tranexamic acid (trans-4-(aminomethyl)cyclohexanecarboxylic acid) and has the following chemical formula: Chem. 2f: *-NH-CH2-(C6H10)-CO-* or Chem. 2f: The linker LP may contain ε-lysine (ε-Lys). The linker LP may contain lysine (Lys). The linker LP may contain Ahx. Ahx is also known as aminohexanoic acid (6-aminohexanoic acid) and is defined as: Chem 2g: *-NH-(CH2)5-CO-* or Chem 2g: The linker LP may contain a Glu diradical, such as... Chem. 2h: The Glu diradical can be contained p times, where p is an integer in the range of 1 to 3. Chem. 2h can also be referred to as γ-Glu, or simply γGlu, because it is the γ-carboxyl group of the amino acid glutamic acid (E), which in this document is used to link to the ε-amino group of the lysine. As explained above, the other linker element can be, for example, another Glu residue or an Ado molecule. The amino group of Glu then forms a amide bond with the carboxyl group of the elongated moiety, or with, for example, the carboxyl group of the Ado molecule (if present), or with, for example, the γ-carboxyl group of another Glu (if present), such as... (Chem. 2i); or (Chem. 2j); or (Chem. 2k).

[0136] In a preferred embodiment, the elongator, taking Chems. 1a-1f as an example, is linked to the polypeptide backbone using a linker (LP) selected from Table 2 below, where "R1" represents a linker site in the hFGF23 antagonistic polypeptide and "P" represents an elongator.

[0137] In a preferred embodiment, the elongators Chem. 1a-1f are linked to the polypeptide backbone using a linker (LP) (referred to as L1 (Chem. 2i) in Table 2 below).

[0138] In a preferred embodiment, the R1 linker is located on the ε-amino group of the polypeptide backbone.

[0139] In one such embodiment, the R1 linker is located on the ε-amino group of the lysine residue at the C-terminus of the polypeptide backbone.

[0140] In another such embodiment, the R1 linker is located on the ε-amino group of the lysine at positions 46, 53, or 57 of the hFGF23 antagonistic peptide.

[0141] Therefore, in a preferred embodiment, the residues at positions 46, 53 and / or 57 are lysine (K).

[0142] In a preferred embodiment, the residue at position 58 is lysine (K).

[0143] Based on the disclosures herein, those skilled in this art will be able to determine the optimal LP linker for the specific hFGF23 antagonistic peptide derivative disclosed herein, after some limited routine experiments, if necessary. surface [2]. Examples of preferred extended connectors (“LP”) Connector (LP) ID structure L1 (Chem. 2i) L2 (Chem. 2j) L3 (Chem. 2k)

[0144] The compounds disclosed herein can be generated by solid-phase peptide synthesis as described in the Examples section below. The Fmoc-Lys(Mtt)-OH attachment at the extension site allows for selective removal of the Mtt- group, followed by attachment of the extension site during peptide synthesis. After synthesis, the resin-bound peptide undergoes full-domain deprotection, followed by reverse-phase HPLC purification.

[0145] The compounds disclosed herein can be produced using recombinant nucleic acid technology. Generally, the nucleic acid sequence of the selected wild-type compound disclosed herein is modified to encode the desired protein. Subsequently, this modified sequence is inserted into an expression vector, which is then transformed or transfected into a host cell.

[0146] The nucleic acid constructs encoding compounds as described herein can appropriately have a genomic body, cDNA, or synthetic origin. Amino acid sequence variations are achieved by modifying the genetic code using well-known techniques.

[0147] DNA sequences encoding compounds as disclosed herein are typically inserted into recombinant vectors, which can be any vector readily subjected to recombinant DNA procedures. The choice of vector usually depends on the host cell to which it is to be introduced. Therefore, the vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plastid. Alternatively, the vector can be a vector that integrates into the genome when introduced into the host cell and replicates along with the chromosome into which it has been integrated.

[0148] The vector is preferably an expression vector in which the DNA sequence encoding the compound disclosed herein is operatively linked to additional segments required for DNA transcription. The term "operatively linked" means that the segments are arranged so that they work together for their intended purpose, such as initiating transcription at a promoter and proceeding via the DNA sequence encoding the polypeptide until it terminates within a terminator.

[0149] Therefore, the expression vector used to represent the compounds disclosed herein will contain a promoter capable of initiating and guiding the transcription of selected genes or cDNA. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein homologous or heterologous to that of the host cell.

[0150] In addition, the expression vector used to represent the compounds disclosed herein will also include a termination sequence, a sequence recognized by the host cell to terminate transcription. The termination sequence is operatively linked 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 this invention.

[0151] The compounds described in this article can target intracellular expression in the cytosol of host cells or be directed to extracellular expression in growth media via the secretory pathway.

[0152] Intracellular expression is a predefined pathway that requires the DNA sequence to sequentially contain a promoter, a DNA sequence encoding the polypeptide of the compound disclosed herein, and a terminator.

[0153] To guide the compounds disclosed herein into the secretory pathway of host cells, a secretory signal sequence (also known as a signal peptide or pre-sequence) is required to extend the N-terminus of the compound. The DNA sequence encoding the signal peptide is attached to the 5' end of the DNA sequence encoding the compound in the correct reading frame. The signal peptide may be associated with a protein or may originate from a gene encoding another secretory protein.

[0154] Procedures for binding the DNA sequence encoding the compound disclosed herein, the promoter, the terminator, and / or the secretion signal sequence, and inserting them into a suitable vector containing the information required 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).

[0155] The host cell into which the DNA sequence encoding the compounds disclosed herein is introduced can be any cell capable of expressing the compounds disclosed herein, either intracellularly or extracellularly. If post-translational modification is required, suitable host cells include yeast, fungi, insects, and higher eukaryotic cells such as mammalian cells.

[0156] This extension can be attached to the polypeptide backbone as follows.

[0157] N-methylpyrrolidone (0.25 × peptide solution volume) was added to a solution of the polypeptide backbone (10-40 mg / mL, 0.25 M Na₂CO₃(aq)) with stirring. To obtain selective reaction conditions for the ε-amino group of lysine, the pH was increased by slowly adding aqueous sodium hydroxide until the pH reached 10.5-11.

[0158] The acetylating agent (2–4 molar equivalents, relative to the polypeptide backbone) was dissolved in N-methylpyrrolidone and added to the stirred peptide solution over 2–60 minutes, while the pH was continuously adjusted to 10.5–11 by adding aqueous sodium hydroxide solution. The final reaction mixture was stirred at room temperature and constant pH until the acetylating agent was depleted (0–4 h). The reaction mixture was neutralized by dropwise addition of acetic acid and purified by reverse-phase HPLC.

[0159] Therefore, the extended portion can be linked to the polypeptide as described above. In other embodiments of the invention, the extended portion can be part of the polypeptide backbone, i.e., a fusion polypeptide dependent on peptidoamine bonds. The fusion polypeptide can be obtained synthetically or by recombination of expression methods known to those skilled in the art.

[0160] The hFGF23 antagonistic peptide derivatives disclosed herein can be prepared into pharmaceutical compositions. In some embodiments, such compositions contain at least one pharmaceutically acceptable excipient.

[0161] Excipients can be used for a variety of purposes, such as as carriers, mediators, fillers, binders, lubricants, flow agents, disintegrants, flow control agents, crystallization inhibitors, solubilizers, stabilizers, colorants, flavoring agents, surfactants, emulsifiers, delivery agents, water-soluble aids or combinations thereof, and / or to improve administration and / or absorption of active pharmaceutical ingredients.

[0162] The amounts of various excipients used can vary within the range known in this technique. Applicable techniques and excipients are described, for example, in Remington: the Science and Practice of Pharmacy, 22nd edition, eds. Remington and Allen, Pharmaceutical Press (2013).

[0163] It can be administered to compositions containing the compounds disclosed herein as a preventative therapy and / or, in some embodiments, as an on-demand therapy. Typically, the composition is administered to an individual suffering from a disease (such as the indications described below) in an amount sufficient to cure, alleviate, or partially block the disease and its complications. The amount suitable for accomplishing this is defined as the "therapeutic effective amount." As will be understood by one of ordinary skill in the art, the effective amount for this purpose will depend on the severity of the disease or injury, as well as the individual's weight and general condition.

[0164] The compounds disclosed in this article, such as hFGF23 antagonistic peptides and hFGF23 antagonistic peptide derivatives, may be administered as appropriate pharmaceutical ingredients via non-enteral administration, such as intravenous, intramuscular, or subcutaneous administration.

[0165] In some embodiments, the dose of the compound to be delivered, for example, by subcutaneous administration, may be from about 0.1 mg to 500 mg of the compound per day, preferably from about 0.5 mg to 150 mg per day, depending on the severity of the condition.

[0166] The appropriate dosage of a particular compound can also be adjusted based on its characteristics, including its in vivo half-life or average residence time in the blood and its biological activity.

[0167] In one embodiment, the present invention relates to an injection device having contents containing the composition.

[0168] In one embodiment, the compound can be used in a low-burden dosing device suitable for home self-delivery or caregiver delivery of medication.

[0169] In one embodiment, the hFGF23 antagonistic peptide or peptide derivative is a cross-species reactive peptide or peptide derivative.

[0170] In one embodiment, the IC50 value of the hFGF23 antagonistic peptide or peptide derivative in the inhibitory efficacy analysis disclosed herein is less than 1 nM.

[0171] In one embodiment, the residual activity % of the hFGF23 antagonistic peptide or peptide derivative at 1 nM was less than 50% in the inhibitory efficacy analysis disclosed herein.

[0172] In one embodiment, the compound of the present invention increases the phosphate content in the body compared to compounds in this art (such as bromosumab).

[0173] [Other Embodiments] [ ] [1.] An hFGF23 antagonistic polypeptide capable of binding to an antigenic determinant on hFGF23 according to SEQ ID NO:1, the antigenic determinant comprising amino acid residues selected from the group consisting of: 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; As measured at 3.5 Å. [2.] The hFGF23 antagonistic peptide according to Example 1 is capable of binding hFGF23 according to SEQ ID NO:1. The antagonistic peptide comprises a binding motif (BM) composed of an amino acid residue sequence selected from the following. i) According to SEQ ID NO:2, XaXbXcAWXdEIXeXfXgPXhLXiDXjQWPAFIEXkLH Among them, each independently, Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; and ii) The amino acid residue sequence has at least 93% similarity to the sequence defined in i). [3.] An hFGF23 antagonistic polypeptide capable of binding hFGF23 according to SEQ ID NO:1, the antagonistic polypeptide comprising a binding motif (BM) composed of an amino acid residue sequence selected from the following i) According to SEQ ID NO:2, XaXbXcAWXdEIXeXfXgPXhLXiDXjQWPAFIEXkLH Among them, each independently, Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; and ii) amino acid residues that have at least 93% identity with the sequence defined in i). sequence. [4.] The hFGF23 antagonistic polypeptide according to Example 2 or 3, wherein the (BM) is selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 and SEQ ID NO:52; It may include 1, 2 or 3 substitutions as needed, wherein the substitutions may occur at any of the positions 1, 2, 3, 6, 9, 10, 11, 13, 15, 17 and / or 25 in the selected (BM) sequence. [5.] The hFGF23 antagonistic polypeptide according to Example 4, wherein the substitution or the substitutions thereon are conservative substitutions. [6.] The hFGF23 antagonistic peptide according to any of the foregoing embodiments, its Includes a binding motif (BM), wherein the polypeptide contains a sequence of amino acid residues selected from the following a) According to SEQ ID NO:3, X1X2X3X4X5X6X7X8-(BM)-X36X37PSQX41X42X43LLX46EARX50LX52X53X54QX56X57X58, Wherein (BM) consists of an amino acid residue sequence as defined in Example 2(i), Among them, each independently, X1 can be G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 can be D, E, H, N, R, or T; X5 can be E, I, L, T, or V; X6 can be F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T or V; X8 can be A, L, or E; X36 can be A, D, or E; X37 is either D or E; X41 can be A, R, or W; X42 represents A, D, or T; X43 is E, N, Q, or S; X46 is A, E, or K; X50 is either Q or R; X52 is either N or E; X53 is D, E, K, or T; X54 is either A or I; X56 is either A or C; X57 is either K or P; X58 is C, K, or E; and b) An amino acid residue sequence that has at least 93% similarity to the sequence defined in a). [7.] An hFGF23 antagonistic polypeptide capable of binding to hFGF23 according to SEQ ID NO:1, wherein the polypeptide comprises an amino acid residue sequence selected from: a) According to SEQ ID NO:3 of Among them, each independently, X1 can be G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 can be D, E, H, N, R, or T; X5 can be E, I, L, T, or V; X6 can be F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T or V; X8 can be A, L, or E; Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; X36 can be A, D, or E; X37 is either D or E; X41 can be A, R, or W; X42 represents A, D, or T; X43 is E, N, Q, or S; X46 is A, E, or K; X50 is either Q or R; X52 is either N or E; X53 is D, E, K, or T; X54 is either A or I; X56 is either A or C; X57 is either K or P; X58 is C, K, or E; and b) An amino acid residue sequence that has at least 93% similarity to the sequence defined in a). [8.] An hFGF23 antagonistic polypeptide that can bind to hFGF23 according to SEQ ID NO:1, comprising an amino acid residue sequence According to SEQ ID NO:3 of Among them, each independently, X1 can be G, I, L, N, or V; X2 is D, E, or Q; X3 is D, E, G, N, or Q; X4 can be D, E, H, N, R, or T; X5 can be E, I, L, T, or V; X6 can be F, W, or Y; X7 is A, G, H, I, L, N, P, Q, R, T or V; X8 can be A, L, or E; Xa is either F or Y; Xb is F, I, L, or V; Xc is either A or Q; Xd can be A, F, H, Y, or W; Xe is either F or Y; Xf is N, Q, or T; Xg is either A or L; Xh can be C, H, N, Q, W, or Y; Xi can be D, N, Q, S, or T; Xj is A, D, E, G, H, L, N, Q, S, T or Y; Xk can be A, Q, or S; X36 can be A, D, or E; X37 is either D or E; X41 can be A, R, or W; X42 represents A, D, or T; X43 is E, N, Q, or S; X46 is A, E, or K; X50 is either Q or R; X52 is either N or E; X53 is D, E, K, or T; X54 is either A or I; X56 is either A or C; X57 is either K or P; X58 is C, K, or E; It may include one, two or three substitutions as needed, wherein the substitutions may occur at any of the aforementioned non-fixed positions. [9.] The hFGF23 antagonistic polypeptide according to Example 8, wherein the substitution or the substitutions thereon are conservative substitutions. [10.] The hFGF23 antagonistic polypeptide according to any one of Examples 6 to 9, wherein, independently of each other, X1 can be 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 either A or L; Xa is Y; Xb is V; Xc is Q; Xd is Y; Xe is either F or Y; Xf is N or Q; Xg is L; Xh is N; Xi is N or T; Xj can be A, N, or Q; Xk is S; X36 is either D or E; X37 is either D or E; X41 is W; X42 is A; X43 is N; X46 is either A or E; X50 is either Q or R; X52 is N; X53 is E; X54 is A; X56 is A; X57 is P; X58 is K. [11.] An hFGF23 antagonistic polypeptide capable of binding hFGF23 according to SEQ ID NO:1, the antagonistic polypeptide having at least 93% identity with hFGF23 antagonistic polypeptides selected from the group consisting of: According to Chem. 197 of SEQ ID NO:168; According to SEQ ID NO:176, Chem. 201; According to SEQ ID NO:234, Chem. 204; According to SEQ ID NO:236, Chem. 206; According to SEQ ID NO:237, Chem. 207; and According to Chem. 208 of SEQ ID NO:238. [12.] The hFGF23 antagonistic peptide according to Example 11 has at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to hFGF23 antagonistic peptides selected from the group consisting of: According to Chem. 197 of SEQ ID NO:168; According to SEQ ID NO:176, Chem. 201; According to SEQ ID NO:234, Chem. 204; According to SEQ ID NO:236, Chem. 206; According to SEQ ID NO:237, Chem. 207; and According to Chem. 208 of SEQ ID NO:238. [13.] The hFGF23 antagonistic peptide according to Example 11 or 12 is capable of binding to the antigenic determinant on hFGF23 according to SEQ ID NO: 1, the antigenic determinant comprising amino acid residues selected from the group consisting of: 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; As measured at 3.5 Å. [14.] The hFGF23 antagonistic polypeptide according to any of Examples 2 to 6, wherein the binding motif (BM) forms part of a triple helical bundle protein domain, which is a variant of protein Z derived from domain B of staphylococcal protein A. [15.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the polypeptide contains 58 amino acid residues. [16.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the polypeptide is selected from the group consisting of the following. Chem. 89; Chem. 106; Chem. 107; Chem. 111; Chem. 197; Chem. 201; Chem. 204; Chem. 206; Chem. 207; and Chem. 208. [17.] The hFGF23 antagonistic peptide according to any of the foregoing embodiments, wherein the peptide is capable of binding hFGF23 with a KD value of less than 100 µM (such as less than 10 µM, such as less than 9 µM, such as less than 8 µM, such as less than 7 µM, such as less than 6 µM, such as less than 5 µM, such as less than 4 µM, such as less than 3 µM, such as less than 2 µM, such as less than 1 µM, such as less than 0.9 µM, such as less than 0.8 µM, such as less than 0.7 µM, such as less than 0.6 µM, such as less than 0.5 µM, such as less than 0.4 µM, such as less than 0.3 µM, such as less than 0.2 µM, such as less than 0.1 µM, such as less than 10 nM, such as less than 1 nM, preferably less than 0.1 µM, such as less than 10 nM, such as less than 2 nM, such as less than 1 nM). [18.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the polypeptide contains a triple-helical bundle protein domain, which is a variant of protein Z derived from domain B of staphylococcal protein A. [19.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the polypeptide is a triple-helix bundle protein domain, which is a variant of protein Z derived from domain B of staphylococcal protein A. [20.] The hFGF23 antagonistic peptide according to any of the foregoing embodiments, wherein the peptide is affibody® or derived from affibody®. [21.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the polypeptide comprises an elongated portion. [22.] The hFGF23 antagonistic polypeptide according to any of the foregoing embodiments, wherein the extended portion is covalently linked to the amino acid residue side chain group on the polypeptide. [23.] The hFGF23 antagonistic polypeptide according to any of Examples 1 to 21, wherein the extended portion is covalently fused to the polypeptide via a peptidamide bond. [24.] The hFGF23 antagonistic polypeptide according to Example 21 or 22, wherein the extended portion contains at least two acidic groups, one of which is located at the end. [25.] hFGF23 antagonistic polypeptide according to Examples 21, 22 or 24, wherein the extended portion comprises C16, C17, C18, C19, C20, C21 or C22 fatty acids, such as C16, C17, C18, C19 or C20 fatty acids. [26.] The hFGF23 antagonistic polypeptide according to Examples 21, 22, 24 or 25, wherein the elongated portion comprises an elongator "P" selected from the group consisting of: (Chem. 1a); (Chem. 1b); (Chem. 1c); (Chem. 1d); (Chem. 1e); QRLMEDICLPRWGCLWEDDF-* (Chem. 1f); and *-QRLMEDICLPRWGCLWEDDF (Chem. 1f); The asterisk (*) indicates a linker to the linker (LP) or the hFGF23 antagonistic polypeptide. [27.] The hFGF23 antagonistic polypeptide according to any one of Examples 1 to 22 or 24 to 26, wherein the polypeptide is a polypeptide derivative comprising an elongated portion, wherein the elongated portion comprises a linker "LP" selected from the group consisting of: *―NH-(CH2)2-(O-(CH2))kO-(CH2)n-CO-* (chem. 2a), where 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-* (Chem. 2e); *-NH-CH2-(C6H10)-CO-* (Chem. 2f); *-NH-(CH2)5-CO-* (Chem. 2g); (Chem. 2h); (Chem. 2i); (Chem. 2j); (Chem. 2k). [28.] The hFGF23 antagonistic polypeptide according to any of Examples 21 to 27, wherein the polypeptide is a polypeptide derivative comprising an elongated portion, wherein the elongated portion is linked to an N-terminal amino acid residue of the polypeptide. [29.] The hFGF23 antagonistic polypeptide according to any of Examples 21 to 27, wherein the polypeptide is a polypeptide derivative comprising an elongated portion, wherein the elongated portion is linked to a C-terminal amino acid residue of the polypeptide. [30.] The hFGF23 antagonistic polypeptide according to any of Examples 21 to 22 or 24 to 29, wherein the polypeptide is a polypeptide derivative comprising an elongated portion, wherein the elongated portion is covalently linked to a side chain of a cysteine ​​residue or lysine residue in the polypeptide. [31.] A compound selected from the group consisting of: ; ; ; . [32.] An hFGF23 antagonistic peptide derivative capable of binding hFGF23 represented by the following structure: [33.] An hFGF23 antagonistic polypeptide derivative capable of binding to hFGF23 represented by the following structure: [ ] [34.] An hFGF23 antagonistic peptide derivative capable of binding to hFGF23 represented by the following structure: [35.] An hFGF23 antagonistic peptide derivative capable of binding to hFGF23 represented by the following structure: [36.] The compound according to Example 31, wherein the compound is an hFGF23 antagonistic polypeptide derivative capable of binding hFGF23. [37.] The hFGF23 antagonistic polypeptide or polypeptide derivative according to any of the foregoing embodiments, wherein the polypeptide or polypeptide derivative is a cross-species reactive polypeptide or polypeptide derivative. [38.] The hFGF23 antagonistic peptide or peptide derivative according to the foregoing embodiments, wherein the peptide or peptide derivative can bind to one or more of the cynomolgus monkey FGF23, canine FGF23 and porcine FGF23. [ ] [39.] A pharmaceutical composition comprising an hFGF23 antagonistic polypeptide, polypeptide derivative or compound according to any of the foregoing embodiments and one or more pharmaceutically acceptable excipients. [ ] [40.] The hFGF23 antagonistic polypeptide derivative, polypeptide derivative, compound or composition according to any of the foregoing embodiments is used in medicine. [ ] [41.] The hFGF23 antagonistic polypeptide derivative, polypeptide derivative, compound or composition according to any of the foregoing embodiments is used to treat a disease characterized by elevated hFGF23 concentration (compared to a healthy individual). [ ] [42.] The hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39 is used to treat hypophosphatemia. [43.] The hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39 is used to treat tumor-induced osteomalacia, fibrous dysplasia, Marko's-Abbott syndrome, autosomal dominant hypophosphatemic rickets (ADHR) or X-linked hypophosphatemic rickets (XLH). [44.] The hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39 is used to treat X-linked hypophosphatemia rickets (XLH). [45.] A method for inhibiting hFGF23-induced signal transduction in a patient in need, comprising administering to the individual an hFGF23 antagonistic polypeptide, polypeptide derivative, compound, or composition according to any of the embodiments of Examples 1 to 39. [46.] A method for treating hypophosphatemia characterized by an elevated hFGF23 concentration compared to that observed in healthy individuals, the method comprising administering to the individual an hFGF23 antagonistic polypeptide, polypeptide derivative, compound, or composition according to any of Examples 1 to 39. [ ] [47.] A method for increasing serum phosphate concentration in an individual suffering from hypophosphatemia, characterized by an elevated FGF23 concentration compared to that observed in a healthy individual, the method comprising administering to the individual an hFGF23 antagonistic polypeptide, polypeptide derivative, compound, or composition according to any of Examples 1 to 39. [ ] [48.] A method of treating an individual suffering from a hypophosphatemic disease, such as tumor-induced osteomalacia, fibrous dysplasia, Marko's-Abbott syndrome, autosomal dominant hypophosphatemic rickets (ADHR), or X-linked hypophosphatemic rickets (XLH), the method comprising administering to the individual an hFGF23 antagonistic polypeptide, polypeptide derivative, compound, or composition according to any of the embodiments of Examples 1 to 39. [ ] [49.] A method of treating an individual suffering from X-linked hypophosphatemia rickets (XLH), comprising administering to the individual an hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of the embodiments of Examples 1 to 39. [ ] [50.] The use of the hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39 is for the manufacture of a medicament for the treatment of an individual in need. [51.] Use of the hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39 for the manufacture of an agent for the treatment of hypophosphatemia, such as X-linked hypophosphatemia rickets (XLH). [52.] A kit comprising an hFGF23 antagonistic polypeptide, polypeptide derivative, compound or composition according to any of Examples 1 to 39, and instructions for use. [53.] A polynucleotide encoding an hFGF23 antagonistic polypeptide, polypeptide derivative or compound according to any of Examples 1 to 38. [54.] A representation vector encoding a polynucleotide according to the foregoing embodiments. [55.] A host cell that produces an hFGF23 antagonistic polypeptide, polypeptide derivative or compound according to any of Examples 1 to 38. [56.] A method for producing an hFGF23 antagonistic polypeptide or polypeptide derivative or compound according to any one of Examples 1 to 38, comprising: a) Culture the host cells according to the foregoing examples under conditions that allow for the expression of the polypeptide encoded by the expression vector of Example 54. b) The polypeptide was isolated. c) If necessary, the extended portion can be linked to the isolated polypeptide. [57.] An hFGF23 antagonistic polypeptide, polypeptide derivative or compound that can be obtained by the method of Example 56. [Example] [ ]

[0174] The following experimental sections begin with a list of abbreviations and are followed by sections on general methods for preparing compounds and methods for measuring the exposure profile-related properties and other properties of the compounds. For the purpose of illustrating the invention, several specific examples are included in each section. All example compounds were prepared according to the general methods described herein.

[0175] [abbreviation] [ ] Ado: 8-amino-3,6-dioxanoic acid Boc: Tertiary butoxycarbonyl Trimethylpyridine: 2,4,6-Trimethylpyridine DCM: Dichloromethane DIC: N,N'-Diisopropylcarbodiimide DMF: N,N-Dimethylformamide DTT: 1,4-Dithiothreitol FBS: Fetal bovine serum Fmoc: 9-Genomethoxycarbonyl HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid HFIP: 1,1,1,3,3,3-Hexafluoro-2-propanol or hexafluoroisopropanol HPLC: High Performance Liquid Chromatography iv: Intravenous LC: Liquid Chromatography LCMS: Liquid Chromatography-Mass Spectrometry MeCN: Acetonitrile MQ: Milli-Q MS: Mass Spectrometry MSA: Multiple sequence alignment Mtt: 4-Methyltriphenylmethyl NMP: N-methyl-2-pyrrolidone OtBu: Tertiary butoxy Oxyma Pure®: Ethyl Cyano(hydroxyamino)acetate OVA: Ovalbumin Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PBS: Phosphate-buffered saline RP: reverse phase RP-HPLC: Reversed-phase high-performance liquid chromatography RT: Room temperature RU: Reaction Unit sc: subcutaneous SPPS: Solid-phase peptide synthesis SPR: Surface Plasmon Resonance tBu: Tertiary Butyl TFA: Trifluoroacetic acid TIPS: Triisopropylsilane Trt: Triphenylmethyl (trityl) UPLC: Ultra-high performance liquid chromatography UV: Ultraviolet Methods for peptide synthesis, purification, and analysis: [ ]

[0176] Unless otherwise specified, the Fmoc-protected amino acid derivatives used are: 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, and Fmoc-His(Trt)-OH. Fmoc-Glu-OtBu and the resin system used for solid-phase peptide synthesis were obtained from Gyros Protein Technologies AB. Fmoc-Glu-OtBu and the resin system used for solid-phase peptide synthesis were obtained from Novabiochem (Merck Millipore). Fmoc-Ado-OH was obtained from Bachem. Triisopropylsilane was obtained from Sigma. Fmoc-8-amino-3,6-dioxanoic acid (Fmoc-Ado-OH) was obtained from Flamma Group, Italy. 16-(tributoxy)-16-sideoxyhexadecanoic acid was purchased from ABCR, 18-(tributoxy)-18-sideoxyoctadecanoic acid was purchased from Solvias, and 20-(tributoxy)-20-sideoxyarachidic acid was prepared as known in this art, for example, as described in WO 2010102886 A1. Oxyma was purchased 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.

[0177] Automated Fmoc SPPS was performed on a SymphonyTMX (Gyros Protein Technologies AB). Fmoc protecting groups were removed by treatment with 20 vol% piperidine for 2 × 10 min in DMF containing 0.1 M Oxyma. Peptide coupling was performed using DIC / trimethylpyridine. An amino acid / Oxyma solution (0.3 M / 0.3 M in DMF, 5 molar excess) was added to the resin, followed by an equimolar equivalent of DIC (1.5 M in DMF) and an equimolar equivalent of trimethylpyridine (1.5 M in DMF). Unless otherwise specified, the coupling procedure was allowed to proceed for 60 min.

[0178] Following polypeptide backbone synthesis, the elongation moiety is selectively attached to the desired site on the resin-bound polypeptide via orthogonally protected amino acids, such as Fmoc-Lys(Mtt)-OH. The Mtt groups are selectively removed by treatment with HFIP / DCM / TIPS (75:22.5:2.5) (3 × 15 min). Subsequently, the resin-bound polypeptide is treated with 20 vol% piperidine / 0.1 M Oxyma / DMF (1 × 5 min). After deprotection, the resin is then washed with DCM and DMF, followed by the introduction of the elongation moiety using the standard SPPS procedure described above.

[0179] Following synthesis, the resin-bound peptides underwent overall deprotection of the resin, followed by washing with DCM and then treatment with TFA / TIPS / DTT / water (95:2.5:2.5:2.5, v / v / v / v) for 2 to 3 hours. After treatment, precipitation was carried out in ice-cold diethyl ether and collected by centrifugation. The aggregated particles were resuspended in diethyl ether and then centrifuged again. The washed aggregated particles were dissolved in a suitable mixture of water, MeCN, and acetic acid, and then purified by reverse-phase preparative HPLC.

[0180] The crude peptide containing cysteine ​​at positions Xh and X56 was folded and then purified as follows: the crude peptide was dissolved in DMSO (10 ml), followed by the addition of 30 mM HEPES buffer (40 ml, pH 7.8, aqueous solution) and stirred overnight. The pH was lowered to 3 using trifluoroacetic acid, and then purified by reverse-phase preparative HPLC.

[0181] The purification of peptides and their final analogues was performed using a Waters™ Deltaprep 4000 preparative chromatography system with a reverse-phase HPLC column. A linear gradient was used for dissociation using a mobile phase consisting of 0.1 vol% TFA / Milli-Q® water (solvent A) and 0.1 vol% TFA / HPLC-grade acetonitrile (solvent B).

[0182] Dissociation was performed using a mobile phase consisting of the following linear gradient: 0.1 vol% TFA, 10 vol% acetonitrile / Milli-Q water (solvent A), and 0.1 vol% TFA / HPLC-grade acetonitrile (solvent B). The dissociated fractions were evaluated by LC-MS, and the purity of the collected fractions was assessed by analytical HPLC or UPLC. The fractions containing the purified target peptides were collected and lyophilized. Analysis program surface [3]. LC-MS conditions Method M64: [system] LC System: Waters™ Acquity UPLC Class H Detector: Waters Xevo G2-XS Qtof Detection λ = 214 nm [Detector Configuration] Ionization method: Electrospray Scan range: 50-4000 m / z Operating mode: MS parsing mode (positive) Voltage: Capillary 3.00 kV, Sampling cone 40 V, Ion source 80 V Temperature: Ion source 150℃, desolvation 350℃ Scan time: 0.500 s Inter-scan delay: 0.014 s [Lockspray™] [calibration] A solution of leucine enkephalin in 50 vol% acetonitrile / Milli-Q water (0.2 mg / L) was used as the lock mass for data calibration, and samples were taken every 20 seconds. Lock mass: 556.2771 m / z [Soluble agent] Solvent A: 100 vol% Milli-Q water Solvent B: 100 vol% acetonitrile Solvent C: 97.9 vol% Milli-Q water, 2 vol% formic acid, 0.1 vol% TFA [LC] [condition] [C18] [Tube Column] [(M64)] Column: WatersTM Acquity BEH, C18, 1.7 µm, 2.1 mm × 50 mm Flow rate: 0.4 mL / min Gradient: 5% to 95% B over 4 minutes, maintaining a composition of 5% C throughout. Column oven temperature: 60℃ Method: M111

[0183] LCMS111 (M111) was performed using the following configuration: a Waters Acquity UPLC system with an adjustable UV detector (TUV) and a Waters Acquity QDa mass spectrometer (QDa). Dissociation reagents: A: water containing 0.05% trifluoroacetic acid; B: acetonitrile containing 0.05% trifluoroacetic acid. Analysis was performed at 40°C by injecting an appropriate volume (preferably 2–10 µl) of sample into the column and dissociating it using a gradient of A and B. UPLC conditions, detector settings, and mass spectrometer settings: Column: Waters Acquity UPLC BEH, C18, 1.7 µm, 2.1 mm × 50 mm. Gradient: linear 10%–90% acetonitrile over 1.6 min at 0.9 ml / min. UV detection: 214 nm, using a TUV detector. MS detection: Atmospheric pressure ionized electrospray (API-ES), scan range 100-1250 Da, using QDa detector, operating in positive mode. [Compound] [ ]

[0184] Table 4 below lists the Chem no., SEQ ID NO, complete main chain sequence, R1 residues connected to the linker, linkers, and elongators. Selected non-limiting examples are shown below: Chem. 4, SEQ ID NO: 53, VDNREFNAYLQAWYEIFNLPNLNDNQWPA FIESLHEDPSQWANLLAEARRLNDAQAKE, R1: 57K, Linker: L1, Elongator: Chem 1b. The structure is shown below, wherein the linker is attached to the ε-amino group of the lysine at position 57. Chem. 106, SEQ ID NO:138,LDEEEFQAYLQAWYEIFNLPNLNDNQWPAFI ESLHEDPSQWANLLAEARRLNDAQAPK,58K, linker: L1, elongator: Chem 1c. The structure is shown below, wherein the linker is attached to the ε-amino group of the lysine at position 58. Chem. 246, SEQ ID NO:274, VDQEEFQAYLQAWYEIFNLPCLNDNQWPAFI ESLHEDPSQWDNLLEEARRLNDAQCPK,58K, Linker: L1, Elongator: Chem 1b. The structure is shown below, wherein the linker is attached to the ε-amino group of the lysine at position 58. Furthermore, when two cysteines (C) are present at positions Xh and X56 in the sequence, those skilled in the art will understand that a disulfide bridge bond is formed between the two cysteines, as shown in the following structure.

[0185] Examples of the compounds of the present invention are disclosed in Table 4 below. This table provides the amino acid residue sequences and information about the elongated portions and their linkage sites to the hFGF23 antagonistic peptide. The linker and elongator columns provide the names of the linkers and elongators used, and see also Tables 1 and 2 above.

[0186] Table 5 below provides technical information about the compounds, such as molecular weight and m / z value. surface [4]. hFGF23 antagonistic peptide derivatives Chem. SEQ ID NO sequence R1 Connector ("LP") Elongator ("P") Chem. 3 10 VDNREFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPE 4 53 VDNREFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAKE 57K L1 1b 5 54 VDNTLYPL [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNKAQAPE 53K L1 1b 6 55 VDNREFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNKAQAPE 53K L1 1b 7 56 VDNTLYPL [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLKEARRLNDAQAPE 46K L1 1b 8 57 VDNREFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLKEARRLNDAQAPE 46K L1 1b 9 58 LDNDEFQL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQAPK 58K L1 1b 10 59 LDNDEFTL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQAPK 58K L1 1b 11 60 LDNTEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQAPK 58K L1 1b 12 61 LDNDTFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQAPK 58K L1 1b 13 62 LDNDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]ADPSQWANLLEEARRLNDAQAPK 58K L1 1b 14 63 VDNDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]ADPSQWANLLAEARRLNDAQAPK 58K L1 1b 15 64 LDNDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]ADPSQWANLLAEARRLNDAQAPK 58K L1 1b 16 65 IDNDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 17 66 NDNDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 18 67 VDQDEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 19 68 VDNTEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 20 69 VDNNEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 21 70 VDNHEFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 22 71 VDNREFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 23 72 VDNDTFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 24 73 VDNDVFIL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 25 74 VDNDEFQL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 26 75 VDNDEFNL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 27 76 VDNDEFLL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 28 77 VDNDEFAL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 29 78 VDNDEFRL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 30 79 VDNDEFGL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 31 80 VDNDEFPL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 32 81 VDNDEFHL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 33 82 VDNDEFVL [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 34 83 LDNDEFIL [YLQAWYEIFNLPWLNDNQWPAFIESLH]ADPSQWANLLEEARRLNDAQAPK 58K L1 1b 35 84 LDNDEFIL [YLQAWYEIFNLPYLNDNQWPAFIESLH]ADPSQWANLLEEARRLNDAQAPK 58K L1 1b 36 85 LDNDEFIE [YLQAWYEIFNLPNLNDNQWPAFIESLH]DDPSQRANLLAEARRLNDAQAPK 58K L1 1b 37 86 LDNDEFIL [YLQAWYEIFQLPNLNDNQWPAFIESLH]DDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 38 87 LDNDEFIL [YLQAWYEIFNLPWLNDNQWPAFIESLH]DDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 39 88 LDNDEFIL [YLQAWYEIFNLPWLNDNQWPAFIESLH]ADPSQWAQLLEEARRLNDAQAPK 58K L1 1b 40 89 VEGDEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 41 90 VEGEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 42 91 VDNTIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 43 92 VDNTVFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 44 93 VDNDLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 45 94 VDNDIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 46 95 VDNDVFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 47 96 VENTLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 48 97 VENTIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 49 98 VENTVFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 50 99 VDNTLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 51 100 VDNTLFNA [YLQAWWEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 52 101 VDNTLFNA [YLQAWFEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 53 102 VDNTLFNA [YLQAWHEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 54 103 VDNTEFIL [YLQAWWEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 55 104 VDNTEFIL [YLQAWFEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 56 105 VDNTEFIL [YLQAWHEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 57 106 VDNTLFIL [YLQAWWEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQAPK 58K L1 1b 58 107 VEGDEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 59 108 VEGEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 60 109 VDNTLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 61 110 VDNTIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 62 111 VDNTVFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 63 112 VDNDLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 64 113 VDNDIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 65 114 VDNDVFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 66 115 VENTLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 67 116 VENTIFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 68 117 VDNTLFNA [YLQAWWEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 69 118 VDNTLFNA [YLQAWHEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 70 119 VDNTEFIL [YLQAWWEIFNLPNLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQAPK 58K L1 1b 71 120 LDNEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 72 121 LDNREFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 73 122 VDQEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 74 123 VDEEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 75 124 VDNEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 76 125 LDNELFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 77 126 LDQTEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 78 127 LDETEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 79 128 LDDEEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 80 129 LDNEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 81 130 VDETLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 82 131 VDQTEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 83 132 VDQEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 84 133 VDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 85 134 VDNTLFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 86 135 LDNELFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 87 136 LDQTLFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 88 137 LDQEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 89 138 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 90 139 LDQELFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 91 140 VDQTLFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 92 141 LDQELFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 93 142 LDEELFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 94 143 LDQTLFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 95 144 LDDTLFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 96 145 VDNDEFNA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 97 146 VDNREFIA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 98 147 VDNREFLA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 99 148 VDNREFNL [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 100 149 LDNEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWDNLLEEARRLNDAQAPK 58K L1 1b 101 150 VDQEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWDNLLAEARRLNDAQAPK 58K L1 1b 102 151 VDNREFVA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 103 152 VDNREFNA [YLAAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 104 153 VDNTEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 105 154 GEGTEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 106 138 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1c 107 155 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 108 156 LENEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 109 157 LENEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 110 158 LENEEFQL [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 111 159 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARQLNDAQAPK 58K L1 1b 112 160 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWTNLLEEARQLNDAQAPK 58K L1 1b 113 161 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWTSLLEEARQLNDAQAPK 58K L1 1b 114 162 LDEEEFQA [YLQAWYEIFNLPHLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 115 163 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIEALH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 116 164 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQAANLLEEARRLNDAQAPK 58K L1 1b 117 165 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIEQLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 118 166 LDEEEFQA [YLQAWYEIFQLPNLNDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 119 167 LDEEEFQA [YLQAWYEIFNLPNLQDNQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 120 168 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1b 121 169 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWAQLLAEARRLNDAQAPK 58K L1 1b 122 170 LDEEEFQA [YLQAWYEIFNLPNLNDDQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 123 171 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 124 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 125 173 LDEEEFQA [YLQAWYEIFNLPNLNDSQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 126 174 LDEEEFQA [YLQAWYEIFNLPNLNDTQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 127 175 LDEEEFQA [YLQAWYEIFNLPNLNDHQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 128 176 LDEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 129 177 LDEEEFQA [YLQAWYEIFNLPNLNDLQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 130 178 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 131 179 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 132 180 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 133 181 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 134 182 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 135 183 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 136 184 LDEEEFQA [YLQAWYEIFNLPYLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 137 185 LDEEEFQA [YLQAWYEIFNLPNLTDNQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 138 186 LDEEEFQA [YLQAWYEIFNLPNLTDNQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 139 187 LDEEEFQA [YLQAWYEIFNLPNLTDEQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 140 188 LDEEEFQA [YLQAWYEIFNLPNLTDEQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 141 189 LDEEEFQA [YLQAWYEIFNLPNLTDEQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 142 190 LDEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 143 191 LDEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 144 192 LDEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 145 193 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1b 146 194 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EEPSQWAELLEEARRLNDAQAPK 58K L1 1b 147 195 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EEPSQWAQLLEEARRLNDAQAPK 58K L1 1b 148 196 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1b 149 197 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EEPSQWAELLEEARRLNDAQAPK 58K L1 1b 150 198 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EEPSQWAQLLEEARRLNDAQAPK 58K L1 1b 151 199 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1b 152 200 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EEPSQWAELLEEARRLNDAQAPK 58K L1 1b 153 201 LDEEEFQA [YLQAWYEIFNLPNLNDDQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 154 202 LDEEEFQA [YLQAWYEIFNLPNLSDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 155 203 LDEEEFQA [YLQAWYEIFNLPNLNDSQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 156 204 LDEEEFQA [YLQAWYEIFNLPNLNDTQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 157 205 LDEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 158 206 LDEEEFQA [YLQAWYEIFNLPNLNDLQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 159 207 LDEEEFQA [YLQAWYEIFNLPNLNDGQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1b 160 208 LDEEEFQA [YLQAWYEIFNLPNLNDDQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 161 209 LDEEEFQA [YLQAWYEIFQLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 162 210 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWASLLEEARRLNDAQAPK 58K L1 1b 163 211 LDEEEFQA [YLQAWYEIFNLPNLNDSQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 164 212 LDEEEFQA [YLQAWYEIFNLPNLNDTQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 165 213 LDEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 166 214 LDEEEFQA [YLQAWYEIFNLPNLNDLQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 167 215 LDEEEFQA [YLQAWYEIFNLPNLNDGQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1b 168 216 LDEEEFAA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 169 217 LDEEEFQA [YVQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 170 218 LDEEEFQA [YIQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 171 219 LDEEEFQA [YFQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 172 220 LDEEEFQA [YLQAWFEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 173 221 LDEEEFQA [YLQAWWEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 174 222 LDEEEFQA [YLQAWHEIFNLLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 175 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 176 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1a 177 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L3 1b 178 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L3 1c 179 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L3 1a 180 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L2 1b 181 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L2 1c 182 172 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L2 1a 183 209 LDEEEFQA [YLQAWYEIFQLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 184 223 LDEEEFQA [YLQAWYEIFNLPQLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 185 224 LDEEEFQA [YLQAWYEIFNLPNLQDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 186 179 LDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1c 187 225 VDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 188 226 VDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 189 227 LEEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1b 190 228 LEEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1b 191 229 LEEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1b 192 225 VDEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 193 226 VDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 194 227 LEEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 195 228 LEEEEFQA [YLQAWYEIFNLPNLNDNQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1c 196 229 LEEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1c 197 168 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1c 198 230 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1c 199 171 LDEEEFQA [YLQAWYEIFNLPNLNDEQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 200 231 LDEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1c 201 176 LDEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 202 232 LDEEEFQA [YLQAWYEIFNLPNLQDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 203 233 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWAQLLAEARRLNDAQAPK 58K L1 1c 204 234 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLAEARQLNDAQAPK 58K L1 1c 205 235 LDEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 206 236 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLAEARRLNEAQAPK 58K L1 1c 207 237 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNEAQAPK 58K L1 1c 208 238 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLAEARQLNEAQAPK 58K L1 1c 209 190 LDEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 210 192 LDEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1c 211 239 LDEEEFQA [YLQAWYEIFNLPNLDDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 212 240 LDEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARQLNDAQAPK 58K L1 1c 213 241 LDEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 214 242 LDEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1c 215 243 LDEEEFQA [YLQAWYEIFNLPNLDDQQWPAFIESLH]EDPSQWANLLAEARRLNDAQAPK 58K L1 1c 216 244 LDEEEFQA [YLQAWYEIFNLPNLDDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 217 245 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 218 246 LQEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 219 247 LEEEEFQA [YLQAWYEIFNLPHLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 220 248 LEEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 221 249 LEEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 222 250 LEEEEFQA [YLQAWYEIFNLPNLNDYQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 223 251 LEEEEFQA [YLQAWYEIFNLPNLNDLQWPAFIESLH]EDPSQWANLLEEARRLNDAQAPK 58K L1 1c 224 252 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWASLLEEARRLNDAQAPK 58K L1 1c 225 253 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1c 226 254 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNEAQAPK 58K L1 1c 227 255 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNTAQAPK 58K L1 1c 228 256 LEEEEFQA [YLQAWYEIFNLPNLNDAQWPAFIESLH]EDPSQWANLLEEARRLNDIQAPK 58K L1 1c 229 257 LEEEEFQA [YLQAWYEIFNLPHLTDQQWPAFIESLH]EEPSQWAELLEEARRLNEAQAPK 58K L1 1c 230 258 LEEEEFQA [YLQAWYEIFNLPHLTDQQWPAFIESLH]EEPSQWAQLLEEARRLNEAQAPK 58K L1 1c 231 259 LEEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1c 232 260 LEEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EDPSQWAELLEEARRLNDAQAPK 58K L1 1c 233 261 LEEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EEPSQWAELLEEARRLNDAQAPK 58K L1 1c 234 262 LEEEEFQA [YLQAWYEIFNLPNLTDYQWPAFIESLH]EEPSQWANLLEEARRLNDAQAPK 58K L1 1c 235 263 LEEEEFQA [YLQAWYEIFNLPNLTDQQWPAFIESLH]EDPSQWAQLLEEARRLNEAQAPK 58K L1 1c 236 264 LEEEEFQA [YLQAWYEIFNLPNLNDQQWPAFIESLH]EDPSQWAQLLEEARRLNEAQAPK 58K L1 1c 237 265 LEEEEFQA [YLQAWYEIFNLPNLTDAQWPAFIESLH]EDPSQWAQLLEEARRLNEAQAPK 58K L1 1c 238 266 LEEEEFQA [YLQAWYEIFNLPNLTDYQWPAFIESLH]EDPSQWANLLEEARRLNEAQAPK 58K L1 1c 239 267 LEEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EEPSQWAQLLEEARRLNEAQAPK 58K L1 1c 240 268 LEEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EEPSQWAQLLEEARRLNDAQAPK 58K L1 1c 241 269 LEEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EEPSQWANLLEEARRLNEAQAPK 58K L1 1c 242 270 LEEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EDPSQWAQLLEEARRLNEAQAPK 58K L1 1c 243 271 LEEEEFQA [YLQAWYEIFNLPHLNDQQWPAFIESLH]EDPSQWAQLLEEARRLNDAQAPK 58K L1 1c 244 272 LEEEEFQA [YLQAWYEIFTLPHLTDQQWPAFIESLH]EEPSQWAELLEEARRLNEAQAPK 58K L1 1c 245 273 LEEEEFQA [YLQAWYEIFTLPHLTDQQWPAFIESLH]EEPSQWAQLLEEARRLNEAQAPK 58K L1 1c 246 274 VDQEEFQA [YLQAWYEIFNLPCLNDNQWPAFIESLH]EDPSQWDNLLEEARRLNDAQCPK 58K L1 1b 247 275 LDNEEFQA [YLQAWYEIFNLPCLNDNQWPAFIESLH]EDPSQWDNLLEEARRLNDAQCPK 58K L1 1b 248 276 LDNDEFIL [YLQAWYEIFNLPCLNDNQWPAFIESLH]DDPSQWANLLAEARRLNDAQCPK 58K L1 1b 249 277 LDNDEFIL [YLQAWYEIFNLPCLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQCPK 58K L1 1b 250 278 LDNDEFQL [YLQAWYEIFNLPCLNDNQWPAFIESLH]DDPSQWANLLEEARRLNDAQCPK 58K L1 1b 251 279 LDEEEFQA [YLQAWYEIFNLPCLNDQQWPAFIESLH]EDPSQWANLLEEARRLNDAQCPK 58K L1 1c 252 280 LDEEEFQA [YLQAWYEIFNLPCLTDSQWPAFIESLH]EDPSQWANLLEEARRLNDAQCPK 58K L1 1c [Combined Model] [(BM)] with [Bold] Highlighted in the sequence of Table 4. surface [5]. Technical and analytical information on hFGF23 antagonistic peptide derivatives Chem. Japanese style m / z experimental value LCMS method 3 C309H448N84O95 m / 7: 980.8 (average) M111 4 C345H514N88O107 m / 7: 1087.5 (average) M111 5 C349H522N84O104 m / 7: 1080.6 (average) M111 6 C346H516N88O105 m / 8: 949.4 (average) M111 7 C350H522N84O106 m / 7: 1086.9 (average) M111 8 C347H516N88O107 m / 7: 1091.1 (average) M111 9 C349H517N85O109 m / 7: 1093.6 (average) M111 10 C348H516N84O109 m / 7: 1089.7 (average) M111 11 C350H522N84O107 m / 7: 1089.4 (average) M111 12 C349H520N84O107 m / 7: 1087.3 (average) M111 13 C349H520N84O106 m / 7: 1085.1 (average) M111 14 C346H516N84O104 m / 7: 1074.8 (average) M111 15 C347H518N84O104 m / 7: 1076.7 (average) M111 16 C348H518N84O106 m / 7: 1083.0 (average) M111 17 C346H513N85O107 m / 7: 1083.0 (average) M111 18 C348H518N84O106 m / 7: 1082.9 (average) M111 19 C347H518N84O105 m / 7: 1078.9 (average) M111 20 C347H517N85O105 m / 7: 1080.7 (average) M111 twenty one C349H518N86O104 m / 7: 1084.0 (average) M111 twenty two C349H523N87O104 m / 7: 1086.7 (average) M111 twenty three C346H516N84O105 m / 7: 1076.8 (average) M111 twenty four C347H518N84O104 m / 7: 1076.6 (average) M111 25 C346H513N85O107 m / 7: 1082.9 (average) M111 26 C345H511N85O107 m / 7: 1081.0 (average) M111 27 C347H516N84O106 m / 7: 1080.9 (average) M111 28 C344H510N84O106 m / 7: 1074.8 (average) M111 29 C347H517N87O106 m / 7: 1087.0 (average) M111 30 C343H508N84O106 m / 7: 1072.8 (average) M111 31 C346H512N84O106 m / 7: 1078.6 (average) M111 32 C347H512N86O106 m / 7: 1084.3 (average) M111 33 C346H514N84O106 m / 7: 1078.9 (average) M111 34 C356H524N84O105 m / 7: 1095.2 (average) M111 35 C354H523N83O106 m / 7: 1091.9 (average) M111 36 C342H516N86O108 m / 7: 1080.8 (average) M111 37 C352H524N84O108 m / 7: 1095.1 (average) M111 38 C358H526N84O107 m / 7: 1103.4 (average) M111 39 C357H526N84O105 m / 7: 1097.1 (average) M111 40 C342H506N84O106 m / 7: 1070.9 (average) M111 41 C343H508N84O106 m / 7: 1072.9 (average) M111 42 C344H513N85O104 m / 7: 1072.7 (average) M111 43 C343H511N85O104 m / 7: 1070.7 (average) M111 44 C344H511N85O105 m / 7: 1074.7 (average) M111 45 C344H511N85O105 m / 7: 1074.7 (average) M111 46 C343H509N85O105 m / 7: 1072.7 (average) M111 47 C345H515N85O104 m / 7: 1074.7 (average) M111 48 C345H515N85O104 m / 7: 1074.7 (average) M111 49 C344H513N85O104 m / 7: 1072.7 (average) M111 50 C344H513N85O104 m / 7: 1072.7 (average) M111 51 C346H514N86O103 m / 7: 1076.0 (average) M111 52 C344H513N85O103 m / 7: 1070.5 (average) M111 53 C341H511N87O103 m / 7: 1069.0 (average) M111 54 C349H519N85O104 m / 7: 1082.2 (average) M111 55 C347H518N84O104 m / 7: 1076.6 (average) M111 56 C344H516N86O104 m / 7: 1075.1 (average) M111 57 C350H523N85O102 m / 7: 1079.9 (average) M111 58 C344H508N84O108 m / 7: 1079.1 (average) M111 59 C345H510N84O108 m / 7: 1081.2 (average) M111 60 C346H515N85O106 m / 7: 1081.0 (average) M111 61 C346H515N85O106 m / 7: 1081.0 (average) M111 62 C345H513N85O106 m / 7: 1079.0 (average) M111 63 C346H513N85O107 m / 7: 1083.0 (average) M111 64 C346H513N85O107 m / 7: 1083.0 (average) M111 65 C345H511N85O107 m / 7: 1081.0 (average) M111 66 C347H517N85O106 m / 7: 1083.0 (average) M111 67 C347H517N85O106 m / 7: 1083.1 (average) M111 68 C348H516N86O105 m / 7: 1084.3 (average) M111 69 C343H513N87O105 m / 7: 1077.3 (average) M111 70 C351H521N85O106 m / 7: 1090.4 (average) M111 71 C345H511N85O107 m / 7: 1081.0 (average) M111 72 C347H518N88O105 m / 7: 1086.8 (average) M111 73 C345H511N85O107 m / 7: 1081.0 (average) M111 74 C345H510N84O108 m / 7: 1081.1 (average) M111 75 C345H511N85O107 m / 7: 1081.0 (average) M111 76 C346H515N85O105 m / 7: 1078.7 (average) M111 77 C345H513N85O106 m / 7: 1079.0 (average) M111 78 C345H512N84O107 m / 7: 1079.2 (average) M111 79 C345H510N84O108 m / 7: 1081.1 (average) M111 80 C346H513N85O107 m / 7: 1083.0 (average) M111 81 C345H514N84O105 m / 7: 1074.9 (average) M111 82 C345H513N85O106 m / 7: 1079.0 (average) M111 83 C346H513N85O107 m / 7: 1083.0 (average) M111 84 C346H512N84O108 m / 7: 1083.1 (average) M111 85 C345H515N85O104 m / 7: 1074.7 (average) M111 86 C347H517N85O105 m / 7: 1080.7 (average) M111 87 C346H517N85O104 m / 7: 1076.7 (average) M111 88 C347H515N85O107 m / 7: 1085.0 (average) M111 89 C347H514N84O108 m / 7: 1085.1 (average) M111 90 C347H517N85O105 m / 7: 1080.7 (average) M111 91 C346H517N85O104 m / 7: 1076.8 (average) M111 92 C348H519N85O105 m / 7: 1082.7 (average) M111 93 C348H518N84O106 m / 7: 1082.9 (average) M111 94 C347H519N85O104 m / 7: 1078.7 (average) M111 95 C346H516N84O105 m / 7: 1076.9 (average) M111 96 C343H507N85O107 m / 7: 1077.0 (average) M111 97 C347H519N87O104 m / 7: 1082.8 (average) M111 98 C347H519N87O104 m / 7: 1082.7 (average) M111 99 C348H520N88O105 m / 7: 1088.9 (average) M111 100 C349H515N85O111 m / 7: 1097.5 (average) M111 101 C347H513N85O109 m / 7: 1089.3 (average) M111 102 C346H517N87O104 m / 7: 1080.7 (average) M111 103 C343H511N87O104 m / 7: 1074.7 (average) M111 104 C344H511N85O106 m / 7: 1077.6 (average) M111 105 C340H504N84O105 m / 6: 1242.7 (average) M111 106 C349H518N84O108 m / 6: 1269.969 (monoisotope) M64 107 C349H516N84O110 m / 6: 1274.967 (monoisotope) M64 108 C347H515N85O107 m / 7: 1085.8 (average) M111 109 C349H517N85O109 m / 7: 1093.8 (average) M111 110 C352H523N85O109 m / 6: 1281.82 (monoisotope) M64 111 C348H512N82O111 m / 7: 1090.2 (average) M111 112 C349H514N82O112 m / 7: 1094.2 (average) M111 113 C348H513N81O112 m / 7: 1089.8 (average) M111 114 C351H517N85O109 m / 7: 1096.6 (average) M111 115 C349H516N84O109 m / 7: 1091.8 (average) M111 116 C341H511N83O110 m / 7: 1077.4 (average) M111 117 C351H519N85O110 m / 7: 1099.8 (average) M111 118 C348H516N84O108 m / 7: 1087.6 (average) M111 119 C348H516N84O108 m / 7: 1087.6 (average) M111 120 C348H516N84O108 m / 7: 1087.6 (average) M111 121 C348H516N84O108 m / 7: 1087.7 ​​(average) M111 122 C349H515N83O111 m / 7: 1094.0 (average) M111 123 C350H517N83O111 m / 7: 1095.8 (average) M111 124 C350H518N84O110 m / 7: 1095.5 (average) M111 125 C348H515N83O110 m / 7: 1090.1 (average) M111 126 C349H517N83O110 m / 7: 1092.2 (average) M111 127 C351H517N85O109 m / 7: 1097.4 (average) M111 128 C348H515N83O109 m / 7: 1088.0 (average) M111 129 C351H521N83O109 m / 7: 1093.7 (average) M111 130 C350H517N83O111 m / 7: 1095.6 (average) M111 131 C350H518N84O110 m / 7: 1095.4 (average) M111 132 C351H518N82O112 m / 7: 1097.6 (average) M111 133 C351H519N83O111 m / 7: 1097.5 (average) M111 134 C351H519N83O111 m / 7: 1097.4 (average) M111 135 C351H520N84O110 m / 7: 1097.3 (average) M111 136 C354H519N83O110 m / 7: 1100.5 (average) M111 137 C350H518N82O111 m / 7: 1093.6 (average) M111 138 C350H519N83O110 m / 7: 1093.5 (average) M111 139 C350H518N82O111 m / 7: 1093.5 (average) M111 140 C351H519N81O112 m / 7: 1095.8 (average) M111 141 C351H520N82O111 m / 7: 1095.6 (average) M111 142 C350H519N83O110 m / 7: 1093.5 (average) M111 143 C351H520N82O111 m / 7: 1095.6 (average) M111 144 C351H521N83O110 m / 7: 1095.5 (average) M111 145 C350H518N84O110 m / 7: 1095.7 (average) M111 146 C351H519N83O111 m / 7: 1097.7 (average) M111 147 C351H520N84O110 m / 7: 1097.4 (average) M111 148 C351H519N83O111 m / 7: 1097.7 (average) M111 149 C352H520N82O112 m / 7: 1099.6 (average) M111 150 C352H521N83O111 m / 7: 1099.5 (average) M111 151 C351H520N84O110 m / 7: 1097.5 (average) M111 152 C352H521N83O111 m / 7: 1099.6 (average) M111 153 C350H517N83O111 m / 7: 1095.7 (average) M111 154 C348H515N83O110 m / 7: 1089.5 (average) M111 155 C349H517N83O110 m / 7: 1091.7 (average) M111 156 C350H519N83O110 m / 7: 1093.7 (average) M111 157 C349H517N83O109 m / 7: 1089.4 (average) M111 158 C352H523N83O109 m / 7: 1095.3 (average) M111 159 C348H515N83O109 m / 7: 1087.4 (average) M111 160 C350H516N82O112 m / 7: 1095.7 (average) M111 161 C350H518N84O110 m / 7: 1095.4 (average) M111 162 C348H515N83O110 m / 7: 1089.4 (average) M111 163 C349H516N82O111 m / 7: 1091.8 (average) M111 164 C350H518N82O111 m / 7: 1093.6 (average) M111 165 C349H516N82O110 m / 7: 1089.3 (average) M111 166 C352H522N82O110 m / 7: 1095.5 (average) M111 167 C348H514N82O110 m / 7: 1087.6 (average) M111 168 C347H513N83O109 m / 7: 1085.1 (average) M111 169 C348H514N84O110 m / 7: 1091.5 (average) M111 170 C349H516N84O110 m / 7: 1093.5 (average) M111 171 C352H514N84O110 m / 7: 1098.3 (average) M111 172 C349H516N84O109 m / 7: 1091.1 (average) M111 173 C351H517N85O109 m / 7: 1096.8 (average) M111 174 C346H514N86O109 m / 7: 1089.7 (average) M111 175 C352H522N84O110 m / 7: 1099.5 (average) M111 176 C348H514N84O110 m / 7: 1091.4 (average) M111 177 C344H507N83O107 m / 7: 1074.7 (average) M111 178 C346H511N83O107 m / 7: 1078.7 (average) M111 179 C342H503N83O107 m / 7: 1070.5 (average) M111 180 C362H540N86O116 m / 7: 1136.9 (average) M111 181 C364H544N86O116 m / 7: 1140.7 (average) M111 182 C360H536N86O116 m / 7: 1133.0 (average) M111 183 C352H522N84O110 m / 7: 1099.5 (average) M111 184 C352H522N84O110 m / 7: 1099.5 (average) M111 185 C352H522N84O110 m / 7: 1099.4 (average) M111 186 C352H522N84O110 m / 7: 1099.4 (average) M111 187 C348H514N84O110 m / 7: 1091.4 (average) M111 188 C349H516N84O110 m / 7: 1093.5 (average) M111 189 C350H518N84O110 m / 7: 1095.4 (average) M111 190 C351H520N84O110 m / 7: 1097.5 (average) M111 191 C352H522N84O110 m / 7: 1099.4 (average) M111 192 C350H518N84O110 m / 7: 1095.4 (average) M111 193 C351H520N84O110 m / 7: 1097.5 (average) M111 194 C352H522N84O110 m / 7: 1099.4 (average) M111 195 C353H524N84O110 m / 7: 1101.5 (average) M111 196 C354H526N84O110 m / 7: 1103.4 (average) M111 197 C350H520N84O108 m / 6: 1272.297 (single isotope) M64 198 C350H519N83O109 m / 6: 1272.458 (single isotope) M64 199 C352H521N83O111 m / 5: 1538.34 (single isotope) M64 200 C348H517N83O107 m / 6: 1262.8 (single isotope) M64 201 C350H519N83O109 m / 6: 1272.458 (single isotope) M64 202 C353H524N84O110 m / 6: 1284.307 (single isotope) M64 203 C351H522N84O108 m / 6: 1274.633 (single isotope) M64 204 C349H516N82O109 m / 6: 1267.617 (single isotope) M64 205 C354H523N85O109 m / 6: 1285.802 (single isotope) M64 206 C351H522N84O108 m / 6: 1274.642 (single isotope) M64 207 C353H524N84O110 m / 6: 1284.305 (single isotope) M64 208 C350H518N82O109 m / 5: 1523.761 (single isotope) M64 209 C352H523N83O110 m / 5: 1535.63 (single isotope) M64 210 C353H525N83O110 m / 6: 1282.137 (single isotope) M64 211 C350H518N82O110 m / 6: 1272.64 (single isotope) M64 212 C351H518N82O111 m / 5: 1532.57 (single isotope) M64 213 C350H520N82O109 m / 6: 1270.306 (single isotope) M64 214 C351H522N82O109 m / 6: 1272.656 (single isotope) M64 215 C350H519N83O109 m / 7: 1090.853 (single isotope) M64 216 C352H521N83O111 m / 7: 1100.0 (average) M111 217 C351H521N83O109 m / 8: 957.8 (average) M111 218 C351H522N84O108 m / 7: 1093.7 (average) M111 219 C353H522N84O108 m / 7: 1097.1 (average) M111 220 C351H522N82O109 m / 7: 1091.5 (average) M111 221 C353H524N84O110 m / 7: 1101.7 (average) M111 222 C357H525N83O110 m / 7: 1107.0 (average) M111 223 C354H527N83O109 m / 7: 1099.8 (average) M111 224 C350H520N82O109 m / 7: 1089.8 (average) M111 225 C352H522N82O110 m / 8: 959.2 (average) M111 226 C352H523N83O109 m / 7: 1095.9 (average) M111 227 C351H523N83O108 m / 7: 1091.8 (average) M111 228 C354H527N83O109 m / 7: 1100.3 (average) M111 229 C358H531N83O110 m / 7: 1109.3 (average) M111 230 C358H532N84O109 m / 7: 1109.3 (average) M111 231 C354H526N82O111 m / 7: 1101.9 (average) M111 232 C352H523N81O110 m / 7: 1094.0 (average) M111 233 C353H525N81O110 m / 7: 1096.4 (average) M111 234 C358H528N82O110 m / 7: 1107.2 (average) M111 235 C355H529N83O110 m / 7: 1104.0 (average) M111 236 C355H528N84O110 m / 7: 1106.0 (average) M111 237 C353H526N82O109 m / 8: 959.5 (average) M111 238 C358H528N82O110 m / 7: 1107.2 (average) M111 239 C358H531N85O109 m / 7: 1111.2 (average) M111 240 C357H529N85O109 m / 7: 1109.1 (average) M111 241 C357H529N85O109 m / 7: 1109.1 (average) M111 242 C357H529N85O109 m / 7: 1109.0 (average) M111 243 C356H527N85O109 m / 7: 1107.5 (average) M111 244 C358H532N82O110 m / 7: 1107.6 (average) M111 245 C358H533N83O109 m / 7: 1107.5 (average) M111 246 C348H512N84O110S2 m / 5: 1539.578 (monoisotope) M64 247 C348H512N84O110S2 m / 5: 1539.578 (single isotope) M64 248 C347H515N83O105S2 m / 5: 1518.781 (single isotope) M64 249 C349H517N83O107S2 m / 5: 1530.586 (single isotope) M64 250 C348H514N84O108S2 m / 5: 1533.58 (single isotope) M64 251 C351H519N83O109S2 m / 5: 1542.014 (single isotope) M64 252 C349H517N81O109S2 m / 5: 1531.2 (monoisotope) M64 (Average) refers to the average molecular mass peak value, and (single isotope) refers to the single isotope peak value in the MS spectrum. [Example] [1] [:Compounds and binding motifs] [, (BM) , ] [Design] [ ]

[0187] The hFGF23 antagonistic peptide revealed in this paper is a molecule developed and designed based on the triple-helical bundle domain of the Z domain of Staphylococcus aureus protein A, which binds to the Fc region of immunoglobulin. A large library was constructed by randomly grouping selected amino acids from residues 9 to 35 on the two first helices constituting the Fc binding surface, and strong hFGF23 binders were isolated from it using various methods. Fc binding surface residues from residue 9 to 35 were then randomly grouped, and peptide variants were subsequently selected to replace the Fc binding capacity with hFGF23 binding capacity (Option #1). The binding motif (BM) thus corresponds to residues 9 to 35. After analyzing the results of Option #1, a new library was designed by randomly grouping other residues within the binding motif (BM) to further improve the hFGF23 binding capacity (Option #2). Subsequently, by substituting amino acids inside and outside the binding motif (BM), the peptide variant derived from select #2 was optimized (select #3) according to efficacy, thereby generating the hFGF23 antagonistic peptide disclosed herein. By identifying the position within the peptide backbone, the peptide variants derived from select #1 and select #2 were optimized based on in vivo half-life, in which extended portions could be linked while maintaining hFGF23 binding capacity.

[0188] The hFGF23 antagonistic peptide of the present invention can be functionalized by gene fusion with the protein module of interest or by linking a side chain of a covalent amino acid residue to a functional part (such as a toxin, an imaging agent, and / or one or more extended parts). [Example] [2] [In serum] [-] [reaction] [-] [In the analysis of luciferase reporter genes, regarding] [hFGF23] [Inhibition effect of induced signal transduction] [ ]

[0189] The purpose of this analysis is to demonstrate and characterize the extent to which the hFGF23 antagonistic peptide derivative ("peptide") provides inhibitory effects on hFGF23-induced signaling mediated by FGFR1c as a receptor and α-croxoprotein (KL) as a co-receptor. hFGF23 is known to specifically transmit signals via the MAPK / ERK pathway through this trimeric complex of FGFR1c / KL / hFGF23. hFGF23 treatment of cells induces luciferase expression, ultimately producing a luminescent signal upon the addition of a detection reagent. The compounds of this invention inhibit hFGF23-induced signaling with an IC50 < 1 nM or reduce hFGF23-induced signaling by >50% at 1 nM. Analysis Principles

[0190] To determine the ability of peptide compounds to inhibit signal transduction, a reporter gene analysis was established. We used luciferase under the control of serum response elements (SREs) to measure typical hFGF23-induced signal transduction.

[0191] HEK293 cells were first stably transfected with human KL phenoplasts encoding the zeocin resistance gene for mammalian selection. After generating a single HEK293 / KL cell line from a single pure line in the presence of 50 µg / ml zeocin (Invitrogen #R25001), the cells were stably transfected with a pGL4.33-based reporter plastid [luc2P / SRE / Hygro] (Promega #E1340), which encodes a luciferase under SRE control. The HEK293 / KL / SRE cell line was then obtained from a single pure line named 17-3 under 200 µg / ml hygromycin selection. HEK293 / KL / SRE cells were maintained at 37°C in DMEM-based complete medium (Gibco #31966-021) under standard cell culture conditions. This complete medium was supplemented with 1% penicillin-streptomycin (Gibco #15140-122), 10% FBS (Thermo Fisher Scientific #10091148), 50 µg / ml genomic mycoplasma, and 200 µg / ml hygromycin (Invitrogen #10687010).

[0192] To evaluate the inhibitory effect of the peptide compound on hFGF23-induced signaling, HEK293 / KL / SRE cells were seeded at a density of 10,000 cells per well in black 384-well dishes (Corning #354663) coated with poly-D-lysine in starvation medium. The starvation medium was based on DMEM supplemented with 0.5% human serum albumin and 1% penicillin-streptomycin. The following day, the peptide compound and recombinant hFGF23 (SEQ ID NO:5) (RnD Systems #2604-FG-025) were diluted in the starvation medium and co-incubated at 22°C for 1 hour before being added to the cells. The cells were incubated at 37°C for 4 hours, then lysed and luminescence was detected using an ultrasensitive EnVision 2104 disc reader (PerkinElmer).

[0193] The peptide compound was tested using either a single-dose or dose-response assay. In the single-dose assay, the peptide compound was applied at a final concentration of 1 nM. In the dose-response assay, the peptide compound was serially diluted and applied to cells at concentrations ranging from 50 nM to 0.032 nM. Both assay configurations applied hFGF23 at a concentration of 45 pM corresponding to hFGF23 EC80. In the single-dose assay, the compound response data were normalized relative to the minimum and maximum responses occurring in the absence and presence of hFGF23, respectively. Results are expressed as "residual activity %". The following formula was used: y = Luminescence reaction of polypeptide compounds Minimum value = No hFGF23 processing Maximum value = hFGF23 processing The purpose of the dose-response experiment was to determine the half-maximal inhibitory concentration (IC50) to compare the in vitro efficacy of the compounds. For this purpose, a four-parameter logarithmic equation (4PL fitting) was applied: Y Y = reaction, which decreases as x increases. x = concentration of polypeptide compound IC50 = half-maximum inhibitory concentration Hill Slope = Steepness of the linear portion of the dose-response curve

[0194] The analysis of single-dose and dose-response experiments was performed using Datalab with Python commands. Material: Genomicon (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) Black 384-well tray coated with poly-D-lysine (Corning #354663) Recombinant human FGF23 (amino acid 25-251) (RnD Systems #2604-FG-025) result

[0195] The results are shown in Table 6, which summarizes the inhibitory efficacy in the form of the IC50 value of the compounds of the present invention or the residual hFGF23 activity at a compound concentration of 1 nM. An IC50 value less than 1 nM or a residual activity % less than 50% at 1 nM indicates that the compounds have unexpectedly strong inhibitory activity under the experimental conditions. surface [6]. hFGF23 inhibitory efficacy (IC50) and residual activity % at 1 nM [Chem.] [IC] 50 [ (nM)] [1 nM] [Residual activity below] [%] [Chem.] [IC, 50 , (nM)] [1 nM] [Residual activity below] [%] 4 0.803 129 0.286 5 0.646 130 0.262 6 0.893 131 0.413 7 0.497 132 0.432 8 0.722 133 0.344 9 21 134 0.412 10 38 135 0.172 11 46 136 0.300 12 38 137 0.283 13 41 138 0.215 14 46 139 0.290 15 38 140 0.112 16 46 141 0.237 17 49 142 0.142 18 44 143 0.181 19 47 144 0.163 20 47 145 0.591 21 47 146 0.428 22 30 147 0.917 23 33 148 0.407 24 48 149 29 25 26 150 0.514 26 42 151 0.465 27 32 152 0.391 28 39 153 0.204 29 30 154 0.372 30 44 155 0.362 31 42 156 0.298 32 48 157 0.614 33 38 158 0.363 34 37 159 0.546 35 39 160 0.591 36 49 161 25 37 40 162 13 38 42 163 11 39 34 164 17 40 42 165 0.239 41 42 166 0.322 42 44 167 0.860 43 42 168 0.550 44 32 169 39 45 27 170 23 46 41 171 37 47 46 172 0.221 48 47 173 0.223 49 47 174 0.325 50 31 175 0.093 51 30 176 0.146 52 43 177 9 53 41 178 11 54 41 179 0.259 55 43 180 0.238 56 49 181 0.198 57 45 182 0.456 58 41 183 20 59 44 184 34 60 29 185 15 61 44 186 0.134 62 41 187 0.231 63 40 188 21 64 37 189 12 65 37 190 0.334 66 49 191 14 67 48 192 0.264 68 38 193 0.503 69 45 194 0.140 70 40 195 0.206 71 27 196 0.204 72 30 197 0.055 73 29 198 0.159 74 37 199 0.105 75 35 200 0.081 76 34 201 0.106 77 22 202 0.742 78 24 203 0.077 79 27 204 0.083 80 20 205 0.087 81 34 206 0.076 82 27 207 0.074 83 24 208 0.109 84 29 209 0.050 85 28 210 0.039 86 27 211 0.137 87 36 212 0.093 88 18 213 0.035 89 0.084 214 0.034 90 21 215 0.196 91 25 216 0.641 92 20 217 0.657 93 17 218 0.583 94 24 219 0.379 95 25 220 0.101 96 41 221 0.195 97 40 222 0.258 98 47 223 0.340 99 48 224 0.273 100 39 225 0.293 101 49 226 0.216 102 41 227 0.483 103 47 228 0.305 104 0.527 229 0.226 105 0.343 230 0.307 106 0.083 231 0.104 107 0.127 232 0.149 108 0.166 233 0.166 109 0.391 234 0.473 110 0.258 235 0.218 111 0.332 236 0.273 112 0.681 237 0.200 113 0.722 238 0.558 114 0.329 239 0.158 115 0.320 240 0.182 116 0.556 241 0.185 117 0.413 242 0.372 118 0.804 243 0.100 119 0.625 244 0.274 120 0.328 245 0.467 121 0.263 246 0.171 122 0.212 247 0.051 123 0.307 248 0.882 124 0.176 249 0.511 125 0.212 250 0.158 126 0.261 251 0.172 127 0.352 252 0.083 128 0.169 3 0.296

[0196] Data analysis shows that the compounds disclosed in this paper can inhibit hFGF23-induced signal transduction. Compounds with high sequence homology (e.g., at least 93% sequence homology) were demonstrated to maintain inhibitory activity against hFGF23. These compounds are therefore suitable for the treatment of hypophosphatemia, such as X-linked hypophosphatemia rickets (XLH). [Example] [3] [Surface plasma resonance] [(SPR)] [right] [hFGF23] [Antagonistic peptides and] [hFGF23] [Characteristics of the interactions between them] [ ]

[0197] The purpose of this analysis was to demonstrate that the hFGF23 antagonistic peptide ("peptide") of this invention binds to hFGF23 and to determine the binding affinity. Binding studies were performed on Biacore™ T200, Biacore™ 8K, or Biacore™ 8K+ (all derived from Cytiva), instruments designed to utilize surface plasma resonance (SPR) for instantaneous quantitative molecular interactions. Samples were maintained at 10°C in a sample compartment while experiments were conducted at 10°C or 25°C. The signal reported by the SPR instrument (expressed in reaction units (RU)) was directly correlated with the mass within eight parallel channels on the sensor wafer surface, each containing two tandem flow channels.

[0198] The procedure begins with the use of an amine coupling kit (Cytiva), following manufacturer guidelines, to covalently immobilize a self-produced anti-HPC4 monoclonal antibody onto two channels of the S-series sensor chip CM4 (Cytiva). Subsequently, HPC4-labeled recombinant hFGF23 (SEQ ID NO:6) is captured by injection into channel 2. This creates an active target surface in channel 2 and a reference surface in channel 1, which is immobilized solely with the anti-HPC4 antibody.

[0199] By injecting analytes into two flow cells to examine the binding of peptides to captured targets, comparative analysis of the binding of different compounds to captured targets (relative to a reference surface) can be achieved. The compounds of this invention are serially diluted in electrophoresis buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20) and injected at a rate of 50 µl min⁻¹ for 240 seconds, allowing dissociation for 900 seconds. After each analyte injection cycle, the CM4 surface is regenerated by injection of 10 mM glycine pH 1.5. This step removes the captured targets and any bound peptides from the antibody-attached surface, preparing it for subsequent sample pair interactions. The regeneration process does not remove the covalently attached anti-HPC4 capture antibody from the wafer surface.

[0200] The peptide binding curves were processed by subtracting the reference surface signal and injecting blank buffer into the captured hFGF23. This method allows for correction for instrument noise, volume shift, and drift during sample injection. The peptide association and dissociation rate constants, ka (association rate) and kd (dissociation rate), were extracted using Biacore Insight evaluation software (version 5.0.18.22102) by fitting a 1:1 Langmuir model to the data on an overall scale. The affinity between the compound and the target was quantified based on the equilibrium dissociation constant (KD), which was calculated kinetically from the equation KD = kd / ka. The results are presented in Table 7 below. surface [7]. Affinity value [Chem.] [K] D [ (nM)] 89 1.61 175 0.99 197 1.05 201 1.67 204 0.81 205 0.86 206 1.80

[0201] Data analysis shows that these compounds exhibit the desired binding to hFGF23. Further evidence demonstrates that compounds with high sequence identity percentages bind to hFGF23 with an affinity (KD) of 2 nM or less, particularly 1.8 nM or less. [Example] [4] [Surface plasma resonance] [(SPR)] [right] [hFGF23] [Antagonistic peptides and those from non-human species] [FGF23] [Characteristics of the interactions between them] [ ]

[0202] The purpose of this analysis was to demonstrate that the hFGF23 antagonistic peptide ("peptide") of the present invention binds to FGF23 from a non-human species and to determine the binding affinity. The analysis was performed similarly to that in Example 3, except that the hFGF23 was replaced with FGF23 from dogs, pigs, or monkeys. The results are presented in Table 8 below. surface [8] Affinity value [Chem.] [K] D [ (nM) [,dog] [)] [K] D [ (nM) [,pig] [)] [K] D [ (nM) [,monkey] [)] 89 7.63 1.10 3.12 175 4.12 0.66 1.65 197 4.32 0.84 1.84 201 6.24 0.61 2.61 204 2.92 0.56 0.39 205 3.32 0.56 1.33

[0203] Data analysis shows that these compounds exhibit the required cross-species reactive binding to FGF23 from dogs, pigs, and monkeys. [Example] [5] Pharmacokinetic studies in miniature pigs [1] [ ]

[0204] The research objective is to determine the blood half-life of certain compounds of the present invention after intravenous administration.

[0205] The study was conducted in female Göttingen minipigs (Ellegaard Minipigs A / S, Dalmose, Denmark) weighing approximately 25 kg. The peptide was administered intravenously to the minipigs at a dose of 10 nmol / kg (n=3 per group). The formulation contained 50 mM phosphate and 70 mM sodium chloride as mediators, at pH 7.4.

[0206] Blood samples were collected over 312 hours via an indwelling permanent venous catheter and collected in EDTA tubes (Sarstedt, Germany). Plasma was separated and compound concentrations were analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0207] Non-compartmental (NCA) pharmacokinetic analysis was performed using Phoenix® WinNonlin® v. 8.1 (Certara LP Princeton, NJ, USA). Terminal elimination time was fitted via linear regression using uniform weights. NCA was performed using nominal sampling time and actual dose.

[0208] The concentration of peptides in miniature pig plasma was assessed by plasma protein precipitation followed by turbulent flow liquid chromatography-mass spectrometry (TF-LC-MS). Calibration standards and quality control samples were prepared by incorporating compounds into blank plasma and then serially diluting them. All plasma samples were prepared by adding four volumes of ethanol to one volume of plasma sample. The precipitation solvent contained 20 nM internal standard. After centrifugation at 4 °C, the supernatant was diluted with one volume of purified water containing 1% formic acid. The precipitated plasma sample was first washed and concentrated on a TurboFlow Cyclone column (0.5 × 50 mm, Thermo Fisher Scientific) at 30 °C and then transferred to an analytical column. The analytical column was an XBridge Peptide BEH C18 column (2.1 × 50 mm, 3.5 µm particle size, Waters) operated at 60 °C. Analytical separation was performed using a linear gradient. Mobile phase A consisted of water containing 5% organic solvent (acetonitrile / methanol (1:1, by volume)) and 1% formic acid, while mobile phase B consisted of 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 from Thermo Fisher Scientific). The mass spectrometer was operated in positive ionization mode, with the selected reaction monitored on the TSQ Altis and parallel reactions monitored on the Q Exactive. Sample quantification was performed relative to the reaction from the calibration standard using linear regression with a 1 / X2 weighting. The reaction was calculated as the analyte peak area divided by the internal standard peak area. The concentrations of the quality control samples and calibration standards were measured within ±15% of the nominal concentration, except for one quality control sample which was -17% of the nominal concentration. The limits of quantitation were between 0.250 nM and 1.00 nM. result

[0209] The results are shown in Table 9 below, where column T1 / 2 represents the blood half-life (T1 / 2). A high half-life value is desired. surface [9]. Pharmacokinetic parameters after intravenous administration [Chem.] [T½ (h)] [Extended son] 197 47.1 C20 diacid 111 47.9 C18 diacid 107 49.6 C18 diacid 89 51.6 C18 diacid 204 51.9 C20 diacid 201 58.8 C20 diacid 106 59.7 C20 diacid

[0210] No adverse clinical observations were observed that were thought to be related to the test compound during the study.

[0211] The results showed that extending the sequence at position 58 with C18 or C20 diacid resulted in a clinically significant blood half-life for polypeptide compounds with high sequence consistency.

[0212] A longer half-life indicates that the specified compound can provide long-term inhibitory efficacy after administration, compared to compounds with relatively short half-lives. A longer half-life allows for a lower frequency of dosing, which is necessary to provide enhanced convenience to patients. [Example] [6] Pharmacokinetics and pharmacodynamics in dogs [ ]

[0213] The objective of this study is to evaluate the pharmacokinetics and pharmacodynamics of the compound of the present invention in male Beagle dogs following a single subcutaneous (sc) injection.

[0214] Male Beagle dogs were administered a single subcutaneous injection of Chem. 206, Chem. 207, and Chem. 208 (n=3 / compound, target dose 10 nmol / kg). The composition of the injectable formulation is listed below: 20 mM L-histidine; 150 mM L-arginine salt; pH=6.49-6.51.

[0215] The study included fifteen male Beagles from Marshall, Beijing, China, aged between 2.60 and 9.10 years and weighing between 11.3 and 15.0 kg. During the study, the dogs were housed in groups, and each animal was identified by a unique tattoo number. The enclosures were maintained at a temperature of 16–25°C and a humidity level of 40–70%. A 12-hour light-dark cycle was used. During the study period, the dogs were fed commercially available dog food (medium-sized adult dry dog ​​food, Royal Canin®, Shanghai, China; 150–225 g / dog / day) once daily at 13:00. Animals were fasted overnight for morning sampling. The animals had free access to quality drinking water.

[0216] On the day the experiment began, dogs received a single subcutaneous bolus injection of the test compound at approximately 9:00 AM. To measure the plasma concentration of the test compound and plasma phosphate, blood samples (1.2 mL at each time point) were collected in EDTA-coated tubes at the following time points: before administration (0 min), 5, 15, 30, 45 min, 1, 1.5, 2, 4, 7, 10 h, 24, 30, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, and 336 h. Blood samples were stored on ice until centrifugation (4 min, 4°C, 4000 rpm). Immediately after centrifugation, two sets of 100 μL EDTA-coated plasma samples (one for quantification of the test compound and one for quantification of phosphate) from each sampling time point were transferred to microtubes and stored at -20°C until analysis. Plasma analysis of test compounds

[0217] The plasma concentrations of Chem. 206, Chem. 207, and Chem. 208 were analyzed by plasma protein precipitation followed by turbulent liquid chromatography-mass spectrometry (TF-LC-MS). Calibrators were prepared by incorporating Chem. 206, Chem. 207, and Chem. 208 in the range of 0.5 to 2000 nM into canine blank plasma. Calibrators were prepared by adding three volumes of ethanol containing 50 nM internal standard (ISTD) (Chem. 175) to one volume of sample, followed by centrifugation at 4000 rpm for 60 min at 4°C, and then protein precipitation. Blank plasma or study samples were used for TF-LC-MS. One volume of the supernatant was mixed with two volumes of water (containing 0.1% formic acid). The mixture was analyzed by TF-LC-MS using a Cyclone turbulent flow column (0.5 × 50 mm, ThermoFisher Scientific) and an Agilent poroshell 120 SB-C18 column (2.1 × 50 mm, 2.7 μm particles). Gradient dissociation was performed using mobile phase A (composed of milli-Q water containing 0.1% formic acid and 5% methanol / acetonitrile (50 / 50)) and mobile phase B (composed of methanol / acetonitrile (50 / 50) containing 0.1% formic acid and 5% milli-Q water). The mass spectrometer was operated in positive ionization mode. An Orbitrap Exploris 240 mass spectrometer (ThermoFisher Scientific) was used as the detector in parallel reaction monitoring mode (Chem. 206: m / z 1912.4600@1748.5375; Chem. 207: m / z 1926.9600@1767.5369; Chem. 208: m / z 1905.4500@2211.0012; Chem. 175: m / z 1923.4500@1762.8643). Concentrations in plasma samples were calculated using linear calibration curves (weighted 1 / x2). Quality control samples were included. The deviation between the nominal and calculated concentrations in the calibrator and quality control samples was less than 15%. The limit of quantitation (LLOQ) was 2.0 nM. Pharmacokinetic analysis of the test compounds

[0218] Enter the plasma concentration values ​​of the test compound at each sampling time point into Phoenix® WinNonlin® version 8.4.0.6172 for non-compartmental analysis (NCA) using the Linear Up Log Down method and a weight of 1 / (Y*Y). NCA calculations use the actual sampling time. Individual concentration values ​​below the LLOQ are treated as zero at time points before the first detectable exposure and as missing values ​​at time points after Tmax. NCA analysis uses the actual dose, calculated using the actual concentration of the compound.

[0219] The results are shown in Table 10 below. surface

[10] . Pharmacokinetic parameters after subcutaneous administration [Chem.] [T½ (h)] [Extended son] 206 77 C20 diacid 207 74 C20 diacid 208 88 C20 diacid

[0220] At the test dose, the compound was well tolerated, and no side effects were observed in test animals. Results showed that the C20 diacid extension at position 58 resulted in a clinically significant blood half-life for the sequence-consistent peptide compound. [Example] [7] [Plasma phosphate content in miniature pigs and dogs] [ ]

[0221] To determine the in vivo efficacy of the compounds of the present invention, plasma phosphate levels were measured in the PK studies described in Examples 5 and 6. Serum Pi method:

[0222] Plasma phosphate was measured using the PHOS2 Inorganic Phosphate Analyzer v2.0 (Roche) on a Cobas Pro C503 analytical instrument. Animals were administered a single intravenous dose of 10 nmol / kg (miniature pigs, Example 5) or a single subcutaneous dose of 10 nmol / kg (Beagle dogs, Example 6). Plasma samples were collected for analysis at administration (0), at 5, 15, and 30 minutes post-administration, and at 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 30, 48, 72, 96, and 144 hours post-administration. Data are reported in Figure 1 / Table 11 and Figure 2 / Table 12, showing plasma phosphate levels in mmol / L. surface

[11] . Plasma phosphate levels (mmol / L) in miniature pigs after intravenous administration. [time] [Chem. 197] [Chem. 201] [Chem. 204] [Mediator] 0 min 1.44 1.45 1.44 1.59 5 min 1.44 1.56 1.43 1.55 15 min 1.57 1.66 1.61 1.39 30 min 1.59 1.57 1.59 1.52 1 h 1.63 1.60 1.71 1.67 1.5 h 1.64 1.66 1.66 1.50 2 h 1.50 1.48 1.48 1.53 3 h 1.47 1.39 1.42 1.51 4 h 1.62 1.49 1.48 1.48 6 h 1.73 1.65 1.66 1.62 8 h 1.73 1.62 1.70 1.59 10 h 1.68 1.63 1.71 1.78 24 h 2.07 2.24 2.18 1.77 30 h 2.49 2.39 2.31 1.62 48 h 2.78 2.50 2.31 1.83 72 h 2.46 2.29 2.17 1.75 96 h 2.04 1.83 1.79 1.75 144 h 1.50 1.42 1.58 1.86 surface

[12] . Plasma phosphate levels in dogs after subcutaneous drug administration (mmol / L) [time] [Chem. 204] [Chem. 206] [Chem. 207] [Chem. 208] 0 min 1.13 1.12 1.16 0.97 5 min 1.12 1.12 1.13 0.99 15 min 1.16 1.14 1.16 0.99 30 min 1.18 1.16 1.17 1.06 1 h 1.19 1.20 1.16 1.06 1.5 h 1.22 1.22 1.15 1.01 2 h 1.22 1.18 1.17 1.07 3 h 1.24 1.23 1.14 1.04 4 h 1.21 1.07 1.10 1.04 6 h 1.32 1.32 1.24 1.19 8 h 1.65 1.62 1.56 1.47 10 h 1.23 1.40 1.26 1.23 24 h 1.38 1.48 1.33 1.34 30 h 1.34 1.46 1.18 1.21 48 h 1.27 1.49 1.17 1.10 72 h 1.29 1.30 1.19 1.15 96 h 1.12 1.25 1.05 1.00 144 h 1.09 1.20 1.06 1.03

[0223] The results showed that the compounds of this invention can increase plasma phosphate levels in vivo. [Example] [8] [:] [Chem. 3] [Crystallization and Use] [X] [Antigenic determinant localization achieved by radiographic crystallography] [ ]

[0224] To determine the position where the compound of the present invention binds to hFGF23, crystallization studies were conducted to identify antigenic determinant residues on hFGF23. hFGF23 and test compounds used for complex purification and initial crystallization

[0225] The complex consisting of the hFGF23 core domain (SEQ ID NO:4) and Chem. 3 (SEQ ID NO:10) was prepared as follows: Chem. 3 (SEQ ID NO:10) in excess at 1.5 moles was mixed with the hFGF23 core domain (SEQ ID NO:4), incubated at 4°C for 2 hours, and purified by size exclusion chromatography (SEC) using a Superdex 75 16 / 60 column in 10 mM HEPES pH 7.5 and 500 mM NaCl. The purified complex was collected from the SEC peak fraction, concentrated to 3.7–7.4 mg / mL, and used for crystallization assays in a 96-well settling drop (using a commercially available crystallization sieve) and incubated at 18°C. Initial crystals were obtained in droplets containing 0.18 µl of stock solution (0.2 M potassium formate, 20% (w / v) PEG 3350) and 0.36 µl of protein solution. Seed stock solution was prepared using the initial crystals for subsequent seeding experiments. The seed stock solution was prepared as follows: 1) the initial crystals in the droplet were crushed; 2) the droplet containing the crushed crystals was transferred to a seed bead tube (HR2-320 Hampton research) containing 50 µl of stock solution (0.2 M potassium formate, 20% (w / v) PEG 3350); and 3) the droplet was shaken on a vortex mixer for 2 minutes. Crystallization and structural determination of the complex of hFGF23 core domain (SEQ ID NO:4) and Chem. 3

[0226] Crystals were obtained at 18 °C by inoculating 35 nl of seed stock solution into droplets containing 0.36 µl of protein solution and 0.36 µl of stock solution (0.2 M calcium acetate hydrate, 20% w / v polyethylene glycol 3,350). The crystals were cryoprotected in glycerol and then frozen in liquid nitrogen. Synchrotron X-ray diffraction data were collected at the Swiss Light Source and processed in XDS / XSCALE until a resolution of 1.8 Å was achieved in space group 4 (P21). The structure was determined in Phenix by molecular substitution using the following pdb inputs as the search model: 2P39 and 5DJT (B chain only). The solution derived from the molecular substitution was found in space group 4 (P21) and contained two complexes (complex 1 and complex 2) in the asymmetric unit. The structure was refined to a resolution of 1.8 Å in Phenix. A brief explanation and interpretation of the structure

[0227] The two complexes in the asymmetric unit show high structural similarity, with RMSDs of 0.658 Å (main chain) and 1.058 Å (all atoms) calculated using "Align" in PyMOL, excluding outliers. The overall structure of the hFGF23 core domain (SEQ ID NO:4) is similar to the aforementioned crystal structure of hFGF23 (pdb input 2P39), with a main chain RMSD of 1.304 Å calculated using "Align" in PyMOL, excluding outliers.

[0228] The interaction between hFGF23 and its receptor (a heterodimer of FGFR1c and α-crosox protein) has previously been characterized by X-ray crystallography (pdb input 5W21). Structural comparison of the hFGF23 / receptor complex (5W21) and the Chem. 3 / hFGF23 core domain (SEQ ID NO:4) complex revealed significant overlap between the binding sites of the hFGF23 receptor and Chem. 3 on hFGF23, confirming that Chem. 3 directly prevents the interaction between hFGF23 and its receptor, thereby inhibiting hFGF23 function. Determination of the antigenic determinants of Chem. 3

[0229] The antigenic determinant residues (the distance between any non-hydrogen atom in Chem. 3 and hFGF23 is 3.5 Å) include the following residues in hFGF23 (SEQ ID NO:1): M74, R76, T86, R91, F108, P110, R114, L166, I167 and N170. [Example] [9] [Computer-simulated antigen determinant assay] [ ]

[0230] Computer simulations were performed using Alphafold v. 2.3 (https: / / www.biorxiv.org / content / 10.1101 / 2021.10.04.463034v2) to simulate the folding of hFGF23 (SEQ NO:1) with Chem. 206, Chem. 207, and Chem. 208. For multiple sequence alignment, a simplified database setup was applied, and the number of iterations was set to 5. All other model parameters were set to default.

[0231] The identified antigenic determinant, determined at a distance of 3.5 Å between the test compound and any non-hydrogen atom in hFGF23, contains the following residues from hFGF23: Chem. 206: [R76] [T86] [R91], Y93, Y107 [R114], F169 and N170. Chem. 207: W36 [R76] [T86] [R91], Y107 [R114], I167, F169 and N170. Chem. 208: [R76] [T86] [R91]、 [R114], L166 and F169.

[0232] Referring to Example 8, all tested compounds bound to the antigenic determinants on hFGF23 containing amino acid residues R76, T86, R91, and R114. These residues (highlighted in bold above) are therefore considered key antigenic determinant residues of the compounds of this invention. [Example]

[10] Pharmacokinetic studies in miniature pigs [2]

[0233] The research objective is to determine the blood half-life of certain compounds of the present invention after intravenous administration.

[0234] The study was conducted in female Ellegaard Minipigs A / S, Dalmose, Denmark, weighing approximately 25 kg. The peptide was administered intravenously to the minipigs at a dose of 14 nmol / kg, n=3 per group. The formulation contained 50 mM phosphate, 70 mM sodium chloride, and 0.007% polysorbate 20, at pH 7.4.

[0235] Blood samples were collected over 312 hours via an indwelling permanent venous catheter and collected in EDTA tubes (Sarstedt, Germany). Plasma was separated and compound concentrations were analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0236] Non-compartmental (NCA) pharmacokinetic analysis was performed using Phoenix® WinNonlin® v. 8.1 (Certara LP Princeton, NJ, USA). Terminal elimination time was fitted via linear regression using uniform weights. NCA was performed using nominal sampling time and actual dose.

[0237] The concentration of peptides in miniature pig plasma was assessed by plasma protein precipitation followed by turbulent flow liquid chromatography-mass spectrometry (TF-LC-MS). Calibration standards and quality control samples were prepared by incorporating compounds into blank plasma and then serially diluting them. All plasma samples were prepared by adding four volumes of ethanol to one volume of plasma sample. The precipitation solvent contained 20 nM internal standard. After centrifugation at 4 °C, the supernatant was diluted with one volume of purified water containing 1% formic acid. The precipitated plasma sample was first washed and concentrated on a TurboFlow Cyclone column (0.5 × 50 mm, Thermo Fisher Scientific) at 30 °C and then transferred to an analytical column. The analytical column was an XBridge Peptide BEH C18 column (2.1 × 50 mm, 3.5 µm particle size, Waters) operated at 60 °C. Analytical separation was performed using a linear gradient. Mobile phase A consisted of water containing 5% organic solvent (acetonitrile / methanol (1:1, by volume)) and 1% formic acid, while mobile phase B consisted of 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 from Thermo Fisher Scientific). The mass spectrometer was operated in positive ionization mode, with the selected reaction monitored on the TSQ Altis and parallel reactions monitored on the Q Exactive. Sample quantification was performed relative to the reaction from the calibration standard using linear regression with a 1 / X2 weighting. The reaction was calculated as the analyte peak area divided by the internal standard peak area. The concentrations of the quality control samples and calibration standards were measured within ±15% of the nominal concentration, except for one quality control sample which was -17% of the nominal concentration. The limits of quantitation were between 0.250 nM and 1.00 nM. result

[0238] The results are shown in Table 13 below, where T The [½] column displays the blood half-life (T). [½]). A high half-life value is required. surface

[13] . Pharmacokinetic parameters after intravenous administration [Chem.] [T½ (h)] [Extended son] 206 61.5 C20 diacid

[0239] No adverse clinical observations were observed that were thought to be related to the test compound during the study.

[0240] The results showed that the use of C20 diacid at position 58 resulted in a clinically significant blood half-life extension.

[0241] A longer half-life indicates that the specified compound can provide long-term inhibitory efficacy after administration, compared to compounds with relatively short half-lives. A longer half-life allows for a lower frequency of dosing, which is necessary to provide enhanced convenience to patients. [Example]

[11] [Plasma phosphate content in miniature pigs] [ ]

[0242] To determine the in vivo efficacy of the compound of the present invention (referred to herein as Chem. 206), plasma phosphate levels were measured in the PK study described in Example 10. Serum Pi method:

[0243] Plasma phosphate was measured using the PHOS2 Inorganic Phosphate Analyzer v2.0 (Roche) on a Cobas Pro C503 analyzer. Animals were administered a single intravenous dose of 14 nmol / kg of the compound. Plasma samples were collected for analysis at administration (0), at 5, 15, and 30 minutes post-administration, and at 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 30, 48, 72, 96, and 144 hours post-administration. Data are reported in Figure 3 / Table 14, showing plasma phosphate levels in mmol / L. surface

[14] . Plasma phosphate levels (mmol / L) in miniature pigs after intravenous administration. [time] [Chem. 206] 0 min 1.78 5 min 15 min 1.83 30 min 1.90 1 h 1.95 1.5 h 1.82 2 h 1.80 3 h 1.80 4 h 1.76 6 h 1.83 8 h 1.70 10 h 2.39 24 h 2.49 30 h 2.58 48 h 2.26 72 h 2.04 96 h 1.72 144 h 1.50

[0244] The results showed that the compounds of this invention can increase plasma phosphate levels in vivo. [ ] [***]

[0245] While certain features of the invention have been described and illustrated herein, many modifications, substitutions, variations, and equivalents will readily occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations that fall within the true spirit of the invention. [ ]

[0246] TW202629583A_114138947_SEQL.xml

Claims

1. A compound selected from the group consisting of:

2. The compound of claim 1, wherein the compound is an hFGF23 antagonistic polypeptide derivative capable of binding hFGF23 according to SEQ ID NO:

1.

3. An hFGF23 antagonistic polypeptide derivative, represented by the following formula:

4. A pharmaceutical composition comprising a compound as claimed in claim 1 or 2 or an hFGF23 antagonistic polypeptide derivative as claimed in claim 3 and one or more pharmaceutically acceptable excipients.

5. A pharmaceutical comprising a compound as claimed in claim 1 or 2 or an hFGF23 antagonistic polypeptide derivative as claimed in claim 3.

6. The use of a medicine as claimed in claim 5, wherein the medicine is used to manufacture a pharmaceutical product for treating X-linked hypophosphatemia rickets (XLH).

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