New compound targeting musk and use thereof

Abstract

The purpose of the present invention is to provide a new compound for activating nerve-to-muscle signal transduction in a neuromuscular junction, and a new therapeutic agent for myasthenia gravis. The present invention provides is a cyclic peptide having the structure of formula (I) or a complex thereof, or a pharmaceutically acceptable salt thereof. Formula (I): -X1-Asn-X2-Val- (in the formula, X1 represents Thr or Val and X2 represents Val or Ile) The present invention provides a therapeutic agent for myasthenia gravis, the therapeutic agent comprising the aforementioned cyclic peptide or a complex thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Description

Novel compounds targeting MuSK and uses thereof REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2023-88120 (filing date: May 29, 2023), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to novel compounds that activate signal transduction at the neuromuscular junction and to therapeutic agents for myasthenia gravis. The present invention also relates to a method for selecting ubiquitin molecules onto which a cyclic peptide pharmacophore has been grafted.

[0003] Myasthenia gravis (MG) is an autoimmune disease known to involve autoantibodies directly in the pathology. It is characterized by generalized muscle weakness and fatigue, particularly eye symptoms such as ptosis and diplopia (Non-Patent Document 1). It is believed that myasthenia gravis occurs when autoantibodies destroy or inhibit the function of several molecules present on the muscle side (signal receiver) at the junction between peripheral nerves and muscles (neuromuscular junction), preventing signals from being transmitted from nerves to muscles, resulting in muscle weakness. The most common target of autoantibodies is the acetylcholine receptor (AChR), accounting for approximately 85% of cases, followed by muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (LRP4), accounting for a few percent of cases. It is also known that there are patients with a seronegative type who are negative for all of these autoantibodies.

[0004] Commonly used symptomatic treatments for myasthenia gravis are steroid therapy and immunosuppressant therapy. However, patients are forced to bear various side effects as a cost of treatment, and many cases are difficult to treat. Recently, the monoclonal antibody "eculizumab (trade name Soliris)" targeting the complement protein C5 has been marketed as a molecularly targeted drug for the treatment of myasthenia gravis, and the cyclic peptide "zirucoplan" with a similar mechanism of action is currently under clinical development (see Patent Documents 1 and 2). While these drugs are intended to treat anti-acetylcholine receptor antibody-positive myasthenia gravis, neither is suitable for myasthenia gravis that is not associated with an increased risk of infection with Neisseria meningitidis or complement activation, nor for myasthenia gravis other than anti-acetylcholine receptor antibody-positive.

[0005] Special table 2019-517473 publication Special table 2022-508952 publication

[0006] Gilhus NE & Verschuuren J. J, Lancet Neurol. 2015 Oct;14(10):1023-36.

[0007] The present invention aims to provide a novel compound that activates signal transmission from nerves to muscles at the neuromuscular junction and a new therapeutic agent for myasthenia gravis. Another objective of the present invention is to provide a new method for selecting a ubiquitin molecule having a cyclic peptide pharmacophore grafted thereon.

[0008] The present invention provides the following: [1] A cyclic peptide or a pharmaceutically acceptable salt thereof comprising the structure of the following formula (I): -X １ -Asn-X ２ -Val- (I) (wherein X １ represents Thr or Val, and X ２ represents Val or Ile) [2] The cyclic peptide or a pharmaceutically acceptable salt thereof according to [1] above, wherein the number of amino acid residues forming the cyclic structure of the cyclic peptide is 8 to 20. [3] The cyclic structure of the cyclic peptide is N—CO—CH ２

[0022] The cyclic peptide or pharmaceutically acceptable salt thereof according to [1] or [2] above, which contains an -S structure. [4] The cyclic peptide or pharmaceutically acceptable salt thereof according to [3] above, wherein N is the amino group of D-tyrosine. [5] The cyclic peptide or pharmaceutically acceptable salt thereof according to [3] or [4] above, wherein S is the thiol group of cysteine. [6] The cyclic peptide or pharmaceutically acceptable salt thereof according to any of [1] to [5] above, wherein the cyclic peptide has a structure selected from the group consisting of the following formula (1) (SEQ ID NO: 2), formula (2) (SEQ ID NO: 3), and formula (3) (SEQ ID NO: 4). (In the above formula, the carboxyl group of the cysteine ​​residue (C) may form an acid amide.) [7] A cyclic peptide complex or a pharmaceutically acceptable salt thereof, comprising two cyclic peptides according to any one of [1] to [6] above, wherein one cyclic peptide is bonded to the other cyclic peptide via a linker. [8] A cyclic peptide complex or a pharmaceutically acceptable salt thereof according to [7] above, wherein the two cyclic peptides are the same cyclic peptide. [9] A cyclic peptide complex or a pharmaceutically acceptable salt thereof according to [7] or [8] above, wherein the linker has a length of 20 to 100 Å.

[10] A cyclic peptide complex or a pharmaceutically acceptable salt thereof according to any one of [7] to [9] above, wherein the linker has a polyethylene glycol structure as at least a part of its structure.

[11] A cyclic peptide complex or a pharmaceutically acceptable salt thereof according to any one of [7] to

[10] above, wherein the linker has at least one saturated hydrocarbon group having 8 to 20 carbon atoms as its side chain.

[12] A part or all of the loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule has the following structure (A): (In the above formula, the MuSK binding region is YX ２１ SYHTNVVASLKR(X ２１is P or L) (SEQ ID NO: 11), spacer 1 consists of an amino acid sequence selected from the group consisting of IQPR (SEQ ID NO: 12), KPGV (SEQ ID NO: 13), WDRG (SEQ ID NO: 14), KPGT (SEQ ID NO: 15), PFGV (SEQ ID NO: 16), YFVG (SEQ ID NO: 17) and LEGM (SEQ ID NO: 18), and spacer 2 consists of an amino acid sequence selected from the group consisting of IRY, MMP, QGI, FMP, TGPQ (SEQ ID NO: 19), WNTP (SEQ ID NO: 20) and IRPQ (SEQ ID NO: 21), or a MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof, wherein the peptide of structure (A) is inserted into the loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule.

[13] The MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof according to

[12] above, wherein the ubiquitin molecule is a protein selected from the group consisting of: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1, (b) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1, and (c) a protein consisting of an amino acid sequence in which one or more amino acids have been modified in the amino acid sequence of SEQ ID NO: 1.

[14] The MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof according to

[12] or

[13] above, wherein structure (A) has an amino acid sequence selected from the group consisting of:(Ub006) IQPRYPSYHTNVVASLKRIRY (SEQ ID NO: 22) (Ub007) KPGVYPSYHTNVVASLKRMMP (SEQ ID NO: 23) (Ub008) WDRGYPSYHTNVVASLKRQGI (SEQ ID NO: 24) (Ub009) KPGTYPSYHTNVVASLKRFMP (SEQ ID NO: 25) (Ub010) PFGVYPSYHTNVVASLKRTGPQ (SEQ ID NO: 26) (Ub011) YFVGYPSYHTNVVASLKRWNTP (SEQ ID NO: 27) (Ub012) LEGMYLSYHTNVVASLKRIRPQ (SEQ ID NO: 28) (Ub013) LEGMYPSYHTNVVASLKRIRPQ (SEQ ID NO: 29)

[15] The MuSK-binding domain-grafted ubiquitin molecule according to any of

[12] to

[14] above, or a pharmaceutically acceptable salt thereof, wherein the loop structure region is a region from the 7th amino acid residue to the 10th amino acid residue of the ubiquitin molecule.

[16] A MuSK-binding domain-grafted ubiquitin molecule complex or a pharmaceutically acceptable salt thereof, comprising two or more (e.g., 2 to 6, preferably 2 to 4) MuSK-binding domain-grafted ubiquitin molecules according to any of

[12] to

[15] above, wherein one MuSK-binding domain-grafted ubiquitin molecule is linked to another MuSK-binding domain-grafted ubiquitin molecule via a linker.

[17] The MuSK-binding domain-grafted ubiquitin molecular complex according to

[16] above, or a pharmaceutically acceptable salt thereof, wherein the two or more MuSK-binding domain-grafted ubiquitin molecules are the same molecule.

[18] The MuSK-binding domain-grafted ubiquitin molecular complex according to

[16] or

[17] above, or a pharmaceutically acceptable salt thereof, wherein the linker has a length of 20 to 100 Å.

[19] The MuSK-binding domain-grafted ubiquitin molecular complex according to any of

[16] to

[18] above, or a pharmaceutically acceptable salt thereof, wherein the linker is a peptide chain.

[20] A pharmaceutical composition or a therapeutic agent for myasthenia gravis, comprising as an active ingredient the cyclic peptide according to any one of [1] to [6] above or a pharmaceutically acceptable salt thereof, the cyclic peptide complex according to any one of [7] to

[11] above or a pharmaceutically acceptable salt thereof, the MuSK-binding domain-grafted ubiquitin molecule according to any one of

[12] to

[15] above or a pharmaceutically acceptable salt thereof, or the MuSK-binding domain-grafted ubiquitin molecule complex according to any one of

[16] to

[19] above or a pharmaceutically acceptable salt thereof. [20-1] A method for treating myasthenia gravis, comprising administering a therapeutically effective amount of the cyclic peptide described in any of [1] to [6] above or a pharmaceutically acceptable salt thereof, the cyclic peptide complex described in any of [7] to

[11] above or a pharmaceutically acceptable salt thereof, the MuSK-binding domain-grafted ubiquitin molecule described in any of

[12] to

[15] above or a pharmaceutically acceptable salt thereof, or the MuSK-binding domain-grafted ubiquitin molecule complex described in any of

[16] to

[19] above or a pharmaceutically acceptable salt thereof, or a therapeutic agent or pharmaceutical composition containing the same to a subject in need thereof. [20-2] The cyclic peptide or a pharmaceutically acceptable salt thereof according to any one of [1] to [6] above, the cyclic peptide complex or a pharmaceutically acceptable salt thereof according to any one of [7] to

[11] above, the MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof according to any one of

[12] to

[15] above, or the MuSK-binding domain-grafted ubiquitin molecular complex or a pharmaceutically acceptable salt thereof according to any one of

[16] to

[19] above, for use as a pharmaceutical. [20-3] The cyclic peptide or a pharmaceutically acceptable salt thereof according to [20-2] above, the cyclic peptide complex or a pharmaceutically acceptable salt thereof, the MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof, or the MuSK-binding domain-grafted ubiquitin molecular complex or a pharmaceutically acceptable salt thereof, for use in the treatment of myasthenia gravis.[20-4] Use of the cyclic peptide or a pharmaceutically acceptable salt thereof according to any of [1] to [6] above, the cyclic peptide complex or a pharmaceutically acceptable salt thereof according to any of [7] to

[11] above, the MuSK-binding domain-grafted ubiquitin molecule or a pharmaceutically acceptable salt thereof according to any of

[12] to

[15] above, or the MuSK-binding domain-grafted ubiquitin molecule complex or a pharmaceutically acceptable salt thereof according to any of

[16] to

[19] above, in a method for treating myasthenia gravis for the manufacture of a medicine or a therapeutic agent for myasthenia gravis, or as a medicine or a therapeutic agent for myasthenia gravis.

[21] A method for presenting a cyclic peptide having binding activity to a desired molecule (preferably a MuSK molecule) together with two spacer sequences on a loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule, wherein the cyclic peptide has a chemical crosslinking structure for forming an intramolecular cyclic structure, and the method comprises replacing the chemical crosslinking structure of the cyclic peptide with two amino acid residues constituting the loop structure via the spacer sequence, thereby fusing the cyclic peptide to a ubiquitin molecule while retaining its binding activity to the desired molecule (preferably a MuSK molecule), and the spacer sequence consists of a randomly generated amino acid sequence of 0 to 4 residues (preferably 3 or 4 residues).

[22] The desired molecule is a MuSK molecule, and the cyclic peptide and spacer sequence presented on the loop structure region of the ubiquitin molecule have the following structure (B): (wherein spacer 3 is a peptide chain consisting of any 0 to 4 (preferably 3 or 4) amino acids, spacer 4 is a peptide chain consisting of any 0 to 4 (preferably 3 or 4) amino acids, and the MuSK-binding region is a region present in the cyclic peptide and having MuSK molecule-binding activity.)

[23] The presentation method according to

[21] or

[22] above, wherein the loop structure region of the ubiquitin molecule is the loop structure region between the β1 domain and the β2 domain.

[24] The presentation method according to

[23] above, wherein the two amino acid residues constituting the loop structure region are the 7th and 10th amino acid residues of the ubiquitin molecule.

[25] The MuSK-binding region is a region represented by the formula: YX ２２ SYHTNVVASLKR(X ２２ is P or L) (SEQ ID NO: 11).

[26] A method for producing a modified protein, comprising fusing a cyclic peptide having binding activity to a desired molecule (preferably a MuSK molecule) together with two spacer sequences to a loop structure region of a ubiquitin molecule to display the cyclic peptide on the protein, the cyclic peptide having a chemical crosslinking structure for forming an intramolecular cyclic structure, selecting a partial amino acid sequence to be displayed on the protein from the amino acid sequence of the cyclic peptide, selecting a nucleotide sequence corresponding to the partial amino acid sequence, selecting nucleotide sequences corresponding to two amino acid residues that constitute the loop structure region of the ubiquitin molecule, deleting bases present between the selected nucleotide sequences as necessary, and inserting a nucleotide sequence corresponding to the partial amino acid sequence of the selected cyclic peptide, and translating the nucleic acid, wherein the spacer sequence consists of a randomly generated amino acid sequence of 0 to 4 residues (preferably 3 or 4 residues).

[27] The desired molecule is a MuSK molecule, and the cyclic peptide and spacer sequence displayed on the loop structure region of the ubiquitin molecule have the following structure (B): (wherein spacer 3 is a peptide chain consisting of any 0 to 4 (preferably 3 or 4) amino acids, spacer 4 is a peptide chain consisting of any 0 to 4 (preferably 3 or 4) amino acids, and the MuSK binding region is a region present in the cyclic peptide and having MuSK molecule binding activity.)

[28] The production method according to

[26] or

[27] above, wherein the loop structure region of the ubiquitin molecule is the loop structure region between the β1 domain and the β2 domain.

[29] The production method according to

[28] above, wherein the two amino acid residues constituting the loop structure region are the 7th and 10th amino acid residues of the ubiquitin molecule.

[30] The MuSK binding region is a region represented by the formula: YX ２２ SYHTNVVASLKR(X ２２ is P or L) (SEQ ID NO: 11).

[31] A method for screening for a modified protein capable of binding to a desired molecule (preferably a modified protein capable of binding to a MuSK molecule), comprising: designing an mRNA library containing a base sequence encoding a ubiquitin molecule in which a cyclic peptide sequence having binding activity to a desired molecule (preferably a MuSK molecule) and spacer sequences consisting of randomly generated amino acid sequences of 0 to 4 residues (preferably 3 or 4 residues) linked upstream and downstream of the cyclic peptide sequence are fused to a loop structure region between the β1 domain and the β2 domain; preparing modified proteins based on the designed mRNA library according to the production method described in

[28] above; and selecting a modified protein capable of binding to the desired molecule (preferably a modified protein capable of binding to a MuSK molecule) from the obtained modified proteins using binding activity to the desired molecule (preferably MuSK-binding activity) as an index.

[32] A peptide or a pharmaceutically acceptable salt thereof comprising the structure of formula (I): -X １ -Asn-X ２ -Val- (I) (wherein X １ represents Thr or Val, and X ２(represents Val or Ile)

[33] The peptide according to

[32] above, or a pharmaceutically acceptable salt thereof, wherein the peptide consists of 8 to 20 amino acid residues.

[34] The peptide according to

[32] or

[33] above, or a pharmaceutically acceptable salt thereof, grafted into a biomolecule.

[0009] The cyclic peptide and MuSK-binding domain-grafted ubiquitin molecule and their complexes provided by the present invention can directly enhance signals essential for the formation and maintenance of neuromuscular junctions. Thus, the present invention is advantageous in that it provides a novel modality molecule that enables the treatment of myasthenia gravis.

[0010] Figure 1 is a schematic diagram illustrating the mechanism of MuSK homodimerization and subsequent acetylcholine receptor clustering. Figure 2 is a schematic diagram illustrating the mechanism of MuSK homodimerization and subsequent acetylcholine receptor clustering upon binding of a MuSK-binding dimeric peptide to the extracellular domain of MuSK. Figure 3A is a schematic diagram of an approach to generate a library of thioether-cyclic peptides using the flexible in vitro translation (FIT) system and rapidly search for target-binding cyclic peptides through RaPID screening. Figure 3B is a schematic diagram of an approach to graft the pharmacophore region of a specific cyclic peptide selected through RaPID screening onto a scaffold protein using the Lasso grafting method, and to create a library of constructed mRNA fusion proteins by placing spacers upstream and downstream of the grafted peptide. Figure 4 shows the results of selecting peptide sequences identified from the initial RaPID screening for affinity to MuSK. The progress of affinity selection was expressed as the percentage of mRNA bound to MuSK-bead complexes (positive) or simply to beads (negative) relative to the total number of mRNAs used in each round of affinity selection. Figure 5 shows SPR sensorgrams of MuSK-binding monomer peptides analyzed on a Biacore T200 over a concentration range of 1.6 to 1000 nM. The fitted curves are also shown. Figure 6-1 shows the structure of homodimers of cyclic peptides (MuSK4, MuSK7, and MuSK9). Figure 6-2 shows the structure of homodimers of linear peptides (MuSK1Ac). Figure 7 shows the chemical structure of a MuSK7 homodimer (MuSK7 PEG11 dimer) using Bis-Mal-dPEG (trademark) 11 as a linker. Figure 8 shows the chemical structure of a MuSK7 homodimer into which a palmitoyl group has been introduced. Figure 9A shows that the cyclic peptide complex of the present invention (MuSK7 PEG11 dimer) induces acetylcholine receptor clustering in a concentration-dependent manner at the cultured myotube cell level.Figure 9B is a dose-response curve of the cyclic peptide complex of the present invention (MuSK7 PEG11 dimer) with respect to the number of acetylcholine receptor clusters. Figure 9C is a graph showing the effect of AST-487 on acetylcholine receptor cluster-inducing activity. Figure 10A is a schematic diagram showing the two molecules used in the NanoBiT assay. Interaction between the extracellular regions of two human MuSKs (extracellular portions of human MuSK) results in binding of LgBiT and SmBiT to form a luminescent enzyme, and luminescence can be observed upon addition of a substrate. Figure 10B is an evaluation result using the NanoBiT assay, showing a dose-response curve of the cyclic peptide complex of the present invention (MuSK7-pal) with respect to MuSK dimerization. Figure 11 is a graph showing the results of an evaluation (weight change) of the therapeutic effect of the cyclic peptide complex of the present invention (high dose) on a myasthenia gravis model animal. Figure 12 is a graph showing the results of an evaluation (movement score) of the therapeutic effect of the cyclic peptide complex of the present invention (high dose) on a myasthenia gravis model animal. 13 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (high dose) of the present invention on a myasthenia gravis model animal (latency to fall in the Hanging Wire Test). FIG. 14 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (high dose) of the present invention on a myasthenia gravis model animal (grip strength). FIG. 15 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (low dose) of the present invention on a myasthenia gravis model animal (body weight change). FIG. 16 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (low dose) of the present invention on a myasthenia gravis model animal (movement score). FIG. 17 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (low dose) of the present invention on a myasthenia gravis model animal (latency to fall in the Hanging Wire Test). FIG. 18 is a graph showing the results of evaluation of the therapeutic effect of a cyclic peptide complex (low dose) of the present invention on a myasthenia gravis model animal (grip strength).Figure 19a is a schematic diagram of the approach to lasso-grafting MET-binding macrocyclic peptides (aMD4 and aMD5) obtained using the RaPID system onto ubiquitin molecules (β1-β2 loop). Figure 19b is a schematic diagram of the strategy for lasso-grafting aMD4 and aMD5 onto ubiquitin molecules by adding spacer sequences of 0 to 3 amino acid residues upstream and downstream of the pharmacophore sequences. Figure 19c is a schematic diagram of the mRNA display strategy, showing the translation products (aMD4 or aMD5 lasso-grafted ubiquitin molecules) bound to mRNA via puromycin. In this specification, the ubiquitin molecule obtained by grafting the binding active region is referred to as a U-body, and the ubiquitin molecule may be abbreviated as Ub. Figure 20 shows the results of selection of ubiquitin molecules lasso-grafted with aMD4 (Figure 20a) or aMD5 (Figure 20b) (population % in the recovered library after selection, K measured by SPR). Ｄ The figure shows the values ​​of "K D "w / mutation" refers to the K of U-bodies that have a mutation in the ubiquitin molecule (a mutation relative to the wild-type ubiquitin molecule). ＤThe values ​​are shown. Flanking spacers are shown for each ubiquitin molecule. The notation for amino acid mutations, from left to right, refers to the amino acid residue before mutation, the mutation position, and the amino acid residue after mutation. Figure 21 is a diagram illustrating an approach in which, when preparing a ubiquitin molecule grafted with a MET-binding region, 0 to 4 consecutive residues in the β1-2 loop are replaced with a pharmacophore sequence flanked upstream and downstream using various flanking spacer sequences (randomized 0 to 3 spacer residues, indicated as X in the figure). Figure 22 is a diagram illustrating an approach in which, when preparing a ubiquitin molecule grafted with a MuSK-binding region, 7 to 10 amino acid residues in the β1-2 loop are replaced with a pharmacophore sequence (MuSK7) flanked upstream and downstream using various flanking spacer sequences (randomized spacer residues of 3 or 4 residues, indicated as X in the figure). Figure 23 shows the results of peptide sequence selection identified in RaPID screening performed to optimize the flanking spacer sequence of the MuSK-binding domain-grafted ubiquitin molecule shown in Figure 22. The progress of affinity selection is expressed as the percentage (recovery rate) of mRNA bound to MuSK-bead complexes (positive) or to simple beads (negative) relative to the total number of mRNAs used in each round of affinity selection. Figure 24 shows the results of evaluation using the NanoBiT assay, showing dose-response curves for MuSK dimerization of the MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention (dimeric U-bodies), a cyclic peptide complex (dimeric peptide / MuSK7-pal), and a dimer of ubiquitin molecules (dimeric Ub).Figure 25 shows the results of an evaluation test of AChR cluster formation using myotube cells, illustrating the concentration-dependent effects of homodimers (dimeric U-bodies) of the MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention and ubiquitin molecule dimers (dimeric Ub) on acetylcholine receptor cluster induction. Figure 26A shows the results of an evaluation using a NanoBiT assay, illustrating dose-response curves for MuSK dimerization of homodimers (dimeric U-bodies) and homotetramers (tetrameric U-bodies) of the MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention. Figure 26B shows the results of an evaluation test of AChR cluster formation using myotube cells, illustrating the concentration-dependent effects of homotetramers (tetrameric U-bodies) of the MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention on acetylcholine receptor cluster induction. Figure 27A shows the results of evaluation of the therapeutic effect (body weight change) of a homotetramer of a MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention on a myasthenia gravis model animal (Example 4(6)). Figure 27B shows the results of evaluation of the therapeutic effect (body weight change) of a homotetramer of Ub012 with a C-terminal GG deletion (Example 4(6)). Figure 28A shows the results of evaluation of the therapeutic effect (motion score) of a homotetramer of a MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention on a myasthenia gravis model animal (Example 4(6)). Figure 28B shows the results of evaluation of the therapeutic effect (motion score) of a homotetramer of Ub012 with a C-terminal GG deletion (Example 4(6)). Figure 29 shows the results of evaluating the therapeutic effect (latency to fall in the Hanging Wire Test) of a homotetramer of a MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention on myasthenia gravis animal models (Example 4(6)). Figure 30 shows the results of evaluating the therapeutic effect (grip strength) of a homotetramer of a MuSK-binding domain-grafted ubiquitin molecule (Ub012) of the present invention on myasthenia gravis animal models (Example 4(6)). Specific Description of the Invention

[0011] [Definition] In this specification, the term "pharmaceutically acceptable salt" includes, for example, salts with pharmaceutically acceptable bases or acids. Specific, non-limiting examples of pharmacologically acceptable salts include addition salts with inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts with organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acids, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts with inorganic bases (ammonium hydroxide or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.

[0012] [Cyclic Peptide] The cyclic peptide of the present invention has the unit structure of formula (I) as part of its structure. Formula (I) includes the following four structures: -Thr-Asn-Val-Val- (Ia) (SEQ ID NO: 30) -Thr-Asn-Ile-Val- (Ib) (SEQ ID NO: 31) -Val-Asn-Val-Val- (Ic) (SEQ ID NO: 32) -Val-Asn-Ile-Val- (Id) (SEQ ID NO: 33)

[0013] As used herein, the term "cyclic peptide" refers to a peptide having at least a cyclic structure formed by six or more amino acids within the molecule. The molecular structure of a cyclic peptide may have, in addition to a cyclic structure, a chain structure in which amino acids are linked by peptide bonds, or a structure other than a peptide structure. As used herein, the term "cyclic structure" refers to a closed ring structure formed within a molecule of a linear peptide by bonding two amino acids separated by four or more amino acid residues, either directly or via a linker or the like. As used herein, "separated by four or more amino acid residues" means that there are at least four amino acid residues between the two amino acids that form the closed ring structure.

[0014] In the present specification, unless otherwise specified, a peptide specified by its amino acid sequence has its N-terminus on the left and its C-terminus on the right. Furthermore, in the present specification, the order or positional relationship of amino acids from the N-terminus to the C-terminus in a peptide may be referred to as "downstream," and the order or positional relationship of amino acids from the C-terminus to the N-terminus may be referred to as "upstream."

[0015] The closed ring structure in the cyclic structure is not particularly limited, and may be formed by covalently bonding two amino acids, optionally via a linker or the like. Examples of the covalent bond between two amino acids include a disulfide bond, a peptide bond, an alkyl bond, an alkenyl bond, an ester bond, a thioester bond, an ether bond, a thioether bond, a phosphonate ether bond, an azo bond, a C-S-C bond, a C-N-C bond, a C=N-C bond, an amide bond, a lactam bridge, a carbamoyl bond, a urea bond, a thiourea bond, an amine bond, and a thioamide bond. The covalent bond between two amino acids may also be formed by bonding the side chains of the two amino acids, the main chains of the two amino acids, or the side chains and main chains of the two amino acids. When two amino acids are bonded at the main chains of the amino acids, a closed ring structure is formed by a peptide bond.

[0016] The cyclic structure is not limited to the bond between the N-terminal and C-terminal amino acids of a linear peptide, but may also be formed by the bond between a terminal amino acid and an amino acid other than the terminal amino acid, or by the bond between amino acids other than the terminal amino acid. When one of the amino acids bonded to form the cyclic structure is a terminal amino acid and the other is a non-terminal amino acid, the cyclic peptide has a structure in which a linear peptide is attached to the cyclic structure like a tail. When a linear peptide is attached to the cyclic structure like a tail, it is preferable that the linear peptide is attached to the C-terminus of the amino acid constituting the cyclic structure in the linear peptide. In other words, it is preferable that the cyclic structure is formed by the bond between the N-terminus of the linear peptide and an amino acid other than the C-terminus.

[0017] Amino acids that form a cyclic structure include not only naturally occurring amino acids but also artificial amino acid variants and / or derivatives, such as naturally occurring proteinogenic L-amino acids, unnatural amino acids, and chemically synthesized compounds having properties known in the art as characteristic of amino acids.

[0018] Proteinogenic amino acids can be identified by the following three-letter or single-letter symbols well known in the art: Alanine: Ala A Arginine: Arg R Asparagine: Asn N Aspartic acid: Asp D Cysteine: Cys C Glutamine: Gln Q Glutamic acid: Glu E Glycine: Gly G Histidine: His H Isoleucine: Ile I Leucine: Leu L Lysine: Lys K Methionine: Met M Phenylalanine: Phe F Proline: Pro P Serine: Ser S Threonine: Thr T Tryptophan: Trp W Tyrosine: Tyr Y Valine: Val V

[0019] Non-proteinogenic amino acids refer to natural or unnatural amino acids other than proteinogenic amino acids. Examples of unnatural amino acids include amino acids whose main chain structure differs from that of natural amino acids (such as α,α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl amino acids, D-amino acids, β-amino acids, γ-amino acids, δ-amino acids, and α-hydroxy acids); amino acids whose side chain structure differs from that of natural amino acids (such as norleucine and homohistidine); amino acids having an extra methylene in the side chain (such as "homo" amino acids, homophenylalanine, and homohistidine); and amino acids whose carboxylic acid functional group in the side chain is substituted with a sulfonic acid group (such as cysteic acid). Specific examples of unnatural amino acids include the amino acids described in WO 2015 / 030014.

[0020] The number of amino acid residues forming the cyclic structure is not particularly limited as long as it is 6 or more, but the lower limit (or more) can be, for example, 7, 8, 9, 10, 11, 12, or 13, and the upper limit (or less) can be 30, 25, 20, 19, 18, 17, 16, or 15. These lower and upper limits can be arbitrarily combined to determine the range of the number of amino acid residues forming the cyclic structure, and can be, for example, 6 to 30, 7 to 25, 8 to 20, 12 to 18, or 13 to 17.

[0021] In the present invention, the cyclic peptide may be modified by one or more of phosphorylation, methylation, acetylation, adenylylation, ADP-ribosylation, glycosylation, polyethylene glycol addition, and fatty acid addition, or may be fused with other peptides and / or proteins. Furthermore, the cyclic peptide may be biotinylated via a suitable linker.

[0022] Furthermore, in the present invention, the cyclic peptide may be a dimer having two cyclic structures within a molecule, in which two cyclic peptides having one cyclic structure are linked via a linker structure, or may have an intramolecular lactam bridge structure in which a lactam structure is formed within a molecule. The intramolecular lactam bridge structure may be formed by bonding the side chains of the amino acids constituting the cyclic peptide. For example, an intramolecular lactam structure may be formed by bonding the amino group on the side chain of Lys with the carboxyl group on the side chain of Asp or Glu to form a peptide bond. Such a cyclic peptide has another cyclic structure as a bridge structure within the molecule. For example, DAP, DAB, or Orn may be bonded to Asp or Glu instead of Lys. In this specification, a cyclic peptide dimer in which two cyclic peptides containing a cyclic structure represented by the above formula (I) are linked via a linker structure is referred to as a "cyclic peptide complex" as described below.

[0023] A preferred example of the cyclic peptide of the present invention is a cyclic peptide having a structure represented by the following formula (II): (Z １ ) m-X １-Asn-X ２ -Val-(Z ２ ) n (II) (wherein X １ and X ２ is the same as defined in formula (I), and Z １ represents any amino acid, Z ２ represents any amino acid, m is an integer of 1 or more, and n is an integer of 1 or more).

[0024] The above formula (II) is a structure represented by formula (I) having (Z １ ) m is bound to the C-terminus, and (Z ２ In formula (II), (Z １ ) m and (Z ２ The amino acids constituting (Z)n each independently represent any amino acid. １ ) m and one amino acid of (Z ２ ) n is preferably bonded directly or via a linker to form a cyclic structure. １ is the C-terminal Z ２ and the N-terminal Z １ is a non-C-terminal Z ２ and a non-N-terminal Z １ is the C-terminal Z ２ and a non-N-terminal Z １ is a non-C-terminal Z ２ It may be formed by bonding with

[0025] Non-terminal Z １ or Z ２ is involved in the bond, the cyclic peptide has a structure in which a linear peptide is attached to the cyclic structure like a tail. １ but non-C-terminal Z ２ may bond to form a cyclic structure. １ and Z ２ and may form a cyclic structure via a linker.

[0026] The sum of m and n (m+n) is an integer of 2 or greater, but the lower limit (or greater) of m+n can be, for example, 2, 3, 4, 5, 6, 7, 8, or 9, and the upper limit (or less) can be 26, 21, 16, 15, 14, 13, 12, or 11. These lower and upper limits can be arbitrarily combined to create a numerical range for m+n, for example, 2 to 26, 3 to 24, 4 to 16, 8 to 14, or 9 to 13.

[0027] (Z １ ) m and (Z ２ )n may include, in addition to amino acids involved in forming a cyclic structure, amino acids having a branched side chain such as Leu, Val, and Ile, amino acids having a hydroxyl group in the side chain such as Ser and Thr, basic amino acids, and aromatic amino acids, and preferably includes Leu or Ser.

[0028] Cyclic peptides are N—CO—CH ２ -S structure (i.e., -NH-CO-CH ２ -S- structure), and N-CO-CH ２ A cyclic structure may be formed by the -S structure. The cyclic structure is preferably formed by bonding an acetyl group, the hydrogen atom of which is substituted with a leaving group L and which is bonded to the amino group at the N-terminus of the peptide before the cyclic structure is formed, to a thiol group in cysteine ​​(Cys) on the C-terminus side. Examples of the leaving group L include halogen atoms such as chlorine, bromine, fluorine, and iodine atoms. The acetyl group in which the hydrogen atom is substituted with a leaving group L is not particularly limited, and examples thereof include a chloroacetyl group. N-CO-CH ２ As a cyclic peptide having the structure of formula (II) in which a cyclic structure is formed by the -S structure, for example, in the peptide represented by the following formula (III), ２ For example, a ring structure is formed by bonding CO— with the thiol group (HS—) of Cys. ２ CO-(Z ３ )-(Z ４ ) p-X １ -Asn-X ２ -Val-(Z ５ )q-Cys-(Z６ ) r (III) (wherein X １ and X ２ is the same as defined in formula (I), and Z ３ , Z ４ , Z ５ and Z ６ represents any amino acid, p is an integer of m-1, q is an integer of n-1, and r is an integer of 0 or 1 or more. m and n are defined as in formula (II).

[0029] In formula (III), Z ３ , (Z ４ ) p, (Z ５ ) q and (Z ６ ) The amino acids constituting r each independently represent any amino acid. ２ The peptide of formula (III) forms a cyclic structure by bonding between CO— and the thiol group (HS—) of Cys.

[0030] The sum of p and q (p+q) is an integer of 0 or greater, but the lower limit (or greater) of p+q can be, for example, 0, 1, 2, 3, 4, 5, 6, or 7, and the upper limit (or less) can be 24, 19, 14, 13, 12, 11, 10, or 9. These lower and upper limits can be arbitrarily combined to form a numerical range for p+q, for example, 0 to 24, 1 to 22, 2 to 14, 6 to 12, or 7 to 11.

[0031] r is not particularly limited as long as it is an integer of 0 or more, but its lower limit (or more) can be, for example, 1, 2, 3, 4, 5, or 6, and its upper limit (or less) can be 20, 15, 14, 13, 12, 11, or 10. These lower and upper limits can be arbitrarily combined to form a numerical range for r, for example, 0 to 20 or 1 to 15. In the present specification, when r is 0, the cyclic peptide is (Z ６ )r in the cyclic peptide, and when r is an integer of 1 or more, ６ ) r corresponds to a chain structure in which amino acids are linked by peptide bonds.

[0032] Z ３is preferably Tyr, and may be in either the D- or L-form.

[0033] (Z ４ ) p and (Z ５ ) q may include an amino acid having a branched side chain such as Leu, Val, or Ile, an amino acid having a hydroxyl group in the side chain such as Ser or Thr, a basic amino acid, or an aromatic amino acid, and preferably includes Leu or Ser.

[0034] Z ６ are preferably independently Gly or Ser. ６ An example of r is a structure represented by (-Gly-Ser-)ra (ra is an integer of 0 to 10, and when ra is 2 or more, it represents a repeating structure consisting of -Gly-Ser-, with the proviso that the C-terminus is -Ser).

[0035] The cyclic structure in a cyclic peptide is N—CO—CH ２ The cyclic structure may be other than the cyclic structure formed by the -S structure. For example, a cyclic peptide can be formed by cyclizing an amino acid having functional group 1 shown in Table 1 below and an amino acid having the corresponding functional group 2. In this embodiment, for example, when the cyclic structure includes the structure represented by formula (II) above, at least one Z １ is an amino acid having functional group 1, and at least one Z ２ may be an amino acid having the corresponding functional group 2, or at least one Z １ is an amino acid having functional group 2, and at least one Z ２ may be an amino acid having the corresponding functional group 1.

[0036] Functional group 1 and functional group 2 may be located at the N-terminus and C-terminus (either may be the N-terminus), one may be located at a terminal amino acid and the other at a non-terminal amino acid, or both may be located at a non-terminal amino acid. The bond formed by functional group 1 and functional group 2 becomes a chemical crosslinking structure for forming a molecular cyclic structure in a cyclic peptide.

[0037] In the chemical structural formula of Table 1, X １is a leaving group, and Ar is an aromatic ring which may have a substituent. Examples of the leaving group include halogen atoms such as chlorine atom, bromine atom, fluorine atom, and iodine atom.

[0038] Examples of the amino acid having the functional group (A-1) include chloroacetylated amino acids, such as N-chloroacetyl-L-alanine, N-chloroacetyl-L-phenylalanine, N-chloroacetyl-L-tyrosine, N-chloroacetyl-L-tryptophan, N-3-(2-chloroacetamido)benzoyl-L-phenylalanine, N-3-(2-chloroacetamido)benzoyl-L-tyrosine, N-3-(2-chloroacetamido)benzoyl-L-tryptophan, β-N-chloroacetyl-L-diaminopropanoic acid, γ-N-chloroacetyl-L-diaminobutyric acid, δ-N-chloroacetyl-L-ornithine, ε-N-chloroacetyl-L-lysine, and their corresponding D-amino acid derivatives. As the amino acid having a functional group (A-1), N-chloroacetyl-L-tyrosine and N-chloroacetyl-D-tyrosine are preferably used.

[0039] Examples of the amino acid having the functional group (A-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid, etc. Cysteine ​​is preferably used as the amino acid having the functional group (A-2).

[0040] Examples of the cyclization method using an amino acid having a functional group (A-1) and an amino acid having a functional group (A-2) include those described in Kawakami, T. et al., Nature Chemical Biology 5, 888-890 (2009); Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009); Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008); Goto, Y. et al., ACS Chemical Biology 3, 120-129 (2008); Kawakami T. et al., Chemistry & Biology 15, 32-42 (2008), and WO 2008 / 117833.

[0041] Examples of amino acids having the functional group (B-1) include propargylglycine, homopropargylglycine, 2-amino-6-heptynoic acid, 2-amino-7-octynoic acid, and 2-amino-8-nonynoic acid. 4-pentynoylated or 5-hexynoylated amino acids may also be used. Examples of 4-pentenoylated amino acids include N-(4-pentenoyl)-L-alanine, N-(4-pentenoyl)-L-phenylalanine, N-(4-pentenoyl)-L-tyrosine, N-(4-pentenoyl)-L-tryptophan, N-3-(4-pentynoylamido)benzoyl-L-phenylalanine, N-3-(4-pentynoylamido)benzoyl-L-tyrosine, N-3-(4-pentynoylamido)benzoyl-L-tryptophan, β-N-(4-pentenoyl)-L-diaminopropanoic acid, γ-N-(4-pentenoyl)-L-diaminobutyric acid, σ-N-(4-pentenoyl)-L-ornithine, ε-N-(4-pentenoyl)-L-lysine, and the corresponding D-amino acid derivatives thereof. Examples of 5-hexynoyl amino acids include amino acids in which the 4-pentynoyl group in the compounds exemplified as 4-pentynoyl amino acids has been substituted with a 5-hexynoyl group.

[0042] Examples of amino acids having the functional group (B-2) include azidoalanine, 2-amino-4-azidobutanoic acid, azidoptonorvaline, azidonorleucine, 2-amino-7-azidoheptanoic acid, and 2-amino-8-azidooctanoic acid. Azidoacetylated or 3-azidopentanoylated amino acids can also be used. Examples of azidoacetylated amino acids include N-azidoacetyl-L-alanine, N-azidoacetyl-L-phenylalanine, N-azidoacetyl-L-tyrosine, N-azidoacetyl-L-tryptophan, N-3-(4-pentynoylamido)benzoyl-L-phenylalanine, N-3-(4-pentynoylamido)benzoyl-L-tyrosine, N-3-(4-pentynoylamido)benzoyl-L-tryptophan, β-N-azidoacetyl-L-diaminopropanoic acid, γ-N-azidoacetyl-L-diaminobutyric acid, σ-N-azidoacetyl-L-ornithine, ε-N-azidoacetyl-L-lysine, and the corresponding D-amino acid derivatives thereof. Examples of 3-azidopentanoylated amino acids include amino acids in which the azidoacetyl group in the compounds exemplified as azidoacetylated amino acids has been substituted with a 3-azidopentanoyl group.

[0043] Examples of the cyclization method using an amino acid having a functional group (B-1) and an amino acid having a functional group (B-2) include the methods described in Sako, Y. et al., Journal of American Chemical Society 130, 7932-7934 (2008) and WO 2008 / 117833.

[0044] Examples of amino acids having a functional group (C-1) include N-(4-aminomethyl-benzoyl)-phenylalanine (AMBF) and 3-aminomethyltyrosine.

[0045] An example of the amino acid having a functional group (C-2) is 5-hydroxytryptophan (WOH).

[0046] Examples of the cyclization method using an amino acid having a functional group (C-1) and an amino acid having a functional group (C-2) include the methods described in Yamagishi, Y. et al., ChemBioChem 10, 1469-1472 (2009) and WO 2008 / 117833.

[0047] Examples of amino acids having a functional group (D-1) include 2-amino-6-chloro-hexynoic acid, 2-amino-7-chloro-heptynoic acid, and 2-amino-8-chloro-octynoic acid.

[0048] Examples of the amino acid having a functional group (D-2) include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid.

[0049] Examples of the cyclization method using an amino acid having a functional group (D-1) and an amino acid having a functional group (D-2) include the method described in WO 2012 / 074129.

[0050] Examples of the amino acid (E-1) include N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophan, and the corresponding D-amino acid derivatives thereof.

[0051] Examples of the (E-2) amino acid include cysteine, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid.

[0052] The cyclization method using an amino acid having a functional group (E-1) and an amino acid having a functional group (E-2) can be carried out with reference to, for example, the cyclization method using (A-1) and (A-2) or the cyclization method using (D-1) and (D-2).

[0053] A more preferred example of the cyclic peptide of the present invention is a cyclic peptide having the structure of the following formula (IV): (In the above formula, Tyr may be in the D-form or the L-form, preferably in the D-form; S represents a thiol group of Cys; the portion referred to as the "variable region" is a peptide chain containing at least the unit structure of formula (I) (the amino acid length can be 4 to 28, 5 to 26, 6 to 18, 10 to 16, or 11 to 15); -(Z ４ ) p-X １ -Asn-X ２ -Val-(Z ５ )q-, and (Z ６ ) r has the same meaning as defined in formula (III).

[0054] In the present specification and drawings, -CH in the above structure ２ The structure represented by —C(═O)— is sometimes written as “Ac”.

[0055] In the above formula (IV), the "variable region" can be represented by the following formula (V): -(Z ４ ) p-X １ -Asn-X ２ -Val-(Z ５ )q- (V) (wherein X １ and X ２is the same as defined in formula (I), and Z ４ and Z ５ and p and q have the same meanings as defined in formula (III), and specific explanations and preferred embodiments are as explained in formula (III).

[0056] Specific examples of suitable cyclic peptides represented by the above formula (IV) include those having any of the structures represented by the following formulas (1) (MuSK7 / SEQ ID NO: 2), (2) (MuSK4 / SEQ ID NO: 3), and (3) (MuSK9 / SEQ ID NO: 4). However, in the following formulas (1) to (3), (Z ６ ) r is omitted. Therefore, in the following formulas (1) to (3), -SCH ２ The C (Cys) bonded to - is ６ ) r may be bonded (Z ６ and r are as defined in formula (III).

[0057] In the above formulas (1) to (3), the carboxyl group (-C(=O)-OH) of C(Cys) is substituted with an acid amide (-C(=O)-NH ２ Such compounds may be represented by the following formulae (1a), (2a) and (3a):

[0058] The cyclic peptides of the present invention have high affinity for MuSK and can bind strongly to MuSK, making them usable as MuSK agonists. Furthermore, as described below, complexes containing the cyclic peptides of the present invention bind to the extracellular domain of MuSK, thereby inducing MuSK homodimerization and selectively inducing downstream MuSK signaling, thereby activating nerve-to-muscle signaling at the neuromuscular junction. Therefore, unlike the previously studied approach of targeting anti-acetylcholine receptor autoantibodies, the cyclic peptides of the present invention are advantageous in that they can potently activate signaling at the neuromuscular junction, thereby providing therapeutic and research methods that differ from existing techniques. Specifically, the cyclic peptides of the present invention and pharmaceutically acceptable salts thereof are useful as pharmaceuticals and research reagents. The present invention provides pharmaceutical compositions containing the cyclic peptides of the present invention and pharmaceutically acceptable salts thereof, as well as the use of the cyclic peptides of the present invention and pharmaceutically acceptable salts thereof as pharmaceuticals.

[0059] In another aspect of the present invention, there is provided a peptide or a pharmaceutically acceptable salt thereof, comprising the structure of formula (I): -X １ -Asn-X ２ -Val- (I) (wherein X １ represents Thr or Val, and X ２ represents Val or Ile)

[0060] The lower limit (or not less than) of the number of amino acid residues in the peptide of the present invention can be, for example, 7, 8, 9, 10, 11, 12, or 13, and the upper limit (or not more than) can be 30, 25, 20, 19, 18, 17, 16, or 15. These lower and upper limits can be arbitrarily combined to define a range of the number of amino acid residues forming the peptide, and can be, for example, 6 to 30, 7 to 25, 8 to 20, 12 to 18, or 13 to 17.

[0061] The peptide of the present invention can be a peptide consisting of the same amino acid sequence as the amino acid sequence of the cyclic peptide of the present invention or a part of the amino acid sequence thereof, and is preferably a peptide containing an amino acid sequence constituting a region having MuSK molecule binding activity (i.e., MuSK binding region) present in the cyclic peptide of the present invention, more preferably YX ２２ SYHTNVVASLKR(X ２２ is P or L) (SEQ ID NO: 11). The peptide of the present invention described above may also be a non-cyclic peptide.

[0062] The peptides of the present invention can be used by grafting them onto a desired biomolecule, such as the MuSK-binding domain-grafted ubiquitin molecule described below. Examples of biomolecules to be grafted include biological proteins such as ubiquitin molecules. The peptides of the present invention can be peptides that have the ability to bind to a desired molecule, and can be grafted onto a biomolecule so that the peptide exhibits binding properties. When the peptides of the present invention are peptides containing an amino acid sequence that constitutes a region having MuSK molecule-binding activity present in the cyclic peptides of the present invention, they can be grafted onto a biomolecule so that the peptide exhibits MuSK binding properties. The peptides of the present invention grafted onto a biomolecule are within the scope of the present invention, so long as the peptide exhibits binding properties to a desired molecule. Regarding grafting into a biomolecule, for example, the peptides of the present invention can be grafted into a loop structure region between two consecutive domain structures in a protein, such as the ubiquitin molecule described below.

[0063] [Method for producing cyclic peptides] The cyclic peptides of the present invention can be suitably produced by translational synthesis using a cell-free translation system. Specifically, they can be produced by preparing a nucleic acid encoding the cyclic peptide and translating the nucleic acid in a cell-free translation system. Nucleic acids encoding cyclic peptides can be appropriately designed by those skilled in the art using genetic codes used in biological translation systems, reprogrammed genetic codes, or a combination thereof. The nucleic acid may be DNA or RNA.

[0064] According to a method using a cell-free translation system, a tRNA aminoacylated with a non-natural amino acid can be used to efficiently introduce non-natural amino acids into peptides in addition to natural amino acids. For example, by using the artificial aminoacyl-tRNA synthetase Flexizyme developed by the present inventors, it is possible to aminoacylate a tRNA having any anticodon with any natural or non-natural amino acid. Therefore, using this technology, the genetic code consisting of an mRNA triplet can be reprogrammed to encode an amino acid different from that encoded by the translation system of a living organism (see, for example, WO 2008 / 059823).

[0065] [Cyclic Peptide Complex] The cyclic peptide complex of the present invention comprises two cyclic peptides of the present invention, and has a structure in which one cyclic peptide is bound to the other cyclic peptide via a linker.

[0066] As shown in Figure 1, Agrin binds to the first β-propeller structure of its receptor, LRP4. MuSK binds to another propeller structure of LRP4 in its extracellular domain (the Ig1 region, which is the immunoglobulin-like region). Therefore, it is thought that the binding of Agrin to LRP4 forms an Agrin-LRP4-MuSK complex, causing homodimerization of MuSK. In addition, the formation of this complex and the action of the intracellular factor Dok-7 cause the intracellular kinase region of MuSK to transition to an active state, which is known to activate downstream signaling through autophosphorylation of MuSK (Zong Y. et al., GENES & DEVELOPMENT 2012; 26:247-258). It is also known that activation of downstream signaling by MuSK leads to the formation of acetylcholine receptor clusters at postsynaptic sites (Xing G. et al., Neurosci Lett, 2020 Jul 13;731:135013). The cyclic peptide complex of the present invention contains two cyclic peptides with MuSK-binding domains. Therefore, each cyclic peptide in the cyclic peptide complex binds to the extracellular domain of MuSK, potentially inducing MuSK homodimerization (Figure 2). Therefore, the cyclic peptide complex of the present invention is believed to activate downstream signaling via MuSK autophosphorylation, thereby inducing acetylcholine receptor clustering. As a result, the cyclic peptide complex of the present invention is believed to activate signal transmission from nerve to muscle at the neuromuscular junction. Therefore, the cyclic peptide complex of the present invention can be useful as a therapeutic agent for myasthenia gravis, which is caused by abnormalities in signal transmission at the neuromuscular junction. In other words, the cyclic peptide complexes of the present invention and pharmaceutically acceptable salts thereof are useful as pharmaceuticals, and the present invention provides pharmaceutical compositions containing the cyclic peptide complexes of the present invention and pharmaceutically acceptable salts thereof, and the use of the cyclic peptide complexes of the present invention and pharmaceutically acceptable salts thereof as pharmaceuticals.

[0067] The cyclic peptide complex of the present invention can exhibit the above-mentioned effect by containing two cyclic peptides of the present invention, one of which is linked to the other cyclic peptide via a linker, and the two cyclic peptides may be the same or different. From the viewpoint of further promoting the homodimerization of MuSK, it is preferable that the two cyclic peptides in the peptide complex are the same.

[0068] The linker connecting the two cyclic peptides in the cyclic peptide complex is not particularly limited, and any linker with a structure well known in the field of peptide synthesis as a linker connecting peptides can be used. However, from the viewpoint of further promoting homodimerization of MuSK, it is preferable that the length of the linker be 20 Å or more and 100 Å or less.

[0069] Non-limiting examples of linkers connecting two cyclic peptides in a peptide conjugate include polymers such as polyethylene glycol (PEG), peptides, nucleic acids, sugars, and combinations thereof. PEG-containing linkers include those in which two PEGs are bonded to small molecules having two or more functional groups, and a non-limiting example is a carboxylic acid-modified PEG bonded to the amino group of 2,3-diaminopropionic acid. PEG-containing linkers also include PEGs having two functional groups, which may be the same or different, at both ends, and a non-limiting example is a PEG having two maleimide groups at both ends. PEG-containing linkers include those having an ethylene glycol structure (-CH ２ O—), and the polymerization number of ethylene glycol may be 2 to 20 or 5 to 15.

[0070] To further improve blood stability, the peptide conjugates of the present invention may have at least one saturated hydrocarbon group having 8 to 20 carbon atoms (preferably 12 to 18 or 14 to 18 carbon atoms) as a side chain. This saturated hydrocarbon group can preferably be introduced into a linker. For example, when the linker is a small molecule having two or more functional groups to which two PEGs are bonded, a saturated fatty acid may be bonded directly or indirectly to the third functional group of the small molecule. Non-limiting examples include a linker in which a carboxylic acid-modified PEG is bonded to the amino group of 2,3-diaminopropionic acid, and a saturated fatty acid having 8 to 20 carbon atoms (preferably 12 to 18 or 14 to 18 carbon atoms) is bonded indirectly to the carboxyl group of the 2,3-diaminopropionic acid. Non-limiting examples of saturated fatty acids having 8 to 20 carbon atoms include palmitic acid (16 carbon atoms) and myristyl acid (14 carbon atoms).

[0071] [Method for Producing Cyclic Peptide Conjugates] The cyclic peptide conjugates of the present invention can be produced by linking the cyclic peptides of the present invention with an appropriate linker. The step of linking the cyclic peptides of the present invention with a linker can be carried out by those skilled in the art using known methods for linking linkers and peptides, or methods equivalent thereto, depending on the type of linker used. For example, if the linker is a linker in which two PEGs are each bonded to a small molecule having two or more functional groups, an amino group can be introduced into each end of the two PEGs in advance, and the amino group can be linked to the C-terminal carboxyl group of the cyclic peptide of the present invention via an amide bond to prepare a peptide conjugate. Furthermore, if the linker is a PEG having two functional groups (e.g., maleimide groups) at both ends, which may be the same or different, the peptide conjugate can be prepared by chemically bonding the two functional groups to the C-terminal functional group of the cyclic peptide of the present invention (e.g., a cysteine ​​thiol group). Furthermore, when the linker is a peptide, two nucleic acids, one encoding the cyclic peptide of the present invention and the other encoding a linker peptide, can be linked together, and a fusion protein of the cyclic peptide of the present invention and the linker peptide can be expressed to prepare a peptide complex.

[0072] [MuSK-binding domain-grafted ubiquitin molecule] The MuSK-binding domain-grafted ubiquitin molecule of the present invention is one in which part or all of the loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule is replaced with a peptide chain containing the MuSK-binding domain. The MuSK-binding domain-grafted ubiquitin molecule of the present invention also is one in which a peptide chain containing the MuSK-binding domain is inserted into the loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule. In the present invention, a ubiquitin molecule in which the entire loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule is replaced with a peptide of structure (A) is preferred.

[0073] Ubiquitin is a protein commonly found in eukaryotes, known to be highly evolutionarily conserved and thermostable. Ubiquitin also contains two α-helices and five β-sheets, specifically, from the N-terminus to the C-terminus, the α-helices and β-sheets are arranged in the following order: β1 (β-sheet 1), β2 (β-sheet 2), α1 (α-helix 1), β3 (β-sheet 3), β4 (β-sheet 4), α2 (α-helix 2), and β5 (β-sheet 5). The regions connecting the β-sheets and the α-helices and β-sheets are called loops, and the ubiquitin molecule of the present invention is characterized in that a peptide chain containing a MuSK-binding domain is grafted into the loop structure region between β1 and β2.

[0074] The ubiquitin molecule to be grafted (i.e., the ubiquitin molecule serving as a scaffold) can be, for example, a protein (human ubiquitin molecule) consisting of the amino acid sequence of SEQ ID NO: 1. β1 corresponds to the region from amino acid 1 to amino acid 6 in the amino acid sequence of SEQ ID NO: 1, the β1-β2 loop corresponds to the region from amino acid 7 to amino acid 10 in the amino acid sequence of SEQ ID NO: 1, and β2 corresponds to the region from amino acid 11 to amino acid 17 in the amino acid sequence of SEQ ID NO: 1. Hereinafter, the ubiquitin molecule of the present invention will be described using an example in which the ubiquitin molecule to be grafted has the amino acid sequence of SEQ ID NO: 1, but the ubiquitin molecule of the present invention is not limited thereto.

[0075] When the ubiquitin molecule to be grafted has the amino acid sequence of SEQ ID NO: 1, "the entire loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule is replaced with the peptide of structure (A)" means that the region from amino acid 7 to amino acid 10 of the amino acid sequence of SEQ ID NO: 1 has been replaced with the peptide of structure (A). In this case, the ubiquitin molecule of the present invention has the peptide of structure (A) downstream of the 6th amino acid and upstream of the 11th amino acid in the amino acid sequence of SEQ ID NO: 1 (see Figure 23).

[0076] When the ubiquitin molecule to be grafted has the amino acid sequence of SEQ ID NO: 1, the phrase "a part of the loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule is substituted with the peptide of structure (A)" means that any of the following amino acids in the amino acid sequence of SEQ ID NO: 1: the 7th amino acid, the 8th amino acid, the 9th amino acid, the 10th amino acid, the region from the 7th to the 8th, the region from the 8th to the 9th, the region from the 9th to the 10th, the region from the 7th to the 9th, and the region from the 8th to the 10th, is substituted with the peptide of structure (A). For example, when the region from the 8th to the 9th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with the peptide of structure (A), the ubiquitin molecule of the present invention will have the peptide of structure (A) downstream of the 7th amino acid and upstream of the 10th amino acid in the amino acid sequence of SEQ ID NO: 1.

[0077] When the ubiquitin molecule to be grafted has the amino acid sequence of SEQ ID NO: 1, the phrase "having a peptide of structure (A) inserted in the loop structure region between the β1 domain and the β2 domain of the ubiquitin molecule" means that the peptide of structure (A) is inserted between the 6th and 7th amino acids, between the 7th and 8th amino acids, between the 8th and 9th amino acids, between the 9th and 10th amino acids, or between the 10th and 11th amino acids in the amino acid sequence of SEQ ID NO: 1. For example, when the peptide of structure (A) is inserted between the 8th and 9th amino acids in the amino acid sequence of SEQ ID NO: 1, the ubiquitin molecule of the present invention will have the peptide of structure (A) downstream of the 8th amino acid and upstream of the 9th amino acid in the amino acid sequence of SEQ ID NO: 1.

[0078] The ubiquitin molecule to be grafted may be a protein consisting of an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1, as long as its binding activity to MuSK is not impaired. In the present invention, for example, a protein consisting of an amino acid sequence having 90% or more sequence identity (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more) with the amino acid sequence of SEQ ID NO: 1 can be used as the ubiquitin molecule to be grafted. Here, "identity" refers to, for example, the degree of identity when the sequences to be compared are appropriately aligned, and refers to the occurrence rate (%) of exact amino acid matches between the sequences. When calculating identity, for example, the presence of gaps in the sequence and the properties of the amino acids are taken into consideration (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The alignment can be performed, for example, using any algorithm. Specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), or Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated, for example, using a publicly available homology search program such as the above. For example, identity can be calculated using the default parameters of the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0079] In the present invention, the ubiquitin molecule to be grafted can also be a protein having one or more amino acid modifications selected from the group consisting of deletion, substitution, insertion, and addition in the amino acid sequence of SEQ ID NO: 1. The number of modified amino acids can be the same as that produced by known methods such as site-directed mutagenesis, or the same as that occurring naturally. The number of modified amino acids can be, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. When there are multiple modifications, the types of modifications may be the same or different. That is, the modifications may be multiple modifications of the same type (e.g., multiple substitutions) or multiple modifications of different types (e.g., a combination of one or more deletions and one or more substitutions).

[0080] Amino acid modifications in the amino acid sequence of SEQ ID NO: 1 may be conservative modifications (e.g., conservative mutations). A "conservative modification" or "conservative mutation" refers to modifying or mutating one or more amino acids so as not to substantially alter the function of the protein. The amino acid substitution may also be conservative substitution. A "conservative substitution" refers to substituting one or more amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. In a conservative substitution, the substituted amino acid and the substituted amino acid preferably have similar properties and / or functions, for example, chemical properties such as hydrophobicity and hydrophilicity index, polarity, and charge, or physical properties such as secondary structure. Amino acids or amino acid derivatives with similar properties and / or functions are known in the art. Non-limiting examples are as follows: Nonpolar amino acids (hydrophobic amino acids) include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, lysine, etc. Examples of negatively charged amino acids (acidic amino acids) include aspartic acid, glutamic acid, etc.

[0081] The amino acid modification (preferably a substitution, more preferably a conservative modification or conservative substitution) in the amino acid sequence of SEQ ID NO: 1 may also be present in a region other than the β1-β2 loop (the region from positions 7 to 10 of the amino acid sequence of SEQ ID NO: 1) (i.e., the region from positions 1 to 6 and the region from positions 11 onwards of the amino acid sequence of SEQ ID NO: 1), preferably in a region other than β1 (the region from positions 1 to 6 of the amino acid sequence of SEQ ID NO: 1) and the β1-β2 loop (the region from positions 7 to 10 of the amino acid sequence of SEQ ID NO: 1) (i.e., the region from positions 11 onwards of the amino acid sequence of SEQ ID NO: 1), and more preferably in a region other than β1 (the region from positions 1 to 6 of the amino acid sequence of SEQ ID NO: 1), the β1-β2 loop (the region from positions 7 to 10 of the amino acid sequence of SEQ ID NO: 1), and β2 (the region from positions 11 to 17 of the amino acid sequence of SEQ ID NO: 1) (i.e., the region from positions 18 onwards of the amino acid sequence of SEQ ID NO: 1).

[0082] Non-limiting examples of positions of amino acid modifications in the amino acid sequence of SEQ ID NO: 1 include positions 11 (K), 12 (T), 37 (P), 54 (R), 57 (S), 62 (Q), and 65 (S), with positions 11 (K), 54 (R), and 57 (S) being preferred. The amino acid sequence of SEQ ID NO: 1 may have modifications (preferably substitution mutations) at one, two, or three of the above modification positions.

[0083] Non-limiting examples of amino acid modifications in the amino acid sequence of SEQ ID NO: 1 include substitution mutations of K11M, K11E, K11T, T12L, T12K, P37R, R54S, R54G, S57T, Q62K, and S65T, and the amino acid sequence of SEQ ID NO: 1 may have one, two, or three of these mutations.

[0084] Other non-limiting examples of positions for amino acid modification in the amino acid sequence of SEQ ID NO: 1 include positions 75 (G) and 76 (G), and one or two of the above modification positions may have a modification (preferably a deletion).

[0085] The substitution or insertion position of the peptide of structure (A) in a ubiquitin molecule consisting of an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1 can be identified by appropriately aligning the amino acid sequence of the ubiquitin molecule to be grafted with the amino acid sequence of SEQ ID NO: 1. That is, the amino acid sequence of the ubiquitin molecule to be grafted is aligned with the amino acid sequence of SEQ ID NO: 1 using the aforementioned homology search software or program, and the region corresponding to positions 1 to 6 in the amino acid sequence of SEQ ID NO: 1 can be designated as β1, the region corresponding to positions 7 to 10 in the amino acid sequence of SEQ ID NO: 1 can be designated as a β1-β2 loop structure, and the region corresponding to positions 11 to 17 in the amino acid sequence of SEQ ID NO: 1 can be designated as β2.

[0086] The peptide having the structure (A) to be grafted onto the ubiquitin molecule consists of a MuSK binding domain sandwiched between two spacer domains. The MuSK binding domain is YX ２１ SYHTNVVASLKR(X ２１ is P or L), and is a sequence derived from MuSK7 of formula (1). Spacer 1 can be selected from the group consisting of IQPR (SEQ ID NO: 12), KPGV (SEQ ID NO: 13), WDRG (SEQ ID NO: 14), KPGT (SEQ ID NO: 15), PFGV (SEQ ID NO: 16), YFVG (SEQ ID NO: 17) and LEGM (SEQ ID NO: 18), and spacer 2 can be selected from the group consisting of IRY, MMP, QGI, FMP, TGPQ (SEQ ID NO: 19), WNTP (SEQ ID NO: 20) and IRPQ (SEQ ID NO: 21).

[0087] According to the present invention, ubiquitin molecules grafted with MuSK-binding domains having spacer regions of several amino acid residues at both ends unexpectedly had higher MuSK-binding activity than ubiquitin molecules grafted with only the MuSK-binding domain. From the viewpoint of MuSK-binding activity, it is preferable that the MuSK-binding domain has spacer sequences 1 and 2 upstream and downstream thereof in any combination of 1 to 7 in Table 2 below.

[0088]

[0089] Non-limiting examples of peptides of structure (A) include peptides consisting of any of the following amino acid sequences: (Ub006) IQPRYPSYHTNVVASLKRIRY (SEQ ID NO: 22) (Ub007) KPGVYPSYHTNVVASLKRMMP (SEQ ID NO: 23) (Ub008) WDRGYPSYHTNVVASLKRQGI (SEQ ID NO: 24) (Ub009) KPGTYPSYHTNVVASLKRFMP (SEQ ID NO: 25) (Ub010) PFGVYPSYHTNVVASLKRTGPQ (SEQ ID NO: 26) (Ub011) YFVGYPSYHTNVVASLKRWNTP (SEQ ID NO: 27) (Ub012) LEGMYLSYHTNVVASLKRIRPQ (SEQ ID NO: 28) (Ub013) LEGMYPSYHTNVVASLKRIRPQ (SEQ ID NO: 29)

[0090] Whether a ubiquitin molecule grafted with the peptide of structure (A) has binding activity to MuSK can be determined according to the Examples described below. For example, surface plasmon resonance (SPR) can be used to quantitatively measure the binding kinetics of the molecule to MuSK immobilized on a sensor chip, and the binding affinity K Ｄ For example, when the binding activity to MuSK is 1 μM or less or 500 nM or less, it can be determined that the molecule has binding activity to MuSK. Note that the extracellular domain (e.g., amino acids 1-493) of human MuSK isoform 1 precursor (GenBank accession number NP_005583.1) can be used as the MuSK molecule.

[0091] According to a preferred aspect of the present invention, there is provided a MuSK-binding domain-grafted ubiquitin molecule, in which a part or all of the loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule has been replaced with a peptide of structure (A), or a peptide of structure (A) (preferably any of the peptides Ub006 to 013) has been inserted into the loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule, wherein the ubiquitin molecule is: a protein consisting of the amino acid sequence of SEQ ID NO: 1; a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1; or a protein in which, in the amino acid sequence of SEQ ID NO: 1, an alteration selected from the group consisting of deletion, substitution, insertion, and addition of one or more amino acids.

[0092] The MuSK-binding domain-grafted ubiquitin molecules of the present invention have high affinity for MuSK, can bind strongly to MuSK, and can be used as MuSK agonists. Furthermore, as described below, complexes containing the ubiquitin molecules of the present invention bind to the extracellular domain of MuSK, causing MuSK homodimerization and thereby selectively inducing downstream MuSK signaling, thereby activating nerve-to-muscle signaling at the neuromuscular junction. Therefore, unlike the previously studied approach of targeting anti-acetylcholine receptor autoantibodies, the MuSK-binding domain-grafted ubiquitin molecules of the present invention are advantageous in that they can potently activate signaling at the neuromuscular junction, thereby providing therapeutic and research methods that differ from existing methods. In other words, the MuSK-binding domain-grafted ubiquitin molecules of the present invention and pharmaceutically acceptable salts thereof are useful as pharmaceuticals and research reagents, and the present invention provides pharmaceutical compositions comprising the MuSK-binding domain-grafted ubiquitin molecules of the present invention and pharmaceutically acceptable salts thereof, and the use of the MuSK-binding domain-grafted ubiquitin molecules of the present invention and pharmaceutically acceptable salts thereof as pharmaceuticals.

[0093] [Method for producing grafted ubiquitin molecules] The MuSK-binding domain-grafted ubiquitin molecules of the present invention can be suitably produced by translational synthesis using a cell-free translation system. They can be prepared by preparing a nucleic acid encoding the ubiquitin molecule of the present invention and translating the nucleic acid in a cell-free translation system. Nucleic acids encoding the ubiquitin molecules of the present invention can be appropriately designed by those skilled in the art using genetic codes used in biological translation systems, reprogrammed genetic codes, or a combination thereof. The nucleic acid may be DNA or RNA.

[0094] As described above, in the method using a cell-free translation system, unnatural amino acids can be efficiently introduced into peptides in addition to natural amino acids by using tRNA aminoacylated with the unnatural amino acid.

[0095] [Grafted Ubiquitin Molecular Conjugate] The grafted ubiquitin molecular conjugate of the present invention comprises a plurality of ubiquitin molecules of the present invention, and has a structure in which a plurality of grafted ubiquitin molecules are linked to each other via linkers.

[0096] As mentioned above, Agrin binds to the first β-propeller structure of its receptor, LRP4, causing the homodimerization of MuSK, which is bound to another β-propeller structure of LRP4, and is known to activate downstream signaling through the autophosphorylation of MuSK (see FIG. 1). It is also known that activation of downstream signaling of MuSK leads to the formation of acetylcholine receptor clusters at postsynaptic sites. The grafted ubiquitin molecular complex of the present invention comprises multiple pharmacophore structures corresponding to the MuSK binding region, and it is believed that each pharmacophore region in the grafted ubiquitin molecular complex binds to the extracellular domain of MuSK, thereby inducing homodimerization of MuSK (FIG. 2). Therefore, it is believed that the grafted ubiquitin molecular complex of the present invention can activate downstream signaling through the autophosphorylation of MuSK and induce clustering of acetylcholine receptors. As a result, it is believed that the grafted ubiquitin molecular conjugates of the present invention can activate signal transmission from nerve to muscle at the neuromuscular junction. Therefore, it can be said that the grafted ubiquitin molecular conjugates of the present invention are useful as therapeutic agents for myasthenia gravis caused by abnormalities in signal transmission at the neuromuscular junction. That is, the grafted ubiquitin molecular conjugates of the present invention and pharmaceutically acceptable salts thereof are useful as pharmaceuticals, and the present invention provides pharmaceutical compositions containing the grafted ubiquitin molecular conjugates of the present invention and pharmaceutically acceptable salts thereof, and uses of the grafted ubiquitin molecular conjugates of the present invention and pharmaceutically acceptable salts thereof as pharmaceuticals.

[0097] The grafted ubiquitin molecule complex of the present invention contains a plurality of ubiquitin molecules of the present invention, and can exhibit the above-mentioned effect as long as the plurality of ubiquitin molecules of the present invention are linked to each other via a linker, and the plurality of grafted ubiquitin molecules may be the same or different. From the viewpoint of further promoting the homodimerization of MuSK, it is preferable that the plurality of grafted ubiquitin molecules in the complex are the same.

[0098] In the grafted ubiquitin molecule complex of the present invention, the linker connecting multiple ubiquitin molecules is not particularly limited, and any linker with a structure well known in the field of peptide synthesis as a linker connecting peptides or proteins can be used. However, from the viewpoint of further promoting homodimerization of MuSK, it is preferable that the length of the linker be 20 Å or more and 100 Å or less.

[0099] Non-limiting examples of linkers connecting multiple ubiquitin molecules in the grafted ubiquitin molecule complex of the present invention include peptides, polymers such as polyethylene glycol (PEG), nucleic acids, sugars, and combinations thereof. Peptide-containing linkers include peptides having a repeating PA structure (e.g., a peptide of A(PA)v, where v is an integer from 6 to 8, preferably 7) and peptides having a repeating EAAAK structure (e.g., a peptide of (EAAAK)x, where x is an integer from 2 to 4, preferably 3). When a peptide is used as a linker connecting multiple grafted ubiquitin molecules, taking a dimer as an example, the C-terminus of one grafted ubiquitin molecule can be linked to the N-terminus of another grafted ubiquitin molecule via the peptide. A similar structure can also be used for complexes of trimers or more. This structure allows grafted ubiquitin molecules to be expressed as a complex protein from a single nucleic acid. In addition to this, the grafted ubiquitin molecule of the present invention can use a linker containing PEG, and specifically, those described in the cyclic peptide complex of the present invention can be used.

[0100] [Method for producing a grafted ubiquitin molecular conjugate] The grafted ubiquitin molecular conjugate of the present invention can be produced by linking the ubiquitin molecules of the present invention via a peptide. In this case, the grafted ubiquitin molecular conjugate of the present invention can be suitably produced by a translational synthesis method using a cell-free translation system. The grafted ubiquitin molecular conjugate of the present invention can be produced by preparing a nucleic acid encoding the grafted ubiquitin molecular conjugate of the present invention and translating the nucleic acid in a cell-free translation system. The nucleic acid encoding the grafted ubiquitin molecular conjugate of the present invention can be appropriately designed by those skilled in the art using the genetic code used in the translation system of a living organism, a reprogrammed genetic code, or a combination thereof. The nucleic acid may be DNA or RNA.

[0101] As described above, in the method using a cell-free translation system, unnatural amino acids can be efficiently introduced into peptides in addition to natural amino acids by using tRNA aminoacylated with the unnatural amino acid.

[0102] The grafted ubiquitin molecule conjugates of the present invention can also be prepared using linkers other than peptides. The step of linking the grafted ubiquitin molecule conjugates of the present invention with linkers can be carried out by those skilled in the art using known methods for connecting linkers and peptides, or methods equivalent thereto, depending on the type of linker used. Specifically, they can be prepared according to the procedures described in the method for producing the cyclic peptide conjugates of the present invention.

[0103] [Pharmaceutical Compositions] The therapeutic agents and pharmaceutical compositions of the present invention comprise at least one active ingredient selected from the group consisting of the cyclic peptides of the present invention and pharmaceutically acceptable salts thereof, the cyclic peptide complexes of the present invention and pharmaceutically acceptable salts thereof, the MuSK-binding domain-grafted ubiquitin molecules of the present invention and pharmaceutically acceptable salts thereof, and the MuSK-binding domain-grafted ubiquitin molecule complexes of the present invention and pharmaceutically acceptable salts thereof (hereinafter, these compounds and their salts are referred to as the "active ingredients of the present invention").

[0104] As described above, the active ingredients of the present invention are thought to induce MuSK homodimerization, activate downstream signal transduction via MuSK autophosphorylation, and induce acetylcholine receptor clustering. Therefore, since the active ingredients of the present invention can activate signal transmission from nerves to muscles at the neuromuscular junction, they can be used as therapeutic agents for myasthenia gravis, which is caused by abnormal signal transmission at the neuromuscular junction. That is, the therapeutic agents and pharmaceutical compositions of the present invention can preferably be used for the treatment of myasthenia gravis. As used herein, "treatment" includes therapeutic and prophylactic treatment and refers to alleviating or eliminating the cause of a disease, delaying or halting its progression, and alleviating, mitigating, improving, or eliminating its symptoms, and / or suppressing the worsening of its symptoms in a patient with the disease.

[0105] Myasthenia gravis patients include anti-acetylcholine receptor antibody-positive patients, anti-MuSK antibody-positive patients, anti-LRP4 antibody-positive patients, and antibody-negative (seronegative) patients who are negative for all of these autoantibodies. The active ingredient of the present invention can activate MuSK and induce acetylcholine receptor clusters at the postsynaptic site of the neuromuscular junction, and is therefore advantageous in that it can treat not only anti-acetylcholine receptor antibody-positive myasthenia gravis, but also anti-MuSK antibody-positive myasthenia gravis, anti-LRP4 antibody-positive myasthenia gravis, and antibody-negative patients.

[0106] When the active ingredient of the present invention is administered to a subject, the route of administration is not particularly limited as long as the desired therapeutic effect is obtained, and oral administration or parenteral administration (e.g., intravenous administration, subcutaneous administration) can be selected. The therapeutic agent and pharmaceutical composition of the present invention may be formulated by adding a pharmaceutically acceptable carrier, etc. in addition to the active ingredient.

[0107] Examples of dosage forms of pharmaceutical preparations include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, troches, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art (e.g., known methods described in the General Provisions for Preparations of the Japanese Pharmacopoeia, 18th Edition, etc.).

[0108] The formulation can be carried out by a conventional method using, as appropriate, additives such as excipients, binders, disintegrants, lubricants, solubilizers, solubilizing agents, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, wetting agents, dispersants, and antioxidants. The additives are not particularly limited, but examples thereof include purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

[0109] The liquid preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. The liquid preparation may contain wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizing aids, preservatives, etc. The tablets or pills may be coated tablets coated with sugar coating, gastric or enteric substances, etc.

[0110] The dosage of the active ingredient of the present invention can be determined depending on the type of active ingredient, the sex, age, and body weight of the subject, symptoms, dosage form, and route of administration. When the active ingredient of the present invention is administered for the purpose of treating myasthenia gravis, the dosage per adult can be determined, for example, within the range of 0.0001 mg to 1000 mg / kg body weight, but is not limited thereto. Furthermore, the above dosage of the active ingredient can be administered once a day or in two to four divided doses. The active ingredient of the present invention can be administered not only to humans in need thereof, but also to non-human mammals (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, and monkeys).

[0111] According to the present invention, there is provided a method for treating myasthenia gravis, which comprises administering to a subject in need thereof a therapeutically effective amount of at least one selected from the group consisting of the cyclic peptide of the present invention and a pharmaceutically acceptable salt thereof, the cyclic peptide complex of the present invention and a pharmaceutically acceptable salt thereof, the MuSK-binding domain-grafted ubiquitin molecule of the present invention and a pharmaceutically acceptable salt thereof, and the MuSK-binding domain-grafted ubiquitin molecule complex of the present invention and a pharmaceutically acceptable salt thereof, or a therapeutic agent or pharmaceutical composition containing the same.

[0112] The present invention also provides use of at least one selected from the group consisting of the cyclic peptide of the present invention and a pharmaceutically acceptable salt thereof, the cyclic peptide complex of the present invention and a pharmaceutically acceptable salt thereof, the MuSK binding domain-grafted ubiquitin molecule of the present invention and a pharmaceutically acceptable salt thereof, and the MuSK binding domain-grafted ubiquitin molecule complex of the present invention and a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic agent for myasthenia gravis, in a method for treating myasthenia gravis, or as a therapeutic agent for myasthenia gravis.

[0113] [Method for displaying cyclic peptides and method for producing ubiquitin molecules with displayed cyclic peptides] The present invention provides a method for displaying cyclic peptides on ubiquitin molecules together with adjacent spacer sequences. The present invention also provides a method for fusing cyclic peptides to proteins, including a method for producing modified proteins in which the cyclic peptides are fused to ubiquitin molecules and the cyclic peptides are displayed on ubiquitin molecules. Conventional genetic engineering techniques can be used to fuse the cyclic peptides to ubiquitin molecules together with the spacer sequences. The cyclic peptides can be those that have binding activity to a desired molecule, and are preferably cyclic peptides that have binding activity to MuSK molecules.

[0114] As the cyclic peptide, it is preferable to use a cyclic peptide selected by an mRNA display method such as the RaPID method or the TRAP method or a phage display method, and it is more preferable to use an mRNA display method. By using the mRNA display method, the base sequence of the partial amino acid sequence to be inserted into the cyclic peptide can be easily determined.

[0115] The presentation and production methods of the present invention utilize LassoGraft Technology to present cyclic peptides with MuSK-binding activity on the loop structure of a ubiquitin molecule. LassoGrafting is a technique for grafting cyclic peptides with the activity of binding to pharmaceutical targets, such as receptors, onto proteins. For implementation of the presentation and production methods of the present invention, see Mihara, E. et al., Nat Commun 2021, 12:1543 and Japanese Patent No. 6,598,344. This technology embeds (grafts) the internal sequence of only a dozen or so amino acids of a cyclic peptide obtained by the RaPID method into a loop structure on the surface of a protein in a lasso-like fashion, thereby imparting the extremely high specificity and affinity of the original cyclic peptide for pharmaceutical target proteins to the base protein.

[0116] The method of the present invention is characterized in that, when a cyclic peptide having binding activity for a desired molecule (preferably MuSK-binding activity) is presented on the loop structure of a ubiquitin molecule, randomly generated spacer sequences consisting of an amino acid sequence of 0 to 4 residues (preferably 3 or 4 residues) are added to the upstream and downstream sides of the cyclic peptide. That is, one of the two spacer sequences can be placed upstream of the cyclic peptide, and the other spacer sequence can be placed downstream of the cyclic peptide. Furthermore, the cyclic peptide having MuSK-binding activity can be selected from the cyclic peptides of the present invention, and preferably can be selected from the cyclic peptides of formulas (1) to (3).

[0117] In a preferred embodiment of the method of the present invention, when the desired molecule is a MuSK molecule, the cyclic peptide and spacer sequence presented on the loop structure region of the ubiquitin molecule can be structure (B) above. In structure (B), spacer 3 is preferably a peptide chain consisting of any 3 or 4 amino acids, and spacer 4 is preferably a peptide chain consisting of any 3 or 4 amino acids. The MuSK-binding region is a region present in the cyclic peptide that has MuSK molecule-binding activity, and can be selected, for example, from the MuSK molecule-binding active regions specified by the cyclic peptides of formulas (1) to (3), and can be a sequence omitting the C-terminal cysteine ​​involved in the cyclic structure. The MuSK-binding region is preferably YX ２２ SYHTNVVASLKR(X ２２ is P or L) (SEQ ID NO: 11).

[0118] In the presentation method and production method of the present invention, the loop structure region of the ubiquitin molecule can be any of: - a loop structure region between the β1 domain and the β2 domain; - a loop structure region between the β2 domain and the α1 domain; - a loop structure region between the α1 domain and the β3 domain; - a loop structure region between the β3 domain and the β4 domain; - a loop structure region between the β4 domain and the α2 domain; and - a loop structure region between the α2 domain and the β5 domain, and is preferably a loop structure region between the β1 domain and the β2 domain.

[0119] The loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule corresponds to the 7th to 10th amino acid residues of the amino acid sequence of SEQ ID NO: 1. The ubiquitin molecule from which a cyclic peptide is presented in the method of the present invention is not limited to the amino acid sequence of SEQ ID NO: 1. That is, the ubiquitin molecule from which a cyclic peptide is presented can be a protein consisting of an amino acid sequence that shares 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more) sequence identity with the amino acid sequence of SEQ ID NO: 1, or a protein having a modification selected from the group consisting of deletion, substitution, insertion, and addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1. In such cases, the substitution or insertion position of the cyclic peptide in a ubiquitin molecule consisting of an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1 can be identified by appropriately aligning the amino acid sequence of the ubiquitin molecule with the amino acid sequence of SEQ ID NO: 1. When the method of the present invention is carried out on a ubiquitin molecule having an amino acid sequence other than that of SEQ ID NO: 1, reference can be made to the description of the grafted ubiquitin molecule of the present invention.

[0120] In the presentation methods and production methods of the present invention, the two amino acid residues constituting the loop region between the β1 domain and the β2 domain can be selected as the 7th and 10th amino acid residues of a ubiquitin molecule. In this case, the nucleotide sequence of a modified protein in which a cyclic peptide is presented in the form of a ubiquitin molecule can be prepared as follows: In a nucleotide sequence encoding a ubiquitin molecule, nucleotide sequences corresponding to the two amino acid residues constituting the loop region between the β1 domain and the β2 domain of the ubiquitin molecule can be selected, bases present between the selected nucleotide sequences can be deleted as necessary, and a nucleic acid can be prepared having a nucleotide sequence incorporating the partial amino acid sequence of the selected cyclic peptide and nucleotide sequences corresponding to spacer sequences upstream and downstream thereof.

[0121] In the present invention, the method of selecting a base sequence corresponding to two amino acid residues constituting a loop structure and deleting bases present between the selected base sequences as needed, and the method of preparing a nucleic acid having a base sequence incorporating a base sequence corresponding to a partial amino acid sequence of a selected cyclic peptide, are not particularly limited and can be carried out by employing a conventionally known method. Furthermore, since the method of translating the prepared nucleic acid is already well known in the technical field to which the present invention pertains, the nucleic acid can be translated by appropriately applying such a well-known method.

[0122] The modified proteins obtained by the display methods and production methods of the present invention, in which a cyclic peptide having binding activity for a desired molecule (preferably MuSK-binding activity) is displayed on a protein using a ubiquitin molecule as a scaffold, display the cyclic peptide while maintaining the structure and activity of the cyclic peptide, and can therefore be used as reagents, pharmaceuticals, etc. based on the activity of the cyclic peptide. Furthermore, because the modified proteins fused with a cyclic peptide exhibit the activity of the cyclic peptide in addition to the activity of ubiquitin itself, they can also be used as MuSK-binding proteins that are stable in vivo (e.g., in blood or tissues).

[0123] [Screening method] As shown in the examples below, combinations of randomly generated spacer sequences and MuSK-binding regions are designed (see Figures 22 and 23), a library of mRNAs encoding those amino acid sequences is prepared, mRNAs from the mRNA library are translated to prepare modified proteins, and MuSK-binding modified proteins are selected from the obtained modified proteins using MuSK-binding activity as an index, thereby enabling screening for MuSK-binding modified proteins. Specifically, the present invention provides a method for screening for a modified protein capable of binding to a desired molecule (preferably a modified protein capable of binding to a MuSK molecule), comprising: designing an mRNA library containing a base sequence encoding a ubiquitin molecule in which a sequence of a cyclic peptide having binding activity to a desired molecule (preferably a cyclic peptide having binding activity to a MuSK molecule) and randomly generated spacer sequences consisting of 0 to 4 amino acid residues (preferably 3 or 4 residues) linked upstream and downstream of the cyclic peptide are fused to the loop structure region between the β1 domain and the β2 domain; preparing modified proteins by translating mRNA from the mRNA library; and selecting a modified protein capable of binding to the desired molecule (preferably a modified protein capable of binding to a MuSK) from the obtained modified proteins using MuSK-binding activity as an indicator. The steps of preparing the mRNA library (i.e., preparing nucleic acids to be translated) and translating nucleic acids from the mRNA library to prepare modified proteins can be carried out according to the production method of the present invention. The screening method of the present invention can also be carried out in other ways according to the proposed method and production method of the present invention.

[0124] When screening for MuSK-binding modified proteins in the screening methods of the present invention, whether a ubiquitin molecule grafted with a MuSK-binding cyclic peptide (preferably a peptide of structure (B)) has binding activity to MuSK can be determined according to the Examples described below. For example, surface plasmon resonance (SPR) can be used to quantitatively measure the binding kinetics of the molecule to MuSK immobilized on a sensor chip, and when the binding affinity KD measured by SPR is, for example, 1 μM or less or 500 nM or less, it can be determined that the molecule has binding activity to MuSK. The extracellular region (e.g., amino acids 1-493) of human MuSK isoform 1 precursor (GenBank accession number NP_005583.1) can be used as the MuSK molecule.

[0125] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0126] Example 1: Preparation of cyclic peptides and evaluation of MuSK binding activity (1) Preparation of cyclic peptide library and selection of cyclic peptides that bind to MuSK Two thioether-cyclic peptide libraries (L-library and D-library) were constructed using the flexible in vitro translation (FIT) system. N-(2-chloroacetyl)-L-tyrosine (L-ClAcTyr) and N-(2-chloroacetyl)-D-tyrosine (D-ClAcTyr) were introduced into the L-library and D-library, respectively, using a previously reported method (Kawakami, T.; Murakami, H.; Suga, H., Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides. Chem Biol 2008, 15 (1), 32-42.; Morimoto, J.; Hayashi, Y.; Suga, H., Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew Chem Int Ed Engl 2012, 51 (14), 3423-7.; Goto, Y.; Katoh, T.; Suga, H., Flexizymes for genetic code reprogramming. Nat Protoc 2011, 6 (6), 779-90.; Hayashi, Y.; Morimoto, J.; Suga, H., In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 2012, 7 (3), 607-13.).

[0127] The mRNA library corresponding to the thioether-cyclic peptide library was designed to have, in order, an AUG initiation codon encoding L-ClAcTyr or D-ClAcTyr, 8 to 15 NNK random codons (N is G, C, A, or U; K is G or U) encoding random proteinogenic amino acid residues, and a UGC codon encoding Cys. After in vitro translation, a thioether bond was spontaneously formed between the acetyl chloride group of the N-terminal Tyr residue and the sulfhydryl group (thiol group) of the downstream cysteine ​​residue.

[0128] Selection of cyclic peptides that bind to human MuSK (hereinafter also referred to as "affinity selection") was performed using the RaPID (random non-standard peptides integrated discovery) system in combination with the flexible in vitro translation (FIT) system (Figure 3A) (Hayashi, Y.; Morimoto, J.; Suga, H., In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 2012, 7 (3), 607-13.; Hipolito, CJ & Suga, H. Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems. Curr Opin Chem Biol. 16, 196-203, 2012). In this example, cyclic peptides were selected from a D-library.

[0129] More specifically, a library of cyclic peptides was prepared and cyclic peptides that bind to MuSK were selected as follows.

[0130] mRNA library and ClAc-L-Tyr-tRNA fMet CAU, and ClAc-D-Tyr-tRNA fMet CAU was prepared as previously reported (Hayashi, Y.; Morimoto, J.; Suga, H., In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem Biol 2012, 7 (3), 607-13.; Hipolito, CJ & Suga, H. Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems. Curr Opin Chem Biol. 16, 196-203, 2012).

[0131] 1 μM of the mRNA library was ligated with 1.5 μM of puromycin linker using T4 RNA ligase at 25°C for 30 minutes. The puromycin linker DNA was attached to the 3'-terminal constant region of the mRNA library. After purification by phenol-chloroform extraction and ethanol precipitation, 1.2 μM of the resulting mRNA-puromycin conjugate was mixed with 50 μM of ClAc-L-Tyr-tRNA. fMet CAU , or ClAc-D-Tyr-tRNA fMet CAUThe mRNA-cDNA hybrid was then reverse transcribed using RNase-H minus reverse transcriptase (Promega) at 42°C for 1 hour to tag the mRNA-cDNA hybrid with a cyclic peptide. An equal volume of blocking buffer (100 mM Tris-HCl (pH 7.6), 300 mM NaCl, 0.1 v / v% Tween-20, 0.2 w / v% acetylated bovine serum albumin) was then added to the reaction mixture.

[0132] The resulting peptide-mRNA / cDNA conjugate library was then subjected to affinity selection. First, as a control selection, the peptide-mRNA / cDNA conjugate library was passed three times over Dynabeads Protein G (Thermo Fisher Scientific) (each pass was performed for 10 minutes at 4°C). This process, called negative selection, removes undesired bead binding. Next, the peptide-mRNA / cDNA conjugate library was subjected to affinity selection for 30 minutes at 4°C using the above-mentioned beads to which 200-400 nM of human MuSK protein (extracellular domain) had been immobilized. This process is called positive selection.

[0133] The cDNA was eluted from the beads by heating at 95°C for 5 minutes, and a portion of the eluted cDNA was evaluated by quantitative PCR using Sybr Green I and a thermal cycler (LightCycler (Roche)). The remaining cDNA was amplified by PCR and used to transcribe an mRNA library for the next round of affinity selection.

[0134] Human MuSK protein (extracellular domain) was prepared as a recombinant protein as follows. DNA corresponding to the extracellular domain (amino acids 1-493) was amplified by PCR using a Flexi clone (FXC31275, Promega) encoding human MuSK isoform 1 precursor (NCBI Reference Sequence: NP_005583.1) as a template. XhoI and EcoRI restriction enzyme recognition sequences were added to both the 5' and 3' ends of the amplified DNA sequence, respectively, and this was used for insertion into the multicloning site of the pCAG-Neo hIgG1-Fc plasmid (Fujifilm Wako Pure Chemical Industries, Ltd.). This plasmid (pCAG-Neo hMuSK-Fc) enables the expression of a chimeric protein (hMuSK-Fc: molecular weight approximately 200 kDa) of the secretory extracellular domain of human MuSK and human IgG1 Fc in animal cells. In practice, the pCAG-Neo hMuSK-Fc plasmid was transfected into Expi293F cells (Invitrogen) using ExpiFectamine 293 reagent (Invitrogen), and the cells were incubated at 8% CO ２ The cells were cultured with shaking in an incubator for 6 days. The culture supernatant was then collected, centrifuged, and sterilized by filtration using a 0.22 μm filter. The resulting culture supernatant was purified using Protein G to finally obtain a purified hMuSK-Fc preparation with a purity of 99.96% (Akta-FPLC analysis).

[0135] Library preparation, negative selection, and positive selection were counted as one round and each round was repeated four times. After four rounds, significant enrichment of cDNA was observed (compared to the positive selection in which the MuSK protein (extracellular domain) was not immobilized on beads) (Figure 4). The recovered cDNA was amplified by PCR, purified using a column (Nucleospin (Machery-Nagel)), and then sequenced using a high-throughput sequencer (MiSeq (Illumina)). Data analysis was performed using software (CLC Sequence Viewer 7 (Qiagen)).

[0136] An example of a structure of a cyclic peptide or the like that binds to MuSK, selected by the RaPID system, is shown below.

[0137] (In the above formulas, the sequence numbers are as follows: MuSK1 and MuSK1Ac: SEQ ID NO: 5, MuSK1tr: SEQ ID NO: 6, MuSK3tr: SEQ ID NO: 7, MuSK6tr: SEQ ID NO: 8, MuSK4: SEQ ID NO: 3, MuSK7: SEQ ID NO: 2, MuSK9: SEQ ID NO: 4, MuSK13: SEQ ID NO: 9, MuSK13tr: SEQ ID NO: 10.)

[0138] (2) Chemical Synthesis of Cyclic Peptides From the sequence-determined cyclic peptides, sequences with high abundance in the L-library and D-library (MuSK1, MuSK1Ac, MuSK4, MuSK7, and MuSK9) were selected and prepared by chemical synthesis. Specifically, cyclic peptides were synthesized by Fmoc solid-phase peptide synthesis (SPPS) using a Syro Wave automated peptide synthesizer (Biotage) in accordance with a known method (Ito, K. et al., Artificial human Meta agonists based on macrocycle scaffolds. Nature Communications 6, 6372, 2015). After automated synthesis of each peptide, the resulting product was capped with an N-terminal chloroacetyl cap by final coupling with chloroacetic acid. The peptide was cleaved from the resin with a solution of 92.5% trifluoroacetic acid (TFA), 2.5% TIPS (triisopropylsilane), 2.5% EDT (ethanedithiol), and 2.5% water for 3 hours. The peptide product was precipitated from ice-cold ether. After a 3-fold dilution with ice-cold ether, the pellet was then resuspended in 4 / 1 DMSO / H2O. ２ The resulting mixture was suspended in 20 mL of 1H O (30 mL for a 75 μmol scale). 60 μL of triethylamine (or an amount sufficient to achieve a pH of >8) was added, and the reaction was carried out at 42°C (approximately 1 hour). The cyclization reaction was monitored by MALDI / TOFMS (α-cyano4-hydroxycinnamic acid matrix). After confirming completion of the reaction, the reaction was quenched with TFA.

[0139] The cyclized peptides were purified by reverse-phase HPLC (Shimadzu Prominence LC-20AP with a Merck Chromolith Prep column, 200 × 25 mm id). The mobile phases were water containing 0.1 v / v% TFA and acetonitrile containing 0.1 v / v% TFA. Fractions containing the target peptide were identified by MALDI / TOFMS (α-cyano 4-hydroxycinnamic acid matrix), and then lyophilized. For peptides used in cell assays, the following procedure was repeated two or three times: dissolving the dried peptide in a 50 v / v% acetonitrile-water mixture containing 5 mM HCl and lyophilizing.

[0140] (3) Evaluation of Cyclic Peptides The cyclic peptides obtained as described above (MuSK1, MuSK1Ac, MuSK4, MuSK7, and MuSK9) were evaluated for affinity to MuSK (binding activity test).

[0141] The binding kinetics of the cyclic peptide to MuSK immobilized on a sensor chip via an Fc tag (binding affinity (dissociation constant) K Ｄ ) was quantitatively measured using surface plasmon resonance (SPR) to evaluate the binding strength between MuSK and the cyclic peptide.

[0142] The binding affinities of each monomeric peptide, dimeric peptide, monomeric U-body, and dimeric U-body to the target proteins human MuSK, mouse MuSK, and rat MuSK were analyzed by surface plasmon resonance (SPR) at 25°C using a Biacore T200 (Cytiva). Fc-tagged human, mouse, and rat MuSK were immobilized on a Protein G chip or Protein A chip (Cytiva) according to the standard immobilization protocol provided by the manufacturer. The running buffer for the monomeric and dimeric peptides contained 0.010 M phosphate, 0.14 M NaCl, 0.0027 M KCl, 0.05% (v / v) Tween 20, and 0.1% (v / v) DMSO. The running buffer for the monomeric / dimeric U-bodies contained 0.010 M phosphate, 0.14 M NaCl, 0.0027 M KCl, and 0.05% (v / v) Tween 20. A series of concentrations of each monomeric peptide, dimeric peptide, monomeric U-body, and dimeric U-body were injected for analysis. A 1:1 binding fitting analysis was performed using Biacore evaluation software to determine the k, kd, and KD values.

[0143] Fc-tagged human MuSK was prepared according to the procedure described in Example 1(1). Fc-tagged mouse MuSK was prepared by expressing a nucleotide sequence encoding the Fc-tagged mouse MuSK extracellular domain (SEQ ID NO: 34). Fc-tagged rat MuSK was prepared using Recombinant Rat MuSK Fc Chimera Protein (catalog number: 9847-MK-050, R&D Systems), a chimeric protein of rat MuSK and Fc protein.

[0144] The SPR response of each peptide to human MuSK is shown in Figure 5. The binding affinity K Ｄ is shown in Table 3.

[0145]

[0146] As shown in FIG. 5 and Table 3, all of the peptides had high binding activity to MuSK.

[0147] Example 2: Verification of the effectiveness of cyclic peptide complexes against the onset mechanism of myasthenia gravis (1) Preparation of cyclic peptide homodimers In this example, human MuSK-binding cyclic peptides MuSK4, MuSK7, and MuSK9, and human MuSK-binding linear peptide MuSK1Ac were linked with two types of linkers to prepare homodimerized compounds. For MuSK1Ac, a complex in which the N-terminal ends were linked with a linker and a complex in which the C-terminal ends were linked with a linker were prepared. Specifically, the preparations were carried out according to the following procedure.

[0148] First, MuSK1Ac, MuSK4, MuSK7, and MuSK9 were prepared by chemical synthesis. Specifically, cyclic peptides were synthesized by Fmoc solid-phase peptide synthesis (SPPS) using a Syro Wave automated peptide synthesizer (Biotage) according to a known method (Ito, K. et al., Artificial human Meta agonists based on macrocycle scaffolds. Nature Communications 6, 6372, 2015). Specifically, a di-t-butyl-disulfide (StBu) protecting group was introduced at the terminal Cys, and the peptides were prepared on Rink amide resin. After automated synthesis of each peptide, the resulting products were capped with an N-terminal chloroacetyl cap by final coupling with chloroacetic acid. The peptides were cleaved from the resin for 3 hours using a solution of 88% trifluoroacetic acid (TFA), 5% phenol, 5% water, and 2% TIPS. The peptide products were precipitated with ice-cold ether. Dilute 3-fold with ice-cold ether, then resuspend the pellet in 4 / 1 DMSO / H ２The resulting mixture was suspended in 10 mL of HCl (30 mL for a 75 μmol scale). 60 μL of triethylamine (or an amount sufficient to achieve a pH > 8) was added, and the reaction was allowed to proceed at 42°C (approximately 1 hour). The cyclization reaction was monitored by MALDI / TOFMS (α-cyano 4-hydroxycinnamic acid matrix). After confirming completion, the reaction was quenched with TFA. The pH was then checked and adjusted to approximately 7 with sodium hydroxide. 1.2 mL of tributylphosphine (for a 75 μmol scale) was added in two portions: 0.6 mL at first and 0.6 mL after 2 hours. The mixture was stirred vigorously at room temperature for a total of 4 hours to complete the reductive cleavage of the StBu group from the terminal Cys.

[0149] The deprotection reaction of the StBu group was monitored by MALDI / TOFMS (α-cyano4-hydroxycinnamic acid matrix). After completion of the reaction was confirmed, the reaction was quenched with TFA. The deprotected cyclic peptide was purified by reverse-phase HPLC (Shimadzu Prominence LC-20AP with a Merck Chromolith Prep column, 200 × 25 mm id). The mobile phases were water containing 0.1 v / v% TFA and acetonitrile containing 0.1 v / v% TFA. After identifying the fractions containing the target peptide using MALDI / TOFMS (α-cyano4-hydroxycinnamic acid matrix), the fractions were lyophilized. For peptides used in cell assays, the dried peptide was dissolved in a 50 v / v% acetonitrile-water mixture containing 5 mM HCl and lyophilized, a procedure that was repeated two or three times.

[0150] For MuSK4, MuSK7, and MuSK9 used in the linking treatment to prepare the complex, another cysteine ​​residue (the carboxyl group forms an acid amide) for linking with a linker was introduced downstream of the C-terminal cysteine ​​residue. For MuSK1Ac, in which the N-terminuses are linked together via a linker, a cysteine ​​residue (the amino group is acetylated) for linking with a linker was introduced upstream of the N-terminal D-tyrosine residue. For MuSK1Ac, in which the C-terminuses are linked together via a linker, a cysteine ​​residue (the carboxyl group forms an acid amide) for linking with a linker was introduced downstream of the C-terminal glutamic acid residue.

[0151] Next, the lyophilized cyclic peptide monomer (prepared as described above) was resuspended in 1 / 1 ACN / (50 mM sodium phosphate buffer, pH 7.5) at a concentration of approximately 2 mg / mL. While stirring, 0.5 equivalents of Bis-Mal-dPEG™ 11 (Quanta Biodesign) or Bis-Mal-dPEG™ 3 (Quanta Biodesign) as a 100 mM solution in DMSO were slowly added dropwise. Consumption of starting material was monitored by UPLC / LCMS, and additional reactant was added as needed (dimerization was complete within 2 h). In the cases of MuSK1Ac, MuSK4, and MuSK7, the mixture was directly purified by HPLC. In the case of MuSK9, the dimer precipitated from the solution. After the reaction, the precipitate was isolated by centrifugation. The resulting pellet was redissolved in 100% DMSO, loaded onto an HPLC, and purified according to standard procedures. For reference, the structures of the prepared cyclic peptide homodimers are shown in Figures 6-1 and 6-2. Also, the chemical structure of the MuSK7 homodimer using Bis-Mal-dPEG (trade name) 11 as the linker is shown in Figure 7.

[0152] (2) Evaluation of MuSK binding activity of cyclic peptide homodimers The affinity of each of the MuSK1Ac, MuSK4, MuSK7, and MuSK9 homodimers (10 types) obtained according to the procedure described in (1) above was evaluated for affinity to human MuSK (binding activity test). The evaluation test was performed according to the procedure described in Example 1 (3). The results are shown in Table 4.

[0153]

[0154] (3) Preparation of Cyclic Peptide Homodimer Having Palmitoyl Group Introduced A MuSK7 homodimer having palmitoyl group introduced therein (Figure 8 / MuSK7-pal) was prepared according to the following scheme. Specifically, a cyclic peptide was prepared by introducing a linker to the carboxyl group of the C-terminal cysteine ​​residue of MuSK7 synthesized in Example 1(2). Two molecules of the cyclic peptide were subjected to a condensation reaction with a linking unit having palmitic acid in the presence of a coupling reagent by a standard method to obtain a complex. The protecting group (PG) was then removed from the resulting complex, and the complex was purified to obtain a MuSK7 homodimer having a palmitoyl group (R) introduced therein.

[0155]

[0156] (4) Evaluation of MuSK binding activity of palmitoyl-introduced cyclic peptide homodimers The palmitoyl-introduced MuSK7 homodimer (MuSK7-pal), obtained according to the procedure described in (3) above, was subjected to an evaluation of affinity (binding activity test) for human, rat, and mouse MuSK. The test was performed according to the procedure described in Example 1 (3). The results are shown in Table 5.

[0157]

[0158] (5) Evaluation of the effect on acetylcholine receptor clustering A. Myotube cell culture Mouse myoblast-derived cultured cell line C2C12 (CRL-1772) was purchased and used from the American Type Culture Collection. For subculture, Dulbecco's Modified Eagle Medium (DMEM, high glucose, GlutaMAX supplement, pyruvate; GIBCO) was used with 20% fetal bovine serum (FBS; GIBCO). At the time of subculture, the culture medium was removed by aspiration, and the adherent cells were washed with phosphate buffered saline (PBS(-)), and then cultured in a medium containing 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid.２＋ , Ca ２＋ Add CO2-free HBSS (GIBCO) and ２ Incubator (37°C, 5% CO ２ The cells were incubated in a centrifuge (Sorvall X4R Pro, Thermo Fisher Scientific) for 5 minutes. The detached cells were collected and centrifuged at 1000 rpm at 4°C for 5 minutes (Sorvall X4R Pro, Thermo Fisher Scientific). The supernatant was removed by aspiration, and the cells were suspended in fresh culture medium. 100 μL of the supernatant was seeded on a 96-well plate (BioCoat Collagen I 96-well Black / Clear plate, Corning). The cells were incubated in a centrifuge for 5 minutes in a centrifuge (Sorvall X4R Pro, Thermo Fisher Scientific). The supernatant was removed by aspiration, and the cells were suspended in fresh culture medium. 100 μL of the supernatant was seeded on a 96-well plate (BioCoat Collagen I 96-well Black / Clear plate, Corning) and incubated for 5 minutes in a centrifuge (Sorvall X4R Pro, Thermo Fisher Scientific). ２ Incubator (37°C, 5% CO ２ The cells were cultured in a 500-well plate (200 mL) for approximately 72 hours until they reached confluence. The culture medium was then removed, and the first medium change was performed with DMEM containing 2% horse serum (GIBCO) for inducing myotube differentiation. A second medium change was performed 48 hours later, and the next day, the success or failure of differentiation into myotubes was confirmed by bright-field observation using a general-purpose inverted microscope.

[0159] B. Induction of AChR clusters and its quantitative analysis. Differentiation medium containing an appropriate amount of the cyclic peptide MuSK7 homodimer (MuSK7 PEG11 dimer) was prepared in advance in a 96-well plate (NunclonΔSurface, 167008, Nunc Corporation) and used at 100 μL / well during the second medium change during the myotube differentiation induction process. 20 hours after the addition of the cyclic peptide MuSK7 homodimer, the medium was removed, the cells were washed with PBS(-), and then the cells were fixed with PBS(-) containing 4% formaldehyde for 15 minutes at room temperature. After washing several times with 1% BSA-PBS(-), fluorescently labeled α-bungarotoxin (α-Btx, Alexa Fluor 488 conjugate; Invitrogen) was added to the wells at a final concentration of 100 nM in 1% BSA-PBS(-), and the mixture was left to stand at room temperature for 30 minutes in the dark. After washing the cells several times with 1% BSA-PBS(-), 100 μL of PBS(-) was added to all wells, and the cells were subjected to fluorescence observation using a fluorescence imaging device. An EVOS M7000 imaging system (Thermo Fisher Scientific) was used to observe the fluorescently labeled AChR clusters. A 10x objective lens was used for fluorescence imaging, and a 1mm ２Tiff images of the area corresponding to the area were acquired. Three tiff images were acquired per well, and structures with a major axis of 10 μm or more that exhibited Alexa-488 fluorescent signals were defined as AChR clusters and counted. The average number of AChR clusters counted in the three tiff images from the same well was calculated and plotted on a graph. The results are shown in Figures 9A and 9B. With increasing amounts of the cyclic peptide MuSK7 homodimer added, the number of AChR clusters increased dose-dependently in the concentration range from pM to 1 μM, indicating that acetylcholine receptor clusters were induced. On the other hand, when the compound concentration exceeded 1 μM, the number of clusters tended to decrease. We speculate that this bell-shaped dose-response curve reflects the characteristics of the homodimer structure of the cyclic peptide MuSK7 homodimer. From the above, it was confirmed that the human MuSK-binding cyclic peptide homodimer promotes acetylcholine receptor clustering at the muscle cell level.

[0160] C. Efficacy Evaluation of MuSK Agonists Using MuSK Inhibitors We searched for candidate MuSK inhibitors in the KinaseNET database (http: / / www.kinasenet.ca / ), an open resource released by Kinexus (Canada), and identified three tyrosine kinase inhibitors (AST-487, foretinib, and sorafenib). Next, we prepared C2C12-derived myotubes treated with 10 pM recombinant rat agrin protein (550-AG, R&D Systems) alone or in combination with various tyrosine kinase inhibitors (3 nM). 20 hours after compound addition, we detected and observed Agrin-induced AchR clusters (crizotinib, linifanib, AST-1306, dovitinib, and BMS-754807, five of which were non-MuSK-inhibitory tyrosine kinase inhibitors). As a result, in the group to which ATS-487 was added simultaneously with Agrin, no AChR cluster formation was observed even at low concentrations of 1 nM. It was suggested that foretinib and sorafenib have lower MuSK inhibitory effects than AST-487. Meanwhile, the number of AChR clusters induced by the other five tyrosine kinase inhibitors (crizotinib, linifanib, AST-1306, dovitinib, and BMS-754807) was comparable to that of the negative control group (DMSO only). These findings demonstrate for the first time at the cellular level that AST-487 is a tyrosine kinase inhibitor that exhibits MuSK selectivity.

[0161] Next, we investigated the effect of AST-487 on the AChR cluster-inducing activity of the cyclic peptide MuSK7 homodimer. The results are shown in Figure 9C. Although AChR cluster formation by the cyclic peptide MuSK7 homodimer was observed in the absence of AST-487, no AChR clusters were detected in the presence of AST-487. This demonstrates at the cellular level that the AChR cluster induction process by the cyclic peptide MuSK7 homodimer is dependent on MuSK kinase activity, similar to that of the natural MuSK activator Agrin.

[0162] (6) Evaluation of MuSK dimerization activity (NanoBiT assay) A. Culture of non-muscle cell lines Expi293 cells were purchased from Thermo Fisher Scientific and used. For subculture, DMEM (high glucose, GlutaMAX supplement, pyruvate; GIBCO) was used with 10% fetal bovine serum (FBS; GIBCO). At the time of subculture, the culture medium was removed by aspiration, and the adherent cells were washed with phosphate-buffered saline (PBS(-)), and then cultured in a medium containing 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid. ２＋ , Ca ２＋ Add CO2-free HBSS (GIBCO) and ２ Incubator (37°C, 5% CO ２ The cells were incubated in a centrifuge (Sorvall X4R Pro, Thermo Fisher Scientific) for 5 minutes. The detached cells were collected and centrifuged at 1000 rpm at 4°C for 5 minutes (Sorvall X4R Pro, Thermo Fisher Scientific). The supernatant was removed by aspiration, and the cells were suspended in fresh culture medium and expanded in a new culture vessel.

[0163] (b) Evaluation of MuSK dimerization activity of MuSK agonists. LgBiT (17.6 kDa) and SmBiT (11 amino acids) are split luciferases designed to exhibit luciferase activity when they form a complex (Dixon, AS et al. ACS Chem. Biol. 2016, 11, 400-8). At steady state, the affinity between the two is approximately 190 μM, and even in the presence of substrate, the amount of luminescence is negligible. However, when the two proteins come into close proximity, they form a complex that exhibits luciferase activity. The NanoBiT assay utilizes this property to evaluate the interaction between any two proteins. Because dimerization between two MuSK molecules is thought to be one of the requirements for activation of the receptor tyrosine kinase MuSK, we employed the NanoBiT assay to evaluate the MuSK-activating ability of MuSK7-pal (Fig. 10A). Specifically, SmBiT or LgBiT was fused downstream of the extracellular domain (amino acids 1-493) of a human MuSK cDNA clone (FXC31275, Promega) and synthetic DNA containing six histidine residues at the C-terminus was prepared and inserted into the EBMulti-Neo plasmid (Fujifilm Wako Pure Chemical Industries). Using FuGENE6 (Promega), two expression plasmids, MuSK-SmBiT and MuSK-LgBiT, were simultaneously transfected into Expi293 cells. After culturing for 48-72 hours in Opti-MEM medium (Invitrogen), the culture supernatant was collected. Next, multiple solutions of the test compound, MuSK7-pal, were prepared at arbitrary dilutions. 50 μL of the collected culture supernatant and 50 μL of the diluted MuSK agonist solution were added to a 96-well microplate (white, Nunc) and placed in a shaker (inVitro Shaker Mix-EVR, TAITEC). The plates were then shaken at room temperature for 15 minutes (200 rpm). According to the Nano-Glo Live Cell Assay (Promega) manual, 25 μL of substrate solution consisting of the provided buffer and substrate was added to each well, and then luciferase luminescence in the wells was immediately detected using a GloMax Navigator (Promega) (measurement time: 0.5 sec / well).The results are shown in Figure 10B. As the amount of MuSK7-pal added increased, the amount of luminescence increased dose-dependently in the concentration range from pM to 40 nM, indicating that MuSK dimerization was promoted. Conversely, when the compound concentration exceeded 40 nM, MuSK dimerization tended to decrease. We speculate that this bell-shaped dose-response curve reflects the characteristics of the homodimeric structure of MuSK7-pal. Combining these results with those in (5) above, it was confirmed that the homodimer of the human MuSK-binding cyclic peptide promotes MuSK kinase activity-dependent acetylcholine receptor clustering.

[0164] Example 3: Evaluation of therapeutic effect of cyclic peptide complex on myasthenia gravis model animal (1) Evaluation of therapeutic effect on myasthenia gravis model animal (1) A. Objective The effects of a dimer (MuSK7-pal / Example 2(3)) (high dose) of the cyclic peptide (p7) of formula (1) on various disease parameters in a rat PTMG model using an anti-acetylcholine receptor monoclonal antibody (mAb35) were evaluated.

[0165] B. Test Method: Five-week-old female rats (n=20, LEW / CrlCrlj, 4 weeks old at time of delivery, Jackson Laboratory Japan) were divided into four groups (Groups A, B, C, and D) based on body weight measurements. Vehicle was intravenously administered to Group A, and the test compound MuSK7-pal dissolved in vehicle was intravenously administered to Groups B (3 mg / kg), C (2 mg / kg), and D (1 mg / kg) (Day 0). Two hours after intravenous administration, an anti-nicotinic acetylcholine receptor monoclonal antibody (mAb35, National Hospital Organization Nagasaki Kawatana Medical Center) dissolved in PBS (pH 7.4) was intraperitoneally administered to all animals at 200 μg / rat as a drug to induce myasthenia gravis (Day 0). The vehicle used as the solvent for the compound was phosphate buffered saline (PBS) containing 20% ​​hydroxypropyl-β-cyclodextrin and 0.02% polysorbate 80 at pH 7.4 (the same applies to Example 3(2)).

[0166] The body weight of the animals was then measured before antibody administration (pre) and at 0, 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration. The movement, muscle strength, and grip strength of the animals were evaluated using the following tests.

[0167] <Movement score> The movement of the animals was evaluated using a 6-level scale (score) at 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration. The evaluation criteria were as follows: 0: normal; 1: decreased grip strength of the forelimbs and hind limbs; 2: incomplete paralysis of the hind limbs; 3: complete paralysis of the hind limbs and inability to stand; 4: moribund; 5: death.

[0168] The average score of the rats in each group was calculated. Transport agar (Oriental Yeast Co., Ltd.) and crushed pellet food were placed in cages containing rats judged to have a score of 2 or higher.

[0169] <Wire-hang Test> Approximately 30 hours after antibody administration, the muscle strength of the rats was measured using the lid (made of wire mesh) of a plastic cage with a lid (Clean 200-TPX cage; CL-0108-2, CLEA Japan). Specifically, the rat was left to rest for at least 15 minutes, and after confirming that it was in a resting state, it was placed on the cage lid. Next, the cage lid was gently shaken approximately three times so that the rat could grasp the cage lid, and then the cage lid was turned upside down. The turned lid was held at a height (approximately 60 cm from the floor) that was high enough for the rat to be unable to climb down easily, but would not cause injury if it fell. The latency time for the rat to fall from the cage lid was measured up to a maximum of 120 seconds.

[0170] Grip Strength Test Approximately 30 hours after antibody administration, rat muscle strength was measured using a mouse grip strength meter (GRIP STRENGTH METER FOR MICE, MODEL MK-380M, Muromachi Kikai Co., Ltd.). Specifically, the rat was left stationary for 15 minutes or more, and after confirming that it was in a resting state, the rat's forelimbs were allowed to cling to the mouse grip strength meter. Next, the rat's body was pulled, and the scale on the grip strength meter was read when the rat released the wire mesh (first time). The above procedure was repeated once more (second time). If the difference between the first and second measurements was 20% or less, the average value was used as the measured value. If the difference was 20% or more, the above procedure was repeated once more (third time), and the average of the two closest values ​​out of the three measurements was used as the measured value.

[0171] C. Results The weight measurement results for each group are shown in Figure 11. Specifically, the weight change in the control group A was observed from 0 hours to 57 hours, and then increased slightly. Groups B and C also showed weight changes similar to those in group A, but the degree of weight loss was slower than in group A. Group D showed almost no significant weight loss, as seen in the other groups.

[0172] The movement score measurement results for each group are shown in Figure 12. Specifically, the score results for the control group A showed that onset began at 9 hours, peaked at 33 hours, and then declined. In groups B and C, onset was not observed in most individuals, and overall scores were low at all measurement points. In group D, all individuals scored 0 at all measurement points, and no pathological onset was observed.

[0173] The results of measuring the latency to fall (Latency) in the Hanging Wire Test for each group are shown in Figure 13. Specifically, the mean latency to fall for the control group A was 0.0 seconds (±0.00 SEM), meaning that the rats fell immediately upon the start of the test. The mean latency to fall for each test compound administered group was 17.6 seconds (±10.46 SEM) for group B, 29.2 seconds (±22.89 SEM) for group C, and 69.6 seconds (±23.78 SEM) for group D, which were longer than the latency of group A, and were significantly longer in groups B and D compared to group A (Kruskal-Wallis test).

[0174] The results of the grip strength measurement for each group in the Grip Strength Test are shown in Figure 14. That is, the average grip strength of the control group A was 142 g (±16.28 SEM), while the grip strength of group B was 467 g (±35.54 SEM), the grip strength of group C was 474 g (±43.03 SEM), and the grip strength of group D was 577 g (±24.89 SEM), which were significantly stronger than the grip strength of group A (one-way analysis of variance (p<0.0001) and Dunnett's test (p<0.0001)).

[0175] D. Discussion As stated in C. above, the myasthenia gravis-like symptoms observed in the control group (Group A) became milder in Groups B and C, and were barely observed in Group D, indicating that all test compounds were effective against the symptoms of myasthenia gravis, with the effect being particularly pronounced in Group D.

[0176] (2) Evaluation of the therapeutic effect on myasthenia gravis model animals (2) A. Objective The effects of MuSK7-pal (Example 2(3)) (low dose) on various disease parameters in a rat PTMG model using an anti-acetylcholine receptor monoclonal antibody (mAb35) were evaluated.

[0177] B. Test Method: Five-week-old female rats (n=24, LEW / CrlCrlj, 4 weeks old at time of delivery, Jackson Laboratory Japan) were divided into four groups (Groups A, B, C, and D) based on body weight measurements. Vehicle was intravenously administered to Group A, and the test compound MuSK7-pal dissolved in vehicle was intravenously administered to Groups B (0.5 mg / kg), C (0.25 mg / kg), and D (0.1 mg / kg) (Day 0). Two hours after intravenous administration, an anti-nicotinic acetylcholine receptor monoclonal antibody (mAb35, National Hospital Organization Nagasaki Kawatana Medical Center) dissolved in PBS (pH 7.4) was intraperitoneally administered to all animals at 200 μg / rat as a drug to induce myasthenia gravis (Day 0).

[0178] The body weight of the animals was then measured before antibody administration (pre) and at 0, 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration. The movement, muscle strength, and grip strength of the animals were evaluated using the following tests.

[0179] <Movement Score> The movement of the animals was evaluated at 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration using a 6-level evaluation scale (score). The evaluation criteria were the same as those in (1) (a) above.

[0180] <Wire-hang Test> Approximately 30 hours after antibody administration, a wire-hang test was performed using the same procedure as in (1) above.

[0181] Grip Strength Test Approximately 30 hours after antibody administration, a grip strength test was performed using the same procedure as in (1) above.

[0182] C. Results The weight measurement results for each group are shown in Figure 15. Specifically, the control group A showed weight loss from 0 to 57 hours, and then a slight increase. Groups B, C, and D also showed weight changes similar to those of group A, but the degree of weight loss was slower than that of group A.

[0183] The movement score measurement results for each group are shown in Figure 16. Specifically, the score results for the control group A showed onset starting at 24 hours, peaked at 48 hours, and then declined. In group B, most individuals did not show onset, and overall scores were low at all measurement points. In group B, scores were significantly lower than in group A at all measurement points from 24 hours onwards (Dunn's test). In group C, like group B, most individuals did not show onset, and scores were low at all measurement points. In group C, scores were significantly lower than in group A at all measurement points except 9 hours and 57 hours (Dunn's test). In group D, half of the group did not show onset, and overall scores were lower than the control group. No significant differences were observed between group D and group A at any measurement point (Dunn's test).

[0184] The results of the measurement of the latency to fall (latency) in the Hanging Wire Test for each group are shown in Figure 17. Specifically, the mean latency to fall for the control group A was 20.0 seconds (±20.00 SEM), but all individuals, except for a few animals, immediately fell at the start of the test. The mean latency to fall for each test compound administration group was 11.3 seconds (±3.91 SEM) for group B, 32.3 seconds (±18.04 SEM) for group C, and 25.3 seconds (±19.07 SEM) for group D. Group B had a shorter latency to fall than group A, but no animals, except for animal number D-01, immediately fell at the start of the test, although this did not reach statistical significance (Kruskal-Wallis test; H(3) = 6.63, n.s.).

[0185] The results of the Grip Strength Test for each group are shown in Figure 18. That is, the average grip strength of the control group A was 177 g (± 60.05 SEM), while the grip strength of group B was 461 g (± 27.61 SEM), the grip strength of group C was 476 g (± 25.51 SEM), and the grip strength of group D was 452 g (± 86.32 SEM). Although the grip strengths of groups B, C, and D were stronger than that of group A, the difference was not statistically significant (Kruskal-Wallis test).

[0186] D. Discussion As mentioned in C. above, although weight loss and decreased muscle strength, which were symptoms of myasthenia gravis observed in the control group (Group A), were also observed in the test compound administration groups, the movement scores were mild in Group D and suppressed in Groups B and C, confirming the efficacy of all test compounds against the symptoms of myasthenia gravis.

[0187] (3) Summary The results of Example 2(4) and the above (1) and (2) demonstrated that MuSK7-pal, one of the cyclic peptides of the present invention, not only has MuSK binding activity but also actually exerts a therapeutic effect in myasthenia gravis model animals.

[0188] Example 4: Grafting of cyclic peptide pharmacophore sequences onto ubiquitin molecules and evaluation of MuSK binding activity of artificial ubiquitin molecules (1) Verification of the effectiveness of the lasso grafting method A. Overview The internal sequences of aMD4 and aMD5 (Ito, K. et al., Nat Commun 2015, 6 (1): 6373.), which are MET (hepatic growth factor receptor)-binding macrocyclic peptides, were grafted onto ubiquitin molecules using the lasso grafting method (Mihara, E. et al., Nat Commun 2021, 12:1543.), and it was confirmed that the MET binding activity of the ubiquitin molecules to which the macrocyclic peptides were grafted was weaker than that of the original macrocyclic peptides. Therefore, randomized spacer sequences of 0 to 3 amino acid residues were added to the N- and C-termini of the pharmacophore sequence of the macrocyclic peptide to be grafted, while the pharmacophore sequence of the macrocyclic peptide was left unchanged, and a library was constructed and selected to evaluate the MET binding activity. Specifically, the procedure was as follows.

[0189] Ubiquitin molecules as scaffolds for the lasso-grafting method. A family of ubiquitin-based ligands was prepared by lasso-grafting pharmacophore sequences onto the loop structure region between the β1 and β2 domains of the ubiquitin molecule (β1-β2 loop) (Fig. 19a). The pharmacophore sequence of aMD4 (YRQFNRRTHEVWNLD; SEQ ID NO: 35) or the pharmacophore sequence of aMD5 (YWYYAWDQTYKAFP; SEQ ID NO: 36) was sandwiched between "G" or "GG" spacers and replaced the L8-T9 or T9-G10 residues in the β1-2 loop (Ub-aMD4_n1-4 and Ub-aMD5_n1-4, naive U-bodies, Fig. 20a, b).

[0190] The dissociation constants and binding rate constants of each U-body construct to the human MET ectodomain were measured by SPR at 25°C using a Biacore 8K instrument (Cytiva). His- and hFc-tagged human MET ectodomain was purchased from Sino Biological (10692-H03H). The running buffer was HBS-EP+ buffer (Cytiva) containing 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% Surfactant P20. His- and hFc-tagged human MET ectodomain was immobilized on a Series S SensorChip Protein G (Cytiva) according to the standard immobilization protocol provided by the manufacturer. The dissociation constants of the native U-body constructs were determined by multi-cycle analysis in parallel mode. Eight concentrations of each U-body construct were injected at a flow rate of 30 μL / min, and the resulting binding responses were analyzed using Biacore insight evaluation software and fitted with a steady-state affinity model.

[0191] The results showed that the naive U-bodies bound to MET, but with significantly weaker affinities than the parent macrocyclic peptides in all cases (Figure 20a, b). Furthermore, the naive U-body constructs exhibited circular dichroism spectra nearly identical to those of wild-type Ub, indicating that the scaffold ubiquitin molecule was correctly folded in the naive U-body constructs. These results suggest that the decreased affinity was not due to misfolding but rather to a conformational change in the lasso-grafted pharmacophore. Rather, we speculated that the decreased binding activity was due to an inappropriate distance between the N- and C-terminal regions of the pharmacophore for lasso grafting. Taking into account the spacer residues that may affect the macrocyclic active form of the pharmacophore peptide, we hypothesized that optimizing the spacer at the grafting site could address the decreased binding activity (see Figure 3B).

[0192] C. Optimization of flanking spacer sequences by mRNA display. To optimize the flanking sequences of the pharmacophore, we used mRNA display to reselect new flanking spacer sequences with superior binding ability on the ubiquitin scaffold (Fig. 19b, c). Two mRNA libraries were constructed, encoding aMD4-grafted U-body (Ub-aMD4) or aMD5-grafted U-body (Ub-aMD5). Various flanking spacer sequences were used to replace 0–4 consecutive residues in the β1-2 loop with pharmacophore sequences flanked upstream and downstream by 0–3 randomized spacer residues (Fig. 19b and Fig. 21). The theoretical diversity of the libraries was 9.9 × 10 ８ The number of practical variants translated by the in vitro translation cocktail (>10 １１ Therefore, all possible combinations were exhaustively explored. Specifically, the following steps were taken:

[0193] Preparation of mRNA Library The DNA template for the mRNA library was prepared by assembling DNA oligos in two steps. In the first step, forward and reverse primers were mixed and an extension reaction was performed to obtain double-stranded DNA encoding the β1-β2 loop sequence, spacer sequence, and pharmacophore sequence (the spacer sequence was randomized with NNK codons). The extension reaction was performed in a 30 μL reaction scale using 1x KOD One PCR Master Mix -Blue- (Toyobo) and 0.3 μM forward and reverse primers.

[0194] In the second step, the extension products were mixed and amplified by PCR to add the constant region. PCR was performed in a 300 μL reaction using 1× KOD One PCR Master Mix -Blue- (Toyobo), 10% (v / v) of the mixed extension products, and 0.3 μM forward and reverse primers. The PCR product was extracted with phenol / chloroform, precipitated with ethanol, dissolved in water, and used for in vitro transcription in a 660 μL reaction at 37°C overnight. The transcription reaction was performed in 40 mM Tris-HCl (pH 8.0), 1 mM spermidine, 0.01% (v / v) Triton X-100, 10 mM DTT, and 30 mM MgCl. ２ The solution contained 5 mM NTP, 30 mM KOH, template DNA solution, 0.1% (v / v) RNasin ribonuclease inhibitor (Promega), and 0.12 μM T7 RNA polymerase. Additionally, 76 μL of DNase buffer (400 mM Tris-HCl (pH 8.0), 100 mM MgSO ) was added. ４ , 10 mM CaCl ２ ) and 20 μL RQ1 RNase-free DNase (Promega) were added, and the mixture was incubated at 37°C for 1 hour. The resulting mRNA transcripts were precipitated by adding 132 μL EDTA (pH 8.0), 89 μL 3 M NaCl, and 785 μL isopropanol, followed by centrifugation. After washing with 70% (v / v) ethanol, the pellet was dissolved in 150 μL water and resuspended in an equal volume of 2x RNA loading buffer (8 M urea, 2 mM NaCl).２ EDTA-2H ２ 0, 2 mM Tris-HCl, pH 7.5) was added. mRNA was purified on a 6% (v / v) polyacrylamide gel containing 8 M urea, extracted and precipitated with 0.3 M NaCl solution, and dissolved in water to a final concentration of 10 μM. The mRNA library was covalently linked to a puromycin linker using T4 RNA ligase before translation.

[0195] Selection by mRNA display The translation system used to generate the U-body library was 50 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 12.3 mM magnesium acetate, 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 20 mM phosphocreatine, 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolate, 2 mM spermidine, 1 mM dithiothreitol, 1.5 mg / mL E. coli total tRNA, 1.2 μM E. coli ribosome, 0.6 μM methionyl-tRNA formyltransferase, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 0.26 μM EF-G, 10 μM EF-Tu, 0.66 μM EF-Ts, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 4 μg / ml, creatine kinase, 3 μg / ml myokinase, 0.1 μM inorganic pyrophosphatase, 0.1 μM nucleotide diphosphate kinase, 0.1 μM T7 RNA polymerase, 0.73 μM AlaRS, 0.03 μM ArgRS, 0.38 μM AsnRS, 0.13 μM AspRS, 0.02 μM CysRS, 0.06 μM GlnRS, 0.23 μM GluRS, 0.09 μM GlyRS, 0.02 μM HisRS, 0.40 μM IleRS, 0.04 μM LeuRS, 0.11 μM The Ub-body library was prepared using 2.5 μL of a translation system containing 0.03 μM LysRS, 0.68 μM MetRS, 0.16 μM ProRS, 0.04 μM SerRS, 0.09 μM ThrRS, 0.03 μM TrpRS, 0.02 μM TyrRS, and 0.02 μM ValRS, 0.5 mM each of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, and 1.5 μM mRNA linked to a puromycin linker. The Ub-body library was translated in a 2.5 μL translation system at 37°C for 30 minutes.

[0196] The reaction mixture was then incubated at 25°C for 7 minutes. Afterwards, 0.5 μL of 500 mM EDTA (pH 8.0) was added, followed by an additional 5 minutes of incubation at 37°C to induce ribosome dissociation from the mRNA-protein conjugates. Reverse transcription was performed at 42°C for 30 minutes using a reverse primer (SSG2an13.R36 for the Ub-aMD4 library and SGS2an13.R36 for the Ub-aMD5 library) and M-MLV reverse transcriptase lacking RNase H activity (Promega). The cDNA-mRNA-protein conjugates were then mixed with 2.5 μL of Dynabeads Protein G (Thermo Fisher) bearing human IgG1 Fc on their surface and incubated at 4°C for 5 minutes, a process known as negative selection, in triplicate. The supernatant from the negative selection was collected and mixed with Dynabeads Protein G immobilized with MET (see (1)B) at 4°C for 10 minutes, after which the beads were washed three times (first round) or six times (second to sixth rounds) with 100 μL of TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.6).

[0197] Next, 100 μL of 1× PCR buffer [120 mM Tris-HCl (pH 8.0), 2.5 mM MgCl ２ , 6 mM (NH ４ ) ２ SO ４One microliter of the following buffer (10 mM KCl, 0.5 mM dNTPs, 0.001% (w / v) AcBSA, 0.1% (v / v) Triton X-100, 0.25 μM T7G10Ub6.F63, and 0.25 μM reverse primer (SSG2an13.R36 for the Ub-aMD4 library and SGS2an13.R36 for the Ub-aMD5 library)) was added to the beads, and the cDNA was eluted by heating the mixture at 95°C for 5 minutes. One microliter of the eluate was mixed with 19 μL of 1x PCR buffer containing SYBR Green I and KOD DNA polymerase, and the amount of cDNA was quantified by real-time PCR (LightCycler Nano, Roche). The remaining eluate was diluted 8-fold with 1x PCR buffer containing KOD DNA polymerase, amplified by PCR, extracted with phenol / chloroform, precipitated with ethanol, dissolved in 40 μL of 50 mM KCl, and used for in vitro transcription using the same procedure as for library preparation. The transcribed mRNA library was precipitated and dissolved in water to a final concentration of 10 μM. If necessary, the transcribed mRNA library was precipitated and then purified using a 4% (v / v) polyacrylamide gel containing 8 M urea.

[0198] After six rounds of selection, the entire cDNA library was sequenced using a next-generation sequencer (NGS) as follows.

[0199] The recovered cDNA was PCR amplified using KOD One PCR Master Mix -Blue- (Toyobo) with 0.5 μM Rd1T7g10M.F70 and an13Rd2.R49 as primers according to the manufacturer's instructions. The products were then subjected to a second PCR step, and sequence barcodes were generated for each sample using KOD One PCR Master Mix -Blue- and Nextera XT v2 Set (Illumina sequence) primers. The PCR products were then combined, extracted with phenol / chloroform, and column purified using a NucleoSpin kit (TaKaRa) according to the manufacturer's instructions. The concentration of the cDNA samples was measured using a Qubit (ThermoFisher) dsDNA HS kit. The resulting DNA was appropriately diluted, denatured, and combined with denatured PhiX Control v3 (Illumina). The denatured DNA was sequenced on an Illumina MiSeq instrument using the v3 kit in paired-end read 2x300 cycle mode, and data were collected in fastq format after stitching.

[0200] Sequence analysis yielded U-bodies with MET binding activity comparable to that of the parent macrocyclic peptide (Fig. 20a, b).

[0201] e. Discussion As described above, when grafting a cyclic peptide pharmacophore onto a ubiquitin molecule, it was shown to be effective to introduce randomized spacer sequences upstream and downstream of the pharmacophore sequence and to perform selection while optimizing the spacer sequence using the mRNA display method.

[0202] (2) Grafting of a cyclic peptide onto a ubiquitin molecule and selection of a grafted ubiquitin molecule (U-body) that binds to MuSK Based on the findings in (1) above, we investigated lasso-grafting the pharmacophore of MuSK7 (Example 1(1)) onto the β1-2 loop of a ubiquitin molecule. The spacer sequences adjacent to the upstream and downstream sides of the pharmacophore were optimized as follows. Specifically, to optimize the adjacent sequences of the pharmacophore, we used the mRNA display method to reselect new adjacent spacer sequences with excellent binding ability on the ubiquitin scaffold. An mRNA library encoding a portion of the amino acid sequence of MuSK7 (YPSYHTNVVASLKR) (SEQ ID NO: 37) was constructed. For construction, the β1-2 loop (T7-G10) sequence was replaced with "random sequence (3 or 4 amino acids)-YPSYHTNVVASLKR-random sequence (3 or 4 amino acids)" as shown in Figure 22. The theoretical diversity of the library was 2.8 x 10 １０ The number of practical variants translated by the in vitro translation cocktail (>10 １１ ) was lower than the MuSK-binding activity. Therefore, all possible combinations were exhaustively explored. Preparation of the mRNA library and selection by mRNA display using MuSK-binding activity as an index were performed in the same manner as in (1) (c) above.

[0203] The recovery rates (%) for each of the six rounds are shown in Figure 23. After six rounds of selection, the entire cDNA library was sequenced using a next-generation sequencer. Sequencing by next-generation sequencing was performed in the same manner as in (1) (c) above.

[0204] As a result of sequence analysis, the following ubiquitin molecules (SEQ ID NOS: 38 to 45) (hereinafter sometimes simply referred to as Ub006 to Ub013) to which the MuSK binding domain (including the spacer domain) was grafted were obtained. (Ub006) IQPRYPSYHTNVVASLKRIRY (SEQ ID NO: 22) (Ub007) KPGVYPSYHTNVVASLKRMMP (SEQ ID NO: 23) (Ub008) WDRGYPSYHTNVVASLKRQGI (SEQ ID NO: 24) (Ub009) KPGTYPSYHTNVVASLKRFMP (SEQ ID NO: 25) (Ub010) PFGVYPSYHTNVVASLKRTGPQ (SEQ ID NO: 26) (Ub011) YFVGYPSYHTNVVASLKRWNTP (SEQ ID NO: 27) (Ub012) LEGMYLSYHTNVVASLKRIRPQ (SEQ ID NO: 28) (Ub013) LEGMYPSYHTNVVASLKRIRPQ (SEQ ID NO: 29)

[0205] (3) Preparation of MuSK-binding domain-grafted ubiquitin molecules. Homodimers and homotetramers of Ub006-013 and Ub012 were prepared by expression from recombinant microorganisms. The Ub012 homodimer and homotetramer were prepared by connecting the C-terminus and N-terminus of Ub012 with a peptide linker (A(PA) ７ As a control for the complex, a dimer of ubiquitin molecules was prepared. Specifically, the C-terminus of one ubiquitin molecule was linked to the N-terminus of the other ubiquitin molecule with a peptide linker (A(PA) ７ ) to prepare a dimer of ubiquitin molecules.

[0206] Polynucleotides encoding Ub006 to 013 (SEQ ID NOs: 38 to 45), Ub012 homodimer (SEQ ID NO: 54), Ub012 homotetramer (SEQ ID NO: 56), and ubiquitin molecule dimer (SEQ ID NO: 58) were each cloned into the pET22b(+) vector and expressed with a His6-tag at the N-terminus. The amino acid sequences of the expressed proteins and the polynucleotides encoding them were as follows: - Amino acid sequences of expressed Ub006 to 013: amino acid sequences of SEQ ID NOs: 38 to 45 with "MHHHHHH" added to the N-terminus - DNA sequences of expressed Ub006 to 013: SEQ ID NOs: 46 to 53 - Amino acid sequence of expressed Ub012 homodimer: amino acid sequence of SEQ ID NO: 54 with "MHHHHHHAS" added to the N-terminus and "TS" added to the C-terminus - DNA sequence of expressed Ub012 homodimer: SEQ ID NO: 55 - Amino acid sequence of expressed Ub012 homotetramer: amino acid sequence of SEQ ID NO: 56 with "MHHHHHHAS" added to the N-terminus and "TS" added to the C-terminus - DNA sequence of expressed Ub012 homotetramer: SEQ ID NO: 57 Amino acid sequence of the expressed homodimer of ubiquitin molecule: amino acid sequence in which "MHHHHHHAS" is added to the N-terminus of SEQ ID NO: 58 and "TS" is added to the C-terminus. DNA sequence of the expressed homodimer of ubiquitin molecule: SEQ ID NO: 59

[0207] All proteins were expressed in E. coli BL21-gold (DE3) cells (Agilent). The cells were grown in lysogeny broth medium (Miller) containing 100 μg / mL ampicillin to an OD600 > 0.4 and induced with 0.4 mM IPTG for 24 hours at 16°C. Cells were lysed by sonication in lysis buffer [50 mM Tris-HCl, 20 mM imidazole-HCl (pH 7.2) in 150 mM NaCl (pH 7.6)], and proteins were captured on Ni Sepharose 6 Fast Flow (Cytiva). Sepharose beads were washed with wash buffer [50 mM Tris-HCl, 20 mM imidazole-HCl (pH 7.2) in 1 M NaCl (pH 7.6)] and the lysis buffer. Proteins were eluted from the Sepharose beads with elution buffer [50 mM Tris-HCl, 150 mM NaCl (pH 7.6) containing 500 mM imidazole-HCl (pH 7.2)].

[0208] The homodimers and homotetramers of Ub006-013 and Ub012 were further purified by size-exclusion chromatography on a Superdex 75 increase 10 / 300 GL (Cytiva) or HiLoad 16 / 600 Superdex 200 pg (Cytiva) column [running buffer: 50 mM Tris-HCl, 150 mM NaCl (pH 7.6)] on an AKTA avant 25 (Cytiva) or AKTA pure (Cytiva) system. The purified proteins were stored at -80°C for further use. Protein concentrations were determined by UV absorbance at 280 nm using a NanoDrop 2000c or NanoDrop One C spectrophotometer (Thermo Fisher Scientific).

[0209] (4) Evaluation of the binding affinity of MuSK-binding domain-grafted ubiquitin molecules and their complexes to MuSK (in vitro test) The U-bodies and their complexes obtained as described above were evaluated for their affinity to human, rat, and mouse MuSK (binding activity test). The test was performed according to the procedure described in Example 1(3). However, the U-body complexes were subjected to surface plasmon resonance (SPR) analysis using Biacore 8K (Cytiva). The results are shown in Table 6.

[0210]

[0211] (5) Evaluation of the effect of U-bodies and their complexes on acetylcholine receptor clustering (cellular level test) The effect of U-bodies and their complexes on acetylcholine receptor clustering was evaluated. The test to evaluate AChR cluster formation using the mouse myoblast-derived cultured cell line C2C12 was performed according to the procedure described in Example 2(5). The test to evaluate using the NanoBiT assay was performed according to the procedure described in Example 2(6). The results are shown in Figures 24 to 26.

[0212] Figure 24 shows the results of a NanoBiT assay. Figure 24 confirms that Ub012 homodimers (dimeric U-bodies) have stronger MuSK dimerization activity than cyclic peptide homodimers (dimeric peptide / MuSK7-pal). Figure 25 shows the results of an evaluation test of AChR cluster formation using myotube cells. Figure 25 confirms that Ub012 homodimers (dimeric U-bodies) have a concentration-dependent promoting effect on AChR cluster formation. Figure 26A shows the results of a NanoBiT assay. Figure 26A confirms that Ub012 homotetramers (tetrameric U-bodies) have stronger MuSK dimerization activity than Ub012 homodimers (dimeric U-bodies). Figure 26B shows the results of an evaluation test of AChR cluster formation using myotube cells. FIG. 26B confirmed that Ub012 homotetramers (tetrameric U-bodies) have a concentration-dependent promoting effect on AChR cluster formation.

[0213] (6) Evaluation of the therapeutic effect of the U-body complex on a myasthenia gravis animal model A. Objective: The effects of the U-body complex (tetramer) on various disease parameters in a rat PTMG model using an anti-acetylcholine receptor monoclonal antibody (mAb35) were evaluated. In this example, the C-terminus and N-terminus of Ub012 were linked by a peptide linker (EAAAK). ３ The C-terminus and N-terminus of a Ub012 homotetramer (SEQ ID NO: 60) linked by the nucleotide sequence GG1 ... ３ A homotetramer (SEQ ID NO: 61) of Ub012(ΔGG) linked by the above amino acid sequence was prepared and used in the following tests.

[0214] Test method: Five-week-old female rats (LEW / CrlCrlj, 4 weeks old at the time of delivery, Jackson Laboratory Japan) were divided into three groups (Group A, Group B, and Group C) based on their body weights. The test compound (tetramer of Ub012 (EAAAK)) dissolved in a vehicle was administered. ３Group A (5 mg / kg) (n=5) received the test compound (tetramer (EAAAK) of Ub012 (ΔGG) dissolved in vehicle) ３ ) was intravenously administered to Group B (5 mg / kg) (n=5), and the vehicle was administered to Group C (n=5) (Day 0). Two hours after the intravenous administration, 200 μg / animal of anti-nicotinic acetylcholine receptor monoclonal antibody (mAb35, National Hospital Organization Nagasaki Kawatana Medical Center) dissolved in PBS (pH 7.4) was intraperitoneally administered to all animals as a drug to induce myasthenia gravis (Day 0).

[0215] The body weight of the animals was then measured before antibody administration (pre) and at 0, 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration. The movement, muscle strength, and grip strength of the animals were evaluated using the following tests.

[0216] <Movement Score> The movement of the animals was evaluated at 9, 24, 33, 48, 57, and 72 hours (hr) after antibody administration using a 6-level evaluation scale (score). The evaluation criteria were the same as those in Example 3(1)B.

[0217] <Wire-hang Test> Approximately 30 hours after antibody administration, a wire-hang test was performed using the same procedure as in Example 3(1)(a).

[0218] Grip Strength Test Approximately 30 hours after antibody administration, a grip strength test was performed using the same procedure as in Example 3(1)(a).

[0219] C. Results The weight measurement results for each group are shown in Figure 27. Specifically, the control group C showed a weight loss from 0 to 57 hours. Groups A and B also showed a similar weight loss to group C, but the degree of weight loss was significantly slower than that of group C (Tukey test; groups A and B: 0 hour > 33 hours, 48 ​​hours, 57 hours, p < 0.05).

[0220] The movement score measurement results for each group are shown in Figure 28. Specifically, the score results for the control group C showed that the onset of pathology began at 9 hours, peaked at 48 hours, and then declined. Similar to group C, groups A and B showed the onset of pathology from 9 hours, but the peak scores for groups A and B were lower than the peak score for group C. However, no significant differences were observed between the groups at any measurement point (Kruskal-Wallis test).

[0221] The results of the measurement of the latency to fall (latency) in the Hanging Wire Test for each group are shown in Figure 29. Specifically, the mean latency to fall for the control group C was 0.2 seconds (±0.20 SEM), with four out of five cases falling immediately upon the start of the test. On the other hand, the mean latency to fall for groups A and B was 25.2 seconds (±23.72 SEM) and 26.8 seconds (±23.46 SEM), respectively. Two or more cases did not fall immediately upon the start of the test, and although the mean latency to fall was slightly longer than that of the control group, no statistically significant difference was observed between the groups (Kruskal-Wallis test; H(9) = 13.20, n.s.).

[0222] The results of the Grip Strength Test for each group are shown in Figure 30. The average grip strength of the control group C was 89 g (±22.24 SEM). On the other hand, the grip strengths of groups A and B were 233 g (±65.16 SEM) and 264 g (±111.10 SEM), respectively, which were stronger than group C, but no statistically significant differences were observed between the groups (Kruskal-Wallis test; H(9) = 8.72, n.s.).

[0223] As mentioned in C above, myasthenia gravis-like symptoms observed in the control group (Group C), such as weight loss, increased score, and decreased muscle strength, were also observed in the test compound administration group. However, although no statistically significant difference was observed, the pathological conditions of Groups A and B were clearly milder than those of the control group, confirming the efficacy of the two test compounds against the symptoms of myasthenia gravis.

Claims

1. A cyclic peptide or a pharma- ceutical acceptable salt thereof, comprising the structure of the following formula (I): １ -Asn-X ２ -Val- (I) (wherein X １ represents Thr or Val, X ２ represents Val or Ile) 2. The cyclic peptide or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the number of amino acid residues forming the cyclic structure of the cyclic peptide is 8 to 20.

3. The cyclic structure of the cyclic peptide is N-CO-CH ２ The cyclic peptide according to claim 1 or 2, which comprises an -S structure, or a pharma- ceutically acceptable salt thereof.

4. The cyclic peptide of claim 3 or a pharma- ceutically acceptable salt thereof, wherein N is an amino group of tyrosine.

5. The cyclic peptide or a pharma- ceutically acceptable salt thereof according to claim 3 or 4, wherein S is a thiol group of cysteine.

6. The cyclic peptide of claim 1 or 3, or a pharma- ceutically acceptable salt thereof, wherein the cyclic peptide has a structure selected from the group consisting of the following formula (1) (sequence number 2), formula (2) (sequence number 3) and formula (3) (sequence number 4). (In the above formula, the carboxyl group of the cysteine ​​residue (C) may form an acid amide.) 7. A cyclic peptide complex or a pharma- ceutically acceptable salt thereof, comprising two cyclic peptides according to claim 1, one of which is bound to the other cyclic peptide via a linker.

8. The cyclic peptide conjugate of claim 7, or a pharma- ceutically acceptable salt thereof, wherein the two cyclic peptides are the same cyclic peptide.

9. The cyclic peptide conjugate or a pharma- ceutically acceptable salt thereof according to claim 7 or 8, wherein the linker has a length of 20 to 100 Å.

10. The cyclic peptide conjugate or a pharma- ceutically acceptable salt thereof according to claim 9, wherein the linker has a polyethylene glycol structure as at least a part of its structure.

11. The cyclic peptide conjugate or a pharma- ceutical acceptable salt thereof according to claim 7 or 10, wherein the linker has at least one saturated hydrocarbon group having 8 to 20 carbon atoms as a side chain thereof.

12. A part or all of the loop structure region between the β1 domain and the β2 domain of a ubiquitin molecule has the following structure (A): (In the above formula, the MuSK binding region is YX ２１ SYHTNVVASLKR(X ２１ is P or L) (SEQ ID NO:11), spacer 1 comprises an amino acid sequence selected from the group consisting of IQPR (SEQ ID NO:12), KPGV (SEQ ID NO:13), WDRG (SEQ ID NO:14), KPGT (SEQ ID NO:15), PFGV (SEQ ID NO:16), YFVG (SEQ ID NO:17) and LEGM (SEQ ID NO:18), and spacer 2 comprises an amino acid sequence selected from the group consisting of IRY, MMP, QGI, FMP, TGPQ (SEQ ID NO:19), WNTP (SEQ ID NO:20) and IRPQ (SEQ ID NO:21).

13. The MuSK binding domain-grafted ubiquitin molecule or a pharma- ceutically acceptable salt thereof according to claim 12, wherein the ubiquitin molecule is a protein selected from the group consisting of: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 1; and (c) a protein consisting of an amino acid sequence in which one or more amino acids have been modified in the amino acid sequence of SEQ ID NO:

1.

14. The MuSK binding domain-grafted ubiquitin molecule of claim 12 or 13, wherein structure (A) has an amino acid sequence selected from the group consisting of: (Ub006) IQPRYPSYHTNVVASLKRIRY (SEQ ID NO: 22) (Ub007) KPGVYPSYHTNVVASLKRMMP (SEQ ID NO: 23) (Ub008) WDRGYPSYHTNVVASLKRQGI (SEQ ID NO: 24) (Ub009) KPGTYPSYHTNVVASLKRFMP (SEQ ID NO: 25) (Ub010) PFGVYPSYHTNVVASLKRTGPQ (SEQ ID NO: 26) (Ub011) YFVGYPSYHTNVVASLKRWNTP (SEQ ID NO: 27) (Ub012) LEGMYLSYHTNVVASLKRIRPQ (SEQ ID NO: 28) (Ub013) LEGMYPSYHTNVVASLKRIRPQ (SEQ ID NO: 29) 15. The MuSK binding domain-grafted ubiquitin molecule or a pharma- ceutically acceptable salt thereof according to claim 12 or 13, wherein the loop structure region is a region from the 7th amino acid residue to the 10th amino acid residue of the ubiquitin molecule.

16. A MuSK-binding domain-grafted ubiquitin molecule complex or a pharma- ceutically acceptable salt thereof, comprising two or more MuSK-binding domain-grafted ubiquitin molecules according to claim 12 or 13, wherein one MuSK-binding domain-grafted ubiquitin molecule is linked to another MuSK-binding domain-grafted ubiquitin molecule via a linker.

17. The MuSK binding domain-grafted ubiquitin molecule conjugate of claim 16, or a pharma- ceutically acceptable salt thereof, wherein the two or more MuSK binding domain-grafted ubiquitin molecules are the same molecule.

18. The MuSK binding domain-grafted ubiquitin molecule complex or a pharma- ceutically acceptable salt thereof according to claim 16 or 17, wherein the length of the linker is 20 to 100 Å.

19. The MuSK binding domain-grafted ubiquitin molecule conjugate or a pharma- ceutically acceptable salt thereof according to claim 16 or 18, wherein the linker is a peptide chain.

20. A pharmaceutical composition or a therapeutic agent for myasthenia gravis comprising as an active ingredient a cyclic peptide described in claim 1 or a pharma- ceutically acceptable salt thereof, a cyclic peptide complex described in claim 7 or a pharma- ceutically acceptable salt thereof, a MuSK binding domain-grafted ubiquitin molecule described in claim 12 or a pharma- ceutical acceptable salt thereof, or a MuSK binding domain-grafted ubiquitin molecule complex described in claim 16 or a pharma- ceutical acceptable salt thereof.

21. A method for presenting a cyclic peptide having binding activity to a desired molecule (preferably a MuSK molecule) on a loop structure region of a ubiquitin molecule together with two spacer sequences, the cyclic peptide having a chemical cross-linking structure for forming an intramolecular cyclic structure, the method comprising: replacing the chemical cross-linking structure of the cyclic peptide with two amino acid residues constituting the loop structure via a spacer sequence, thereby fusing the cyclic peptide to a ubiquitin molecule while retaining its binding activity to the desired molecule (preferably a MuSK molecule), the spacer sequence consisting of a randomly generated amino acid sequence of 0 to 4 residues.

22. The desired molecule is a MuSK molecule, and the cyclic peptide and spacer sequence presented on the loop structure region of the ubiquitin molecule have the following structure (B): (In the above formula, spacer 3 is a peptide chain consisting of any 0 to 4 amino acids, spacer 4 is a peptide chain consisting of any 0 to 4 amino acids, and the MuSK binding region is a region present in the cyclic peptide and having MuSK molecule binding activity.) The presentation method according to claim 21.

23. The method of displaying according to claim 21 or 22, wherein the loop structure region of the ubiquitin molecule is the loop structure region between the β1 domain and the β2 domain.

24. The display method according to claim 23, wherein the two amino acid residues constituting the loop structure region are the 7th and 10th amino acid residues of the ubiquitin molecule.

25. The MuSK binding domain is YX ２２ SYHTNVVASLKR(X ２２ The method of claim 22, wherein the peptide chain has the amino acid sequence of (SEQ ID NO: 11):

26. A method for producing a modified protein, comprising fusing a cyclic peptide having binding activity to a desired molecule (preferably a MuSK molecule) together with two spacer sequences onto a loop structure region of a ubiquitin molecule to present the cyclic peptide on the protein, the method comprising: selecting a partial amino acid sequence to be presented on the protein from the amino acid sequence of the cyclic peptide, selecting a base sequence corresponding to the partial amino acid sequence, selecting a base sequence corresponding to two amino acid residues that constitute the loop structure region of the ubiquitin molecule, deleting bases present between the selected base sequences as necessary, and inserting and incorporating a base sequence corresponding to the partial amino acid sequence of the selected cyclic peptide; and translating the nucleic acid, wherein the spacer sequence consists of a randomly generated amino acid sequence of 0 to 4 residues.

27. The desired molecule is a MuSK molecule, and the cyclic peptide and spacer sequence presented on the loop structure region of the ubiquitin molecule have the following structure (B): (In the above formula, spacer 3 is a peptide chain consisting of any 0 to 4 amino acids, spacer 4 is a peptide chain consisting of any 0 to 4 amino acids, and the MuSK binding region is a region present in the cyclic peptide and having MuSK molecule binding activity.) The manufacturing method described in claim 26.

28. The method of claim 26 or 27, wherein the loop structure region of the ubiquitin molecule is the loop structure region between the β1 domain and the β2 domain.

29. The method according to claim 28, wherein the two amino acid residues constituting the loop structure region are the 7th and 10th amino acid residues of the ubiquitin molecule.

30. The MuSK binding domain is YX ２２ SYHTNVVASLKR(X ２２ The method of claim 27, wherein the peptide chain has the amino acid sequence of:

31. A method for screening for a modified protein capable of binding to a desired molecule (preferably a modified protein capable of binding to a MuSK molecule), comprising: designing an mRNA library containing a base sequence encoding a ubiquitin molecule in which a cyclic peptide sequence having binding activity to the desired molecule (preferably a MuSK molecule) and a spacer sequence consisting of a randomly generated amino acid sequence of 0 to 4 residues linked upstream and downstream of the cyclic peptide sequence are fused to a loop structure region between the β1 domain and the β2 domain; preparing modified proteins based on the designed mRNA library according to the production method described in claim 28; and selecting a modified protein capable of binding to the desired molecule (preferably a modified protein capable of binding to a MuSK molecule) from the obtained modified proteins using binding activity to the desired molecule (preferably MuSK binding activity) as an index.

32. A peptide or a pharma- ceutically acceptable salt thereof comprising the structure of the following formula (I): -X １ -Asn-X ２ -Val- (I) (wherein X １ represents Thr or Val, X ２ represents Val or Ile) 33. The peptide according to claim 32, or a pharma- ceutically acceptable salt thereof, wherein the peptide consists of 8 to 20 amino acid residues.

34. A peptide or a pharma- ceutically acceptable salt thereof according to claim 32 or 33, grafted into a biomolecule.

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