Modified ferritin and method for producing the same

Abstract

The present invention provides a technique in which the number of modifying groups introduced on the surface of a ferritin can be easily controlled. More specifically, the present invention provides a modified ferritin comprising a human ferritin H chain, wherein the human ferritin H chain contains a modifying group specifically covalently bonded to a cysteine residue at positions 91 and / or 103 on the basis of the reference position of a wild-type human ferritin H chain.

Description

[Technical Field]

[0001] The present invention relates to modified ferritin and a method for producing the same. [Background technology]

[0002] Ferritin is a globular protein with a lumen composed of multiple monomers, found universally in plants, animals, and microorganisms. In animals such as humans, ferritin is composed of two types of monomers: H chains and L chains. It is also known that ferritin is a multimer composed of 24 monomers (often a mixture of H and L chains), with a cage-like structure with an outer diameter of 12 nm and a lumen of 7 nm. The N-terminus of the monomers constituting ferritin is exposed on the surface of the 24-mer, while the C-terminus of each monomer is not exposed on the surface and resides within the lumen. Ferritin can hold iron within its lumen and plays a role in physiological functions such as iron transport and storage, thus being deeply involved in the homeostasis of iron elements in living organisms and cells. Furthermore, because ferritin can dissociate and associate relatively easily, it can exist in the form of a 24-mer, monomers, or a combination thereof, depending on the conditions of the solvent containing it. Therefore, by appropriately adjusting the conditions of the solvent containing ferritin, the 24-mer can be dissociated into monomers, and the dissociated monomers can be reassembled into the 24-mer. It is also known that during reassembly, by coexisting a predetermined substance with the ferritin monomer in the solvent, the substance can be encapsulated in the lumen of the reassembled 24-mer.

[0003] Currently, techniques for modifying the ferritin surface are known for purposes such as altering ferritin delivery properties and improving stability. For example, techniques utilizing modifying groups that can specifically react with amino groups (-NH3) in the side chains of lysine residues exposed on the ferritin surface (Non-Patent Documents 1 and 2), and techniques utilizing modifying groups that can specifically react with carboxyl groups (-COOH) in the side chains of aspartic acid and glutamic acid residues exposed on the ferritin surface (Patent Document 1) have been reported.

[0004] Furthermore, techniques for modifying the ferritin surface are known, utilizing ferritin monomers into which mutations enabling surface modification have been introduced. For example, such techniques include a technique utilizing a peptide attached to the N-terminus of a ferritin monomer (Non-Patent Literature 3), and a technique utilizing a thiol group (-SH) in a cysteine ​​residue introduced into a ferritin monomer (Non-Patent Literature 4). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 2008 / 0292545 [Non-patent literature]

[0006] [Non-Patent Document 1] Xin Lin et al.,Nano Letters(2011),11(2),814-819 [Non-Patent Document 2] JOHN R. FEAGLER et al., J. Cell Biology (1974), 60 (3), 541-53 [Non-Patent Document 3] Dong Men et al.,ACS Nano(2015),9(11),10852-10860 [Non-Patent Document 4] Tuo Zhang et al.,Adv.Mater.Research(2014),887-888,596-600 [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors of the present invention have focused on the fact that in techniques utilizing modifying groups that can specifically react with amino groups (-NH3) in the side chains of lysine residues exposed on the ferritin surface, or carboxyl groups (-COOH) in the side chains of aspartic acid and glutamic acid residues exposed on the ferritin surface (Non-Patent Documents 1, 2, and Patent Document 1), it may be difficult to control the number of modifying groups introduced to the ferritin surface. This is because ferritin contains numerous lysine, aspartic acid, and glutamic acid residues, and since these amino acid residues are exposed on the ferritin surface, numerous modifying groups can be introduced, making it difficult to control the number of modifying groups. In the clinical application of ferritin, a high level of quality control is required, so modified ferritin with excellent surface modification uniformity is preferable to modified ferritin with non-uniform modification. Therefore, the development of a technique that can reliably produce modified ferritin with excellent surface modification uniformity is desired. However, if the number of modifying groups cannot be easily controlled, it is not easy to reliably produce modified ferritin with excellent surface modification uniformity.

[0008] Therefore, the object of the present invention is to provide a technology that allows for easy control of the number of modifying groups introduced to the ferritin surface. [Means for solving the problem]

[0009] As a result of diligent research, the inventors have discovered that by using the thiol group (-SH) in the side chain of the cysteine ​​residue of the human ferritin H chain, which is a ferritin monomer, as the reaction target, it is possible to selectively modify the thiol residue in the side chain of the cysteine ​​residue at positions 91 and / or 103, which correspond to the reference position of the natural human ferritin H chain, and consequently, the number of modifying groups introduced to the ferritin surface can be easily and highly controlled. Non-patent documents 1 and 2, and patent document 1 neither teach nor suggest the technical concept of controlling the number of modifying groups introduced to the ferritin surface. Therefore, based on these findings, the inventors have succeeded in reproducibly providing modified ferritin with excellent uniformity of surface modification, and have completed the present invention.

[0010] In other words, the present invention is as follows: [1] Contains human ferritin H chain, Modified ferritin, wherein the human ferritin H chain contains a modifying group that is specifically covalently bound to the cysteine ​​residues at positions 91 and / or 103, following the reference position of the natural human ferritin H chain. [2] The modified ferritin of [1], wherein the modified ferritin contains one or two modifying groups per human ferritin H chain. [3] Modified ferritin of [1] or [2], wherein the modified ferritin is a 24-mer containing 24 human ferritin H chains. [4] The modified ferritin of [3], wherein the modified ferritin contains 24 or 48 modifying groups. [5] Modified ferritin of any of the following [1] to [4], wherein the human ferritin H chain is as follows: (A) Proteins containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (B) A protein having the ability to form a 24-mer, which includes an amino acid sequence in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 that contains mutations of one or more amino acid residues selected from the group consisting of substitutions, deletions, insertions, and additions of amino acid residues; or (C) A protein that contains an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and that has the ability to form a 24-mer. [6] A modified ferritin of any of the following types [1] to [5], wherein the human ferritin H chain is a natural human ferritin H chain that may have a deletion of the methionine residue at position 1. [7] A modified ferritin of any of the following types [1] to [6], wherein the human ferritin H chain has a functional peptide inserted into a flexible linker region consisting of amino acid residues at positions 78 to 96, which follows the reference position of the natural human ferritin H chain. [8] A modified ferritin of [7], wherein the functional peptide is a peptide that has the ability to bind to a target material. [9] A modified ferritin according to any of [1] to [8], wherein the modifying group contains a reactive group and the modified ferritin is a ferritin with a reactive group added. The modified ferritin of [9], wherein the reactive group is a bioorthogonal functional group for proteins. The modified ferritin of

[10] , wherein the bioorthogonal functional group contains one or more partial structures selected from the group consisting of a maleimide moiety, an azide moiety, a ketone moiety, an aldehyde moiety, a thiol moiety, an alkene moiety, an alkyne moiety, a halogen moiety, a tetrazine moiety, a nitrone moiety, a hydroxylamine moiety, a nitrile moiety, a hydrazine moiety, a boronic acid moiety, a cyanobenzothiazole moiety, an allyl moiety, a phosphine moiety, a disulfide moiety, a thioester moiety, an α-halocarbonyl moiety, an isonitrile moiety, a sydnone moiety, and a selenium moiety. The modified ferritin according to any one of [1] to [8], wherein the modifying group contains a functional substance and the modified ferritin is a functional substance-added ferritin. The modified ferritin of

[12] , wherein the functional substance contains one or more parts selected from the group consisting of a peptide, a protein, a nucleic acid, a low-molecular organic compound, a chelator, a sugar chain, a lipid, a polymer, and a metal. The modified ferritin according to any one of [1] to

[13] , wherein the modified ferritin has a substance in the lumen. A method for producing a reactive group-added ferritin, comprising: reacting human ferritin with a thiol-modifying reagent to produce a reactive group-added ferritin, wherein the human ferritin and the reactive group-added ferritin contain the human ferritin H chain, and the human ferritin H chain contained in the reactive group-added ferritin specifically covalently binds to the cysteine residue at position 91 and / or 103 according to the reference position of the native human ferritin H chain, and the method includes a modifying group containing a reactive group. The method of

[15] , wherein the thiol-modifying reagent contains one or more partial structures selected from the group consisting of a maleimide moiety, a benzyl halide moiety, an α-haloamide moiety, an α-haloketone moiety, an alkene moiety, an alkyne moiety, a fluoroaryl moiety, a nitroaryl moiety, a methylsulfonyloxadiazole moiety, and a disulfide moiety. A method for producing a functional substance-added ferritin, comprising: Reacting human ferritin with a functional substance to produce a functional substance-added ferritin, where the human ferritin and the functional substance-added ferritin contain the human ferritin H chain, and the human ferritin H chain contained in the functional substance-added ferritin specifically covalently binds to the cysteine residues at positions 91 and / or 103 according to the reference positions of the native human ferritin H chain, and includes a modified group containing a functional substance.

[18] A method for producing a functional substance-added ferritin, (1) Reacting human ferritin with a thiol-modifying reagent to produce a reactive group-added ferritin; and (2) Reacting the reactive group-added ferritin with a functional substance to produce a functional substance-added ferritin, where the human ferritin, the reactive group-added ferritin, and the functional substance-added ferritin contain the human ferritin H chain, the human ferritin H chain contained in the reactive group-added ferritin specifically covalently binds to the cysteine residues at positions 91 and / or 103 according to the reference positions of the native human ferritin H chain, and includes a modified group containing a reactive group, and the human ferritin H chain contained in the functional substance-added ferritin specifically covalently binds to the cysteine residues at positions 91 and / or 103 according to the reference positions of the native human ferritin H chain, and includes a modified group containing a functional substance. [Advantages of the Invention]

[0011] According to the present invention, it is possible to reproducibly provide a modified ferritin with excellent surface modification uniformity. Further, by using native human ferritin as ferritin, immunogenicity can be avoided in clinical applications to humans. [Brief Description of the Drawings]

[0012] [Figure 1]Figure 1 shows (A) the amino acid sequence of the natural human ferritin H chain (with methionine at position 1) (SEQ ID NO: 1), (B) the nucleotide sequence of the polynucleotide encoding the natural human ferritin H chain (with methionine at position 1) (SEQ ID NO: 2), and (C) the amino acid sequence of the natural human ferritin H chain (without methionine at position 1) (SEQ ID NO: 3). [Figure 2] Figure 2 shows the ESI-TOFMS analysis of the natural human ferritin H chain specifically modified with ethyl maleimide (6 equivalents). [Figure 3] Figure 3 shows the MS spectrum of the modification site of the natural human ferritin H chain specifically modified with ethyl maleimide. [Figure 4] Figure 4 shows the CID spectrum of the modification site of the natural human ferritin H chain specifically modified with ethyl maleimide. [Figure 5] Figure 5 shows the ESI-TOFMS analysis of the natural human ferritin H chain specifically modified with p-azidophenacylbromide (6 equivalents). [Figure 6] Figure 6 shows the ESI-TOFMS analysis of the natural human ferritin H chain specifically modified with p-azidophenacylbromide (12 equivalents). [Figure 7] Figure 7 shows the analysis (MS spectrum) of the modification site of the natural human ferritin H chain specifically modified with p-azidophenacylbromide. [Figure 8] Figure 8 shows the analysis (CID spectrum) of the modification site of the natural human ferritin H chain specifically modified with p-azidophenacylbromide. [Figure 9] Figure 9 shows a comparison of peak area values ​​at positions 91, 103, and 131 of the human ferritin H chain based on LC-MS / MS analysis of the modification sites of the human ferritin H chain. [Figure 10] Figure 10 shows the ESI-TOFMS analysis of the natural human ferritin H chain specifically modified with p-azidophenacyl bromide (6 equivalents). [Figure 11]Figure 11 shows the ESI-TOFMS analysis of natural human ferritin H chains specifically modified with DBCO-PEG4-maleimide (6 equivalents). [Figure 12] Figure 12 shows the ESI-TOFMS analysis of natural human ferritin H chains specifically modified with 1,3-dichloroacetone (6 equivalents) and aminooxy-PEG5-azide (10 equivalents). [Figure 13] Figure 13 shows the MS spectrum (measured value: m / z 853.37134, theoretical value: 853.37194, trivalent) of the peptide fragment KPDCDDWESGLNAMECALHLEK (SEQ ID NO: 9), a peptide consisting of 22 amino acid residues containing one modification site (1,3-dichloroacetone modification (+54.011Da)) on a cysteine ​​residue by trypsin digestion of the human ferritin H chain (Example 9). [Figure 14] Figure 14 shows the CID spectrum of the product ion with m / z 1206.78 (theoretical value: 1206.50), corresponding to divalent b21, which shows 1,3-dichloroacetone modification (+54.011Da) of the cysteine ​​residues at amino acid positions 90 and 102 of the human ferritin H chain (Example 9). [Figure 15] Figure 15 shows the results of calculating the peak area of ​​the extracted chromatogram of a peptide containing 1,3-dichloroacetone modification (+54.011 Da) to a cysteine ​​residue from the trypsin digest of human ferritin H chain using Xcalibur Qual Browser (Example 9). The horizontal axis shows the cysteine ​​residue contained in the human ferritin H chain, and the vertical axis shows the peak area. [Figure 16] Figure 16 shows the amide group modification by ESI-TOFMS. [Figure 17]Figure 17 shows the MS spectrum (measured value: m / z 873.38158, theoretical value: 873.38273, trivalent) of the peptide fragment KPDCDDWESGLNAMECALHLEK (SEQ ID NO: 9), a peptide consisting of 22 amino acid residues containing one modification site (α-iodoacetamide modification (+57.021Da)) on a cysteine ​​residue by trypsin digestion of the human ferritin H chain (Example 11). [Figure 18] Figure 18 shows the CID spectrum of the product ion with m / z 1236.85 (theoretical value: 1236.52), corresponding to divalent b21, which exhibits α-iodoacetamide modification (+57.021Da) of the cysteine ​​residues at amino acid positions 90 and 102 of the human ferritin H chain (Example 11). [Figure 19] Figure 19 shows the results of calculating the peak area of ​​the extracted chromatogram of a peptide containing α-iodoacetamide modification (+57.021 Da) to a cysteine ​​residue in the trypsin digest of human ferritin H chain using Xcalibur Qual Browser (Example 11). The horizontal axis shows the cysteine ​​residue contained in the human ferritin H chain, and the vertical axis shows the peak area. [Figure 20] Figure 20 shows the dispersibility of ethylmaleimide-modified ferritin by DLS. [Figure 21] Figure 21 shows the dispersibility of ethylmaleimide-modified ferritin by size exclusion column chromatography. [Figure 22] Figure 22 shows the stability of doxorubidin-encapsulated ethylmaleimide-modified ferritin as determined by size exclusion column chromatography. [Figure 23] Figure 23 shows the encapsulation of a fluorescently modified peptide into ethylmaleimide-modified ferritin by size exclusion column chromatography. [Figure 24] Figure 24 shows the modification of fluorescently modified peptide-encapsulated ferritin with ethyl maleimide by ESI-TOFMS. [Figure 25]Figure 25 shows that even when ferritin containing fluorescently modified peptides was modified with ethyl maleimide, the peptides encapsulated in the ferritin were maintained, as demonstrated by size exclusion column chromatography. [Figure 26] Figure 26 shows the modification of double-stranded DNA encapsulating ferritin with ethyl maleimide by ESI-TOFMS. [Figure 27] Figure 27 shows that even when ferritin containing double-stranded DNA was modified with ethyl maleimide by size exclusion column chromatography, the double-stranded DNA contained within the ferritin was maintained. [Figure 28] Figure 28 shows the modification of peptides with PEG-6 as a linker (YGRKKRRQRRR, TAT, SEQ ID NO: 13) or (RRRRRRRRR, R9, SEQ ID NO: 14) by ESI-TOFMS. [Figure 29] Figure 29 shows the dispersion of azide-treated FTH according to DLS. [Figure 30] Figure 30 shows the dispersibility of azide-modified FTH by size exclusion column chromatography. [Figure 31] Figure 31 shows that the azide-modified peptide was encapsulated within the column using size exclusion column chromatography. [Figure 32] Figure 32 shows electrophoretic images of samples of native FTH and azidized FTH modified with siRNA, obtained by SDS-PAGE. [Figure 33] Figure 33 shows the dispersibility of nucleic acid-modified ferritin by DLS. [Figure 34] Figure 34 shows the dispersibility of nucleic acid-modified ferritin by size exclusion column chromatography. [Figure 35] Figure 35 shows the modification of the peptide-inserted human ferritin H chain with p-azidophenacyl bromide by ESI-TOFMS. [Figure 36]Figure 36 shows the modification of the surface of natural FTH with N-Succinimidyl 3-(Acetylthio)propionate by ESI-TOFMS. [Modes for carrying out the invention]

[0013] This invention comprises human ferritin H chains, The present invention provides modified ferritin, in which the human ferritin H chain contains a modifying group that is specifically covalently bonded to the cysteine ​​residue at positions 91 and / or 103.

[0014] The positions of amino acid residues in the human ferritin H chain (e.g., the methionine residue at position 1, the cysteine ​​residue at position 91, and the cysteine ​​residue at position 103) are determined according to the reference positions of the natural human ferritin H chain. The natural human ferritin H chain is typically a polypeptide containing the amino acid sequence of SEQ ID NO: 1. In this invention, the natural human ferritin H chain includes not only the polypeptide containing the amino acid sequence of SEQ ID NO: 1, but also naturally occurring variants thereof. Preferably, the natural human ferritin H chain is a polypeptide containing the amino acid sequence of SEQ ID NO: 1.

[0015] The human ferritin H chain contained in the modified ferritin of the present invention may be a natural human ferritin H chain that may have a methionine residue at position 1 deleted. A natural human ferritin H chain that does not have a methionine residue at position 1 corresponds to a polypeptide containing the amino acid sequence of SEQ ID NO: 1. A natural human ferritin H chain that has a methionine residue at position 1 corresponds to a polypeptide containing the amino acid sequence of SEQ ID NO: 3.

[0016] In certain embodiments, the human ferritin H chain may be as follows: (A) Proteins containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (B) A protein having the ability to form a 24-mer, which includes an amino acid sequence in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 that contains mutations of one or more amino acid residues selected from the group consisting of substitutions, deletions, insertions, and additions of amino acid residues; or (C) A protein that contains an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and that has the ability to form a 24-mer.

[0017] In protein (B), one or more amino acid residues can be modified by one, two, three, or four types of mutations selected from the group consisting of deletion, substitution, addition, and insertion of amino acid residues. Amino acid residue mutations may be introduced into one region of the amino acid sequence or into multiple different regions. In this invention, substitution of amino acid residues from non-cysteine ​​amino acid residues to cysteine ​​residues is not intended. Similarly, addition and insertion of amino acid residues, or addition and insertion of cysteine ​​residues or cysteine ​​residue-containing regions, are not intended. The term "one or several" indicates a number that does not impair the ability to form a 24-mer. The number indicated by the term "one or several" is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5 (e.g., 1, 2, 3, 4, or 5).

[0018] For protein (C), the degree of identity with respect to the target amino acid sequence is preferably 92% or higher, more preferably 95% or higher, even more preferably 97% or higher, and most preferably 98% or higher or 99% or higher. The amino acid sequence identity percentage can be calculated using GENETYX Ver13.1.1 software from Genetics Co., Ltd., using the full length of the polypeptide portion encoded in the ORF, after performing Muscle alignment, ClustalW alignment, or Multiple sequence alignment, and then calculating the value with the setting "Gaps are taken into account".

[0019] The location of the amino acid residue in the amino acid sequence in which mutations should be introduced is obvious to those skilled in the art, but it may also be identified by further reference to sequence alignment. Specifically, those skilled in the art can 1) compare multiple amino acid sequences, 2) identify relatively conserved and relatively unconserved regions, and then 3) predict from the relatively conserved and relatively unconserved regions which regions may play an important role in function and which may not, respectively, thus recognizing the correlation between structure and function. Therefore, those skilled in the art can identify the location in the amino acid sequence in which mutations should be introduced by utilizing sequence alignment, and can also identify the location of the amino acid residue in the amino acid sequence in which mutations should be introduced by using known secondary and tertiary structural information in combination. Introducing mutations to amino acid residues in the C-terminal region and other regions located inside the cage-like structure of ferritin under normal conditions (amino acid residues that are not exposed on the surface and therefore have a low risk of causing immunogenicity) is also preferable from the viewpoint of avoiding the risk of causing immunogenicity. Furthermore, mutations in cysteine ​​residues or cysteine ​​residue-containing peptides (e.g., substitution, insertion, and addition to the N-terminal region) may be introduced at positions not exposed on the ferritin surface, but it is also preferable that no mutations in cysteine ​​residues or cysteine ​​residue-containing peptides (e.g., substitution, insertion, and addition to the N-terminal region) are introduced.

[0020] When an amino acid residue is mutated by substitution, the substitution may be a conservative substitution. As used herein, the term "conservative substitution" means substituting a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the conservative amino acid substitutions may be substitutions between aspartic acid and glutamic acid, between arginine, lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0021] In certain embodiments, the human ferritin H chain in the proteins of (B) and (C) above may be a human ferritin H chain further containing a functional peptide that does not contain a cysteine ​​residue. In the present invention, genetic modifications other than those for introducing modifying groups to the ferritin surface are permitted, and therefore, genetic modifications for further containing a functional peptide are permitted. The N-terminus of the ferritin monomer in higher organisms is exposed on the surface of the 24-mer. In the human ferritin H chain, which is the ferritin monomer of higher organisms, various functional peptides can be inserted into its N-terminus while maintaining the ability to form a 24-mer. Furthermore, the ferritin monomer in higher organisms has six α-helices that are highly conserved among various higher organisms. In human ferritin H chains, which are ferritin monomers in higher organisms, there are six α-helices: 1) the first region (also called the A region) consisting of amino acid residues from positions 15 to 42, 2) the second region (also called the B region) consisting of amino acid residues from positions 50 to 77, 3) the third region (also called the C region) consisting of amino acid residues from positions 97 to 124, 4) the fourth region (also called the D region) consisting of amino acid residues from positions 128 to 137, 5) the fifth region (also called the D region) consisting of amino acid residues from positions 139 to 159, and 6) the sixth region (also called the E region) consisting of amino acid residues from positions 165 to 174. The regions between these α-helices are called flexible linker regions, and it has been reported that various functional peptides can be inserted between these flexible linker regions while maintaining the ability to form a 24-mer (e.g., International Publication No. 2019 / 163871; U.S. Patent Application Publication No. 2016 / 0060307; Jae Og Jeon et al., ACS Nano (2013), 7(9), 7462-7471; Sooji Kim et al., Biomacromolecules (2016), 17(3), 1150-1159; Young Ji Kang et al., Biomacromolecules (2012), 13(12), 4057-4064). For example, the α-helices of the B and C regions in ferritin monomers are well known in the art, and those skilled in the art can appropriately determine the positions of the α-helices of the B and C regions in ferritin monomers derived from various organisms.Therefore, the flexible linker region between the α-helices of regions B and C into which the functional peptide is inserted in the present invention is also well known in the art and can be appropriately identified by those skilled in the art. For example, the flexible linker region between the second and third regions, positions 78-96 (preferably 83-91), can be suitably used as such an insertion site for the functional peptide into the human ferritin H chain (e.g., International Publication No. 2019 / 163871).

[0022] Functional peptides can be those that, when fused with a target protein, can impart a desired function to the target protein. Examples of such peptides include peptides that have binding ability to target materials, protease-degradable peptides, cell-permeable peptides, and stabilizing peptides.

[0023] The functional peptide inserted into the region described above may be a single peptide having the desired function, or it may be several peptides of the same or different species (e.g., two, three, or four) having the desired function. When the functional peptide is several such peptides, the multiple functional peptides can be inserted in any order and fused with the ferritin monomer. Fusion can be achieved via an amide bond. Fusion may be achieved directly by an amide bond, or indirectly by an amide bond mediated by a peptide (peptide linker) consisting of one amino acid residue or several amino acid residues (e.g., 2 to 20, preferably 2 to 10, more preferably 2, 3, 4, or 5). Various peptide linkers are known, and such peptide linkers can be used in the present invention. Preferably, the total length of the peptide inserted into the region described above is 20 amino acid residues or less.

[0024] When using a functional peptide that has the ability to bind to a target material, the target material can be, for example, a bioorganic molecule, a protein purification tag (e.g., histidine tag, maltose-binding protein tag, glutathione-S-transferase), or a material that can interact with it (e.g., nickel, maltose, glutathione), or a labeling substance (e.g., radioactive material, fluorescent material, dye). Ferritin is known to be taken up by transferrin receptor-presenting cells (e.g., L. Li et al. Proc Natl Acad Sci USA. 2010;107(8):3505-10). When using a functional peptide that has the ability to bind to a bioorganic molecule, ferritin can be delivered to cells and organs that express bioorganic molecules to which ferritin would not normally bind.

[0025] Examples of bioorganic molecules include proteins (e.g., oligopeptides or polypeptides), nucleic acids (e.g., DNA or RNA, or nucleosides, nucleotides, oligonucleotides, or polynucleotides), carbohydrates (e.g., monosaccharides, oligosaccharides, or polysaccharides), and lipids. Bioorganic molecules may also be cell surface antigens (e.g., cancer antigens, cardiac disease markers, diabetes markers, neurological disease markers, immune disease markers, inflammatory markers, hormones, and infectious disease markers). Bioorganic molecules may also be disease antigens (e.g., cancer antigens, cardiac disease markers, diabetes markers, neurological disease markers, immune disease markers, inflammatory markers, hormones, and infectious disease markers). Various peptides have been reported to have binding ability to such bioorganic molecules.For example, peptides that can bind to proteins (see F. Danhier et al., Mol. Pharmaceutics, 2012, vol.9, No.11, p.2961., CH. Wu et al., Sci. Transl. Med., 2015, vol.7, No.290, 290ra91., L. Vannucci et al. Int. J. Nanomedicine. 2012, vol.7, p.1489, J. Cutrera et al., Mol. Ther. 2011, vol.19(8), p.1468, R. Liu et al., Adv. Drug Deliv. Rev. 2017, vol.110-111, p.13), peptides that can bind to nucleic acids (see R. Tan See et.al. Proc.Natl.Acad.Sci.USA, 1995, vol.92, p.5282; R.Tan et.al. Cell, 1993, vol.73, p.1031; R.Talanian et.al. Biochemistry. 1992, vol.31, p.6871; peptides with carbohydrate binding ability (e.g., K.Oldenburg et.al., Proc.Natl.Acad.Sci.USA, 1992, vol.89, No.12, pp.5393-5397; K.Yamamoto et.al., J.Biochem., 1992, vol.111, p.436; A.Baimiev) Various peptides have been reported, including peptides that have the ability to bind to lipids (see et al., Mol. Biol. (Moscow), 2005, vol. 39, No. 1, p. 90.) and peptides that have the ability to bind to lipids (see, for example, O. Kruse et al., B Z. Naturforsch., 1995, vol. 50c, p. 380, O. Silva et al., Sci. Rep., 2016, vol. 6, 27128., A. Filoteo et al., J. Biol. Chem., 1992, vol. 267, No. 17, p. 11800).

[0026] When protease-degradable peptides are used as functional peptides, the proteases include, for example, cysteine ​​proteases such as caspases and cathepsins (D. McIlwain et al., Cold Spring Harb Perspect Biol., 2013, vol.5, a008656, V. Stoka et al., IUBMB Life. 2005, vol.57, No.4-5 p.347), collagenases (G. Lee et al., Eur J Pharm Biopharm., 2007, vol.67, No.3), p.646), thrombin and factor Xa (R. Jenny et al., Protein Expr. Purif., 2003, vol.31, p.1, H. Xu et al., J. Virol., Examples include (2010, vol.84, No.2, p.1076) and virus-derived proteases (C. Byrd et al., Drug Dev. Res., 2006, vol.67, p.501). Various protease-degradable peptides are known (e.g., E. Lee et al., Adv. Funct. Mater., 2015, vol. 25, p. 1279; G. Lee et al., Eur J Pharm Biopharm., 2007, vol. 67, No. 3, p. 646; Y. Kang et al., Biomacromolecules, 2012, vol. 13, No. 12, p. 4057; R. Talanian et al., J. Biol. Chem., 1997, vol. 272, p. 9677; Jenny et al., Protein Expr. Purif., 2003, vol. 31, p. 1; H. Xu et al., J. Virol., 2010, vol. 84, No. 2, p. 1076).In the present invention, various protease-degradable peptides can be used (e.g., E. Lee et al., Adv. Funct. Mater., 2015, vol. 25, p. 1279; G. Lee et al., Eur J Pharm Biopharm., 2007, vol. 67, No. 3, p. 646; Y. Kang et al., Biomacromolecules, 2012, vol. 13, No. 12, p. 4057; R. Talanian et al., J. Biol. Chem., 1997, vol. 272, p. 9677; Jenny et al., Protein Expr. Purif., 2003, vol. 31, p. 1; H. Xu et al., J. Virol., 2010, vol. 84, No. 2, p. 1076).

[0027] When stabilized peptides are used as functional peptides, various types of stabilized peptides can be used (e.g., X. Meng et al., Nanoscale, 2011, vol.3, No.3, p.977; E. Falvo et al., Biomacromolecules, 2016, vol.17, No.2, p.514).

[0028] When cell-permeable peptides are used as functional peptides, various cell-permeable peptides can be used (e.g., Z. Guo et al. Biomed. Rep., 2016, vol. 4, No. 5, p. 528).

[0029] As functional peptides, peptides having the ability to bind to target materials are preferred. Of the peptides having the ability to bind to target materials, peptides having the ability to bind to bioorganic molecules are preferred, and peptides having the ability to bind to proteins are more preferred.

[0030] Human ferritin H chains may be modified in their N-terminal and / or C-terminal regions. The N-terminus of animal ferritin monomers, such as human ferritin monomers, is exposed on the surface of the multimer, while its C-terminus cannot be exposed. Therefore, the peptide portion attached to the N-terminus of animal ferritin monomers is exposed on the surface of the multimer and can interact with target materials present outside the multimer, but the peptide portion attached to the C-terminus of animal ferritin monomers is not exposed on the surface of the multimer and cannot interact with target materials present outside the multimer (e.g., International Publication No. 2006 / 126595). However, it has been reported that the C-terminus of animal ferritin monomers can be used for encapsulating drugs into the lumen of a multimer by modifying its amino acid residues (e.g., see YJKang, Biomacromolecules. 2012, vol. 13(12), 4057).

[0031] In certain embodiments, the human ferritin H chain may have a peptide moiety added to its N-terminus as a modification to its N-terminal region. Examples of peptide moieties to be added include the functional peptides described above. Alternatively, examples of peptide moieties to be added include peptide components that improve the solubility of the target protein (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factors), peptide components with other functions (e.g., full-length proteins or parts thereof), and linkers. The peptide moiety added to the N-terminus of the human ferritin H chain can be the same as or different from the functional peptide inserted in the region between the second and third α-helices, but it is also preferable to use a different peptide from the viewpoint of realizing interactions with different target materials. Preferably, the peptide moiety added to the N-terminus of the human ferritin H chain is a functional peptide as described above. It is also preferable that the peptide moiety added to the N-terminus includes an amino acid residue corresponding to the start codon (e.g., a methionine residue) at the N-terminus. Such a design can promote the translation of the human ferritin H chain.

[0032] In another specific embodiment, the human ferritin H chain may be modified in its C-terminal region by substituting an amino acid residue in the C-terminal region with a reactive amino acid residue, inserting a reactive amino acid residue in the C-terminal region, or adding a reactive amino acid residue or a peptide containing it (e.g., a peptide consisting of 2 to 12, preferably 2 to 5, amino acid residues) to the C-terminus. For example, in the case of the human ferritin H chain, such a C-terminal region is the region consisting of amino acid residues from positions 175 to 183 (preferably from positions 179 to 183). Such modification allows the reactive amino acid residue to react with a predetermined substance (e.g., a drug, a labeling substance), thereby encapsulating the predetermined substance in the lumen of the polymer via covalent bonding. Examples of such reactive amino acid residues include cysteine ​​residues having a thiol group, lysine residues having an amino group, arginine residues, asparagine residues, and glutamine residues, but cysteine ​​residues are preferred. Preferably, the modification of the C-terminal region of the fusion protein of the present invention is the addition of a reactive amino acid residue or a peptide containing it to the C-terminus.

[0033] In a preferred embodiment, if the human ferritin H chain further comprises a functional peptide, the functional peptide may be a human-derived peptide. When the functional peptide is a human-derived peptide, the risk of immunogenicity in humans can be reduced.

[0034] Preferably, the human ferritin H chain contained in the modified ferritin of the present invention is a natural-type human ferritin H chain that may have a methionine residue at position 1 deleted.

[0035] The modified ferritin of the present invention is a multimer containing the human ferritin H chain as described above. Human ferritin can form a 24-mer containing 24 ferritin monomers (H chain and L chain). Naturally occurring ferritin in humans is usually a hetero-24-mer, which is a mixture of H chains and L chains, while experimentally prepared ferritin is usually a homo-24-mer. This is because preparing a hetero-24-mer experimentally requires separately preparing ferritin H chains and L chains and then mixing them, so a homo-24-mer is prepared to reduce the experimental burden. Therefore, the modified ferritin of the present invention may be a homo-24-mer containing only human ferritin H chains, or a hetero-24-mer containing both human ferritin H chains and L chains. In this case, the human ferritin L chain may contain the mutations described above in the human ferritin H chain (e.g., mutations that further include functional peptides, and modifications of the N-terminus and C-terminus). From the standpoint of simple preparation, a homo-24-mer containing only human ferritin H chains is preferred.

[0036] Multimers containing human ferritin H chains can be obtained by using host cells containing polynucleotides encoding human ferritin H chains to induce host cells to produce human ferritin H chains. Examples of host cells for producing human ferritin H chains include cells derived from animals, insects, plants, or microorganisms. As animals, mammals or birds (e.g., chickens) are preferred, with mammals being more preferred. Examples of mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), and domestic and working mammals (e.g., cattle, pigs, sheep, goats, horses).

[0037] In preferred embodiments, the host cells are human cells or cells commonly used for the production of human proteins (e.g., Chinese hamster ovary (CHO) cells, human fetal kidney-derived HEK293 cells). From the viewpoint of clinical application in humans, it is preferable to use such host cells.

[0038] In another preferred embodiment, the host cell is a microorganism. Such a host cell may be used from the viewpoint of mass production of fusion proteins, etc. Examples of microorganisms include bacteria and fungi. As bacteria, any bacteria used as host cells can be used, for example, bacteria of the genus Bacillus (e.g., Bacillus subtilis), bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum), bacteria of the genus Escherichia (e.g., Escherichia coli), and bacteria of the genus Pantoea (e.g., Pantoea ananatis). As fungi, any fungi used as host cells can be used, for example, fungi of the genus Saccharomyces (e.g., Saccharomyces cerevisiae). Examples include fungi of the genus Cerevisiae and Schizosaccharomyces (e.g., Schizosaccharomyces pombe). Alternatively, filamentous fungi may be used as microorganisms. Examples of filamentous fungi include bacteria belonging to the genera Acremonium / Talaromyces, Trichoderma, Aspergillus, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, and Hypocrea.

[0039] A host cell can be designed to contain an expression unit that includes a polynucleotide encoding the human ferritin H chain, in addition to a promoter operably linked to the polynucleotide. The term "expression unit" refers to a unit that enables the transcription of a polynucleotide, and consequently the production of the protein encoded by the polynucleotide, by including a predetermined polynucleotide to be expressed as a protein and a promoter operably linked to it. The expression unit may further include elements such as a terminator, a ribosome binding site, and a drug resistance gene. The expression unit may be DNA or RNA, but DNA is preferred. The expression unit can be contained in a genomic region (e.g., a natural genomic region which is a natural locus in which the polynucleotide encoding the protein is intrinsically located, or a non-natural genomic region which is not such a natural locus) or a non-genomic region (e.g., within the cytoplasm) in a microorganism (host cell). The expression unit may be contained within a genomic region at one or more different locations (e.g., 1, 2, 3, 4, or 5). Specific forms of expression units contained in a non-genomic region include plasmids, viral vectors, phages, and artificial chromosomes.

[0040] The modified ferritin of the present invention may further contain human ferritin H chains that do not contain a modifying group, as long as it contains one or more human ferritin H chains that contain a modifying group specifically covalently bonded to the cysteine ​​residue at position 91 and / or 103. Such modified ferritin can be prepared by reacting ferritin with a reactive substance (e.g., thiol modification reagents and functional substances) in a low ratio. Such modified ferritin can also be prepared by separately preparing a 24-mer containing a human ferritin H chain that contains a modifying group specifically covalently bonded to the cysteine ​​residue at position 91 and / or 103, and a 24-mer containing a human ferritin H chain that does not contain such a modifying group, and then dissociating and reassociating these 24-mers in a solution containing these 24-mers in a predetermined ratio. Since ferritin can dissociate and associate relatively easily, it may exist in the form of a 24-mer, monomer, or a combination thereof, depending on the conditions such as the solvent in which it is contained. Therefore, by appropriately adjusting the conditions of the solvent containing ferritin, the 24-mer can be dissociated into monomers, and the dissociated monomers can be reassociated into the 24-mer. Such dissociation and reassociation are used when encapsulating various substances in the lumen of ferritin (see, for example, WO2020 / 090708; Chinese Patent Application Publication No. 106110333; Journal of Controlled Release 196(2014) 184-196; Biomacromolecules 2016,17,514-522; Proc Natl Acad Sci USA. 2014 111(41):14900-5). If the modified ferritin of the present invention further comprises human ferritin H chains that do not contain the modifying group, the molar ratio of the human ferritin H chains containing the modifying group and the human ferritin H chains that do not contain the modifying group in the modified ferritin of the present invention can be controlled according to the ratio of the reactive substance to the ferritin, or these ratios before re-association in solution. Preferably, the modified ferritin of the present invention is a 24-mer comprising 24 human ferritin H chains, each containing a modifying group specifically covalently bonded to the cysteine ​​residue at positions 91 and / or 103.

[0041] The human ferritin H chain contained in the modified ferritin of the present invention contains a modifying group that is specifically covalently bonded to the cysteine ​​residue at position 91 and / or 103. "Specific" covalent bonding in the modified ferritin of the present invention means that the selectivity for covalent bonding to the cysteine ​​residue at position 91 and / or 103 (more precisely, to the sulfur atom in the side chain of the cysteine ​​residue) is significantly higher than that for other amino acid residues. Preferably, the "specific" covalent bonding in the modified ferritin of the present invention is covalently bonded only to the cysteine ​​residue at position 91 and / or 103, and not to any other amino acid residues.

[0042] The amino acid residues modified in this invention are the cysteine ​​residues at positions 91 and / or 103 in the human ferritin H chain. In human ferritin, the cysteine ​​residues at positions 91 and 103 are in close proximity to each other. The modified ferritin of this invention may contain one or two modifying groups per human ferritin H chain at these positions. Therefore, the present invention can provide modified ferritin comprising a human ferritin H chain containing either (A) or (B) below: (A) A human ferritin H chain containing one or two modifying groups specifically covalently bonded to one or two sulfur atoms in the side chain of one or both cysteine ​​residues at position 91 or 103; or (B) A human ferritin H chain containing one modifying group that specifically cross-links two sulfur atoms in the side chains of cysteine ​​residues at positions 91 and 103 by a covalent bond. Modified ferritin containing such human ferritin H chains can be prepared by setting the molar ratio of human ferritin to the thiol-modifying reagent or functional substance described later (human ferritin: thiol-modifying reagent or functional substance) to be, for example, within the range of 1:1 to 1:30, preferably 1:1 to 1:20, and more preferably 1:1 to 1:15.

[0043] Preferably, the modified ferritin of the present invention can contain 24 or 48 modifying groups. This is because the modified ferritin of the present invention is a 24-mer that can contain one or two modifying groups per human ferritin H chain at the cysteine ​​residues at positions 91 and 103.

[0044] The modified ferritin of the present invention may also contain two or more modifying groups (e.g., two or more reactive groups or modifying groups containing functional substances). According to the present invention, the number of modifying groups introduced into modified ferritin can be highly controlled. For example, by introducing 24 first modifying groups into ferritin, and then introducing 24 second modifying groups into the resulting modified ferritin, modified ferritin containing two or more modifying groups with a highly controlled number can be obtained. This is because the modified ferritin of the present invention is a 24-mer that can contain one or two modifying groups per human ferritin H chain at the cysteine ​​residues at positions 91 and 103.

[0045] In one embodiment, the modified ferritin of the present invention may be a reactive group-added ferritin in which the modifying group includes a reactive group. In other words, in this case, Contains human ferritin H chains, A reactive ferritin is provided, which contains a reactive modifying group, specifically covalently bonded to the cysteine ​​residue at position 91 and / or 103 of the human ferritin H chain. For example, such reactive group-added ferritins are useful as synthetic intermediates for functional substance-added ferritins.

[0046] Reactive group-added ferritin can be produced by reacting human ferritin with a thiol-modifying reagent. The ferritin used in the reaction may be in either a 24-mer or monomeric state, but the 24-mer state is preferred. The thiol-modifying reagent used in the present invention is a reactive substance containing at least one reaction site for a thiol group, and has the ability to add a modifying group containing a reactive group to human ferritin through the reaction. Any reagent capable of catalyzing such a reaction can be used as the thiol-modifying reagent. From the viewpoint of improving reaction efficiency, the thiol-modifying reagent is preferably one that contains one or more substructures selected from the group consisting of maleimide moiety, benzyl halide moiety, α-haloamide moiety, α-haloketone moiety, alkene moiety, alkyne moiety, fluoroaryl moiety, nitroaryl moiety, methylsulfonyloxadiazole moiety, and disulfide moiety.

[0047] The reactive group can be appropriately configured to react with the functional substance. Therefore, the reactive group is not particularly limited as long as it contains a portion that can react with a predetermined functional group in the functional substance (the functional substance may be derivatized to have a predetermined functional group that can react with the reactive group). The number of reactive groups in the modifying group can be one or more, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. When the number of reactive groups in the modifying group is multiple, the reactive groups may be of the same type or different types. For example, when reaction with multiple functional substances of the same type is desired, from the viewpoint of adopting a simple structure, the same type of reactive group is preferred. On the other hand, when reaction with multiple functional substances of different types is desired, from the viewpoint of ensuring selectivity of the reaction, different types of reactive groups are preferred.

[0048] In certain embodiments, the reactive group may be a bioorthogonal functional group to the protein. In such cases, the reaction between the reactive group-added ferritin and the functional substance can be promoted while suppressing the reaction between the reactive group-added ferritin and the protein. A bioorthogonal functional group is a group that does not react with or reacts poorly with biological components (e.g., amino acids, nucleic acids, lipids, sugars, phosphates), but reacts readily with components other than biological components, exhibiting reaction selectivity. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)). Therefore, bioorthogonal functional groups for proteins refer to groups that cannot react with the side chains of the 20 naturally occurring amino acid residues that make up proteins, or that react poorly with those side chains, but readily react with targets other than those side chains, thus possessing reaction selectivity. The 20 naturally occurring amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inert to normal reactions.Therefore, bioorthogonal functional groups to proteins are functional groups that cannot react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, as well as, if necessary, the side chain of cysteine ​​(e.g., if the cysteine ​​residues at positions 91 and / or 103 of the human ferritin H chain are not sufficiently modified), in addition to the side chains of these amino acids which have side chains that are inactive to normal reactions.

[0049] Examples of bioorthogonal functional groups for proteins include maleimide moieties, azide moieties, ketone moieties, aldehyde moieties, thiol moieties, alkene moieties (in other words, any moiety that has a vinyl (ethenyl) moiety or vinylene (ethenylene) moiety, which is the smallest unit having an intercarbon double bond; the same applies herein), alkyne moieties (in other words, any moiety that has an ethynyl moiety or ethynylene moiety, which is the smallest unit having an intercarbon triple bond; the same applies herein), halogen moieties (e.g., fluoride) Examples of substructures include groups comprising one or more substructures selected from the group consisting of a moiety, chloride moiety, bromide moiety, iodide moiety, tetrazine moiety, nitrone moiety, hydroxylamine moiety, nitrile moiety, hydrazine moiety, boronic acid moiety, cyanobenzothiazole moiety, allyl moiety, phosphine moiety, disulfide moiety, thioester moiety, α-halocarbonyl moiety (e.g., a carbonyl moiety having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α position), isonitrile moiety, cydonone moiety, and selenium moiety. Proteins include proteins that may contain free thiols in the side chain of cysteine ​​residues (e.g., proteins other than antibodies) and proteins that may not contain free thiols (e.g., antibodies, proteins that do not contain cysteine ​​residues, and ferritin, such as that provided in the present invention, which includes a human ferritin H chain in which the cysteine ​​residues at positions 91 and / or 103 are sufficiently modified). In proteins that may not contain free thiols, the thiol functions as a bioorthogonal functional group. Therefore, in principle, the bioorthogonal functional groups for proteins include thiol moieties. However, if the cysteine ​​residues at positions 91 and / or 103 are not sufficiently modified (e.g., if the average number of cysteine ​​residue modifications per human ferritin H chain is 1), the bioorthogonal functional groups for proteins do not include thiol moieties.

[0050] The reactive group can also be configured to react specifically with a particular functional group in the functional substance (e.g., peptides, proteins, nucleic acids, lipids, low-molecular-weight organic compounds) that may subsequently react with the reactive group.

[0051] A reactive group only needs to be able to react with a functional substance. Therefore, a reactive group may have substituents as long as it includes a substructure that can react with a predetermined functional group in the functional substance. For example, to simplify the structure of the portion that can react with a predetermined functional group in the functional substance, it is preferable that the substructure does not have substituents. However, if the portion that can react with a predetermined functional group in the functional substance has a substituted hydrogen atom, it is also preferable that the substructure has substituents. The number of such substituents varies depending on factors such as the number of substituted hydrogen atoms, and is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. Examples of such substituents include monovalent hydrocarbon groups, monovalent heterocyclic groups, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), hydroxyl groups, nitro groups, sulfate groups, sulfonic acid groups, cyano groups, and combinations of two or more of these (for example, 2 to 6, preferably 2 to 5, more preferably 2 to 4, even more preferably 2 or 3, and particularly preferably 2).

[0052] Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups. A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure and does not contain a cyclic structure in the main chain. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched. As alkyl groups, alkyl groups having 1 to 12 carbon atoms are preferred, alkyl groups having 1 to 6 carbon atoms are more preferred, and alkyl groups having 1 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkyl groups having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl. As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl. As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl. Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0053] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic. As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl. As for cycloalkynyls, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl. As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0054] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl. Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0055] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups, with alkyl being more preferred.

[0056] A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As monovalent aromatic heterocyclic groups, those having 1 to 15 carbon atoms are preferred, those having 1 to 9 carbon atoms are more preferred, and those having 1 to 6 carbon atoms are even more preferred. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonal, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl. As monovalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 2 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 2 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 2 to 6 carbon atoms are even more preferred. The number of carbon atoms in the above carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil. Among these, a 5-membered or 6-membered heterocyclic group is preferred as the monovalent heterocyclic group.

[0057] The reaction for producing reactive ferritin can be carried out under conditions (mild conditions) that do not cause denaturation or decomposition of ferritin (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in a suitable reaction system, such as a buffer solution, at a temperature that does not cause denaturation or decomposition of ferritin (e.g., about 15 to 60°C, preferably 15 to 45°C). The pH of the buffer solution is, for example, 3.0 to 12.0, preferably 5.0 to 9.0, and more preferably 6.0 to 8.0. The buffer solution may contain components such as a catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0058] In another embodiment, the modified ferritin of the present invention may be a functional substance-added ferritin in which the modifying group contains a functional substance. In other words, in this case, Contains human ferritin H chains, A functional substance-modified ferritin is provided, which contains a modification group containing a functional substance, in which the human ferritin H chain is specifically covalently bonded to the cysteine ​​residue at positions 91 and / or 103. For example, ferritin with such functional substances attached is useful for the prevention, treatment, or diagnosis of specific diseases in humans.

[0059] The functional substance is not particularly limited as long as it is a substance that confers any function to ferritin, and examples include drugs, labeling substances, and stabilizers, but is preferably a drug or labeling substance. The functional substance may also be a single functional substance or a substance in which two or more functional substances are linked together.

[0060] The drug may be a drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cerebrovascular diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the orthopedic field (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive organ diseases). The drug may be a preventive or therapeutic drug for the disease or a drug for alleviating side effects.

[0061] More specifically, the drug is an anticancer agent. Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes or substances containing them. Examples of chemotherapeutic agents include DNA-damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA-binding inhibitors, tubulin-binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., 153 H), radioisotopes of carbon atoms (e.g., 14 C), radioisotopes of phosphorus atoms (e.g., 32 P), radioisotopes of sulfur atoms (e.g., 35 S ), radioisotopes of yttrium (e.g., 90 Y), radioisotopes of technetium (e.g., 99m Tc), radioisotopes of indium (e.g., 111 In), radioisotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131 I), radioisotopes of samarium (e.g., 153Sm), radioactive isotopes of rhenium (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212 Bi) is one example. More specifically, drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calichemycin, duocalmycin, eribulin, anthracycline, dmDNA31, and tubulisin.

[0062] A targeting substance is an affinity substance for a target (e.g., tissue, cell, or substance). Examples of targeting substances include antibodies against the target substance or fragments thereof that have the ability to bind to the target substance, ligands that have the ability to bind to receptors, proteins that have the ability to form complexes (e.g., adapter proteins), proteins that have the ability to bind to glycans (lectins), glycans that have the ability to bind to proteins, and nucleic acids that have the ability to bind to complementary sequences (e.g., natural nucleic acids such as DNA and RNA, or artificial nucleic acids).

[0063] Labeling substances are substances that enable the detection of targets (e.g., tissues, cells, materials). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those mentioned above), or substances containing them.

[0064] Stabilizers are substances that enable the stabilization of ferritin. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0065] Functional substances may also be peptides, proteins (e.g., antibodies), nucleic acids (e.g., DNA, RNA, and artificial nucleic acids), small organic compounds (e.g., small organic compounds described later), chelators, glycans, lipids, high molecular weight polymers, and metals (e.g., gold).

[0066] Functionalized ferritin can be produced by reacting human ferritin with a functional substance. The functional substance used in such a reaction is a reactive substance having at least one reaction site for a thiol group, and has the ability to add a modifying group containing the functional substance to human ferritin through the reaction. Any functional substance having a functional group that readily reacts with a thiol group can be used as the functional substance used in such a reaction. Alternatively, if the functional substance does not have a functional group that readily reacts with a thiol group, a functional substance that has been derivatized to have such a functional group can be used. From the viewpoint of improving reaction efficiency, the functional substance (which may be derivatized) is preferably one that contains one or more substructures selected from the group consisting of maleimide moiety, benzyl halide moiety, α-haloamide moiety, α-haloketone moiety, alkene moiety, alkyne moiety, fluoroaryl moiety, nitroaryl moiety, methylsulfonyloxadiazole moiety, and disulfide moiety.

[0067] Functionally modified ferritin can also be produced by reacting human ferritin with a thiol-modifying reagent, and then further reacting the reactive group-modified ferritin obtained from the reaction with a functional substance. The step of reacting human ferritin with a thiol-modifying reagent can be carried out as described above. The functional substance used in the step of further reacting reactive group-modified ferritin with a functional substance is a reactive substance having at least one reaction site for the reactive group in the reactive group-modified ferritin, and has the ability to add a modifying group containing the functional substance to human ferritin through the reaction. Any functional substance having a functional group that readily reacts with the reactive group can be used as the functional substance used in such a reaction. Alternatively, if the functional substance does not have a functional group that readily reacts with the reactive group, a functional substance that has been derivatized to have such a functional group can be used. Therefore, the functional group in the functional substance used in the reaction can be appropriately determined according to the type of reactive group.

[0068] Derivatization of functional substances is common technical knowledge in the art (e.g., International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be carried out using a crosslinking agent as described above. Alternatively, derivatization may be carried out using a specific linker having the desired functional group. For example, such a linker may be capable of separating the functional substance and the antibody by cleavage in a suitable environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers that are degraded by specific proteases (e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory proteases)) (e.g., U.S. Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at locally acidic sites present in the body (e.g., U.S. Patent Nos. 5,622,929, 5,122,368; 5,824,805). Linkers may also be self-immolative (e.g., International Publication No. 02 / 083180, International Publication No. 04 / 043493, International Publication No. 05 / 112919). In this invention, derivatized functional substances can also be simply referred to as "functional substances."

[0069] In certain embodiments, the functional substance may be a functional substance having a functional group that readily reacts with a reactive group that is a bioorthogonal functional group to a protein, or a functional substance that has been derivatized to have a functional group that readily reacts with said reactive group. The functional group that readily reacts with a bioorthogonal functional group may also vary depending on the specific type of bioorthogonal functional group. Those skilled in the art can appropriately select a suitable functional group as a functional group that readily reacts with a bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Examples of functional groups that readily react with a bioorthogonal functional group include, but are not limited to, maleimide groups and disulfide groups when the bioorthogonal functional group corresponds to a thiol moiety, azide groups when the bioorthogonal functional group corresponds to an alkyne moiety, and hydrazine groups when the bioorthogonal functional group corresponds to an aldehyde or ketone moiety.

[0070] The reaction for producing functionally modified ferritin can be carried out under conditions (mild conditions) that do not cause denaturation or decomposition of ferritin (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in a suitable reaction system, such as a buffer solution, at a temperature that does not cause denaturation or decomposition of ferritin (e.g., about 15 to 60°C, preferably 15 to 45°C). The pH of the buffer solution is, for example, 3.0 to 12.0, preferably 5.0 to 9.0, and more preferably 6.0 to 8.0. The buffer solution may contain components such as a catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0071] The modified ferritin of the present invention may contain a substance in its lumen. Encapsulation of a substance into the lumen of modified ferritin can be achieved by utilizing the ferritin's property of being able to incorporate substances. Human ferritin forms a cage-like structure with a lumen of approximately 12 nm in outer diameter (7 nm in inner diameter). Therefore, the size of the substance can be such that it can be encapsulated within this lumen. Various inorganic and organic substances have been reported to be encapsulated within the lumen of ferritin (see, e.g., WO2020 / 090708; Chinese Patent Application Publication No. 106110333; Journal of Controlled Release 196(2014) 184-196; Biomacromolecules 2016,17,514-522; Proc Natl Acad Sci USA. 2014 111(41):14900-5). The encapsulation of a substance into the lumen of modified ferritin can be performed before or after the modification reaction of ferritin. The substance is preferably a low molecular weight organic compound. A low molecular weight organic compound is defined as an organic compound with a molecular weight of 1500 or less. Low molecular weight organic compounds can be natural or synthetic compounds. The molecular weight of the low molecular weight organic compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of the low molecular weight organic compound may also be 30 or more, 40 or more, or 50 or more. The low molecular weight organic compound may be a drug or labeling substance as described above. Examples of low molecular weight organic compounds include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and their salts.

[0072] If the modified ferritin of the present invention has a substance in its lumen, it may be provided as a set of several different types of modified ferritin containing several different types of substances (e.g., two, three, or four types). For example, if the modified ferritin of the present invention is provided as a set of two modified ferritins each containing two substances, such a set can be obtained by combining a first modified ferritin containing a first substance and a second modified ferritin containing a second substance different from the first substance, each prepared separately.

[0073] The formation of modified ferritin (e.g., ferritin with reactive groups or functional substances) can be confirmed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), and / or mass spectrometry, depending on the type and molecular weight of the modifying group. Regioselectivity can be confirmed by peptide mapping, for example. Peptide mapping can be performed by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as the protease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Modified ferritin can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography). [Examples]

[0074] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0075] Example 1: Preparation of human ferritin H chain The DNA encoding the human ferritin H chain (FTH (SEQ ID NO: 1)) (SEQ ID NO: 2) was totally synthesized. When the expression plasmid of this DNA is introduced into E. coli, the human ferritin H chain is expressed in E. coli. The methionine at the start codon is converted to N-formylmethionine and removed by post-translational modification. As a result, the human ferritin H chain defined by the amino acid sequence of SEQ ID NO: 3 is obtained, with the methionine residue at position 1 of the amino acid sequence of SEQ ID NO: 1 removed (Figure 1). Hereafter, the positions of the amino acid residues of human ferritin will be identified based on the typical natural human ferritin H chain defined by the amino acid sequence of SEQ ID NO: 1. The positions of the amino acid sequences of SEQ ID NO: 3 may also be shown as needed.

[0076] PCR was performed using synthetic DNA encoding human ferritin H chain as a template, with 5'-GAAGGAGATATACATATGACGACCGCGTCCACCTCG-3' (SEQ ID NO: 4) and 5'-CTCGAATTCGGATCCTTAGCTTTCATTATCACTGTC-3' (SEQ ID NO: 5) as primers. PCR was also performed using pET20 (Merck) as a template, with 5'-TTTCATATGTATATCTCCTTCTTAAAGTTAAAC-3' (SEQ ID NO: 6) and 5'-TTTGGATCCGAATTCGAGCTCCGTCG-3' (SEQ ID NO: 7) as primers. After purifying the obtained PCR products using the Wizard DNA Clean-Up System (Promega), an expression plasmid (pET20-FTH) containing the gene encoding FTH was constructed by in-fusion enzyme treatment with the In-Fusion HD Cloning Kit (Takara Bio) at 50°C for 15 minutes.

[0077] Next, Escherichia coli BL21(DE3) cells into which the constructed pET20-FTH had been introduced were incubated in 100 ml of LB medium (containing 10 g / l Bacto-typtone, 5 g / l Bacto-yeast extract, 5 g / l NaCl, and 100 mg / l ampicillin) in a flask at 37°C for 24 hours. After sonication of the resulting cells, the supernatant was heated at 60°C for 20 minutes. After heating, the supernatant was cooled and centrifuged. The supernatant was then injected into a HiPerp Q HP column (Cytiva) equilibrated with 50 mM TrisHCl buffer (pH 8.0), and the target protein was separated and purified by applying a salt concentration gradient with 50 mM TrisHCl buffer (pH 8.0) containing 0 mM to 500 mM NaCl, based on differences in surface charge. The solvent in the protein-containing solution was replaced with 10 mM TrisHCl buffer (pH 8.0) by centrifugal ultrafiltration using a Vivaspn 20-100K (Cytiva). This solution was injected into a HiPrep 26 / 60 Sephacryl S-300 HR column (Cytiva) equilibrated with 10 mM TrisHCl buffer (pH 8.0) to separate and purify FTH. The FTH-containing solution was concentrated by centrifugal ultrafiltration using a Vivaspn 20-100K (Cytiva), and the protein concentration was determined using a protein assay CBB solution (Nacalai Tesque) with bovine albumin as the standard. As a result, 1 ml of solution containing 5 mg / ml of FTH was obtained per 100 ml of culture medium.

[0078] Example 2: Specific modification of human ferritin H chains with ethyl maleimide and ESI-TOFMS analysis (2-1) Specific modification of human ferritin H chains by ethyl maleimide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. Ethyl maleimide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of ethyl maleimide (based on molar amount; the same applies hereafter) were added to the aqueous solution containing the human ferritin H chain and stirred, then shaken at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. The starting material, human ferritin H chain, showed a peak at 21093, while the product showed a peak at 21344, indicating the introduction of two ethyl maleimide molecules (molecular weight 125) (Figure 2).

[0079] Example 3: Peptide mapping of ethylmaleimide-modified human ferritin H chains (3-1) Trypsin treatment of ethylmaleimide-transformed human ferritin H chains In a 1.5 mL low-adsorption microtest tube, 10 μL of the sample solution (containing the ethyl maleimide derivative of human ferritin heavy chain, prepared in Example 2), 30 μL of 50 mM Tris-HCl buffer / 8 M urea, and 10 μL of trifluoroethanol were added and stirred. Then, 15 μL of 48 mM dithiothreitol aqueous solution was added and the mixture was heated at 65°C for 1 hour. After that, 15 μL of 120 mM iodoacetamide aqueous solution was added and the mixture was reacted at room temperature for 60 minutes under the protection of light. After the reaction, overreaction was suppressed by adding 5 μL of 48 mM dithiothreitol aqueous solution, 150 μL of 50 mM Tris-HCl buffer was added and stirred, and 2.5 μL of 100 ng / μL trypsin aqueous solution (Proteomics Grade, Code No. T6567-5x20 μg (SIGMA)) was added and allowed to stand at 37°C for 1 hour. Then, another 2.5 μL of 100 ng / μL trypsin aqueous solution was added and enzymatic digestion was carried out at 37°C for 15 hours. After digestion, 15 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction, and the solution was diluted 10-fold with 0.1% trifluoroacetic acid aqueous solution and subjected to LC-MS / MS measurement.

[0080] (3-2) LC-MS / MS measurement conditions for human ferritin heavy chain (Analyzer) Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific) Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0081] (HPLC analysis conditions) Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific) Analytical column: ESI-column (NTCC-360 / 75-3-125, 75μm × 12.5cm, 3μm (Nikkyo Technos Co., Ltd.)) Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300nL / min Sample injection volume: 1 μL Gradient conditions (B%): 2% (0.0-0.5 min), 2% → 30% (0.5-23.5 min), 30% → 75% (23.5-25.5 min), 75% (25.5-35.0 min)

[0082] (Mass spectrometer analysis conditions) Ionization method: ESI, Positive mode Scan type: Data-dependent acquisition Activation Type: Collision Induced Dissociation(CID) Data acquisition was performed using the included software, Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

[0083] (3-3) Analysis conditions for the modification site of the human ferritin H chain Modification site analysis of LC-MS / MS measurement results was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). Analysis using BioPharma Finder was performed with the S / N Threshold set to 20 and the MS Noise Level set to 1000. The digestive enzyme was set to Trypsin, and Specificity to High. Furthermore, the Mass Accuracy for peptide identification was set to 5 ppm. For Dynamic Modifications, methionine oxidation (+15.995 Da) and modifications to cysteine ​​residues (N-ethylmaleimide derivative (+125.048 Da), carbamide methylation with iodoacetamide (+57.021 Da)) were selected. Additionally, a filter was applied to include only samples with a Confidence Score of 80 or higher and for which MS / MS was observed. Furthermore, the amino acid sequence shown in SEQ ID NO: 1 was used as the data for the amino acid sequence to be searched for modification sites.

[0084] (3-4) Results of LC-MS / MS analysis of modification sites in human ferritin H chains Analysis using LC-MS / MS revealed the MS spectrum (measured value: m / z 903.67727, theoretical value: 903.67738, quadrivalent) of the peptide fragment IFLQDIKKPDCDDWESGLNAMECALHLEK (SEQ ID NO: 8), which consists of 29 amino acid residues and includes modification sites (N-ethylmaleimide introducer (+125.048Da)) on two cysteine ​​residues, generated by trypsin digestion of the specific modification of the human ferritin heavy chain with ethyl maleimide in Example 2 (Figure 3). The CID spectrum confirmed a product ion with m / z 1118.24 (theoretical value: 1117.85) corresponding to trivalent y27, indicating modification of cysteine ​​residues at positions 91 and 103 of the human ferritin H chain (positions 90 and 102 in the amino acid sequence of SEQ ID NO: 3) (Figure 4).

[0085] Example 4: Specific modification of human ferritin H chain with p-azidophenacyl bromide and ESI-TOFMS analysis (4-1) Specific modification of human ferritin H chains by 6 equivalents of p-azidophenacyl bromide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. p-azidophenacyl bromide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of p-azidophenacyl bromide were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 for the starting human ferritin H chain, and for the product, the starting peak and peaks at 21252 (with one p-azidophenacyl molecule, molecular weight 160) and 21412 (with two p-azidophenacyl molecules) were confirmed (Figure 5).

[0086] (4-2) Specific modification of human ferritin H chains by 12 equivalents of p-azidophenacylbromide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. p-azidophenacyl bromide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. 12 equivalents of p-azidophenacyl bromide were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 for the starting material human ferritin H chain, and peaks at 21252 (with one p-azidophenacyl molecule, molecular weight 160) and 21412 (with two p-azidophenacyl molecules) were observed for the product (Figure 6).

[0087] Example 5: Peptide mapping of p-azidophenacyl-transformed human ferritin H chains (5-1) Trypsin treatment of p-azidofenasil-transformed human ferritin H chains The human ferritin heavy chain ethyl maleimide derivative was subjected to LC-MS / MS measurement in the same manner as in Example 3(3-1), except that the sample solution containing the human ferritin heavy chain p-azidophenacyl derivative, prepared in Example 4(4-2), was used instead of the sample solution containing the human ferritin heavy chain ethyl maleimide derivative, prepared in Example 2.

[0088] (5-2) LC-MS / MS measurement conditions for human ferritin H chain The analytical instrument, HPLC analysis conditions, and mass spectrometer analysis conditions are the same as in Example 3(3-2).

[0089] (5-3) Analysis conditions for the modification site of the human ferritin H chain Modification site analysis of LC-MS / MS measurement results was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). Analysis using BioPharma Finder was performed with the S / N Threshold set to 20 and the MS Noise Level set to 1000. The digestive enzyme was set to Trypsin, and Specificity to High. Furthermore, the Mass Accuracy for peptide identification was set to 5 ppm. For Dynamic Modifications, methionine oxidation (+15.995 Da) and modifications to cysteine ​​residues (p-azidophenacyl derivative (+159.043262 Da), p-aminophenacyl derivative (+133.052764 Da), and carbamide methylation with iodoacetamide (+57.021 Da)) were selected. Additionally, a filter was applied to include only samples with a Confidence Score of 80 or higher and for which MS / MS was observed. Furthermore, the amino acid sequence shown in SEQ ID NO: 1 was used as the data for the amino acid sequence to be searched for modification sites. Furthermore, during digestion, reductive alkylation occurs with dithiothreitol and iodoacetamide, so the p-azidophenacyl derivative is partially reduced to the p-aminophenacyl derivative.

[0090] (5-4) Results of LC-MS / MS analysis of modification sites of human ferritin H chain Analysis using LC-MS / MS revealed the MS spectrum (measured value: m / z 907.67977, theoretical value: 907.67992, tetravalent) of the peptide fragment IFLQDIKKPDCDDWESGLNAMECALHLEK (SEQ ID NO: 8), which consists of 29 amino acid residues and includes modification sites on two cysteine ​​residues (p-aminophenacyl introduced product (+133.052764Da) reduced by dithiothreitol) generated by trypsin digestion of the p-azidophenacyl-specifically modified ferritin heavy chain of Example 4 (4-2) (Figure 7). The CID spectrum confirmed a product ion with m / z 842.98 (theoretical value: 842.64) corresponding to tetravalent y27, indicating modification of cysteine ​​residues at positions 91 and 103 of the human ferritin H chain (positions 90 and 102 in the amino acid sequence of SEQ ID NO: 3) (Figure 8).

[0091] Based on the analysis results obtained in (5-4), the peak area values ​​of positions 91, 103, and 131 of the human ferritin H chain (positions 90, 102, and 130 in the amino acid sequence of SEQ ID NO: 3) were compared (Figure 9). It can be seen that positions 91 and 103 of the human ferritin H chain (positions 90 and 103 in the amino acid sequence of SEQ ID NO: 3) are selectively modified.

[0092] Example 6: Specific modification of human ferritin H chains with Cy5-PEG-maleimide and ESI-TOFMS analysis (6-1) Specific modification of human ferritin H chains by Cy5-PEG-maleimide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. Cy5-PEG-maleimide (Broad Pharm, BP-23037) was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of Cy5-PEG-maleimide were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak at 21094 was observed for the starting material human ferritin H chain, and a peak at 22836 was confirmed for the product, with the starting material peak and two Cy5-PEG-maleimide (molecular weight 871) molecules introduced (Figure 10).

[0093] Example 7: Specific modification of human ferritin H chains with DBCO-PEG4-maleimide and ESI-TOFMS analysis (7-1) Specific modification of human ferritin H chains by DBCO-PEG4-maleimide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. DBCO-PEG4-maleimide (Broad Pharm, BP-22294) was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of DBCO-PEG4-maleimide were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21094 for the starting human ferritin H chain, and a peak at 22443 was confirmed for the product, with the starting peak and two DBCO-PEG4-maleimide (molecular weight 673) molecules introduced (Figure 11).

[0094] Example 8: Specific modification of human ferritin H chains with 1,3-dichloroacetone, oxime ligation with aminooxy-PEG5-azide, and ESI-TOFMS analysis (8-1) Specific modification of human ferritin H chains with 1,3-dichloroacetone and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. 1,3-dichloroacetone was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of 1,3-dichloroacetone were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak at 21094 was observed for the starting material human ferritin H chain, and a peak at 21148 was confirmed for the product, in which one acetone molecule (molecular weight 54) was introduced (Figure 12).

[0095] (8-2) Specific modification of oxime ligation by aminooxy-PEG5-azide and ESI-TOFMS analysis The human ferritin H-chain acetone modified compound obtained in (8-1) was dissolved in acetate buffer at pH 4.7 to a concentration of 2.0 mg / ml. Aminooxy-PEG5-azide (Broad Pharm, BP-23194) was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Ten equivalents of aminooxy-PEG5-azide were added to the aqueous solution containing the human ferritin H-chain acetone modified compound and stirred, then the reaction was carried out by shaking at 37°C for 16 hours. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21148 for the starting material, human ferritin H-chain acetone modified compound, and a peak at 21452 was confirmed for the product, in which one aminooxy-PEG5-azide (molecular weight 304) was introduced (Figure 12).

[0096] Example 9: Peptide mapping of a 1,3-dichloroacetone-transformed human ferritin H chain. (9-1) Trypsin treatment of 1,3-dichloroacetone-derived human ferritin H chains 5 μL of the sample solution (containing the 1,3-dichloroacetone derivative of human ferritin H chain, prepared in Example 8), 35 μL of 50 mM Tris-HCl buffer / 8 M urea, and 10 μL of trifluoroethanol were added to a 1.5 mL low-adsorption microtest tube and stirred. Then, 15 μL of 48 mM dithiothreitol aqueous solution was added and the mixture was heated at 65°C for 1 hour. After that, 15 μL of 120 mM iodoacetamide aqueous solution was added and the mixture was reacted at room temperature for 1 hour under light protection. After the reaction, overreaction was suppressed by adding 5 μL of 48 mM dithiothreitol aqueous solution, 150 μL of 50 mM Tris-HCl buffer was added and stirred, and 2.5 μL of 100 ng / μL trypsin aqueous solution was added and the mixture was allowed to stand at 37°C for 1 hour. Then, another 2.5 μL of 100 ng / μL trypsin aqueous solution was added and the mixture was enzymatically digested at 37°C for 15 hours. After digestion, 15 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction, and the sample was diluted 10-fold with 0.1% formic acid aqueous solution before being subjected to LC-MS / MS analysis.

[0097] (9-2) LC-MS / MS measurement conditions for human ferritin H chain (Analyzer) Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific) Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0098] (HPLC analysis conditions) Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific) Analytical column: ESI-column (NTCC-360 / 75-3-125, 75μm × 12.5cm, 3μm (Nikkyo Technos Co., Ltd.)) Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300nL / min Sample injection volume: 1 μL Gradient conditions (B%): 2% (0.0-0.5 min), 2% → 30% (0.5-23.5 min), 30% → 75% (23.5-25.5 min), 75% (25.5-35.0 min)

[0099] (Mass spectrometer analysis conditions) Ionization method: ESI, Positive mode Scan type: Data-dependent acquisition Activation Type: Collision Induced Dissociation(CID) MS1:Orbitrap MS2: Ion Trap Data acquisition was performed using the included software, Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

[0100] (9-3) Analysis conditions for the modification site of the human ferritin H chain Modification site analysis of LC-MS / MS measurement results was performed using Xcalibur 3.0 (Thermo Fisher Scientific). In Xcalibur, extracted chromatograms were created within a range of ±0.005 from the theoretical peptide value, and the peak area was automatically calculated using software. When calculating the theoretical peptide value, the possibility of oxidation of methionine residues (+15.995 Da), 1,3-dichloroacetone modification of cysteine ​​residues (+54.011 Da), and carbamide methylation by iodoacetamide (+57.021 Da) was considered, and the amino acid sequence data used was that of the human ferritin H chain shown in Sequence ID No. 3.

[0101] (9-4) Analysis results of modification sites of human ferritin H chain by LC-MS / MS Analysis using LC-MS / MS revealed the MS spectrum (measured value: m / z 853.37134, theoretical value: 853.37194, trivalent) of the peptide fragment KPDCDDWESGLNAMECALHLEK (SEQ ID NO: 9), a peptide consisting of 22 amino acid residues containing one cysteine ​​residue modification (1,3-dichloroacetone modification (+54.011Da)), which was generated by trypsin digestion of the 1,3-dichloroacetone-transformed human ferritin H chain of Example 8 (Figure 13). From the CID spectrum, a product ion with m / z 1206.78 (theoretical value: 1206.50), corresponding to the divalent b21, was confirmed, indicating 1,3-dichloroacetone modifications at cysteine ​​residues at amino acid positions 90 and 102 (Figure 14). Furthermore, a comparison of peak areas showed that modifications to the cysteine ​​residues at amino acid positions 90 and 102 occurred with high selectivity (Figure 15).

[0102] Example 10: Construction of amide-modified ferritin (10-1) Specific modification of human ferritin H chains by α-iodoacetamide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 1 was dissolved in HEPES buffer at pH 8.5 to a concentration of 5.0 mg / ml. α-iodoacetamide was dissolved in N,N-dimethylformamide to a concentration of 50 mM. 20 equivalents of α-iodoacetamide were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 for the starting human ferritin H chain, and for the product, the starting peak and peaks 21151 (with one p-azidophenacil molecule, molecular weight 160) and 21208 (with two p-azidophenacil molecules) were confirmed (Figure 16).

[0103] Example 11: Peptide mapping of α-iodoacetamide derivatives of human ferritin H chains. (11-1) Trypsin treatment of α-iodoacetamide derivatives of human ferritin H chains 5 μL of the sample solution (containing the α-iodoacetamide derivative of human ferritin H chain, prepared in Example 10), 35 μL of 50 mM Tris-HCl buffer / 8 M urea, and 10 μL of trifluoroethanol were added to a 1.5 mL low-adsorption microtest tube and stirred. Then, 15 μL of 48 mM dithiothreitol aqueous solution was added and the mixture was heated at 65°C for 1 hour. After that, 15 μL of 120 mM iodoacetic acid aqueous solution was added and the mixture was reacted at room temperature for 1 hour under light protection. After the reaction, overreaction was suppressed by adding 5 μL of 48 mM dithiothreitol aqueous solution, 150 μL of 50 mM Tris-HCl buffer was added and stirred, and 2.5 μL of 100 ng / μL trypsin aqueous solution was added and the mixture was allowed to stand at 37°C for 1 hour. Then, another 2.5 μL of 100 ng / μL trypsin aqueous solution was added and the mixture was enzymatically digested at 37°C for 15 hours. After digestion, 15 μL of 20% trifluoroacetic acid aqueous solution was added to stop the reaction, and the sample was diluted 10-fold with 0.1% formic acid aqueous solution before being subjected to LC-MS / MS analysis.

[0104] (11-2) LC-MS / MS measurement conditions for human ferritin H chain The LC-MS / MS measurement conditions for human ferritin H chains were the same as in Example 9.

[0105] (11-3) Analysis conditions for the modification site of the human ferritin H chain Modification site analysis of LC-MS / MS measurement results was performed using Xcalibur 3.0 (Thermo Fisher Scientific). In Xcalibur, extracted chromatograms were created within a range of ±0.005 from the theoretical peptide value, and the peak area was automatically calculated using software. When calculating the theoretical peptide value, the possibility of oxidation of methionine residues (+15.995 Da), α-iodoacetamide modification of cysteine ​​residues (+57.021 Da), and carboxymethylation by iodoacetic acid (+58.005 Da) was considered, and the amino acid sequence data used was that of the human ferritin H chain shown in Sequence ID No. 3.

[0106] (11-4) Analysis results of modification sites of human ferritin H chain by LC-MS / MS Analysis using LC-MS / MS revealed the MS spectrum (measured value: m / z 873.38158, theoretical value: 873.38273, trivalent) of the peptide fragment KPDCDDWESGLNAMECALHLEK (SEQ ID NO: 9), a peptide consisting of 22 amino acid residues containing two cysteine ​​residue modifications (α-iodoacetamide modifications (+57.021Da)), generated by trypsin digestion of the α-iodoacetamide transderm of human ferritin H chain from Example 10 (Figure 17). The CID spectrum confirmed a product ion with m / z 1236.85 (theoretical value: 1236.52) corresponding to the divalent b21, indicating α-iodoacetamide modifications at amino acid positions 90 and 102 (Figure 18). Furthermore, peak area comparison showed that the modifications to the cysteine ​​residues at amino acid positions 90 and 102 occurred with high selectivity (Figure 19).

[0107] Example 12: Physical property analysis of ethylmaleimide-modified ferritin FTH (ethylmaleimide-modified FTH with 24-mer stoichiometric volume 512256) constructed in Example 2, in which ethylmaleimide was introduced at the cysteine ​​positions 91 and 103, was suspended in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 1 mg / ml and left at 25°C for 30 minutes. The solution dispersibility and size of the amidated FTH contained in the solution were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern). Measurements were performed on 50 μl of solution at 25°C. As a result, good dispersion with a peak at 11.7 nm was observed (Figure 20), indicating that the cage-like structure of ferritin was maintained even after modification.

[0108] The size of the ethylmaleimide-modified FTH was also evaluated by size exclusion column chromatography. 10 μl of a solution suspended in D-PBS(-) to a final concentration of ethylmaleimide-modified ferritin of 1 mg / ml was loaded onto a Superdex200 increase 16 / 30 (Cytiva), eluted in PBS (Takara Bio) at a flow rate of 0.8 ml / min, and the protein was detected by absorbance at 280 nm. As a result, ethylmaleimide-modified FTH was found to elute at the same time as natural FTH (15 min), without aggregation, and without any change in size (Figure 21).

[0109] Furthermore, the surface charge of ethylmaleimide-modified ferritin was evaluated. Ethylmaleimide-modified FTH was suspended in D-PBS(-) to a final concentration of 0.1 g / l, and the surface charge in the buffer at 25°C was measured using a Zetasizer Nano ZS (Malvern). The measurement was performed using a DTS1070 cell with 750 μl of sample, Material settings (RI: 1.450, Absorption: 0.001), Dispersant settings (Temperature: 25°C, Viscosity: 0.8872 cP, RI: 1.330, Dielectric constant: 78.5), and a Smoluchowski model (Fκa value: 1.50). As a result, the surface charge of ethylmaleimide-modified FTH was -17mV, which is equivalent to the surface charge of FTH without the ethylmaleimide group introduced in D-PBS(-) (-17mV), indicating that the modification did not significantly alter the FTH surface.

[0110] Example 13: Intemoturing ethylmaleimide-modified ferritin (1) In Example 2, a low-molecular-weight anticancer drug was encapsulated in ethyl maleimide-modified FTH. Doxorubicin hydrochloride (DOX, CAS number 25316-40-9) was mixed with 1 ml of 50 mM Tris hydrochloride buffer (pH 9) containing ethyl maleimide-modified FTH at a final concentration of 1 mg / ml to a final concentration of 0.3 mg / l, and the mixture was left to stand at 60°C for 60 minutes. After standing, the mixture was centrifuged (15,000 rpm, 1 minute), and the supernatant was subjected to a desalting column PD-10 (Sephadex G-25 packed, Cytiva) equilibrated with 10 mM Tris hydrochloride buffer (pH 8) to separate the unencapsulated drug and protein. Of the total volume of the solution (3.0 ml), 1.0 ml was loaded onto Superdex200 increase 16 / 30 (Cytiva), and eluted in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min. The protein was purified using absorbance at 280 nm as an indicator to obtain doxorubicin-encapsulated ethyl maleimide-modified FTH (DOX-ethyl maleimide-modified FTH). To investigate the stability of the obtained DOX-ethylmaleimide-modified FTH, 1 ml of D-PBS(-) containing 0.2 mg / ml of ethylmaleimide-modified ferritin was left at room temperature for 24 hours. Then, it was loaded onto a Superdex200 increase 16 / 30 (Cytiva) and simultaneously measured absorbance at 280 nm and 480 nm while separating and purifying the FTH by size in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min. As a result, an absorbance peak at 480 nm, presumed to be derived from DOX, was observed at the same elution position as FTH without DOX, indicating that DOX and ethylmaleimide-modified FTH are strongly compounded (Figure 22).

[0111] Furthermore, the surface charge of DOX-ethylmaleimide-modified FTH was evaluated. FTH was suspended in 50 mM Tris hydrochloride buffer (pH 8.0) to a final concentration of 0.1 g / l, and the surface charge in this buffer at 25°C was measured using a Zetasizer Nano ZS (Malvern). The measurement was performed by placing 800 μl of the sample into a DTS1070 cell and using the following settings: Material setting (RI: 1.450, Absorption: 0.001), Dispersant setting (Temperature: 25°C, Viscosity: 0.8872 cP, RI: 1.330, Dielectric constant: 78.5), and Smoluchowski model (Fκa value: 1.50). As a result, the surface charge of DOX-ethylmaleimide-modified FTH was -10.9 ± 0.5 mV (mean ± standard deviation), which is equivalent to the surface charge of un-ethylmaleimide-modified FTH under the same conditions (-11 mV), indicating that the FTH surface was not significantly altered by DOX inclusion.

[0112] Example 14: Intemoration of ethylmaleimide-modified ferritin (2) Ethyl maleimide-modified ferritin constructed in Example 2 was encapsulated with a fluorescently modified peptide [(5(6)-FAM-CGGPKKKRKVG (SEQ ID NO: 10), FAM-SV40, formula weight 1516)]. 0.1 ml of 16 mM hydrochloric acid (pH 1.6) containing amidated FTH at a final concentration of 3 mg / ml and FAM-SV40 at a final concentration of 0.1 mM was left at room temperature for 15 minutes. Then, 50 μl of 1 M Tris hydrochloride buffer (pH 9.0) was added to neutralize the solution, and it was left at room temperature for 3 hours. After standing, the volume was increased to 1 ml with water, and the ethyl maleimide-modified ferritin and the unencapsulated peptide were separated and purified by size using a Superdex200 increase 16 / 30 (Cytiva) equilibrated with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) at a flow rate of 0.8 ml / min. As a result, in ferritin subjected to the FAM-SV40 encapsulation procedure, absorbance at 480 nm, which is not observed in the protein alone, was confirmed at the same elution position as in ferritin 24-mer, indicating that FAM-SV40 was encapsulated within ethylmaleimide-modified ferritin (Figure 23).

[0113] Example 15: Surface modification of encapsulated ferritin (1) (15-1) Chemical modification of the ferritin surface by encapsulating molecules in the internal cavity of ferritin First, we constructed FTH molecules containing either a fluorescently modified peptide (5(6)-FAM-CGGPKKKRKVG (SEQ ID NO: 10), FAM-SV40, formula weight 1516) or double-stranded DNA (5'-AACAGGTAGGTTCAGTGT-3' (SEQ ID NO: 11) and 5'-ACACTGAACCTACCTGTT-3' (SEQ ID NO: 12), dsDNA, formula weight 10996). To construct ferritin encapsulating FAM-SV40 (FAM-SV40-encapsulated FTH), 1 ml of 50 mM glycine hydrochloride buffer (pH 2.3) containing 3 mg / ml of FTH (24-mer molecular weight 506260) and 0.1 mM of peptide was left at room temperature for 15 minutes. Then, 10 μl of 1 M Tris hydrochloride buffer (pH 9.0) was added to neutralize the mixture, and it was left at room temperature for 3 hours. Ten samples with the same reaction composition were combined, and FAM-SV40-encapsulated FTH was separated and purified by size using a Superdex200 increase 16 / 30 (Cytiva) equilibrated with D-PBS(-) (Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min. The obtained samples were concentrated to 16 mg / ml as FTH using an ultrafiltration membrane (Amicon Ultra 0.5, 100 kDa, Merck) and then subjected to the modification reaction. Furthermore, to construct ferritin encapsulating dsDNA (dsDNA-encapsulated FTH), 0.1 ml of 50 mM glycine hydrochloride buffer (pH 2.3) containing 3 mg / ml of FTH (24-mer molecular weight 506260) and 0.1 mM of dsDNA was left at room temperature for 15 minutes. Then, 10 μl of 1 M Tris hydrochloride buffer (pH 9.0) was added to neutralize the mixture, and it was left at room temperature for 3 hours. Ten samples with the same reaction composition were combined, and dsDNA-encapsulated FTH was separated and purified by size using a Superdex200 increase 16 / 30 (Cytiva) equilibrated with D-PBS(-) (Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min. The obtained samples were concentrated to 25 mg / ml of FTH using an ultrafiltration membrane (Amicon Ultra 0.5, 100 kDa, Merck) and subjected to modification reactions.

[0114] (15-2) Modification of fluorescently modified peptide-encapsulated ferritin with ethyl maleimide and analysis by ESI-TOFMS Next, the two types of encapsulated ferritin obtained above were modified with ethyl maleimide. FAM-SV40-encapsulated FTH was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. Ethyl maleimide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of ethyl maleimide were added to the aqueous solution containing FAM-SV40-encapsulated FTH and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 for the human-derived ferritin H chain in the starting material, and a peak at 21344 was confirmed for the product, indicating the introduction of two ethyl maleimide molecules (molecular weight 125) (Figure 24). The retention of FAM-SV40 encapsulation by FTH within FAM-SV40 after modification was confirmed by size exclusion column chromatography. 10 μl of a solution suspended in D-PBS(-) to a final FTH concentration of 2 mg / ml was loaded onto a Superdex200 increase 16 / 30 (Cytiva) and eluted in PBS (Takara Bio) at a flow rate of 0.8 ml / min. Protein was detected at 280 nm and FAM at 480 nm. As a result, absorbance at 480 nm derived from FAM was confirmed at the same elution position as the ferritin 24-mer, indicating that FAM-SV40 is maintained within ferritin even after the modification reaction (Figure 25).

[0115] (15-3) Modification of double-stranded DNA-encapsulated ferritin with ethyl maleimide and analysis by ESI-TOFMS dsDNA-encapsulated FTH was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. Ethyl maleimide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. Six equivalents of ethyl maleimide were added to the aqueous solution containing dsDNA-encapsulated FTH and stirred, then the reaction was carried out by shaking at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 for the human ferritin H chain in the starting material, and a peak at 21344 was confirmed for the product, indicating the introduction of two ethyl maleimide molecules (molecular weight 125) (Figure 26). The retention of dsDNA encapsulation in dsDNA-encapsulated FTH after modification was confirmed by size exclusion column chromatography. To identify the encapsulated contents of the modified FTH, the higher-order structure of amidated FTH was disrupted with 100 mM glycine hydrochloride buffer (pH 2.3) to release the encapsulated contents, and then neutralized with 200 mM Tris hydrochloride buffer (pH 9.0). 10 μl of this sample was suspended in D-PBS(-) to a final concentration of 2 mg / ml as FTH, loaded onto a Superdex200 increase 16 / 30 (Cytiva), and eluted in PBS (Takara Bio) at a flow rate of 0.8 ml / min. Protein and dsDNA were detected at 280 nm, respectively. As a result, no peak was observed at the elution site of dsDNA (20.5 min) before structural disruption with glycine hydrochloride buffer (Figure 27). This suggests that dsDNA was released from inside FTH by the acid, indicating that dsDNA was maintained within ferritin even after the modification reaction.

[0116] Example 16: Surface modification of encapsulated ferritin (2) The surface of the FAM-SV40-encapsulated FTH constructed in Example 15 was modified with a peptide having a PEG6-maleimide group at its C-terminus (YGRKKRRQRRR-PEG6-maleimide, TAT-Mal, molecular weight 2046, SEQ ID NO: 13) or (RRRRRRRRR-PEG6-maleimide, R9-Mal, molecular weight 1910, SEQ ID NO: 14). The FAM-SV40-encapsulated FTH was dissolved in PBS buffer (pH 7.4) to a concentration of 5.0 mg / ml. TAT or R9 was dissolved in N,N-dimethylformamide to a concentration of 100 mM. Ten equivalents each of TAT-Mal or R9-Mal were added to the FAM-SV40-encapsulated FTH solution and stirred, then the reaction was carried out at room temperature for 1 hour by shaking. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21094 in the human-derived ferritin H chain used as the raw material, and in the product, a peak at 25187 was confirmed with the introduction of two TAT-Mal units, and a peak at 24915 was confirmed with the introduction of two R9-Mal units (Figure 28).

[0117] Example 17: Purification of azide-modified ferritin Example 4(4-2) The obtained azid group-modified ferritin (azidated FTH) was diluted with water to a concentration of 1.0 mg / ml, 1.0 ml was loaded onto Superdex200 increase 16 / 30 (Cytiva), and eluted in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min. The protein was purified using absorbance at 280 nm as an indicator to obtain azidated FTH.

[0118] Example 18: Physical property analysis of azide-modified FTH FTH (azidated FTH, 24-mer molecular weight 513888) purified in Example 17, in which both cysteine ​​at positions 91 and 103 were azed, was suspended in 50 mM Tris hydrochloride buffer (pH 8.0) to a final concentration of 0.5 mg / ml and left at 25°C for 30 minutes. The solution dispersibility and size of azidated FTH contained in the solution were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern). Measurements were performed on 50 μl of solution at 25°C. As a result, good dispersion with a peak at 16 nm (PDI 0.217) was observed (Figure 29), indicating that the cage-like structure of ferritin was maintained even after modification.

[0119] The maintenance of azide-containing FTH size was also confirmed by size exclusion column chromatography. 10 μl of a solution suspended in D-PBS(-) to a final concentration of 1 mg / ml of azide-containing FTH was loaded onto a Superdex200 increase 16 / 30 (Cytiva), eluted in PBS (Takara Bio) at a flow rate of 0.8 ml / min, and detected by absorbance at 280 nm and 480 nm. As a result, azidized FTH was found to have eluted at the same time as natural FTH (15 min), indicating that its size had not changed due to aggregation or other factors (Figure 30). Furthermore, because it did not contain pigments or other components, no light absorption at 480 nm was observed.

[0120] Furthermore, the surface charge of azidated FTH was evaluated. FTH was suspended in 0 mM Tris hydrochloride buffer (pH 8.0) to a final concentration of 0.1 g / l, and the surface charge in this buffer at 25°C was measured using a Zetasizer Nano ZS (Malvern). The measurement was performed by placing 750 μl of the sample into a DTS1070 cell and using the following settings: Material (RI: 1.450, Absorption: 0.001), Dispersant (Temperature: 25°C, Viscosity: 0.8872 cP, RI: 1.330, Dielectric constant: 78.5), and Smoluchowski model (Fκa value: 1.50). As a result, the surface charge of azidated FTH was -9.9 ± 0.9 mV, which is equivalent to the surface charge of unazidated FTH (-11 mV), indicating that the FTH surface did not change significantly due to azidation.

[0121] Example 19: Low molecular weight encapsulation into azide-modified ferritin A fluorescent dye was encapsulated in the azidated FTH purified in Example 17. Uranine (CAS No. 518-47-8) was mixed with 1 ml of 50 mM acetate buffer (pH 5) containing azidated FTH at a final concentration of 3 mg / ml to a final concentration of 100 mM, and the mixture was left to stand at 40°C for 60 minutes. After standing, the mixture was centrifuged (15,000 rpm, 1 minute), and the supernatant was subjected to a desalting column PD-10 (Sephadex G-25 packed, Cytiva) equilibrated with D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Industries, Ltd.) to separate the unencapsulated drug and protein. The entire solution (3.0 ml) was ultrafiltered and concentrated using Vivaspin 20 (MW100 kDa), then reduced to 1.0 ml and loaded onto a Superdex200 increase 16 / 30 (Cytiva). The protein was eluted in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Corporation) at a flow rate of 0.8 ml / min, and the protein was purified using absorbance at 280 nm as an indicator. During purification, absorbance at 480 nm, which is not observed in protein alone, was confirmed at the same elution position as ferritin 24-mer, indicating that uranine-encapsulated azidized FTH was obtained (Figure 31).

[0122] Example 20: Surface modification of medium-molecule ferritin The surfaces of native FTH and azidized FTH were modified with siRNA. First, siRNAs (maleimide-PPIB or DBCO-PPIB) were synthesized with A(M)C(M)AGC(M)AAAU(M)U(M)C(M)C(M)AU(M)C(M)GU(M)GU(M) (SEQ ID NO: 15, A(M), C(M), and U(M) are 2'-OMe-modified RNA) modified at the 5' end with 6-FAM and at the 3' end with a maleimide group or dibenzocyclooctyne group via an Amino C6 linker, and 5'-AC(F)AC(F)GAU(F)GGAAU(F)U(F)U(F)GC(F)U(F)GU(F)UU-3' (SEQ ID NO: 16, C(F) and U(F) are 2'-Fluoro-modified RNA) as the sense strand, and 5'-AC(F)AC(F)GAU(F)GGAAU(F)U(F)U(F)GC(F)U(F)GU(F)UU-3' (SEQ ID NO: 16, C(F) and U(F) are 2'-Fluoro-modified RNA) as the antisense strand. Native FTH was dissolved in PBS buffer at pH 7.4 to a concentration of 3.0 mg / ml. The siRNA (maleimide-PPIB) synthesized above, which has a maleimide group at its 3' end via an Amino C6 linker, was dissolved in N,N-dimethylformamide to a concentration of 7.5 mM. 7.5 equivalents of siRNA were added to an aqueous solution containing native ferritin and stirred, then shaken at room temperature for 18 hours. The reaction mixture was purified using an NAP column (Cytiva), mixed with sample loading buffer (Bio-rad), and allowed to stand at 90°C for 2 minutes. SDS-PAGE was then performed using a Mini-Protean TGX gel (Bio-rad). In Figure 32, lane 1 shows native FTH and lane 2 shows the product. In lane 2, two types of native FTH were identified: one with two sense strands attached and another with two dsDNAs attached. The azide-modified FTH obtained in Example 4(4-2) was dissolved in PBS buffer at pH 7.4 to a concentration of 3.0 mg / ml. The siRNA (DBCO-PPIB) synthesized above, having a DBCO group at its 3' end via an Amino C6 linker, was dissolved in N,N-dimethylformamide to a concentration of 7.5 mM. 7.5 equivalents of siRNA were added to an aqueous solution containing natural ferritin and stirred, then shaken at room temperature for 18 hours. The reaction mixture was purified using an NAP column (Cytiva), mixed with sample loading buffer (Bio-rad), and allowed to stand at 90°C for 2 minutes. SDS-PAGE was then performed using a Mini-Protean TGX gel (Bio-rad). In Figure 32, lane 3 shows azide-modified FTH and lane 4 shows the product. In lane 4, two types of FTH were identified: one with two sense strands attached to the natural FTH, and another with two dsDNAs attached.

[0123] Example 21: Physical property analysis of moderately modified ferritin The surface-modified native FTH and azidized FTH constructed in Example 20 were suspended in D-PBS(-) (pH 7.4, Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.5 mg / ml and left at 25°C for 30 minutes. The dispersibility and size of the amidated FTH in the solution were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern). Measurements were performed on 50 μl of solution at 25°C. As a result, good dispersion with peaks at 16.5 nm or 50.7 nm was observed (Figure 33), suggesting that the supramolecular structure of ferritin was maintained even after modification.

[0124] The size of these nucleic acid-modified FTH molecules was also evaluated by size exclusion column chromatography. 10 μl of solutions containing siRNA-modified native FTH and azidized FTH suspended in D-PBS(-) to a final concentration of 0.5 mg / ml each were loaded onto a Superdex200 increase 16 / 30 (Cytiva) and eluted in PBS (Takara Bio) at a flow rate of 0.8 ml / min. Proteins were detected by absorbance at 280 nm. As a result, elution was observed at a faster time (13 min) than with natural FTH, and although aggregation did not occur, the size was found to be increased due to the nucleic acid modification on the surface (Figure 34). In addition, a higher absorbance at 260 nm was observed than with natural FTH, indicating that the absorbance at 260 nm was improved due to the nucleic acid modification.

[0125] Example 22: Preparation of peptide-inserted human ferritin H chains DNA encoding a human ferritin H chain (FTH-BC-GBP (SEQ ID NOs. 18 and 19)) was totally synthesized in which a gold-recognizing peptide (GBP1:GMHGKTQATSGTIQSG (SEQ ID NO: 17)) was inserted and fused into the flexible linker region between the second and third α-helices from the N-terminus of the six α-helices constituting the ferritin monomer. When this DNA expression plasmid is introduced into E. coli, the human ferritin H chain is expressed in E. coli. The methionine at the start codon becomes N-formylmethionine, which is removed by post-translational modification. As a result, a peptide-inserted human ferritin H chain is obtained, defined by the amino acid sequence of SEQ ID NO: 20, in which the methionine residue at position 1 in the amino acid sequence of SEQ ID NO: 19 is removed. PCR was performed using synthetic DNA encoding FTH-BC-GBP as a template, with 5'-GAAGGAGATATACATATGACGACCGCGTCCACCTCG-3' (SEQ ID NO: 4) and 5'-CTCGAATTCGGATCCTTAGCTTTCATTATCACTGTC-3' (SEQ ID NO: 5) as primers. PCR was also performed using pET20 (Merck) as a template, with 5'-TTTCATATGTATATCTCCTTCTTAAAGTTAAAC-3' (SEQ ID NO: 6) and 5'-TTTGGATCCGAATTCGAGCTCCGTCG-3' (SEQ ID NO: 7) as primers. After purifying the obtained PCR products using the Wizard DNA Clean-Up System (Promega), an expression plasmid (pET20-FTH-BC-GBP) containing the gene encoding FTH was constructed by in-fusion enzyme treatment with the In-Fusion HD Cloning Kit (Takara Bio) at 50°C for 15 minutes. Next, Escherichia coli BL21(DE3) cells into which the constructed pET20-FTH-BC-GBP had been introduced were incubated in 100 ml of LB medium (containing 10 g / l Bacto-typtone, 5 g / l Bacto-yeast extract, 5 g / l NaCl, and 100 mg / l ampicillin) in a flask at 37°C for 24 hours. After sonication of the resulting cells, the supernatant was heated at 60°C for 20 minutes. After heating, the supernatant was cooled and centrifuged. The supernatant was then injected into a HiPerp Q HP column (Cytiva) equilibrated with 50 mM TrisHCl buffer (pH 8.0), and the target protein was separated and purified by applying a salt concentration gradient with 50 mM TrisHCl buffer (pH 8.0) containing 0 mM to 500 mM NaCl, based on differences in surface charge. The solvent in the protein-containing solution was replaced with 10 mM TrisHCl buffer (pH 8.0) by centrifugal ultrafiltration using a Vivaspn 20-100K (Cytiva). This solution was injected into a HiPrep 26 / 60 Sephacryl S-300 HR column (Cytiva) equilibrated with 10 mM TrisHCl buffer (pH 8.0) to separate and purify FTH-BC-GBP. The solution containing FTH-BC-GBP was concentrated by centrifugal ultrafiltration using a Vivaspn 20-100K (Cytiva), and the protein concentration was determined using a protein assay CBB solution (Nacalai Tesque) with bovine albumin as the standard. As a result, 1 ml of solution containing 1 mg / ml of FTH was obtained per 100 ml of culture medium.

[0126] Example 23: Specific modification of peptide-inserted human ferritin H chains with p-azidophenacyl bromide and ESI-TOFMS analysis (23-1) Specific modification of peptide-inserted human ferritin H chains with 2.2 equivalents of p-azidophenacylbromide and ESI-TOFMS analysis The human ferritin H chain expressed in Example 22 was dissolved in PBS buffer at pH 7.4 to a concentration of 5.0 mg / ml. p-azidophenacyl bromide was dissolved in N,N-dimethylformamide to a concentration of 20 mM. 2.2 equivalents of p-azidophenacyl bromide were added to the aqueous solution containing the human ferritin H chain and stirred, then shaken at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 22637 for the starting material FTH-BC-GBP, and a peak at 22955 was observed for the product, showing the peak of the starting material and two p-azidophenacyl (molecular weight 160) molecules introduced (Figure 35).

[0127] Reference Example 1: Modification of lysine residues in the human ferritin H chain by NHS-active esters. The human ferritin H chain expressed in Example 1 was dissolved in PBS buffer at pH 8.5 to a concentration of 2.5 mg / ml. N-Succinimidyl 3-(Acetylthio)propionate (TCI) was dissolved in N,N-dimethylformamide to a concentration of 50 mM. 20 equivalents of N-Succinimidyl 3-(Acetylthio)propionate were added to the aqueous solution containing the human ferritin H chain and stirred, then the reaction was carried out at room temperature for 1 hour by shaking. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS. A peak was observed at 21093 in the human ferritin H chain of the raw material, and in the product, the peak of the raw material and peaks at 21223 (with one 3-(Acetylthio)propionate (molecular weight 130) introduced), 21354 (with two), 21484 (with three), 21614 (with four), and 21744 (with five) were confirmed (Figure 36).

Claims

1. Contains human ferritin H chain, A modified ferritin comprising a human ferritin H chain containing a modifying group specifically covalently bonded to the cysteine ​​residues at positions 91 and / or 103, corresponding to the reference position of the natural human ferritin H chain, The cysteine ​​residues at positions 91 and / or 103, corresponding to the reference position of the natural human ferritin H chain, are exposed on the surface of modified ferritin. The reference position of the natural human ferritin H chain corresponds to the position of the amino acid residue in the amino acid sequence of SEQ ID NO: 1, and (a) The modifying group contains a reactive group comprising one or more substructures selected from the group consisting of maleimide moiety, azide moiety, ketone moiety, aldehyde moiety, thiol moiety, alkene moiety, alkyne moiety, halogen moiety, tetrazine moiety, nitrone moiety, hydroxylamine moiety, nitrile moiety, hydrazine moiety, boronic acid moiety, cyanobenzothiazole moiety, allyl moiety, phosphine moiety, disulfide moiety, thioester moiety, α-halocarbonyl moiety, isonitrile moiety, cydonone moiety, and selenium moiety, or (b) Modified ferritin comprising a functional substance selected from the group consisting of drugs, labeling substances and stabilizers.

2. The modified ferritin according to claim 1, wherein the modified ferritin contains one or two modifying groups per human ferritin H chain.

3. The modified ferritin according to claim 1 or 2, wherein the modified ferritin is a 24-mer containing 24 human ferritin H chains.

4. The modified ferritin according to claim 3, wherein the modified ferritin comprises 24 or 48 modifying groups.

5. The modified ferritin according to any one of claims 1 to 4, wherein the human ferritin H chain is as follows: (A) Proteins containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; (B) A protein having the ability to form a 24-mer, comprising an amino acid sequence in which mutations of 1 to 15 amino acid residues are introduced, selected from the group consisting of substitutions, deletions, insertions, and additions of amino acid residues, in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or (C) A protein that contains an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and that has the ability to form a 24-mer.

6. The modified ferritin according to any one of claims 1 to 5, wherein the human ferritin H chain is a protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

3.

7. In the human ferritin H chain, a functional peptide is inserted into the flexible linker region consisting of amino acid residues from positions 78 to 96, which follows the reference position of the natural human ferritin H chain. Here, if the human ferritin H chain is a protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, then the amino acid residues at positions 78-96 that correspond to the reference position of the native human ferritin H chain correspond to (a) the amino acid residues at positions 78-96 in the amino acid sequence of SEQ ID NO: 1, or (b) the amino acid residues at positions 77-95 in the amino acid sequence of SEQ ID NO:

3. The modified ferritin according to any one of claims 1 to 6, wherein the functional peptide is a peptide having binding ability to a target material, a protease-degradable peptide, a cell-permeable peptide, or a stabilizing peptide.

8. The modified ferritin according to any one of claims 1 to 7, wherein the functional substance comprises one or more parts selected from the group consisting of peptides, proteins, nucleic acids, low molecular weight organic compounds having a molecular weight of 30 to 1500, chelators, glycans, lipids, high molecular weight polymers, and metals.

9. A modified ferritin according to any one of claims 1 to 7, wherein the functional substance comprises an organic compound.

10. The modified ferritin according to any one of claims 1 to 9, wherein the modified ferritin has a substance in its lumen.

11. A method for producing reactive group-added ferritin, This includes reacting human ferritin with a thiol-modifying reagent to produce reactive group-added ferritin, Human ferritin and reactive group-added ferritin contain human ferritin H chains, The human ferritin H chain contained in reactive group-modified ferritin contains a modified group that includes a reactive group, specifically covalently bonded to the cysteine ​​residue at positions 91 and / or 103, which correspond to the reference position of the natural human ferritin H chain. The cysteine ​​residues at positions 91 and / or 103, corresponding to the reference position of the natural human ferritin H chain, are exposed on the surface of modified ferritin. The reference position of the natural human ferritin H chain corresponds to the position of the amino acid residue in the amino acid sequence of SEQ ID NO: 1, and A method comprising a reactive group comprising one or more substructures selected from the group consisting of maleimide moiety, azide moiety, ketone moiety, aldehyde moiety, thiol moiety, alkene moiety, alkyne moiety, halogen moiety, tetrazine moiety, nitrone moiety, hydroxylamine moiety, nitrile moiety, hydrazine moiety, boronic acid moiety, cyanobenzothiazole moiety, allyl moiety, phosphine moiety, disulfide moiety, thioester moiety, α-halocarbonyl moiety, isonitrile moiety, cydonone moiety, and selenium moiety.

12. The method according to claim 11, wherein the thiol-modifying reagent comprises one or more substructures selected from the group consisting of maleimide moiety, benzyl halide moiety, α-haloamide moiety, α-haloketone moiety, alkene moiety, alkyne moiety, fluoroaryl moiety, nitroaryl moiety, methylsulfonyloxadiazole moiety, and disulfide moiety.

13. A method for producing ferritin with added functional substances, This includes reacting human ferritin with a functional substance to produce ferritin with a functional substance, Human ferritin and functional substance-added ferritin contain human ferritin H chains, The human ferritin H chain contained in functional substance-modified ferritin is specifically covalently bonded to the cysteine ​​residues at positions 91 and / or 103, which correspond to the reference positions of the natural human ferritin H chain, and contains a modification group containing a functional substance. The cysteine ​​residues at positions 91 and / or 103, corresponding to the reference position of the natural human ferritin H chain, are exposed on the surface of modified ferritin. The reference position of the natural human ferritin H chain corresponds to the position of the amino acid residue in the amino acid sequence of SEQ ID NO: 1, and A method for selecting a functional substance from the group consisting of drugs, labeling substances, and stabilizers.

14. A method for producing ferritin with added functional substances, (1) Reacting human ferritin with a thiol-modifying reagent to produce reactive group-added ferritin; and (2) The process includes reacting reactive group-added ferritin with a functional substance to produce functional substance-added ferritin, Human ferritin, ferritin with reactive groups, and ferritin with functional substances include human ferritin H chains, The human ferritin H chain contained in reactive group-modified ferritin contains a modified group that includes a reactive group, specifically covalently bonded to the cysteine ​​residue at positions 91 and / or 103, which correspond to the reference position of the natural human ferritin H chain. The human ferritin H chain contained in functional substance-modified ferritin is specifically covalently bonded to the cysteine ​​residues at positions 91 and / or 103, which correspond to the reference positions of the natural human ferritin H chain, and contains a modification group containing a functional substance. The cysteine ​​residues at positions 91 and / or 103, corresponding to the reference position of the natural human ferritin H chain, are exposed on the surface of modified ferritin. The reference position of the natural human ferritin H chain corresponds to the position of the amino acid residue in the amino acid sequence of SEQ ID NO: 1, and The reactive group comprises one or more substructures selected from the group consisting of maleimide moiety, azide moiety, ketone moiety, aldehyde moiety, thiol moiety, alkene moiety, alkyne moiety, halogen moiety, tetrazine moiety, nitrone moiety, hydroxylamine moiety, nitrile moiety, hydrazine moiety, boronic acid moiety, cyanobenzothiazole moiety, allyl moiety, phosphine moiety, disulfide moiety, thioester moiety, α-halocarbonyl moiety, isonitrile moiety, cydonone moiety, and selenium moiety. A method for selecting a functional substance from the group consisting of drugs, labeling substances, and stabilizers.

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