阅读说明：本技术 胶囊蛋白质及其多聚体组合物以及使用其的医药组合物 (Capsule protein, polymer composition thereof, and pharmaceutical composition using same ) 是由 乾隆 于 2020-05-19 设计创作，主要内容包括：目前有在具有桶状结构的脂质运载蛋白型前列腺素D合成酶中收容药物、在α螺旋H2和E-F环间中导入二硫键、圈住药物的设想,但有时药物从桶状结构的开口的间隙被释放。以人脂质运载蛋白型前列腺素D合成酶的活性中心的半胱氨酸取代为丙氨酸、β链D的至少1个氨基酸取代为隔断氨基酸为特征的胶囊蛋白质通过设于构成桶状结构的开口的β链D的隔断氨基酸,抑制了收容的药物的释放。(At present, it is assumed that a drug is contained in a lipocalin-type prostaglandin D synthase having a barrel structure, a disulfide bond is introduced between the α -helix H2 and the E-F ring, and the drug is trapped, but the drug is sometimes released from the gap of the opening of the barrel structure. The capsule protein characterized in that cysteine at the active center of human lipocalin-type prostaglandin D synthase is substituted with alanine and at least 1 amino acid of beta-chain D is substituted with a blocking amino acid suppresses the release of the drug contained therein by the blocking amino acid of beta-chain D provided in the opening constituting the barrel structure.)

1. A capsule protein characterized in that cysteine of the active center of human lipocalin-type prostaglandin D synthase is substituted with alanine, and at least 1 amino acid of beta-chain D is substituted with a blocked amino acid.

2. The capsule protein of claim 1, wherein a disulfide bond is introduced between the E-F loop and alpha helix H2.

3. The capsule protein of claim 1 or 2, wherein the N-terminus or C-terminus of the capsule protein is bound with an identifier peptide.

4. The capsule protein of any one of claims 1 to 3, wherein said blocked amino acid is at least 1 amino acid selected from the group consisting of lysine (K), histidine (H), tryptophan (W), tyrosine (Y), phenylalanine (F).

5. A multimeric composition of capsule proteins, characterized in that a plurality of said capsule proteins are bound.

6. The multimeric composition of a capsule protein of claim 5, wherein said multimeric composition is a 4-mer or an 8-mer.

7. The multimeric composition of claim 6, wherein said encapsulated protein binds to streptavidin via a 4-mer of biotin.

8. A pharmaceutical composition comprising the capsule protein according to any one of claims 1 to 7 and a drug.

9. The pharmaceutical composition of claim 8, which is lyophilized.

10. A processed food comprising the capsule protein according to any one of claims 1 to 7 and a complex containing a compound other than a drug.

Technical Field

The present invention relates to a capsule protein that can be used as a Drug Delivery System (DDS) and a polymer composition thereof, and more particularly, to a capsule protein that can dissolve a poorly water-soluble drug and release the drug to the affected area after administration, and a pharmaceutical composition and a processed food using the same.

Background

In DDS, the development of drug carriers is a key technology. Liposomes, microparticles, nanophase-related substances, drug-polymer conjugates, and the like have been studied. Among them, polymeric micelles have been attracting attention as a favorable vehicle for the delivery of poorly water-soluble drugs. For example, a micelle-forming composition comprising a hydrophobic core surrounded by a hydrophilic shell composed of PVP (N-vinyl-2-pyrrolidone) (patent document 1).

Patent document 2 discloses that a capsule protein in which a lipocalin-type prostaglandin D synthase (hereinafter referred to as "L-PGDS") as a biological product is modified can exhibit the effect of a drug by dissolving a poorly water-soluble drug in water and administering the solution. Since the capsule protein of the modified L-PGDS is an in vivo product, is not an antigen, and is not a toxic substance to humans, it can be said to be a safe and reliable drug carrier.

Patent document 3 discloses a protein in which a marker for recognizing cells in an affected area is attached to a modified L-PGDS capsule protein. For example, it is considered that the capsule protein for modifying L-PGDS having a marker peptide that specifically binds to cancer cells is concentrated on the cancer cells in the affected area, and the therapeutic effect is improved.

Patent document 4 discloses a protein in which tryptophan at 34 th and 92 th positions from the N-terminus of a capsule protein modified with L-PGDS is substituted with cysteine, and a lid is provided by opening a closed disulfide bond in a redox atmosphere. The aim is to improve the retention of the drug by opening the closed disulfide bonds.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2004-501180

Patent document 2: japanese patent laid-open No. 2008-120793

Patent document 3: japanese patent laid-open publication No. 2011-207830

Patent document 4: japanese patent laid-open No. 2013-162760

Disclosure of Invention

Technical problem to be solved by the invention

The capsule protein of L-PGDS can easily dissolve poorly water-soluble drugs, and is a biological product, thus having high safety. However, the barrel structure holding the drug is in a container-like shape without a lid. Therefore, there is a problem that the drug once taken is released during transportation. In order to solve this problem, in patent document 4, disulfide bond-based immobilization is used for the capsule protein using L-PGDS.

However, it is known that the opening of the barrel-shaped structure cannot be closed in the immobilization of the opening of the disulfide-bond-based encapsulated protein using L-PGDS.

Technical scheme for solving technical problem

The present invention has been made in view of the above problems, and an object of the present invention is to provide a capsule protein in which an amino acid serving as a bulky partition is provided in an opening of a barrel structure, and which can prevent accidental release of a drug.

More specifically, the capsule protein of the present invention is characterized in that,

cysteine of the active center of human lipocalin-type prostaglandin D synthase is substituted by alanine, and at least 1 amino acid of the beta chain D is substituted by a blocking amino acid.

ADVANTAGEOUS EFFECTS OF INVENTION

In the capsule protein of the present invention, since the amino acid as a partition is provided in the opening of the barrel structure formed by the beta chain of the mutant L-PGDS, the drug put in the barrel structure is less released during transportation, and the drug can efficiently reach the affected site cell.

Furthermore, by using the capsule protein of the present invention as a multimeric composition, an Enhanced Permeability and Retention (EPR) effect can be exhibited, and the effect of inhibiting the influence on normal cells can be expected while the specificity of cancer cell invasion is increased and the effect of a drug is improved.

Drawings

FIG. 1 is a diagram showing the crystal structure of the mutant L-PGDS.

FIG. 2 shows the amino acid sequence of the mutant L-PGDS.

FIG. 3 is a diagram showing a modeled structure of a D-clip mutant into which a D-clip (disulfide bond) has been introduced.

FIG. 4 is a diagram showing a model structure of a disrupted mutant to which a disrupted amino acid has been added.

FIG. 5 is a diagram showing the concept of a multimeric composition.

FIG. 6 is a diagram showing the modeled structure of mutant L-PGDS identified as mutant L-PGDS.

FIG. 7 is a diagram showing a modeled structure of a truncated D-clip variant identified as a truncated D-clip variant.

FIG. 8 is a graph of the retention and release capacity of capsules containing SN-38 in their proteins.

FIG. 9 is a graph for investigating the retention and release capacity of the capsule protein (mutant with cleavage) when SN-38 is included.

Figure 10 is a graph investigating the retention of dipyridamole in capsule proteins (mutants with interruptions).

FIG. 11 is a graph for studying tumor growth inhibitory ability when SN-38-containing capsule protein was administered to mice that developed human prostate cancer.

FIG. 12 is a graph for studying tumor growth inhibitory ability when an octamer composition containing the encapsulating protein of SN-38 was administered to a mouse having generated human prostate cancer.

FIG. 13 is a graph showing the change in body weight of the mouse of FIG. 12.

Detailed Description

The capsule protein of the present invention will be described below with reference to the drawings and examples. The following description illustrates an embodiment and an example of the present invention, and the present invention is not limited to the following description. The following description may be changed within a scope not departing from the technical idea of the present invention.

The capsule protein of the present invention is based on human lipocalin-type prostaglandin D synthase (L-PGDS). In Table 1, the amino acid sequence of L-PGDS (SEQ ID NO: 1) is shown. L-PGDS is composed of 168 amino acids from alanine at the N-terminus to glutamine at 168 th.

[ Table 1]

L-PGDS obtained by transformation of Escherichia coli is referred to as "mutant L-PGDS" or simply as "mutant". In Table 2, the amino acid sequence of the mutant L-PGDS is shown (SEQ ID NO: 2). For ease of artificial production, glycine-serine (GS) was added to the N-terminus, and 2 amino acids were added as compared with the case of table 1. Hereinafter, the amino acid sequence of the mutant L-PGDS is represented by a sequence obtained by adding glycine-serine (GS) to the N-terminus of the sequence of L-PGDS.

[ Table 2]

The mutant L-PGDS has the cysteine (C) at the 45 th position (43 th position in SEQ ID NO: 1) from the N-terminus replaced with alanine (A) due to the loss of the enzyme active site. In addition, to prevent the generation of incorrect disulfide bonds, cysteine (C) at the 147 th position (145 th position in SEQ ID NO: 1) was also substituted with alanine (A). In table 2 (the same applies to the sequence of the capsule protein below), 2 are surrounded by a rectangle. The mutant L-PGDS is also referred to as "C45A/C147A".

The crystal structure of the mutant L-PGDS is shown in FIG. 1. Fig. 1(b) shows a state where fig. 1(a) is rotated by 90 °. In addition, L-PGDS is also the same shape. In addition, FIG. 2 shows the correspondence of the amino acid sequence (SEQ ID NO: 2) of the mutant L-PGDS (C45A/C147A) to the beta chain and alpha helix.

The mutant L-PGDS has 8 beta strands from the symbol A to H, and an alpha helix from the symbol H1 to the symbol H3. In addition, there are loops between the beta strands, short strands I and short helices H4, H5.

The beta strands A to H are arranged in a helical shape so as to surround a space in the center. This is referred to as a barrel structure. When the mutant L-PGDS retains the drug, the drug is considered to be contained in the barrel structure. Referring to fig. 1(b), a large opening 10 for a barrel structure is formed between the β -strand D and the α -helix H2.

If the opening 10 is opened in this way, even if the drug is once stored, the drug is highly likely to be released before reaching the predetermined cell. Then, a cover is provided to the opening 10 (patent document 4). Here, a disulfide bond is created between the loop connecting β chain E and β chain F (referred to as "E-F loop") and α helix H2.

Referring again to FIG. 2, the E-F loop is 4 amino acids from proline (P) at position 90 to glycine (G) at position 93 of the N-terminus of the mutant L-PGDS. Similarly, the α -helix H2 is 10 amino acids from the N-terminus to the 32 th serine (S) to the 41 th alanine (a). By changing one of these to cysteine (C), a disulfide bond can be obtained. It is called a "disulfide clip" or "D clip".

For example, FIG. 3 shows the structure of a mutant L-PGDS in which lysine (K at 38. sup. th from the N-terminus) of the. alpha. helix H2 and histidine (H) of the E-F loop were substituted with cysteine (C), respectively. It is known that a part of the opening 10 is closed by a disulfide loop 12 formed between cysteines (C). This mutant L-PGDS is referred to as the "D-clip variant". Patent document 4 discloses a D-clip variant.

However, it is known that the disulfide paperclip 12 sandwiches the end of the opening 10, and the drug contained inside the barrel structure is released from the gap 14 between the disulfide paperclip 12 and the β -strand D. Thus, in order to block the gap 14 in the beta strand D, a blocking amino acid is provided in the present invention. The blocked amino acid is an amino acid that can reduce the area of the opening 10. By providing the blocking amino acid, not only the gap 14 is blocked, but also the opening 10 can be effectively narrowed.

Referring again to fig. 2, the β -strand D is an 11 amino acid sequence of glutamine (Q) at the 68 th to proline (P) at the 78 th from the N-terminus of the mutant L-PGDS. The blocking amino acid desirably has a sterically extensive functional group. This is to block the gap 14 as much as possible. Furthermore, it is necessary that the crystal structure of the mutant L-PGDS is not greatly changed by introducing a blocking amino acid into the beta-strand D. This is due to the need to maintain the barrel structure.

For example, lysine (K), histidine (H), tryptophan (W), tyrosine (Y), phenylalanine (F), etc. can be suitably used as the blocking amino acid. Furthermore, the substitution may be more than 1. In addition, the blocking amino acid may be inserted into an amino acid sequence constituting the β chain.

FIG. 4 shows the structure of a mutant L-PGDS in which methionine (M: 74 th from the N-terminus, see FIG. 2) in the beta-strand D is substituted with tryptophan (W). Shown as "74W" in the figure. The situation is shown where the gap 14 is blocked by the arrangement of tryptophan on the beta strand D. Such a mutant L-PGDS having a blocked amino acid is referred to as a "blocked mutant".

In addition, disulfide clips may also be provided in the cleaved mutants. The mutant L-PGDS provided with the blocked amino acid and disulfide loop needle is called a D loop needle mutant with the block.

Furthermore, as shown in patent document 3, a peptide (marker peptide) recognizing a target cell may be added to the N-terminus or the C-terminus or both of the N-terminus and the C-terminus. Further, not only the end portion may be attached, but also a portion may be overlapped. The marker peptide is not particularly limited, and for example, a peptide sequence NGR that specifically binds to a membrane protein (CD13) expressed in neovascular endothelial cells can be selected. Further, the motif may be an internalized-Arg-Gly-Asp (iRGD) motif that recognizes α v β 3 and α v β 5 integrins, or a Cys-Arg-Gly-Asp-Lys (CRGDK) motif that recognizes neuropilin 1.

In addition, Lys-Leu-Pro (KLP) motif (Cancer Res, 97, 1075-81, 2006) recognizing peritoneal tumors of gastric Cancer, Asn-Val-Val-Arg-Gln (NVVRQ) motif (Cl in Cancer Res, 14, 5494-502, 2008) recognizing metastatic Cancer cells, Phe-Gln-His-Pro-Ser-Phe-I le (FQHPSFI) motif (Mol Med, 13, 246-54, 2007) recognizing liver Cancer cells, and the like can also be suitably used.

A mutant in which a labeled peptide is added to a mutant with a cleavage site as a capsule protein of the present invention is referred to as a "labeled mutant with a cleavage site". In addition, a disulfide clip is added to the needle, and the needle is called "marked D-clip variant with a partition". In addition, in the capsule protein of the present invention, for convenience of production by gene recombination, a plurality of amino acids may be bonded to the N-terminus or the C-terminus in addition to GS described above.

The capsule protein of the present invention can be prepared by placing a compound having a size of about 800Da in a barrel structure. By compound is meant herein a drug or other compound. Here, the other compound means a compound serving as an auxiliary nutrient, and may also mean a compound derived from a natural product.

In addition, it is known that in DDS, because of epr (enhanced Permeability and retention) effect, selectivity for invasion into cancer cells is improved, the remaining period is prolonged, and long-term drug effect is expected. Therefore, the isolated mutant of the capsule protein of the present invention can be used as a multimeric composition. In addition, the overall size may be 10nm or more in order to expect the EPR effect. In addition, a marker peptide may be added to the multimeric composition.

When the capsule protein is formed into a multimeric composition, a multimeric composition in which a plurality of capsule proteins are bonded to each other at their C-terminus and N-terminus and connected linearly can be suitably used. However, it is more preferable if the capsule proteins can be connected radially. A conceptual diagram of the structure of the multimeric composition of the capsule protein employed in the present invention is shown in FIG. 5.

A conceptual diagram of the multimeric composition of the capsule protein (hereinafter simply referred to as "multimeric composition") is shown in fig. 5 (a). FIG. 5(b) is a partial exploded view of the multimeric composition 21. The multimeric composition 21 is in a form in which a dimer 36 is bound to a tetramer 32 of streptavidin 30 via biotin 38, and the dimer 36 is a dimer in which the capsule protein 34 is bound via a linker 35. Thus, the multimeric composition 21 forms an octameric composition of the capsule protein 34.

The capsule protein 34 may be suitably a mutant L-PGDS, or a mutant with a cut-off. Here, the case of using a mutant with a partition will be described.

If the dissociation constant (Kd) of the binding of biotin 38 and streptavidin 30 is 10^-15M, this is the strongest of the non-covalent binding interactions between the known protein and ligand. In addition, the interaction is formed very rapidly and is not easily affected by pH, temperature, denaturants, organic solvents, and the like after formation. Furthermore, since biotin is a low molecule, the functionality of the modified molecule is not hindered. Therefore, the octamer shown in FIG. 5(a) is considered to exist very stably.

FIG. 5(c) shows a multimeric composition 22 in which a monomer of a capsule protein 34 is bound to a tetramer 32 of streptavidin 30 via biotin 38. This forms a tetramer of the capsule protein 34.

As described later, the diameter of the octamer polymer composition 21 of the capsule protein 34 is 10nm or more, which is larger than that of the tetramer or monomer. Therefore, the blood vessels in the cancer cells, which have a larger gap between endothelial cells than in the normal cells, can enter the cancer cells, but are less likely to leak out of the normal cells. Therefore, the EPR selectively invades into cancer cells and remains inside the cancer cells, thereby exhibiting the so-called EPR effect.

In the polymer composition 21 of the present invention (in which the capsule protein 34 forms an octamer), it is considered that the monomers of the capsule protein exhibit significantly different effects in vivo experiments described later, and the EPR effect is exhibited.

The capsule protein is also soluble upon the addition of a drug or other compound. As a multimeric composition, the effect is unchanged. Therefore, the capsule protein can be suitably used for solubilizing a poorly soluble drug, a poorly soluble vitamin, or the like. For example, SN-38, vitamin A, D, E, K, thyroid hormones, steroid hormones, isoflavones and the like can be suitably used. In addition, since a poorly soluble substance can be solubilized, the threshold for solubilization in drug development can be lowered.

The pharmaceutical composition containing a drug in the capsule protein of the present invention (hereinafter, simply referred to as "pharmaceutical composition") can be provided in a liquid form. In addition, it can also be provided as a powder by lyophilization. In the case of lyophilization, the effect is maintained as shown in patent document 3.

Thus, when the pharmaceutical composition of the present invention is used for therapy, it can be administered orally or parenterally (for example, by intravenous, subcutaneous, or intramuscular injection, topical, rectal, transdermal, or nasal injection). Examples of the composition for oral administration include tablets, capsules, pills, granules, powders, liquids, and suspensions.

Further, examples of the composition for parenteral administration include an aqueous injection preparation, an oily preparation, an ointment, a cream, an emulsion, an aerosol, a suppository, and a patch. These preparations are prepared by a conventionally known technique, and may contain a nontoxic and inert carrier or excipient which is generally used in the field of preparations.

In addition, a complex in which a drug or a compound other than a drug is incorporated into a capsule protein can be provided as a processed food. Examples of the processed food include not only general processed foods such as confectionery, chewing gum, jelly, biscuit, cookie, rice cake, bread, noodles, fish and livestock meat products, tea, soft drink, coffee drink, milk drink, whey drink, lactic acid bacteria drink, yogurt, ice cream, pudding and the like, but also health functional foods such as specific health foods or nutritional functional foods prescribed in the health functional food system of japan ministry of health and labor, and nutritional supplementary foods (supplements), feeds, food additives and the like. Furthermore, the insoluble substance can be used as an industrial product or an industrial raw material by utilizing its property of being soluble in water.

The processed food of the present invention can be prepared by adding capsule protein (complex) containing other compounds to the raw material of these processed foods. In addition, since the capsule protein is a mutant with a protein partition and is coated with other compounds, it is easily decomposed by heat. Therefore, the processed food of the present invention is preferably completed by a step without performing a heating step after the compound is added.

Examples

As a capsule protein for a sample, produced

(1) Mutant L-PGDS

(2) Identified mutant L-PGDS

(3) D-shaped needle mutant with partition

(4) Marked D-clip with partition

These four types.

The mutant L-PGDS is a protein capsule represented by SEQ ID No. 2 in which the 45 th cysteine (C) was substituted with alanine (A) from the N-terminus and the 147 th cysteine (C) was substituted with alanine (A) ("C45A/C147A"). The mutant L-PGDS inactivates the active site and is made by substituting cysteine (C) at position 147 with alanine (A) so as not to generate an incorrect disulfide bond.

The labeled mutant L-PGDS was prepared by attaching an iRGD peptide (CRGDKGPDC: SEQ ID NO: 3) capable of achieving both tumor-targeting and cell membrane penetration to the C-terminus of the mutant L-PGDS as a label.

Wherein the addition is performed so as to overlap with a part of the C-terminus of the mutant L-PGDS. This is to eliminate antigenicity to mice in subsequent in vivo experiments on mice. The amino acid sequence of the identified mutant L-PGDS is shown as sequence number 4 in Table 4. The portion of the tag peptide is denoted by "■". In addition, the modeled structure of the mutant L-PGDS identified as mutant L-PGDS is shown in FIG. 6. [ Table 3]

[ Table 4]

In the truncated D-clip variant, methionine (M) on beta-strand D is first substituted with tryptophan (W) as the truncated amino acid. Methionine is the 74 th from the N-terminus of the mutant L-PGDS.

The D-shaped needle mutant with the partition is formed by further adding a D-shaped needle. The D-clip was obtained by substituting the 38 th lysine (K) for cysteine (C) and the 91 th histidine (H) for cysteine (C) from the N-terminus of the mutant L-PGDS. The amino acid sequence is shown in Table 5 (SEQ ID NO: 5). In Table 5, the blocked amino acid is indicated by "tangle-solidup", and the D-loop (disulfide bond) is indicated by "major". A disulfide bond is produced between 2 cysteines (C) represented by ″ -. In addition, disulfide bonds are separated in a reducing environment, and the opening 10 can be opened or closed by a D-clip.

[ Table 5]

The identified blocked D-clip mutants were obtained by superimposing the iRGD peptide (CRGDKGPDC) on the C-terminus of the blocked D-clip mutants (SEQ ID NO: 5). In Table 6, the blocked amino acids are indicated by "tangle-solidup", and the D-loop (disulfide bond) is indicated by "major". In addition, the portion of the tag peptide is denoted by "■". FIG. 7 is a diagram showing a modeled structure of a truncated D-clip variant identified as a truncated D-clip variant.

[ Table 6]

Each capsule protein was prepared by preparing a designed expression plasmid by the super primer method, and then transformed into E.coli (Escherichia coli) BL21(DE3) strain to express it as a fusion protein with glutathione S-transferase (glutathione S-transferase).

The protein-expressing strain was cultured in LB/Amp test tube medium under shaking at 37 ℃ for 8 hours, then in 2 XYT/Amp automatic shaking (auto induction) medium for subculture, and under shaking at 37 ℃ for 16 hours. The obtained cells were subjected to ultrasonic pulverization, and the supernatant of the pulverized liquid was subjected to affinity chromatography on a Glutathione-agarose (Glutathione-Sepharose)4B column.

The fusion protein adsorbed on the column was reacted with 165 units of thrombin (thrombin) overnight, and the target protein was eluted and purified.

Next, these capsule proteins were allowed to contain SN-38. SN-38 is an abbreviation for poorly water soluble anticancer agent 7-ethyl-10-hydroxycamptothecin. It is known to exhibit a low dose and a high antitumor effect as compared with irinotecan hydrochloride, which is a prodrug of SN-38, currently used in clinical practice.

< drug Release ability >

5mL each of SN-38/mutant L-PGDS, SN-38/disrupted D-clip mutant, and SN-38/disrupted D-clip variant, SN-38/disrupted D-clip variant identified with disruption, each having an SN-38 concentration of 50. mu.M and a capsule protein concentration of 50. mu.M, was dialyzed with a Dialysis Membrane (diamond Membrane, Size 27 Wako molecular weight cutoff: 14,000) in an incubator at 37 ℃ for 72 hours using 150mL of PBS as an external solution.

500. mu.L of the external solution at 0, 1, 3, 6, 8, 12, 24, 36, 48, 60, 72 hours after the start of dialysis was sampled and replaced with the same amount of PBS. The SN-38 concentration in the sampled external liquid was measured using a spectrofluorometer F-7000(HITACHI) (excitation wavelength: 365nm, measurement wavelength: 380-600nm, measurement temperature: 37 ℃ C.).

Next, the drug release function of the blocked D-clip mutants and the identified blocked D-clip mutants in response to the reducing environment were evaluated by the same equilibrium dialysis method as described above. The external solution was dialyzed with PBS and 10mM DTT/PBS, and the oxidative environment was defined in the case of PBS as the external solution, and the reductive environment was defined in the case of DTT addition.

FIG. 8 shows the result of plotting the change in the SN-38 concentration in the dialysate over time against the dialysis time. The horizontal axis represents the reaction time (hours), and the vertical axis represents the SN-38 concentration (nM). Error bars are expressed as mean ± standard deviation (n ═ 3). In addition, in the abbreviations in the figures, "SN-38/L-PGDS (" ● ")" means SN-38/mutant L-PGDS, "SN-38/Capsule (. tangle-solidup)" means SN-38/disrupted D-looper mutant, "SN-38/Cap-sCRGDK (■)" means SN-38/disrupted D-looper mutant. In addition, the phrase "SN-38/Capsule" and "SN-38/Cap-sCRGDK" used herein means that "+ 10mM DTT (". DELTA. "and" □ ")" is added to each of the pharmaceutical composition solutions at a concentration of 10mM DTT. The solution is made into a strongly reducing environment by the addition of DTT (Dithiothreitol).

Referring to FIG. 8, in the case where the external fluid was PBS, the SN-38 concentration of the external fluid in the disrupted D-clip mutant (SN-38/Capsule) and the identified disrupted D-clip mutant (SN-38/Cap-sCRGDK) was significantly lower than that of the mutant L-PGDS (SN-38/L-PGDS) at all times from the start of dialysis, and it was judged that the release of SN-38 from SN-38/the disrupted D-clip mutant and SN-38/the identified disrupted D-clip mutant was inhibited as compared with the release from SN-38/the mutant L-PGDS.

On the other hand, in the case of the external liquid DTT/PBS, the concentration of SN-38 in the external liquid was increased in the blocked D-clip mutant (SN-38/Capsule: ". DELTA."), and in the marked blocked D-clip mutant (SN-38/Cap-sCRGDK: "□").

The above shows that the blocked D-clip mutants, and the identified blocked D-clip mutants, retain SN-38 in an oxidizing environment for a longer period of time than the mutant L-PGDS, releasing SN-38 in response to a reducing environment. Furthermore, it was shown that there was no difference in drug release pattern between the blocked D-clip mutants and the identified blocked D-clip mutants, and thus the addition of the identification peptide had no effect on controlling drug release.

Next, the same experiment was performed for the disrupted mutant (M74W) and the mutant L-PGDS without the D-clip. Mutant L-PGDS repeated experiments were performed in the case of FIG. 8. The results are shown in FIG. 9. FIG. 9(a) shows the case of the mutant L-PGDS, and FIG. 9(b) shows the case of the mutant with a partition (M74W). Referring to FIGS. 9(a) and 9(b), the horizontal axis represents reaction time (hours) and the vertical axis represents SN-38 concentration (. mu.M).

Referring to FIG. 9(a), the SN-38 concentration at the reaction time in the case where the external liquid was PBS (black circle "●") was almost the same as that in the case of FIG. 8, and it was found that the experiment of FIG. 8 was reproduced well. In the mutant L-PGDS, in the case where the external solution was DTT/PBS (white circles ". smallcircle"), the SN-38 concentration reached 2.0. mu.M in a short reaction time, and almost stationary phase was observed.

On the other hand, referring to FIG. 9(b), in the case where the external solution was PBS (black circle "●"), the SN-38 concentration did not exceed 1.0. mu.M even after the reaction time passed, and the release of SN-38 was suppressed as compared with the case of the mutant L-PGDS, the blocked D-clip mutant, or the marked blocked D-clip mutant shown in FIG. 8.

On the other hand, in the case where the external solution was DTT/PBS (white circle ". smallcircle"), the SN-38 concentration reached almost 2.0. mu.M within 48 hours of the dialysis time. This value is almost the same concentration as in the case of the blocked D-clip variants or the marked blocked D-clip variants shown in fig. 8. In conclusion, it was found that the mutant with a partition had superior drug retention ability than the D-clip mutant with a partition or the identified D-clip mutant with a partition. In addition, it was confirmed that the mutant with a partition can release the contained drug almost as much as other capsule proteins under a reducing atmosphere.

Next, the result of the same experiment conducted with SN-38 changed to dipyridamole, which is a poorly water-soluble antianginal drug, is shown in fig. 10. In addition, the external liquid was PBS. Referring to fig. 10, the horizontal axis represents reaction time (hours) and the left vertical axis represents dipyridamole concentration (nM). The right vertical axis represents the axis in which the left vertical axis is converted into the release rate (%). The dipyridamole concentration increased with reaction time in the mutant L-PGDS, whereas in the blocking mutant (M74W), the dipyridamole concentration increased only to near the detection limit.

Thus, it was found that the mutant with a partition has excellent ability to hold the contained drug, and can be held with little leakage depending on the drug. As described above, it can be said that the drug contained in the DDS can be continuously retained until the inside of the cell is reduced, and the DDS is highly efficient.

< in vivo Effect >

Male BALB/C-nu/nu mice (Japanese SLC) aged 4 weeks were allowed to drink and feed water freely in an animal room controlled at room temperature and with 12-hour light-dark cycle, and were allowed to breed for 1 week for acclimatization. Thereafter, 100 μ L of 5X 10 were administered subcutaneously in the right flank⁷Prostate cancer cell PC-3 per mL (PBS: matrigel ═ 1: 1) of human prostate cancer, and a prostate cancer model mouse was prepared.

Tumor volume (approximate: calculated by { (major diameter) × (minor diameter) 2 }/2) reached 250mm³Day (2) of administration, mice were randomly classified into each administration group of PBS, SN-38/mutant L-PGDS (2.0mg/kg/D), SN-38/labeled mutant L-PGDS (2.0mg/kg/D), SN-38/blocked D-clip variant (2.0mg/kg/D), and SN-38/labeled blocked D-clip variant (2.0mg/kg/D), and each sample was administered from the caudal vein every other day for a total of 8 times. In addition, the control group was given only PBS.

The results of the in vivo antitumor experiment are shown in figure 11. The horizontal axis represents the number of days elapsed from day 0 of administration (day), and the vertical axis represents the tumor volume (mm)³). The abbreviations in the diagrams are as follows.

PBS: control group

SN-38/L-PGDS: SN-38/mutant L-PGDS

SN-38/L-PGDS-sCRGDK: SN-38/identified mutant L-PGDS

SN-38/Capsule: SN-38/D-clip with partition

SN-38/Cap-sCRGDK: marked D-clip with partition

In the PBS-administered group, the antitumor effect was not confirmed, and the tumor volume continued to increase from the day of the start of administration. On the other hand, significant tumor growth inhibition was confirmed in the groups administered with SN-38/L-PGDS, SN-38/L-PGDS-sCRGDK, SN-38/Capsule, and SN-38/Cap-sCRGDK.

In addition, in the SN-38/Capsule and SN-38/L-PGDS-sCRGDK-administered groups, no significant tumor growth inhibition was observed compared with the SN-38/L-PGDS-administered group. Thus, it was determined that when only one of the targeting and release control functions was added, a significantly higher antitumor activity was not obtained as compared with the mutant L-PGDS.

On the other hand, SN-38/Cap-sCRGDK (identified D-clip variant with a partition) showed significantly high antitumor activity compared to SN-38/L-PGDS (mutant L-PGDS). From the above results, it is understood that tumor growth can be significantly inhibited by a synergistic effect of 2 functions of the drug release control function (blocking amino acids and D-clip) and the cancer targeting function (marker peptide).

< multimer >

Next, the production of a polymer composition of capsule proteins (octamer composition) will be described. As illustrated in fig. 5, the octamer composition is in a shape formed by binding biotin 38 to dimer 36 on tetramer 32 of streptavidin 30, wherein dimer 36 is a dimer of capsule protein 34 bound via linker 35.

Thus, after the biotinylated dimer composition of biotin bound to the dimer of the encapsulated protein is produced, it is bound to the separately produced tetramer of streptavidin to obtain an octamer composition of encapsulated protein. The linker was encoded by the nucleotide sequence shown in Table 7 (SEQ ID NO: 7). Streptavidin was encoded by the nucleotide sequence shown in Table 8 (SEQ ID NO: 8).

[ Table 7]

[ Table 8]

< preparation of dimer L-PGDS expression vector >

The gene sequence of the disrupted mutant was amplified by PCR using a forward primer containing a BamHI recognition site and a reverse primer containing an Eco RI recognition site and a linker sequence (GGGGS: SEQ ID NO: 7) and subjected to agarose gel electrophoresis. To this, Bam HI and Eco RI were used, and the restriction enzyme-treated sequence was inserted as a mutation with a block.

In addition, in the mutant with a cleavage, the 45 th cysteine (C) was substituted with alanine (a) from the N-terminus and the 147 th cysteine (C) was substituted with alanine (a) ("C45A/C147A"), and the cleavage amino acid was substituted with methionine (M) on the β -chain D with tryptophan (W) ("M74W"). Methionine is the 74 th from the N-terminus of the mutant L-PGDS. The amino acid sequence of the mutant with a block (SEQ ID NO: 9) is shown in Table 9.

[ Table 9]

In the same manner, a restriction enzyme-treated plasmid (pGEX4T-2) was prepared. The blocked mutant insert and the restriction enzyme-treated pGEX4T-2 were subjected to agarose gel electrophoresis, respectively.

After staining with ethidium bromide, the Gel was excised, and DNA was extracted with an Agarose Gel Extraction Kit (Jene Bioscience) and ligated. In this procedure, the nucleotide sequence encoding the disrupted mutant and the sequence binding to the linker was inserted into plasmid pGEX 4T-2. This plasmid is called a disrupted mutant expression vector.

The obtained mutant expression vector with a partition was used to transform E.coli strain DH5 alpha (DE3) and inoculated into LB/Amp plate medium. For colonies grown in the plate medium, colony direct PCR was performed. Colonies in which the presence of the disrupted mutant was confirmed were inoculated into LB/Amp test tube medium (5mL) and cultured at 37 ℃ for 16 hours. Thereafter, mini prep chromatography (ミニプレップ) was performed using SV miniprep (promega) to purify the mutant expression vector with the cleavage site.

Sequencing of the resulting mutant expression vector with the cleavage was performed, and it was confirmed that the nucleotide sequences encoding the mutant with the cleavage and the linker were inserted.

In this disrupted mutant expression vector, the amplified disrupted mutant insert was integrated using primers containing Eco RI and SalI recognition sites by the same procedure. Thereafter, E.coli strain DH5 alpha (DE3) was transformed in the same manner, cultured, amplified and purified. The plasmid includes a base sequence encoding a truncated mutant of the dimeric portion. Thus, this plasmid is referred to as a dimer band-interrupted mutant expression vector. The obtained dimer with partition mutant expression vector was sequenced to confirm the base sequence.

< preparation of modified dimer L-PGDS expression vector >

Next, the 15 amino acid residues (Avitag) specifically recognized by the encoding biotin ligase (BirA) were added^TMHereinafter referred to as "Av". ) The annealing was performed using oligo DNAs complementary to SEQ ID Nos. 10 and 11. Biotin ligase binds biotin at the lysine residue of the peptide Av. The nucleotide sequences are shown in Table 10 and Table 11, respectively. Thereafter, the annealed product was purified using a purification column FastGene Gel/PCR extraction kit (Nippon genetics, Tokyo). This base sequence is referred to as the Av base sequence.

[ Table 10]

[ Table 11]

The dimer-cleaved mutant expression vector was subjected to restriction enzyme cleavage using Bam HI (37 ℃ C., 3 hours), and then subjected to agarose gel electrophoresis. Then, the DNA of the mutant expression vector with the dimer cleaved was extracted, and the Av base sequence was inserted into the cleavage site of Bam HI using In Fusion (registered trademark) HD cloning kit (Clontech). A vector obtained by inserting an Av base sequence into a dimer segment-interrupted mutant expression vector is referred to as a modified dimer segment-interrupted mutant expression vector. The obtained vector was sequenced to confirm the insertion of the target sequence.

< dimer-disrupted mutant expression Strain >

And transforming the E.coli strain AVB101 expressing BirA by using the modified dimer with separated mutant expression vector to obtain a dimer with separated mutant expression strain. The dimer with cut-off mutant expression strain was inoculated into 5ml of LB/Amp/Chl liquid medium and cultured overnight at 37 ℃ with shaking. Next, the cells were passaged in 1L 2 XYT/Amp/Chl medium and cultured at 37 ℃.

In addition, the E.coli strain AVB101 for BirA was an E.coli strain into which an expression vector having a nucleotide sequence encoding BirA was inserted.

< production of biotinylated dimer band-disrupted mutant >

IPTG (final concentration 0.1mM) and biotin (final concentration 50. mu.M) were added at the time when the OD600 value of the culture solution of the mutant expression strain with dimer partition reached 0.6-1.0. By this operation, both the modified dimer band-disrupted mutant and BirA proteins were expressed in vivo in escherichia coli. Then, BirA allowed additional peptide Av to bind to biotin in the dimer band-disrupted mutant. As a result, biotinylation of the dimer band-disrupted mutant was induced.

Thereafter, the cells were cultured at 37 ℃ for 6 hours, and the culture broth was centrifuged (8, 400 Xg, 10 minutes, 4 ℃) to collect the cells. Then, the cells were washed with PBS, and the bacteria were collected by centrifugation and then ultrasonically pulverized.

A15 ml affinity column to which glutathione-Sepharose 4B (GE healthcare bioscience, UK) was dispensed was equilibrated with 5 times the column volume of PBS passed through a 0.22 μm filter, to give a supernatant of the fraction passed through the 0.22 μm filter.

After washing with 3 times the column volume of 1% Triton X-100/PBS and 5 times the column volume of PBS, 165 units of thrombin (thrombin) (Sigma) was added thereto, and the mixture was stirred well and allowed to stand at room temperature for 12 hours or more. Elution was carried out with PBS in an amount 5 times the column volume, and centrifugation was repeated using a concentrator having a molecular weight cut-off of 10kDa (8400g, 20 minutes, 4 ℃ C.) to concentrate the eluate to 4 ml.

Next, the mixture was degassed with 5mM Tris-HCl (pH8.0) through a 0.22 μm filter for 10 minutes, and equilibrated with Superdex 7516/600 (GE healthcare bioscience, UK) through 2-fold amounts of the same buffer. The concentrate was passed through a 0.22 μm filter and added to Superdex 7516/600. While monitoring the absorbance at 280nm, the eluate was retained at a flow rate of 0.5 ml/min to 1.5ml each, and a fraction corresponding to the peak of biotinylated dimer L-PGDS was recovered.

The recovered fractions were combined, dialyzed against 20mM sodium acetate (Na-acetate) buffer (pH5.5), and then concentrated to 4ml by repeated centrifugation using a concentrator having a molecular weight cut-off of 10kDa (8400g, 20 minutes, 4 ℃).

This was supplied to a column packed with SP Sepharose FF (GE healthcare bioscience, UK) and subjected to cation exchange chromatography by the linear gradient method using 20mM sodium acetate (Na-acetate) buffer (pH5.5) → 1M NaCl/20mM sodium acetate buffer (pH 5.5).

The eluates were collected at a flow rate of 1.0 ml/min while monitoring the absorbance at a UV wavelength of 280nm, and the eluates were cut off at 1.5ml each. After that, SDS-PAGE analysis was performed to recover a single band portion of the mutant which was confirmed to be a biotinylated dimer band-disrupted mutant.

< purification of streptavidin >

Coli strain BL21(DE3) was transformed with a Streptavidin expression vector (pET21a-Streptavidin-Alive, Addgene) to obtain a Streptavidin expression strain. The streptavidin-expressing strain was inoculated into 5ml of LB/Amp liquid medium and cultured overnight at 37 ℃ with shaking. Then, the cells were subcultured in 2 XYT/Amp medium 1L at 37 ℃. Then, when the OD600 reached 0.6-1.0, IPTG was added so that the final concentration became 0.1mM, and expression was induced. Then, after culturing at 18 ℃ for 24 hours, the culture broth was centrifuged (8400 Xg, 10 minutes, 4 ℃), and the cells were collected.

PBS was added to the obtained cells, and the cells were suspended, and 1. mu.l of 100mg/ml Lysozyme (Lysozyme) was added to 1g of the cells, followed by stirring in ice water. Then, the cells were ultrasonically crushed while stirring in ice (7 groups of cells were subjected to 1 minute of ultrasound and 2 minutes of rest), and the crushed liquid was centrifuged (4 ℃ C., 15000rpm) to obtain a supernatant.

A10 ml affinity column (GE healthcare bioscience, UK) into which Ni Sepharose was dispensed was equilibrated with a 5-fold amount of 20mM imidazole/20 mM sodium phosphate buffer (pH7.0) by volume of the column passed through a 0.22 μm filter, to give a disruption supernatant passed through the 0.22 μm filter. The washing was carried out with 20mM sodium phosphate buffer (pH7.0) at each imidazole concentration (20, 50, 100mM), and eluted with 300mM imidazole/sodium phosphate buffer (pH 7.0). Thereafter, centrifugation was repeated using a concentrator having a molecular weight cut-off of 10kDa (8400g, 20 minutes, 4 ℃ C.) to concentrate it to 4 ml.

Subsequently, PBS (pH7.4) passed through the 0.22 μm filter was degassed for 10 minutes, and equilibrated with Superdex 7516/600 by passing 2 times the amount of the same buffer. The concentrate was passed through a 0.22 μm filter and added to Superdex 7516/600. While monitoring the absorbance at 280nm, the eluates were retained at a flow rate of 0.5 ml/min, and analyzed by non-reducing SDS-PAGE to obtain streptavidin tetramers.

< preparation of octamer composition >

The purified dimer band-blocked mutant (in PBS, pH7.4) and streptavidin tetramer (in PBS, pH7.4) were mixed at a molar ratio of 4: 1, and allowed to stand at room temperature for 15 minutes. Thereafter, centrifugation was repeated using a concentrator having a molecular weight cut-off of 50kDa (8400g, 20 minutes, 4 ℃ C.) to concentrate it to 4 ml.

Subsequently, PBS (pH7.4) passed through a 0.22 μm filter was degassed for 10 minutes, and equilibrated with Superdex 20016/600 (GE healthcare bioscience, UK) by passing 2 times the amount of the same buffer. The concentrate was passed through a 0.22 μm filter and added to Superdex 20016/600. While monitoring the absorbance at 280nm, the eluates were cut into 1.5ml portions at a flow rate of 0.5 ml/min, and analyzed by SDS-PAGE to obtain an octamer composition.

In addition, a mutant expression vector with a partition prior to the preparation of a mutant expression vector with a dimer partition was directly introduced into E.coli to obtain a monomer of a mutant with a partition. In addition, a monomer of a labeled cleavage-associated mutant in which an iRGD peptide was added to a cleavage-associated mutant was prepared. The amino acid sequences of the identified truncated mutants are shown in Table 12 (SEQ ID NO: 12). The method for attaching the iRGD peptide is the same as in the case of sequence No. 6.

[ Table 12]

In addition, the mutant expression vector with Av base sequence inserted into the mutant expression vector with partition and modified monomer with partition was used to transform the E.coli strain AVB101 strain expressing BirA, and a tetramer composition with 4 monomers bound by streptavidin could also be obtained.

< size of octamer composition >

< size of DLS-based octamer composition >

< size of SAXS-based octamer >

The size of the resulting octamer composition was determined by X-ray small Angle Scattering (SAXS: Smal Angle X-ray Scattering). In addition, for comparison, the size of the mutant L-PGDS was also determined.

The SAXS assay was performed in the Beamline BL40B2 from SPring-8 (Bingkun county, Zusajun, Japan) as a large irradiation facility. The X-ray wavelength was adjusted to 1.000A (angstroms) and the camera length was adjusted to 2.193m, and the experiment was performed at 25 ℃. Each measurement was exposed for 20-50 seconds, and scattered light was detected using PILATUS-2M (Rickipedia, Tokyo).

For maximum scattering of X-rays, a sample cell (sample cell) of 3.0mm thickness is used, and a 0.02mm quartz plate is used for the cell window. To avoid measurement errors, the protein sample and buffer were measured alternately. The cell was filled with 25. mu.L of the sample, and the measurement was performed, after which the sample was removed from the cell and washed 3 times with the buffer. Then, the subsequent measurement was performed.

The scattering patterns of the protein sample and the buffer recorded two-dimensionally in the detector were converted into one-dimensional data by ring averaging, and the data of the buffer was subtracted from the data of the protein sample. The scattering curve in the small angle region was analyzed by the Guinier approximation formula for monodisperse systems. The scattering intensity I (S, C) is a function of the scattering vector S and the protein concentration C, and the origin scattering intensity I (0, C) and the radius of inertia R can be used_g(C) (radius of gyration) is expressed in the form of formula (1).

[ mathematical formula 1]

In addition, the first and second substrates are,

further, 2 θ here is a scattering angle, and λ represents the wavelength of the X-ray.

Radius of inertia calculated from the Guinier region of the resulting scattering curveR_g1.8. + -. 0.04nm in the monomer (mutant L-PGDS), approximately 3 times this in the octamer composition, 6.0. + -. 0.69nm, the radius of inertia R being known_gIncreased by octamer. The measurement results are shown in Table 13. Molecular weight (Mw) calculated from the scattering curve^exp) The theoretical values (Mw) for the molecular weights of the monomeric (mutant L-PGDS) and the octameric compositions^calc) Therefore, it was judged that octamers of the blocking mutants, which are considered to have almost the same size as the monomer (mutant L-PGDS), had indeed progressed. Thus, the octamer composition can be expected to have tumor-aggregating properties based on the EPR effect due to the shape of particles having a sufficient size exceeding 10 nm.

[ Table 13]

	Rg(nm)	Dmax(nm)	Mwexp(kDa)	Mwcalc(kDa)
					Monomer	1.8±0.04	4.75	16	19
Octameric composition	6.0±0.69	22.4	231	217

< containing of drug >

Subsequently, monomers, the isolated mutants, the identified isolated mutants and the octamer composition were allowed to contain SN-38. As already mentioned, SN-38 is an abbreviation for the poorly water soluble anticancer agent 7-ethyl-10-hydroxycamptothecin. It is known to exhibit a low dose and a high antitumor effect as compared with irinotecan hydrochloride, which is a prodrug of SN-38, currently used in clinical practice.

To a PBS suspension of SN-38 incubated at 37 ℃ were added PBS solutions of the monomer (mutant L-PGDS), the mutant with a partition, the identified mutant with a partition, or the octamer composition so that the final concentrations reached 1. mu.M, 0.25. mu.M, and 0.125. mu.M, respectively, and the mixture was stirred at 37 ℃ for 6 hours. After completion of the stirring, free SN-38 was removed by ultrafiltration to prepare a sample containing the drug in the capsule protein.

< in vivo Effect >

Male BALB/C-nu/nu mice (Japanese SLC) aged 4 weeks were allowed to drink and feed water freely in an animal room controlled at room temperature and with 12-hour light-dark cycle, and were allowed to breed for 1 week for acclimatization. Thereafter, 100 μ L of 5X 10 were administered subcutaneously in the right flank⁷Prostate cancer cell PC-3 per mL (PBS: matrigel ═ 1: 1) of human prostate cancer, and a prostate cancer model mouse was prepared.

Tumor volume (approximate: by { (major diameter) × (minor diameter)²}/2 calculation) to 250mm³On day 0 of the administration, mice were randomly classified into groups of PBS, the monomer (2.0mg SN-38/kg/d), the mutant with a block (2.0mg SN-38/kg/d), the identified mutant with a block (2.0mg SN-38/kg/d), and the octamer composition (2.0mg SN-38/kg/d), and the monomer, the mutant with a block, the identified mutant with a block, and the octamer composition were administered 4 times every 4 days. In addition, only PBS was given every 4 days in the control group for a total of 4 times.

The results of the in vivo antitumor experiment are shown in figure 12. The horizontal axis represents the number of days elapsed from day 0 of administration (day), and the vertical axis represents the tumor volume (mm)³). The abbreviations in the diagrams are as follows.

PBS: control group

SN-38/L-PGDS: monomer (SN-38/mutant L-PGDS)

SN-38/M74W: SN-38/mutant with a partition

SN-38/M74W-sCRGDK: SN-38/identified mutant with a partition

SN-38/M74W-octamer: SN-38/octamer compositions with a disrupted mutant

In comparison with the case of fig. 11, there is no clip with D.

The identified blocking mutant had the C-terminus of the blocking mutant added with Cys-Arg-Gly-Asp-Lys (CRGDK) motif that recognizes neuropilin 1 (reference numeral 12).

Referring to fig. 12, in the PBS-administered group, the antitumor effect was not confirmed, and the tumor volume continued to increase from the day of administration. In contrast, the SN-38/L-PGDS-administered group showed an effect of suppressing the increase of tumors. Moreover, SN-38/M74W, a mutant with a disruption, further inhibited tumor growth.

On the other hand, surprisingly, no tumor growth was observed at all in SN-38/M74W-sCRGDK (identified mutant with a truncation (without D clip)) and SN-38/M74W-octamer (octamer composition of truncation mutants) from the beginning of the experiment. Since SN-38 is considered to be a drug that inhibits the proliferation of cancer cells but does not induce apoptosis, it can be said that the effect of SN-38 is sufficiently exerted.

Furthermore, although the administration was performed 4 times every 4 days, the tumor growth was also completely inhibited temporarily after the 15 th day on which the administration was not performed. Therefore, the drug remained in the cell tissue and was not excreted by the lymphatic system, and the EPR effect that exhibited the drug effect for a long period of time was confirmed.

Fig. 13 is a graph showing the average body weight of mice when the experiment of fig. 12 was performed. The horizontal axis represents the number of days (days) elapsed from day 0 of administration, and the vertical axis represents the body weight ratio (%) relative to the body weight of the mouse at the start of administration. The average of each administration group (5 individuals) is shown. The abbreviation in the graph is the same as in the case of fig. 12. The PBS-administered group as a control group in fig. 12 was not included.

If reference is made to FIG. 13, the disrupted mutant (SN-38/M74W) which showed very effective inhibition of cancer cell growth showed signs of less than 80% body weight on day 20, and the experiment was discontinued. Considering that the weight of the newly inactivated L-PGDS (SN-38/L-PGDS) did not decrease below 80% despite the reduction in body weight, it is considered that the mutant with a block was excellent in the retention of SN-38 (drug), but due to the release of SN-38 in normal cells, side effects occurred.

On the other hand, in the identified blocking mutant (SN-38/M74W-sCRGDK) and the octamer (SN-38/M74W-octamer) of the blocking mutant, which showed a significant effect on the inhibition of cancer cell proliferation, the body weight was slightly reduced, and SN-38 tended to increase even 15 days after the administration was stopped.

Thus, it is considered that the identified cleaved mutant (SN-38/M74W-sCRGDK) and the cleaved mutant octamer (SN-38/M74W-octamer) specifically recognize cancer cells and release drugs in the cancer cells.

In addition, if a multimer is made to exert an EPR effect, the drug can be delivered regardless of cancer. In particular, it is considered to be very useful that the drug can be selectively injected into only cancer cells regardless of the metastatic site without affecting normal cells in the post-metastatic cancer.

Possibility of industrial utilization

The invention wraps the insoluble drug or other compounds to form soluble matters. Thus, after uptake into cells, disulfide bonds are cleaved in an intracellular reducing environment (intracellular reduced glutathione concentrations of about 0.5-10mM, 1000-fold higher than extracellular concentrations of about 100-. Therefore, it can be suitably used as a DDS capsule for a poorly soluble compound. Furthermore, the insoluble substance can be used as an industrial product or an industrial raw material by utilizing the property of making the insoluble substance soluble.

Description of the symbols

10 (of barrel-like construction) opening

12 disulfide paper clip

14 gap

21 Polymer composition (octamer composition)

22 multimeric composition (tetrameric composition)

30 streptavidin

32 tetramer

35 linking group

34 Capsule protein

36 dimer

38 biotin.

Sequence listing

<110> Osaka, university of justice

<120> capsule protein, multimeric composition thereof, and pharmaceutical composition using same

<130> UO22004PCT

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 168

<212> PRT

<213> human

<400> 1

Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln Asp Lys

1 5 10 15

Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser Ser Trp

20 25 30

Leu Arg Glu Lys Lys Ala Ala Leu Ser Met Cys Lys Ser Val Val Ala

35 40 45

Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu Arg Lys

50 55 60

Asn Gln Cys Glu Thr Arg Thr Met Leu Leu Gln Pro Ala Gly Ser Leu

65 70 75 80

Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp Gly Ser Thr Tyr Ser Val

85 90 95

Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr Ser Gln

100 105 110

Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu Tyr Ser

115 120 125

Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr Ala Phe

130 135 140

Cys Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu Pro Gln

145 150 155 160

Thr Asp Lys Cys Met Thr Glu Gln

165

<210> 2

<211> 170

<212> PRT

<213> Artificial sequence

<220>

<223> enzyme inactivation mutant

<400> 2

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Lys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Met Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Met Thr Glu Gln

165 170

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> iRGD peptide

<400> 3

Cys Arg Gly Asp Lys Gly Pro Asp Cys

1 5

<210> 4

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> identified mutant

<400> 4

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Lys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Met Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Arg Gly Asp Lys Gly Pro Asp Cys

165 170

<210> 5

<211> 170

<212> PRT

<213> Artificial sequence

<220>

<223> D-clip variant with partition

<400> 5

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Cys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Trp Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro Cys Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Met Thr Glu Gln

165 170

<210> 6

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> identified D-clip with partition

<400> 6

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Cys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Trp Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro Cys Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Arg Gly Asp Lys Gly Pro Asp Cys

165 170

<210> 7

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> linker

<400> 7

Gly Gly Gly Gly Ser

1 5

<210> 8

<211> 412

<212> DNA

<213> Streptomyces afficiens

<400> 8

gctgaagctg gtatcaccgg cacctggtac aaccagctgg gatccacctt catcgttacc 60

gctggtgctg acggtgctct gaccggtacc tacgaatccg ctgttggtaa cgctgaatct 120

agatacgttc tgaccggtcg ttacgactcc gctccggcta ccgacggttc cggaaccgct 180

ctgggttgga ccgttgcttg gaaaaacaac taccgtaacg ctcactccgc taccacctgg 240

tctggccagt acgttggtgg tgctgaagct cgtatcaaca cccagtggtt gttgacctcc 300

ggcaccaccg aagccaacgc gtggaaatcc accctggttg gtcacgacac cttcaccaaa 360

gttaaaccgt ccgctgcttc ccatcaccat caccaccatt aataaaagct tg 412

<210> 9

<211> 170

<212> PRT

<213> Artificial sequence

<220>

<223> mutant with partition

<400> 9

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Lys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Trp Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Met Thr Glu Gln

165 170

<210> 10

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Avitag sense primer

<400> 10

gttccgcgtg gatccatgtc tggcctgaac gatattttcg aagcgcagaa aattgaatgg 60

cacgaa 66

<210> 11

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Avitag antisense primer

<400> 11

ctcgggtgcg gatccttcgt gccattcaat tttctgcgct tcgaaaatat cgttcaggcc 60

agacat 66

<210> 12

<211> 174

<212> PRT

<213> Artificial sequence

<220>

<223> identified mutant with partition

<400> 12

Gly Ser Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn Phe Gln Gln

1 5 10 15

Asp Lys Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu Ala Ser Asn Ser

20 25 30

Ser Trp Leu Arg Glu Lys Lys Ala Ala Leu Ser Met Ala Lys Ser Val

35 40 45

Val Ala Pro Ala Thr Asp Gly Gly Leu Asn Leu Thr Ser Thr Phe Leu

50 55 60

Arg Lys Asn Gln Cys Glu Thr Arg Thr Trp Leu Leu Gln Pro Ala Gly

65 70 75 80

Ser Leu Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp Gly Ser Thr Tyr

85 90 95

Ser Val Ser Val Val Glu Thr Asp Tyr Asp Gln Tyr Ala Leu Leu Tyr

100 105 110

Ser Gln Gly Ser Lys Gly Pro Gly Glu Asp Phe Arg Met Ala Thr Leu

115 120 125

Tyr Ser Arg Thr Gln Thr Pro Arg Ala Glu Leu Lys Glu Lys Phe Thr

130 135 140

Ala Phe Ala Lys Ala Gln Gly Phe Thr Glu Asp Thr Ile Val Phe Leu

145 150 155 160

Pro Gln Thr Asp Lys Cys Arg Gly Asp Lys Gly Pro Asp Cys

165 170