Capsular proteins and multimeric compositions thereof and pharmaceutical compositions using the same

Abstract

There is an idea of accommodating a drug in a barrel-shaped structure of a lipocalin-type prostaglandin D synthase, introducing a disulfide bond between an α-helix H2 and an E-F loop, and enclosing the drug, but sometimes the drug is released from a gap of an opening of the barrel-shaped structure. A capsule protein characterized by substitution of cysteine in an active site of a human lipocalin-type prostaglandin D synthase with alanine and substitution of at least one amino acid of a β-strand D with a blocking amino acid suppresses release of an accommodated drug through the blocking amino acid provided in the β-strand D of the opening of the barrel-shaped structure.

Description

Technical Field

[0001] This invention relates to capsule proteins and polymeric compositions thereof that can be used as drug delivery systems (DDS), and more particularly to capsule proteins that can dissolve poorly water-soluble drugs, release drugs at the affected area after administration, and pharmaceutical compositions and processed foods using the same. Background Technology

[0002] In drug delivery systems (DDS), the development of drug delivery carriers is a key technology. Previous studies have explored liposomes, microparticles, nanomaterials, and drug-polymer conjugates. Among these, polymer micelles have attracted attention as advantageous carriers for delivering poorly water-soluble drugs. For example, micelle-forming compositions consisting of a hydrophobic core surrounded by a hydrophilic shell, where the hydrophilic shell is composed of PVP (N-vinyl-2-pyrrolidone) (Patent Document 1).

[0003] Patent document 2 discloses a capsule protein modified with lipid transport protein prostaglandin D synthase (hereinafter referred to as "L-PGDS"), which is a biological product, to exert the drug's effect by dissolving a poorly water-soluble drug in water before administration. Since the L-PGDS-modified capsule protein is an in vivo product, not an antigen, and not toxic to humans, it can be considered a safe and reliable drug delivery carrier.

[0004] Patent document 3 discloses a capsule protein modified with L-PGDS that carries a marker protein that allows cells in the affected area to recognize it. For example, it is believed that capsule proteins modified with L-PGDS containing marker peptides that specifically bind to cancer cells concentrate on cancer cells in the affected area, thereby improving the therapeutic effect.

[0005] Patent document 4 discloses a protein in which the 34th and 92nd tryptophan residues from the N-terminus of a modified L-PGDS capsule protein are replaced with cysteine ​​residues, and a cap is provided that opens and closes disulfide bonds under a redox atmosphere. The aim is to improve drug retention by opening and closing disulfide bonds.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Publication No. 2004-501180

[0009] Patent Document 2: Japanese Patent Application Publication No. 2008-120793

[0010] Patent Document 3: Japanese Patent Application Publication No. 2011-207830

[0011] Patent Document 4: Japanese Patent Application Publication No. 2013-162760 Summary of the Invention

[0012] The technical problem that the invention aims to solve

[0013] L-PGDS-based capsule proteins can readily dissolve poorly water-soluble drugs and, being biological products, offer the advantage of high safety. However, maintaining the drug's barrel-like structure results in an open container shape. Consequently, there is a risk of once-ingested drug being released during transport. To address this issue, Patent Document 4 employs disulfide bond-based immobilization for L-PGDS-based capsule proteins.

[0014] However, in the fixation of the opening of capsule proteins using L-PGDS based on disulfide bonds, it is known that the opening of the barrel structure cannot be closed.

[0015] Technical solutions adopted to solve technical problems

[0016] The present invention was made in view of the above-mentioned problems, and its object is to provide a capsule protein having an amino acid as a large volume partition at the opening of a barrel-shaped structure, which can prevent accidental release of drugs.

[0017] More specifically, the capsule protein of the present invention is characterized in that,

[0018] In human lipid transport protein prostaglandin D synthase, cysteine ​​in the active site is replaced by alanine, and at least one amino acid in the β-chain D is replaced by a cleaved amino acid.

[0019] The effects of the invention

[0020] The capsule protein of the present invention has amino acids that act as septa in the opening of the barrel-shaped structure formed by the β chain of the mutant L-PGDS. The drug placed in the barrel-shaped structure is less released during delivery, which can efficiently deliver the drug to the cells of the affected area.

[0021] Furthermore, by making the capsule protein of the present invention into a polymeric composition, an EPR (Enhanced Permeability and Retention) effect can be achieved, which can increase the specificity of cancer cell invasion and improve the efficacy of the drug while inhibiting the effect on normal cells, and can be expected to have the effect of inhibiting side effects. Attached Figure Description

[0022] Figure 1 This is a diagram showing the crystal structure of the mutant L-PGDS.

[0023] Figure 2 This is a diagram representing the amino acid sequence of the mutant L-PGDS.

[0024] Figure 3This is a diagram showing the modeling structure of the D-paperclip mutant with imported D-paperclip (disulfide bond).

[0025] Figure 4 This is a diagram representing the modeling structure of a septate mutant endowed with septate amino acids.

[0026] Figure 5 This is a diagram illustrating the concept of a polymeric composition.

[0027] Figure 6 This is a diagram representing the modeling structure of the mutant L-PGDS, identified as the mutant L-PGDS.

[0028] Figure 7 This is a diagram representing the modeling structure of the D-paperclip mutant identified as a septate D-paperclip mutant.

[0029] Figure 8 This is a chart showing the retention and release capabilities of capsule proteins containing SN-38.

[0030] Figure 9 It is a graph showing the retention and release capabilities of capsule proteins (septate mutants) containing SN-38.

[0031] Figure 10 This is a graph showing the retention capacity of dipyridamole in a capsule protein (a septate mutant).

[0032] Figure 11 This is a graph showing the tumor growth inhibition ability of mice with human prostate cancer when given capsules containing SN-38.

[0033] Figure 12 This is a graph showing the tumor growth inhibition ability of an octamer composition containing SN-38 capsule protein when administered to mice with human prostate cancer.

[0034] Figure 13 It means Figure 12 A graph showing the weight changes of mice. Detailed Implementation

[0035] The capsule protein of the present invention will be described below with reference to the accompanying drawings and embodiments. Furthermore, the following description illustrates one embodiment and one example of the invention, and the invention is not limited to the following description. Changes to the following description may be made without departing from the technical spirit of the invention.

[0036] The capsule protein of this invention is based on human lipid transporter prostaglandin D synthase (L-PGDS). Table 1 shows the amino acid sequence of L-PGDS (Sequence Number 1). L-PGDS consists of 168 amino acids, starting from the 168th amino acid, alanine at the N-terminus, up to glutamine.

[0037] [Table 1]

[0038]

[0039] The L-PGDS obtained by transforming *E. coli* is referred to as "mutant L-PGDS" or simply "mutant". Table 2 shows the amino acid sequence of mutant L-PGDS (sequence number 2). For ease of artificial manufacture, glycine-serine (GS) is added to the N-terminus, increasing the number of amino acids by two compared to the case in Table 1. Hereinafter, when specifying the amino acid sequence of mutant L-PGDS, the sequence with glycine-serine (GS) added to the N-terminus of the L-PGDS sequence will be used.

[0040] [Table 2]

[0041]

[0042] The mutant L-PGDS loses its enzyme active site, with the 45th cysteine ​​(C) from the N-terminus (43rd in sequence number 1) replaced by alanine (A). Furthermore, to prevent incorrect disulfide bond formation, the 147th cysteine ​​(C) (145th in sequence number 1) is also replaced by alanine (A). These two instances are enclosed in rectangles in Table 2 (the same applies to the sequences of the capsule proteins below). This mutant L-PGDS is also known as "C45A / C147A".

[0043] Figure 1 The crystal structure of the mutant L-PGDS is shown in the image. Figure 1 (a) The state after rotating 90° is shown in Figure 1 (b). Additionally, L-PGDS also have the same shape. Furthermore, Figure 2 The image shows the amino acid sequence (sequence number 2) of the mutant L-PGDS (C45A / C147A) and its correspondence with the β chain and α helix.

[0044] The mutant L-PGDS has eight β strands from symbol A to H and α helices from symbol H1 to symbol H3. In addition, there are loops between the β strands, and short strand I and short helices H4 and H5 are present.

[0045] The β-chains A through H are arranged in a helical shape, enclosing a space at the center. This is called a barrel structure. In the L-PGDS mutant, the drug is considered to be contained within this barrel structure. Furthermore, refer to... Figure 1 (b) A large opening 10 for the barrel structure is formed between the β chain D and the α helix H2.

[0046] If opening 10 is left open, there is a high probability that even if the drug is initially contained, it will be released before reaching the intended cells. Therefore, a cap is provided at opening 10 (Patent Document 4). Here, a disulfide bond is formed between the ring connecting the β chain E and the β chain F (called the "EF ring") and the α helix H2.

[0047] Refer again Figure 2 The EF ring in the mutant L-PGDS consists of four amino acids, from the 90th proline (P) to the 93rd glycine (G) from the N-terminus. Similarly, the α-helix H2 consists of ten amino acids, from the N-terminus to the 32nd serine (S) to the 41st alanine (A). By replacing one of these with cysteine ​​(C), a disulfide bond is formed. This is called a "disulfide paperclip" or "D-paperclip."

[0048] For example, Figure 3 The modeling structure of the mutant L-PGDS, in which the lysine (38th K from the N-terminus) of the α-helix H2 and the histidine (H) of the EF ring are replaced with cysteine ​​(C), is shown. It is known that a portion of the opening 10 of the disulfide paperclip 12 formed between the cysteine ​​(C) residues is closed. This mutant L-PGDS is called the "D paperclip mutant". Patent Document 4 discloses the D paperclip mutant.

[0049] However, it is known that the disulfide paperclip 12 clamps the end of the opening 10, and the drug contained inside the barrel-shaped structure is released from the gap 14 between the disulfide paperclip 12 and the β chain D. Therefore, in order to block the gap 14 on the β chain D, the present invention includes a blocking amino acid. A blocking amino acid is an amino acid that reduces the area of ​​the opening 10. By including the blocking amino acid, not only is the gap 14 blocked, but the opening 10 can also be effectively narrowed.

[0050] Refer again Figure 2 The β-chain D is the 11-amino acid sequence of the mutant L-PGDS, from the 68th glutamine (Q) to the 78th proline (P) from the N-terminus. Ideally, the blocking amino acid should have a large spatially occupied functional group. This is to block the gap 14 as much as possible. Furthermore, by introducing the blocking amino acid into the β-chain D, the crystal structure of the mutant L-PGDS should not change significantly. This is because a barrel-shaped structure needs to be maintained.

[0051] For example, suitable amino acids used as septum substitutes include lysine (K), histidine (H), tryptophan (W), tyrosine (Y), and phenylalanine (F). Furthermore, there can be more than one substitution. Additionally, the septum amino acid can be inserted into the amino acid sequence that constitutes the β-chain.

[0052] Figure 4 The image shows methionine (M: the 74th from the N-terminus) on the D chain of the β chain. Figure 2 The modeling structure of the L-PGDS mutant with tryptophan (W) replaced by β-chain D is shown in the figure as "74W". This illustrates the state in which gap 14 is blocked by placing tryptophan on the β-chain D. The L-PGDS mutant with this blocking amino acid is referred to as the "blocked mutant".

[0053] In addition, septate mutants may also contain disulfide paperclips. The mutant L-PGDS, which contains both septate amino acids and disulfide paperclips, is called the "septate D paperclip mutant".

[0054] Furthermore, as shown in Patent Document 3, a peptide (marker peptide) that recognizes target cells can be attached to the N-terminus, C-terminus, or both ends. In addition, it can be attached not only to the terminal portion but also partially overlapped. There are no particular limitations on the marker peptide; for example, the peptide sequence NGR that specifically binds to the membrane protein (CD13) expressed in neovascular endothelial cells can be selected. Furthermore, it can be the internalization-Arg-Gly-Asp (iRGD) motif that recognizes αvβ3 and αvβ5 integrins, or the Cys-Arg-Gly-Asp-Lys (CRGDK) motif that recognizes neurofeline 1.

[0055] In addition, the Lys-Leu-Pro (KLP) motif for recognizing peritoneal tumors of gastric cancer (CancerRes, 97, 1075-81, 2006), the Asn-Val-Val-Arg-Gln (NVVRQ) motif for recognizing metastatic cancer cells (Cl inCancer Res, 14, 5494-502, 2008), and the Phe-Gln-His-Pro-Ser-Phe-I le (FQHPSFI) motif for recognizing liver cancer cells (Mol Med, 13, 246-54, 2007) motifs may also be appropriately used.

[0056] The capsule protein of the present invention, to which a marker peptide is attached to a septate mutant, is referred to as a "marked septate mutant". Furthermore, the capsule protein to which a disulfide paperclip is also attached is referred to as a "marked septate D-paperclip mutant". In addition, in order to facilitate manufacturing via gene recombination, in addition to the aforementioned GS, multiple amino acids may be attached to the N-terminus or C-terminus of the capsule protein of the present invention.

[0057] The capsule protein of this invention can house compounds up to approximately 800 Da in a barrel-shaped structure. Here, "compound" refers to a drug or other compound. "Other compounds" here refers to compounds used as supplementary nutrients, or compounds derived from natural sources.

[0058] Furthermore, it is known that in DDS, due to the EPR (Enhanced Permeability and Retention) effect, the selectivity for cancer cell invasion is improved while the retention period is prolonged, and long-term drug effects can be expected. Therefore, the septate mutant of the capsule protein of the present invention can also be used to formulate a multimeric composition. In addition, in order to expect the EPR effect, the overall size can be set to 10 nm or more. Furthermore, a label peptide can also be attached to the multimeric composition.

[0059] When forming a polymeric composition of capsule proteins, a polymeric composition in which multiple capsule proteins are linearly linked together at their C-termini and N-termini is suitable. However, radial linking of the capsule proteins is more preferred. Figure 5 The diagram shows a conceptual structure of the multipolymer composition of the capsule protein used in this invention.

[0060] Figure 5 (a) shows a conceptual diagram of a multimeric composition of capsule proteins (hereinafter referred to as the "multimeric composition"). Figure 5 (b) is a partial exploded view of the polymeric composition 21. The polymeric composition 21 is formed by binding a dimer 36 to a tetramer 32 of streptavidin 30 via biotin 38, wherein the dimer 36 is a dimer of the capsule protein 34 bound together by a linker 35. Thus, the polymeric composition 21 forms an octamer composition of the capsule protein 34.

[0061] Capsule protein 34 can be suitably expressed using either the L-PGDS mutant or a septate mutant. The use of a septate mutant will be explained here.

[0062] If the dissociation constant (Kd) of the binding of biotin 38 and streptavidin 30 is 10 -15 M represents the strongest known non-covalent interaction between a protein and its ligand. Furthermore, this interaction forms very rapidly and is not easily affected by pH, temperature, denaturing agents, or organic solvents. Additionally, because biotin is a low-molecular-weight substance, it does not impede the functionality of the modified molecule. Therefore, it is considered... Figure 5 The octamer shown in (a) exists very stably.

[0063] in addition, Figure 5 In (c), a multimeric composition 22 is shown in which a monomer of capsule protein 34 is bound to a tetramer of streptavidin 30 via biotin 38. This forms a tetramer of capsule protein 34.

[0064] As described later, the octamer of capsule protein 34, in its multimeric composition 21, has a diameter greater than 10 nm, which is larger than that of the tetramer or monomer. Therefore, it can enter cancer cells from blood vessels in cancer cells, which create larger gaps between endothelial cells compared to normal cells, but is less likely to penetrate and leak out of normal cells. Thus, it selectively invades and remains within cancer cells, exerting the so-called EPR effect.

[0065] In the multipolymer composition 21 of the present invention (formed by the formation of an octamer of capsule protein 34), it is believed that the monomers of the capsule protein showed significantly different effects in the in vivo experiments described later, thus exerting an EPR effect.

[0066] Capsule proteins remain soluble even after containing drugs or other compounds. This property remains unchanged as a polymeric composition. Therefore, capsule proteins are suitable for soluble drugs or vitamins. For example, SN-38, vitamins A, D, E, and K, thyroid hormones, steroid hormones, isoflavones, etc., can be suitably used. Furthermore, because they can soluble substances, the solubility threshold in drug development can be lowered.

[0067] The pharmaceutical composition (hereinafter referred to as "pharmaceutical composition") containing a drug in the capsule protein of the present invention can be provided in liquid form. Alternatively, it can be provided as a powder by lyophilization. In the case of lyophilization, the effect is maintained as shown in Patent Document 3.

[0068] Therefore, when the pharmaceutical composition of the present invention is used for treatment, it can be administered orally or non-orally (e.g., intravenous, subcutaneous, or intramuscular injection; local, rectal, transdermal, or nasal). Examples of compositions for oral administration include tablets, capsules, pills, granules, powders, liquids, and suspensions.

[0069] Furthermore, examples of compositions intended for non-oral administration include aqueous or oily formulations for injection, ointments, creams, emulsions, aerosols, suppositories, and adhesives. These formulations are prepared using conventionally known techniques and may contain non-toxic and inert carriers or excipients commonly used in the pharmaceutical industry.

[0070] Furthermore, complexes containing drugs or other compounds within capsule proteins can be provided as processed foods. These processed foods include not only general processed foods such as candy, chewing gum, jelly, biscuits, cookies, rice cakes, bread, noodles, fish and meat products, tea, soft drinks, coffee drinks, milk drinks, whey drinks, lactic acid bacteria drinks, yogurt, ice cream, and pudding—foods for personal enjoyment or health—but also health function foods such as foods for specific health purposes or nutritional functional foods as defined in the Japanese Ministry of Health, Labour and Welfare's Health Function Food Regulations. Processed foods also include nutritional supplements, animal feed, and food additives. Moreover, utilizing the property of making poorly soluble substances soluble, they can also be used as industrial products or industrial raw materials.

[0071] The processed foods of this invention can be prepared by adding encapsulated proteins (complexes) containing other compounds to the raw materials of these processed foods. However, since the encapsulated proteins are septate mutants of proteins encapsulating other compounds, they are easily decomposed by heat. Therefore, it is ideal for the processed foods of this invention to be prepared by a process that does not involve heating after the addition of the complex.

[0072] Example

[0073] Capsule proteins were manufactured as samples.

[0074] (1) Mutant L-PGDS

[0075] (2) The identified mutant L-PGDS

[0076] (3) The D paperclip mutant with septum

[0077] (4) Identified septate D paperclip mutant

[0078] These four.

[0079] The mutant L-PGDS is created by replacing the 45th cysteine ​​(C) from the N-terminus with alanine (A) and the 147th cysteine ​​(C) with alanine (A) in the capsule protein shown in sequence number 2 (“C45A / C147A”). The mutant L-PGDS inactivates the active site; during its creation, the 147th cysteine ​​(C) is replaced with alanine (A) to avoid the formation of incorrect disulfide bonds.

[0080] The identified mutant L-PGDS is formed by attaching an iRGD peptide (CRGDKGPDC: sequence number 3) to the C-terminus of the mutant L-PGDS as a marker, which enables both accumulation in the tumor and penetration of the cell membrane.

[0081] The peptide is attached by overlapping a portion of the C-terminus of the mutant L-PGDS. This is to eliminate antigenicity in mice in subsequent in vivo experiments. The amino acid sequence of the identified mutant L-PGDS is shown in Table 4 as sequence number 4. The peptide is indicated by "■". Additionally, Figure 6 The modeling structure of the mutant L-PGDS, identified as the mutant L-PGDS, is shown in Table 3.

[0082]

[0083] [Table 4]

[0084]

[0085] In the D-paperclip mutant with a septum, the first septum amino acid, methionine (M), is replaced by tryptophan (W) on the D chain of the β chain. Methionine is the 74th amino acid from the N-terminus in the L-PGDS mutant.

[0086] The D-paperclip mutant with septum is obtained by further adding D-paperclips. The D-paperclip is formed by replacing the 38th lysine (K) from the N-terminus of the L-PGDS mutant with cysteine ​​(C) and the 91st histidine (H) with cysteine ​​(C). The amino acid sequence is shown in Table 5 (Sequence Number 5). In Table 5, septum amino acids are indicated by "▲", and D-paperclips (disulfide bonds) are indicated by "★". A disulfide bond is formed between the two cysteine ​​(C) residues indicated by "★". Furthermore, the disulfide bond is separated under reducing conditions, and opening 10 can be opened or closed by the D-paperclip.

[0087] [Table 5]

[0088]

[0089] The identified septate-bound D-paperclip mutant was formed by overlapping an iRGD peptide (CRGDKGPDC) at the C-terminus of the septate-bound D-paperclip mutant (sequence number 5). In Table 6, the septate amino acid is indicated by "▲", and the D-paperclip (disulfide bond) is indicated by "★". Furthermore, the peptide marker is indicated by "■". Figure 7 This is a diagram representing the modeling structure of the D-paperclip mutant identified as a septate D-paperclip mutant.

[0090] [Table 6]

[0091]

[0092] After the designed expression plasmids of each capsule protein were created using the superprimer method, they were transformed into Escherichia coli BL21(DE3) strain to express them as fusion proteins with glutathione S-transferase.

[0093] The protein expression strain was cultured in LB / Amp test tubes at 37°C with shaking for 8 hours, followed by subculture in 2×YT / Amp auto-induction medium at 37°C with shaking for 16 hours. The resulting bacterial cells were ultrasonically homogenized, and the supernatant of the homogenate was fed onto a glutathione-Sepharose 4B column for affinity chromatography.

[0094] The fusion protein adsorbed on the column was reacted with 165 units of thrombin overnight, the target protein was eluted, and the protein was purified.

[0095] Next, these capsule proteins are infused with SN-38. SN-38 is the abbreviation for 7-ethyl-10-hydroxycamptothecin, a poorly soluble water-soluble anticancer agent. It is known to exhibit high antitumor efficacy at lower doses compared to irinotecan hydrochloride, currently used clinically as a prodrug of SN-38.

[0096] <Drug release capacity>

[0097] For 5 mL each of SN-38 at a concentration of 50 μM, SN-38 / mutant L-PGDS with capsule protein concentration adjusted to 50 μM, SN-38 / septate D-paperclip mutant, and SN-38 / labeled septate D-paperclip mutant, dialysis was performed using a dialysis membrane (Size 27 Wako molecular weight cutoff: 14,000) with 150 mL of PBS as the external solution in a constant temperature incubator at 37°C for 72 hours.

[0098] Sampling was performed at 0, 1, 3, 6, 8, 12, 24, 36, 48, 60, and 72 hours after the start of dialysis. An equal volume of PBS was added to replace the 500 μL of the external dialysis fluid. The concentration of SN-38 in the sampled external dialysis fluid was determined using a spectrophotometer F-7000 (HITACHI) (excitation wavelength: 365 nm, measurement wavelength: 380-600 nm, measurement temperature: 37 °C).

[0099] Next, the drug release function of the septated D-paperclip mutant and the labeled septated D-paperclip mutant in a reducing environment was evaluated using the same balanced dialysis method as described above. As the external solution, PBS and 10 mM DTT / PBS were used for dialysis; PBS was used as the oxidizing environment, and DTT was added as the reducing environment.

[0100] The results of plotting the change in SN-38 concentration in the dialysate over time relative to dialysis time are shown below. Figure 8 The horizontal axis represents reaction time (hours), and the vertical axis represents SN-38 concentration (nM). Error bars are expressed as mean ± standard deviation (n=3). Furthermore, in the figures, "SN-38 / L-PGDS ("●")" indicates SN-38 / mutant L-PGDS, "SN-38 / Capsule (▲)" indicates SN-38 / septate D-paperclip mutant, and "SN-38 / Cap-sCRGDK (■)" indicates SN-38 / identified septate D-paperclip mutant. Additionally, in "SN-38 / Capsule" and "SN-38 / Cap-sCRGDK", "+10mM DTT ("△" and "□")" refers to the addition of 10mM DTT to the respective pharmaceutical composition solution. The addition of DTT (Dithiothreitol) creates a strongly reducing environment in the solution.

[0101] Reference Figure 8 With PBS as the external solution, the SN-38 concentration in the external solution of the septated D-clips mutant (SN-38 / Capsule) and the labeled septated D-clips mutant (SN-38 / Cap-sCRGDK) was significantly lower than that of the mutant L-PGDS (SN-38 / L-PGDS) throughout all time from the start of dialysis. This indicates that the release of SN-38 from SN-38 / septated D-clips mutant and SN-38 / labeled septated D-clips mutant was inhibited compared to the release from SN-38 / mutant L-PGDS.

[0102] On the other hand, when the external solution was DTT / PBS, the concentration of SN-38 in the external solution increased compared to the case of PBS, for the septated D-clips mutant (SN-38 / Capsule: "△") and the identified septated D-clips mutant (SN-38 / Cap-sCRGDK: "□").

[0103] The above results show that, compared to the L-PGDS mutant, the septated D-paperclip mutant and the labeled septated D-paperclip mutant retain SN-38 for a longer period in an oxidizing environment and release SN-38 in response to a reducing environment. Furthermore, it was shown that there was no difference in drug release patterns between the septated D-paperclip mutant and the labeled septated D-paperclip mutant; therefore, the addition of the label peptide has no effect on controlling drug release.

[0104] Next, the same experiment was performed on the septate mutant (M74W) without the D paperclip and the mutant L-PGDS. The mutant L-PGDS was then subjected to... Figure 8 The experiment was repeated under the same conditions. The results are shown in... Figure 9 . Figure 9 (a) is the case of the mutant L-PGDS. Figure 9 (b) is the case for the septate mutant (M74W). See reference... Figure 9 (a) and Figure 9 (b) The horizontal axis represents the reaction time (hours), and the vertical axis represents the SN-38 concentration (μM).

[0105] Reference Figure 9 (a) The reaction time of SN-38 with respect to the external solution being PBS (black circle "●") and... Figure 8 The situation is almost the same, so it can be known that Figure 8 The experiment was well reproduced. In the mutant L-PGDS, with DTT / PBS as the external solution (white circle "○"), the concentration of SN-38 reached 2.0 μM within a short reaction time, and it was observed to have almost reached the stationary phase.

[0106] In contrast, refer to Figure 9 (b) When the external solution was PBS (black circle "●"), the concentration of SN-38 did not exceed 1.0 μM even after the reaction time, which is consistent with... Figure 8 Compared to the L-PGDS mutant, the D-paperclip mutant with septum, or the identified D-paperclip mutant with septum, the release of SN-38 was suppressed.

[0107] On the other hand, with the external solution being DTT / PBS (white circle "○"), the SN-38 concentration reached almost 2.0 μM within 48 hours of dialysis. This value is comparable to... Figure 8The septated D-paperclip mutant and the labeled septated D-paperclip mutant were shown at almost the same concentration. In summary, it is evident that the septated mutant exhibits superior drug retention compared to the septated D-paperclip mutant or the labeled septated D-paperclip mutant. Furthermore, it was confirmed that under a reducing atmosphere, the septated mutant can release the contained drug almost in the same manner as other capsule proteins.

[0108] Next, the same experiment was conducted by changing the drug from SN-38 to dipyridamole, a poorly water-soluble antianginal drug. The results are shown below. Figure 10 Additionally, the external solution is PBS. (See reference...) Figure 10 The horizontal axis represents reaction time (hours), and the left vertical axis represents dipyridamole concentration (nM). Additionally, the right vertical axis is a conversion of the left vertical axis to release rate (%). In the L-PGDS mutant, dipyridamole concentration increased with reaction time; conversely, in the septate mutant (M74W), dipyridamole concentration only increased to near the detection limit.

[0109] This demonstrates that septate mutants possess excellent drug retention capabilities, maintaining almost no leakage depending on the drug. Thus, it can be said that the contained drug is continuously retained within the body until a reducing atmosphere is achieved within the cell, making it highly efficient as a DDS (Drug Dispenser).

[0110] <Internal Effects>

[0111] Four-week-old male BALB / c-nu / nu mice (Japanese SLC) were allowed free access to water and food for one week in an animal room with controlled room temperature and a 12-hour light-dark cycle to allow them to acclimatize. Afterwards, 100 μL of 5×10⁻⁶ solution was administered subcutaneously to the right abdomen. 7 A mouse model of prostate cancer was created using human prostate cancer cells PC-3 at a concentration of 1 cell / mL (PBS:Matrix gel = 1:1).

[0112] The tumor volume (approximate formula: calculated using {(major diameter) × (minor diameter)²} / 2) reached 250 mm. 3 On day 0 of the dosing regimen, mice were randomly assigned to one of the following groups: PBS, SN-38 / mutant L-PGDS (2.0 mg / kg / d), SN-38 / labeled mutant L-PGDS (2.0 mg / kg / d), SN-38 / septate D-paperclip mutant (2.0 mg / kg / d), and SN-38 / labeled septate D-paperclip mutant (2.0 mg / kg / d). Each sample was administered via tail vein every other day for a total of 8 times. The control group received only PBS.

[0113] Figure 11The results of in vivo antitumor experiments are shown. The horizontal axis represents the number of days elapsed from day 0 of drug administration, and the vertical axis represents the tumor volume (mm²). 3 The abbreviations in the chart are as follows.

[0114] PBS: Control group

[0115] SN-38 / L-PGDS: SN-38 / mutant L-PGDS

[0116] SN-38 / L-PGDS-sCRGDK: SN-38 / identified mutant L-PGDS

[0117] SN-38 / Capsule: SN-38 / D paperclip mutant with septum

[0118] SN-38 / Cap-sCRGDK: Identified septate D-paperclip mutant

[0119] In the PBS administration group, no antitumor effect could be confirmed, and tumor volume continued to increase from the start of administration. In contrast, significant tumor growth inhibition was confirmed in the SN-38 / L-PGDS, SN-38 / L-PGDS-sCRGDK, SN-38 / Capsule, and SN-38 / Cap-sCRGDK administration groups.

[0120] Furthermore, no significant tumor growth inhibition was observed in the SN-38 / Capsule and SN-38 / L-PGDS-sCRGDK administration groups compared to the SN-38 / L-PGDS administration group. Therefore, it was concluded that adding only either targeting or release control function did not result in significantly higher antitumor activity compared to the mutant L-PGDS.

[0121] On the other hand, compared with SN-38 / L-PGDS (the mutant L-PGDS), SN-38 / Cap-sCRGDK (the labeled septate D-paperclip mutant) showed significantly higher antitumor activity. These results indicate that the synergistic effect of two functions—drug release control (septate amino acids and D-paperclip) and cancer targeting (label peptide)—can significantly inhibit tumor growth.

[0122] <Merizoid>

[0123] Next, the manufacture of the multimeric composition (octamer composition) of the capsule protein will be described. For example... Figure 5 As described, the octamer composition is formed by binding a dimer 36 to a tetramer 32 of streptavidin 30 via biotin 38, wherein the dimer 36 is a dimer formed by binding of capsule protein 34 via a linker 35.

[0124] Therefore, after generating a biotinylated dimer composition by binding biotin to the dimer of the capsule protein, it is combined with a separately generated tetramer of streptavidin to obtain an octamer composition of the capsule protein. Furthermore, the linker is encoded by the base sequence shown in Table 7 (Sequence Number 7). Additionally, streptavidin is encoded by the base sequence shown in Table 8 (Sequence Number 8).

[0125] [Table 7]

[0126]

[0127] [Table 8]

[0128]

[0129] <Preparation of Dimeric L-PGDS Expression Vectors>

[0130] For the gene sequence of the septate mutant, PCR amplification was performed using a forward primer containing a Bam HI recognition site and a reverse primer containing an Eco RI recognition site and a linker sequence (GGGGS: sequence number 7), followed by agarose gel electrophoresis. Sequences treated with restriction enzymes using Bam HI and Eco RI were considered as septate mutant insertions.

[0131] In addition, in the septate mutant, the 45th cysteine ​​(C) from the N-terminus is replaced with alanine (A), and the 147th cysteine ​​(C) is replaced with alanine (A) (“C45A / C147A”). Furthermore, the septate amino acid is formed by replacing methionine (M) on the D chain of the β chain with tryptophan (W) (“M74W”). Methionine is the 74th amino acid from the N-terminus in the L-PGDS mutant. The amino acid sequence of the septate mutant (Sequence Number 9) is shown in Table 9.

[0132] [Table 9]

[0133]

[0134] Similarly, a restriction enzyme-treated plasmid (pGEX4T-2) was prepared. The septate mutant insertion and the restriction enzyme-treated pGEX4T-2 were subjected to agarose gel electrophoresis.

[0135] After ethidium bromide staining, DNA was extracted from the gel using an agarose gel extraction kit (Jene Bioscience) and then ligated. In this procedure, the base sequence encoding the septate mutant and the ligation sequence was inserted into the plasmid pGEX4T-2. This plasmid is called the septate mutant expression vector.

[0136] The obtained septate mutant expression vector was used to transform *E. coli* DH5α(DE3) strain, and the transformed strain was inoculated into LB / Amp agar plates. Colonies grown in the agar plates were subjected to direct colony PCR. Colonies confirming the presence of the septate mutant were inoculated into 5 mL LB / Amp test tubes and incubated at 37°C for 16 hours. Subsequently, the septate mutant expression vector was purified using minipreparative chromatography (SV Minipreps, Promega).

[0137] Sequencing of the obtained septate mutant expression vector confirmed that the base sequence encoding the septate mutant and the linker was inserted.

[0138] In this septate mutant expression vector, primers containing Eco RI and SalI recognition sites were used to integrate the amplified septate mutant insertion using the same procedure. Subsequently, the vector was transformed into *E. coli* DH5α(DE3) strain using the same procedure, cultured, amplified, and the plasmid was purified. This plasmid contains the base sequence encoding the septate mutant dimer. Therefore, this plasmid is called the dimer septate mutant expression vector. The obtained dimer septate mutant expression vector was sequenced to confirm the base sequence.

[0139] <Preparation of modified dimer L-PGDS expression vector>

[0140] Next, 15 amino acid residues specifically recognized by the biotin ligase (BirA) are encoded (Avitag... TM The peptide Av (hereinafter referred to as "Av") was annealed using complementary oligoDNAs of sequences 10 and 11. Biotin ligase binds to biotin at the lysine residues of peptide Av. The base sequences are shown in Tables 10 and 11, respectively. The annealed product was then purified using a FastGene Gel / PCR extraction kit (Nippon Genetics, Tokyo). This base sequence is referred to as the Av base sequence.

[0141] [Table 10]

[0142]

[0143] [Table 11]

[0144]

[0145] The dimer-segmented mutant expression vector was cleaved with Bam HI restriction enzyme (37°C, 3 hours) and then subjected to agarose gel electrophoresis. Subsequently, DNA was extracted from the dimer-segmented mutant expression vector, and an Av base sequence was inserted at the cleavage site using the In Fusion HD cloning kit (Clontech). The vector resulting from the insertion of the Av base sequence into the dimer-segmented mutant expression vector is called a modified dimer-segmented mutant expression vector. The resulting vector was sequenced to confirm the insertion of the target sequence.

[0146] <Mixed mutant expression strain with septate dimer>

[0147] The *E. coli* strain AVB101, expressing BirA, was transformed using a modified dimeric mutant expression vector to obtain a dimeric mutant expression line. The dimeric mutant expression line was inoculated into 5 ml of LB / Amp / Chl liquid medium and cultured overnight at 37°C with shaking. Subsequently, it was passaged in 1 L of 2×YT / Amp / Chl medium and cultured at 37°C.

[0148] In addition, the E. coli strain AVB101 expressing BirA is an E. coli strain injected with an expression vector containing a base sequence encoding BirA.

[0149] <Generation of biotinylated dimer septate mutants>

[0150] At the point when the OD600 value of the culture medium of the dimer-segmented mutant expression strain reached 0.6–1.0, IPTG (final concentration 0.1 mM) and biotin (final concentration 50 μM) were added. This procedure resulted in the expression of the modified dimer-segmented mutant and the BirA protein in *E. coli*. BirA then induced biotin binding to the additional peptide Av in the dimer-segmented mutant. This induced biotinylation of the dimer-segmented mutant.

[0151] Afterwards, the culture was incubated at 37°C for 6 hours, and the culture medium was centrifuged (8, 400×g, 10 minutes, 4°C) to recover the bacterial cells. Then, the bacterial cells were washed with PBS, collected by centrifugation, and the bacterial cells were ultrasonically pulverized.

[0152] An affinity column containing 15 ml of glutathione-agarose 4B (GE Helthcare Bio Science, UK) was equilibrated with 5 times the column volume of PBS that had passed through a 0.22 μm filter, providing a pulverized supernatant that had passed through a 0.22 μm filter.

[0153] After washing with 3 column volumes of 1% Triton X-100 / PBS and 5 column volumes of PBS, 165 units of thrombin (SIGMA) were added and stirred thoroughly. The mixture was then allowed to stand at room temperature for at least 12 hours. Elution was performed with 5 column volumes of PBS. The mixture was then concentrated using a concentrator with a molecular weight cutoff of 10 kDa and centrifuged repeatedly (8400 g, 20 min, 4 °C) to a final volume of 4 ml.

[0154] Next, the solution was degassed for 10 minutes with 5 mM Tris-HCl (pH 8.0) passed through a 0.22 μm filter, and then equilibrated with twice the volume of the same buffer using Superdex 75 16 / 600 (GE Healthcare Biosciences, UK). The concentrate was then added to Superdex 75 16 / 600 through a 0.22 μm filter. While monitoring the absorbance at 280 nm UV wavelength, the eluent was collected at a flow rate of 0.5 ml / min, with each eluent being reduced to 1.5 ml. The peak corresponding to the biotinylated dimer L-PGDS was recovered.

[0155] The recovered portions were combined, dialyzed with 20 mM sodium acetate buffer (pH 5.5), and then centrifuged repeatedly (8400 g, 20 min, 4 °C) using a concentrator with a molecular weight cutoff of 10 kDa to concentrate to 4 ml.

[0156] It was fed into a column packed with SP agarose gel FF (SP sepharose Fast Flow) (GE Healthcare Biosciences, UK) and subjected to cation exchange chromatography using a linear gradient method with 20 mM sodium acetate buffer (pH 5.5) → 1 M NaCl / 20 mM sodium acetate buffer (pH 5.5).

[0157] While monitoring the absorbance at 280 nm ultraviolet wavelength, the eluent was collected in portions of 1.5 ml each at a flow rate of 1.0 ml / min. SDS-PAGE analysis was then performed to recover portions of single bands from mutants confirmed to be biotinylated dimer bands.

[0158] <Purification of Streptavidin>

[0159] Escherichia coli BL21(DE3) strain was transformed with the streptavidin expression vector (pET21a-Streptavidin-Alive, Addgene) to obtain a streptavidin-expressing strain. The streptavidin-expressing strain was inoculated into 5 ml of LB / Amp liquid medium and cultured overnight at 37°C with shaking. Then, it was passaged in 1 L of 2×YT / Amp medium and cultured at 37°C. Subsequently, when the OD600 value reached 0.6-1.0, IPTG was added to achieve a final concentration of 0.1 mM to induce expression. After culturing at 18°C ​​for 24 hours, the culture was centrifuged (8400×g, 10 min, 4°C) to recover the bacterial cells.

[0160] Add PBS to the obtained bacterial cells to suspend them. Add 1 μl of 100 mg / ml lysozyme to each 1 g of bacterial cells and stir in ice water. Then, while stirring in ice, sonicate the bacterial cells (1 minute sonication, 2 minutes rest, 7 sets). Separate the pulverized liquid by centrifugation (4℃, 15000 rpm) and further pulverization to obtain the supernatant.

[0161] A 10 mL aliquot of the Ni agarose (GE Helthcare Bio Science, UK) affinity column was equilibrated with five times the column volume of 20 mM imidazole / 20 mM sodium phosphate buffer (pH 7.0) filtered through a 0.22 μm filter, providing a fragmented supernatant. The column was washed with 20 mM sodium phosphate buffer (pH 7.0) at various imidazole concentrations (20, 50, 100 mM), followed by elution with 300 mM imidazole / sodium phosphate buffer (pH 7.0). The column was then concentrated to 4 mL by repeated centrifugation (8400 g, 20 min, 4 °C) using a concentrator with a 10 kDa molecular weight cutoff.

[0162] Next, the PBS (pH 7.4) passed through a 0.22 μm filter was degassed for 10 minutes, then equilibrated with twice the volume of the same buffer using a Superdex 75 16 / 600. The concentrate was then added to the Superdex 75 16 / 600 through a 0.22 μm filter. While monitoring the absorbance at 280 nm UV wavelength, the eluent was collected at a flow rate of 0.5 mL / min, with each eluent being 1.5 mL. Non-reducing SDS-PAGE analysis yielded streptavidin tetramer.

[0163] <Preparation of Octamer Compositions>

[0164] The purified dimeric mutant with septum (in PBS, pH 7.4) and streptavidin tetramer (in PBS, pH 7.4) were mixed at a molar ratio of 4:1 and allowed to stand at room temperature for 15 minutes. Then, the mixture was repeatedly centrifuged (8400 g, 20 min, 4 °C) using a concentrator with a molecular weight cutoff of 50 kDa to concentrate the solution to 4 ml.

[0165] Next, PBS (pH 7.4) passed through a 0.22 μm filter was degassed for 10 minutes, then equilibrated with twice the volume of the same buffer using Superdex 200 16 / 600 (GE Healthcare Biosciences, UK). The concentrate was then added to the Superdex 200 16 / 600 through a 0.22 μm filter. While monitoring the absorbance at 280 nm UV wavelength, the eluent was collected at a flow rate of 0.5 ml / min, resulting in 1.5 ml eluents. SDS-PAGE analysis yielded the octamer composition.

[0166] In addition, monomers of the septate-bound mutant were obtained by directly introducing the septate-bound mutant expression vector into *E. coli* before preparing the dimeric septate-bound mutant expression vector. Furthermore, monomers of the septate-bound mutant with the iRGD peptide appended were also prepared. The amino acid sequences of the labeled septate-bound mutants (Sequence Number 12) are shown in Table 12. The method for appending the iRGD peptide was the same as in Sequence Number 6.

[0167] [Table 12]

[0168]

[0169] In addition, transforming the BirA-expressing Escherichia coli strain AVB101 with a monoclonal mutant expression vector modified by inserting an Av base sequence into the mutant expression vector with septum can also yield a tetramer composition of four monomers bound by streptavidin.

[0170] <Size of the octamer composition>

[0171] <Size of DLS-based octamer compositions>

[0172] <SAXS-based octamer size>

[0173] The size of the octamer composition was determined using small-angle X-ray scattering (SAXS). Additionally, for comparison, the size of the mutant L-PGDS was also determined.

[0174] SAXS measurements were performed at Beamline BL40B2, a large-scale radiation facility at SPring-8 (Sagao District, Hyogo Prefecture, Japan). The X-ray wavelength was adjusted to 1.000 Å, the camera length to 2.193 m, and the experiments were conducted at 25°C. Each measurement involved an exposure of 20–50 seconds, and the scattered light was detected using a PILATUS-2M (Ricoh Corporation, Tokyo).

[0175] To maximize X-ray scattering, a 3.0 mm thick sample cell was used, with a 0.02 mm quartz slab window. To avoid measurement errors, protein samples and buffer were measured alternately. 25 μL of sample was placed in the cell for measurement, followed by removal of the sample and washing three times with buffer. Subsequent measurements were then performed.

[0176] Ring averaging converts the scattering patterns of the protein sample and buffer, recorded in two dimensions in the detector, into one-dimensional data, and then subtracts the buffer data from the protein sample data. Scattering curves in the small-angle region are analyzed using the Guiner approximation for monodisperse systems. The scattering intensity I(S, C) is a function of the scattering vector S and the protein concentration C, and can be expressed using the origin scattering intensity I(0, C) and the radius of inertia R. g (C) (radius of gyration) is expressed in the form of equation (1).

[0177] [Mathematical Expression 1]

[0178]

[0179] in addition,

[0180] Furthermore, 2θ here is the scattering angle, and λ represents the wavelength of the X-ray.

[0181] The radius of inertia R was calculated from the Guinier region of the obtained scattering curve. g The radius of inertia is 1.8 ± 0.04 nm in the monomer (mutant L-PGDS) and approximately three times that in the octamer composition, at 6.0 ± 0.69 nm. Therefore, the radius of inertia R... g The molecular weight increases through octamerization. The results of each measurement are shown in Table 13. Furthermore, the molecular weight (Mw) calculated from the scattering curves... exp In both monomeric (mutant L-PGDS) and octamer compositions, the molecular weight is consistent with the theoretical value (Mw). calcThe values ​​are close to those of the monomer (mutant L-PGDS), so it is determined that octamerization of the septum mutant, which is considered to be almost the same size as the monomer (mutant L-PGDS), has indeed been performed. Thus, due to the sufficiently large particle shape of more than 10 nm, tumor accumulation based on EPR effect of the octamer composition can be expected.

[0182] [Table 13]

[0183] <![CDATA[R g (nm)]]> <![CDATA[D max (nm)]]> <![CDATA[Mw exp (kDa)]]> <![CDATA[Mw calc (kDa)]]> monomer 1.8±0.04 4.75 16 19 Octamer Composition 6.0±0.69 22.4 231 217

[0184] <Drug Contents>

[0185] Next, the monomer, the septate mutant, the labeled septate mutant, and the octamer were combined to contain SN-38. As already stated, SN-38 is the abbreviation for the water-poorly soluble anticancer agent 7-ethyl-10-hydroxycamptothecin. It is known to exhibit high antitumor efficacy at lower doses compared to irinotecan hydrochloride, currently used clinically as a prodrug of SN-38.

[0186] To a PBS suspension of SN-38 incubated at 37°C, PBS solutions of monomers (mutant L-PGDS), septated mutants, labeled septated mutants, or octamer combinations were added to achieve final concentrations of 1 μM, 0.25 μM, and 0.125 μM, respectively. The mixture was stirred at 37°C for 6 hours. After stirring, free SN-38 was removed by ultrafiltration to prepare samples containing the drug in encapsulated proteins.

[0187] <Internal Effects>

[0188] Four-week-old male BALB / c-nu / nu mice (Japanese SLC) were allowed free access to water and food for one week in an animal room with controlled room temperature and a 12-hour light-dark cycle to allow them to acclimatize. Afterwards, 100 μL of 5×10⁻⁶ solution was administered subcutaneously to the right abdomen. 7 A mouse model of prostate cancer was created using human prostate cancer cells PC-3 at a concentration of 1 cell / mL (PBS:Matrix gel = 1:1).

[0189] Tumor volume (approximate formula: {(major axis) × (minor axis)}) 2 (Calculated by} / 2) reaches 250mm 3Day 0 of the administration was designated as day 0. Mice were randomly assigned to different treatment groups: PBS, monomer (2.0 mg SN-38 / kg / d), septate mutant (2.0 mg SN-38 / kg / d), labeled septate mutant (2.0 mg SN-38 / kg / d), and octamer combination (2.0 mg SN-38 / kg / d). The monomer, septate mutant, labeled septate mutant, and octamer combination were administered via tail vein every 4 days for a total of 4 times. In the control group, PBS was administered only every 4 days for a total of 4 times.

[0190] Figure 12 The results of in vivo antitumor experiments are shown. The horizontal axis represents the number of days elapsed from day 0 of drug administration, and the vertical axis represents the tumor volume (mm²). 3 The abbreviations in the chart are as follows.

[0191] PBS: Control group

[0192] SN-38 / L-PGDS: Monomer (SN-38 / mutant L-PGDS)

[0193] SN-38 / M74W: SN-38 / septate mutant

[0194] SN-38 / M74W-sCRGDK: SN-38 / identified septate mutant

[0195] SN-38 / M74W-octamer: An octamer composition of SN-38 / septate mutants

[0196] and Figure 11 Compared to the previous cases, none of them included a D-shaped paperclip.

[0197] In addition, the septate mutant was created by adding the Cys-Arg-Gly-Asp-Lys (CRGDK) motif that recognizes neurofeline 1 to the C-terminus of the septate mutant (identifier number 12).

[0198] Reference Figure 12 In the PBS-treated group, no antitumor effect could be confirmed, and tumor volume continued to increase from the start of administration. In contrast, the SN-38 / L-PGDS-treated group showed an effect of inhibiting tumor growth. Moreover, the SN-38 / M74W mutant with septum further inhibited tumor growth.

[0199] On the other hand, surprisingly, from the start of the experiment, no tumor growth was observed in either SN-38 / M74W-sCRGDK (the identified septate mutant (without the D paperclip)) or SN-38 / M74W-octamer (an octamer combination of septate mutants). It is believed that SN-38 is a drug that inhibits cancer cell proliferation rather than inducing apoptosis; therefore, it can be said that the effects of SN-38 were fully realized.

[0200] Furthermore, despite administering the drug a total of four times every four days, tumor growth was temporarily and completely suppressed even after the 15th day without drug administration. Therefore, the drug remained within the cellular tissues and was not excreted by the lymphatic system, confirming the long-term efficacy of EPR.

[0201] Figure 13 Indicates to proceed Figure 12 The graph shows the average body weight of the mice at the start of the experiment. The horizontal axis represents the number of days elapsed since day 0 of drug administration, and the vertical axis represents the percentage of body weight relative to the mice's initial body weight at the start of drug administration. The values ​​represent the average for each drug administration group (n=5). Abbreviations in the graph are not included. Figure 12 The situation is the same. (Excluding...) Figure 12 The PBS-treated group served as the control group.

[0202] If reference Figure 13 The septate mutant (SN-38 / M74W), which showed very effective inhibition of cancer cell growth, showed signs of weight loss below 80% on day 20, and the experiment was discontinued. Considering that the newly inactivated L-PGDS (SN-38 / L-PGDS), although its weight decreased, did not drop below 80%, it is believed that although the septate mutant is excellent in maintaining SN-38 (the drug), it produces side effects because SN-38 is also released in normal cells.

[0203] On the other hand, in the septate mutant (SN-38 / M74W-sCRGDK) and the octamer of the septate mutant (SN-38 / M74W-octamer), which showed significant effects in inhibiting cancer cell proliferation, body weight was slightly reduced, and even tended to increase 15 days after SN-38 administration was stopped.

[0204] Therefore, it is believed that the labeled septate mutant (SN-38 / M74W-sCRGDK) and the octamer of the septate mutant (SN-38 / M74W-octamer) specifically recognize cancer cells and release drugs within the cancer cells.

[0205] Furthermore, by creating polymers to achieve the EPR effect, drugs can be delivered regardless of the type of cancer. In particular, for metastatic cancer, regardless of the site of metastasis, drugs can be selectively injected only into cancer cells without affecting normal cells, which is considered extremely useful.

[0206] Industrial applications

[0207] This invention encapsulates poorly soluble drugs or other compounds into soluble forms. Upon ingestion into cells, the disulfide bonds break under the reducing environment within the cells (the intracellular concentration of reduced glutathione is approximately 0.5-10 mM, about 100-1000 times higher than the extracellular concentration), releasing the encapsulated drug or other compound. Therefore, it can be suitable for use as a DDS capsule for poorly soluble compounds. Furthermore, utilizing its property of making poorly soluble substances soluble, it can be used as an industrial product or industrial raw material.

[0208] Symbol Explanation

[0209] 10 (barrel-shaped structure) openings

[0210] 12 Disulfide paperclips

[0211] 14 gaps

[0212] 21. Polymer Compositions (Octamer Compositions)

[0213] 22. Polymer compositions (tetramer compositions)

[0214] 30 Streptavidin

[0215] 32 Tetramer

[0216] 35 Connecting base

[0217] 34 capsules of protein

[0218] 36 Dimer

[0219] 38 Biotin sequence list <110> Public University Corporation Osaka <120> Capsule proteins and their polymeric compositions, and pharmaceutical compositions using the 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 mutants <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> The D-paperclip mutant with septum <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-paperclip mutant with septum <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 avidinii <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> Partitioned Mutant <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 justice 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 septate mutants <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

Claims

1. A capsule protein, characterized in that, Both of the cysteines at positions 45 and 147 from the N-terminus of human lipocalin-type prostaglandin D synthase are substituted with alanine, and the methionine (M) at position 74 from the N-terminus of the beta strand D is substituted with tryptophan (W).

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

3. The capsule protein of claim 1 or 2, wherein An identification peptide is bound to the N-terminus or C-terminus of the capsule protein.

4. A multimeric composition of a capsule protein, characterized in that, A plurality of the capsule proteins are bound, wherein the capsule proteins are the capsule proteins according to any one of claims 1 to 3.

5. The multimeric composition of capsule proteins of claim 4, wherein, The multimeric composition is a tetramer or an octamer.

6. The multimeric composition of capsule proteins of claim 5, wherein, In the multimeric composition, the capsule proteins are bound via a tetramer of biotin and streptavidin.

7. A pharmaceutical composition comprising a compound of the formula: ###0002### or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. The capsule protein according to any one of claims 1 to 6 contains a drug.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition is lyophilized.

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