Drug loading technology using papilloma virus-derived virus-like particles

Abstract

The present invention relates to a drug loading technology using virus-like particles derived from papilloma virus, and by identifying a method to effectively load drugs onto the outside (surface) or inside of virus-like particles (L1 protein) derived from the virus, it can be utilized in the form of a delivery vehicle, therapeutic agent, vaccine, etc., for delivering various drugs such as anticancer agents, proteins, and peptides.

Description

Technology Field

[0001] The present invention relates to a drug loading technology using virus-like particles derived from papilloma virus. Background Technology

[0002] The term "virus-like particles" (VLPs) was established with the advent of the electron microscope. Since the 1950s, when the electron microscope was introduced, spherical, virus-like substances observed in various human tissues began to be named as virus-like particles. In recent years, VLPs have come to refer to forms of virus-derived proteins produced through genetic recombination technology and are generally recognized as multi-binding complexes of virus-derived proteins. VLPs have been developed by removing virus-derived genes responsible for viral pathogenicity and incorporating a capsid or envelope necessary to provide protective immunity or the ability to penetrate cells; they are primarily developed and utilized as vaccines. Structurally, VLPs are very similar to native viruses, allowing them to safely and effectively induce neutralizing antibodies against viruses. Furthermore, VLPs are attracting attention for their potential as vectors due to their characteristic ability to penetrate cells. However, verifying the capabilities of VLPs as vectors requires large quantities, so experimental validation remains insufficient.

[0003] Virus-like particles (VLPs) of the human papillomavirus (HPV) and the hepatitis B virus (HPV) are representative examples of vaccines that have been developed and commercialized. In the case of HPV VLPs, the L1 protein produced through genetic recombination can self-assemble to form a viral capsid. The surface of these VLPs features repeating antigenic sequences (referring to the repetition of amino acid sequences), and this unique repeating structure can highly effectively stimulate immune cells, enabling the development of a superior vaccine.

[0004] Native virions contain viral genes inside the viral capsid, whereas virus-like particles are hollow. Viruses inherently possess structural characteristics that allow them to easily enter cells. Therefore, if the hollow space of virus-like particles is utilized for drug loading, they have high utility value as delivery vehicles. Prior art literature

[0005] Yeast-Based Virus-like Particles as an Emerging Platform for Vaccine Development and Delivery(released on 2023.02.18.)Yeast as an expression system for producing virus-like particles: what factors do we need to consider?(released on 2017.02.01.) The problem to be solved

[0006] The object of the present invention is to provide a composition for delivering a drug or a physiologically active substance comprising, as an active ingredient, a complex comprising a virus-like particle derived from a human papilloma virus and a drug or a physiologically active substance.

[0007] Another objective of the present invention is to provide a composition for delivering a drug or a physiologically active substance, comprising as an active ingredient a complex having a drug or a physiologically active substance loaded on the outside of a virus-like particle, wherein the composition comprises a biotin-labeled virus-like particle; streptavidin that binds to the virus-like particle; and a drug or a physiologically active substance bound to biotin that binds to the streptavidin.

[0008] Another objective of the present invention is to provide a composition for delivering a drug or physiologically active substance, comprising as an active ingredient a complex containing a drug or physiologically active substance inside a virus-like particle, wherein the virus-like particle is disassembled in a buffer containing β-mercaptoethanol, dialyzed with the drug or physiologically active ingredient, and reassembled.

[0009] Another objective of the present invention is to provide a composition for delivering a drug or physiologically active substance, comprising as an active ingredient a complex having a drug or physiologically active substance loaded inside and outside a virus-like particle, wherein the virus-like particle is disassembled in a buffer containing β-mercaptoethanol, dialyzed with the drug or physiologically active ingredient, and reassembled to include a virus-like particle having a drug or physiologically active substance loaded inside, and wherein the virus-like particle is biotinylated on the outside of the virus-like particle, streptavidin that binds to the virus-like particle, and a drug or physiologically active substance bound to biotin that binds to the streptavidin.

[0010] Another objective of the present invention is to provide a method for manufacturing a complex comprising a human papilloma virus-derived virus-like particle and a drug or a physiologically active substance, comprising the step of loading a drug or a physiologically active substance onto the exterior (surface) or interior of the human papilloma virus-derived virus-like particle. means of solving the problem

[0011] To achieve the above objective, the present invention provides a composition for delivering a drug or a physiologically active substance comprising, as an active ingredient, a complex comprising a human papilloma virus-derived virus-like particle and a drug or a physiologically active substance.

[0012] In addition, the present invention provides a composition for delivering a drug or a physiologically active substance, comprising as an active ingredient a complex having a drug or a physiologically active substance loaded on the outside of a virus-like particle, wherein the composition comprises a biotin-labeled virus-like particle; streptavidin that binds to the virus-like particle; and a drug or a physiologically active substance bound to biotin that binds to the streptavidin.

[0013] In addition, the present invention provides a composition for delivering a drug or physiologically active substance comprising, as an active ingredient, a complex containing a drug or physiologically active substance inside a virus-like particle, wherein the virus-like particle is disassembled in a buffer solution containing β-mercaptoethanol, dialyzed with the drug or physiologically active ingredient, and reassembled.

[0014] In addition, the present invention provides a composition for delivering a drug or physiologically active substance comprising, as an active ingredient, a complex having a drug or physiologically active substance loaded inside and outside a virus-like particle, wherein the virus-like particle is disassembled in a buffer solution containing β-mercaptoethanol, dialyzed with a drug or physiologically active ingredient, and reassembled to include a virus-like particle having a drug or physiologically active substance loaded inside, and wherein the virus-like particle is biotinylated on the outside of the virus-like particle, streptavidin that binds to the virus-like particle, and a drug or physiologically active substance bound to biotin that binds to the streptavidin.

[0015] In addition, the present invention provides a human papilloma virus-derived virus-like particle comprising the step of loading a drug or a physiologically active substance onto the exterior (surface) or interior of the human papilloma virus-derived virus-like particle; and a method for manufacturing a complex comprising a drug or a physiologically active substance. Effects of the invention

[0016] According to the present invention, by identifying a method to effectively load a drug onto the outside (surface) or inside of a virus-like particle (L1 protein) derived from a papilloma virus, it can be utilized in the form of a delivery vehicle, therapeutic agent, vaccine, etc., for delivering various drugs such as anticancer agents, proteins, and peptides. Brief explanation of the drawing

[0017] Figure 1 is a schematic diagram of material loading on the outside (surface) of a virus-like particle (hereinafter referred to as VLP). Figure 2 shows the results of analyzing whether substances (streptavidin and biotinylated molecules) are loaded onto the VLP surface (external) according to Example 3-1. Figure 2A is a conceptual diagram of a VLP with a biotinylated surface, Figure 2B shows the binding of streptavidin to the VLP with a biotinylated surface, and Figure 2C shows the binding of a biotinylated molecule (β-Amyloid peptide) to the VLP with a biotinylated surface. Figure 3 shows the results of conjugating streptavidin to biotin-labeled VLP, followed by conjugating biotin-labeled β-Amyloid peptide (A), Ovalbumin protein (45 kDa) (B), human transferrin protein (80 kDa) (C) and Trastuzumab antibody (150 kDa) (D). Figure 4 shows the results of analyzing whether a substance was loaded onto a VLP conjugated with a biotinylated peptide according to Example 3-2. Figure 4A shows the analysis results using Biotin-DRKHC peptide (Sequence No. 2); Figure 4B shows the analysis results using Biotin-DRTKYSHTC peptide (Sequence No. 3); and Figure 4C shows the analysis results using Biotin-DRTKYSHTFLRDHC peptide (Sequence No. 4). Figure 5 shows the results of analyzing whether a substance was loaded onto a VLP bound to an Avtag peptide according to Example 3-3. Figure 5A shows the analysis results using the GLNDIFEAQKIEWHEC peptide (SEQ No. 5); and Figure 5B shows the analysis results using the GLNDIFEAQKIEWHEGGC peptide (SEQ No. 6). Figure 6 is a schematic diagram of material loading inside a VLP. Examples of loading applications include drugs, proteins, antibodies, genes, etc. Figure 7 shows the results of analyzing whether green fluorescent protein (GFP) is loaded inside a VLP according to Example 4-1. Specifically, Figure 7A shows the results of size-exclusion chromatography (SEC) analysis under conditions containing 0.162 M NaCl and various beta-mercaptoethanol concentrations (0, 0.5, 2%), and Figure 7B shows the results of the retention time, area, and height analysis of the elution peak obtained from the analysis results of Figure 7A. Figure 7C shows the results of SEC analysis under conditions containing 0.325 M NaCl and various beta-mercaptoethanol concentrations (0, 2, 5%), and Figure 7D shows the results of the retention time, area, and height analysis of the elution peak obtained from the analysis results of Figure 7C. Figure 8 shows the results of analyzing whether IgG2b and κ antibodies conjugated with FITC are loaded inside the VLP according to Example 4-2. Figure 8A is the SEC analysis result, and Figure 8B is the analysis result of the retention time, area, and height of the elution peak obtained from the SEC analysis result. Figure 9 shows the results of analyzing whether streptavidin bound to Alexa Flour 647 was loaded inside the VLP according to Example 4-3. Figure 9A is the SEC analysis result, Figure 9B is the analysis result of the retention time, area, and height of the elution peak obtained from the SEC analysis result, and Figure 9C is the result of fluorescence analysis of the sample eluted by the SEC analysis using a plate reader. Figure 10 is the result of analyzing whether Cas9 combined with GFP is loaded inside the VLP according to Example 4-4. Figure 10A is the SEC analysis result, and Figure 10B is the result of the retention time, area, and height analysis of the elution peak obtained from the SEC analysis result. Figure 11 shows the results of analyzing whether streptavidin-CpG ODN combined with FITC was loaded inside the VLP according to Examples 4-5. Figure 11A is the SEC analysis result, and Figure 11B is the analysis result of the retention time, area, height, and 260nm / 280nm ratio of the elution peak obtained from the SEC analysis result. Figure 12 is the result of analyzing whether doxorubicin combined with BSA was loaded inside the VLP according to Examples 4-6. Figure 13 shows the results of analyzing whether material is loaded on the exterior (surface) and interior of the VLP. Figure 13A is a schematic diagram of material loading on the exterior (surface) and interior of the VLP, Figure 13B shows the results of analyzing whether material is loaded on the exterior (surface) of the VLP, and Figure 13C shows the results of analyzing whether material is loaded inside the VLP. Figure 14 shows the results of comparing biotin-labeled papillomavirus virus-like particles and Q-beta bacteriophage virus-like particles after streptavidin was bound to each, followed by binding to biotin-labeled β-Amyloid peptide (A), Trastuzumab antibody (150 kDa) (B), and Ovalbumin protein (45 kDa) (C). Figure 15 shows the results of analyzing papillomavirus virus-like particles and Q-beta bacteriophage virus-like particles via HPLC. In the following test, to determine whether they were internally loaded, each virus-like particle was analyzed using an absorbance of 280 nm and fluorescence wavelengths of 488 nm / 520 nm, which correspond to the wavelength range of GFP. Figure 16 shows the results of HPLC analysis after loading GFP inside papillomavirus virus-like particles and Q-beta bacteriophage virus-like particles. When analyzing the results for each wavelength range, it was confirmed that GFP was successfully loaded inside the papillomavirus virus-like particles, but GFP was not loaded inside the Q-beta bacteriophage virus-like particles. Specific details for implementing the invention

[0018] The present invention will be described in more detail below.

[0020] The present invention provides a composition for delivering a drug or a physiologically active substance comprising, as an active ingredient, a complex comprising a virus-like particle derived from a human papilloma virus and a drug or a physiologically active substance.

[0021] The above virus-like particle may be a human papillomavirus L1 protein composed of the amino acid sequence of SEQ ID NO. 1.

[0022] In addition, the average diameter of the virus-like particles may be 25 to 160 nm.

[0023] The above complex may be in a form in which a drug or a physiologically active substance is loaded inside or outside the virus-like particle.

[0024] The above drugs or physiologically active substances may be one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds, but are not limited thereto.

[0026] In addition, the present invention provides a composition for delivering a drug or a physiologically active substance, comprising as an active ingredient a complex having a drug or a physiologically active substance loaded on the outside of a virus-like particle, wherein the composition comprises a biotin-labeled virus-like particle; streptavidin that binds to the virus-like particle; and a drug or a physiologically active substance bound to biotin that binds to the streptavidin.

[0027] The above biotinylation may involve labeling virus-like particles with a biotin linker, a biotin-labeled peptide, or a biotin-labeled Avitag peptide.

[0028] The above biotin linker may be a Sulfo-NHS-Biotin (Sulfosuccinimidyl Biotin) linker or an SMPH (Succinimidyl 6-(β-maleimidopropionamido) hexanoate) linker.

[0029] The above biotin-labeled peptide may contain cysteine ​​at the C-terminus and be labeled with biotin at the N-terminus.

[0030] In addition, the biotin-labeled peptide may consist of one amino acid sequence selected from the group consisting of SEQ ID NOs 2 to 4.

[0031] The above biotin-labeled Avitag peptide may contain cysteine ​​at the C-terminus and may be labeled with biotin on the lysine residue of the Avitag peptide using an enzyme.

[0032] In addition, the above Avitag peptide may consist of one amino acid sequence selected from the group consisting of SEQ ID NOs 5 to 6.

[0033] The above drugs or physiologically active substances may be one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds, but are not limited thereto.

[0034] The above virus-like particle may be composed of L1 protein derived from papillomavirus.

[0036] In addition, the present invention provides a composition for delivering a drug or physiologically active substance comprising, as an active ingredient, a complex containing a drug or physiologically active substance inside a virus-like particle, wherein the virus-like particle is disassembled in a buffer solution containing β-mercaptoethanol, dialyzed with the drug or physiologically active ingredient, and reassembled.

[0037] The above drugs or physiologically active substances may be one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds, but are not limited thereto.

[0038] The above virus-like particle may be composed of L1 protein derived from papillomavirus.

[0040] In addition, the present invention provides a composition for delivering a drug or physiologically active substance comprising, as an active ingredient, a complex having a drug or physiologically active substance loaded inside and outside a virus-like particle, wherein the virus-like particle is disassembled in a buffer solution containing β-mercaptoethanol, dialyzed with a drug or physiologically active ingredient, and reassembled to include a virus-like particle having a drug or physiologically active substance loaded inside, and wherein the virus-like particle is biotinylated on the outside of the virus-like particle, streptavidin that binds to the virus-like particle, and a drug or physiologically active substance bound to biotin that binds to the streptavidin.

[0041] The above drugs or physiologically active substances may be one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds, but are not limited thereto.

[0042] The above virus-like particle may be composed of L1 protein derived from papillomavirus.

[0044] In addition, the present invention provides a human papilloma virus-derived virus-like particle comprising the step of loading a drug or a physiologically active substance onto the exterior (surface) or interior of the human papilloma virus-derived virus-like particle; and a method for manufacturing a complex comprising a drug or a physiologically active substance.

[0045] The step of loading a drug or physiologically active substance into a virus-like particle derived from a human papilloma virus may include: (1) a step of decomposing the virus-like particle by mixing the virus-like particle derived from a human papilloma virus with a buffer containing beta-mercaptoethanol; and (2) a step of loading the drug or physiologically active substance into the virus-like particle decomposed in step (1), and then reassembling the virus-like particle by dialysis in a sodium chloride buffer.

[0046] The step of loading a drug or physiologically active substance onto the outside (surface) of a virus-like particle derived from a human papilloma virus may include: (1) a step of biotinylating the surface of a virus-like particle derived from a human papilloma virus by labeling it with biotin; (2) a step of biotinylating the surface of a drug or physiologically active substance by labeling it with biotin; (3) a step of loading a drug or physiologically active substance onto the outside (surface) of a virus-like particle in the form of a virus-like particle-streptavidin-drug / physiologically active substance complex by combining the virus-like particle with the biotinylated surface of step (1); streptavidin; and the drug or physiologically active substance with the biotinylated surface of step (2).

[0047] The above step (1) may be prepared by mixing the virus-like particles with a linker and reacting them, and then dialyzing them in a sodium chloride solution; or by mixing the virus-like particles with a linker and reacting them, and then dialyzing the virus-like particle-linker conjugate prepared by dialyzing it in a sodium chloride buffer solution, and then mixing it with a peptide and reacting it, and then dialyzing it in a sodium chloride solution.

[0048] The above linker may be a Sulfo-NHS-Biotin (Sulfosuccinimidyl Biotin) linker or an SMPH (Succinimidyl 6-(β-maleimidopropionamido) hexanoate) linker.

[0049] The above peptide may be biotinized by labeling the C-terminus with cysteine ​​and the N-terminus with biotin.

[0050] The above peptide may consist of one amino acid sequence selected from the group consisting of SEQ ID NOs 2 to 6.

[0051] The above drugs or physiologically active substances may be one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds, but are not limited thereto.

[0053] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0055] [Example 1] Production of Virus-like Particles Derived from Papillomavirus

[0056] To produce a Virus-Like Particle (VLP) (L1 protein) of human papilloma virus type 11, a Saccharomyces cerevisiae expression system was utilized. First, the strain was cultured in a yeast culture medium supplemented with galactose at 30°C and then obtained. Subsequently, the intracellular L1 protein was eluted by cell lysis, and impurities in the cell lysate were removed through heating and cooling in the presence of beta-mercaptoethanol. Afterward, the L1 protein was obtained through a chromatography process. The beta-mercaptoethanol in the L1 protein solution was removed by dialysis in a buffer solution that did not contain beta-mercaptoethanol. Afterwards, the L1 protein was reassembled by dialyzing it in a buffer containing 0.325 M sodium chloride (hereinafter referred to as NaCl) and filtered through a 0.22 μm membrane. The final obtained L1 protein was confirmed to be in VLP form and stored at a temperature of -60°C or lower.

[0057] [L1 Protein Sequence] (Sequence No. 1, 501aa)(Genbank Accession no.: MH407282.1)

[0058] MWRPSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVPKVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLLNKYDD VENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNGDCPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYGDRLFFYLRKE QMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSEAQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTRSTNMTLCASVSKSATYTNSDYKEYMRHVEEFDLQFIFQLC SITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQAITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGIKRPAVSKPSTAPKRKRTKTKK

[0060] [Example 2] Biotinization of VLP Surface

[0061] For the biotinylation of the above VLP surface, biotin binding was performed on the VLP surface using a Sulfo-NHS-Biotin (Sulfosuccinimidyl Biotin) linker. Virus-like particles and the Sulfo-NHS-Biotin linker were mixed in an amine-free buffer and reacted on ice for 2 hours or at room temperature for 30 minutes. Afterward, the mixture was dialyzed in an amine-free buffer containing 0.325 M NaCl.

[0063] [Example 3] Material loaded on the exterior (surface) of the VLP

[0064] The possibility of loading a substance onto the exterior (surface) of the above VLP was analyzed. Streptavidin and biotinylated molecules were used as the loaded substances. Specifically, in Example 3-1 below, a substance was loaded onto the exterior of a VLP with a biotinylated surface; in Example 3-2, a VLP with a biotinylated peptide attached; and in Example 3-3, a VLP with an Avtag peptide attached. All of the above VLPs were VLPs with biotinylated surfaces, and a VLP without biotinylated surfaces was set as a control group.

[0066] 3-1. Material loading onto the exterior (surface) of a biotinized VLP

[0067] To conjugate streptavidin and biotinylated molecules to biotinylated VLPs, biotinylated VLPs were coated in amine-free buffer in a 96-well plate. Subsequently, the plates were blocked overnight at 4°C with PBST (phosphate-buffered saline + 0.05% Tween 20) containing 5% skim milk, and reacted with 1 μg / mL of streptavidin at room temperature for 30 minutes. Afterward, a biotinylated peptide or biotinylated antibody (3 μg / mL) was applied and reacted at room temperature for 30 minutes. The biotinylated peptide used was a peptide against human amyloid beta, and the biotinylated antibody used was a humanized anti-HER2 antibody (trastuzumab). Human amyloid beta peptides bound to the terminals were detected using mouse anti-amyloid beta antibodies and anti-mouse IgG HRP antibodies. Additionally, humanized anti-HER2 antibodies bound to the terminals were detected using mouse anti-human IgG and anti-mouse IgG HRP antibodies. At each step, 96-well plates were washed with PBST.

[0069] 3-2. Material loading onto the exterior (surface) of VLPs bound to biotinylated peptides

[0070] VLPs conjugated with biotinylated peptides were prepared using an SMPH (Xuccinimidyl 6-(β-maleimidopropionamido) hexanoate) linker. First, the prepared VLPs and SMPH linkers were mixed in an amine-free buffer and reacted at room temperature for 1 hour, followed by overnight dialyzing in an amine-free buffer containing 0.15 M NaCl at 4°C. Subsequently, the biotinylated peptides and the virus-like particle-SMPH conjugate were mixed, reacted at room temperature for 1 hour, and dialyzed overnight in an amine-free buffer containing 0.325 M NaCl at 4°C.

[0071] The above biotinylated peptides contained cysteine ​​at the C-terminus and biotinylated the N-terminus by biotin labeling, and a total of three types of peptides were synthesized and used (the above peptides can form a bond with lysine on the VLP surface through a linker). The peptide sequences are as shown in Table 1.

[0072] To conjugate streptavidin and biotinylated molecules to the exterior (surface) of the biotinylated peptide-conjugated VLP prepared above, the biotinylated peptide-conjugated VLP was coated in an amine-free buffer in a 96-well plate. Subsequently, it was blocked overnight at 4°C with PBST (phosphate-buffered saline + 0.05% Tween 20) containing 5% skim milk, and reacted with 1 μg / mL of streptavidin at room temperature for 30 minutes. Afterward, the biotinylated peptide, biotinylated ovalbumin protein, biotinylated transferrin protein, or biotinylated antibody were applied (3 μg / mL each) and reacted at room temperature for 30 minutes. The biotinylated peptide used was a peptide against human amyloid beta, and the biotinylated antibody used was a humanized anti-HER2 antibody (trastuzumab). Human amyloid beta peptides bound to the ends were detected using mouse anti-amyloid beta antibodies and anti-mouse IgG HRP antibodies, and ovalbumin proteins bound to the ends were detected using Rabbit anti-ovalbumin antibodies and anti-Rabbit IgG HRP antibodies. Transferrin proteins bound to the ends were detected using mouse anti-transferrin antibodies and anti-mouse IgG HRP antibodies, and humanized anti-HER2 antibodies bound to the ends were detected using mouse anti-human IgG and anti-mouse IgG HRP antibodies. At each step, 96-well plates were washed with PBST.

[0073] Ranking name Sequence number order Biotinylated peptide 1 2 DRKHC Biotinylated peptide 2 3 DRTKYSHTC Biotinylated peptide 3 4 DRTKYSHTFLRDHC Avitag peptide 1 5 GLNDIFEAQKIEWHEC Avitag peptide 2 6 GLNDIFEAQKIEWHEGGC

[0075] 3-3. Material loading onto the exterior (surface) of a VLP bound to Avitag peptide

[0076] VLP conjugated with avitag peptide was prepared using an SMPH linker. First, the prepared VLP and SMPH linker were mixed in an amine-free buffer and reacted at room temperature for 1 hour, followed by overnight dialyzing in an amine-free buffer containing 0.15 M NaCl at 4°C. Subsequently, the avitag peptide was biotinized by reacting at 30°C for 40 minutes according to the test procedure of the BirA kit. After the reaction was complete, the biotinized avitag peptide and the VLP-SMPH conjugate were mixed and reacted at room temperature for 1 hour, followed by overnight dialyzing in an amine-free buffer containing 0.325 M NaCl at 4°C.

[0077] The above Avitag peptides were synthesized and used in a total of two types by including cysteine ​​at the Avitag C-terminus (the above peptides can be bound to the VLP surface using a linker, and Avitag-specific biotinylation is possible by reacting with the BirA enzyme and biotin). The peptide sequences are as shown in Table 1.

[0078] To bind streptavidin and biotinylated molecules to the outside (surface) of the VLP bound to the Avitag peptide prepared above, an experiment was performed in the same manner as in Example 3-2 above.

[0080] [Example 4] Material loaded inside VLP

[0081] 4-1. GFP

[0082] The above VLP was decomposed in buffers containing 0, 0.5, 2%, or 5% beta-mercaptoethanol, mixed with GFP (green fluorescent protein) (approx. 27 kDa), and reassembled by dialyzing in a buffer containing 0.325 M NaCl. The presence of GFP loaded into the VLP was analyzed by size-exclusion chromatography (hereinafter referred to as SEC). Superose-6 was used as the resin for SEC (0.5 × 20 cm, Cytiva), and the peak at the 4-minute mark, which is the region where the VLP elutes, was determined to be a valid peak. The VLP was confirmed at UV 280 nm, and the fluorescence of GFP was confirmed at 488 nm / 520 nm (excitation / emission).

[0084] 4-2. FITC-conjugated IgG2b, κ antibodies

[0085] The above VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol, mixed with an IgG2b,κ antibody (approx. 150 kDa) conjugated with FITC (Fluorescein Isothiocyanate), and then reassembled by dialyzing in a buffer containing 0.325 M NaCl. The presence of the FITC-conjugated IgG2b,κ antibody inside the VLP was analyzed by SEC, and the SEC was performed in the same manner as described in Example 4-1 above.

[0087] 4-3. Streptavidin conjugated with Alexa Flour 647

[0088] The above VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol, mixed with Alexa Flour 647-conjugated streptavidin (approx. 67 kDa), and reassembled by dialyzing in a buffer containing 0.325 M NaCl. The presence of Alexa Flour 647-conjugated streptavidin within the VLP was analyzed via SEC. Superose-6 resin (0.5 × 20 cm) was used for the SEC, and the peak at the 4-minute mark, which is the elution region of the VLP, was eluted and analyzed for fluorescence using a fluorescent plate reader. The VLP was confirmed at UV 280 nm, and the fluorescence of Alexa Flour 647-conjugated streptavidin was confirmed at 640 nm / 668 nm (excitation / emission).

[0090] 4-4. GFP-conjugated Cas9

[0091] The above VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol, mixed with GFP-conjugated Cas9 (approx. 190 kDa), and reassembled by dialyzing in a buffer containing 0.325 M NaCl. The presence of GFP-conjugated Cas9 within the VLP was analyzed using SEC. Superose-6 resin was used for SEC (0.5 × 20 cm), and the peak at the 4-minute mark, which corresponds to the elution of the VLP, was determined to be a valid peak. The VLP was identified at UV 280 nm, and the fluorescence of GFP was identified at 488 nm / 520 nm (excitation / emission).

[0093] 4-5. FITC-conjugated Streptavidin-CpG ODN

[0094] The above VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol. FITC-conjugated streptavidin and biotinylated CpG ODN (Cytosine-phosphate-Guanine Oligodeoxynucleotide) were reacted in a 1:4 molar ratio. Subsequently, the decomposed VLP and FITC-conjugated streptavidin-CpG ODN were mixed and reassembled by dialysis in a buffer containing 0.325 M NaCl. The presence of FITC-conjugated streptavidin-CpG ODN within the VLP was analyzed using SEC. Superose-6 resin (0.5 × 20 cm) was used for SEC, and the peak at the 4-minute mark, which corresponds to the elution of the VLP, was determined to be a valid peak. VLP was confirmed at UV 280 nm, and the fluorescence of FITC-conjugated streptavidin-CpG ODN was confirmed at 260 nm. The 260 nm / 280 nm ratio of VLP was approximately 0.8, and it was confirmed that when FITC-conjugated streptavidin-CpG ODN was loaded inside VLP, the 260 nm / 280 nm ratio was greater than 1.

[0096] 4-6. BSA-conjugated doxorubicin

[0097] The above VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol, mixed with 9.6 mg BSA (Bovine Serum Albumin), and reassembled by dialyzing in a buffer containing 0.325 M NaCl. Free BSA not loaded inside the VLP was removed by eluting using SEC (using Superose-6 as the resin), where the peak at the 4-minute mark, the elution region of the VLP, was identified as a valid peak. The eluted VLP (loaded with BSA) and Doxorubicin were mixed in a molar ratio of approximately 1:5, incubated at 37°C for 2 hours and at room temperature for 2 hours, and then induced binding between Doxorubicin and BSA overnight at 4°C. Unbound free Doxorubicin was removed by dialyzing overnight in a buffer containing 0.325 M NaCl. As a control group, VLP and doxorubicin were mixed in a 1:5 molar ratio, and the reaction was carried out in the same manner as the experimental group. The concentrations of the control and experimental groups with free doxorubicin removed were measured, 100 μg of each was taken, and fluorescence was confirmed at 488 nm / 590 nm (excitation / emission) using a plate reader.

[0099] [Example 5] Material loading on the exterior (surface) and interior of the VLP

[0100] The possibility of loading substances onto the exterior (surface) and interior of the above VLP was analyzed. The VLP was decomposed in a buffer containing 0.162 M NaCl and 0.5% beta-mercaptoethanol, mixed with GFP, and then reassembled by dialyzing in a buffer containing 0.325 M NaCl. The presence of GFP inside the VLP was analyzed using SEC (using superose-6 as the resin), and the peak at the 4-minute mark, which is the elution region of the VLP, was eluted.

[0101] Biotin binding to the exterior (surface) of the eluted GFP-loaded VLP was performed using a Sulfo-NHS-Biotin linker. The prepared VLP and Sulfo-NHS-Biotin linker were mixed in amine-free buffer and reacted on ice for 2 hours or at room temperature for 30 minutes. Subsequently, the mixture was dialyzed in amine-free buffer containing 0.325 M NaCl. The prepared VLP was coated in amine-free buffer in a 96-well plate. It was then blocked overnight at 4°C with PBST (phosphate-buffered saline + 0.05% Tween 20) containing 5% skim milk, and reacted with 1 μg / mL streptavidin at room temperature for 30 minutes.

[0102] Subsequently, biotinylated peptide, biotinylated ovalbumin protein, biotinylated transferrin protein, or biotinylated antibody were applied (3 μg / mL each) and reacted at room temperature for 30 minutes. The biotinylated peptide used was a peptide against human amyloid beta, and the biotinylated antibody used was a humanized anti-HER2 antibody (trastuzumab). Human amyloid beta peptide bound to the terminals was detected using mouse anti-amyloid beta antibody and anti-mouse IgG HRP antibody, and ovalbumin protein bound to the terminals was detected using Rabbit anti-ovalbumin antibody and anti-Rabbit IgG HRP antibody. Transferrin protein bound to the terminals was detected using mouse anti-transferrin antibody and anti-mouse IgG HRP antibody, and humanized anti-HER2 antibody bound to the terminals was detected using mouse anti-human IgG and anti-mouse IgG HRP antibody. At each step, the 96-well plate was washed with PBST.

[0104] [Experimental Example 1] Material loaded on the exterior (surface) of the VLP

[0105] 1-1. Material loading onto the exterior (surface) of a biotinized VLP

[0106] According to Example 3-1, the loading of substances onto the exterior (surface) of the biotinized VLP was confirmed. The loading of streptavidin onto the exterior of the biotinized VLP was confirmed through FITC fluorescence attached to streptavidin. The loading of substances (biotinized molecules) onto the exterior of the biotinized VLP was confirmed through ELISA analysis. VLPs loaded with substances onto the exterior were coated onto ELISA plates, and the loading of substances was confirmed through ELISA analysis using antibodies against the substances. As a result, as shown in Figure 2, it was confirmed that substances (streptavidin and biotinized molecules) were not loaded onto the exterior of the VLP without biotinylation, whereas substances were loaded onto the exterior of the VLP with biotinylation.

[0108] 1-2. Material loading onto the exterior (surface) of the VLP bound to the biotinylated peptide

[0109] Streptavidin was conjugated to biotinylated VLPs, and an ELISA analysis was performed to determine if biotin-labeled peptides [Fig. 3A; β-Amyloid peptide, Fig. 3B; Ovalbumin protein (45kDa), Fig. 3C; human transferrin protein (80kDa), Fig. 3D; Trastuzumab antibody (150kDa)] could be loaded. As a result, as shown in Fig. 3, it was confirmed that while no substances were externally loaded on VLPs that were not biotinylated on the surface, all substances were externally loaded on VLPs that were biotinylated on the surface.

[0111] 1-3. Material loading onto the exterior (surface) of the VLP bound to the biotinylated peptide

[0112] According to Example 3-2, ELISA analysis was performed to confirm whether a substance was loaded onto a VLP bound to a biotinylated peptide using a biotinylated linker. As a result, as shown in Figure 4, it was confirmed that while no substance was externally loaded onto a VLP with a non-biotinized surface, a substance was externally loaded onto a VLP with a biotinylated surface.

[0114] 1-4. Material loading onto the exterior (surface) of a VLP bound to Avitag peptide

[0115] According to Example 3-3, an ELISA analysis was performed to confirm whether a substance was loaded onto the VLP bound to the Avitag peptide (performed in the same manner as in Example 3-1). As a result, as shown in Fig. 5, it was confirmed that the substance was not externally loaded onto the VLP without biotinylation of the surface, whereas the substance was externally loaded onto the VLP with biotinylation of the surface.

[0117] [Experimental Example 2] Material loaded inside VLP

[0118] 2-1. GFP

[0119] According to Example 4-1, the presence of GFP inside the VLP was analyzed. Specifically, beta-mercaptoethanol, used as a reducing agent for GFP loading, was used at various concentrations (0, 0.5, 2, or 5%), and the presence of loading was analyzed via SEC. Since GFP has a significantly smaller size compared to VLP, its elution time is slower than that of VLP in SEC analysis; therefore, utilizing this characteristic, the presence of loading was determined by checking whether GFP was eluted together with VLP at the same time. In addition, experiments were conducted on VLPs under conditions containing 0.162 M NaCl and 0.325 M NaCl. As a result, as shown in Figure 7, it was confirmed that GFP was internally loaded under conditions containing 0.162 M NaCl and 0% beta-mercaptoethanol, and also under conditions containing 0.5–5% beta-mercaptoethanol.

[0121] 2-2. FITC-conjugated IgG2b, κ antibodies

[0122] According to Example 4-2, the presence of IgG2b, κ antibodies conjugated with FITC inside the VLP was analyzed. As a result, as shown in Figure 8, it was confirmed that fluorescence of FITC was detected at the peak in the 4-minute range, which is the elution section of the VLP, thereby confirming that IgG2b, κ antibodies conjugated with FITC were loaded inside the VLP.

[0124] 2-3. Streptavidin conjugated with Alexa Flour 647

[0125] According to Example 4-3, the loading of streptavidin bound to Alexa Flour 647 inside the VLP was analyzed. As a result, as shown in Figure 9, it was confirmed that the fluorescence of Alexa Flour 647 was detected at the peak in the 4-minute range, which is the elution range of the VLP, thereby confirming that streptavidin bound to Alexa Flour 647 was loaded inside the VLP.

[0127] 2-4. GFP-conjugated Cas9

[0128] According to Example 4-4, the presence of Cas9 bound to GFP inside the VLP was analyzed. As a result, as shown in Figure 10, it was confirmed that fluorescence of GFP was detected at the peak in the 4-minute range, which is the elution section of the VLP, thereby confirming that Cas9 bound to GFP was loaded inside the VLP.

[0130] 2-5. FITC-conjugated Streptavidin-CpG ODN

[0131] According to Examples 4-5, the presence of streptavidin-CpG ODN conjugated with FITC inside the VLP was analyzed. As a result, as shown in Figure 11, it was confirmed that fluorescence of FITC was detected at the peak in the 4-minute range, which is the elution range of the VLP, thereby confirming that streptavidin-CpG ODN1 conjugated with FITC was loaded inside the VLP.

[0133] 2-6. BSA-conjugated doxorubicin

[0134] According to Examples 4-6, the presence of doxorubicin bound to BSA inside the VLP was analyzed. As a result, as shown in Fig. 12, it was confirmed that the binding strength with doxorubicin of the VLP loaded with BSA was improved by approximately 42% compared to the VLP without BSA.

[0136] [Experimental Example 3] Material loading on the exterior (surface) and interior of the VLP

[0137] According to Example 5 above, the presence of a substance on the exterior (surface) and interior of the VLP was analyzed (performed in the same manner as the SEC analysis of Experimental Example 1 and the ELISA analysis of Experimental Example 2). As a result, as shown in Fig. 13B, it was confirmed that GFP fluorescence appeared at the peak in the 4-minute range, which is the elution section of the VLP, thereby confirming that GFP was loaded inside the VLP. In addition, as shown in Fig. 13C, it was confirmed that while the substance was not externally loaded on the VLP without biotinylation of the surface, the substance was externally loaded on the VLP with biotinylation of the surface.

[0139] [Experimental Example 4] Comparison of Internal and External Drug Loading Efficacy According to Virus-like Particle Type

[0140] To determine whether there are differences in drug loading efficiency depending on the type of virus, the drug loading efficiencies of human pharylomavirus VLP (hereinafter referred to as HPV VLP) and Qbeta bacteriophage virus-like particles (hereinafter referred to as Qbeta VLP) were compared.

[0141] First, to confirm the loading efficiency on the VLP surface (external), biotinylated peptides, proteins, or antibodies were displayed on the VLP surface, and the loading efficiency was analyzed. The surface-bound peptides, proteins, or antibodies of the prepared drug-loaded VLPs were evaluated via ELISA. As a result, as shown in Figure 14, it was confirmed that HPV VLPs had a higher loading rate than Qbeta VLPs in all experimental groups.

[0142] In addition, to verify the drug loading efficiency inside the VLP, the drug was loaded according to the loading method described above, and the fluorescence wavelength of the Ex488 / Em520 was used to measure GFP. Figures 15 and 16 show the HPLC analysis results before and after loading GFP inside the HPV VLP and Qbeta VLP. Specifically, Figure 15 shows the results of analyzing the characteristics of each virus-like particle before internal loading of GFP; Figure 15A shows the chromatogram for each wavelength range, and Figure 15B shows the results of the peak retention time, area, and height obtained under those conditions. Additionally, Figure 16 shows the results of analyzing the characteristics of each virus-like particle after internal loading of GFP; Figure 16A shows the chromatogram for each wavelength range, and Figure 16B shows the results of the peak retention time, area, and height obtained under those conditions. As a result, it was confirmed that HPV VLP was internally loaded, as the UV peaks before and after loading were identical and GFP fluorescence was detected during the time when HPV VLP was eluting. On the other hand, it was confirmed that Qbeta VLP had different UV peaks before and after loading, and GFP fluorescence was not detected during the time when Qbeta VLP was eluting before loading. In addition, it was confirmed that Qbeta VLP did not reassemble well after disassembly, so GFP was not loaded inside the virus-like particle.

[0143] From the above results, it was confirmed that a multifunctional VLP with dual functions can be successfully implemented by effectively loading functional drugs (chemotherapy drugs, proteins, genes, antibodies, etc.) inside the human papillomavirus VLP and simultaneously displaying peptides, proteins, or antibodies on its surface. In addition, it was confirmed that it exhibits superior efficiency when comparing loading functionality with other types of VLPs other than the human papillomavirus VLP.

[0145] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition for delivering a drug or a bioactive substance comprising, as an active ingredient, a complex composed of a virus-like particle derived from a human papilloma virus and a drug or a bioactive substance. Claim 2 A composition according to claim 1, characterized in that the virus-like particle is a human papillomavirus L1 protein composed of the amino acid sequence of SEQ ID NO.

1. Claim 3 A composition according to claim 1, characterized in that the average diameter of the virus-like particles is 25 to 160 nm. Claim 4 A composition according to claim 1, characterized in that the complex is in the form in which a drug or physiologically active substance is loaded inside or outside the virus-like particle. Claim 5 A composition according to claim 1, characterized in that the drug or physiologically active substance is one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, immunoadjuvants, proteins, peptides, genetic material, vaccine antigens, and low-molecular-weight compounds. Claim 6 A composition for delivering a drug or a bioactive substance comprising, as an active ingredient, a complex having a drug or a bioactive substance loaded on the outside of a virus-like particle, wherein the composition comprises: a biotin-labeled virus-like particle; streptavidin that binds to the virus-like particle; and a biotin-bound drug or a bioactive substance that binds to the streptavidin. Claim 7 A composition according to claim 6, wherein the biotinylation is characterized by labeling virus-like particles with a biotin linker, a biotin-labeled peptide, or a biotin-labeled Avitag peptide. Claim 8 A composition according to claim 7, characterized in that the biotin linker is a Sulfo-NHS-Biotin (Sulfosuccinimidyl Biotin) linker or an SMPH (Succinimidyl 6-(β-maleimidopropionamido) hexanoate) linker. Claim 9 A composition according to claim 6, wherein the biotin-labeled peptide comprises cysteine ​​at the C-terminus and is labeled with biotin at the N-terminus. Claim 10 A composition according to claim 9, characterized in that the biotin-labeled peptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs 2 to 4. Claim 11 A composition according to claim 6, wherein the biotin-labeled Avitag peptide comprises cysteine ​​at the C-terminus and is characterized by labeling the lysine residue of the Avitag peptide with biotin using an enzyme. Claim 12 A composition according to claim 11, wherein the above Avitag peptide is characterized by being composed of one amino acid sequence selected from the group consisting of SEQ ID NOs 5 to 6. Claim 13 A composition according to claim 6, characterized in that the above-mentioned drug or physiologically active substance is one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and low-molecular-weight compounds. Claim 14 A composition according to claim 6, characterized in that the virus-like particles are composed of papillomavirus-derived L1 protein. Claim 15 A composition for delivering a drug or physiologically active substance comprising, as an active ingredient, a complex containing a drug or physiologically active substance loaded inside a virus-like particle, wherein the virus-like particle is disassembled in a buffer containing β-mercaptoethanol, dialyzed with the drug or physiologically active ingredient, and reassembled. Claim 16 A composition according to claim 15, wherein the above-mentioned drug or physiologically active substance is one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds. Claim 17 A composition according to claim 15, characterized in that the virus-like particles are composed of papillomavirus-derived L1 protein. Claim 18 A composition for delivering a drug or physiologically active substance, comprising as an active ingredient a complex having a drug or physiologically active substance loaded inside and outside a virus-like particle, wherein the virus-like particle is disassembled in a buffer containing β-mercaptoethanol, dialyzed with a drug or physiologically active ingredient, and reassembled to include a virus-like particle having a drug or physiologically active substance loaded inside, and wherein the virus-like particle is biotinylated on the outside of the virus-like particle, streptavidin that binds to the virus-like particle, and a drug or physiologically active substance bound to biotin that binds to the streptavidin. Claim 19 A composition according to claim 18, wherein the above-mentioned drug or physiologically active substance is one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, adjuvants, proteins, peptides, genetic material, vaccine antigens, and small molecule compounds. Claim 20 A composition according to claim 18, characterized in that the virus-like particles are composed of papillomavirus-derived L1 protein. Claim 21 A method for manufacturing a complex comprising a human papilloma virus-derived virus-like particle, comprising the step of loading a drug or a physiologically active substance onto the exterior (surface) or interior of the human papilloma virus-derived virus-like particle; and a drug or a physiologically active substance. Claim 22 A method for manufacturing according to claim 21, wherein the step of loading a drug or physiologically active substance into a virus-like particle derived from a human papilloma virus comprises: (1) a step of decomposing the virus-like particle by mixing the virus-like particle derived from a human papilloma virus with a buffer containing beta-mercaptoethanol; (2) a step of loading the drug or physiologically active substance into the virus-like particle decomposed in step (1), and then reassembling the virus-like particle by dialysis in a sodium chloride buffer. Claim 23 A manufacturing method according to claim 21, wherein the step of loading a drug or physiologically active substance onto the outside (surface) of a virus-like particle derived from a human papilloma virus comprises: (1) a step of biotinylating the surface of a virus-like particle derived from a human papilloma virus by labeling it with biotin; (2) a step of biotinylating the surface of a drug or physiologically active substance by labeling it with biotin; (3) a step of loading a drug or physiologically active substance onto the outside (surface) of a virus-like particle in the form of a virus-like particle-streptavidin-drug / physiologically active substance complex by combining the virus-like particle with the biotinylated surface of step (1); streptavidin; and the drug or physiologically active substance with the biotinylated surface of step (2). Claim 24 A method of manufacturing according to claim 23, wherein step (1) is prepared by mixing the virus-like particles with a linker and reacting them, and then dialyzing the resulting solution with a sodium chloride solution; or by mixing the virus-like particles with a linker and reacting them, and then dialyzing the resulting virus-like particle-linker conjugate prepared by dialyzing the resulting solution with a peptide and reacting it, and then dialyzing the resulting solution with a sodium chloride solution. Claim 25 A manufacturing method according to claim 24, characterized in that the linker is a Sulfo-NHS-Biotin (Sulfosuccinimidyl Biotin) linker or an SMPH (Succinimidyl 6-(β-maleimidopropionamido) hexanoate) linker. Claim 26 A method of preparation according to claim 24, characterized in that the peptide has a C-terminus that is cysteine ​​and an N-terminus that is biotinized by labeling it with biotin. Claim 27 A method of manufacturing according to claim 26, characterized in that the peptide comprises one amino acid sequence selected from the group consisting of SEQ ID NOs 2 to 6. Claim 28 A method of manufacturing according to claim 21, characterized in that the above drug or physiologically active substance is one or more selected from the group consisting of anticancer agents, antibodies, enzymes, growth factors, cytokines, carbohydrates, nucleic acids, lipids, vitamins, immunoadjuvants, proteins, peptides, genetic material, vaccine antigens, and low-molecular-weight compounds.

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