Protein cage nano-drug with slow release effect as well as preparation method and application of protein cage nano-drug

Abstract

The invention belongs to the technical field of nano-drugs, and particularly relates to a protein cage nano-drug with a slow release effect as well as a preparation method and application of the protein cage nano-drug. According to the protein cage nano-drug with the slow release effect provided by the invention, the protein binding peptide is modified at the C end of the protein subunit monomer of the nano-protein cage and is bound with the protein with long half-life period in vivo, so that the action time of the drug is prolonged, the curative effect of the drug is improved, and the possible toxic and side effects of the drug are weakened; meanwhile, the protein binding peptide is selected from serum albumin binding peptide with an amino acid sequence as shown in SEQ ID NO.1, so that the protein cage nano-drug provided by the technical scheme can be bound with the most abundant endogenous protein in blood, and the half-life period of the high-concentration protein cage nano-drug can also be prolonged; the technical problems that in the prior art, a sustained-release protein cage nano-drug is lacked, and the drug is difficult to release durably are solved.

Description

Technical Field

[0001] The present application belongs to the field of nanomedicine technology, and in particular relates to a protein cage nanomedicine with a sustained-release effect, a preparation method thereof, and an application thereof. Background Art

[0002] Macromolecule drugs are divided into vaccines, blood and blood products. Based on their sources, peptides, proteins, antibodies, polysaccharides and nucleic acids are clinically classified as biological macromolecule drugs.

[0003] Protein drugs among biomacromolecule drugs have the advantages of high specificity and targeting. For example, anti-PD1 antibodies can precisely bind to specific targets such as tumor cell surface antigens or immune checkpoint molecules, reducing accidental damage to normal cells. However, the structure of protein drugs will be destroyed under the action of proteolysis and acid-base changes in the body; at the same time, nucleic acid drugs have broad prospects in the field of gene therapy. For example, mRNA drugs release mRNA after reaching the cell and translate the target protein to exert the drug effect. siRNA drugs use RNA interference to silence abnormally expressed genes, opening up new paths for disease treatment. However, nucleases such as DNase and RNase present in the blood and cells in the body can quickly decompose nucleic acid drugs.

[0004] Nanomedicine refers to drug delivery systems designed and fabricated using nanotechnology. These innovative drugs deliver drug molecules precisely to lesions via nanoscale carriers, achieving efficient treatment or diagnosis in vivo. Nanomedicines can improve drug stability and protect protein and nucleic acid drugs from degradation. Protein cages are a promising class of nanomedicine delivery vehicles. They are hollow cage-like structures composed of multiple protein subunits, which self-assemble into monomers. The hollow nanoscale cavities within the cages are used to encapsulate drug molecules. Protein cages offer excellent biocompatibility and low immunogenicity, ensuring safe drug delivery. Their uniform size distribution imparts excellent stability and consistency, ensuring that each carrier delivers the drug with the same efficiency, significantly promoting the standardization and effectiveness of clinical treatments. However, with traditional nanomedicine delivery methods, the rapid release rate of protein cage nanomedicines can lead to excessively high local drug concentrations, shortening the duration of drug action, compromising therapeutic efficacy, and even damaging surrounding normal tissues. Therefore, further in-depth research on protein cages is needed to develop sustained-release nanomedicines. Summary of the Invention

[0005] In view of this, the present application provides a protein cage nanomedicine with a sustained-release effect, a preparation method and an application thereof, to solve the technical problem in the prior art of lack of sustained-release protein cage nanomedicine and difficulty in sustained drug release.

[0006] The first aspect of the present application provides a protein cage nanomedicine with a sustained-release effect, comprising a nanoprotein cage and a biomacromolecule drug;

[0007] The internal cavity of the nanoprotein cage encapsulates the biomacromolecule drug;

[0008] a C-terminal modified protein binding peptide of the protein subunit monomer of the nanoprotein cage;

[0009] The protein-binding peptide can bind to a protein with a half-life of no less than 5 days.

[0010] Preferably, the protein binding peptide is selected from at least one of serum albumin binding peptide, transferrin binding peptide, immunoglobulin G binding peptide, immunoglobulin A binding peptide, and immunoglobulin M binding peptide.

[0011] Preferably, the amino acid sequence of the serum albumin binding peptide is shown in SEQ ID NO.1;

[0012] The amino acid sequence of the Fc-binding peptide Fc-III in the immunoglobulin G (IgG antibody) is as follows:

[0013] DCAWHLGELVWCT.

[0014] The amino acid sequence of domain B and the amino acid sequence of domain C in the Fc binding peptide Protein A in the immunoglobulin G (IgG antibody) are as follows:

[0015] Amino acid sequence of domain B: FDGATNQKTFTVTTAAVDTKKNFTEEGGD;

[0016] Amino acid sequence of domain C: FNKEQQNAFYEILHLPNLNEEQRNGFIQSLK.

[0017] The amino acid sequence of domain C1 in the Fc binding peptide Protein G in the immunoglobulin G (IgG antibody) is as follows:

[0018] YKTTGKTYYTNSAVTATNYKTEE.

[0019] The amino acid sequence of domain B1 in the Fc binding peptide Protein L in the immunoglobulin G (IgG antibody) is as follows:

[0020] VPGVYTYTNGKTYEGYTVTVT.

[0021] Preferably, the biomacromolecule drug is selected from tumor therapeutic anti-PD1 antibodies and / or tumor therapeutic mRNA.

[0022] Preferably, the tumor therapeutic anti-PD1 antibody is selected from pembrolizumab, nivolumab, sintilimab or toripalimab;

[0023] The tumor therapeutic mRNA is selected from mRNA-4157 (V940) or BNT111.

[0024] Preferably, the biomacromolecule drug is selected from the combination immunotherapy of tumor therapeutic mRNA and anti-PD1 antibody.

[0025] Preferably, the nano protein cage is selected from the 240-mer protein cage NatQt, with the PDB number 6NJ8.

[0026] Preferably, the C-terminus of the protein subunit monomer of the nanoprotein cage is modified with a protein purification tag, the amino acid sequence of which is HHHHHH.

[0027] Preferably, the C-terminus of the protein subunit monomer of the nanoprotein cage is modified with a protein binding peptide specifically as follows: the C-terminus of the protein subunit monomer of the nanoprotein cage is modified with a protein binding peptide via a flexible linker.

[0028] The second aspect of the present application provides a method for preparing a protein cage nanomedicine with a sustained-release effect, which can be used to prepare the protein cage nanomedicine with a sustained-release effect described in the first aspect. The preparation method comprises the following steps:

[0029] Step S1: By genetic recombination, the gene encoding the protein binding peptide is fused to the 3' end of the gene encoding the protein subunit monomer of the nanoprotein cage to obtain Qt 结合肽 genes;

[0030] Step S2: Qt 结合肽 The gene was subcloned into a vector plasmid, transformed into competent cells, cultured and amplified, induced to express, self-assembled, and purified to obtain the nanoprotein cage Qt 结合肽 ;

[0031] Step S3: After the nano protein cage is depolymerized, the biomacromolecule drug is added thereto for secondary self-assembly to obtain a protein cage nano drug encapsulating the biomacromolecule drug.

[0032] Preferably, in step S1, the nanoprotein cage is selected from the protein subunit monomer of the nanoprotein cage with PDB number 6NJ8;

[0033] The fusion process of the gene encoding the protein binding peptide and the gene encoding the nanoprotein cage uses flexible linker gene fusion;

[0034] The gene encoding the nanoprotein cage is also fused with a gene encoding a protein purification tag;

[0035] Qt 结合肽 Selected from Qt ABP .

[0036] Preferably, in step S2, the vector plasmid is selected from the pet28a plasmid;

[0037] The competent cells are selected from competent Escherichia coli BL21 DE3;

[0038] The inducing agent used in the induced expression is IPTG;

[0039] The purification is carried out by sequentially using metal affinity chromatography and size exclusion chromatography.

[0040] Preferably, in step S3, the adding of the biomacromolecule drug is adding tumor therapeutic anti-PD1 antibodies and / or tumor therapeutic mRNA.

[0041] The third aspect of the present application provides the use of the protein cage nanomedicine with sustained-release effect described in the first aspect in the preparation of tumor treatment drugs.

[0042] Compared with the prior art, the protein cage nanomedicine with sustained release effect provided by this application has at least the following beneficial effects:

[0043] 1. The C-terminal modified protein binding peptide of the protein subunit monomer of the protein cage nanodrug provided in this application gives the protein cage nanodrug a sustained-release effect. After the protein cage nanodrug with a sustained-release effect enters the body, it can bind to proteins with a long half-life in the body, thereby prolonging the drug's action time, improving the drug's efficacy, and reducing possible drug toxicity and side effects.

[0044] 2. The protein-binding peptide modified by the protein cage nanomedicine provided in this application is a serum albumin binding peptide. Serum albumin is the most abundant endogenous protein in the blood. This makes the protein cage nanomedicine provided by this technical solution have a 10 -5 When the concentration of nM~10nM enters the body, it can bind well to serum albumin, and the half-life of high-concentration protein cage nanomedicines can also be extended. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0046] Figure 1The engineered nanoprotein cage Qt is obtained by fusing the gene encoding the protein binding peptide and the gene encoding the protein purification tag to the gene encoding the nanoprotein cage through gene recombination as provided in Example 1 of the present application. ABP Schematic diagram;

[0047] Figure 2 Nanoprotein cage Qt of different concentrations provided in Example 1 of this application ABP Binding performance test results with human serum albumin;

[0048] Figure 3 Nanoprotein cage Qt of different concentrations provided in Example 1 of this application ABP Binding performance test results with mouse serum albumin;

[0049] Figure 4 The test results of the concentration change of the nanoprotein cage in the mice after the nanoprotein cage encapsulating the protein drug provided in Examples 1-2 of the present application was injected into the mice;

[0050] Figure 5 The test results of the concentration change of the nanoprotein cage in the mice after the nanoprotein cage encapsulating the nucleic acid drug provided in Examples 1-2 of the present application was injected into the mice;

[0051] Figure 6 The test results of the changes in the concentration of protein drugs in mice after the nanoprotein cages encapsulating protein drugs provided in Examples 1-2 of the present application were injected into mice;

[0052] Figure 7 The test results of the changes in the concentration of nucleic acid drugs in mice after the nanoprotein cages encapsulating nucleic acid drugs provided in Examples 1-2 of the present application were injected into mice;

[0053] Figure 8 The test results of the number of leukemia cells in mice after the nanoprotein cages encapsulating protein drugs provided in Examples 1-2 of the present application and PBS were injected into leukemia mice;

[0054] Figure 9 The wet weight test results of mouse tumor tissue after melanoma mice were injected with the nanoprotein cages encapsulating protein drugs provided in Examples 1-2 of the present application and PBS;

[0055] Figure 10 The test results of the number of leukemia cells in mice after injecting leukemia mice with the nanoprotein cages encapsulating nucleic acid drugs provided in Examples 1-2 of the present application and PBS;

[0056] Figure 11The test results of the tumor tissue wet weight of the melanoma mice after being injected with the nano-protein cage encapsulating the nucleic acid drug and PBS provided by Embodiments 1-2 of the present application. DETAILED DESCRIPTION

[0057] The present application provides a protein cage nano-drug with a slow-release effect, a preparation method and application thereof, and solves the technical problem that there is a lack of slow-release protein cage nano-drugs in the prior art and it is difficult to release drugs for a long time.

[0058] The technical solutions of the present application will be described clearly and completely in combination with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0059] In view of the fact that there is a lack of further in-depth research on protein cages to develop protein cage nano-drugs with a slow-release effect, the present technical solution provides a protein cage nano-drug with a slow-release effect. The protein cage nano-drug with a slow-release effect provided by the present technical solution comprises a nano-protein cage and a biological macromolecular drug. The interior cavity of the nano-protein cage encapsulates the biological macromolecular drug, and the C-terminal of the protein subunit monomer of the nano-protein cage is modified with a protein binding peptide.

[0060] The protein cage is a hollow cage structure self-assembled by multiple protein subunit monomers, and the N-terminus of the protein subunit monomers is exposed on the inner surface of the protein cage, and the C-terminus is fully exposed on the outer surface of the cage, so that a functional group can be modified on the N-terminus or C-terminus of the protein subunit monomers as required to achieve active targeting of nanodrugs, environment-responsive release or to achieve sustained release effect; the technical solution modifies a protein binding peptide at the C-terminus of the protein subunit monomers of the protein cage, and the modified protein binding peptide can bind to polypeptides or proteins with a half-life of not less than 5 days in serum, binds to these proteins after the protein cage nanodrug enters the blood, prolongs the half-life of the nanoprotem cage and the biological macromolecular drug, significantly prolongs the drug action time, effectively improves the drug efficacy, and reduces the possible drug toxicity and side effects; it can be seen that after one administration of the protein cage nanodrug with sustained release effect provided by the technical solution, the encapsulated drug can maintain an effective concentration in the body for a long time, and frequent administration is not required, which not only improves the patient's compliance, but also avoids the inconvenience and potential risks caused by frequent administration; for example, in the treatment of chronic diseases, stable and persistent drug release can provide more stable support for disease control, and in terms of improving drug efficacy, stable drug release reduces drug concentration fluctuations, avoids the adverse effects of excessively high or low drug concentrations on treatment effectiveness, and also avoids the toxic side effects that may be caused by excessively high drug concentrations; therefore, the stable sustained release of the protein cage nanodrug with sustained release effect provided by the technical solution ensures that the drug is always within the effective therapeutic window, fully plays the therapeutic role of the drug, and enhances the treatment effect.

[0061] As a preferred technical solution, the modified protein binding peptide in the protein cage nanodrug with sustained release effect provided by the technical solution is selected from at least one of an albumin binding peptide, a transferrin binding peptide, an immunoglobulin G binding peptide, an immunoglobulin A binding peptide, and an immunoglobulin M binding peptide; the half-lives of different proteins / polypeptides in the body are different, for example, the half-lives of insulin and glycoprotein interleukin-6 (IL-6) are relatively short, usually only a few minutes or hours, which do not meet the requirements, while the albumin binding peptide, the transferrin binding peptide, the immunoglobulin G binding peptide, the immunoglobulin A binding peptide, and the immunoglobulin M binding peptide provided by the technical solution can bind to serum albumin with a half-life of 19 days, transferrin with a half-life of 7-10 days, immunoglobulin G with a half-life of 21 days, immunoglobulin A with a half-life of 6 days, and immunoglobulin M with a half-life of 5 days, respectively.

[0062] As a preferred technical solution, in the protein cage nanomedicine with a sustained-release effect provided by the present technical solution, the modified protein binding peptide is further selected from albumin binding peptides. The serum includes albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin and other proteins, of which albumin accounts for about 50~60%, γ-globulin accounts for about 12~20%, and γ-globulin includes immunoglobulin G, immunoglobulin A, and immunoglobulin M. It can be seen that the protein binding peptide provided by the present application is bound to the most abundant endogenous protein in the blood, which enables the protein cage nanomedicine provided by the present technical solution to bind to the corresponding serum albumin and prolong the half-life when entering the body at a low concentration. When entering the body at a high concentration, the high-concentration protein cage nanomedicine can also bind to the corresponding serum albumin, which can prolong the half-life of the high-concentration protein cage nanomedicine and achieve a sustained-release effect by virtue of the circulation characteristics of albumin in the body. Therefore, the present technical solution can prolong the half-life of low-concentration and high-concentration protein cage nanomedicines by selecting specific serum albumin binding peptides; such as Figure 2-3 As shown, the protein cage nanomedicine provided by this technical solution is 10 -5 When entering the body at a concentration of nM~10nM, it can bind to serum albumin well. This application modifies the serum albumin binding peptide to make the effective concentration of protein cage nanomedicine wider.

[0063] As a preferred technical solution, in the protein cage nanodrug with a sustained-release effect provided by this technical solution, the amino acid sequence of the modified albumin-binding peptide (SEQ ID NO. 1) is RLIEDICLPRWGCLWEDDF; serum albumin has multiple binding sites, and the albumin-binding peptide with a specific amino acid sequence provided by this technical solution can bind to a specific structural domain of serum blood protein; at the same time: the sequence length of the albumin-binding peptide is relatively short, making it easy to insert into the surface of the protein cage, and will not affect the self-assembly structure of the protein cage and the stability of the drug packaged therein.

[0064] The protein cage nanomedicine with sustained release effect provided by the present technical solution will be specifically described below with reference to the embodiments and experimental examples.

[0065] Example 1

[0066] This embodiment 1 provides a method for preparing a protein cage nano drug with a sustained release effect, the preparation method comprising preparing a codable nano protein cage Qt ABP The steps of preparing the fusion gene, expressing and purifying the fusion gene, and encapsulating the biomacromolecule drug are described.

[0067] Preparation of programmable nanoprotein cage Qt ABPThe steps of the fusion gene include: based on the 240-mer nano protein cage NatQt with PDB number 6NJ8, the C-terminus of the protein subunit monomer structure is fully exposed on the outer surface of the cage, which can be used to install the properties of albumin binding peptide ABP for engineering transformation. The engineering process is as follows Figure 1 As shown; gene recombination technology was used to fuse the gene encoding albumin binding peptide ABP to the 3' end of the NatQt gene through the flexible linker gene to obtain a fusion gene. The fusion gene encodes the C-terminal modified protein binding peptide ABP of the protein subunit monomer of NatQt (amino acid sequence as shown in SEQ ID NO.1), which was named nanoprotein cage Qt ABP At the same time, in order to facilitate downstream purification, the purification tag 6×His tag (HHHHHH) gene was also fused to the 3' end of the gene encoding the albumin binding peptide ABP, so that Qt ABP The outermost surface presents 240 His tags.

[0068] The steps of expressing and purifying the fusion gene include: encoding the nanoprotein cage Qt ABP The fusion gene sequence was subcloned into the pet28a plasmid, and the plasmid was transformed into competent Escherichia coli BL21 DE3. BL21 DE3 was then amplified using LB medium, and IPTG was added to induce protein expression. The bacterial precipitate was collected, and the E. coli was disrupted by ultrasound. The supernatant was collected by centrifugation. The supernatant contained the nano-protein cages self-assembled by the protein subunits, which were then purified by Co-NTA metal affinity chromatography. ABP The specific binding between the purified surface tags on the surface of the nanoparticles was separated, and then purified by size exclusion chromatography, and the outflow fractions were collected at a specified volume to obtain the purified nanoprotein cage Qt ABP The purified samples were stored at -20°C for long-term storage and used in subsequent experiments.

[0069] The steps of encapsulating biomacromolecule drugs include: adjusting pH to encapsulate nanoprotein cage Qt ABP The protein cage nanomedicine encapsulating the tumor therapeutic anti-PD1 antibody or tumor therapeutic mRNA was obtained by disassembly, followed by the addition of tumor therapeutic anti-PD1 antibody (pembrolizumab) or tumor therapeutic mRNA (mRNA-4157 (V940)), and the pH was adjusted for secondary self-assembly. ABP _IgG4 Pro The protein cage nanomedicine encapsulating tumor therapeutic mRNA is named Qt ABP _IgG4 mRNA .

[0070] Example 2

[0071] This Example 2 provides a method for preparing a protein cage nanomedicine, which serves as a comparative example of Example 1. The preparation method includes the steps of preparing a fusion gene, expressing and purifying the fusion gene, and encapsulating a biomacromolecule drug.

[0072] The steps of preparing the fusion gene include: based on the 240-mer nanoprotein cage NatQt with PDB number 6NJ8, engineering modification is performed by utilizing the property that the C-terminus of its protein subunit monomer structure is fully exposed on the outer surface of the cage and can be used to install a purification tag 6×His tag (HHHHHH); using gene recombination technology to directly fuse the gene encoding the purification tag 6×His tag (HHHHHH) to the 3' end of the gene of the nanoprotein cage to obtain a fusion gene, and after the fusion gene encodes the NatQt protein subunit monomer, the C-terminus is modified with a purification tag, and the nanoprotein cage NatQt is named, and the outermost surface of the nanoprotein cage NatQt presents 240 His tags.

[0073] The steps for expressing and purifying the fusion gene include subcloning the NatQt fusion gene sequence into the pet28a plasmid, transforming the plasmid into competent Escherichia coli BL21 DE3 cells, amplifying the BL21 DE3 cells using LB medium, and then inducing protein expression by adding IPTG. The bacterial pellet is collected, the E. coli cells are disrupted by sonication, and the supernatant is collected by centrifugation. The supernatant contains the nanocages self-assembled from the protein subunits. The supernatant is then purified using Co-NTA metal affinity chromatography, which utilizes the specific binding of cobalt ions to the purification surface tag on the NatQt nanocages for separation. The supernatant is then purified using size exclusion chromatography, and the effluent fraction is collected at a specified volume to obtain the purified NatQt nanocages. The purified sample is stored at -20°C for long-term use in subsequent experiments.

[0074] The steps of encapsulating biomacromolecule drugs include: adjusting the pH to disaggregate the nanoprotein cage NatQt, then adding a tumor therapeutic anti-PD1 antibody (pembrolizumab) or a tumor therapeutic mRNA (mRNA-4157 (V940), and adjusting the pH for secondary self-assembly to obtain a protein cage nanodrug encapsulating the tumor therapeutic anti-PD1 antibody or tumor therapeutic mRNA. The protein cage nanodrug encapsulating the tumor therapeutic anti-PD1 antibody is named Qt_IgG4. Pro The protein cage nanomedicine encapsulating tumor therapeutic mRNA is named Qt_IgG4 mRNA .

[0075] Experimental Example 1

[0076] This experimental example 1 is a nano protein cage Qt provided in Example 1-2. ABPThe nanoprotein cage NatQt was subjected to a performance test, and the performance test was a test of its binding ability with serum albumin.

[0077] The test process includes: -5 nM~10nM nanoprotein cage Qt ABP The nanoprotein cage NatQt was incubated with human serum albumin and mouse serum albumin in vitro, respectively. After centrifugation to remove unbound nanoprotein cages, the absorbance was measured at a wavelength of 450 nm using an ultraviolet spectrophotometer.

[0078] The test results are as follows Figure 2-3 As shown; Figure 2 The concentration is 10 -5 nM~10nM nanoprotein cage Qt ABP and the binding performance test results of the nanoprotein cage NatQt and human serum albumin; Figure 3 The concentration is 10 -5 nM~10nM nanoprotein cage Qt ABP The binding performance test results of nanoprotein cage NatQt and mouse serum albumin; Figure 2-3 It can be seen that the binding ability of the nano protein cage NatQt provided in Example 2 to human serum albumin and mouse serum albumin is relatively weak, and the absorbance measured by ultraviolet spectrophotometer at a wavelength of 450nm is relatively low, while the nano protein cage Qt provided in Example 1 is relatively weak. ABP It has a strong binding ability with human serum albumin and mouse serum albumin, and at a concentration of 10 -5 The nano-protein cage Qt provided in Example 1 can bind to serum albumin in the range of low to high concentrations from nM to 10nM. ABP After encapsulating the drug, it is expected that the half-life of high-concentration protein cage nanomedicines will be extended.

[0079] Experimental Example 2

[0080] In this Experimental Example 2, the performance of the protein cage nanomedicine provided in Example 1-2 was tested. The performance test was an in vivo sustained-release effect test.

[0081] The sustained release effect test of protein cage in vivo during the test process includes: dividing mice into two groups, each group of mice is further divided into two groups, among which Qt ABP _IgG4 Pro The mouse is Qt ABP _IgG4 Pro Group, tail vein injection of Qt ABP _IgG4 mRNA The mouse is Qt ABP _IgG4 mRNA Group, tail vein injection of Qt_IgG4Pro The mice were Qt_IgG4 Pro Group, tail vein injection of Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA Group: After the tail vein injection of 50 μg / mouse protein cage nanodrug, the concentration changes of nano protein cage in mouse serum were regularly detected by ELISA.

[0082] The sustained-release effect test of protein drugs and nucleic acids in vivo included the following steps: mice were divided into two groups, each group of mice was further divided into two groups, among which Qt ABP _IgG4 Pro The mouse is Qt ABP _IgG4 Pro Group, tail vein injection of Qt ABP _IgG4 mRNA The mouse is Qt ABP _IgG4 mRNA Group, tail vein injection of Qt_IgG4 Pro The mice were Qt_IgG4 Pro Group, tail vein injection of Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA Group: After the protein cage nanodrugs were injected into the tail vein, the concentration changes of protein drugs and nucleic acids in the mouse serum were regularly detected by ELISA.

[0083] The results of the sustained release effect test of the protein cage in vivo are as follows Figure 4-5 shown; among them, Figure 4 Nanoprotein cage Qt for encapsulating protein drugs ABP _IgG4 Pro and Qt_IgG4 Pro The test results of the concentration change of nano protein cage in mice after tail vein injection, Figure 5 Nanoprotein cage Qt for encapsulating nucleic acid drugs ABP _IgG4 mRNA and Qt_IgG4 mRNA The concentration change test results of nano protein cages in mice after tail vein injection; Figure 4-5 It can be seen that the concentration of the nanoprotein cage encapsulating protein drugs or nucleic acid drugs provided in Example 2 decreases faster in vivo, while the concentration of the nanoprotein cage encapsulating protein drugs or nucleic acid drugs provided in Example 1 decreases slower in vivo and has a prolonged half-life.

[0084] The results of sustained release test of protein drugs and nucleic acids in vivo are as follows Figure 6-7 shown; among them, Figure 6Nanoprotein cage Qt for encapsulating protein drugs ABP _IgG4 Pro and Qt_IgG4 Pro The test results of the concentration change of protein drugs in mice after tail vein injection, Figure 7 Nanoprotein cage Qt for encapsulating nucleic acid drugs ABP _IgG4 mRNA and Qt_IgG4 mRNA The test results of the concentration change of nucleic acid drugs in mice after tail vein injection; Figure 6-7 It can be seen that the nanoprotein cages encapsulating protein drugs or nucleic acid drugs provided in Example 2 have a faster rate of decrease in the concentration of protein drugs or nucleic acid drugs in vivo, while the nanoprotein cages encapsulating protein drugs or nucleic acid drugs provided in Example 1 have a slower rate of decrease in the concentration of protein drugs or nucleic acid drugs in vivo, and the half-life of the drugs is extended, which is conducive to their continued effect in mice.

[0085] Experimental Example 3

[0086] In this experimental example 3, the protein cage nanomedicine provided in Example 1-2 was subjected to a performance test, and the performance test was a test for the treatment of hematological tumors and solid tumors.

[0087] The blood tumor treatment test process includes: using NALM6 leukemia cells, stably transfected with PDL1 and ffluc, named PDL1-NALM6 ffluc Severely immunodeficient NCG mice were first inoculated with human PBMCs via the tail vein to reshape the human immune system; 14 days later, the mice with reshaped immune systems were injected with PDL1-NALM6 via the tail vein. ffluc , to construct a mouse leukemia model; seven days later, the mouse leukemia model was divided into three groups, among which 50 μg / mouse protein cage nanodrug Qt was injected into the tail vein. ABP _IgG4 Pro The mouse is Qt ABP _IgG4 Pro Group A, tail vein injection of protein cage nanodrug Qt_IgG4 Pro The mice were Qt_IgG4 Pro group, and mice injected with the same volume of sterile PBS through the tail vein served as the negative control group; 13 days later, the number of leukemia cells in mice was observed by small animal in vivo imaging. The more leukemia cells there were, the higher the relative fluorescence intensity was, and the greater the tumor burden of the mice.

[0088] The solid tumor treatment test process includes: using A375 melanoma cells stably transfected with PDL1, named PDL1-A357; C57BL / 6 mice were first subcutaneously injected with PDL1-A375 to establish a mouse melanoma model; 15 days later, the mouse melanoma model was divided into three groups, of which the protein cage nanodrug Qt was injected into the tail vein. ABP _IgG4 Pro The mouse is Qt ABP _IgG4 Pro Group A, tail vein injection of protein cage nanodrug Qt_IgG4 Pro The mice were Qt_IgG4 Pro group, and mice injected with PBS into the tail vein served as the negative control group; 14 days later, the tumor tissues of the mice were collected and the wet weight of the tumors was measured.

[0089] Hematologic malignancy treatment test results and solid tumor treatment test results are as follows Figure 8-9 shown; from Figure 8 It can be seen that the protein cage nano drug Qt_IgG4 was injected into the tail vein Pro Compared with mice injected with protein cage nanoparticles Qt ABP _IgG4 Pro The mice with leukemia cells in the body were less, the relative fluorescence intensity was lower, and the tumor burden was smaller. Figure 9 It can be seen that the protein cage nano drug Qt_IgG4 was injected into the tail vein Pro Compared with mice injected with protein cage nanoparticles Qt ABP _IgG4 Pro The wet weight of melanoma in mice injected with PBS was lower; while the number of leukemia cells in mice injected with PBS was the largest and the wet weight of melanoma was the largest.

[0090] Experimental Example 4

[0091] This Experimental Example 4 performs a performance test on the protein cage nanomedicine provided in Example 1-2, and the performance test is a test for the treatment of hematological tumors and solid tumors.

[0092] The blood tumor treatment test process includes: using NALM6 leukemia cells, stably transfected with PDL1 and ffluc, named PDL1-NALM6ffluc, and first inoculating human PBMCs through the tail vein of severely immunodeficient NCG mice to reshape the human immune system; 14 days later, the mice with reshaped immune systems were injected with PDL1-NALM6ffluc through the tail vein to establish a mouse leukemia model; seven days later, the mouse leukemia model was divided into three groups, of which the protein cage nanodrug Qt was injected into the tail vein. ABP _IgG4 mRNA The mouse is Qt ABP _IgG4 mRNAGroup A, tail vein injection of protein cage nanodrug Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA group, and mice injected with PBS in the tail vein served as the negative control group; 13 days later, the number of leukemia cells in mice was observed by in vivo imaging of small animals. The more leukemia cells there were, the higher the relative fluorescence intensity was, and the greater the tumor burden of the mice.

[0093] The solid tumor treatment test process includes: using A375 melanoma cells stably transfected with PDL1, named PDL1-A357; C57BL / 6 mice were first subcutaneously injected with PDL1-A375 to establish a mouse melanoma model; 15 days later, the mouse melanoma model was divided into three groups, of which the protein cage nanodrug Qt was injected into the tail vein. ABP _IgG4 mRNA The mouse is Qt ABP _IgG4 mRNA Group A, tail vein injection of protein cage nanodrug Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA group, and mice injected with PBS into the tail vein served as the negative control group; 14 days later, the tumor tissues of the mice were collected and the wet weight of the tumors was measured.

[0094] Hematologic malignancy treatment test results and solid tumor treatment test results are as follows Figure 10-11 shown; from Figure 10 It can be seen that the protein cage nano drug Qt_IgG4 was injected into the tail vein mRNA Compared with mice injected with protein cage nanoparticles Qt ABP _IgG4 mRNA The mice with leukemia cells in the body were less, the relative fluorescence intensity was lower, and the tumor burden was smaller. Figure 11 It can be seen that the protein cage nano drug Qt_IgG4 was injected into the tail vein mRNA Compared with mice injected with protein cage nanoparticles Qt ABP _IgG4 mRNA The wet weight of melanoma in mice injected with PBS was lower; while the number of leukemia cells in mice injected with PBS was the largest and the wet weight of melanoma was the largest.

[0095] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A protein cage nanomedicine with sustained release effect, characterized in that: including nanoprotein cages and biomacromolecule drugs; The internal cavity of the nanoprotein cage encapsulates the biomacromolecule drug; a C-terminal modified protein binding peptide of the protein subunit monomer of the nanoprotein cage; The protein-binding peptide can bind to a protein with a half-life of no less than 5 days.

2. The protein cage nanomedicine with sustained release effect according to claim 1, characterized in that: The protein binding peptide is selected from at least one of serum albumin binding peptide, transferrin binding peptide, immunoglobulin G binding peptide, immunoglobulin A binding peptide, and immunoglobulin M binding peptide.

3. The protein cage nanomedicine with sustained release effect according to claim 2, characterized in that: The amino acid sequence of the serum albumin binding peptide is shown in SEQ ID NO.

1.

4. The protein cage nanomedicine with sustained release effect according to claim 1, characterized in that: The biomacromolecule drug is selected from tumor therapeutic anti-PD1 antibodies and / or tumor therapeutic mRNA.

5. The protein cage nanomedicine with sustained release effect according to claim 1, characterized in that: The nano protein cage is selected from the 240-mer protein cage NatQt, with the PDB number 6NJ8.

6. The protein cage nanomedicine with sustained release effect according to claim 1, characterized in that: The C-terminus of the protein subunit monomer of the nanoprotein cage is modified with a protein purification tag.

7. The protein cage nanomedicine with sustained release effect according to claim 1, characterized in that: The C-terminal modified protein binding peptide of the protein subunit monomer of the nano protein cage is specifically: the C-terminal of the protein subunit monomer of the nano protein cage is modified with a flexible linker to form a protein binding peptide.

8. The method for preparing a protein cage nanomedicine with sustained release effect according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step S1: By genetic recombination, the gene encoding the protein binding peptide is fused to the 3' end of the gene encoding the protein subunit monomer of the nanoprotein cage to obtain Qt 结合肽 genes; Step S2: Qt 结合肽 The gene was subcloned into a vector plasmid, transformed into competent cells, cultured and amplified, induced to express, self-assembled, and purified to obtain the nanoprotein cage Qt 结合肽 ; Step S3: After the nano protein cage is depolymerized, the biomacromolecule drug is added thereto for secondary self-assembly to obtain a protein cage nano drug encapsulating the biomacromolecule drug.

9. The method for preparing a protein cage nanomedicine with sustained release effect according to claim 8, characterized in that: In step S2, the vector plasmid is selected from the pet28a plasmid; The competent cells are selected from competent Escherichia coli BL21 DE3; The inducing agent used in the induced expression is IPTG; The purification is carried out by sequentially using metal affinity chromatography and size exclusion chromatography.

10. Use of the protein cage nanomedicine with sustained release effect according to any one of claims 1 to 7 in the preparation of tumor therapeutic drugs.

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