Protein cage nanodrug with sustained-release effect and preparation method and application thereof

Abstract

The application belongs to the technical field of nano drugs, and particularly relates to a protein cage nano drug with a slow-release effect and a preparation method and application thereof. The protein cage nano drug with the slow-release effect is provided in the application, a protein binding peptide is modified at a C end of a protein subunit monomer of a nano protein cage, and is combined with a protein with a long half-life in a body, so that a drug action time is prolonged, a drug efficacy is improved, and a possible drug toxic side effect is weakened. Meanwhile, the protein binding peptide is selected from a serum albumin binding peptide with an amino acid sequence as shown in SEQ ID NO. 1, so that the protein cage nano drug provided in the technical solution can be combined with the most abundant endogenous protein in blood, and the half-life of the high-concentration protein cage nano drug can also be prolonged. The technical problem that there is a lack of slow-release protein cage nano drugs and it is difficult to release drugs for a long time in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine, and particularly relates to a protein cage nanomedicine with a slow-release effect and a preparation method and application thereof. BACKGROUND

[0002] Macromolecular drugs are divided into vaccines, blood and blood products, and polypeptides, proteins, antibodies, polysaccharides and nucleic acids are classified as biological macromolecular drugs according to sources.

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

[0004] Nanomedicine refers to a drug delivery system designed and prepared by using nanotechnology, which precisely delivers drug molecules to lesions through nanoscale carriers, and realizes efficient treatment or diagnosis in vivo, and nanomedicine can improve the stability of drug molecules, protect protein drugs and nucleic acid drugs from degradation; protein cage is a highly potential nanomedicine delivery carrier, protein cage is a hollow cage-like structure self-assembled by multiple protein subunits as monomers, the hollow nanoscale cavity in the cage-like structure is used for packaging drug molecules, meanwhile, protein cage has good biocompatibility and low immunogenicity, ensuring the safety of drug molecule delivery, in addition, the uniform size distribution of protein cage endows it with good stability and consistency, ensuring that each carrier transports drugs with the same efficiency, which effectively promotes the standardization and effectiveness of clinical treatment; however, under the traditional nanomedicine administration mode, the drug release speed of protein cage nanomedicine is too fast, which may lead to excessive local drug concentration, shorten the drug action time, affect the treatment effect, and even cause damage to the surrounding normal tissues, therefore, it is necessary to further study the protein cage to develop a protein cage nanomedicine with a slow-release effect. SUMMARY

[0005] Therefore, the application provides a protein cage nanomedicine with a slow-release effect and a preparation method and application thereof, which solves the technical problem that there is a lack of slow-release protein cage nanomedicine in the prior art, and it is difficult to release drugs for a long time.

[0006] The first aspect of the application provides a protein cage nanodrug with a sustained-release effect, comprising a nanometer protein cage and a biological macromolecular drug;

[0007] The internal cavity of the nanometer protein cage encapsulates the biological macromolecular drug;

[0008] The C-terminal of the protein subunit monomer of the nanometer protein cage is modified with a protein binding peptide;

[0009] The protein binding peptide can bind to a protein with a half-life of not less than 5 days.

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

[0011] Preferably, the amino acid sequence of the serum albumin binding peptide is as 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 sequences of domain B and domain C of the Fc binding peptide Protein A in the immunoglobulin G (IgG antibody) are as follows:

[0015] The amino acid sequence of domain B is: FDGATNQKTFTVTTAAVDTKKNFTEEGGD.

[0016] The amino acid sequence of domain C is: FNKEQQNAFYEILHLPNLNEEQRNGFIQSLK.

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

[0020] VPGVYTYTNGKTYEGYTVTVT.

[0021] Preferably, the biological macromolecular drug is selected from a tumor therapeutic anti-PD1 antibody and / or a tumor therapeutic mRNA.

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

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

[0024] Preferably, the biological macromolecular drug is selected from the group consisting of tumor therapeutic mRNA and anti-PD1 antibody for immunotherapy combination.

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

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

[0027] Preferably, the C-terminal of the protein subunit monomer of the nano protein cage is modified with a protein binding peptide, and the C-terminal of the protein subunit monomer of the nano protein cage is modified with a protein binding peptide through a flexible Linker.

[0028] The second aspect of the present application provides a preparation method of a protein cage nano drug with sustained release effect, which can prepare the protein cage nano drug with sustained release effect of the first aspect. The preparation method comprises the following steps:

[0029] Step S1, fusing a gene encoding a protein binding peptide to the 3' end of a gene encoding a protein subunit monomer of a nano protein cage through genetic recombination to obtain a gene encoding a nano protein cage Qt 结合肽 ;

[0030] Step S2, subcloning the gene encoding Qt 结合肽 to a vector plasmid in sequence, transforming to a competent cell, cell culture amplification, induction of expression, self-assembly, and purification to obtain a nano protein cage Qt 结合肽 ;

[0031] Step S3, adding a biological macromolecular drug to the nano protein cage after depolymerization to perform secondary self-assembly to obtain a protein cage nano drug encapsulating the biological macromolecular drug inside.

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

[0033] The fusion process of the gene encoding the protein binding peptide and the gene encoding the nano protein cage uses a flexible Linker gene fusion;

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

[0035] the Qt 结合肽 selected from the group consisting of Qt ABP .

[0036] Preferably, in step S2, the carrier plasmid is selected from a pet28a plasmid.

[0037] The competent cells are selected from competent E. coli BL21 DE3.

[0038] The inducer used for inducing expression is IPTG.

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

[0040] Preferably, in step S3, the biological macromolecular drug added is a tumor therapeutic anti-PD1 antibody and / or a tumor therapeutic mRNA.

[0041] The third aspect of the present application provides a use of the protein cage nanodrug with a sustained-release effect in the preparation of a tumor treatment drug.

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

[0043] 1. The C-terminal of the protein subunit monomer of the protein cage nanodrug provided by the present application is modified with a protein binding peptide, so that the protein cage nanodrug has a sustained-release effect. After the protein cage nanodrug with a sustained-release effect enters the body, it can be combined with a protein with a long half-life in the body, thereby prolonging the drug action time, improving the drug efficacy, and reducing possible drug toxicity and side effects.

[0044] 2. The protein binding peptide modified by the protein cage nanodrug provided by the present application is a serum albumin binding peptide. Serum albumin is the most abundant endogenous protein in blood, which makes the protein cage nanodrug provided by the present technical solution be able to combine with serum albumin at a concentration of 10 -5 nM~10nM when entering the body, and the half-life of the high-concentration protein cage nanodrug can also be prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0046] Figure 1The genetically recombined gene encoding the protein binding peptide and the gene encoding the protein purification tag are fused to the gene encoding the nanopro protein cage Qt ABP schematic diagram

[0047] Figure 2 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application ABP and human serum albumin binding performance test results

[0048] Figure 3 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application ABP and mouse serum albumin binding performance test results

[0049] Figure 4 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0050] Figure 5 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0051] Figure 6 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0052] Figure 7 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0053] Figure 8 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0054] Figure 9 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[0055] Figure 10 The nanopro protein cage Qt of different concentrations provided in Example 1 of the present application

[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 exerts the therapeutic effect 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 sustained-release effect provided by this technical solution, the modified protein-binding peptide is further selected from albumin-binding peptides. Serum includes various proteins such as albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin, with albumin accounting for approximately 50-60% and γ-globulin accounting for approximately 12-20%. γ-globulin includes immunoglobulin G, immunoglobulin A, and immunoglobulin M. It is evident that the protein-binding peptide provided by this application binds to the most abundant endogenous proteins in the blood. This allows the protein cage nanomedicine provided by this technical solution to bind to the corresponding serum albumin and prolong its half-life when entering the body at low concentrations. Similarly, when entering the body at high concentrations, the high-concentration protein cage nanomedicine can also bind to the corresponding serum albumin, prolonging the half-life of the high-concentration protein cage nanomedicine. Furthermore, by utilizing the circulatory characteristics of albumin in the body, a sustained-release effect is achieved. Therefore, this technical solution, by selecting specific serum albumin-binding peptides, can prolong the half-life of both low-concentration and high-concentration protein cage nanomedicines. Figures 2-3 As shown, the protein cage nanomedicine provided by this technical solution has a concentration of 10... -5 When these concentrations of nM to 10nM enter the body, they can bind well to serum albumin. This application modifies the serum albumin-binding peptide to broaden the effective concentration of the protein cage nanomedicine.

[0063] As a preferred technical solution, in the protein cage nanomedicine with sustained-release effect provided by this technical solution, the amino acid sequence (SEQ ID NO.1) of the modified albumin-binding peptide 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 albumin; 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 without affecting the self-assembly structure of the protein cage and the stability of the packaged drug.

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

[0065] Example 1

[0066] This embodiment 1 provides a method for preparing a protein cage nanomedicine with a sustained-release effect. The preparation method includes preparing a codeable nanoprotein cage Qt. ABP The steps involved in gene fusion, gene expression and purification, and encapsulation of biological macromolecular drugs.

[0067] Preparation of codeable nanoprotein cages Qt ABPThe steps of fusing the gene include: based on the 240-mer nanocage NatQt with PDB number 6NJ8, the C-terminal of the protein subunit monomer structure of NatQt is fully exposed on the outer surface of the cage, which can be used for installation of the albumin binding peptide ABP, and the engineering process is as shown in Figure 1 The gene encoding the albumin binding peptide ABP is fused to the 3' end of the gene of NatQt by a flexible Linker gene, to obtain a fusion gene, which encodes a modified protein binding peptide ABP (amino acid sequence as shown in SEQ ID NO. 1) at the C-terminal of the protein subunit monomer of NatQt after coding, and is named as nanocage Qt ABP ; and in order to facilitate downstream purification, a purification tag 6×His tag (HHHHHH) gene is also fused to the 3' end of the gene encoding the albumin binding peptide ABP, so that the Qt ABP outermost surface presents 240 His tags.

[0068] The steps of expressing and purifying the fusion gene include: subcloning the fusion gene sequence that can encode the nanocage Qt ABP into a pet28a plasmid, then transforming the plasmid into competent Escherichia coli BL21 DE3, followed by amplifying the BL21 DE3 using LB medium, then adding IPTG for protein induction expression, collecting bacterial precipitate, ultrasonically crushing the Escherichia coli, and centrifuging to collect the supernatant, wherein the supernatant contains the nanocage obtained by self-assembly of the protein subunit, then purifying it by Co-NTA metal affinity chromatography, and separating it by specific binding between cobalt ions and the purification surface tag on the Qt ABP , and then purifying it by size exclusion chromatography, and collecting the effluent components in a specified volume to obtain the purified nanocage Qt ABP . The purified sample is stored at -20℃ for a long time for subsequent experiments.

[0069] The steps of encapsulating biological macromolecular drugs include: adjusting the pH to depolymerize the nanocage Qt ABP , 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 the tumor therapeutic mRNA in the inside, the protein cage nanodrug encapsulating the tumor therapeutic anti-PD1 antibody is named as Qt ABP _IgG4 Pro , and the protein cage nanodrug encapsulating the tumor therapeutic mRNA is named as Qt ABP _IgG4 mRNA .

[0070] Example 2

[0071] The present embodiment 2 provides a preparation method of a protein cage nanodrug, which is a comparative example of embodiment 1. The preparation method comprises the steps of preparing a fusion gene, expressing and purifying the fusion gene, and encapsulating a biological macromolecular drug.

[0072] The step of preparing the fusion gene comprises the following steps: based on the 240-mer nano protein cage NatQt with PDB number 6NJ8, the C-terminus of the protein subunit monomer structure of the nano protein cage is fully exposed on the outer surface of the cage, which can be used for engineering modification of the property of installing a purification tag 6×His tag (HHHHHH); the gene encoding the purification tag 6×His tag (HHHHHH) is directly fused to the 3' end of the gene of the nano protein cage by using genetic recombination technology to obtain a fusion gene. The fusion gene codes for the C-terminus of the protein subunit monomer of the modified NatQt purification tag, which is named as the nano protein cage NatQt. The outermost surface of the nano protein cage NatQt presents 240 His tags.

[0073] The step of expressing and purifying the fusion gene comprises the following steps: the fusion gene sequence of the nano protein cage NatQt is subcloned into a pet28a plasmid, and then the plasmid is transformed into competent Escherichia coli BL21 DE3. Subsequently, the BL21 DE3 is amplified by using LB medium, and then IPTG is added for protein induction expression. The bacterial precipitate is collected, and the Escherichia coli is broken by ultrasonic. The supernatant is collected by centrifugation. The nano protein cage obtained by self-assembly of the protein subunit is contained in the supernatant. Then, the nano protein cage is purified by using Co-NTA metal affinity chromatography. The specific binding between cobalt ions and the purification surface tag on the nano protein cage NatQt is used to realize separation. Then, the nano protein cage NatQt is purified by using size exclusion chromatography. The elution components are collected in a specified volume. The purified nano protein cage NatQt is obtained. The purified sample is stored at -20°C for a long time and used for subsequent experiments.

[0074] The step of encapsulating a biological macromolecular drug comprises the following steps: the nano protein cage NatQt is depolymerized by adjusting the pH, and then a tumor therapeutic anti-PD1 antibody (pembrolizumab) or a tumor therapeutic mRNA (mRNA-4157 (V940) is added. The pH is adjusted for secondary self-assembly. The protein cage nanodrug encapsulating the tumor therapeutic anti-PD1 antibody or the tumor therapeutic mRNA is obtained. The protein cage nanodrug encapsulating the tumor therapeutic anti-PD1 antibody is named as Qt_IgG4 Pro , and the protein cage nanodrug encapsulating the tumor therapeutic mRNA is named as Qt_IgG4 mRNA .

[0075] Experimental example 1

[0076] In this experimental example 1, the nano protein cage Qt ABPNatQt were tested for performance, and the performance test was a serum albumin binding ability test.

[0077] The test process included: the nanoprotem cage Qt -5 was incubated with human serum albumin and mouse serum albumin in vitro at a concentration of 10 ABP nM~10nM, and after centrifugation to remove unbound nanoprotem cages, the absorbance was measured at 450 nm using a UV spectrophotometer.

[0078] The test results are shown in Figures 2-3 ; Figure 2 The binding performance test results of the nanoprotem cage Qt -5 and the nanoprotem cage NatQt with human serum albumin at a concentration of 10 ABP nM~10nM; Figure 3 The binding performance test results of the nanoprotem cage Qt -5 and the nanoprotem cage NatQt with mouse serum albumin at a concentration of 10 ABP nM~10nM; Figures 2-3 It can be seen that the nanoprotem cage NatQt provided in Example 2 has weaker binding ability with human serum albumin and mouse serum albumin, and the absorbance measured at 450 nm by the UV spectrophotometer is lower, while the nanoprotem cage Qt ABP provided in Example 1 has stronger binding ability with human serum albumin and mouse serum albumin, and can bind with serum albumin at a low concentration of 10 -5 nM~10nM to a high concentration, so that the nanoprotem cage Qt ABP provided in Example 1 is expected to prolong the half-life of high-concentration nanoprotem drug after encapsulating drugs.

[0079] Experimental Example 2

[0080] This experimental example 2 tests the performance of the nanoprotem drug provided in Examples 1-2, and the performance test is an in vivo sustained release effect test.

[0081] The in vivo sustained release effect test of the nanoprotem cage during the test includes: the mice are divided into two groups, and each group is divided into two groups, wherein the mice injected with Qt ABP _IgG4 Pro in the tail vein are Qt ABP _IgG4 Pro group, and the mice injected with Qt ABP _IgG4 mRNA in the tail vein are Qt ABP _IgG4 mRNA group, and the mice injected with Qt_IgG4Pro The mice were Qt_IgG4 Pro Group 1, Qt_IgG4 injected via tail vein mRNA The mice were Qt_IgG4 mRNA Group; after tail vein injection of 50 μg / mouse of protein cage nanomedicine, the concentration of nanoprotein cages in mouse serum was detected periodically by ELISA.

[0082] The testing process for the sustained-release effects of protein drugs and nucleic acid drugs in vivo included: dividing mice into two large groups, and then further dividing each group into two subgroups. In one subgroup, mice were injected via tail vein with Qt. ABP _IgG4 Pro The mice are Qt ABP _IgG4 Pro Group, tail vein injection of Qt ABP _IgG4 mRNA The mice are Qt ABP _IgG4 mRNA Group 1, Qt_IgG4 injected via tail vein Pro The mice were Qt_IgG4 Pro Group 1, Qt_IgG4 injected via tail vein mRNA The mice were Qt_IgG4 mRNA Group; after tail vein injection of protein cage nanomedicine, the concentration changes of protein drugs and nucleic acids in mouse serum were detected periodically by ELISA.

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

[0084] Results of in vivo sustained-release effect tests for protein drugs and nucleic acid drugs are as follows: Figures 6-7 As shown; where, Figure 6Nano protein cage Qt for encapsulating protein drugs ABP _IgG4 Pro and Qt_IgG4 Pro The test results of the concentration change of the protein drugs in the mice after tail vein injection of the mice, Figure 7 Nano protein cage Qt for encapsulating nucleic acid drugs ABP _IgG4 mRNA and Qt_IgG4 mRNA The test results of the concentration change of the nucleic acid drugs in the mice after tail vein injection of the mice; from Figures 6-7 It can be seen that the nano protein cage for encapsulating protein drugs or nucleic acid drugs provided in Example 2 has a faster concentration decrease rate of the protein drugs or nucleic acid drugs in the body, while the nano protein cage for encapsulating protein drugs or nucleic acid drugs provided in Example 1 has a slower concentration decrease rate of the protein drugs or nucleic acid drugs in the body, and the half-life of the drugs is prolonged, which is beneficial to the continuous effect in the mice.

[0085] Experimental Example 3

[0086] The performance test of the protein cage nano drug provided in Examples 1-2 is carried out in this experimental example 3, and the performance test is a blood tumor and solid tumor treatment test.

[0087] The blood tumor treatment test process includes: using NALM6 leukemia cells, stably transfected with PDL1 and ffluc, named PDL1-NALM6 ffluc , a severe immunodeficient NCG mouse is first inoculated with human PBMCs via tail vein to reconstruct the human immune system; 14 days later, the mouse with a reconstructed immune system is injected with PDL1-NALM6 ffluc via tail vein to construct a mouse leukemia model; seven days later, the mouse leukemia model is divided into three groups, wherein the mouse injected with 50 μg of protein cage nano drug Qt ABP _IgG4 Pro per mouse via tail vein is the Qt ABP _IgG4 Pro group, the mouse injected with protein cage nano drug Qt_IgG4 Pro per mouse via tail vein is the Qt_IgG4 Pro group, and the mouse injected with the same volume of sterile PBS via tail vein is the negative control group; 13 days later, the number of leukemia cells in the mouse is observed by small animal live imaging, and the more the number of leukemia cells, the higher the relative fluorescence intensity, and the greater the tumor burden of the mouse.

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

[0089] The blood tumor treatment test results and the solid tumor treatment test results are as shown in Figures 8-9 ; it can be seen from Figure 8 that, compared with the mice injected with the protein cage nanodrug Qt_IgG4 Pro in the tail vein, the mice injected with the protein cage nanodrug Qt ABP _IgG4 Pro in the tail vein have fewer leukemia cells, lower relative fluorescence intensity, and smaller tumor load; it can be seen from Figure 9 that, compared with the mice injected with the protein cage nanodrug Qt_IgG4 Pro in the tail vein, the mice injected with the protein cage nanodrug Qt ABP _IgG4 Pro have lower melanoma wet weight; and the mice injected with PBS in the tail vein have the most leukemia cells and the largest melanoma wet weight.

[0090] Experimental Example 4

[0091] The protein cage nanodrug provided in Examples 1-2 is tested for performance in this experimental example 4, and the performance test is a blood tumor and solid tumor treatment test.

[0092] The blood tumor treatment test process includes: using NALM6 leukemia cells, stably transfected with PDL1 and ffluc, named PDL1-NALM6ffluc, and first injecting human PBMC into the tail vein of a severe immunodeficient NCG mouse to reconstruct a human immune system; 14 days later, the mouse with the reconstructed immune system is injected with PDL1-NALM6ffluc in the tail vein to construct a mouse leukemia model; seven days later, the mouse leukemia model is divided into three groups, wherein the mice injected with the protein cage nanodrug Qt ABP _IgG4 mRNA in the tail vein are Qt ABP _IgG4 mRNAGroup, tail vein injection of protein cage nanomedicine Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA In the control group, mice injected with PBS via the tail vein served as the negative control group. After 13 days, the number of leukemia cells in mice was observed by in vivo imaging of small animals. The higher the number of leukemia cells, the higher the relative fluorescence intensity, and the greater the tumor burden in the mice.

[0093] The solid tumor treatment test process included: using A375 melanoma cells, which were stably transfected with PDL1 and 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, among which, the protein cage nanomedicine Qt was injected via tail vein. ABP _IgG4 mRNA The mice are Qt ABP _IgG4 mRNA Group, tail vein injection of protein cage nanomedicine Qt_IgG4 mRNA The mice were Qt_IgG4 mRNA In the control group, mice injected with PBS via the tail vein served as the negative control group; 14 days later, tumor tissues were collected from the mice, and the wet weight of the tumors was measured.

[0094] Results of hematologic malignancy treatment trials and results of solid tumor treatment trials, as follows Figures 10-11 As shown; from Figure 10 It can be seen that, compared with tail vein injection of the protein cage nanomedicine Qt_IgG4 mRNA Compared to mice, tail vein injection of the protein cage nanodrug Qt ABP _IgG4 mRNA The mice showed fewer leukemia cells, lower relative fluorescence intensity, and smaller tumor burden; Figure 11 It can be seen that, compared with tail vein injection of the protein cage nanomedicine Qt_IgG4 mRNA Compared to mice, tail vein injection of the protein cage nanodrug Qt ABP _IgG4 mRNA In mice, the wet weight of melanoma was lower; while mice injected with PBS via the tail vein had the highest number of leukemia cells and the largest wet weight of melanoma.

[0095] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A protein cage nanodrug with a sustained release effect, characterized in that, The nano-protein cage and a biological macromolecular drug are included; An internal cavity of the nano-protein cage encapsulates the biological macromolecular drug; A C-terminal of a protein subunit monomer of the nano-protein cage is modified with a protein binding peptide; The protein binding peptide can bind to a protein with a half-life of no less than 5 days; The protein binding peptide is selected from a serum albumin binding peptide; An amino acid sequence of the serum albumin binding peptide is shown in SEQ ID NO.

1. 2.The protein cage nanodrug with sustained-release effect according to claim 1, characterized in that, The biological macromolecular drug is selected from a tumor therapeutic anti-PD1 antibody and / or a tumor therapeutic mRNA. 3.The protein cage nanodrug with sustained-release effect according to claim 1, characterized in that, The nano-protein cage is selected from a 240-mer protein cage NatQt, with a PDB number of 6NJ8. 4.The protein cage nanodrug with sustained-release effect according to claim 1, characterized in that, A C-terminal of a protein subunit monomer of the nano-protein cage is modified with a protein purification tag.

5. The protein cage nanodrug with sustained release effect according to claim 1, characterized in that, The C-terminal of the protein subunit monomer of the nano-protein cage is modified with a protein binding peptide through a flexible Linker.

6. The method for preparing the protein cage nanodrug with sustained release effect according to any one of claims 1-5, characterized in that, The method comprises the following steps: Step S1, fusing the gene encoding the protein binding peptide to the 3' end of the gene encoding the protein subunit monomer of the nanoprotem cage by genetic recombination, to obtain Qt 结合肽 gene; Step S2, subclone the genes of Qt 结合肽 in turn into a vector plasmid, transform into competent cells, cell culture amplification, induction of expression, self-assembly, purification, obtain nano protein cage Qt 结合肽 ; Step S3, after the nano-protein cage is depolymerized, a biological macromolecular drug is added for secondary self-assembly to obtain a protein cage nano-drug encapsulating the biological macromolecular drug inside.

7. The method for preparing a protein cage nanomedicine with a sustained-release effect according to claim 6, characterized in that, In step S2, the carrier plasmid is selected from a pet28a plasmid; The competent cell is selected from a competent Escherichia coli BL21 DE3; The inducer used for inducing expression is IPTG; The purification is performed by sequentially using metal affinity chromatography and size exclusion chromatography.

8. Use of a protein cage nano-drug with a sustained release effect according to any one of claims 1-5 in the preparation of a tumor therapeutic drug.

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