A protein cage nanodrug targeting t cells 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 targeting T cells and a preparation method and application thereof; the protein cage nano drug targeting T cells provided by the application is modified by a specific nucleic acid binding peptide at an N terminal and a CD8 VHH at a C terminal; the encapsulated nucleic acid drug can be precisely targeted and delivered to T cells to kill malignant tumor cells, and the memory effect of the T cells can also reduce the risk of recurrence of malignant tumors; the coupling efficiency of the process of coupling modification of the CD8 VHH is high, the irreversible amide bond formed by coupling enables the CD8 VHH to be stably connected to the C terminal of the protein cage, and the specific nucleic acid binding peptide of the specific amino acid sequence improves the effect of the encapsulated nucleic acid drug; and thus the technical problem of the lack of the protein cage nano drug targeting T cells in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanomedicine, and particularly relates to a protein cage nanomedicine targeting T cells 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 in clinic according to sources.

[0003] Nucleic acid drugs in biological macromolecular drugs have broad prospects in the field of gene therapy. For example, mRNA drugs release mRNA after reaching cells, and translate target proteins to exert drug effects. siRNA drugs silence abnormal gene expression by RNA interference, opening up a new path for disease treatment. However, DNase and RNase existing in blood and cells in vivo can quickly decompose nucleic acid drugs.

[0004] Nanomedicine refers to a drug delivery system designed and prepared by nanotechnology, which precisely delivers drug molecules to lesions through nanoscale carriers, and realizes efficient treatment or diagnosis in vivo. Nanomedicine can improve the stability of drug molecules and protect nucleic acid drugs from degradation. Lipid nanoparticles and polymer nanoparticles are delivery tools for nanomedicine. However, these delivery tools generally have the defect of insufficient targeting. They are difficult to precisely deliver mRNA to specific cell types, resulting in inaccurate distribution of drugs in vivo, and some drugs cannot reach the intended target, which limits the further development and application of nucleic acid drugs. In addition to lipid nanoparticles and polymer nanoparticles, protein cages are also an important nanomedicine delivery tool. Protein cages are hollow cage-like structures self-assembled by multiple protein subunits as monomers. The hollow nanoscale cavity in the cage-like structure is used to package drug molecules, overcoming the defect that nucleic acid drugs are easily degraded by nucleases. Meanwhile, protein cages have good biocompatibility and low immunogenicity, ensuring the safety of drug molecule delivery. In addition, the regular space inside the protein cage is suitable for different nucleic acid molecules, realizing efficient encapsulation and delivery of nucleic acid drugs and providing key technical support for gene therapy. It can be seen that protein cages are a kind of extremely potential delivery tool for nucleic acid drugs. However, the traditional nanomedicine administration mode generally has the defect of insufficient targeting, which makes it difficult for nanomedicine to precisely deliver drugs to specific cell types, resulting in inaccurate distribution of drugs in vivo, and some drugs cannot reach the intended target, which limits the further development and application of nanomedicine. SUMMARY

[0005] Therefore, the present application provides a protein cage nanomedicine targeting T cells and a preparation method and application thereof, to solve the technical problem of lack of protein cage nanomedicine targeting T cells in the prior art.

[0006] The first aspect of the application provides a T cell targeted protein cage nanomedicine, comprising a nanoprotem cage and a CAR mRNA;

[0007] The N-terminus of the protein subunit monomer of the nanoprotem cage is modified with a specific nucleic acid binding peptide;

[0008] The internal cavity of the nanoprotem cage encapsulates the CAR mRNA;

[0009] The C-terminus of the protein subunit monomer of the nanoprotem cage is modified with a T cell targeted nanobody.

[0010] Preferably, the T cell targeted nanobody is selected from CD8 VHH.

[0011] Preferably, the C-terminus of the protein subunit monomer of the nanoprotem cage is modified with a T cell targeted nanobody, specifically: the C-terminus of the protein subunit monomer of the nanoprotem cage is connected with SpyTag, the N-terminus of CD8 VHH is connected with SpyCatcher, and the covalent binding of SpyTag and SpyCatcher makes the C-terminus of the protein subunit monomer of the nanoprotem cage modified with a T cell targeted nanobody.

[0012] Preferably, the amino acid sequence of the specific nucleic acid binding peptide is shown in SEQ ID NO. 1.

[0013] Preferably, the CAR mRNA is selected from CD19CAR mRNA.

[0014] Preferably, the nanoprotem cage is selected from the nanoprotem cage with PDB number 6NJ8.

[0015] Preferably, the C-terminus of the protein subunit monomer of the nanoprotem cage is connected with a protein purification tag, and the amino acid sequence of the protein purification tag is HHHHHH.

[0016] The second aspect of the application provides a T cell targeted protein cage nanomedicine, which can be prepared according to the first aspect of the application; the preparation method comprises the following steps:

[0017] Fusing the gene encoding the specific nucleic acid binding peptide to the 5' end of the gene encoding the protein subunit monomer of the nanoprotem cage, and fusing the gene encoding SpyTag to the 3' end, to obtain a nanoprotem cage fusion gene;

[0018] Subcloning the nanoprotem cage fusion gene and the CAR mRNA to the downstream of the first promoter and the downstream of the second promoter of the double promoter plasmid in turn, transforming into competent cells, cell culture amplification, induction of co-expression, self-assembly, and purification to obtain a nanoprotem cage encapsulating CAR mRNA.

[0019] fusing a gene encoding SpyCatcher at the 3' end of the gene to a gene encoding a nanobody targeting T cells at the 5' end of the gene to obtain a nanobody fusion gene;

[0020] subcloning the nanobody fusion gene into a vector plasmid in turn, transforming into competent cells, cell culture amplification, inducing expression, purification to obtain a nanobody;

[0021] carrying out a coupling reaction on the nanobody and the nanoprotein cage encapsulating CAR mRNA to obtain a protein cage nanodrug targeting T cells.

[0022] Preferably, in the process of obtaining the nanoprotein cage encapsulating CAR mRNA through genetic engineering, the 3' end of the gene encoding SpyTag of the nanoprotein cage fusion gene is fused with a gene encoding a protein purification tag.

[0023] Preferably, the double-promoter plasmid is selected from pACYCDuet 1;

[0024] The competent cells are selected from competent Escherichia coli BL21 (DE3);

[0025] The culture medium used in the cell culture amplification is LB culture medium;

[0026] The inducer used in the induction of co-expression is an inducer acting on the lactose operon, and the inducer is selected from IPTG;

[0027] The purification is carried out by using metal affinity chromatography and size exclusion chromatography in turn.

[0028] Preferably, in the process of obtaining the nanobody fusion gene through genetic engineering, the 3' end of the gene encoding SpyCatcher of the nanobody fusion gene is fused with a gene encoding a protein purification tag.

[0029] The vector plasmid is selected from pET-28a (+) plasmid;

[0030] The competent cells are selected from competent Escherichia coli BL21 DE3;

[0031] The culture medium used in the cell culture amplification is LB culture medium;

[0032] The inducer used in the induction of expression is IPTG;

[0033] The purification is carried out by using metal affinity chromatography and size exclusion chromatography in turn.

[0034] Preferably, the coupling reaction process is as follows: the nanoprotein cage encapsulating the CAR mRNA and the nanobody are mixed in a TBST buffer solution, and the coupling reaction is carried out at 4°C overnight to obtain the protein cage nanodrug targeting T cells.

[0035] The third aspect of the present application provides an application of the protein cage nanodrug targeting T cells in the first aspect in the preparation of a tumor treatment drug.

[0036] Compared with the prior art, the protein cage nanodrug targeting T cells provided by the present application has at least the following beneficial effects:

[0037] 1. The protein cage nanodrug targeting T cells provided by the present application can efficiently and specifically encapsulate the target mRNA by modifying the specific nucleic acid binding peptide at the N-terminus of the protein subunit monomer of the protein cage and co-expressing the gene encoding the protein cage and the gene encoding the CD19CAR mRNA, thus simplifying the complicated production process of the traditional protein cage nanodrug which needs to synthesize the nucleic acid drug before encapsulation; at the same time, the CD8 VHH is modified at the C-terminus of the protein subunit monomer of the protein cage, so that it can accurately target the encapsulated nucleic acid drug to T cells for killing malignant tumor cells, and utilize the specific cells targeted and the memory effect of T cells to reduce the risk of malignant tumor recurrence.

[0038] 2. The protein cage nanodrug targeting T cells provided by the present application improves the stability of the CD8 VHH by forming an irreversible amide bond between the SpyTag and the SpyCatcher during the coupling; at the same time, the coupling process is simple and the coupling efficiency is as high as more than 90%.

[0039] 3. The protein cage nanodrug targeting T cells provided by the present application has the amino acid sequence (SEQ ID NO. 1) of the specific nucleic acid binding peptide GNAKTRRRERRAEKQAQWKAAN, which can specifically bind to the CD19CAR mRNA, so that the protein cage nanodrug encapsulates more CD19CAR mRNA in the internal cavity. BRIEF DESCRIPTION OF DRAWINGS

[0040] 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.

[0041] Figure 1A process diagram for preparing a nano-protein cage encapsulating CAR mRNA by engineering the inner surface of the protein cage for Example 1 of the present application is shown in FIG. 1A.

[0042] Figure 2 A process diagram for preparing a T cell targeted protein cage nanodrug with CD8 VHH coupled to the outer surface of the protein cage for Example 1 of the present application is shown in FIG. 1B.

[0043] Figure 3 SpyTag-Qt_CAR provided for Example 1 of the present application mRNA Electrophoresis diagram of coupling of SpyCatcher-CD8 VHH;

[0044] Figure 4 Test results of the performance of the protein cage nanodrug in targeting T cells in vitro provided for Examples 1-2 of the present application are shown in FIG. 2A.

[0045] Figure 5 Test results of the killing of hematological tumor cells by the protein cage nanodrug in vitro provided for Examples 1-2 of the present application are shown in FIG. 2B.

[0046] Figure 6 Test results of the killing of solid tumor cells by the protein cage nanodrug in vitro provided for Examples 1-2 of the present application are shown in FIG. 2C.

[0047] Figure 7 Test results of the performance of the protein cage nanodrug in targeting T cells in vivo provided for Examples 1-2 of the present application are shown in FIG. 3A.

[0048] Figure 8 Test results of the killing of hematological tumor cells by the protein cage nanodrug in vivo provided for Examples 1-2 of the present application are shown in FIG. 3B.

[0049] Figure 9 Test results of the killing of solid tumor cells by the protein cage nanodrug in vivo provided for Examples 1-2 of the present application are shown in FIG. 3C. DETAILED DESCRIPTION

[0050] The present application provides a T cell targeted protein cage nanodrug, as well as a preparation method and applications thereof, to solve the technical problem of the lack of T cell targeted protein cage nanodrugs in the prior art.

[0051] The technical solutions of the present application will be described clearly and completely below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0052] In view of the current lack of protein cage nanomedicines targeting T cells, the present application provides a protein cage nanomedicine targeting T cells, which is composed of a nano protein cage and a CAR mRNA; wherein the N-terminus of the protein subunit monomer of the nano protein cage is modified with a specific nucleic acid binding peptide, so that the internal cavity of the nano protein cage encapsulates the CAR mRNA, and the C-terminus of the protein subunit monomer of the nano protein cage is modified with a nano antibody targeting T cells.

[0053] Traditional nanomedicine administration methods, such as ferritin cage nanomedicines, take advantage of the fact that malignant tumor cells require a large amount of iron due to rapid proliferation, and the expression of transferrin receptor TfR1 on their surface is significantly up-regulated, allowing ferritin cage nanomedicines to target and deliver the drugs encapsulated in their internal cavities to malignant tumor cells by binding to TfR1. However, such protein cages are limited in their ability to encapsulate nucleic acid drugs due to the negative charge of the internal cavity. In addition, ferritin cage nanomedicines directly target malignant tumor cells, which can inhibit tumor growth or induce apoptosis, but due to the lack of memory effect, malignant tumors can recur. Therefore, although ferritin cage nanomedicines have natural targeting properties, they are not ideal for encapsulating CAR mRNA and other nucleic acid drugs, and their targeting properties are insufficient, allowing them to deliver drugs only to malignant tumor cells with significantly up-regulated expression of transferrin receptor TfR1, but not to other specific cell types, such as T cells. T cells can differentiate into memory T cells, which continuously monitor and eliminate residual malignant tumor cells, reducing the risk of malignant tumor recurrence. The protein cage nanomedicine targeting T cells provided by the present application can target T cells, guide T cells to express CAR proteins using the CAR mRNA encapsulated in its internal cavity, allowing them to specifically recognize and accurately kill malignant tumor cells, while taking advantage of the memory effect of T cells to continuously monitor and eliminate residual malignant tumor cells, reducing the risk of malignant tumor recurrence. Therefore, the development of protein cage nanomedicines targeting T cells can provide patients with a better treatment method to prevent malignant tumor recurrence. In addition, the protein cage nanomedicine targeting T cells provided by the present application also uses N-terminal modification of specific nucleic acid binding peptides to encapsulate more CAR mRNA in its internal cavity, allowing more T cells to express CAR proteins and improve treatment efficacy. Therefore, the protein cage nanomedicine targeting T cells provided by the present application, based on the specific properties of targeting T cells, is expected to promote the wider clinical application of protein cage nanomedicines and exert greater therapeutic potential.

[0054] As preferred, in the protein cage nanomedicine for targeting T cells provided in the application, the C-terminal of the protein subunit monomer of the protein cage is connected with SpyTag, the N-terminal of the nanobody for targeting T cells is connected with SpyCatcher, and the irreversible amide bond formed between SpyTag and SpyCatcher is used to modify the nanobody for targeting T cells at the C-terminal of the protein subunit monomer of the nanoprotem cage. The form of the irreversible amide bond makes the nanoprotem cage nanomedicine modified nanobody for targeting T cells have better stability, and the nanobody for targeting T cells is not easy to fall off. The nanobody for targeting T cells is selected from CD8 VHH, and by targeting CD8 positive T cells, the precise recognition of malignant tumor cells by the responsible cellular immune T cells can be utilized.

[0055] As preferred, in the protein cage nanomedicine for targeting T cells provided in the application, the specific nucleic acid binding peptide amino acid sequence (SEQ ID NO. 1) is GNAKTRRRERRAEKQAQWKAAN, and the CAR mRNA is selected from CD19CAR mRNA; the nucleic acid binding peptide with the amino acid sequence as shown in SEQ ID NO. 1 can specifically bind to the sequence element on the CD19CAR mRNA, realize one-step packaging in vivo, and make the protein cage nanomedicine encapsulate more CD19CAR mRNA in the internal cavity.

[0056] The protein cage nanomedicine for targeting T cells provided in the technical solution will be specifically described below in combination with examples and experimental examples.

[0057] Example 1

[0058] The example 1 provides a preparation method of the protein cage nanomedicine for targeting T cells. The preparation method comprises the steps of preparing a nanoprotem cage fusion gene and a nanobody fusion gene, preparing a nanoprotem cage encapsulating CAR mRNA, preparing a nanobody, and coupling the nanoprotem cage encapsulating CAR mRNA and the nanobody.

[0059] The step of preparing the nanoprotem cage fusion gene and the nanobody fusion gene comprises the following steps: based on the 240-mer nanoprotem cage NatQt with the PDB number of 6NJ8, the C-terminal of the protein subunit monomer structure is fully exposed on the outer surface of the cage, and the N-terminal is fully exposed on the inner surface of the cage, which can be respectively used for installing the nanobody for targeting T cells and installing the specific nucleic acid binding peptide for encapsulating CAR mRNA. The inner surface and the outer surface of the protein cage are engineered, and the engineering process is as follows: Figures 1-2As shown, a gene encoding a specific nucleic acid-binding peptide (amino acid sequence as shown in SEQ ID NO. 1) was fused to the 5' end of the gene encoding the protein subunit monomer of the nanoprotein cage using gene recombination. A gene encoding SpyTag was fused to the 3' end of the SpyTag gene, and a gene encoding a protein purification tag was further fused to the 3' end of the SpyTag gene to obtain the nanoprotein cage fusion gene. Simultaneously, the SpyCatcher gene was fused to the 5' end of the CD8 VHH gene using gene recombination, and a gene encoding a protein purification tag was fused to the 3' end of the CD8 VHH gene to obtain the nanobody fusion gene.

[0060] The steps for preparing nanocages encapsulating CAR mRNA included: subcloning the nanocage fusion gene and the gene sequence encoding CD19CAR mRNA downstream of the first and second promoters of the dual promoter plasmid pACYCDuet 1, respectively. The CD19CAR mRNA was then purified by transformation into competent BL21 (DE3) cells, LB medium cell culture expansion, IPTG-induced co-expression, self-assembly, metal affinity chromatography, and size exclusion chromatography. One-step specific packaging of CD19CAR mRNA was achieved during the self-assembly of the nanocages. The packaging system was named SpyTag-Qt_CAR. mRNA .

[0061] The steps for preparing nanobodies include: subcloning the nanobodies fusion gene into the pET-28a(+) plasmid, transforming it into competent BL21(DE3) cells, amplifying the cells in LB medium, inducing expression with IPTG, self-assembly, purification by metal affinity chromatography and size exclusion chromatography, and obtaining the nanobodies, named SpyCatcher-CD8 VHH.

[0062] The steps for conjugating CAR mRNA nanocages and nanobodies include: conjugating purified SpyTag-Qt_CAR... mRNA SpyCatcher-CD8 VHH was mixed with SpyCatcher-CD8 VHH at a molar ratio of 1:1.2 in neutral pH TBST buffer and incubated overnight at 4°C to obtain a protein cage nanomedicine targeting T cells with CD8 VHH conjugated to its outer surface, named Qt. CD8VHH _CAR mRNA ,from Figure 3 As can be seen from the first and second columns of the electrophoresis diagram, SpyTag-Qt_CAR mRNA The SpyTag-Qt_CAR has higher purity than SpyCatcher-CD8 VHH, as can be seen from the third column. mRNA Coupling with SpyCatcher-CD8VHH resulted in a new stripe Qt. CD8VHH _CARmRNA , adding a small amount of SpyTag-Qt_CAR mRNA , and the coupling efficiency is more than 90%. The coupling reaction is simple and efficient.

[0063] Example 2

[0064] This example 2 provides a preparation method of a protein cage nanodrug, which is a comparative example of example 1. The preparation method comprises the steps of preparing a nanoprogram cage fusion gene and preparing a nanoprogram cage encapsulating CAR mRNA.

[0065] The step of preparing a nanoprogram cage fusion gene comprises: based on the 240-mer nanoprogram cage NatQt with PDB number 6NJ8, the N-terminus of the protein subunit monomer structure is fully exposed on the inner surface of the cage, which can be used to install a specific nucleic acid binding peptide for encapsulating CAR mRNA. The inner surface of the protein cage is engineered based on the property. The engineering process uses gene recombination to fuse the gene encoding the specific nucleic acid binding peptide (amino acid sequence SEQ ID NO. 1) at the 5' end of the gene encoding the protein subunit monomer of the nanoprogram cage, and the gene encoding the protein purification tag at the 3' end, to obtain the nanoprogram cage fusion gene.

[0066] The step of preparing a nanoprogram cage encapsulating CAR mRNA comprises: subcloning the nanoprogram cage fusion gene and the gene sequence of CD19 CAR mRNA into the downstream of the first and second promoters of the double-promoter plasmid pACYCDuet 1, respectively, through transformation to competent cells BL21 (DE3), cell culture amplification in LB medium, IPTG induction of co-expression, self-assembly, metal affinity chromatography and size exclusion chromatography purification, realizing one-step specific packaging of CD19 CAR mRNA in the process of protein cage self-assembly, and the packaging system is named Qt_CAR mRNA .

[0067] Experimental example 1

[0068] This experimental example 1 tests the performance of the protein cage nanodrug Qt CD8 VHH _CAR mRNA provided in example 1 and the protein cage nanodrug Qt_CAR mRNA provided in example 2, which includes in vitro targeting of mouse T cells and in vitro performance testing of hematoma and solid tumor treatment effect.

[0069] The process of in vitro performance testing of mouse T cells comprises: adding the same molar amount of Qt_CAR mRNA and Qt CD8 VHH _CARmRNA respectively, and the percentage of CD19 CAR positive CD8 T cells was detected by flow cytometry after 24 hours of co-incubation with fresh isolated and activated mouse T cells in vitro, to determine the percentage of CD19 CAR expressing T cells, and the results are shown in Figure 4 .

[0070] The process of performance test of in vitro hematoma and solid tumor treatment effect includes: co-incubating Qt_CAR mRNA and Qt CD8 VHH _CAR mRNA respectively, with fresh isolated and activated mouse T cells in vitro for 24 hours, and then co-culturing with A375 melanoma cells and NALM6 leukemia cells respectively which stably express CD19 gene overnight, and then detecting A375 melanoma cells and NALM6 leukemia cells by flow cytometry to evaluate the killing effect on A375 melanoma cells and leukemia tumor cells, and the results are shown in Figures 5-6 .

[0071] As can be seen from Figure 4 , when Qt_CAR mRNA is co-incubated with mouse T cells in vitro for 24 hours, only about 6.70% of mouse T cells express CD19 CAR, while Qt CD8 VHH _CAR mRNA is co-incubated with mouse T cells in vitro for 24 hours, 63.00% of mouse T cells express CD19 CAR, which shows that the target T cell protein cage nanodrug Qt CD8 VHH _CAR mRNA provided in Example 1 can precisely target mouse T cells in vitro, and precisely deliver the CD19 CAR mRNA nucleic acid drug encapsulated therein to most mouse CD8 T cells.

[0072] As can be seen from Figure 5 , when Qt_CAR mRNA is co-cultured with NALM6 leukemia cells which stably express CD19 gene overnight, less than 20% of leukemia cells are killed, while Qt CD8 VHH _CAR mRNA is co-cultured with NALM6 leukemia cells which stably express CD19 gene overnight, about 60% of leukemia cells are killed, and the tumor growth inhibition effect is obvious, and as shown in Figure 6 , Qt CD8 VHH _CAR mRNA can also effectively kill A375 melanoma cells when co-cultured with A375 melanoma cells which stably express CD19 gene overnight, and significantly inhibit tumor growth.

[0073] Experimental Example 2

[0074] Example 2 provides the T cell targeted protein cage nanodrug Qt CD8 VHH _CAR mRNA and the protein cage nanodrug Qt_CAR provided in Example 2 mRNA Performance tests were carried out, including in vivo T cell targeting performance test in mice and in vivo hematoma and solid tumor treatment effect performance test.

[0075] The process of the in vivo T cell targeting performance test in mice included tail vein injection of the same molar amount of Qt_CAR mRNA and Qt CD8 VHH _CAR mRNA into mice respectively, 48 hours after injection, the spleen of the mice was isolated, and then the proportion of CD19 CAR positive CD8 T cells was detected by flow cytometry to determine the proportion of T cells expressing CD19 CAR, and the results are shown in Figure 7 .

[0076] The process of the in vivo hematoma and solid tumor treatment effect performance test included first tail vein injection of human PBMC into severe immunodeficient NCG mice to reconstruct the human immune system; 14 days later, the tail vein of the immune system reconstructed mice was inoculated with CD19-NALM6 ffluc (CD19 and ffluc stably transfected NALM6 leukemia cells are named CD19-NALM6 ffluc ), to construct a mouse leukemia model; 5 days later, the same molar amount of Qt_CAR mRNA , Qt CD8 VHH _CAR mRNA and PBS were injected into the tail vein of the leukemia mice; 14 days later, the tumor burden of the mice was observed by small animal live imaging, and the results are shown in Figure 8 .

[0077] The process of the in vivo solid tumor treatment effect performance test included stably transfecting CD19 into A375 melanoma cells, named CD19-A375. C57BL / 6 mice were first subcutaneously inoculated with CD19-A375 to construct a mouse melanoma model; 10 days later, the same molar amount of Qt_CAR mRNA , Qt CD8 VHH _CAR mRNA and PBS were injected into the tail vein of the tumor mice and the tail vein of the leukemia mice; 14 days later, the tumor volume of the mice was measured, and the tumor inhibition rate was calculated, and the results are shown in Figure 9 .

[0078] As can be seen from Figure 7 , Qt CD8 VHH _CAR mRNAThe proportion of CD19CAR-expressing cells in the CD8 T cell subset of treated mice was approximately 3-4%, which was much higher than that of Qt_CAR. mRNA The treated mice, Qt_CAR mRNA In the treated mice, only a very small number of CD8T cells expressed CD19CAR.

[0079] from Figure 8 It can be seen that when using Qt_CAR mRNA After inducing tail vein leukemia in mice, the number of leukemia cells was observed. A higher number of leukemia cells indicated a lower inhibition rate. The inhibition rate was found to be less than 20%, indicating a large tumor burden in the mice. However, the use of Qt... CD8 VHH _CAR mRNA After inducing tail vein leukemia in mice, the inhibition rate of leukemia cells exceeded 60%, the leukemia cell count was significantly suppressed, and the tumor burden in the mice was significantly reduced; simultaneously, Figure 9 As shown, when Qt CD8 VHH _CAR mRNA After treatment of tail vein melanoma mice, the melanoma cells were significantly inhibited and the melanoma volume was significantly reduced.

[0080] 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 T-cell targeted protein cage nanomedicine, characterized in that, The nano protein cage and the CD19 CAR mRNA are encapsulated in the internal cavity of the nano protein cage. The N-terminal of the protein subunit monomer of the nano protein cage is modified with a specific nucleic acid binding peptide. The N-terminal of the protein subunit monomer of the nano protein cage is modified with a specific nucleic acid binding peptide. The N-terminal of the protein subunit monomer of the nano protein cage is modified with a specific nucleic acid binding peptide. The N-terminal of the protein subunit monomer of the nano protein cage is modified with a specific nucleic acid binding peptide. The amino acid sequence of the specific nucleic acid binding peptide is shown in SEQ ID NO.

1. The nano protein cage is selected from the nano protein cage with PDB number 6NJ8.

2. The method of claim 1, wherein the method of preparing a T cell targeted protein cage nanomedicine is characterized by, The C-terminal of the protein subunit monomer of the nano protein cage is connected with a protein purification tag. The steps include: Fusing the gene encoding the specific nucleic acid binding peptide to the 5' end of the gene encoding the protein subunit monomer of the nano protein cage and fusing the gene encoding SpyTag to the 3' end of the gene, to obtain a nano protein cage fusion gene; Subcloning the nano protein cage fusion gene and the CD19 CAR mRNA to the downstream of the first and second promoters of the double-promoter plasmid in turn, transforming into competent cells, cell culture amplification, induction of co-expression, self-assembly, and purification to obtain the nano protein cage encapsulating the CD19 CAR mRNA; Fusing the gene encoding SpyCatcher to the 3' end of the gene encoding the nanobody CD8 VHH targeting T cells to the 5' end of the gene, to obtain a nanobody fusion gene; Subcloning the nanobody fusion gene to the vector plasmid in turn, transforming into competent cells, cell culture amplification, induction of expression, and purification to obtain the nanobody CD8 VHH; 3. The method of claim 2, wherein the protein cage nanomedicine targeting T cells is prepared by the steps of: a) providing a protein cage nanomedicine targeting T cells; b) providing a targeting moiety; c) conjugating the targeting moiety to the protein cage nanomedicine targeting T cells. Coupling the nano protein cage encapsulating the CD19 CAR mRNA and the nanobody CD8 VHH to obtain the protein cage nanomedicine targeting T cells. The coupling reaction process is as follows: adding the nano protein cage encapsulating the CD19 CAR mRNA and the nanobody CD8 VHH into TBST buffer solution for mixing, and performing coupling reaction at 4°C overnight to obtain the protein cage nanomedicine targeting T cells.

4. The use of the protein cage nanomedicine targeting T cells of claim 1 in the preparation of a tumor treatment drug.

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