CN118222627A

CN118222627A - Pharmaceutical composition and application thereof

Info

Publication number: CN118222627A
Application number: CN202410347147.1A
Authority: CN
Inventors: 张灿; 鞠曹云; 李静雯; 吴梦同; 薛玲静
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2024-03-26
Filing date: 2024-03-26
Publication date: 2024-06-21

Abstract

The invention relates to a pharmaceutical composition and application thereof, in particular to a pharmaceutical composition comprising a delivery vector and a nucleic acid expression vector, wherein the expression vector comprises the following elements: a transposon 5 'end repeat, a promoter, a coding exogenous receptor sequence, a nuclear targeting sequence, and a transposon 3' end repeat; the nuclear targeting sequence is selected from one or more of nfkb nuclear targeting sequence, glucocorticoid response element sequence and SV40 nuclear targeting sequence; the delivery vehicle comprises adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, wherein the neutral phospholipid is selected from dioleoyl phosphatidylethanolamine, the adamantane tail chain lipid is selected from compound shown in formula (AD 8), and the ionizable lipid is selected from compound shown in formula (A7). The pharmaceutical composition provided by the invention can mediate efficient transfection of exogenous genes into host cells and efficient and stable expression in the host cells.

Description

Pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a pharmaceutical composition and application thereof.

Background

Chimeric antigen receptor T-cell (CAR-T) therapy refers to a novel immunotherapy in which T cells are engineered in vitro to express CARs capable of specifically recognizing and binding tumor cell surface antigens, and the CARs are amplified in vitro and then infused back into a patient for killing tumor cells. Since the first CAR-T product Kymriah (nowa) was marketed in the world after FDA approval in 2017, by 2024, 6 CAR-T products have been FDA approved. Five CAR-T therapies of alemtujose, rayleigh omelanSai, naloxone, iyatomoxef and ze Wo Jiao renSai were also approved sequentially in 2021 in China. The number of clinical trials of CAR-T therapies has also shown explosive growth in recent years, up to 3 months 2024, and as high as 1119 clinical trials of CAR-T cell therapies registered on clinical trials. A large amount of clinical data suggests that CAR-T shows great potential and distinct advantage ^[1] in the treatment of various tumors, particularly relapsed or refractory lymphomas and myelomas, compared to traditional tumor therapies.

CAR gene transfection is the most critical step in CAR-T therapy, and this is also the rate limiting step limiting the rapid development of CAR-T therapy. Most of the current studies utilize lentiviruses or gamma-retroviruses to deliver CAR-encoding nucleic acids into T cells for transfection. Of the eleven CAR-T products marketed, seven (tembloc, li Jimai, sidaoram, iyakaoram, rayaloram, nalaoram and ze Wo Jiao ram) used lentivirus and the remaining two (alkiram, brikiaoram) used gamma retrovirus. Nearly 95% of current global CAR-T clinical studies use viral vectors to construct CAR-T ^[2] in research projects. Viral vectors suffer from a number of limitations including small entrapment capacity, high production costs, high immunogenicity, insertional mutagenesis, risk of carcinogenesis, etc., while the complex procedures and high costs involved limit the large-scale production and use ^[3] of CAR-T. Thus, safer, less costly non-viral delivery means are increasingly being focused on by researchers and used for the construction of CAR-T cells.

For example, the use of electroporation to deliver plasmid DNA or mRNA directly into cells for in vitro construction of CAR-T, there are currently more than ten relevant clinical studies (NCT 03288493, NCT03741127, NCT04250324, NCT03927261, etc.). Although electroporation has a wide application range, simple operation and high transfection efficiency, many defects still exist. Electroporation is only suitable for in vitro use and causes irreversible cell damage during the preparation process, resulting in significant reduction ^[4] in cell proliferation capacity and viability. Therefore, there is a need to develop a more safe and efficient novel T cell gene transfection strategy, which is of great clinical significance in driving CAR-T further development.

Lipid nanoparticles (Lipid nanoparticle, LNP) are a novel class of lipid non-viral vectors that are receiving increasing attention for safety and efficacy. Three LNP drugs are currently approved by the FDA for sale, including 1 siRNA drug (Onpattro) and 2 mRNA vaccines (BNT 162b2 and mRNA-1273). LNP has been demonstrated to exert better gene delivery effects on a variety of cells, but its use in CAR-T cell construction is still relatively small.

Most of the current research is the use of LNP to deliver CAR-mRNA into T cells for transient expression of CAR molecules. For example, daniel j.siegwart research team utilized spleen-targeted SORT lipid nanoparticle delivery of CD19 CAR-mRNA to construct CAR-T in situ in mice with 7% ^[5] in-spleen T cell positive rate. Mitchell research team obtains a lipid nanoparticle capable of efficiently delivering CAR-mRNA to primary human T cells by screening a novel ionizable lipid pool, the transfection efficiency is comparable to the electrotransfection efficiency, and the cell viability (> 75%) is significantly better than electroporation (31%) ^[6]. Xu Qiaobing the research team designed and synthesized a class of ionizable lipids containing imidazole headgroups, and the optimal lipid nanoparticle formulation was obtained by screening and optimization and used for in vivo delivery of cre-mRNA with a T cell positive rate of about 8% ^[7]. The CD 5-targeted lipid nanoparticles were used by the p.sup. Jonathan A Epstein team to deliver optimized FAP CAR-mRNA for in vivo production of transiently expressed CAR-T cells with a T cell positive rate of about 80% ^[8] after 48h transfection. Although many studies have shown that LNP exhibits superior efficacy in delivering mRNA into T cells, mRNA can only be transiently expressed in cells, and target protein expression drops rapidly to low levels within days, so repeated dosing is often required to maintain long-term anti-tumor effects, which may lead to unpredictable cytotoxicity.

A transposon plasmid system is a genetic vector capable of achieving stable transgene, and is generally composed of a plasmid carrying CAR (transposon) and a plasmid carrying transposon enzyme, and can achieve continuous and efficient transgene expression through safe and effective genome integration. The transposon plasmid has simple preparation, low price and high safety, and provides an innovative and potential treatment scheme for a series of gene diseases. There have been studies reporting that the LNP delivery transposon plasmid system is used for stably transferring CAR-T construction, but the transfection efficiency in vitro is only 4.57% ^[7] at the highest, which cannot meet the requirements of disease treatment. The inefficiency of transfection is currently a great bottleneck in the construction of CAR-T by impeding the entry of LNP delivery plasmids into T cells, the main reasons of which include the following three: (1) LNP is difficult to efficiently enter T cells: endocytosis of LNP into T cells is a prerequisite for transfection, but T cells act as a non-phagocytic lymphocyte, which is difficult to phagocytose exogenous substances, thereby limiting LNP entry into T cells for transfection ^[9]; (2) insufficient endosomal escape efficiency: gene medicine needs to escape from endosomes into cytoplasm to avoid degradation or excretion, but the currently reported escape efficiency of LNP endosomes is not more than 15%, and the difficulty of endosome escape is a key factor ^[10] which affects the transfection efficiency of LNP at present; (3) plasmid nuclear difficulty: the transposon plasmid used for stably constructing the CAR-T cells has larger volume and is difficult to enter the nucleus for transcriptional expression.

Reference is made to:

[1]LU J,JIANG G.The journey of CAR-T therapy in hematological malignancies[J].Mol Cancer,2022,21(1):194.

[2]MICHELS A,HO N,BUCHHOLZ C J.Precision medicine:In vivo CAR therapy as a showcase for receptor-targeted vector platforms[J].Mol Ther,2022,30(7):2401-15.

[3]HIGH K A,RONCAROLO M G.Gene Therapy[J].N Engl J Med,2019,381(5):455-64.

[4]DITOMMASO T,COLE J M,CASSEREAU L,et al.Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo[J].Proc Natl Acad Sci U S A,2018,115(46):E10907-E14.

[5]ALVAREZ-BENEDICTO E,TIAN Z,CHATTERJEE S,et al.Spleen SORT LNP Generated in situ CAR T Cells Extend Survival in a Mouse Model of Lymphoreplete B Cell Lymphoma[J].Angew Chem Int Ed Engl,2023,62(44):e202310395.

[6]BILLINGSLEY M M,SINGH N,RAVIKUMAR P,et al.Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering[J].Nano Lett,2020,20(3):1578-89.

[7]ZHOU J E,SUN L,JIA Y,et al.Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6knockdown in vivo[J].J Control Release,2022,350:298-307.

[8]RURIK J G,TOMBACZ I,YADEGARI A,et al.CAR T cells produced in vivo to treat cardiac injury[J].Science,2022,375(6576):91-6.

[9]OLDEN B R,CHENG E,CHENG Y,et al.Identifying key barriers in cationic polymer gene delivery to human T cells[J].Biomater Sci,2019,7(3):789-97.

[10]WITTRUP A,AI A,LIU X,et al.Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown[J].Nat Biotechnol,2015,33(8):870-6.

Disclosure of Invention

In view of the defects in the prior art, the inventor makes extensive and intensive studies to construct a pharmaceutical composition comprising a delivery vector and a nucleic acid expression vector, wherein the pharmaceutical composition can mediate efficient transfection of exogenous genes into host cells, and the nucleic acid expression vector can be efficiently and stably expressed in the host cells.

In order to solve the problems, the application adopts the following technical scheme:

In one aspect, the invention provides a pharmaceutical composition comprising a delivery vehicle and a nucleic acid expression vector, wherein,

The nucleic acid expression vector comprises the following elements: a transposon 5 'end repeat, a promoter, a coding exogenous receptor sequence, a nuclear targeting sequence, and a transposon 3' end repeat; the nuclear targeting sequence is selected from one or more of nfkb nuclear targeting sequence, glucocorticoid response element sequence and SV40 enhancer sequence;

The delivery vehicle comprises adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, wherein the neutral phospholipid is selected from dioleoyl phosphatidylethanolamine,

The adamantane tail chain lipid is selected from compounds shown in a formula (AD 8),

The ionizable lipid is selected from compounds represented by formula (A7),

Preferably, the tail of the pegylated lipid is a saturated or unsaturated alkane chain with a length of C6-C20.

Further preferably, the pegylated lipid is selected from one or more of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, distearyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol-polyethylene glycol 2000, polyethylene glycol 2000 ditetradecanol succinate.

Still more preferably, the pegylated lipid is selected from polyethylene glycol 2000 ditetradecyl succinate (PEG ₂₀₀₀-Suc-TA₂) having the following structural formula:

Further preferably, the delivery vehicle comprises a compound represented by formula (AD 8), a compound represented by formula (A7), dioleoyl phosphatidylethanolamine, cholesterol, and polyethylene glycol 2000 ditetradecyl succinate.

Preferably, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 10-20:10-20:30-50:0.5-5.

Further preferably, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 12-18:12-20:40-50:1-3.

Still further preferably, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 15:15:18:50:2.

Preferably, the nuclear targeting sequence is selected from the group consisting of nfkb nuclear targeting sequences comprising a first sequence and a second sequence,

Wherein the nucleic acid sequence of the first sequence comprises any one of the following sets:

(1) A polynucleotide sequence shown in SEQ ID NO. 1;

(2) The nucleotide sequence has more than or equal to 95 percent of the identity with the polynucleotide sequence shown as SEQ ID NO. 1; or alternatively, the first and second heat exchangers may be,

(3) A polynucleotide sequence that is partially or fully complementary to any one of (1) to (2);

the nucleic acid sequence of the second sequence comprises any one of the following sets of:

(4) A polynucleotide sequence shown in SEQ ID NO. 8;

(5) The nucleotide sequence has more than or equal to 95 percent of the identity with the polynucleotide sequence shown in SEQ ID NO. 8; or alternatively, the first and second heat exchangers may be,

(6) A polynucleotide sequence partially or fully complementary to any one of (4) to (5).

Preferably, the first sequence and the second sequence are separate.

Preferably, the first sequence is located upstream of the transposon 5' end repeat.

Preferably, the second sequence is located upstream of the transposon 3' end repeat.

Preferably, the promoter is selected from one or more of the group consisting of CMV promoter, miniCMV promoter, CMV53 promoter, miniSV promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1 alpha promoter, SV40 promoter, ubiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, beta-actin promoter, TK promoter and GRP78 promoter.

Further preferred, the promoter is a CMV promoter and/or an EF 1a promoter.

Still more preferably, the promoter is an EF1 alpha promoter.

Still further preferably, the nucleic acid sequence of the EF 1. Alpha. Promoter is as shown in SEQ ID NO. 3;

Preferably, the exogenous receptor comprises a non-naturally occurring receptor that is a chimeric antigen receptor that targets one or more ：CD19、CD7、CD20、CD22、CD23、CD30、CD33、CD38、CD44v7/8、CD123、CD133、CD138、CD171、AFP、BCMA、CIL-1、CS-1、CEA、CA125、CA199、CLDN18.2、EpCAM、EGFR、EGFRvⅢ、FAP、GPC1、GPC3、HER2、IL-13Ra2、 integrins beta 7, FR, TAG-72, MUC1, MSLN, nectin-1, NY-ESO-1, GD2, cy2C, GMR, PSMA, gp100, VFGFR1, VFGFR2 selected from the following target binding proteins.

Further preferred, the chimeric antigen receptor targets one or more of the target binding proteins selected from the group consisting of: CD19, BCMA, CLDN18.2, MSLN, GPC3, HER2, GD2, PSMA, MUC1, GUCY2C, CD, CD7, CD20, CD30, CD123, CD33.

Still further preferred, the chimeric antigen receptor targets a CD19 target binding protein.

Still further preferably, the nucleic acid sequence encoding CD19 is as set forth in SEQ ID NO. 4.

Preferably, the transposon 5' terminal repeat is as shown in SEQ. ID No. 2.

Preferably, the transposon 3' -terminal repeat is as shown in SEQ. ID No. 9.

Preferably, the nucleic acid expression vector further comprises a transcription termination sequence as shown in SEQ ID NO. 7.

Preferably, the nucleic acid expression vector further comprises a nucleic acid sequence encoding a linker, the sequence encoding the linker being as shown in SEQ ID NO. 5.

Preferably, the nucleic acid expression vector has a nucleic acid sequence as shown in SEQ ID NO. 10.

Preferably, the pharmaceutical composition further comprises a vector capable of expressing a transposase.

In another aspect, the present invention provides the use of a pharmaceutical composition according to any one of the above in the manufacture of a medicament for the prevention and/or treatment of cancer related diseases.

Preferably, the cancer is a hematological malignancy and/or a solid tumor.

ADVANTAGEOUS EFFECTS OF INVENTION

The pharmaceutical composition provided by the invention has the advantages of stronger nuclear insertion capability, higher integration efficiency, higher transfection efficiency and the like. The CAR-T constructed by the pharmaceutical composition provided by the invention has stronger target cell killing capability, and provides a novel tool and means based on a non-viral vector for cell therapy.

Drawings

FIG. 1 is a schematic diagram of the structure of a pPB CD19 CAR-3NF-MC nuclear targeting small loop plasmid constructed according to the present invention.

FIG. 2 shows the result of the XbalI endonuclease cleavage in example 1, showing that pPB CD19 CAR-3NF-MC was detected, lane 1 was a DNA marker, lane 2 was pPB CD19 CAR-3NF-MC, and lane 3 was a cleavage.

FIG. 3 is a schematic representation of the electrotransformation effect of two plasmids (PB and pPB CD19 CAR-3 NF-MC) in T cells under an inverted fluorescence microscope and flow cytometer.

FIG. 4 is a graph showing T cell transfection efficiency of different neutral phospholipid LNPs in example 4.

FIG. 5 is T cell transfection efficiency of Lipo 2000, A7 LNP, A13AD8 LNP, and A7AD8 LNP of example 4.

FIG. 6 is a schematic diagram of the capacity of agarose electrophoresis to examine LNP loaded nucleic acid of example 5, lane 1 is naked DNA, lane 2 is pPB CD19 CAR-3NF-MC/LNP, and lane 3 is pPB/LNP.

FIG. 7 is a schematic representation of the effect of flow cytometry in examining the T cell transfection of optimal LNP in example 5.

FIG. 8 is a bar graph depicting the uptake capacity of pDNA/A7AD8 LNP in human T cells as described in example 6.

FIG. 9 is a schematic diagram of an examination of endosomal escape ability of pDNA/A7AD8 LNP in human T cells in example 6.

Figure 10 is a histogram of a long term stable expression capability study of CAR-T cells in example 7.

FIG. 11 is a graph showing the effect of LNP transfection on T cell proliferation in example 7.

FIG. 12 is a graph showing the effect of LNP transfection on T cell viability in example 7.

FIG. 13 is the effect of LNP transfection on T cell phenotype CD4/CD8 in example 7.

FIG. 14 is a graph showing cytokine secretion after co-incubation of CAR-T cells with target cells in example 7.

FIG. 15 is a graph showing the killing ability of CAR-T cells and T cells to Raji cells and K562 cells in example 8.

Detailed Description

In order to make the technical scheme and the beneficial effects of the application more obvious and understandable, the following detailed description is given by way of example. Wherein the drawings are not necessarily to scale, and wherein local features may be exaggerated or reduced to more clearly show details of the local features; unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The new functional lipid containing adamantane tail chain is designed and synthesized in the early stage of the team (patent number: 2022100961821), namely, one of two hydrophobic alkane chains of a lipid material is replaced by the adamantane tail chain with extremely high rigidity. Experimental data show that the functional lipid can obviously reduce the particle size of LNP, increase endocytosis of cells, and further promote the escape efficiency of LNP endosomes. The adamantane lipid and LNP prepared by the adamantane lipid show good delivery effect in T cell transfection, but the problem of low transfection efficiency in the delivery of CAR plasmids still exists. The main reason for this is that transposon CAR plasmids used for CAR-T construction are large in size and difficult to nuclear.

In order to overcome the problem of difficult nuclear insertion of transposon CAR plasmids, the inventors have made extensive experiments and creative efforts to construct a pharmaceutical composition comprising a delivery vector and a nucleic acid expression vector capable of mediating efficient integration and efficient stable expression of exogenous genes in host cells. The inventor inserts a nuclear targeting sequence which can be identified by a nuclear transcription factor into a traditional transposon plasmid aiming at a nucleic acid expression vector, simultaneously only maintains a functional region sequence in the transposon plasmid, removes a bacterial skeleton part, further reduces the plasmid volume and improves the nuclear localization signal capability of the plasmid, and solves the problems of low efficiency of T cell transfection, difficult T cell endocytosis and insufficient LNP endosome escape efficiency of a transposon pDNA/lipid nanoparticle transfection system; for a delivery carrier, the preparation method can effectively improve the T cell transfection capacity by optimizing the prescription of the lipid nanoparticle containing adamantane tail chain functional lipid obtained by the front-stage synthesis screening of a team. Meanwhile, the invention also provides a CAR-T in-vitro construction method which has the advantage of high transfection efficiency.

The pharmaceutical composition provided by the invention can effectively improve the transfection capacity of T cells, and solves three bottleneck problems of the existing transfection of the pDNA-CAR/lipid nanoparticle into the T cells to a certain extent. Compared with a T cell transfection method commonly used in clinical research, the pDNA-CAR/lipid nanoparticle delivery system is safer and more convenient, and has important significance for developing a novel T cell delivery carrier with independent intellectual property rights in China.

The following is a description of some of the terms involved in the present invention.

As used herein, the term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably referring to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises 1 or more regulatory sequences operably linked, which under compatible conditions direct expression of the coding sequence in a suitable host cell. Expression should be understood to include any step involved in the production of a protein or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, the term "derived from" refers to the origin or source of a molecule and may include naturally occurring, recombinant, unpurified or purified molecules. Nucleic acid or polypeptide molecules are considered "derivatized" when they include moieties or elements that are assembled in a manner that produces functional units. The parts or elements may be assembled from multiple sources as long as they retain evolutionarily conserved functions. In some embodiments, the derivative nucleic acid or polypeptide molecule comprises a sequence that is substantially identical to the source nucleic acid or polypeptide molecule. For example, a derivative nucleic acid or polypeptide molecule of the present disclosure may have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the source nucleic acid or polypeptide molecule.

As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may include ribonucleoside triphosphates such as Adenosine Triphosphate (ATP), uridine Triphosphate (UTP), cytosine Triphosphate (CTP), guanosine Triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP or derivatives thereof. Such derivatives may include, for example, [ aS ] dATP, 7-deaza-dGTP and 7-deaza-dATP, aS well aS nucleotide derivatives that confer nuclease resistance on nucleic acid molecules containing them.

As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to any length of polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) or analogs thereof, in single-stranded, double-stranded, or multi-stranded form. Polynucleotides may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or fragment thereof. The polynucleotide may be DNA or RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may comprise one or more analogs (e.g., altered backbones, sugars, or nucleobases). The nucleotide structure, if present, may be modified before or after assembly of the polymer. Some non-limiting examples of modified nucleotides or analogs include: pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acid, xenogeneic nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, cpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, dialkyside (queuosine), and hurusoside. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNAs (mRNA), transfer RNAs (tRNA), ribosomal RNAs (rRNA), short interfering RNAs (siRNA), short hairpin RNAs (shRNA), micrornas (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes and primers. The nucleotide sequence may be interrupted by non-nucleotide components.

As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or regulating the timing and/or rate of gene transcription. For example, a promoter may be a cis-acting transcriptional control element, including enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5 'and 3' untranslated regions, or intron sequences, that are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to perform (turn on/off, regulate, modulate, etc.) gene transcription.

As used herein, the term "DNA targeting sequence (DNA Nulear Target Sequence, DTS)" refers to a short consensus motif recognized by a specific Transcription Factor (TF), a DNA fragment that mediates targeting of DNA vectors in the cytoplasm into the nucleus through the nuclear pore complex (Nulear Pore Complex, NPC). The DNA targeting sequence comprises a nucleotide sequence complementary to a specific sequence within the target DNA (complementary strand of the target DNA). In other words, the DNA targeting sequence interacts with the target polynucleotide sequence of the target DNA in a sequence-specific manner by hybridization (i.e., base pairing). In this regard, the nucleotide sequence of the DNA targeting sequence can vary, and it determines the location within the target DNA where the polynucleotide of the invention and the target DNA will interact. The DNA targeting sequence may be modified or designed (e.g., by genetic engineering) to hybridize to any desired sequence within the target DNA.

As used herein, "nfkb (nuclear factor- κb)" is a protein complex, an important nuclear transcription factor in cells. Involved in the response of cells to stimuli, such as inflammatory responses, immune responses, modulation of apoptosis, stress responses, etc. NF-. Kappa.B is present in almost all animal cell types.

As used herein, "GRE (glucocorticoid response element)" or "glucocorticoid response element" refers to a DNA sequence that specifically binds to a glucocorticoid receptor.

As used herein, the term "transcription termination sequence" refers to a nucleic acid sequence that is recognized by a polymerase of a host cell and results in termination of transcription. Transcription termination sequences are DNA sequences that provide termination of mRNA transcription or both mRNA transcription and ribosomal translation of an upstream open reading frame at the 3' end of a natural or synthetic gene. Any terminator that is functional in the host cell of choice may be used in the present invention, including but not limited to SV40, hGH, BGH and rbGlob, rBG pA transcription termination sequences and/or polyadenylation signals.

As used herein, the term "linker" refers to a molecule or combination of molecules that links two molecules, such as a DNA binding protein and a random peptide, such that the two molecules are in a preferred configuration, e.g., such that the random peptide binds to the receptor of the DNA binding protein with minimal steric hindrance. As used herein, "linker" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any domain/region/element of the nucleic acid construct of the invention. The linker may be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used when it is necessary to ensure that two adjacent domains do not spatially interfere with each other. The linker may be cleavable or non-cleavable. In some embodiments, the linker sequence comprises an Internal Ribosome Entry Sequence (IRES). In some embodiments, the IRES is an IRES from CVB3 or EV 71. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, porcine teschovirus type 12A (P2A), a echinacea armyworm beta tetrad virus (Thosea asigna virus, T2A), or a combination, variant, and functional equivalent thereof. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

In some embodiments, the nucleic acid construct comprises one or more linker sequences that divide the components of the nucleic acid construct. In some embodiments, one or more of the linker sequences have the same or different sequences.

As used herein, the term "operably linked" refers to a functional relationship between two or more segments, such as nucleic acid segments or polypeptide segments. In general, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.

As used herein, the terms "complementary," "complementary sequence," "complementary," and "complementarity" refer to sequences that are complementary to a given sequence and that are hybridizable. In some cases, the sequence that hybridizes to a given nucleic acid is referred to as the "complement" or "reverse complement" of the given molecule if its base sequence on a given region is capable of complementarily binding to the base of its binding partner, thereby forming, for example, A-T, A-U, G-C and G-U base pairs.

As used herein, the term "sequence identity", such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/embossneedle/nucleic. Html), the BLAST algorithm (see, e.g., the BLAST alignment tool available at BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi, optionally default settings), or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.ukaools/psa/EMBOSS _water/nucleic. Html, optionally default settings). Any suitable parameters of the selected algorithm (including default parameters) may be used to evaluate the optimal alignment.

Complementarity may be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids may mean that the two nucleic acids may form a duplex, wherein each base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementarity may mean that the sequence in one strand is incompletely and/or incompletely complementary to the sequence in the opposite strand, but that sufficient bonding between bases on both strands occurs to form a stable hybridization complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by: the melting temperature (Tm) of the hybridized strand is predicted using sequence and standard mathematical calculations, or the Tm is empirically determined using conventional methods.

As used herein, the term "transposon" is a segment within a chromosome that is translocatable within the genome, also referred to as a "jump gene. There are two different classes of transposons: class I or retrotransposons, mobilized by RNA intermediates and a "copy and paste" mechanism; and class II or DNA transposons, mobilized by excision integration or "cut and paste" mechanisms (Ivics NatMethods 2009). Bacteria, lower eukaryotes (e.g., yeast), and invertebrate transposons appear to be largely species specific and cannot be used for efficient transposition of DNA in vertebrate cells. "Sleeping Beauty transposon" (sleep Beauty, IVICSCELL 1997) is the first active transposon, which is artificially reconstructed by sequence shuffling of inactive TE of fish. This makes it possible to successfully achieve DNA integration by transposition into vertebrate cells, including human cells. The sleeping beauty transposon is a class II DNA transposon belonging to the Tcl/mariner transposon family (Ni Genomics Proteomics 2008). At the same time, additional functional transposons have been identified or reconstituted from different species including Drosophila, frog and even human genomes, all of which have been demonstrated to allow DNA transposition into vertebrate and human host cell genomes. Each of these transposons has advantages and disadvantages related to transposition efficiency, expression stability, genetic payload capacity, etc. Exemplary class II transposases that have been generated include the sleeping American transposon, piggyBac, frog prince transposon, himarl, passport, minos, hAT, toll, to, aciDs, PIF, harbinger, harbinger-DR, and Hsmarl.

As used herein, the term "transposase" is an enzyme that is capable of forming a functional complex with a composition containing a transposon end (e.g., transposon end) and catalyzing the insertion or transposition of the composition containing a transposon end into double stranded DNA incubated with an in vitro transposon reaction. The term "transposon end" means double stranded DNA containing the nucleotide sequences necessary for the formation of a complex with a transposase or integrase that functions in an in vitro transposition reaction ("transposon end sequences").

The transposon end forms a complex or synaptic complex or transposon composition with a transposase or integrase that recognizes and binds the transposon end, and the complex is capable of inserting or transposing the transposon end into target DNA incubated in an in vitro transposition reaction. The transposon end exhibits two complementary sequences consisting of a transferred transposon end sequence or transfer strand and a non-transferred transposon end sequence or non-transfer strand. For example, one transposon end that forms a complex with an hyperactive Tn5 transposase (which is active in an in vitro transposition reaction) comprises: the transfer strand of the transfer transposon end sequence is shown as follows: 5'-AGATGTGTATAAGAGACAG-3'; and a non-transferred strand showing the following "non-transferred transposon end sequences": 5'-CTGTCTCTTATACACATCT-3'. In an in vitro transposition reaction, the 3' end of the transfer strand is ligated or transferred to the target DNA. In an in vitro transposition reaction, the non-transferred strand of the transposon end sequence, which exhibits complementarity to the transferred transposon end sequence, is not ligated or transferred to the target DNA.

"Recombinant" as used herein means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase Chain Reaction (PCR), and/or ligation steps that result in a construct having a structurally encoded or non-encoded sequence that is distinguishable from endogenous nucleic acids found in the natural system. The DNA sequence encoding the polypeptide may be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid capable of being expressed from recombinant transcription units contained in cells or in cell-free transcription and translation systems. Genomic DNA comprising the relevant sequences may also be used in the formation of recombinant genes or transcriptional units. Sequences of the non-translated DNA may be present at the 5 'or 3' end of the open reading frame, where such sequences do not interfere with manipulation or expression of the coding region, and may actually function to regulate production of the desired product by a variety of mechanisms. Thus, for example, the term "recombinant" nucleic acid refers to a nucleic acid that does not occur naturally, e.g., is made by human intervention by artificially combining two otherwise separate segments of sequence. Such artificial combination is often accomplished by chemical synthesis means or by manually manipulating separate segments of nucleic acid (e.g., by genetic engineering techniques). This is typically by ligating together nucleic acid segments having the desired functions to produce the desired combination of functions. Such artificial combination is often accomplished by chemical synthesis means or by manually manipulating separate segments of nucleic acid (e.g., by genetic engineering techniques). When a recombinant polynucleotide encodes a polypeptide, the sequence encoding the polypeptide may be naturally occurring ("wild-type") or may be a variant (e.g., mutant) of the naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence is not naturally occurring. In contrast, a "recombinant" polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide may be naturally occurring ("wild-type") or non-naturally occurring (e.g., variants, mutants, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but may be a naturally occurring amino acid sequence. Where the polynucleotide sequence is known, each polynucleotide molecule may be prepared using methods conventional in the art, or the nucleic acid (DNA or RNA) may be constructed as a product of various combinations of cloning, restriction, polymerase Chain Reaction (PCR) and/or ligation steps into a corresponding vector (e.g., a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering), to obtain a recombinant vector. Recombinant vectors can be constructed using methods well known to those skilled in the art.

As used herein, the term "nucleic acid expression vector" refers to a nucleic acid molecule capable of transporting or mediating expression of a heterologous nucleic acid. As used herein, a plasmid is a species of genus encompassed by the term "nucleic acid expression vector". Generally, expression vectors of utility are often in the form of "plasmids," which refer to circular double-stranded DNA molecules that do not bind to chromosomes in their vector form and typically contain entities for stable or transient expression of the encoded DNA. Other nucleic acid expression vectors useful in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, phage or viral vectors, and such nucleic acid expression vectors can be integrated into the genome of a host or autonomously replicated in a cell. The nucleic acid expression vector may be in the form of DNA or RNA. Other forms of expression vectors known to those of skill in the art that serve equivalent functions, such as, for example, self-replicating extra-chromosomal vectors or nucleic acid expression vectors capable of integration into a host genome, may also be used. Exemplary nucleic acid expression vectors are those capable of autonomously replicating and/or expressing a nucleic acid to which they are linked. A harbor locus is a region within the genome into which additional exogenous or heterologous nucleic acid sequences can be inserted, and the host genome is capable of containing the inserted genetic material. Exemplary safe harbor sites include, but are not limited to: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.

As used herein, "delivery vehicle" refers to a non-viral vector that is capable of stably supporting nucleic acids and delivering nucleic acids, such as RNA, DNA, etc., into cells for gene silencing, such as Lipid Nanoparticles (LNP) as described in this patent, which may act to promote cellular uptake, enhance endosomal escape, etc. And the medicine can realize multiple system administration, is suitable for various injection forms, and is beneficial to more conveniently and effectively playing roles in preventing and treating diseases.

As used herein, a "chimeric antigen receptor" (CAR) is an artificial T cell receptor intended for use as a cancer therapy using a technique known as adoptive cell transfer. The essential antigen binding, signaling and stimulatory functions of the complex have been reduced by genetic recombination methods to a single polypeptide chain, commonly referred to as a Chimeric Antigen Receptor (CAR). See, for example, eshhar, U.S. patent No. 7,741,465; eshhar, U.S. patent application publication 2012/0093842. The CAR is specifically constructed to stimulate T cell activation and proliferation in response to a specific antigen to which the CAR binds. The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides, typically two polypeptides in the simplest embodiment, which when expressed in immune effector cells, provide specificity for the cell against a target cell (typically against a cancer cell) and provide intracellular signaling. In some embodiments, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (which may also be referred to as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule. In some aspects, the set of polypeptides are contiguous with each other. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from conventional costimulatory molecules in the art, such as 4-1BB, i.e. (CD 137), CD27, and/or CD28. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane. Typically "CAR-T cells" are used, which have been engineered substantially to contain T cells of the chimeric antigen receptor. Thus, T lymphocytes carrying such CARs are commonly referred to as CAR-T lymphocytes.

In some cases, the CAR is referred to as a first, second, and/or third generation CAR. In some aspects, the first generation CAR is a CAR that provides only CD3 chain-induced signaling after antigen binding; in some aspects, the second generation CAR is a CAR that provides such signals and co-stimulatory signals, e.g., a CAR that includes an intracellular signaling domain from a co-stimulatory receptor (e.g., CD28 or CD 137); in some aspects, the third generation CAR is in some aspects a CAR comprising multiple co-stimulatory domains of different co-stimulatory receptors. In a first aspect, the present invention provides a nucleic acid construct comprising the following elements:

A transposon 5 'end repeat, a promoter, a coding exogenous receptor sequence, a nuclear targeting sequence, and a transposon 3' end repeat; the nuclear targeting sequence is selected from one or more of nfkb nuclear targeting sequence, glucocorticoid response element sequence (Glucocorticoid Response Elements, GRE) and SV40 nuclear targeting sequence.

In certain embodiments, the nuclear targeting sequence is selected from nfkb nuclear targeting sequences.

In certain embodiments, the consensus sequence comprises a first sequence and a second sequence,

(1) A polynucleotide sequence shown in SEQ ID NO. 1;

(2) More than or equal to 95% identity (particularly preferably 96%, 97%, 98%, 99%) with the polynucleotide sequence shown in SEQ ID NO. 1, or;

SEQID NO:1：

GTACCCTGGGGACTTTCCAGCCTGGGGACTTTCCAGCTGGGACTTTCCAGGCGGTAC

In certain embodiments, the nucleic acid sequence of the first sequence has greater than or equal to 98% identity to the polynucleotide sequence set forth in SEQ ID NO. 1.

In certain embodiments, the nucleic acid sequence of the first sequence has greater than or equal to 99% identity to the polynucleotide sequence set forth in SEQ ID NO. 1.

(4) A polynucleotide sequence shown in SEQ ID NO. 8;

(5) The nucleotide sequence has more than or equal to 95 percent of identity (particularly preferably 96 percent, 97 percent, 98 percent and 99 percent) with the polynucleotide sequence shown in SEQ ID NO. 8; or;

SEQID NO:8：

AGCTTCTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGACTTTCCAGGAGAAGCT

In certain embodiments, the nucleic acid sequence of the second sequence has greater than or equal to 98% identity to the polynucleotide sequence set forth in SEQ ID NO. 8.

In certain embodiments, the nucleic acid sequence of the second sequence has greater than or equal to 99% identity to the polynucleotide sequence set forth in SEQ ID NO. 8.

In certain embodiments, the nucleic acid construct comprises or is operably linked to a single copy or multiple copies of a nuclear targeting sequence.

In certain embodiments, the first sequence and the second sequence are separate.

In certain embodiments, the first sequence is located upstream of the transposon 5' end repeat.

In certain embodiments, the second sequence is located upstream of the transposon 3' end repeat. In certain embodiments, the transposon 5' terminal repeat is selected from the group consisting of a PiggyBac transposon 5' terminal repeat and/or a Sleeping Beauty transposon 5' terminal repeat.

In certain embodiments, the transposon 5 'terminal repeat is a PiggyBac transposon 5' terminal repeat.

In certain embodiments, the 5' -terminal repeat of the PiggyBac transposon is shown in SEQ. ID No. 2.

SEQID NO:2：

TTAACCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATATATTTTCTTGTTATAGATATC

In certain embodiments, the transposon 3' terminal repeat is selected from the group consisting of a PiggyBac transposon 3' terminal repeat and/or a sleeping beauty transposon 3' terminal repeat.

In certain embodiments, the transposon 3 'terminal repeat is a PiggyBac transposon 3' terminal repeat.

In certain embodiments, the 3' -terminal repeat of the PiggyBac transposon is shown in SEQ. ID No. 9.

SEQID NO:9：

TTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAA

In certain embodiments, the promoter is selected from one or more of the CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, yb_tata promoter, EF 1a promoter, SV40 promoter, ubiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, β -actin promoter, TK promoter, and GRP78 promoter.

In certain embodiments, the promoter is a CMV promoter and/or an EF1 a promoter.

In certain embodiments, the promoter is an EF 1a promoter.

In certain embodiments, the nucleic acid sequence of the EF 1. Alpha. Promoter is as set forth in SEQ ID NO. 3.

SEQID NO:3：

GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

In certain embodiments, the exogenous receptor comprises a non-naturally occurring receptor.

In certain embodiments, the non-naturally occurring receptor is a synthetic, modified, recombinant, mutated or chimeric receptor.

In certain embodiments, the non-naturally occurring receptor is a receptor comprising one or more sequences isolated or derived from a T-cell receptor (TCR).

In certain embodiments, the non-naturally occurring receptor is a chimeric antigen receptor.

In certain embodiments, the chimeric antigen receptor targets one or more of the target binding proteins selected from the group consisting of: CD19, CD7, CD20, CD22, CD23, CD30, CD33, CD38, CD44v7/8, CD123, CD133, CD138, CD171, AFP (alpha fetoprotein), BCMA (B cell maturation antigen), CIL-1 (C-type lectin-like molecule-1), CS-1 (signaling lymphocyte activation molecule family 7, SLAMF 7), CEA (carcinoembryonic antigen), CA125 (mucin 16, MUC 16), CA19-9 (carbohydrate antigen), claudin 18.2 (Claudin-18 splice variant 2, CLDN 18.2), epCAM (epithelial cell adhesion molecule), EGFR (epidermal growth factor receptor), EGFRv III (epidermal growth factor receptor variant III), FAP (fibroblast activation protein) GPC1 (glypican 1), GPC3 (glypican 3), HER2 (human EGFR 2), IL-13Ra2 (interleukin 13 receptor subunit alpha-2), integrin beta 7 (integrin beta 7), FR (folate receptor), TAG-72 (tumor associated glycoprotein-72), MUC1 (mucin 1), MSLN (mesothelin), nectin-1 (poliovirus receptor associated protein 1, PVRL 1), NY-ESO-1 (cancer-testis antigen), GD2 (ganglioside 2), GUCY2C (guanylate cyclase C), GMR (granulocyte-macrophage colony stimulating factor receptor alpha chain), PSMA (prostate specific membrane antigen), gp100 (glycoprotein 100), VFGFR1 (vascular endothelial growth factor receptor-1), VFGFR2 (vascular endothelial growth factor receptor-2).

In certain embodiments, the chimeric antigen receptor targets one or more of the target binding proteins selected from the group consisting of: CD19, BCMA, claudin 18.2, MSLN, GPC3, HER2, GD2, PSMA, MUC1, GUCY2C, CD, CD7, CD20, CD30, CD123, CD33.

In certain embodiments, the chimeric antigen receptor targets a CD19 target binding protein.

In certain embodiments, the nucleic acid sequence encoding CD19 is as set forth in SEQ ID NO. 4.

SEQID NO:4：

GCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

In certain embodiments, the nucleic acid construct further comprises a transcription termination sequence.

In certain embodiments, the transcription termination sequence is selected from rBG pA.

In certain embodiments, the nucleic acid sequence encoding rBG pA is as set forth in SEQ ID NO. 7.

SEQID NO:7：

TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC

In certain embodiments, the nucleic acid construct comprises, in order, in the 5'-3' direction: a first sequence, a 5 'terminal repeat, a promoter, a sequence encoding an exogenous receptor, a transcription termination sequence, a second sequence, and a transposon 3' terminal repeat.

In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a linker.

In certain embodiments, the linker is selected from the group consisting of an IRES (internal ribosome entry sequence) element and a 2A polypeptide element.

In certain embodiments, the linker is selected from one or more of P2A, T2A, E2A, F a.

In certain embodiments, the linker is T2A.

In certain embodiments, the nucleic acid sequence of T2A is as set forth in SEQ ID NO. 5.

SEQID NO:5：

GGAAGCGGAGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCC

In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a marker protein.

In certain embodiments, the marker protein is selected from a fluorescent protein selected from one or more of a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, and a blue fluorescent protein.

In certain embodiments, the marker protein is selected from one or more of GFP, eGFP, eYFP, eCFP.

In certain embodiments, the marker protein is eGFP.

In certain embodiments, the nucleic acid sequence of eGFP is as shown in SEQ ID NO. 6.

SEQID NO:6：

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

In certain embodiments, the nucleic acid construct has one or more features selected from the group consisting of:

The transposon 5 'end repetitive sequence is a piggyBac transposon 5' end repetitive sequence;

The promoter is selected from the group consisting of: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1 alpha promoter, SV40 promoter, ubiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, beta-actin promoter, TK promoter and GRP78 promoter;

the coding exogenous receptor sequence comprises a chimeric antigen receptor sequence for expressing a targeted tumor cell;

the nuclear targeting sequence is selected from an nfkb nuclear targeting sequence comprising a first sequence and a second sequence;

the transcription termination sequence is selected from rBG pA;

The transposon 3 '-terminal repeat is a PiggyBac transposon 3' -terminal repeat;

The linker is selected from the group consisting of 2A polypeptide elements;

the marker protein is selected from GFP, eGFP, eYFP, eCFP.

The repeated sequence of the 5' -end of the transposon is shown as SEQ. ID No. 2;

The sequence of the promoter is shown as SEQ ID NO. 3;

the coding exogenous receptor sequence is shown as SEQ ID NO. 4;

The nuclear targeting sequences are shown as SEQ ID NO.1 and SEQ ID NO. 8

The transcription termination sequence is shown as SEQ ID NO. 7;

The 3' -terminal repeated sequence of the transposon is shown as SEQ. ID No. 9;

The linker sequence is shown as SEQ ID NO. 5;

the coded marker protein sequence is shown in SEQ ID NO. 6.

In certain embodiments, the nucleic acid construct is a polynucleotide sequence as set forth in SEQ ID NO. 10.

SEQID NO:10：

CTAGAGCTGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCCTGGGGACTTTCCAGCCTGGGGACTTTCCAGCTGGGACTTTCCAGGCGGTACCTCGCGCGACTTGGTTTGCCATTCTTTAGCGCGCGTCGCGTCACACAGCTTGGCCACAATGTGGTTTTTGTCAAACGAAGATTCTATGACGTGTTTAAAGTTTAGGTCGAGTAAAGCGCAAATCTTTTTTAACCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTCTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACGACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACTCATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATATATTTTCTTGTTATAGATATCATCAACTTTGTATAGAAAAGTTGGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACCCAGCTTTCTTGTACAAAGTGGTGATCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTCTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGACTTTCCAGGAGAAGCTTGGATCCCTCGAGTTAATTAACGAGAGCATAATATTGATATGTGCCAAAGTTGTTTCTGACTGACTAATAAGTATAATTTGTTTCTATTATGTATAGGTTAAGCTAATTACTTATTTTATAATACAACATGACTGTTTTTAAAGTACAAAATAAGTTTATTTTTGTAAAAGAGAGAATGTTTAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTTAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAAATAATAGTTTCTAATTTTTTTATTATTCAGCCTGCTGTCGTCGGAATTCACTAGTCGCGCCCGGGGAGCCCAAGGTTACCCCAGTTGGGGCGGGCCCCCATGGGTCGACGAGCTCCTGCAGGGATCCGATTTAAATTCGAAGCTAGCTCGACT

In a second aspect, the present invention provides a recombinant vector comprising the nucleic acid construct of any one of the first aspects.

In certain embodiments, the recombinant vector is a recombinant vector obtained by recombining the nucleic acid construct of any one of the present invention with a pMC-series vector.

The invention also provides a construction method of the recombinant vector in the second aspect, which comprises the following steps:

(1) Recombining and expressing the nuclear targeting sequence fragment and the transposon plasmid;

(2) Amplifying the expression element of the transposon plasmid inserted with the nuclear targeting sequence by PCR;

(3) PCR amplification to express small-loop plasmid pMC.BESPX-MCS2 skeleton fragment;

(4) And (3) connecting the small loop plasmid PCR fragment in the step (3) and the transposon plasmid PCR fragment in the step (2), and carrying out expression and culture to obtain the recombinant vector.

In the present invention, the expression element in the step (2) includes a transposon 5 'end repetitive sequence, a promoter, a coding expression exogenous receptor sequence, a linker, an expressed fluorescent protein fragment, a nuclear targeting sequence, a transcription termination sequence, and a transposon 3' end repetitive sequence.

In certain embodiments, the construction of the recombinant vector specifically comprises the steps of:

(1) Recombining the nuclear targeting sequence fragment and the transposon plasmid, transferring into an escherichia coli competent cell DH5 alpha, culturing, picking up a monoclonal, carrying out PCR amplification, and carrying out sequencing verification to extract the transposon plasmid inserted into the nuclear targeting sequence fragment.

(2) PCR amplifying the expression element (transposon 5 'end repetitive sequence, promoter, coding expression exogenous receptor sequence, linker, expression fluorescent protein fragment, nuclear targeting sequence, transcription termination sequence and transposon 3' end repetitive sequence) of the transposon plasmid with the inserted nuclear targeting sequence;

(4) Respectively carrying out double enzyme digestion on the amplified expression fragments to expose enzyme digestion sites;

(5) Connecting the small ring plasmid PCR fragment treated in the step (4) with the transposon plasmid PCR fragment, transferring into a competent ZYCY P3S2T, selecting a monoclonal after culturing, enriching and culturing for 10-16h by using a TB liquid culture medium, adding an equal volume of arabinose induction culture medium for culturing for 4-10h, and carrying out plasmid extraction to obtain the recombinant vector.

In a third aspect, the present invention provides a transposon system comprising the nucleic acid construct of any one of the first aspects or the recombinant vector of the second aspect.

In certain embodiments, the transposon system further comprises a vector capable of expressing a transposase.

In certain embodiments, the transposase is a PiggyBac transposase.

In a fourth aspect, the invention provides a host cell comprising or expressing the nucleic acid construct of any of the first aspects or the recombinant vector of the second aspect or the transposon system of the third aspect.

In certain embodiments, the host cell is a mammalian cell.

In certain embodiments, the host cell is selected from the group consisting of an immune cell, a Jurkat cell, a K562 cell, an embryonic stem cell, a tumor cell, a HEK293 cell, and a CHO cell.

In certain embodiments, the host cell is selected from any one or more of T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T Cells (CTLs), dendritic Cells (DCs), tumor Infiltrating Lymphocytes (TILs), macrophages, NK T cells, and γδ T cells.

In certain embodiments, the host cell is a T cell.

In a fifth aspect, the present invention provides a delivery vehicle comprising an adamantane tail lipid, an ionizable lipid, a neutral phospholipid, cholesterol, and a pegylated lipid, wherein the neutral phospholipid is selected from the group consisting of dioleoyl phosphatidylethanolamine,

The ionizable lipid is selected from compounds represented by formula (A7),

In certain embodiments, the pegylated lipid has a tail of a saturated or unsaturated alkane chain of length C ₆-C₂₀.

In certain embodiments, the pegylated lipid is selected from one or more of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol-polyethylene glycol 2000, polyethylene glycol 2000 ditetradecanol succinate.

In certain embodiments, the pegylated lipid is selected from polyethylene glycol 2000 ditetradecyl succinate (PEG ₂₀₀₀-Suc-TA₂), having the following structural formula:

In certain embodiments, the delivery vehicle comprises a compound of formula (AD 8), a compound of formula (A7), dioleoyl phosphatidylethanolamine, cholesterol, and polyethylene glycol 2000 ditetradecyl succinate.

The adamantane tail chain lipid adopted in the invention is disclosed in a patent CN114436994A, the ionizable lipid adopted in the invention is an A7 compound disclosed in a patent CN109503411B, and the polyethylene glycol lipid adopted in the invention is PEG ₂₀₀₀-suc-TA₂ disclosed in a patent CN 114539083A.

In certain embodiments, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 10-20:10-20:30-50:0.5-5.

In certain embodiments, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 12-18:12-20:40-50:1-3.

In certain embodiments, the molar ratio of the adamantane tail lipid, the ionizable lipid, the neutral phospholipid, the cholesterol, and the pegylated lipid is 15:15:18:50:2.

The delivery vehicle is prepared from functional lipid adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid according to a conventional method. Wherein conventional methods include, but are not limited to: ethanol injection method, microfluidic method, T-tube mixing method, and film extrusion method.

In a sixth aspect, the invention provides a pharmaceutical composition comprising the nucleic acid construct of any one of the first aspects or a nucleic acid expression vector comprising the nucleic acid construct of the first aspect and the delivery vector of any one of the fifth aspects.

The pharmaceutical composition is prepared according to a conventional method. Wherein conventional methods include, but are not limited to: ethanol injection method, microfluidic method, T-tube mixing method, and film extrusion method.

In certain embodiments, the pharmaceutical composition is prepared using an ethanol injection process.

In one embodiment, the method of making comprises:

(1) Weighing a proper amount of adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, and dissolving in absolute ethanol to obtain ethanol phase;

(2) The nucleic acid expression vector was dissolved in 10mM citrate buffer (ph=4.0) to obtain an aqueous phase.

(3) Under the condition of intense stirring, the ethanol phase is rapidly injected into the water phase, and the volume ratio of the water phase to the ethanol phase is 1:1 to 5:1. after the injection is completed, the delivery carrier solution can be obtained by dialysis with ultrapure water for 2 to 8 hours at room temperature and is preserved at 4 ℃ for standby.

In certain embodiments, the pharmaceutical composition is prepared using microfluidic methods.

In one embodiment, the method of making comprises:

(3) Two phases at a flow rate of 0.02-6 mL/min and 1:1 to 5:1 are mixed by micro-fluidic equipment at the same time, and dialyzing with ultrapure water for 2-8 hours at room temperature to obtain a delivery carrier solution, and preserving at 4 ℃ for standby.

In a seventh aspect, the present invention provides the use of a nucleic acid construct according to any one of the first aspects or a recombinant vector according to the second aspect or a transposon system according to the third aspect or a cell according to the fourth aspect or a delivery vector according to the fifth aspect or a pharmaceutical composition according to the sixth aspect for the preparation of a medicament for the prevention and/or treatment of a cancer-related disease.

In certain embodiments, the cancer is a hematological malignancy and a solid tumor.

In certain embodiments, the hematological malignancy includes, but is not limited to, B-cell line hematological malignancy, such as acute B-lymphocyte line leukemia, burkkit lymphoma, follicular lymphoma, and the like.

Embodiments of the present invention will be described in detail with reference to examples, wherein "%" represents mass% unless otherwise specified. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, edited by Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 construction of Nuclear targeting Small Loop CD19 CAR plasmids

In the embodiment, the pPB CD19 CAR transposon plasmid (abbreviated as PB transposon plasmid, entrusted to synthesis by yunshu biotechnology limited) is first digested with KpnI endonuclease, and the digested product is subjected to agarose gel electrophoresis and gel recovery is performed by using a gel recovery kit. The ligation of DTS-3NF1[ SEQ ID NO:1, entrusted to Shanghai Biotechnology (Shanghai) Inc. ] and the restriction transposon plasmid (PB transposon plasmid) was performed using T4 DNA ligase, and the ligation product was transferred into DH 5. Alpha. Competent cells, and the plasmid was extracted after the competent cells were expanded and cultured, and the plasmid identified as correct was designated as 3NF1-PB transposon plasmid. And then, enzyme digestion is carried out on the 3NF1-PB plasmid by using Hind III endonuclease, agarose gel electrophoresis is carried out on enzyme digestion products, and gel recovery is carried out by using a gel recovery kit. The DTS-3NF2[ SEQ ID NO:8, designated Shanghai Biotechnology (Shanghai) Co., ltd.) and the digested 3NF1-PB transposon plasmid were ligated using T4 DNA ligase, the ligation product was transferred into DH 5. Alpha. Competent cells, the plasmid was extracted after expansion culture of the competent cells, and the plasmid identified correctly was designated as pPB CD19 CAR-3NF transposon plasmid.

The small loop plasmids pMC (purchased from SBI, USA) and the pPB CD19 CAR-3NF transposon plasmid were amplified linearly using DNA polymerase. EcoRI and XbaI cleavage sites were inserted upstream and downstream of the two linearized fragments, respectively. Double digestion is carried out on the pPB CD19 CAR-3NF plasmid PCR amplified fragment and the small-loop plasmid pMC PCR amplified fragment by using EcoRI endonuclease and XbaI endonuclease, agarose gel electrophoresis is carried out on the digested products, and gel recovery is carried out by using a gel recovery kit. The pPB CD19 CAR-3NF plasmid PCR linearization fragment and the small loop plasmid pMC PCR linearization fragment are connected by using T4 ligase, the connection product is introduced into ZYCY P3S2T competent cells, and positive monoclonal colonies are selected for amplification and cryopreservation after selection.

The frozen bacterial liquid is taken, and after the frozen bacterial liquid is added into Kana ⁺/TB culture medium for enrichment and culture, the same volume of induction culture medium (100 mL of LB culture medium, 8mL of 20% arabinose and 3mL of 1M NaOH solution) is added, and the culture is carried out for 4 hours at 37 ℃ and 200 rpm. The plasmid is extracted by using a plasmid extraction kit, and the small ring nucleus targeting plasmid pPB CD19 CAR-3NF-MC is obtained. Simultaneously, the enzyme digestion verification (FIG. 2) and the sequencing verification were carried out by using XbaI endonuclease, and the preparation of the pPB CD19 CAR-3NF-MC (FIG. 1) was proved.

EXAMPLE 2 transfection efficiency of plasmid vectors on human T cells

The same electrotransfection procedure was used to transfect the pPB CD19 CAR-3NF-MC transposon and the PB transposon plasmid into T cells, respectively, and the effect of transfection of both plasmids in T cells was examined by an inverted fluorescence microscope and a flow cytometer 48h after transfection, and the results are shown in fig. 3. The transfection efficiency of the pPB CD19 CAR-3NF-MC transposon plasmid in T cells is 2 times that of the PB transposon plasmid, the MFI is 3 times that of the PB transposon plasmid, and the result shows that the T cell transfection effect of the nuclear targeting small loop plasmid is obviously improved compared with that of the common transposon plasmid.

Example 3 pDNA/LNP preparation and characterization

First, an ethanol injection method was used to ionize lipids according to the prescription (A7 or a13 disclosed in CN109503411B literature): adamantane tail chain lipids (AD 1-AD8 disclosed in CN114436994a literature): DOPE: cholesterol: PEG ₂₀₀₀-Suc-TA₂ = 15:15:18:50:1 (mol: mol) different Lipid Nanoparticles (LNP), N/P (nitrogen to phosphorus ratio) =6 were prepared. The prescribed amounts of adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol, and pegylated lipid were dissolved in absolute ethanol as the ethanol phase.

GFP model plasmid PmaxGFP was dissolved in 10mM citrate buffer (ph=4.0) as an aqueous phase and Lipofectamine 2000 (Lipo 2000) was used as a control. Under the condition of intense stirring, the ethanol phase is rapidly injected into the water phase, and the volume ratio of the water phase to the ethanol phase is 5:1. after the injection is completed, the sample is dialyzed for 4 hours at room temperature by ultrapure water to obtain LNP solution, and the LNP solution is preserved at 4 ℃ for standby. Meanwhile, LNP without adamantane tail lipid was prepared as a control, prescribed as ionizable lipid (A7 or a 13): DOPE: cholesterol: PEG ₂₀₀₀-Suc-TA₂ = 30:18:50:1 (mol: mol), N/p=6. The particle size, potential and polydispersity of LNP were measured by dynamic light scattering and the results are shown in table 1. As shown in Table 1, A7AD8 LNP has good safety and meets the requirement of transfection as a gene vector.

TABLE 1

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LNPs containing different neutral phospholipids were prepared as described above. The neutral phospholipid is one of DOPE, DSPC, DMPC, DOPC, DPPE, DSPE, SPC, and the prescription proportion is ionizable lipid (A7): adamantane tail chain lipid (AD 8): neutral phospholipids: cholesterol: PEG ₂₀₀₀-Suc-TA₂ = 15:15:18:50:1 (mol: mol), N/p=6. The particle size potential and polydispersity index of LNP were measured by dynamic light scattering and the results are shown in table 2.

TABLE 2

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Agarose gel electrophoresis was used to examine the loading capacity of A7AD8 LNP on the plasmid. The results showed that the A7AD8 LNP payload plasmid did not leak, indicating good load stability.

Example 4 pDNA/LNP transfection efficiency on human T cells

Peripheral blood of healthy people is singly collected, and mononuclear cells of the peripheral blood of the people are separated. Human CD3 ⁺ T cells were extracted from human peripheral blood mononuclear cells using a magnetic bead sorting kit, cells were resuspended in T cell medium containing human IL-2 and human CD3/CD28 antibodies, and plated at 1X 10 ⁶/mL in six well plates.

LNP prepared in example 3 above was added to 24-well plates inoculated with human T cells, each well containing 1. Mu.g of total plasmid, incubated in a cell incubator at 37℃with 5% CO ₂, and the expression of GFP was observed 2 days after transfection using an inverted fluorescence microscope, and the GFP ⁺ T cell positive rate and average fluorescence intensity (MFI) of GFP were examined by flow cytometry, the T cell transfection efficiencies of different neutral phospholipid LNPs were shown in FIG. 4, and the transfection results indicated that the efficiency of LNP transfected T cells was highest when neutral phospholipid was DOPE. T cell transfection results of LNP obtained with different combinations of ionizable lipids and adamantane tail lipids showed that pmaxGFP/A7AD8 LNP had the highest transfection efficiency for human T cells as shown in FIG. 5. In conclusion, the transfection effect of the combined application of A7 and AD8 is obviously better than that of the combination of the purely ionizable lipid, other ionizable lipids and adamantane tail chain lipid, and is obviously better than that of positive control Lipo2000.

Example 5 pDNA/A7AD8 LNP preparation and characterization

The small loop nuclear targeting transposon plasmid pPB CD19 CAR-3NF-MC prepared in example 1 was loaded using the preferred LNP recipe A7AD8 LNP described above. Preparation of pPB CD19 CAR-3NF-MC/A7AD8 LNP using microfluidic method, prescribed ratio was ionizable lipid (A7): adamantane tail chain lipid (AD 8): DOPE: cholesterol: PEG ₂₀₀₀-Suc-TA₂ = 15:15:18:50:1 (mol: mol), N/p=6. Precisely weighing required adamantane tail chain lipid, ionizable lipid, neutral phospholipid, cholesterol and polyethylene glycol lipid, and dissolving in absolute ethanol as ethanol phase. The pPB CD19 CAR-3NF-MC small loop plasmid and hyPBase transposase plasmid (available from yunshu biotechnology limited) were dissolved in 10mM citrate buffer (ph=4.0) at a mass ratio of 4:1 as aqueous phases. The two phases are mixed by a microfluidic device at a total flow rate of 6mL/min, and dialyzed with ultrapure water at room temperature for 4 hours to obtain lipid nanoparticle solution, and the lipid nanoparticle solution is preserved at 4 ℃ for standby. PB/MC3AD8 LNP and PB/A7AD8 LNP were prepared simultaneously as controls, in the same manner as for the preparation and for the pPB CD19 CAR-3NF-MC/A7AD8 LNP.

The loading capacity of A7AD8 LNP on the plasmid was examined by agarose gel electrophoresis, which is shown in FIG. 6, using a naked plasmid as a positive control. The results show that the pPB CD19 CAR-3NF-MC/A7AD8 LNP payload plasmid does not leak, indicating good load stability.

LNPs prepared as described above were transfected into human T cells as in example 4, and the transfection efficiencies of pPB CD19 CAR-3NF-MC/A7AD8 LNP with non-optimized PB/MC3AD8 LNP and PB/A7AD8 LNP are shown in FIG. 7.

In conclusion, the delivery efficiency of the optimized optimal LNP and pPB CD19 CAR-3NF-MC combination in T cells is remarkably improved, 10 times of that of an unoptimized prescription (PB/MC 3AD8 LNP) and 3 times of that of PB/A7AD8 LNP, and the necessity and the effectiveness of nuclear targeting small-loop plasmid transformation are proved.

Example 6 intracellular transport of pDNA/LNP in human T cells

In order to examine the mechanism of high transfection efficiency of pDNA/A7AD8 LNP prepared by the present invention on human T cells, intracellular transport process was also studied in this example. Cy5-pmaxGFP/A7AD8 LNP was prepared as described in example 5 using Cy5 fluorescent-labeled pmaxGFP as a model plasmid.

The uptake capacity of human T cells for pDNA/A7AD8 LNP was first examined. T cells were transfected as in example 4, and the positive rate of Cy5 ⁺ T cells and MFI of Cy5 were measured for each group using a flow cytometer, with non-transfected T cells as negative controls (fig. 8). The results show that the uptake of A7AD8 LNP by T cells is significantly increased, with an uptake rate of about 99% which is much higher than that of the commercially available positive control Lipo 2000.

The endosomal escape ability of pDNA/A7AD8 LNP in human T cells was subsequently studied. Human CD3 ⁺ T cells were extracted and activated as in example 4, seeded at a density of 1X 10 ⁶/mL in a twelve well plate, 1mL of medium was added to each well followed by 200. Mu.L of Cy5-pmaxGFP/A7 AD8 LNP, the cells were placed in a cell incubator for 6h, each group of T cells was collected, washed with PBS, 1mL of T cell medium was added, and culturing was continued for 0h and 2h. Each group of T cells was collected, and T cell culture medium containing lysosome green fluorescent probe (Lyso-TRACKER GREEN) and Hurst fluorescent dye 33342 (Hoechst 33342) was added thereto, and after incubating at 37℃for 30min, the cells were collected by centrifugation, and the intracellular transport process was observed and photographed using a laser confocal microscope (FIG. 9). The results show that the A7AD8 LNP can effectively escape from human T cell endosomes into cytoplasm after transfection for 6h, which is beneficial to subsequent plasmid expression.

EXAMPLE 7 physiological Activity study of CAR-T constructed based on pPB CD19 CAR-3NF-MC/LNP

In order to evaluate that CAR-T constructed using pPB CD19 CAR-3NF-MC/A7AD8 LNP still maintains normal physiological activity, the present invention was studied in terms of CAR expression duration, cell proliferation, cell viability, cell phenotype, and cytokine secretion. CD19 CAR-T was first prepared as in example 5. The expression of GFP was then observed with an inverted fluorescence microscope at 2, 5, 7, 10 and 15 days after transfection, respectively, and the GFP ⁺ T cell positive rate and the average fluorescence intensity of GFP were examined by flow cytometry (FIG. 10). The results indicate that CAR-T cells constructed with pPB CD19 CAR-3NF-MC/A7AD8 LNP can continue to express CAR for at least 15 days. Meanwhile, CAR-T cells after transfection were counted at 0, 2, 5, 7, 10 days, respectively, and in vitro expansion fold of cells (=cell number after transfection/cell number before transfection) was calculated, and three duplicate wells were set for each time point, with non-transfected T cells as a blank control group (fig. 11). The results show that the proliferative capacity of the constructed CAR-T cells is not significantly different from T cells.

Subsequently, the viability of CAR-T cells was examined. After 48h of T cell transfection, using an Annexin V PE-7AAD apoptosis kit for staining, using a flow cytometer to examine apoptosis conditions of the cells, and setting three compound wells. Untransfected CD3 ⁺ T cells served as control (fig. 12). The results indicate that the pPB CD19 CAR-3NF-MC/LNP does not affect the viability of T cells.

In addition, to avoid that the pPB CD19 CAR-3NF-MC/A7AD8 LNP transfection would affect the phenotype of T cells, the CD4/CD8 phenotype of constructing CAR-T cells based on pPB CD19 CAR-3NF-MC/A7AD8 LNP was also studied. After 48h of T cell transfection, CAR-T cells were collected and stained with PBS solution containing PE anti-human CD8 antibody, APC anti-human CD4 antibody for 30min, and then washed 2 times with PBS. The ratio of CD4/CD8 of CAR-T was examined using a flow cytometer. Untransfected CD3 ⁺ T cells served as a blank (fig. 13). The results show that pPB CD19 CAR-3NF-MC/A7AD8 LNP does not affect the CD4/CD8 phenotype of T cells.

Finally, the efficacy of CAR-T cells constructed based on pPB CD19 CAR-3NF-MC/A7AD8LNP was investigated using cytokine secretion capacity as an index. Human lymphoma Raji cells were plated in 96-well plates at a density of 1×10 ⁴ cells/well, 1×10 ⁵ CAR-T cells were added to each well, followed by co-incubation in a 5% co ₂ cell incubator at 37 ℃ for 24h, and all cells were collected. After staining for 30min with a PBS solution containing FITC anti-human CD3 antibody, the washing was performed 2 times with PBS. After cells were fixed by PFA, triton was perforated, stained with PBS solution containing PE/Cy7 anti-human GzmB antibody, PE anti-human TNFα antibody, APC anti-human IL-2 antibody for 30min, and washed 2 times with PBS. Cytokine secretion of CAR-T cells was measured using a flow cytometer with untransfected T cells as a control (fig. 14). The results indicate that CAR-T cells constructed based on pPB CD19 CAR-3NF-MC/A7AD8LNP can secrete cytokines under target cell antigen stimulation without affecting the efficacy of CAR-T.

The results show that after the pPB CD19 CAR-3NF-MC/A7AD8 LNP transfects the T cells, the normal physiological functions of the T cells are not affected, and the pPB CD19 NF-MC/A7AD8 LNP can be used for subsequent application.

Example 8 specific killing of target cells by CAR-T constructed based on pPB CD19 CAR-3NF-MC/LNP

CD19 CAR-T was prepared as in example 5. Human lymphoma Raji cells expressing CD19 are used as antigen-positive target cells, human chronic myelogenous leukemia K562 cells not expressing CD19 are used as antigen-negative target cells, and untransfected T cells are used as controls. Target cells were plated in 96-well plates at a density of 1×10 ⁴/well, a corresponding number of CAR-T cells were added per well in an effective target ratio, followed by co-incubation in a 5% co ₂ cell incubator at 37 ℃. At the same time, a sample control well with only tumor cells and a control well with maximum enzyme activity for subsequent lysis were set. Before reaching the preset detection time point for 1h, taking out a ninety-six pore plate from the cell culture box, and adding an LDH release reagent into the 'sample maximum enzyme activity control pore', wherein the addition amount is 10% of the volume of the original culture medium. After adding the LDH releasing reagent, repeatedly and evenly mixing the mixture by blowing the mixture for a plurality of times, and then continuously incubating the mixture in a cell incubator. After a predetermined time, the cell culture plates were centrifuged for 5min with 400g in a multi-well plate centrifuge. The supernatant from each well was taken at 120. Mu.L and added to the corresponding well of a new 96-well plate. mu.L of LDH detection working solution was added to each well. Mixing, and incubating at room temperature for 30min in dark place. Absorbance was measured at 490 nm. The dual wavelength measurement was performed using 600nm as the reference wavelength. Cell death rate (%) = (absorbance of treated sample-absorbance of sample control well)/(absorbance of maximum enzyme activity of cell-absorbance of sample control well) ×100. As shown in fig. 15, CAR-T constructed based on pPB CD19 CAR-3NF-MC/A7AD8 LNP had good specific killing ability against CD19 positive target cells, whereas killing ability against target cells that did not express CD19 was comparable to non-transfected T cells.

It should be understood that the above examples are illustrative and are not intended to encompass all possible implementations encompassed by the claims. Various modifications and changes may be made in the above embodiments without departing from the scope of the disclosure. Likewise, the individual features of the above embodiments can also be combined arbitrarily to form further embodiments of the invention which may not be explicitly described. Therefore, the above examples merely represent several embodiments of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A pharmaceutical composition comprising a delivery vehicle and a nucleic acid expression vector, wherein,

The ionizable lipid is selected from compounds represented by formula (A7),

2. The pharmaceutical composition of claim 1, wherein the pegylated lipid has a tail of saturated or unsaturated alkane chain of length C ₆-C₂₀.

3. The pharmaceutical composition according to claim 2, wherein the pegylated lipid is selected from one or more of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol-polyethylene glycol 2000, polyethylene glycol 2000 ditetradecanol succinate;

And/or the delivery vehicle comprises a compound shown in a formula (AD 8), a compound shown in a formula (A7), dioleoyl phosphatidylethanolamine, cholesterol and polyethylene glycol 2000 bitetradecyl succinate.

4. The pharmaceutical composition of claim 1, wherein the nuclear targeting sequence is selected from the group consisting of NF- κB nuclear targeting sequences comprising a first sequence and a second sequence,

(1) A polynucleotide sequence shown in SEQ ID NO. 1;

(2) The nucleotide sequence with SEQ ID NO. 1 is more than or equal to 95 percent identical; or alternatively, the first and second heat exchangers may be,

(4) A polynucleotide sequence shown in SEQ ID NO. 8;

(5) The nucleotide sequence with SEQ ID NO. 8 is more than or equal to 95 percent identical; or alternatively, the first and second heat exchangers may be,

5. The pharmaceutical composition of claim 4, wherein the first sequence and the second sequence are separate;

and/or, the first sequence is located upstream of the transposon 5' end repeat sequence;

and/or, the second sequence is located upstream of the transposon 3' end repeat.

6. The pharmaceutical composition of claim 1, wherein the promoter is selected from one or more of the group consisting of CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, yb_tata promoter, EF 1a promoter, SV40 promoter, ubiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, β -actin promoter, TK promoter, and GRP78 promoter;

And/or the exogenous receptor comprises a non-naturally occurring receptor that is a chimeric antigen receptor that targets one or more ：CD19、CD7、CD20、CD22、CD23、CD30、CD33、CD38、CD44v7/8、CD123、CD133、CD138、CD171、AFP、BCMA、CIL-1、CS-1、CEA、CA125、CA199、CLDN18.2、EpCAM、EGFR、EGFRvⅢ、FAP、GPC1、GPC3、HER2、IL-13Ra2、 integrins beta 7, FR, TAG-72, MUC1, MSLN, nectin-1, NY-ESO-1, GD2, cy2C, GMR, PSMA, gp100, VFGFR1, VFGFR2 selected from the following target binding proteins.

7. The pharmaceutical composition of claim 1, wherein the transposon 5' terminal repeat is represented by SEQ ID No. 2;

and/or, the transposon 3' terminal repeat is shown as SEQ ID NO. 9;

and/or the nucleic acid sequence of the promoter is shown as SEQ ID NO. 3;

And/or the nucleic acid expression vector further comprises a transcription termination sequence, wherein the transcription termination sequence is shown as SEQ ID NO. 7;

And/or the nucleic acid expression vector further comprises a nucleic acid sequence for encoding a linker, wherein the sequence for encoding the linker is shown as SEQ ID NO. 5.

8. The pharmaceutical composition of claim 1, wherein the nucleic acid expression vector has a nucleic acid sequence as set forth in SEQ ID No. 10.

9. The pharmaceutical composition of any one of claims 1-8, further comprising a vector capable of expressing a transposase.

10. Use of the pharmaceutical composition according to any one of claims 1 to 9 in a medicament for the prevention and/or treatment of cancer related diseases.