CN114106203A

CN114106203A - Nucleic acid construct containing targeted Her-2CAR sequence and application thereof

Info

Publication number: CN114106203A
Application number: CN202111419541.4A
Authority: CN
Inventors: 陈伟; 刘昊; 胡立强; 谢尚志
Original assignee: Hangzhou Qianhe Cell Biotechnology Co ltd
Current assignee: Hangzhou Qianhe Cell Biotechnology Co ltd
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-03-01

Abstract

A nucleic acid construct containing a targeted Her-2CAR sequence and application thereof, wherein the nucleic acid construct is composed of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signaling domain which are connected in series, the tumor-associated antigen binding region is a single-chain antibody FRP5 scFv targeted to Her-2, and the nucleic acid and polypeptide sequences of the nucleic acid construct are respectively shown as SEQ ID No.3 and SEQ ID No. 4. The nucleic acid construct can be used for specifically identifying Her-2 high-expression tumor cells, activating immune cells and enabling the immune cells to release cytokines such as IFN-gamma and the like to kill the tumor cells.

Description

Nucleic acid construct containing targeted Her-2CAR sequence and application thereof

Technical Field

The invention relates to the field of tumor immunotherapy, in particular to a nucleic acid construct containing a targeted Her-2CAR sequence and application thereof.

Background

Chimeric Antigen Receptor (CAR) immune cell therapy is one of the most promising current tumor immunotherapies. A fusion protein of a Single chain antibody (scFv) for recognizing a tumor-associated antigen and an immune cell activation sequence, namely a Chimeric Antigen Receptor (CAR), is expressed on the surface of an immune cell by a foreign gene transfection technology, so that the scFv capable of specifically recognizing the tumor-associated antigen is coupled with an activation proliferation signal domain in the immune cell through a transmembrane region. CAR-expressing immune cells (e.g., T cells, NK cells) bind tumor antigens in an antigen-dependent, but not MHC-restricted manner, initiating and activating a specific killing tumor response.

After the CAR is combined with tumor-associated antigens on tumor cells, an activation signal is provided for immunity to cause immune cell activation, which is expressed by CARs-dependent killing, proliferation and cytokine release, so that the CAR plays a role in killing the tumor cells. These effector functions can be used to design synthetic novel CARs such as CARs comprising multiple chimeric activation regions, such as

generations

1, 2, and 3 CARs with 1, 2, or 3 signaling motifs within the cell. Early CARs were designed to be relatively simple, with only one intracellular signaling region, also known as CAR generation 1, signaling mainly mediated through the immunoreceptor tyrosine kinase activation motif (ITAM) on CD3 ζ, with only 3 ITAMs compared to CD3 molecule and lacking the 2 nd signal of costimulatory molecule transduction, so that CARs generation 1 had only weak antitumor effect, and T cells hardly proliferated after binding tumor antigen due to lack of the 2 nd signal of T cell proliferation, thus having poor effect in clinical application; the 2 generation CARs is to add an intracellular signal region on the basis of the 1 generation, and the signal region provides costimulatory signals (such as CD28 and CD 137); the 3 rd generation CARs add two co-stimulatory signal regions in series within the cell. In addition, there have been 4 th generation CARs (also known as TRUCKs) in recent years, which use vectors encoding CARs and/or CAR-responsive promoters to construct CARs, and which generate potent signals under the action of transgenically produced cytokines that recruit other components of the immune system to amplify the anti-tumor immune effect.

Unlike CAR-T cells, CAR-NK cells retain the intrinsic ability to recognize and target tumor cells through their natural receptors, so that the likelihood that tumor cells can escape killing is reduced when treated by CAR targeting. Second, CAR-NK cell therapy, in multiple clinical trials, showed no immune rejection within days to weeks. Thus, CAR-NK cell therapy does not exhibit similar safety issues, such as the absence of the trouble of cytokine release syndrome, as compared to many clinical trials of CAR-T cell therapy. Finally, NK cells do not require strict HLA matching and therefore do not cause graft versus host disease, which is a great advantage of CAR-NK cell therapy and also a significant risk for CAR-T cell immunotherapy.

HER2(Human epidermal growth factor receptor 2) belongs to the epidermal growth factor receptor family and is a transmembrane protein with tyrosine kinase activity. Studies have shown that HER2 gene overexpression is present in patients with approximately third-generation breast cancer. In these patients, the malignancy is high, the drug resistance is high, the recurrence and metastasis rate is high, and the disease-free survival of the patients is inversely proportional to the overall survival and the expression of the HER2 gene. During the pathological process of breast cancer, the change of the expression level of HER2 plays a crucial role, so HER2 becomes an important target for scientists to develop a breast cancer treatment drug.

Disclosure of Invention

Based on the background technology, the invention aims to provide a nucleic acid construct containing a targeted Her-2CAR sequence and application thereof, wherein the nucleic acid construct can be used for specifically recognizing tumor cells expressing high expression of Her2, activating immune cells and enabling the immune cells to release cytokines such as IFN-gamma and the like to kill the tumor cells.

Specifically, the present invention is directed to solving the above-mentioned problems of the prior art, and the present invention specifically provides a nucleic acid construct comprising a targeted Her-2CAR sequence, wherein the nucleic acid construct is composed of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain connected in series.

Preferably, the nucleic acid construct further comprises a Signal peptide Sequence (SP), which is a short (5-30 amino acids in length) peptide chain responsible for directing the newly synthesized protein to different membrane structure-containing subcellular organelles of the cell, typically at the N-terminus of the protein. The signal peptide may be selected from the group of signal peptides commonly used for recombinant proteins such as Human IgKVIII, Mouse Ig Kappa, Human IL-2, Human insulin and the like, and it is preferred according to the present invention to provide a CD8a signal peptide comprising a nucleic acid construct targeting Her-2CAR sequences, the nucleic acid and polypeptide sequences of which are shown in SEQ ID No.1 and SEQ ID No.2, respectively.

Preferably, the antigen binding region is capable of tightly binding to a tumor associated antigen expressed on the surface of a tumor cell, determines targeting of the CAR structure, and is the core structure that determines the effect of the modified immune cell. Currently, the extracellular antigen-binding domain of most chimeric antigen receptors is derived from single chain antibodies (scFv) formed by the connection of light (VL) and heavy (VH) chains of monoclonal antibodies targeting specific antigens of interest, and a flexible hinge (linker) in the middle. The heavy or light chain of a single-chain antibody is connected to a signal peptide and a hinge region, respectively. The nucleic acid and polypeptide sequences of the Her-2 targeting single chain antibody (FRP5) scFv (VH-linker-VL) of the Her-2 targeting nucleic acid construct containing the Her-2CAR sequence according to the preferred embodiment of the present invention are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

Preferably, the hinge region connecting the extracellular antigen-binding region and the transmembrane domain, the hinge region of most CARs is derived from the IgG hinge or the CD8 a/CD 28 extracellular region. The length of the hinge region depends on the location of the epitope on the target cell and the degree of exposure. Several studies have shown that CAR-T cell activation is related to hinge region length. Adjusting the length of the hinge region allows the CAR-T cell to be at an optimal distance from the target cell, avoiding the effects of large phosphatases to attenuate CAR signals during antigen-antibody binding. However, in some cases, the epitope may be relatively inaccessible and a longer hinge region may be required so that the scFv can overcome steric hindrance and effectively bind the antigen. Thus, the epitopes of the antigen are different and the optimal length of the hinge region is also different, and when targeting a neo-antigen, the length of the hinge region may need to be adjusted accordingly. The extracellular hinge region may be a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, etc., or IgG1, IgG4 (with or without the CH2CH3 region). According to various preferred embodiments of the present invention there is provided CD8 Hinge (CD 8H), CD28 Hinge (CD 28H), and IgG4 Hinge-CH2-CH3 comprising nucleic acid constructs targeting Her-2CAR sequences, the nucleic acid and polypeptide sequences of which are set forth in SEQ ID nos. 5 and 6, 7 and 8, and 9 and 10, respectively.

Preferably, the transmembrane domain connects the extracellular domain of the CAR with the intracellular signaling domain and anchors the receptor to the immune cell membrane. Transmembrane domain (TMD) may be derived from the nucleic acid, polypeptide sequence of the following protein transmembrane domains: the α, β or ζ chain of a T cell receptor, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, CTLA. For example, the CD3 zeta transmembrane domain enables the CAR to form a homodimer or heterodimer with an endogenous TCR, enhancing CAR-T cell activity, but is also being phased out as it does not require binding to endogenous TCRs to highly activate T cells. The transmembrane domains of CD8a and CD28 are currently employed in most clinical trials due to their ability to promote CAR expression on the cell surface. CD8, CD28, and NKG2D fransmembrane domain containing nucleic acid constructs targeting Her-2CAR sequences provided according to various preferred embodiments of the present invention have nucleic acid and polypeptide sequences as set forth in SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, and SEQ ID NO.15 and SEQ ID NO.16, respectively.

Preferably, the intracellular signaling Domain comprises or does not comprise a Costimulatory Domain (CD), the Costimulatory Domain. The co-stimulatory domain is typically from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD 27). The co-stimulation domain can realize the dual activation of the co-stimulation molecules and signals in cells, so that the T cells can continuously proliferate and release cytokines, and the anti-tumor capacity of the T cells is improved. For example, the CD28 co-stimulatory domain makes the CAR-T cell dependent on glycolysis metabolism, causing the CAR-T cell to differentiate towards effector T cells. While the 4-1BB co-stimulatory domain promotes mitochondrial generation, enhances respiration and fatty acid oxidation, and after antigen stimulation, CAR-T cells preferentially differentiate into central memory T cells. The co-stimulatory domain may be derived from one or more of the following different combinations of nucleic acid, polypeptide sequences of intracellular functional signaling junction domains of proteins: OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137) and 2B4 or functional variants thereof. CD137 Costimmulation Domain, CD28 Costimmulation Domain, DAP10 Costimmulation Domain, and 2B4 Costimmulation Domain containing nucleic acid constructs targeting Her-2CAR sequences provided according to various preferred embodiments of the present invention have the nucleic acid and polypeptide sequences shown in SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, and SEQ ID NO.23 and SEQ ID NO.24, respectively.

Preferably, the signal transduction Domain (SD) is typically a T cell receptor TCR/CD3 zeta chain or an immunoglobulin Fc receptor fcepsilon RI gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAMs). Plays a cell signal transduction function. According to the preferred embodiment of the present invention, there is provided a CD3 ζ Signaling Domain comprising a nucleic acid construct targeting a Her-2CAR sequence, the nucleic acid and polypeptide sequences of which are set forth in SEQ ID No.25 and SEQ ID No.26, respectively.

It is another object of the present invention to provide a plurality of preferred sequence combinations of nucleic acid constructs comprising a targeted Her-2CAR sequence, each combination being as follows, with specific nucleic acid and polypeptide sequences as described above:

a)CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD8 TMD-CD137 CD-CD3ζSD；

b)CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD3ζSD；

c)CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD137 CD-CD3ζSD；

d)CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-DAP10 CD-CD3ζSD；

e)CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-CD3ζSD；

f)CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-DAP10 CD-CD3ζSD；

g)CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-CD137 CD-2B4 CD-CD3ζSD。

it is another object of the invention that the immune effector cells used for CAR introduction can be T cells, NK cells or macrophages and their origin can be autologous, allogeneic, stem cell differentiation or specific cell lines when using the nucleic acid constructs containing targeted Her-2CAR sequences provided according to various preferred embodiments of the invention. Preferably, the immune cells are selected from NK cells, but the invention is not limited thereto. The extracellular antigen binding region is derived from a monoclonal antibody targeting a specific target antigen, and thus can be transfected into immune cells (e.g., T cells, NK cells) for tumor immunotherapy to assist the patient in eliminating or reducing tumor cells in vivo.

When the immune effector cells used for CAR introduction are NK cells, the CAR-NK cells retain the intrinsic ability to recognize and target tumor cells through their natural receptors, such that upon CAR-targeted therapy, the likelihood that tumor cells can escape killing is reduced, and CAR-NK cell therapy, in multiple clinical trials, shows no immune rejection within days to weeks, is safer than CAR-T cell therapy, e.g., without the trouble of cytokine release syndrome, while NK cells do not require strict HLA matching, and therefore do not cause graft-versus-host disease.

It is another object of the invention to provide a nucleic acid construct comprising a Her-2 targeting CAR sequence, the transmembrane domain of which can be derived from CD8a and CD28, thereby enabling the expression of the CAR on the cell surface.

Another objective of the invention is to provide a nucleic acid construct containing a Her-2CAR targeting sequence, wherein the signal transduction domain is T cell receptor TCR/CD3 zeta chain or immunoglobulin Fc receptor FceRI gamma chain, and the nucleic acid construct contains immunoreceptor tyrosine-based activation motifs (ITAMs) for exerting cell signal transduction function.

It is another object of the invention to provide a nucleic acid construct containing a targeted Her-2CAR sequence that can be adapted for lentiviral (Lentivirus) vector transfection with near 100% transduction efficiency, greater transgene load, and less genotoxic.

Another object of the present invention is to provide a nucleic acid construct containing a targeted Her-2CAR sequence, which is suitable for transfection of lentiviral vectors, thereby differentiating common retroviral vectors, which has an infectious capacity for both dividing and non-dividing cells, and can effectively integrate foreign genes into host chromosomes, thereby achieving persistent expression, and in addition, can effectively infect various types of cells such as neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, and the like in terms of infectious capacity, thereby achieving a good gene therapy effect, and having a broad application prospect.

It is another object of the invention to provide a nucleic acid construct containing a targeted Her-2CAR sequence that can be adapted for lentiviral vector transfection, allowing the escape of Rev-assisted unspliced viral mRNA in conjunction with the use of an HIV-1 Rev Response Element (RRE); the 3' terminal truncated LTR removes the U3 region, eliminates the possibility of self-replication of the lentiviral vector and greatly improves the safety; promoters that drive expression of downstream CAR structural genes, such as the human own ubiquitin C promoter, EF1a promoter, EFs promoter, etc.; and cPPT and WPRE elements, to improve transduction efficiency and transgene expression efficiency.

It is another object of the present invention to provide lentiviral vectors, which require an envelope plasmid (envelope plasmid) and a packaging plasmid (packaging plasmid), in addition to a shuttle plasmid (transfer plasmid), for the preparation of nucleic acid constructs containing targeted Her-2CAR sequences, and packaging methods thereof. For the Envelope plasmid, VSV-G is usually chosen because lentiviruses containing this Envelope protein can infect a variety of hosts. However, resting T, B, NK and hematopoietic stem cells hardly express LDLR gene, so they are very difficult to infect VSV-G enveloped lentiviruses, and Baboon enveloped pseudotyped viral vectors with Baboon as the envelope are reported in literature to significantly improve the transduction efficiency of NK cells.

The transfer plasmid takes pCDH-UBC-DSRED-LUC-EF1 Hygro (addendum #129437) as an initial vector, and an EFS-containing promoter is constructed, wherein the EFS-containing promoter is used for starting a lentiviral vector for expressing a foreign gene. The reason is because the EFS promoter has a shorter sequence and has a higher transduction efficiency for primary NK cells.

The transfer plasmid contains the prokaryotic replicon pUC Ori sequence for plasmid replication; the ampR sequence of the ampicillin-containing resistance gene is used for the mass amplification of a target strain; a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells; elements for lentiviral packaging include: an RSV promoter for initiating transcription of lentiviral mRNA; lentivirus 5 terminal truncated LTR, 3 terminal truncated LTR, RRE cis element, env cis element, cPPT cis element; an EFS promoter for eukaryotic transcription of a target gene, a downstream TurboRFP and ZsGreen1 fluorescent protein sequence thereof, a T2A 'self-shearing' peptide sequence for connecting the co-transcription expression fluorescent protein and a WPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element for enhancing the expression efficiency of transgenosis.

The method for constructing the transfer plasmid comprises the following steps:

1) constructing a vector containing EFS-TurboRFP-T2A-ZsGreen1, ClaI enzyme and KpnI enzyme sites, wherein the nucleic acid sequence of the EFS-TurboRFP-T2A-ZsGreen1 is shown in SEQ ID NO. 27;

2) the pCDH-UBC-DSRED-LUC-EF1 Hygro vector (addendum #129437) is cut by ClaI enzyme and KpnI enzyme, and the linearized vector fragment is recovered;

3) the vector containing the EFS-TurboRFP-T2A-ZsGreen1 fragment is digested by ClaI enzyme and KpnI enzyme, and the digested fragment is recovered.

4) The recovered products of 2) and 3) are connected, transformed and identified to obtain the plenti-EFS-TurboRFP-T2A-Zsgreen vector.

The preparation method of the transfer plasmid containing the targeted Her-2CAR sequence comprises the following steps:

1) constructing a Her-2 CAR-targeting nucleic acid construct, preferably the Her-2 CAR-targeting nucleic acid construct having a nucleic acid sequence as set forth in SEQ ID No. 29;

2) utilizing Xbal enzyme + XhoI enzyme to carry out enzyme digestion on the plenti-EFS-TurboRFP-T2A-Zsgreen carrier, and recovering a linearized carrier fragment;

3) digesting the vector containing the nucleic acid construct containing the targeted Her-2CAR with the Xbal + XhoI enzyme and recovering the fragment;

4) connecting, transforming and identifying the recovered products obtained in the steps 2) and 3) to obtain pHer2-CAR1, namely: a transfer plasmid containing a sequence targeted to Her-2 CAR.

Preferably, the Envelope plasmid takes pMD2.G (addendum #12259) as an initial vector, and the specific construction method is as follows:

1) constructing a vector containing a BaEVRless fragment, wherein the nucleic acid sequence of the BaEVRless fragment is shown as SEQ ID NO. 28;

2) the pMD2.G vector is cut by HindIII enzyme and NotI enzyme, and a linearized vector fragment is recovered;

3) carrying out enzyme digestion on the vector containing the BaEVRless fragment by using HindIII enzyme and NotI enzyme, and recovering the fragment;

4) connecting the recovered products obtained in the steps 2) and 3), converting and identifying to obtain pBaEVR, namely the Envelope plasmid.

The packaging method for preparing the lentiviral vector containing the nucleic acid construct targeting the Her-2CAR sequence specifically comprises the following steps:

1) plasmids

·pHer2-CAR1

·VSV-G：pMD2.G(Addgene#12259)or pBaEVR

·Rev：pRSV-Rev(Addgene#12253)

·Gag/Pol：pMDLg/pRRE(Addgene#12251)

·Tat：pCEP4-tat(Addgene#22502)

2) Cell lines

Low passage 293T cells

3) Reagent

293T Medium: high-glucose DMEM (sodium pyruvate, glutamine) + 10% FBS + GlutaMAX.

Virus harvest medium: 50ml of 293T medium were taken, 0.5g BSA (Sigma A9418) and HEPES at a final concentration of 10-15mM were added, and 0.22 μm filtration was carried out.

Opti-MEM Medium: for mixing the transfection complexes.

Transfection reagents: X-tremeGENE HP DNA (or other low toxicity and high efficiency reagents).

4) Packaging process

Day one: 293T cells were grown according to the following table.

The next day-morning: a mix of Opti-MEM medium, plasmid and transfection reagent was prepared according to the following table. Fresh medium was changed for 293T cells. The mixture was allowed to stand at room temperature for 15-30min, added dropwise to 293T cell culture system, and shaken gently.

	6-well	10-cm	15-cm
				Seeding number	8.75x10⁵	5.5x10⁶	1.8x10⁷
Medium volume	1.5-2mL	5-6mL	15-17mL
				Transfer plasmid	1.2μg	6μg	16.6μg
VSV-G	0.24μg	1.2μg	3.4μg
				Rev	0.12μg	0.6μg	1.7μg
Gag/Pol	0.12μg	0.6μg	1.7μg
				Tat	0.12μg	0.6μg	1.7μg
Opti-MEM	200μL	1mL	2mL
				X-tremeGENE	5.4μL	27μL	74.5μL

Next day-afternoon: after 6-8hr of transfection, the medium was gently changed to virus harvest medium.

Day four: 48hr after transfection, cell culture supernatant was centrifuged at 400 Xg for 4min, filtered at 0.45 μm, added with 5 XPEG 8000 solution by volume, mixed every 20-30min for 3-5 times, and left at 4 ℃ for 6h or overnight. Centrifuging at 4 deg.C and 4000g for 20min, sucking off supernatant, standing for 1-2min, and sucking off residual liquid. Adding a proper amount of virus dissolving solution to dissolve virus precipitate, dividing virus solution into tubes, placing the tubes into a refrigerator at the temperature of 80 ℃ below zero for freezing, and taking one tube after one night to further measure the virus titer.

HT-1080 type 24-well plates, 5 wells, 42000cells per well, after adherence, virus infection was performed. 10ml of polybrene MEM medium (8 ug/ml) was prepared. 15ul of virus stock +135ul of medium. Then gradually diluting, taking 15ul +135ul, and carrying out 4 times. Then 50ul of each group is taken and added into 450ul of culture medium holes, and the dilution level is respectively 10^ -2,10^ -3,10^ -4 and 10^ -5. After 72h, the cells were digested for flow analysis of GFP%.

Another object of the present invention is to provide Her-2 CAR-NK cells targeted, the method of making Her-2 CAR-NK cells comprising:

a) purifying and amplifying NK cells;

b) and transfecting the NK cells by using viruses of a lentiviral vector containing a nucleic acid construct of the targeted Her-2CAR sequence, and obtaining the targeted Her-2 CAR-NK cells after expression and identification.

Preferably, said a) is in particular the isolation of Peripheral Blood Mononuclear Cells (PBMCs) with a lymph separation (purchased from GE) taking fresh anticoagulated blood from healthy volunteers. After counting the separated cells, the cells are cultured in a CD16 coated 6-well plate for 72h at 2.5X10^ 6/well, and then the culture is continued to expand for 72h by replacing the common 6-well plate. Purified cells (purchased from Miltenyi Biotec) are sorted by NK magnetic beads, 1640 (purchased from Thermo Scientific) culture medium containing 10% FBS +200IU/ml IL-2 is added for continuous induction culture to obtain purified NK cells, and the phenotype proportion is detected by flow, and the detection result is shown in the figure, which indicates that the proportion of the NK cells prepared by the method is more than 90%.

Preferably, said b) is specifically the purified NK cells, seeded at about 2.5X10^6NK cells per well in 24 well plates (BD Biosciences) and mixed with an appropriate amount of viral supernatant in the presence of final concentrations Protamine sulfate 8ug/ml (Sigma-Aldrich) and BX7951.5 uM (Sigma-Aldrich) at a final volume of not more than 1 ml. Cytokines were supplemented and the plates were centrifuged at 1000 g for 1 hour at room temperature. After centrifugation, without removal of the viral supernatant, the plates were incubated at 37 ℃ for 4-6 hours under 5% CO 2. After the incubation was completed, a second centrifugation was performed at 1000. multidot.g for 1 hour at room temperature, and then from the wells and 1ml of fresh NK cell growth medium. After the cells are maintained in the culture medium with the cytokines added every day for 2 days, the Her-2 CAR-NK-expressing cells are obtained and further detected and identified.

Preferably, the expression and identification method of the chimeric tumor antigen receptor is to extract proteins of NK cells after transfection, perform SDS-PAGE gel electrophoresis, perform semidry membrane transfer, incubate a mouse anti-human CD3 zeta antibody at 4 ℃ overnight, incubate a horseradish peroxidase-labeled anti-mouse secondary antibody at 37 ℃ for lh, and finally add an ECL developing solution on the membrane for developing color.

The invention also aims to provide application of the nucleic acid construct containing the Her-2CAR targeting sequence in targeted killing of tumor cells, in particular to targeted killing of Her-2 positive tumor cells.

The invention also aims to provide the application of the nucleic acid construct or the lentiviral vector in preparing medicines for treating or killing tumor cells or treating cancer patients.

Preferably, said tumor cell or said patient is Her-2 positive.

The invention provides a nucleic acid construct containing a targeted Her-2CAR sequence, which is suitable for being transfected into immune cells to perform tumor immunotherapy and has a polypeptide sequence selected from at least one group of a) to f) or a combination thereof, wherein the transfected immune cells obtain the capability of specifically recognizing Her-2 protein expression positive tumor cells, and the killing activity on the tumor cells is obviously enhanced.

Drawings

FIG. 1 is a flow chart of the construction of pCDH-EFS-TurboRFP-T2A-Zsgreen vector;

FIG. 2 is a PCR identification map of pCDH-EFS-TurboRFP-T2A-Zsgreen vector;

FIG. 3 is a PCR identification map of the pBaEVR vector;

FIG. 4 is a schematic representation of pHer2-CAR 1;

FIG. 5 is a PCR identification map of pHer2-CAR 1;

FIG. 6 is a lentiviral packaging and titer determination flow cytogram;

FIG. 7 is a flow cytogram of NK cell purification;

FIG. 8 is a transfection efficiency characterization;

FIG. 9 is the in vitro killing of tumor cells by targeted Her-2 CAR-NK cells;

FIG. 10 is IFN- γ secretion levels after co-culture of CAR-NK cells with different cells.

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

Example 1 nucleic acid constructs containing Targeted Her-2CAR sequences

The targeted Her-2CAR provided by the invention is composed of a tumor associated antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain with or without a costimulatory domain, which are sequentially connected in series.

Wherein the signal peptide can be selected from the group consisting of signal peptides commonly used for recombinant proteins such as Human IgKVIII, Mouse Ig Kappa, Human IL-2, Human insulin and the like, and preferably according to the present invention is CD8a signal peptide of the provided nucleic acid construct comprising a targeted Her-2CAR sequence, the nucleic acid and polypeptide sequences of which are shown in SEQ ID No.1 and SEQ ID No.2, respectively;

secondly, the antigen binding region can be tightly combined with tumor-associated antigens expressed on the surface of tumor cells, determines the targeting property of the CAR structure, and is a core structure for determining the effect of modified immune cells. Currently, the extracellular antigen-binding domain of most chimeric antigen receptors is derived from single chain antibodies (scFv) formed by the connection of light (VL) and heavy (VH) chains of monoclonal antibodies targeting specific antigens of interest, and a flexible hinge (linker) in the middle. The heavy or light chain of a single-chain antibody is connected to a signal peptide and a hinge region, respectively. The nucleic acid and polypeptide sequences of Her-2-targeting single-chain antibody (FRP5) scFv (VH-linker-VL) of the Her-2-targeting nucleic acid construct provided according to the preferred embodiment of the present invention containing the Her-2CAR sequence are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;

as regards the hinge region connecting the extracellular antigen-binding region and the transmembrane domain, most CAR's hinge region is derived from the IgG hinge or the extracellular region of CD8 α/CD 28. The length of the hinge region depends on the location of the epitope on the target cell and the degree of exposure. Several studies have shown that CAR-T cell activation is related to hinge region length. Adjusting the length of the hinge region allows the CAR-T cell to be at an optimal distance from the target cell, avoiding the effects of large phosphatases to attenuate CAR signals during antigen-antibody binding. However, in some cases, the epitope may be relatively inaccessible and a longer hinge region may be required so that the scFv can overcome steric hindrance and effectively bind the antigen. Thus, the epitopes of the antigen are different and the optimal length of the hinge region is also different, and when targeting a neo-antigen, the length of the hinge region may need to be adjusted accordingly. The extracellular hinge region may be a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, etc., or IgG1, IgG4 (with or without the CH2CH3 region). CD8 Hinge, CD28 Hinge, and IgG4 Hinge-CH2-CH3 containing nucleic acid constructs targeting Her-2CAR sequences provided according to various preferred embodiments of the present invention have nucleic acid and polypeptide sequences as set forth in SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, and SEQ ID No.9 and SEQ ID No.10, respectively;

furthermore, the transmembrane domain connects the extracellular domain of the CAR with the intracellular signaling domain and anchors the receptor to the immune cell membrane. The transmembrane domain may be derived from the nucleic acid, polypeptide sequence of the transmembrane domains of the following proteins: the α, β or ζ chain of a T cell receptor, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, CTLA. For example, the CD3 zeta transmembrane domain enables the CAR to form a homodimer or heterodimer with an endogenous TCR, enhancing CAR-T cell activity, but is also being phased out as it does not require binding to endogenous TCRs to highly activate T cells. The transmembrane domains of CD8a and CD28 are currently employed in most clinical trials due to their ability to promote CAR expression on the cell surface. CD8, CD28, and NKG2D fransmembrane domain containing nucleic acid constructs targeting Her-2CAR sequences provided according to various preferred embodiments of the present invention have nucleic acid and polypeptide sequences as set forth in SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, and SEQ ID NO.15 and SEQ ID NO.16, respectively;

in addition, the co-stimulatory domain is typically from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD 27). The co-stimulation domain can realize the dual activation of the co-stimulation molecules and signals in cells, so that the T cells can continuously proliferate and release cytokines, and the anti-tumor capacity of the T cells is improved. For example, the CD28 co-stimulatory domain makes the CAR-T cell dependent on glycolysis metabolism, causing the CAR-T cell to differentiate towards effector T cells. While the 4-1BB co-stimulatory domain promotes mitochondrial generation, enhances respiration and fatty acid oxidation, and after antigen stimulation, CAR-T cells preferentially differentiate into central memory T cells.

The co-stimulatory domain may be derived from one or more of the following different combinations of nucleic acid, polypeptide sequences of intracellular functional signaling junction domains of proteins: OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137) and 2B4 or functional variants thereof. CD137 Costimmulation Domain, CD28 Costimmulation Domain, DAP10 Costimmulation Domain, and 2B4 Costimmulation Domain containing nucleic acid constructs targeting Her-2CAR sequences provided according to various preferred embodiments of the present invention have the nucleic acid and polypeptide sequences shown in SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, and SEQ ID NO.23 and SEQ ID NO.24, respectively;

finally, the signal transduction domain is typically a T cell receptor TCR/CD3 zeta chain or an immunoglobulin Fc receptor fcepsilon RI gamma chain, containing an immunoreceptor tyrosine-based activation motif (ITAMs). Plays a cell signal transduction function. According to a preferred embodiment of the present invention, there is provided a CD3 ζ Signaling Domain comprising a nucleic acid construct targeting a Her-2CAR sequence, the nucleic acid and polypeptide sequences of which are set forth in SEQ ID No.25 and SEQ ID No.26, respectively:

preferably, based on the above sequences, the combination of sequences of the nucleic acid constructs comprising the Her-2 CAR-targeting sequences provided according to various preferred embodiments of the invention are as follows, with specific nucleic acid and polypeptide sequences as described above:

CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD8 TMD-CD137 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD137 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-DAP10 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-DAP10 CD-CD3ζSD；

CD8 SP-Her-2 scFv(FRP5)-CD8 H-NKG2D TMD-CD137 CD-2B4 CD-CD3ζSD。

using the nucleic acid constructs containing the targeted Her-2CAR sequences provided according to various preferred embodiments of the invention, the immune effector cells used for CAR introduction may be T cells, NK cells or macrophages, and their origin may be autologous, allogeneic, stem cell differentiation or specific cell lines. Preferably, the immune cells are selected from NK cells, but the invention is not limited thereto.

Example 2 Lentiviral vector construction

2.1 shuttle vector construction

As shown in FIG. 1, a lentiviral vector containing an EFS promoter to promote the expression of a foreign gene was constructed using pCDH-UBC-DSRED-LUC-EF1 Hygro (Addgene #129437) as an initiation vector. The reason is because the EFS promoter has a shorter sequence and has a higher transduction efficiency for primary NK cells.

The shuttle vector contains a prokaryotic replicon pUC Ori sequence for plasmid replication; the ampR sequence of the ampicillin-containing resistance gene is used for the mass amplification of a target strain; a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells; elements for lentiviral packaging include: an RSV promoter for initiating transcription of lentiviral mRNA; lentivirus 5 terminal truncated LTR, 3 terminal truncated LTR, RRE cis element, env cis element, cPPT cis element; an EFS promoter for eukaryotic transcription of a target gene, a downstream TurboRFP and ZsGreen1 fluorescent protein sequence thereof, a T2A 'self-shearing' peptide sequence for connecting the co-transcription expression fluorescent protein, a WPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element for enhancing the expression efficiency of transgenosis,

the specific construction process is as follows:

1) an EFS-TurboRFP-T2A-ZsGreen1 fragment is artificially synthesized, and the nucleic acid sequence of the fragment is shown as SEQ ID NO. 27:

2) the pCDH-UBC-DSRED-LUC-EF1 Hygro vector (addendum #129437), ClaI + KpnI were digested, and the linearized vector fragment was recovered.

3) The synthesized EFS-TurboRFP-T2A-ZsGreen1 fragment vector is cut by enzyme, ClaI + KpnI is released, a fragment with the length of-2335 bp is released, and the fragment is recovered.

4) Connecting the recovered products obtained in the steps 2) and 3), transforming and identifying to obtain pCDH-EFS-TurboRFP-T2A-Zsgreen.

The results of the identification are shown in FIG. 2.

2.2 helper vector construction

Packaging lentiviruses requires the involvement of helper vectors in addition to shuttle vectors. It is reported in literature that Baboon enveloped pseudotyped viral vectors (BaEVRless) with Baboon as the outer shell can obviously improve the transduction efficiency of NK cells.

Based on this, the helper vector of the present invention takes pmd2.g (adddge #12259) as the initial vector, and the specific construction method is as follows:

1) artificially synthesizing a BaEVRless fragment, wherein the nucleic acid sequence of the BaEVRless fragment is shown as SEQ ID NO. 28;

2) digesting pMD2.G vector, HindIII + NotI, releasing a fragment, recovering linearized vector fragment

3) Digesting the synthesized vector containing the BaEVRless fragment, HindIII + NotI, releasing a fragment, and recovering the fragment

4) Connecting, transforming and identifying the recovered products obtained in the steps 2) and 3) to obtain pBaEVR.

The results of the identification are shown in FIG. 3.

Example 3 preparation of shuttle vectors containing Targeted Her-2CAR sequences

1) Artificially synthesizing anti-Her 2CAR, wherein the nucleic acid sequence of the anti-Her 2CAR is shown as SEQ ID NO. 29;

2) carrying out enzyme digestion on plenti-EFS-TurboRFP-T2A-Zsgreen vector and Xbal + XhoI, releasing a fragment, and recovering a linearized vector fragment

3) Cleaving the synthesized vector containing anti-Her 2CAR fragment, Xbal + XhoI, releasing a fragment, recovering the fragment

4) The recovered products of steps 2) and 3) were ligated, transformed, and identified to give pHer2-CAR1, the results are shown in FIGS. 4 and 5.

Example 4 Lentiviral packaging and Titer assay

1) Plasmids

·pHer2-CAR1

·VSV-G：pMD2.G(Addgene#12259)or pBaEVR

·Rev：pRSV-Rev(Addgene#12253)

·Gag/Pol：pMDLg/pRRE(Addgene#12251)

·Tat：pCEP4-tat(Addgene#22502)

2) Cell lines

Low passage 293T cells

3) Reagent

Opti-MEM Medium: for mixing the transfection complexes.

4) Packaging process

Day one: 293T cells were grown according to the following table.

HT-1080 type 24-well plates, 5 wells, 42000cells per well, after adherence, virus infection was performed. 10ml of polybrene MEM medium (8 ug/ml) was prepared. 15ul of virus stock +135ul of medium. Then gradually diluting, taking 15ul +135ul, and carrying out 4 times. Then 50ul of each group is taken and added into 450ul of culture medium holes, and the dilution level is respectively 10^2,10^3,10^4 and 10^ 5. After 72h, the cells were digested for flow analysis of GFP% and the lentivirus titer after packaging was obtained as TU (8.94 × 0.01 × 42000/0.5) × 10000 ═ 7.51E7 TU/ml as shown in fig. 6 and calculated according to the following formula.

Titer (TU/mL) ═ F × C/V × D

Frequency of GFP positive cells (percentage of GFP positive cells/100)

Number of cells per well (42,000 cells) at the time of transduction

Transduction volume (mL) (0.5mL)

D ═ lentiviral dilution factor

Example 5 targeting of Her-2 CAR-NK cells

5.1 NK cell purification and expansion

20ml of fresh anticoagulated blood from healthy volunteers were collected and Peripheral Blood Mononuclear Cells (PBMC) were isolated from lymph isolate (purchased from GE). After counting the separated cells, the cells are cultured in a CD16 coated 6-well plate for 72h at 2.5X10^ 6/well, and then the culture is continued to expand for 72h by replacing the common 6-well plate. Purified cells (purchased from Miltenyi Biotec) were sorted by NK magnetic beads, induction culture was continued with 1640 medium containing 10% FBS +200IU/ml IL-2 (purchased from Thermo Scientific) to obtain purified NK cells, and the phenotype ratio of CD3 and CD56 was measured by flow, as shown in fig. 7, in which the horizontal axis represents CD3, the vertical axis represents CD56, the cell population represented by CD3 negative and CD56 positive was NK cells, indicating that the proportion of NK cells prepared by the method was more than 90%.

5.2 Targeted Her-2 CAR-NK cell preparation

Purified NK cells were obtained, seeded at about 2.5X10^6NK cells per well in 24 well plates (BD Biosciences) and mixed with appropriate amount of virus supernatant in the presence of Protamine sulfate 8ug/ml (Sigma-Aldrich) and BX7951.5uM (Sigma-Aldrich) at a final concentration, with a final volume of no more than 1 ml. Cytokines were supplemented and the plates were centrifuged at 1000 g for 1 hour at room temperature. After centrifugation, without removal of the viral supernatant, the plates were incubated at 37 ℃ for 4-6 hours under 5% CO 2. After the incubation was completed, a second centrifugation was performed at 1000. multidot.g for 1 hour at room temperature, and then from the wells and 1ml of fresh NK cell growth medium. After the cells were maintained in a medium with daily cytokine addition for 2 days, Her-2 CAR-NK expressing cells (hereinafter, abbreviated as CAR-NK) were obtained and further transfection efficiency characterization was performed.

After transfection, NK cells are incubated for 15min by recombinant human Her-2-Fc protein, the temperature is 4 ℃, after washing, the cells are incubated for 15min by PE-labeled mouse anti-human anti-IgG1 antibody, the temperature is 4 ℃, and further flow detection is carried out, the result is shown in figure 8, the abscissa represents scFV expression, the left graph is blank control vector anti-Her2-scFV expression, the right graph is anti-Her2-scFv expression after transfection of NK cells by the vector containing CAR, and the transfection efficiency of the NK cells is about 30%.

Example 6 killing of tumor cells in vitro by Targeted Her-2 CAR-NK cells

6.1 CAR-NK cell lethality assay for tumor cells

Her-2 positive breast cancer cells BT474 were adjusted to 1X10^6/ml in culture medium, labeled by adding Calcein-AM to a final concentration of 5ug/ml, incubated at 37 ℃ for 1h, washed three times with PBS, resuspended in phenol-free red 1640 complete culture medium and counted. 10000/well of tumor cells were adjusted to be added to a 96-well round bottom plate. According to the formula E, T is 10: 1; 5: 1; 2.5: 1; 1.25: 1; 0.625: 1; 0.03125:1 Mock NK cell and CAR-NK cell transfected with empty vector 1X10 were added⁵；5X10⁴；2.5X10⁴；1.25X10⁴；0.625X10⁴；0.03125X10⁴. The tumor cells were then treated with 2% Triton X-100 for 5 minutes at 100g and incubated at 37 ℃ for 3 hours, and then centrifuged at 300g for 5 minutes, 100ul of each well was aspirated and transferred to a 96-flat plate for OD detection.The detection result is shown in fig. 9, and the killing power of the CAR-NK cells on the Her-2 positive breast cancer cells is remarkably higher than that of the control NK cells.

6.2 detection of IFN-gamma secretion levels after Co-culture of CAR-NK cells with different cells

Lymphoblast LCL, Her-2 positive breast cancer cell BT474 and Her-2 negative breast cancer cell MDA-MB-231 are co-cultured with NK cells, empty vector transfected Mock NK cells and CAR-NK cells for 12 hours according to the condition that the ratio of E to T is 2.5:1, then supernatant is taken to carry out ELISA to detect the concentration of IFN-gamma in the supernatant, and the detection result is shown in figure 10, and the CAR-NK cells can obviously improve the secretion level of IFN-gamma.

Sequence listing

<110> Applicant

<120> a nucleic acid construct comprising a targeted Her-2CAR sequence

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 2

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 3

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caggtacaac tgcagcagtc tggacctgaa ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cctctgggta tcctttcaca aactatggaa tgaactgggt gaagcaggct 120

ccaggacagg gtttaaagtg gatgggctgg attaacacct ccactggaga gtcaacattt 180

gctgatgact tcaagggacg gtttgacttc tctttggaaa cctctgccaa cactgcctat 240

ttgcagatca acaacctcaa aagtgaagac tcggctacat atttctgtgc aagatgggag 300

gtttaccacg gctacgttcc ttactggggc caagggacca cggtcaccgt ttcctctggc 360

ggtggcggtt ctggtggcgg tggctccggc ggtggcggtt ctgacatcca gctgacccag 420

tctcacaaat tcctgtccac ttcagtagga gacagggtca gcatcacctg caaggccagt 480

caggatgtgt ataatgctgt tgcctggtat caacagaaac caggacaatc tcctaaactt 540

ctgatttact cggcatcctc ccggtacact ggagtccctt ctcgcttcac tggcagtggc 600

tctgggccgg atttcacttt caccatcagc agtgtgcagg ctgaagacct ggcagtttat 660

ttctgtcagc aacattttcg tactccattc acgttcggct cggggacaaa attggagatc 720

<210> 4

<211> 240

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Pro Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Ser Thr Gly Glu Ser Thr Phe Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp Ser Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Trp Glu Val Tyr His Gly Tyr Val Pro Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser His Lys Phe

130 135 140

Leu Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asp Val Tyr Asn Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val

180 185 190

Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Pro Asp Phe Thr Phe Thr

195 200 205

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln

210 215 220

His Phe Arg Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

225 230 235 240

<210> 5

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 6

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 7

<211> 117

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117

<210> 8

<211> 39

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 9

<211> 687

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 60

tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 120

gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 180

gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 240

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 300

tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 360

gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 420

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 540

gactccgacg gctccttctt cctctacagc aggctcaccg tggacaagag caggtggcag 600

gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 660

aagagcctct ccctgtctct gggtaaa 687

<210> 10

<211> 229

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 11

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

acc 63

<210> 12

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

20

<210> 13

<211> 81

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 14

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 15

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccattttttt tctgctgctt catcgctgta gccatgggaa tccgtttcat tattatggta 60

aca 63

<210> 16

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly Ile Arg Phe

1 5 10 15

Ile Ile Met Val Thr

20

<210> 17

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 18

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 19

<211> 123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 20

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 21

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctgtgcgcac gcccacgccg cagccccgcc caagatggca aagtctacat caacatgcca 60

ggcaggggc 69

<210> 22

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr

1 5 10 15

Ile Asn Met Pro Gly Arg Gly

20

<210> 23

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tggaggagaa agaggaagga gaagcagtca gagaccagtc ccaaggaatt tttgacaatt 60

tacgaagatg tcaaggatct gaaaaccagg agaaatcacg agcaggagca gacttttcct 120

ggagggggga gcaccatcta ctctatgatc cagtcccagt cttctgctcc cacgtcacaa 180

gaacctgcat atacattata ttcattaatt cagccttcca ggaagtctgg ttccaggaag 240

aggaaccaca gcccttcctt caatagcact atctatgaag tgattggaaa gagtcaacct 300

aaagcccaga accctgctcg attgagccgc aaagagctgg agaactttga tgtttattcc 360

<210> 24

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser Pro Lys Glu

1 5 10 15

Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr Arg Arg Asn

20 25 30

His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr Ile Tyr Ser

35 40 45

Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu Pro Ala Tyr

50 55 60

Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly Ser Arg Lys

65 70 75 80

Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu Val Ile Gly

85 90 95

Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser Arg Lys Glu

100 105 110

Leu Glu Asn Phe Asp Val Tyr Ser

115 120

<210> 25

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300

acctacgacg cccttcacat gcaggccctg ccccctcgc 339

<210> 26

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 27

<211> 2338

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

atcgatggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag 60

ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg 120

gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata 180

agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg 240

tcgtgacgcg tctagagcca ccatgagcga gctgatcaag gagaacatgc acatgaagct 300

gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa 360

gccctacgag ggcacccaga ccatgaagat caaggtggtc gagggcggcc ctctcccctt 420

cgccttcgac atcctggcta ccagcttcat gtacggcagc aaagccttca tcaaccacac 480

ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagaat 540

caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagct tccagaacgg 600

ctgcatcatc tacaacgtca agatcaacgg ggtgaacttc ccatccaacg gccctgtgat 660

gcagaagaaa acacgcggct gggaggccaa caccgagatg ctgtaccccg ctgacggcgg 720

cctgagaggc cacagccaga tggccctgaa gctcgtgggc gggggctacc tgcactgctc 780

cttcaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcttcca 840

cttcgtggac cacagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca 900

gcacgagatg gctgtggcca agtactgcga cctccctagc aaactggggc acagactcga 960

ggagggcagg ggaagtcttc taacatgcgg ggacgtggag gaaaatcccg gccccgccca 1020

gagcaagcac ggcctgacca aggagatgac catgaagtac agaatggagg gctgcgtgga 1080

cggccacaag ttcgtgatta ccggcgaggg catcggctac cccttcaagg gcaagcaggc 1140

catcaacctg tgcgtggtgg agggcggccc cctgcccttc gccgaggaca tcctgagcgc 1200

cgccttcatg tacggcaaca gagtgttcac cgagtacccc caggacatcg tggactactt 1260

caagaacagc tgccccgccg gctacacctg ggacagaagc ttcctgttcg aggacggcgc 1320

cgtgtgcatc tgcaacgccg acatcaccgt gagcgtggag gagaactgca tgtaccacga 1380

gagcaagttc tacggcgtga acttccccgc cgacggcccc gtgatgaaga agatgaccga 1440

caactgggag cccagctgcg agaagattat ccccgtgccc aagcagggca tcctgaaggg 1500

cgacgtgagc atgtacctgc tgctgaagga cggcggcaga ctgagatgcc agttcgacac 1560

cgtgtacaag gccaagagcg tgcccagaaa gatgcccgac tggcacttca tccagcacaa 1620

gctgaccaga gaggacagaa gcgacgccaa gaaccagaag tggcacctga ccgagcacgc 1680

catcgccagc ggcagcgccc tgcccgtcga ctagataact gaggatccac gcgtctggaa 1740

caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 1800

tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 1860

tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 1920

gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 1980

tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 2040

tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 2100

gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct 2160

cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 2220

caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 2280

tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggtacc 2338

<210> 28

<211> 2575

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

aagcttgcca ccatgggatt taccaccaaa attatctttc tgtacaatct ggtgctggtg 60

tatgctggct tcgacgatcc aagaaaggct attgagctgg tgcagaaaag gtacggccgg 120

ccctgcgact gctctggggg acaggtctca gagcctccat ctgatcgggt ttctcaggtg 180

acctgcagcg gtaaaacagc gtaccttatg cccgatcaga gatggaagtg caaatctatc 240

cctaaggata cctctccctc aggcccactg caagaatgcc cttgtaactc ttaccagtca 300

tccgtgcaca gttcttgcta caccagctat cagcagtgtc gctccgggaa caagacatac 360

tacactgcca cactcctgaa gacacagact gggggaacat cagatgtcca ggtcctggga 420

agtaccaata aactgattca gtcaccgtgc aacggcatca agggacagtc tatctgctgg 480

tcaaccaccg ctcccattca tgtctcagac gggggtggtc ctctggatac aacaagaatt 540

aaatctgtgc agaggaaact cgaggagatt cataaggcct tgtaccccga gctccagtac 600

catcccttgg ccattcctaa ggttcgcgac aacctgatgg tcgacgcaca gaccttgaat 660

atcctgaatg ccacttacaa tctcctgttg atgtccaaca ccagcctggt tgatgattgt 720

tggttgtgct tgaagctggg accacccacc cctctcgcta ttccaaactt cttgctttcc 780

tatgtgacga ggtcctctga taatattagt tgtctcatta tccccccgtt gctcgttcag 840

ccaatgcagt tctccaactc atcttgtctg ttcagcccca gttataattc cacggaggag 900

atcgacctgg gacatgtggc cttttcaaat tgtaccagca tcaccaacgt gactgggcct 960

atttgtgccg tcaatggtag cgtgttcctg tgtggaaaca acatggcata tacttacttg 1020

ccgaccaatt ggactggttt gtgcgtcctg gcgactctcc tgcctgacat cgacattatt 1080

cccggcgatg agccagtgcc catcccggcc attgaccact tcatctatag gccaaagcgc 1140

gccattcaat tcattcccct cctggccggg ctgggaatca ccgctgcctt tactactgga 1200

gctactgggc ttggcgtgag cgtcacccag tatactaaat tgtctaacca gcttatttct 1260

gatgtgcaga tcttgtcttc caccattcag gatctgcagg accaggtgga ctcattggca 1320

gaagttgtgc tgcagaatag acgcggtctg gatctgctga ccgccgagca ggggggaatt 1380

tgtctggccc tgcaggagaa gtgttgcttt tatgttaata aatcagggat tgtccgcgat 1440

aaaattaaaa ctttgcaaga agagttggag agaagaagaa aggacctcgc ttcaaatcct 1500

ctctggactg gtctgcaggg gttgctgcca tacctgcttc cattcttggg accattgctt 1560

accctgctgc tgctgctcac cattggccca tgtattttca accggctgac cgctttcatt 1620

aatgacaaac tgaatataat ccatgctatg taactcaaat cctgcacaac agattcttca 1680

tgtttggacc aaatcaactt gtgataccat gctcaaagag gcctcaatta tatttgagtt 1740

tttaattttt atgaaaaaaa aaaaaaaaaa cggaattcac cccaccagtg caggctgcct 1800

atcagaaagt ggtggctggt gtggctaatg ccctggccca caagtatcac taagctcgct 1860

ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac tactaaactg 1920

ggggatatta tgaagggcct tgagcatctg gattctgcct aataaaaaac atttattttc 1980

attgcaatga tgtatttaaa ttatttctga atattttact aaaaagggaa tgtgggaggt 2040

cagtgcattt aaaacataaa gaaatgaaga gctagttcaa accttgggaa aatacactat 2100

atcttaaact ccatgaaaga aggtgaggct gcaaacagct aatgcacatt ggcaacagcc 2160

cctgatgcct atgccttatt catccctcag aaaaggattc aagtagaggc ttgatttgga 2220

ggttaaagtt ttgctatgct gtattttaca ttacttattg ttttagctgt cctcatgaat 2280

gtcttttcac tacccatttg cttatcctgc atctctcagc cttgactcca ctcagttctc 2340

ttgcttagag ataccacctt tcccctgaag tgttccttcc atgttttacg gcgagatggt 2400

ttctcctcgc ctggccactc agccttagtt gtctctgttg tcttatagag gtctacttga 2460

agaaggaaaa acagggggca tggtttgact gtcctgtgag cccttcttcc ctgcctcccc 2520

cactcacagt gacccggaat ccctcgacat ggcagtctag cactagtgcg gccgc 2575

<210> 29

<211> 1701

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc 60

acgccgccag gccgcaggta caactgcagc agtctggacc tgaactgaag aagcctggag 120

agacagtcaa gatctcctgc aaggcctctg ggtatccttt cacaaactat ggaatgaact 180

gggtgaagca ggctccagga cagggtttaa agtggatggg ctggattaac acctccactg 240

gagagtcaac atttgctgat gacttcaagg gacggtttga cttctctttg gaaacctctg 300

ccaacactgc ctatttgcag atcaacaacc tcaaaagtga agactcggct acatatttct 360

gtgcaagatg ggaggtttac cacggctacg ttccttactg gggccaaggg accacggtca 420

ccgtttcctc tggcggtggc ggttctggtg gcggtggctc cggcggtggc ggttctgaca 480

tccagctgac ccagtctcac aaattcctgt ccacttcagt aggagacagg gtcagcatca 540

cctgcaaggc cagtcaggat gtgtataatg ctgttgcctg gtatcaacag aaaccaggac 600

aatctcctaa acttctgatt tactcggcat cctcccggta cactggagtc ccttctcgct 660

tcactggcag tggctctggg ccggatttca ctttcaccat cagcagtgtg caggctgaag 720

acctggcagt ttatttctgt cagcaacatt ttcgtactcc attcacgttc ggctcgggga 780

caaaattgga gatcaaagaa ttcaccacga cgccagcgcc gcgaccacca acaccggcgc 840

ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca gcggcggggg 900

gcgcagtgca cacgaggggg ctggacttcg cctgtgatcc attttttttc tgctgcttca 960

tcgctgtagc catgggaatc cgtttcatta ttatggtaac atggaggaga aagaggaagg 1020

agaagcagtc agagaccagt cccaaggaat ttttgacaat ttacgaagat gtcaaggatc 1080

tgaaaaccag gagaaatcac gagcaggagc agacttttcc tggagggggg agcaccatct 1140

actctatgat ccagtcccag tcttctgctc ccacgtcaca agaacctgca tatacattat 1200

attcattaat tcagccttcc aggaagtctg gttccaggaa gaggaaccac agcccttcct 1260

tcaatagcac tatctatgaa gtgattggaa agagtcaacc taaagcccag aaccctgctc 1320

gattgagccg caaagagctg gagaactttg atgtttattc cagagtgaag ttcagcagga 1380

gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag ctcaatctag 1440

gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 1500

gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 1560

agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 1620

acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 1680

tgcaggccct gccccctcgc c 1701

Claims

1. A nucleic acid construct comprising a Her-2 CAR-targeting sequence, wherein the nucleic acid construct is composed of a Her-2-targeting single-chain antibody FRP5 scFv, in which the nucleic acid and polypeptide sequences are set forth in SEQ ID No.3 and SEQ ID No.4, respectively, and the extracellular hinge region, transmembrane domain, and intracellular signaling domain are connected in series.

2. The nucleic acid construct of claim 1, further comprising a signal peptide selected from the group consisting of signal peptides of Human IgKVIII, Mouse Ig Kappa, Human IL-2, Human insulin, CD8 a.

3. The nucleic acid construct of claim 2, wherein the nucleic acid and polypeptide sequences of the CD8a signal peptide are set forth in SEQ ID No.1 and SEQ ID No.2, respectively.

4. The nucleic acid construct of any one of claims 1 to 3, wherein the extracellular hinge region has a hinge region sequence selected from the group consisting of CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 with or without the CH2-CH3 region.

5. The nucleic acid construct of claim 4, wherein the nucleic acid and polypeptide sequences of the CD8, CD28, and IgG4 Hinge-CH2-CH3 extracellular Hinge regions are set forth in SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, and SEQ ID No.9 and SEQ ID No.10, respectively.

6. The nucleic acid construct of any one of claims 1 to 3, wherein the transmembrane domain is derivable from a nucleic acid, polypeptide sequence of a transmembrane domain of a protein: the α, β or ζ chain of a T cell receptor, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, CTLA.

7. The nucleic acid construct of claim 6, wherein the nucleic acid and polypeptide sequences of the CD8 transmembrane domain, CD28 transmembrane domain, and NKG2D transmembrane domain are set forth in SEQ ID No.11 and SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14, and SEQ ID No.15 and SEQ ID No.16, respectively.

8. The nucleic acid construct of any of claims 1-3, wherein the intracellular signaling domain comprises or does not comprise a costimulatory domain derivable from a different combination of one or more of the nucleic acid, polypeptide sequences of intracellular functional signaling junction domains of the following proteins: OX40, CD27, CD28, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, 4-1BB (CD137) and 2B4 or functional variants thereof.

9. The nucleic acid construct of claim 8, wherein the nucleic acid and polypeptide sequences of the CD137 co-stimulatory domain, the CD28 co-stimulatory domain, the DAP10 co-stimulatory domain, and the 2B4 co-stimulatory domain are shown in SEQ ID No.17 and SEQ ID No.18, SEQ ID No.19 and SEQ ID No.20, SEQ ID No.21 and SEQ ID No.22, and SEQ ID No.23 and SEQ ID No.24, respectively.

10. The nucleic acid construct of any one of claims 1-3, wherein the signal transduction domain is typically a T cell receptor TCR/CD3 zeta chain or an immunoglobulin Fc receptor FceRI gamma chain, comprising an immunoreceptor tyrosine activation motif.

11. The nucleic acid construct of claim 10, wherein the Signaling Domain is CD3 ζ Signaling Domain, and wherein the nucleic acid and polypeptide sequences are set forth in SEQ ID No.25 and SEQ ID No.26, respectively.

12. A Her-2CAR sequence-targeting nucleic acid construct consisting of a signal peptide, a tumor associated antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain in tandem, wherein the nucleic acid and polypeptide sequences of each component in the nucleic acid construct are as in claims 1-11, and wherein the Her-2CAR sequence-targeting nucleic acid construct is selected from any one of a) to g) or combinations thereof, wherein each combination is as follows:

a)CD8 SP-Her-2scFv(FRP5)-CD8 H-CD8 TMD-CD137 CD-CD3ζSD；

b)CD8 SP-Her-2scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD3ζSD；

c)CD8 SP-Her-2scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-CD137 CD-CD3ζSD；

d)CD8 SP-Her-2scFv(FRP5)-CD8 H-CD28 TMD-CD28 CD-DAP10 CD-CD3ζSD；

e)CD8 SP-Her-2scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-CD3ζSD；

f)CD8 SP-Her-2scFv(FRP5)-CD8 H-NKG2D TMD-2B4 CD-DAP10 CD-CD3ζSD；

g)CD8 SP-Her-2scFv(FRP5)-CD8 H-NKG2D TMD-CD137 CD-2B4 CD-CD3ζSD。

13. the nucleic acid construct of claim 12, wherein the nucleic acid construct is selected from e) CD8SP-Her-2scFv (FRP5) -CD 8H-NKG 2D TMD-2B4 CD-CD3 ζ SD; the nucleic acid sequence is shown as SEQ ID NO. 29.

14. A lentiviral vector comprising a nucleic acid construct comprising a targeted Her-2CAR sequence according to any one of claims 1-13, wherein the lentiviral vector comprises a shuttle plasmid, an envelope plasmid, and a packaging plasmid.

15. The lentiviral vector of claim 14, wherein the shuttle plasmid is pCDH-UBC-DSRED-LUC-EF1 Hygro as a starting vector, and is constructed to contain an EFS promoter for the lentiviral vector to promote expression of the foreign gene.

16. The lentiviral vector of claim 14, wherein the envelope plasmid is pMD2.G as a starting vector and the vector is constructed to contain a baEVRless fragment having the nucleic acid sequence shown in SEQ ID No. 28.

17. Use of a nucleic acid construct according to any one of claims 1 to 13, or a lentiviral vector according to any one of claims 14 to 16, in the manufacture of a medicament for treating or killing a tumor cell or for treating a patient with cancer.

18. Use according to claim 17, characterized in that the immune effector cells used for the introduction of the nucleic acid construct can be T cells, NK cells or macrophages and their origin can be autologous, allogeneic, stem cell differentiated or specific cell lines.

19. The use of claim 18, wherein said immune effector cell is an NK cell.

20. The use of any one of claims 17-19, wherein said tumor cell or said patient is Her-2 positive.