CN116656614A - Her-2 targeted CAR-NK cell and application thereof - Google Patents

Her-2 targeted CAR-NK cell and application thereof Download PDF

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CN116656614A
CN116656614A CN202210146396.5A CN202210146396A CN116656614A CN 116656614 A CN116656614 A CN 116656614A CN 202210146396 A CN202210146396 A CN 202210146396A CN 116656614 A CN116656614 A CN 116656614A
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陈伟
刘昊
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Hangzhou Qianhe Cell Biotechnology Co ltd
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Hangzhou Qianhe Cell Biotechnology Co ltd
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    • C07K2319/00—Fusion polypeptide
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    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

A Her-2 targeting CAR-NK cell and application thereof, in particular to the CAR-NK is formed by a CAR structure constructed by human NK cell transduction expression artificial design, the nucleotide structure of the CAR is formed by serially connecting a tumor-related antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal transduction domain in sequence, the tumor-related antigen binding region is a single-chain antibody FRP5scFv targeting Her-2, and the nucleic acid and polypeptide sequences of the single-chain antibody FRP5scFv are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4. The CAR-NK cell can be used for specifically recognizing tumor cells expressing Her-2 and activating immune cells to release IFN-gamma and other cytokines to kill the tumor cells.

Description

Her-2 targeted CAR-NK cell and application thereof
Technical Field
The invention relates to the field of tumor immunotherapy, in particular to Her-2-targeted CAR-NK cells and application thereof.
Background
Chimeric antigen receptor (Chimeric antigen receptor, CAR) immune cell therapy is one of the most promising tumor immunotherapy at present. The fusion protein of a single-chain antibody (Single chain fragment variable, scFv) for recognizing tumor-associated antigens and an immune cell activation sequence, namely a Chimeric Antigen Receptor (CAR), is expressed on the surface of immune cells through exogenous gene transfection technology, so that the scFv capable of specifically recognizing tumor-associated antigens is coupled with an activation proliferation signal domain in immune cells through a transmembrane region. Immune cells (e.g., T cells, NK cells) expressing the CAR bind to tumor antigens in an antigen-dependent, but non-MHC-restricted manner, initiating and activating a specific killing tumor response.
The CAR is combined with tumor related antigen on tumor cells to provide an activation signal for immunity to cause activation of immune cells, and the activation is expressed as killing, proliferation and cytokine release dependent on CARs, so that the effect of killing the tumor cells is exerted. These effector functions can be used to design new CARs, such as CARs comprising multiple chimeric activation regions, such as generation 1, generation 2, and generation 3 CARs having 1, 2, or 3 signal motifs in the cell. Early-designed CARs were simpler, had only one intracellular signaling region, also known as generation 1 CAR, signaling was mainly conducted through the immunoreceptor tyrosine kinase activation motif on CD3 ζ (immunoreceptor tyrosine-based activation motif, ITAM), had only 3 ITAMs compared to CD3 molecules, and lacked the 2 nd signal conducted by co-stimulatory molecules, so generation 1 CARs had only a weaker antitumor effect, and T cells were poorly effective in clinical applications because of the lack of the 2 nd signal of T cell proliferation, which was difficult to proliferate further after binding to tumor antigen; generation 2 CARs are based on generation 1 by adding an intracellular signal region that provides a co-stimulatory signal (e.g., CD28, CD 137); the 3 rd generation CARs have two costimulatory signal regions in series added to the cell. In addition, 4 th generation CARs (also known as TRUCKs) have emerged in recent years, i.e., CARs are constructed using vectors encoding CARs and/or CAR-responsive promoters, which produce 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 their inherent ability to recognize and target tumor cells through their natural receptors, so that the likelihood that tumor cells can escape killing is reduced upon CAR-targeted therapy. Second, CAR-NK cell therapy showed no immune rejection for days to weeks in multiple clinical trials. Thus, none of the CAR-NK cell therapies exhibit similar safety issues, such as the absence of the trouble of cytokine release syndrome, compared to the clinical trials of many CAR-T cell therapies. 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 of CAR-T cell immunotherapy.
HER2 (Human epidermal growth factor receptor 2), which belongs to the family of epidermal growth factor receptors, is a transmembrane protein with tyrosine kinase activity. Studies have shown that there is a phenomenon of HER2 gene overexpression in about three breast cancer patients. In such patients, the malignancy of the tumor is high, the tumor has a certain drug resistance, the recurrence and metastasis speed is high, and the disease-free survival time of the patient is inversely proportional to the total survival time and the expression condition of HER2 genes. In the pathological occurrence process of breast cancer, the expression level change of HER2 plays a crucial role, so HER2 becomes an important target point for scientists to develop breast cancer therapeutic drugs.
Disclosure of Invention
Based on the background art, the invention aims to provide Her-2 targeted CAR-NK cells and applications thereof, and specifically, the CAR nucleotide sequence structure in the CAR-NK cells provided by the invention is formed by serially connecting a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal transduction domain in sequence. The CAR-NK cell can be used for specifically recognizing tumor cells expressing Her2 and activating immune cells to release IFN-gamma and other cytokines to kill the tumor cells.
Preferably, the CAR-NK nucleotide sequence 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 into subcellular organelles with different membrane structures, typically at the N-terminus of the protein. The signal peptide may be selected from signal peptides commonly used for recombinant proteins such as Human IgKVGIII, mouse Ig Kappa, human IL-2, human insulin, etc.;
preferably, the signal peptide of the CAR-NK cell is selected from CD8a signal peptide, and the nucleic acid and polypeptide sequences of the signal peptide are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Preferably, the antigen binding region is capable of tightly binding to a tumor-associated antigen expressed on the surface of a tumor cell, determining the targeting of the CAR structure, which is the core structure determining the effect of the modified immune cell. Currently, the extracellular antigen binding regions of most chimeric antigen receptors are single chain antibodies (single chain fragment variable, scFv) derived from the joining of light (VL) and heavy (VH) chains and an intermediate flexible hinge (linker) that target a specific antigen monoclonal antibody of interest. The heavy chain or the light chain of the single-chain antibody is respectively connected with the signal peptide and the hinge region;
preferably, the single-chain antibody of the CAR-NK cell is anti-her 2scFv (VH-linker-VL), and the nucleic acid and polypeptide sequences of the single-chain antibody are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
Preferably, the hinge region connecting the extracellular antigen binding region and the transmembrane domain, most of the hinge region of the CAR is derived from the hinge of IgG or the CD8 a/CD 28 extracellular region. The length of the hinge region depends on the location and exposure of the target cell epitope. Several studies have shown that CAR-T cell activation is related to hinge region length. The length of the hinge region is adjusted to allow the CAR-T cell to be at an optimal distance from the target cell, avoiding the attenuation of CAR signal by the action of large phosphatases during antigen-antibody binding. However, in some cases, the epitope may be relatively inaccessible, requiring the use of a longer hinge region, so that the scFv can overcome steric hindrance and bind antigen efficiently. Thus, the optimal length of the hinge region will vary from epitope to epitope, and in targeting a neoantigen, it may be necessary to adjust the length of the hinge region 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 a CH2CH3 region);
preferably, the Hinge region of the CAR-NK cell is selected from the group consisting of CD8 finger (CD 8H), CD28 finger (CD 28H), and IgG4 finger-CH 2-CH3, the nucleic acid and polypeptide sequences of which are shown 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.
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. The transmembrane domain (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 T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, ctla. Such as the cd3ζ transmembrane domain, is capable of enabling CAR to form homodimers or heterodimers with endogenous TCRs, enhancing CAR-T cell activity, but is increasingly abandoned because it does not require binding to endogenous TCRs to highly activate T cells. The transmembrane domains of CD8a and CD28 are currently used in most clinical trials because of their ability to promote CAR expression on the cell surface.
Preferably, the transmembrane domain of the CAR-NK cell is selected from the group consisting of CD8 transmembrane domain, CD28 transmembrane domain and NKG2D transmembrane domain, the nucleic acid and polypeptide sequences of which are shown in SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively.
Preferably, the intracellular signaling domain comprises or does not comprise a co-stimulatory domain (Costimulatory Domain, CD). The co-stimulatory domain is typically from the CD28 receptor family (CD 28, ICOS) or the tumor necrosis factor receptor family (4-1 BB, OX40, CD 27). The co-stimulatory domain can realize dual activation of co-stimulatory molecules and intracellular signals, so that the T cells continuously proliferate and release cytokines, and the anti-tumor capability of the T cells is improved. Such as the CD28 co-stimulatory domain, allows CAR-T cells to rely on glycolytic metabolism, promoting differentiation of CAR-T cells to effector T cells. Whereas the 4-1BB co-stimulatory domain promotes mitochondrial production, enhances respiration and fatty acid oxidation, and upon antigen stimulation, CAR-T cells preferentially differentiate into central memory T cells. The co-stimulatory domain may be derived from one or more different combinations of nucleic acids, polypeptide sequences of the following intracellular functional signaling domains of proteins: OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), DAP10, DAP12,4-1BB (CD 137) and 2B4 or functional variants thereof.
Preferably, the co-stimulatory domain of the CAR-NK cell is selected from the group consisting of CD137 Costimulatory Domain, CD28 Costimulatory Domain, DAP10 Costimulatory Domain, and 2B4 Costimulatory Domain, the nucleic acid and polypeptide sequences of which 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.
Preferably, the Signaling Domain (SD) is typically the T-cell receptor TCR/CD3 zeta chain or the immunoglobulin Fc receptor FcεRIgamma chain, containing the immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAMs). Exerting cell signaling function.
Preferably, the signal transduction Domain of the CAR-NK cell is CD3 zeta Signaling Domain, and the nucleic acid and polypeptide sequences of the signal transduction Domain are shown as SEQ ID NO.25 and SEQ ID NO.26 respectively.
It is another object of the present invention to provide NK cells expressing a CAR structure consisting of a signal peptide, a tumor associated antigen binding region, an extracellular hinge region, a transmembrane domain and a cascade of intracellular signaling domains comprising a costimulatory domain, the nucleotide structure of which CAR structure can be selected from any one of the groups a) to g) or a combination thereof, each combination having the following specific nucleic acid and polypeptide sequences, respectively, 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。
another object of the present invention is to provide a lentiviral (Lentiviral) vector containing Her-2 targeting CAR sequences, which can be used for lentiviral vector transfection to distinguish general retroviral vectors, which have infectivity to both dividing cells and non-dividing cells, and which can effectively integrate foreign genes into host chromosomes to achieve persistent expression, and which can also effectively infect neuronal cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and other types of cells in terms of infectivity, thereby achieving good gene therapy effect and having wide application prospects.
Preferably, the lentiviral vector comprising Her-2 targeting CAR sequence may comprise HIV-1 Rev Response Element (RRE) allowing Rev-assisted uncombined viral mRNA nucleation; 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 promote expression of downstream CAR structural genes, such as the human ubiquitin C promoter, EF1a promoter, EFs promoter, etc.; and cPPT and WPRE elements, improving transduction efficiency and expression efficiency of transgenes.
Preferably, the lentiviral vector contains, in addition to the shuttle plasmid (transfer plasmid), an envelope plasmid (envelope plasmid) and a packaging plasmid (packaging plasmid). For the Envelope plasmid, VSV-G is generally selected because lentiviruses containing this Envelope protein can infect a variety of hosts. However, resting T, B, NK and hematopoietic stem cells hardly express the LDLR gene, so that they are very difficult to infect VSV-G coat lentiviruses, and Baboon envelope pseudotyped lentiviral vectors using Baboon as a coat has been reported in the literature to significantly improve NK cell transduction efficiency.
The transfer plasmid takes pCDH-UBC-DSRED-LUC-EF1 Hygro (adedge # 129437) as an initial vector, and constructs a slow virus vector containing an EFS promoter for promoting the expression of exogenous genes. The reason is that EFS promoters have shorter sequences and have higher transduction efficiency for primary NK cells.
The transfer plasmid contains a prokaryotic replicon pUC Ori sequence for plasmid replication; ampicillin resistance gene AmpR sequence for mass amplification of the strain of interest; viral replicon SV40Ori sequences for enhancing replication in eukaryotic cells; the element for lentiviral packaging comprises: RSV promoter for initiating transcription of lentiviral mRNA; lentivirus 5terminal 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, zsGreen1 fluorescent protein sequence, a T2A self-shearing peptide sequence for connecting the co-transcription expression fluorescent protein and a WPRE enhanced woodchuck hepatitis B virus posttranscriptional regulatory element for enhancing the expression efficiency of transgenes.
The transfer plasma id construction method comprises the following steps:
1) Constructing a vector containing EFS-TurboRFP-T2A-ZsGreen1 and ClaI enzyme and KpnI enzyme sites, wherein the EFS-TurboRFP-T2A-ZsGreen1 nucleic acid sequence is shown as SEQ ID NO. 27;
2) The pCDH-UBC-DSRED-LUC-EF1 Hygro vector (addgene # 129437) was digested with ClaI enzyme and KpnI enzyme, and the linearized vector fragment was recovered;
3) The vector containing the EFS-TurboRFP-T2A-ZsGreen1 fragment was digested with ClaI enzyme and KpnI enzyme, and the digested fragments were recovered.
4) And (3) connecting the recovered products of the steps 2) and 3), converting, and identifying to obtain the vector of the plenti-EFS-TurboRFP-T2A-Zsgreen.
The preparation method of the transfer plasma id containing the targeting Her-2CAR sequence comprises the following steps:
1) Constructing a targeting Her-2CAR-NK containing sequence, preferably the targeting Her-2CAR-NK containing sequence as shown in SEQ ID No. 29;
2) Cutting the pliti-EFS-TurboRFP-T2A-Zsgreen vector by using Xbal enzyme and XhoI enzyme, and recovering the linearization vector fragment;
3) Using Xbal enzyme+XhoI enzyme to cleave the vector containing the Her-2CAR-NK targeting nucleic acid sequence and recovering fragments;
4) Ligating the recovered products of steps 2) and 3), converting, identifying to obtain pHer2-CAR1, namely: transfer plasma containing Her-2CAR targeting sequences.
Preferably, the envilope plasmid uses pMD2.G (adedge # 12259) as a starting 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 digested by HindIII enzyme and NotI enzyme, and linearized vector fragments are recovered;
3) The vector containing the BaEVRless fragment is digested by HindIII enzyme and NotI enzyme, and fragments are recovered;
4) And (3) connecting the recovered products of the steps 2) and 3), converting, and identifying to obtain pBaEVR, namely, envelope plasmid.
The packaging method for preparing the Her-2 targeting CAR-NK-containing lentiviral vector specifically comprises the following steps:
1) Plasmid(s)
·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
293T cells with low passage number
3) Reagent(s)
293T Medium: high sugar DMEM (sodium pyruvate, glutamine) +10% fbs+glutamax.
Virus harvest medium: 50ml of 293T medium was taken and added with 0.5g BSA (Sigma A9418) and HEPES at a final concentration of 10-15mM, and filtered at 0.22. Mu.m.
Opti-MEM Medium: for mixing transfection complexes.
Transfection reagent: X-tremgeNE HP DNA (or other low toxicity, high efficiency reagents).
4) Packaging process
First day: 293T cells were seeded according to the following table.
The next day-am: the Opti-MEM medium, plasmid and transfection reagents were mixed according to the following table. Fresh medium was exchanged for 293T cells. The mixture was allowed to stand at room temperature for 15-30min, then added dropwise to the 293T cell culture system, and gently shaken well.
The next day-afternoon: after 6-8hr of transfection, gentle exchange fluid was used as virus harvest medium.
Fourth day: 48hr after transfection, taking cell culture supernatant, centrifuging at 400 Xg for 4min, filtering with 0.45 μm, adding 5 XPEG 8000 solution by volume, mixing every 20-30min for 3-5 times, and standing at 4deg.C for 6 hr or overnight. The supernatant was removed by centrifugation at 4000g for 20min at 4℃and the tube was left to stand for 1-2min to remove residual liquid. Adding appropriate amount of virus dissolving solution to dissolve virus precipitate, separating the virus solution, freezing at-80deg.C, and taking one branch for further measurement of virus titer.
HT-1080 24-well plates, 5 wells, 42000cells per well, after adherence, virus infection was performed. 10mL of polybrene MEM medium was prepared at 8. Mu.g/mL. 15. Mu.L of virus stock+135. Mu.L of medium. Then, the mixture was diluted stepwise to 15. Mu.L+135. Mu.L and the mixture was subjected to 4 times. Then 50. Mu.L of each group was added to 450. Mu.L of medium wells, and the dilution levels were 10, respectively -2 ,10 -3 ,10 -4 ,10 -5 . After 72h, the digested cells were subjected to flow analysis for GFP%.
It is another object of the present invention to provide a Her-2 targeted CAR-NK cell preparation method comprising:
a) NK cell sorting and purification;
b) Constructing a virus containing the lentiviral vector of the CAR-NK nucleotide sequence of any one of claims 1 to 12, and transfecting the virus into NK cells purified in step a), and obtaining Her-2 targeted CAR-NK cells after expression and identification.
Preferably, the step a) is to isolate and purify NK cells from healthy people, specifically comprises isolating peripheral blood mononuclear cells from fresh anticoagulants of healthy people by using lymph separation liquid, performing stimulated culture in a 6-well plate coated with CD16, then separating and purifying cells by using NK magnetic beads, and continuing to perform induction culture by using 1640 culture medium containing 10% FBS+200IU/ml IL-2 to obtain purified NK cells.
Preferably, said step b) is specifically obtaining purified NK cells at 2.5X10 per well 6 NK cells were seeded into 24 well plates and mixed with the virus supernatant in the presence of final concentration of 8. Mu.g/ml Protamine sulfate and 1.5. Mu.M BX795, and plates were centrifuged at 1000. Mu.g for 1 hour at room temperature; after centrifugation, without removal of virus supernatant, plates were incubated at 37 ℃,5% co2 for 4-6 hours; after the end of incubation, a second centrifugation at 1000 g at room temperature for 1 hour was performed, and then after the cells were maintained in the medium for 2 days, her-2 expressing CAR-NK cells were obtained.
More preferably, said step a) is specifically the collection of fresh anticoagulants from healthy volunteers, and the isolation of Peripheral Blood Mononuclear Cells (PBMC) with a lympho-isolation solution (purchased from GE company). After isolated cell counts, the cells were counted as per 2.5X10 6 Well density was stimulated in 6-well plates coated with CD16 for 72h, after which the culture expansion was continued for 72h in the same 6-well plates. Purified cells were isolated using NK magnetic beads (from Miltenyi Biotec Co.), and induced culture was continued by adding 1640 medium containing 10% FBS+200IU/ml IL-2 (from Thermo Scientific), to obtain purified NK cells, and the phenotype ratio was examined by flow, and the examination results were shown as a graph, indicating that the NK cell ratio prepared by the method was more than 90%.
More preferably, said step b) is specifically obtaining purified NK cells at about 2.5X10 per well 6 NK cells were seeded into 24 well plates (BD Biosciences) and mixed with an appropriate amount of virus supernatant in the presence of a final concentration of Protamine sulfate. Mu.g/mL (Sigma-Aldrich) and BX 795.5. Mu.M (Sigma-Aldrich) in a final volume of no more than 1mL. Cytokine supplementation and plates were centrifuged at 1000 g for 1 hour at room temperature. After centrifugation, the plates were incubated at 37℃with 5% CO without removal of the viral supernatant 2 Incubate for 4-6 hours. After incubation is finishedA second centrifugation is performed at 1000. Multidot.g for 1 hour at room temperature, then from the wells and 1ml of fresh NK cell growth medium. After the cells were maintained in medium supplemented with cytokines for 2 days per day, her-2 expressing CAR-NK cells were obtained and further examined and identified.
Preferably, the chimeric tumor antigen receptor expression identification method is to extract the protein of transfected NK cells, perform SDS-PAGE gel electrophoresis, semi-dry transfer membrane, incubate with mouse anti-human CD3 zeta antibody at 4 ℃ overnight, incubate with horseradish peroxidase-labeled anti-mouse secondary antibody at 37 ℃ for lh, and finally add ECL color development liquid on the membrane for color development.
Another object of the invention is to provide the use of said CAR-NK cells for the preparation of a medicament for the treatment of Her-2 mediated diseases.
Preferably, the related disease refers to the use in oncological diseases.
Preferably, the tumor is mainly breast cancer.
The Her-2 targeted single-chain antibody FRP5scFv and the CAR-NK cells prepared by the same are suitable for tumor immunotherapy, specifically, the carrier containing at least one group of CAR-NK sequences in a) to f) combination or the combination thereof is selected, and the transfected immune cells obtain the CAR-NK cells which specifically recognize Her-2 proteins, can be used for targeted killing of Her-2 expressed positive tumor cells, and obviously enhance the killing activity on the tumor cells.
Drawings
FIG. 1 is a flow chart of the construction of a 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 pBaEVR vector;
FIG. 4 is a schematic of pHer2-CAR 1;
FIG. 5 is a PCR identification map of pHer2-CAR 1;
FIG. 6 is a flow cytometry plot of lentiviral packaging and titer assays;
FIG. 7 is a flow cytometry map of NK cell purification;
FIG. 8 is a graph of transfection efficiency assays;
FIG. 9 is the killing of tumor cells in vitro by Her-2CAR-NK cells targeted;
FIG. 10 is IFN-gamma secretion levels after co-culturing of CAR-NK cells with different cells.
Detailed Description
The following description is presented to enable one of ordinary skill in the art to make and use the invention. The preferred embodiments in the following description are by way of example only and other obvious variations will occur to those skilled in the art. The basic principles of the invention 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 CAR-NK cells containing Her-2CAR nucleotide sequence targeting
The targeting Her-2CAR nucleotide sequence provided by the invention sequentially comprises a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signaling domain sequence cross-linked with or without a costimulatory domain.
The nucleotide sequence for NK cell transduction CARs also 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 into subcellular organelles with different membrane structures, typically at the N-terminus of the protein. The signal peptide may be selected from signal peptides commonly used for recombinant proteins such as Human IgKVGIII, mouse Ig Kappa, human IL-2, human insulin, etc.;
the signal peptide for NK cell transduction CAR is selected from CD8a signal peptide, and the nucleic acid and polypeptide sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
The antigen binding region can be tightly combined with a tumor-associated antigen expressed on the surface of a tumor cell, determines the targeting of the CAR structure and is a core structure for determining the effect of the modified immune cells. Currently, the extracellular antigen binding regions of most chimeric antigen receptors are single chain antibodies (single chain fragment variable, scFv) derived from the joining of light (VL) and heavy (VH) chains and an intermediate flexible hinge (linker) that target a specific antigen monoclonal antibody of interest. The heavy chain or the light chain of the single-chain antibody is respectively connected with the signal peptide and the hinge region;
the single-chain antibody for NK cell transduction CAR is anti-Her 2scFv (VH-linker-VL), and the nucleic acid and polypeptide sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
The hinge region connecting the extracellular antigen binding region and the transmembrane domain, most of the hinge region of CARs is derived from the hinge of IgG or the CD8 a/CD 28 extracellular region. The length of the hinge region depends on the location and exposure of the target cell epitope. Several studies have shown that CAR-T cell activation is related to hinge region length. The length of the hinge region is adjusted to allow the CAR-T cell to be at an optimal distance from the target cell, avoiding the attenuation of CAR signal by the action of large phosphatases during antigen-antibody binding. However, in some cases, the epitope may be relatively inaccessible, requiring the use of a longer hinge region, so that the scFv can overcome steric hindrance and bind antigen efficiently. Thus, the optimal length of the hinge region will vary from epitope to epitope, and in targeting a neoantigen, it may be necessary to adjust the length of the hinge region 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 a CH2CH3 region);
the Hinge region for NK cell transduction CAR can be selected from CD8 finger (CD 8H), CD28 finger (CD 28H), and IgG4 finger-CH 2-CH3, and the nucleic acid and polypeptide sequences thereof are shown 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.
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 (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 T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, ctla. Such as the cd3ζ transmembrane domain, is capable of enabling CAR to form homodimers or heterodimers with endogenous TCRs, enhancing CAR-T cell activity, but is increasingly abandoned because it does not require binding to endogenous TCRs to highly activate T cells. The transmembrane domains of CD8a and CD28 are currently used in most clinical trials because of their ability to promote CAR expression on the cell surface.
The transmembrane domain for NK cell transduction CAR can be selected from CD8 transmembrane domain, CD28 transmembrane domain and NKG2D transmembrane domain, and the nucleic acid and polypeptide sequences are respectively shown as 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.
The intracellular signaling domain comprises or does not comprise a co-stimulatory domain (Costimulatory Domain, CD). The co-stimulatory domain is typically from the CD28 receptor family (CD 28, ICOS) or the tumor necrosis factor receptor family (4-1 BB, OX40, CD 27). The co-stimulatory domain can realize dual activation of co-stimulatory molecules and intracellular signals, so that the T cells continuously proliferate and release cytokines, and the anti-tumor capability of the T cells is improved. Such as the CD28 co-stimulatory domain, allows CAR-T cells to rely on glycolytic metabolism, promoting differentiation of CAR-T cells to effector T cells. Whereas the 4-1BB co-stimulatory domain promotes mitochondrial production, enhances respiration and fatty acid oxidation, and upon antigen stimulation, CAR-T cells preferentially differentiate into central memory T cells. The co-stimulatory domain may be derived from one or more different combinations of nucleic acids, polypeptide sequences of the following intracellular functional signaling domains of proteins: OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), DAP10, DAP12,4-1BB (CD 137) and 2B4 or functional variants thereof.
The costimulatory domain for NK cell transduction CAR can be selected from the group consisting of CD137 Costimulatory Domain, CD28 Costimulatory Domain, DAP10 Costimulatory Domain, and 2B4 Costimulatory Domain, the nucleic acid and polypeptide sequences of which are shown as 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.
The Signaling Domain (SD) is typically the TCR/CD3 zeta chain of the T cell receptor or the Fc receptor FcεRIgamma chain of the immunoglobulin, containing the immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motifs, ITAMs). Exerting cell signaling function.
The signal transduction structural Domain for NK cell transduction CAR is CD3 zeta Signaling Domain, and the nucleic acid and polypeptide sequences are respectively shown as SEQ ID NO.25 and SEQ ID NO. 26.
Based on the above sequences, the CAR-NK nucleotide structure provided according to various preferred embodiments of the present invention may be selected from any one of the groups a) to g) or a combination thereof, each combination having the following specific nucleic acid and polypeptide sequences, respectively, 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。
EXAMPLE 2 lentiviral vector construction
2.1 shuttle vector construction
As shown in FIG. 1, a lentiviral vector containing the EFS promoter for promoting the expression of a foreign gene was constructed using pCDH-UBC-DSRED-LUC-EF1 Hygro (addgene # 129437) as the starting vector. The reason is that EFS promoters have shorter sequences and have higher transduction efficiency for primary NK cells.
The shuttle vector contains a prokaryotic replicon pUC Ori sequence for plasmid replication; ampicillin resistance gene AmpR sequence for mass amplification of the strain of interest; viral replicon SV40Ori sequences for enhancing replication in eukaryotic cells; the element for lentiviral packaging comprises: RSV promoter for initiating transcription of lentiviral mRNA; lentivirus 5terminal 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, zsGreen1 fluorescent protein sequence, a T2A self-shearing peptide sequence for connecting the co-transcription expression fluorescent protein, a WPRE enhanced woodchuck hepatitis B virus posttranscriptional regulatory element for enhancing the expression efficiency of transgenes,
the specific construction flow is as follows:
1) Artificially synthesizing EFS-TurboRFP-T2A-ZsGreen1 fragment, wherein the nucleic acid sequence of the fragment is shown in SEQ ID NO. 27:
2) The pCDH-UBC-DSRED-LUC-EF1 Hygro vector (addgene # 129437), claI+KpnI was digested and the linearized vector fragment was recovered.
3) And (3) enzyme cutting the vector of the synthesized EFS-TurboRFP-T2A-ZsGreen1 fragment, releasing a fragment with the length of 2335bp by ClaI+KpnI, and recovering the fragment.
4) And (3) connecting the recovered products of the steps 2) and 3), converting, and identifying to obtain the pCDH-EFS-TurboRFP-T2A-Zsgreen.
The results of the identification are shown in FIG. 2.
2.2 construction of auxiliary vector
Packaging lentiviruses requires the participation of helper vectors in addition to shuttle vectors. Baboon envelope pseudotyped lentiviral vectors (BaEVRless) using Baboon as a shell has been reported to significantly improve NK cell transduction efficiency.
Based on the above, the auxiliary vector of the invention takes pMD2.G (addgene # 12259) as an 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) Cleavage of pMD2.G vector, hindIII+NotI, release of a fragment, recovery of linearized vector fragment
3) The vector containing the BaEVRless fragment synthesized above was digested, hindIII+NotI was released to release a fragment, and the fragment was recovered
4) And (3) connecting the recovered products of the steps 2) and 3), converting, and identifying to obtain pBaEVR.
The results of the identification are shown in FIG. 3.
Example 3 preparation of shuttle vector containing Her-2 targeting CAR sequence
1) Artificially synthesizing an anti-Her 2CAR sequence, wherein the nucleic acid sequence of the anti-Her 2CAR sequence is shown as SEQ ID NO. 29;
2) Cutting the plenti-EFS-TurboRFP-T2A-Zsgreen vector, xbal+XhoI, releasing a fragment, recovering the linearized vector fragment
3) The vector containing the anti-Her 2CAR sequence fragment synthesized by the above method is digested with Xbal+XhoI, a fragment is released, and the fragment is recovered
4) The recovered products of steps 2) and 3) were ligated, transformed and identified to yield pHer2-CAR1, the results of which are shown in FIGS. 4 and 5.
EXAMPLE 4 lentiviral packaging and titre determination
1) Plasmid(s)
·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
293T cells with low passage number
3) Reagent(s)
293T Medium: high sugar DMEM (sodium pyruvate, glutamine) +10% fbs+glutamax.
Virus harvest medium: 50mL of 293T medium was added with 0.5g BSA (Sigma A9418) and HEPES at a final concentration of 10-15mM, and filtered at 0.22. Mu.m.
Opti-MEM Medium: for mixing transfection complexes.
Transfection reagent: X-tremgeNE HP DNA (or other low toxicity, high efficiency reagents).
4) Packaging process
First day: 293T cells were seeded according to the following table.
The next day-am: the Opti-MEM medium, plasmid and transfection reagents were mixed according to the following table. Fresh medium was exchanged for 293T cells. The mixture was allowed to stand at room temperature for 15-30min, then added dropwise to the 293T cell culture system, and gently shaken well.
6-well 10-cm 15-cm
Seeding number 8.75x10 5 5.5x10 6 1.8x10 7
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
The next day-afternoon: after 6-8hr of transfection, gentle exchange fluid was used as virus harvest medium.
Fourth day: 48hr after transfection, taking cell culture supernatant, centrifuging at 400 Xg for 4min, filtering with 0.45 μm, adding 5 XPEG 8000 solution by volume, mixing every 20-30min for 3-5 times, and standing at 4deg.C for 6 hr or overnight. The supernatant was removed by centrifugation at 4000g for 20min at 4℃and the tube was left to stand for 1-2min to remove residual liquid. Adding appropriate amount of virus dissolving solution to dissolve virus precipitate, separating the virus solution, freezing at-80deg.C, and taking one branch for further measurement of virus titer.
HT-1080 24-well plates, 5 wells, 42000cells per well, after adherence, virus infection was performed. 10mL of polybrene MEM medium was prepared at 8. Mu.g/mL. 15. Mu.L of virus stock+135. Mu.L of medium. Then diluting step by step, taking 15 mu L+135. Mu.L, performed 4 times. Then 50. Mu.L of each group was added to 450. Mu.L of medium wells, and the dilution levels were 10, respectively 2 ,10 3 ,10 4 ,10 5 . After 72 hours, the digested cells were subjected to flow analysis for GFP% and as a result, as shown in fig. 6, the lentivirus titer after packaging was calculated as tu= (8.94×0.01×42000/0.5) ×10000= 7.51E7TU/mL according to the following formula.
Titer (TU/mL) = (f×c/V) ×d
F=gfp positive cell frequency (GFP positive cell percentage/100)
C=number of cells per well at transduction (42,000 cells)
V=transduction volume (mL) (0.5 mL)
D = lentiviral dilution factor
Example 5 targeting Her-2CAR-NK cells
5.1 NK cell purification and expansion
Fresh anticoagulants were collected from 20mL healthy volunteers, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated with a lymph isolation solution (purchased from GE). After isolated cell counts, the cells were counted as per 2.5X10 6 Well density was stimulated in 6-well plates coated with CD16 for 72h, after which the culture expansion was continued for 72h in the same 6-well plates. Purified cells were sorted using NK magnetic beads (from Miltenyi Biotec Co.), and induced culture was continued with the addition of 1640 (from Thermo Scientific) medium containing 10% FBS+200IU/ml IL-2 to obtain purified NK cells, and the phenotype ratio of CD3 and CD56 was examined by flow assay, the examination results were 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 NK cell ratio prepared by the method was more than 90%.
5.2 preparation of Her-2CAR-NK cell targeting
Purified NK cells were obtained at about 2.5X10 per well 6 NK cells were seeded into 24 well plates (BD Biosciences) and mixed with an appropriate amount of virus supernatant in the presence of a final concentration of Protamine sulfate. Mu.g/mL (Sigma-Aldrich) and BX 795.5. Mu.M (Sigma-Aldrich) in a final volume of no more than 1mL. Cytokine supplementation and plates were centrifuged at 1000 g for 1 hour at room temperature. After centrifugation, the plates were incubated at 37℃with 5% CO without removal of the viral supernatant 2 Incubate for 4-6 hours. After the incubation was completed, a second centrifugation was performed at 1000 g for 1 hour at room temperature, followed by removal of 1ml of fresh NK cell growth medium from the wells. After the cells were maintained in a medium supplemented with cytokines for 2 days per day, her-2CAR-NK cells (hereinafter abbreviated as CAR-NK) were obtained and further transfection efficiency was identified.
After transfection, NK cells were incubated with recombinant human Her-2-Fc protein for 15min at 4 ℃, washed, and then incubated with PE-labeled murine anti-human anti-IgG1 antibody for 15min at 4 ℃, and further subjected to forward flow detection, the results are shown in FIG. 8, the abscissa indicates scFV expression, the left image indicates the blank vector anti-Her2-scFV expression, the right image indicates anti-Her2-scFv expression after transfection of NK cells with the CAR-containing vector, and the transfection efficiency of NK cells was about 30%.
Example 6 targeting Her-2CAR-NK cell killing of tumor cells in vitro
6.1 Detection of killing power of CAR-NK cells on tumor cells
Her-2 positive breast cancer cell BT474 was adjusted to 1X10 with medium 6 Per mL, labeling was performed by adding 5. Mu.g/mL Calcein-AM, incubating at 37℃for 1h, washing three times with PBS, and resuspending the cells with phenol red-free 1640 complete medium, counting. 10000/well tumor cells were adjusted and added to a 96-well round bottom plate. T is 10:1;5:1;2.5:1;1.25:1;0.625:1;0.03125:1 Mock NK cells and CAR-NK cells transfected with empty vector were added respectively 1X10 5 ；5×10 4 ；2.5×10 4 ；1.25×10 4 ；0.625×10 4 ；0.03125×10 4 . Tumor cells were further centrifuged for 5min at 100g for a further 2% Triton X-100 and untreated groups, and after 3h at 37℃for a further 5min at 300g, 100. Mu.L of cells were pipetted into 96-plate plates per well for detection of OD. The detection results are shown in fig. 9, and demonstrate that the killing power of the CAR-NK cells on Her-2 positive breast cancer cells is significantly higher than that of control NK cells.
6.2 detection of IFN- γ secretion levels after Co-culture of CAR-NK cells and 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 and Mock NK cells transfected by empty vector according to E:T of 2.5:1 for 12 hours, and then the supernatant is taken for ELISA to detect the concentration of IFN-gamma in the supernatant, and the detection result is shown in figure 10, and the secretion level of IFN-gamma can be remarkably improved by the CAR-NK cells.
Sequence listing
<110> Hangzhou dry cell Biotech Co., ltd
<120> Her-2 targeting CAR-NK cells and uses thereof
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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
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His Ala Ala Arg Pro
20
<210> 3
<211> 720
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<213> Artificial sequence (Artificial Sequence)
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gctgatgact tcaagggacg gtttgacttc tctttggaaa cctctgccaa cactgcctat 240
ttgcagatca acaacctcaa aagtgaagac tcggctacat atttctgtgc aagatgggag 300
gtttaccacg gctacgttcc ttactggggc caagggacca cggtcaccgt ttcctctggc 360
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caggatgtgt ataatgctgt tgcctggtat caacagaaac caggacaatc tcctaaactt 540
ctgatttact cggcatcctc ccggtacact ggagtccctt ctcgcttcac tggcagtggc 600
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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
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His Phe Arg Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
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Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
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Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
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Phe Pro Gly Pro Ser Lys Pro
35
<210> 9
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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
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<212> PRT
<213> Artificial sequence (Artificial Sequence)
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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 (19)

1. A Her-2 targeted CAR-NK cell, wherein the nucleotide sequence of the CAR comprises the structure: the tumor-associated antigen binding region is single-chain antibody FRP5scFv targeting Her-2, and the nucleic acid and polypeptide sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. The CAR-NK cell of claim 1, wherein the CAR nucleotide sequence further comprises 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 CAR-NK cell of claim 2, wherein the nucleic acid and polypeptide sequences of the CD8a signal peptide are shown in SEQ ID No.1 and SEQ ID No.2, respectively.
4. The CAR-NK cell of claim 1, wherein the extracellular hinge region is selected from the group consisting of a hinge region sequence of CD8, CD28, CTLA4, PD-1, NKG2D, igG1, igG4 with or without a CH2-CH3 region.
5. The CAR-NK cell of claim 4, wherein the nucleic acid and polypeptide sequences of the CD8 extracellular Hinge region, CD28 extracellular Hinge region, and IgG4 finger-CH 2-CH3 extracellular Hinge region are shown 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 CAR-NK cell of claim 1, wherein the transmembrane domain can be derived from a nucleic acid, polypeptide sequence of the following protein transmembrane domain: the α, β or ζ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1, ctla.
7. The CAR-NK cell of claim 6, wherein the nucleic acid and polypeptide sequences of the CD8 transmembrane domain, CD28 transmembrane domain, and NKG2D transmembrane domain are shown 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 CAR-NK cell of claim 1, wherein the intracellular signaling domain comprises or does not comprise a co-stimulatory domain derivable from one or more different combinations of nucleic acids, polypeptide sequences of the following protein intracellular functional signaling domains: OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), DAP10, DAP12,4-1BB (CD 137) and 2B4 or functional variants thereof.
9. The CAR-NK cell 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 CAR-NK cell of claim 1, wherein the signaling domain is typically a T cell receptor TCR/CD3 ζ chain or an immunoglobulin Fc receptor fceriγ chain, comprising an immunoreceptor tyrosine activation motif.
11. The CAR-NK cell of claim 10, wherein the Signaling Domain is cd3ζ Signaling Domain having the nucleic acid and polypeptide sequences shown in SEQ ID No.25 and SEQ ID No.26, respectively.
12. A CAR-NK cell as claimed in any one of claims 1 to 10 which in turn consists of a tandem of signal peptide, tumour associated antigen binding region, extracellular hinge region, transmembrane domain and intracellular signalling domain comprising a co-stimulatory domain, the nucleic acid and polypeptide sequences of each of the above components being as claimed in claims 5 to 14, the CAR-NK structure being selected from any one of the groups a) to g) or a combination thereof, each combination being 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 CAR-NK cell of claim 12 having a structure selected from e) CD8 SP-Her-2scFv (FRP 5) -CD 8H-NKG 2D TMD-2b4 CD-CD3 ζsd; the nucleic acid sequence is shown as SEQ ID NO. 29.
14. The method of preparing a CAR-NK cell as defined in any one of claims 1 to 13, comprising the steps of:
a) NK cell sorting and purification;
b) Constructing a virus containing the lentiviral vector of the CAR-NK nucleotide sequence of any one of claims 1 to 12, and transfecting the virus into NK cells purified in step a), and obtaining Her-2 targeted CAR-NK cells after expression and identification.
15. The method of claim 14, wherein step a) comprises isolating and purifying NK cells from healthy humans, specifically comprising isolating peripheral blood mononuclear cells from fresh anticoagulants from healthy humans using a lymphocyte isolate, stimulating the cells in a CD16 coated 6-well plate, sorting the purified cells with NK magnetic beads, and continuing the induction culture using 1640 medium containing 10% fbs+200iu/ml IL-2 to obtain purified NK cells.
16. The method of claim 14, wherein step b) is performed to obtain purified NK cells at a rate of 2.5X10 per well 6 NK cells were seeded into 24 well plates and mixed with the virus supernatant in the presence of final concentration of 8. Mu.g/ml Protamine sulfate and 1.5. Mu.MBX 795, and plates were centrifuged at 1000. Mu.g for 1 hour at room temperature; after centrifugation, the plates were incubated at 37℃with 5% CO without removal of the viral supernatant 2 Incubating for 4-6 hours; incubation methodAfter the end of the incubation, a second centrifugation at 1000 g was performed for 1 hour at room temperature, and then after the cells were maintained in the medium for 2 days, her-2 expressing CAR-NK cells were obtained.
17. Use of the CAR-NK cells of any one of claims 1-16 for the manufacture of a medicament associated with Her-2 mediated disease.
18. The use according to claim 17, in tumor diseases.
19. The use according to claim 18, wherein the tumour is predominantly breast cancer.
CN202210146396.5A 2022-02-17 2022-02-17 Her-2 targeted CAR-NK cell and application thereof Pending CN116656614A (en)

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