CN114891115A - Nucleic acid construct for CAR cell therapy, lentiviral vector, cell preparation and application thereof - Google Patents

Abstract

The invention discloses a nucleic acid construct for CAR cell therapy, and a lentiviral vector, a cell preparation and application thereof, wherein the nucleic acid construct for CAR cell therapy comprises a signal peptide, an antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal domain which are sequentially connected in series, and the intracellular signal domain consists of DAP10CD, CD137CD and CD3zeta SD. A lentiviral vector comprising a gene of a nucleic acid construct for CAR cell therapy. A cell preparation comprising an immune effector cell into which the lentiviral vector has been introduced. Use of a cell preparation in the treatment of cancer. Compared with immune cell preparations constructed by other CAR structures, the cell preparation prepared by the invention has higher activity, and particularly NK cells modified by the CAR structures have extremely high killing capacity on target tumor cells (such as breast cancer cells expressing Her-2).

Description

Nucleic acid construct for CAR cell therapy, lentiviral vector, cell preparation and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of cellular immunotherapy, in particular to a nucleic acid construct for CAR cell therapy, and a lentiviral vector, a cell preparation and an application thereof.

[ background of the invention ]

Malignant tumors are one of the major diseases that endanger human health. Traditional tumor treatment modalities, such as surgery, radiation therapy, and chemotherapy, have been the primary strategy for tumor treatment in recent decades. However, patients are susceptible to resistance to drug and radiation therapy, resulting in a high frequency of tumor recurrence. In recent years, the tumor cell therapy has attracted great attention due to its advantages of targeting property, remarkable effect, few side effects and the like, and gradually becomes an important means in the comprehensive treatment of tumors, is called as green therapy of tumors in the industry, and is also a hotspot and development direction of the current basic research and clinical application of tumor therapy.

A Chimeric Antigen Receptor (CAR) therapy of tumor refers to a novel precise targeted therapy for treating tumor by using immune cells modified by Chimeric Antigen Receptor, obtains good effect on clinical tumor treatment by optimizing and improving in recent years, has the characteristics of precision, rapidness and high efficiency, is possible to cure cancer, and has a very prospect. In general, chimeric antigen receptor cell therapy of tumors transduces localization-homing devices-chimeric antigen receptors recognizing tumors into immune cells (such as T cells and NK cells) by genetic engineering techniques, recombining to CAR-T cells and CAR-NK cells, respectively. CAR-T cells and CAR-NK cells can specifically recognize tumor cells in vivo by using the transduced CAR structure, and then are activated, and release a large amount of multiple effector factors through immune action, so that the tumor cells are efficiently killed, and the purpose of treating malignant tumors is achieved.

Chimeric antigen receptors are typically 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, in sequential tandem. With the continuous advance of scientific and technical research, CARs have been developed to the fourth generation so far. Among them, the first generation CARs contain an extracellular single-chain variable fragment scFv, a transmembrane region, and a single intracellular activation signal CD3 ζ or fceri γ. The first generation CAR-T cells only cause transient T cell proliferation and less cytokine secretion, their in vivo antitumor activity is greatly limited, and decreased T cell proliferation ultimately leads to T cell apoptosis. The second generation CARs introduce a co-stimulatory molecule on the basis of the first generation CARs, and the tumor killing efficacy is improved. Third-generation CARs carry multiple costimulators, such as CD28, CD134(OX40), and CD137(4-1 BB). The co-stimulatory molecule can activate signal pathways such as JNK, ERK, NF-kB and the like in the T cell, so that the T cell has obvious improvement in the aspects of antitumor activity, proliferation activity, survival period, secretion extension of cytokines (such as IL-2, TNF-alpha and IFN-gamma) and the like. The fourth generation CARs are added with selectable markers and promoters encoding CARs amplification and suicide on the basis of the third generation CARs.

Studies have shown that CAR design and intrinsic properties also affect the expansion and persistence of CAR-modified immune cells. Currently, second generation CARs comprising a CD 3-zeta signaling domain and a costimulatory domain (typically CD28 or 4-1BB) remain the most common nucleic acid constructs in clinical use. Furthermore, it was found that the duration of T cell killing was short although the killing effect was strong when CD28 was used as a co-stimulatory molecule, whereas the killing ability was limited although the duration of T cell killing was long when 4-1BB was used as a co-stimulatory molecule.

Therefore, the existing chimeric antigen receptor immune cell therapy still has many imperfect points for tumor treatment. For example, CAR-T cell therapy can be associated with a variety of adverse effects, including cytokine release syndrome. Moreover, the clinical manifestations of these toxic and side effects are often complicated, varied and rapidly progressed; moreover, each CAR-modified cell preparation has unique and different effect characteristics due to different production processes or CAR structures, and further sufficient knowledge and accurate evaluation are needed.

[ summary of the invention ]

The invention aims to solve the problems in the prior art and provides a nucleic acid construct containing a specific intracellular signal structure combination for CAR cell therapy, a lentiviral vector, a cell preparation and application thereof, wherein the prepared cell preparation has higher activity compared with an immune cell preparation constructed by other CAR structures, and particularly the NK cell modified by the CAR structure has extremely high killing capacity on target tumor cells (such as breast cancer cells expressing Her-2).

In order to achieve the purpose, the invention is realized by the following technical scheme:

a nucleic acid construct for CAR cell therapy comprising, in series, in order, a signal peptide, an antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signal domain;

the signal peptide is Human IgKVIII, Human IL-2 or Human insulin and the like;

the antigen binding region is anti-CD19 scFv, anti-Her-2scFv, anti-ROR1 scFv or anti-BCMA scFv and the like;

the extracellular hinge region is a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 containing a CH2CH3 region or IgG4 not containing a CH2CH3 region, and the like;

the transmembrane domain is a transmembrane domain sequence of an α, β or ζ chain, CD28, CD3 ∈ CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1 or CTLA from a T cell receptor, or the like;

the intracellular domain consists of DAP10CD, CD137CD, and CD3 ζ SD.

Preferably, the signal peptide is CD8 SP, and the nucleic acid sequence and the polypeptide sequence of the CD8 SP are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table.

Preferably, the antigen binding region is Her-2scFv, and the nucleic acid sequence and the polypeptide sequence of the Her-2scFv are respectively shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence table.

Preferably, the extracellular hinge region is CD8H, CD28H or IgG4H-CH2-CH3, the nucleic acid sequence and the polypeptide sequence of the CD8H are respectively shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28H are respectively shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the IgG4H-CH2-CH3 are respectively shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence table.

Preferably, the transmembrane domain is CD8TMD, CD28TMD or NKG2D TMD, the nucleic acid sequence and the polypeptide sequence of the CD8TMD are respectively shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28TMD are respectively shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the NKG2D TMD are respectively shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence table.

Preferably, the nucleic acid sequence and the polypeptide sequence of the DAP10CD are respectively shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD137CD are respectively shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the CD3zeta SD are respectively shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence table.

Preferably, the sequence of the intracellular signal domain is DAP10 CD-CD137CD-CD 3zeta SD, and the nucleic acid sequence and the polypeptide sequence of the DAP10 CD-CD137CD-CD 3zeta SD are respectively shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence table.

A lentiviral vector comprising a gene of the nucleic acid construct for CAR cell therapy as any of the preceding.

A cell preparation is an immune effector cell introduced with the lentiviral vector, wherein the immune effector cell is a T cell, an NK cell or a macrophage, and the immune effector cell is derived from an autologous, allogeneic, stem cell differentiation or specific cell line.

Preferably, the NK cell is a human.

Use of a cell preparation in the treatment of cancer.

The invention has the beneficial effects that:

the invention adopts a nucleic acid construct consisting of a signal peptide, an antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal domain which are sequentially connected in series, and defines the intracellular signal domain to be composed of DAP10CD, CD137CD and CD3zeta SD, and then prepares a corresponding cell preparation by a lentiviral vector technology. Compared with immune cell preparations constructed by other CAR structures, the cell preparation prepared by the invention has higher activity, and particularly NK cells modified by the CAR structures have extremely high killing capacity on target tumor cells (such as breast cancer cells expressing Her-2).

The features and advantages of the present invention will be described in detail by embodiments in conjunction with the accompanying drawings.

[ description of the drawings ]

FIG. 1 is a sequence composition diagram of the CAR of examples one to three;

FIG. 2 shows the results of nucleotide electrophoresis of T-CAR/NK-CAR1/NK-CAR2 sequences on pUC57 vector after digestion with BamHI and XbaI;

FIG. 3 is a vector map of pCDH-SFFV-Luc-T2A-EGFP;

FIG. 4 is a flow cytogram of lentivirus titer assays containing different intracellular structure CAR;

FIG. 5 is a flow cytogram after NK cell purification and expansion;

FIG. 6 is the results of flow cytometry analysis of the identification of transfection efficiency following lentiviral transfection of NK cells with different targeted Her-2 CAR;

FIG. 7 is the killing of breast cancer cells in vitro by Her-2 CAR-NK cells targeted to different intracellular structures;

FIG. 8 is the basis of different sets of CAR-NK cells and the secretion levels of IFN-. gamma.and TNF-. alpha.after co-culture with different breast cancer cells;

FIG. 9 is a vector map of NK-CAR 2.

In fig. 2: lane 1 nucleic acid molecular weight standards; lanes 1-3 correspond to the T-CAR, NK-CAR1 and NK-CAR2 sequence portions, respectively; lane 4 is the cleaved pCDH-SFFV-Luc-T2A-EGFP vector fraction.

[ detailed description ] embodiments

Examples one to three, design and acquisition of a targeted her-2 CAR:

a nucleic acid construct for CAR cell therapy, characterized in that: comprises a signal peptide, an antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal domain which are sequentially connected in series;

the Signal Peptide (SP) is a short (5-30 amino acids in length) peptide chain responsible for guiding newly synthesized protein to subcellular organelles containing different membrane structures of cells, and is generally positioned at the N-terminal of the protein.

The signal peptide is a signal peptide commonly used for recombinant proteins, such as Human IgKVIII, Human IL-2 or Human insulin and the like. Furthermore, the signal peptide is CD8 SP, and the nucleic acid sequence and the polypeptide sequence of the CD8 SP are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table.

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. The antigen binding region is a single chain antibody (scFv) formed by a light chain (VL) and a heavy chain (VH) linked by a flexible hinge (linker) located therebetween, derived from a monoclonal antibody targeting a particular antigen of interest. The heavy or light chain of a single-chain antibody is connected to a signal peptide and a hinge region, respectively.

Wherein the targeted antigen may include alpha-folate receptor (ovarian and epithelial cancers), CAIX (kidney cancer), CD19 (B-cell malignancies, CLL and ALL), CD20 (B-cell malignancies and lymphoma), CD22 (B-cell malignancies), CD23(CLL), CD24 (pancreatic cancer), CD30 (lymphoma), CD33(AML), CD38(NHL), CD44v7/8 (cervical cancer), CEA (colorectal cancer), EGFRvIII (glioblastoma), EGP-2 (multiple malignancies), EGP-40 (colorectal cancer), EphA2 (glioblastoma), Erb-B2 (breast, prostate and colon cancers), FBP (ovarian cancer), GD2 (neuroblastoma and melanoma), GD3 (melanoma), HER2 (pancreatic, ovarian, glioblastoma and osteosarcoma), HMff _ a (melanoma), IL _13Ra and 2 (glioma), KDR (tumor vasculature), kappa light chain (B cell malignancy), L1 (neuroblastoma), MAGE-A1 (melanoma), mesothelioma (mesothelioma), MUC1 (breast and ovarian cancer), MUC16 (ovarian cancer), NY-ES0-1 (multiple myeloma), carcinoembryonic antigen (various tumors), PSCA (prostate cancer), PSMA (prostate cancer), ROR1(B-CLL), TAG-72 (adenocarcinoma), VEGF-R2 (tumor neovasculature), and the like.

The antigen binding region is anti-CD19 scFv, anti-Her-2scFv, anti-ROR1 scFv or anti-BCMA scFv, etc. Furthermore, the antigen binding region is Her-2scFv, and the nucleic acid sequence and the polypeptide sequence of the Her-2scFv are respectively shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence table.

An extracellular Hinge region (Hinge, H) connects the extracellular antigen-binding region and the transmembrane domain. The length of the extracellular hinge region depends on the location and extent of exposure of the epitope to the target cell. Furthermore, several studies have shown that CAR-T cell activation is related to hinge region length. Thus, adjusting the length of the hinge region can allow the CAR-T cell to be at an optimal distance from the target cell, avoiding the effects of large phosphatases to attenuate the CAR signal 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. In summary, due to the different epitopes, the optimal length of the hinge region is different, and when targeting a new antigen, the length of the hinge region needs to be adjusted accordingly.

The extracellular hinge region is a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 containing a CH2CH3 region or IgG4 not containing a CH2CH3 region, and the like. Furthermore, the extracellular hinge region is CD8H, CD28H or IgG4H-CH2-CH3, the nucleic acid sequence and the polypeptide sequence of the CD8H are respectively shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28H are respectively shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the IgG4H-CH2-CH3 are respectively shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence table.

A transmembrane domain (TMD) capable of linking the extracellular domain of the CAR with the intracellular signaling domain and anchoring the receptor to the immune cell membrane.

The transmembrane domain is a transmembrane domain sequence of an alpha, beta or zeta chain from a 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 or CTLA, or the like. Wherein the transmembrane domain of CD3 ζ is capable of homodimerizing a CAR or heterodimerizing with an endogenous TCR, enhancing CAR-T cell activity, and the transmembrane domains of CD8 α and CD28 are capable of promoting expression of a CAR on the cell surface. Furthermore, the transmembrane domain is CD8TMD, CD28TMD or NKG2D TMD, the nucleic acid sequence and the polypeptide sequence of the CD8TMD are respectively shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28TMD are respectively shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the NKG2D TMD are respectively shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence table.

The Costimulatory Domain (CD) can realize dual activation of Costimulatory molecules and intracellular signals, so that T cells can continuously proliferate and release cytokines, and the anti-tumor capacity of T cells is improved.

The intracellular domain consists of DAP10CD, CD137CD, and CD3 ζ SD. Furthermore, the nucleic acid sequence and the polypeptide sequence of the DAP10CD are respectively shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD137CD are respectively shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the CD3zeta SD are respectively shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence table. The sequence of the intracellular signal domain is DAP10 CD-CD137CD-CD 3zeta SD, and the nucleic acid sequence and the polypeptide sequence of the DAP10 CD-CD137CD-CD 3zeta SD are respectively shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence table.

Wherein, the related nucleic acid sequences and polypeptide sequences are shown in the following table 1:

TABLE 1 sequence listing

The various different signal peptides, antigen binding regions, extracellular hinge regions, transmembrane domains and intracellular signal domains are sequentially combined and serially connected to obtain the CAR sequence combinations of embodiments one to three shown in FIG. 1, wherein the specific sequences are SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO. 33.

Example four, construction of lentiviral vectors:

the CAR sequences of examples one to three were synthesized and the synthesized CAR sequences were cloned into pUC57 vectors, named pUC57-T-CAR, pUC57-NK-CAR1 and pUC57-NK-CAR2, respectively. Plasmids pUC57-T-CAR, pUC57-NK-CAR1 and pUC57-NK-CAR2 were extracted and digested with the Quickcut restriction enzymes BamHI and XbaI (Takara). Wherein, the enzyme digestion system is as follows:

pUC57-T-CAR/pUC57-NK-CAR1/pUC57-NK-CAR21 μ g, BamHI and XbaI each 1 μ l, 10 XQuickcut Green Buffer2 μ l, supplemented with water to 20 μ l, water bath at 37 ℃ for 15min, after the enzyme-cleaved product was separated by agarose gel electrophoresis, DNA fragment recovery was performed using agarose gel DNA fragment recovery kit (purchased from Takara Co., Ltd.) (see results in FIG. 2, lanes 1-3 are bands of pUC57-T-CAR/pUC57-NK-CAR1/pUC CAR 57-NK-2 vector cleaved with BamHI and XbaI to release T-CAR/NK-CAR1/NK-CAR 2). The pCDH-SFFV-Luc-T2A-EGFP vector (FIG. 3) was digested with BamHI and XbaI in the same manner, and the digested vector fragment was separated and recovered by agarose gel electrophoresis (see FIG. 2, wherein lane 4 is a digested pCDH-SFFV-Luc-T2A-EGFP vector fraction).

The recovered T-CAR/NK-CAR1/NK-CAR2 fragment and the enzyme-cleaved vector were ligated by T4 ligase (purchased from Takara). Wherein, the reaction system and conditions are as follows:

the T-CAR/NK-CAR1/NK-CAR2 and pCDH-SFFV-Luc-T2A-EGFP vectors which are cut by BamHI and XbaI are respectively quantified, and then according to the vectors, a target band 1: 3 molar ratio was added to the system, 10X T4 DNALigase Buffer 1. mu.l, ligase 1. mu.l, supplemented to 10. mu.l with water, and ligated for 4h or overnight at 16 ℃. The ligation product was transformed into e.coli DH5a competent bacteria, cultured overnight at 37 ℃, single colonies were picked, expanded, and plasmids of positive clones were extracted with a plasmid extraction kit (purchased from Axygene) according to the kit instructions, and the correct vector was named pCDH-T-CAR/pCDH-NK-CAR1/pCDH-NK-CAR2 by enzyme digestion and sequencing detection.

Example five, lentivirus packaging and titer determination:

1) plasmid:

·pCDH-T-CAR/pCDH-NK-CAR1/pCDH-NK-CAR2；

·VSV-G：pMD2.G(Addgene#12259)；

·Rev：pRSV-Rev(Addgene#12253)；

·Gag/Pol：pMDLg/pRRE(Addgene#12251)。

2) cell line:

low passage number 293T cells.

3) Reagent:

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

virus harvest medium: taking 50ml of 293T culture medium, adding 0.5g BSA (SigmaA9418) and HEPES with the final concentration of 10-15 mM, and filtering by 0.22 mu m;

Opti-MEM Medium: for mixing the transfection complexes;

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

4) And (3) packaging process:

day one: 293T cells were grown according to table 2 below;

the next day-morning: preparing a mixed Opti-MEM culture medium, a plasmid and a transfection reagent according to the following table 2, replacing a fresh culture medium for 293T cells, standing the mixture at room temperature for 15-30 min, dropwise adding the mixture into a 293T cell culture system, and gently shaking the mixture uniformly;

TABLE 2 Opti-MEM Medium

The next day-afternoon: after 6-8 h of transfection, the soft change solution is a virus harvesting culture medium;

day four: 48h after transfection, cell culture supernatant was centrifuged at 400 Xg for 4min and filtered at 0.45 μm; adding 5 multiplied PEG8000 solution according to the volume, mixing once every 20-30 min, 3-5 times totally, and standing for 6h or overnight at 4 ℃; centrifuging at 4000g for 20min at 4 deg.CDiscarding the supernatant, standing the tube for 1-2 min, and sucking away residual liquid; adding a proper amount of virus dissolving solution to dissolve virus precipitates, dividing virus solution into tubes, putting 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 types of 24-hole plates, 5 holes, 42000cells per hole, and carrying out virus infection after the plates are attached to the wall; preparing 10ml of 8ug/ml polybrene MEM culture medium; preparing 15ul of virus stock solution and 135ul of culture medium; then diluting step by step, taking 15ul +135ul, and carrying out 4 times; subsequently 50ul of each group was added to 450ul of medium wells at a dilution scale of 10 ² 、10 ³ 、10 ⁴ And 10 ⁵ (ii) a After 72h, the digested cells were subjected to flow analysis for GFP% and the results are shown in FIG. 4, and the packaged lentivirus titer was obtained by calculation according to the following formula:

TU _T-CAR ＝(8.01×0.01×42000/0.5)×10000＝6.73E7 TU/ml

TU _NK-CAR1 ＝(2.95×0.01×42000/0.5)×100000＝2.45E8 TU/ml

TU _NK-CAR2 ＝(8.94×0.01×42000/0.5)×10000＝7.51E7 TU/ml。

wherein, the titer (TU/mL) ═ F × C/V × D;

F-GFP positive cell frequency (percent GFP positive cells/100);

c-number of cells per well at the time of transduction (42,000 cells);

v ═ transduction volume (mL) (0.5 mL);

d ═ lentiviral dilution factor.

Example six, CAR-NK cell preparation:

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 corporation); after counting the isolated cells, 2.5X10 ⁶ Stimulating and culturing the density of each well in a 6-well plate coated with CD16 for 72h, and then changing the common 6-well plate to continue culturing and amplifying for 72 h; purified cells (purchased from Miltenyi Biotec) were sorted using NK magnetic beads, induction culture was continued with addition of 1640 medium containing 10% FBS +200IU/ml IL-2 (purchased from Thermo Scientific) to obtain purified NK cells, and detection of CD3 and CD3 by flow assayPhenotypic ratio of CD 56. The detection result is shown in fig. 5, wherein the horizontal axis represents CD3, the vertical axis represents CD56, the cell population represented by CD3 negative and CD56 positive is NK cells, and the proportion of the NK cells prepared by the method is more than 90%;

2) preparation of targeted Her-2 CAR-NK cells:

purified NK cells were obtained at about 2.5X10 per well ⁶ NK cells were seeded in 24-well plates (BD Biosciences) and mixed with appropriate amounts of viral supernatant in the presence of final concentrations Protamine sulfate 8ug/ml (Sigma-Aldrich) and bx7951.5um (Sigma-Aldrich), with a final volume of no more than 1 ml; cytokine supplementation and plates centrifuged at 1000 · g for 1h at room temperature; after centrifugation, the plates were incubated at 37 ℃ in 5% CO without removing the viral supernatant ₂ Incubating for 4-6 h; after the incubation was completed, a second centrifugation was performed at 1000. g for 1h at room temperature, followed by centrifugation from the wells and 1ml of fresh NK cell growth medium; after maintaining the cells in a culture medium added with cytokines every day for 2 days, obtaining Her-2 CAR-NK cells (hereinafter, abbreviated as CAR-NK), and carrying out further transfection efficiency identification; incubating the transfected NK cells with recombinant human Her-2-Fc protein for 15min, washing at 4 ℃, incubating with a PE-labeled mouse anti-human anti-IgG1 antibody for 15min, and further performing flow detection at 4 ℃; the results are shown in FIG. 6, where the abscissa represents scFv expression.

Example seven, killing of tumor cells by CAR-NK cells:

1) and (3) detecting the killing power of the CAR-NK cells on tumor cells:

adjusting Her-2 positive breast cancer cell BT474 and Her-2 negative breast cancer cell MDA-MB-468 to 1X10 with culture medium ⁶ Adding 5ug/ml Calcein-AM for labeling, incubating at 37 deg.C for 1h, washing with PBS for three times, suspending cells with phenol-free red 1640, and counting; adjusting 10000/hole of tumor cells to be added into a 96-hole round bottom plate; according to E: t is 10: 1. 5: 1. 2.5: 1. 1.25: 1. 0.625 of: 1 and 0.03125: 1 Mock NK cells transfected with empty vectors and corresponding different sets of CAR-NK cells 1X10 were added separately ⁵ 、5X10 ⁴ 、2.5X10 ⁴ 、1.25X10 ⁴ 、0.625X10 ⁴ And 0.03125X10 ⁴ (ii) a Swelling and swelling treating medicineAdding a group of 2% Triton X-100 and untreated group into tumor cells, centrifuging for 5min at 100g, co-culturing for 3h at 37 ℃, centrifuging for 5min at 300g, sucking 100ul of each well, transferring to a 96-flat-bottom plate, and detecting OD value; the detection result is shown in figure 7, and the detection result shows that the killing capacity of CAR-NK cells containing DAP10 CD-137 CD-3 zeta SD intracellular signal domain NK-CAR2 on Her-2 positive breast cancer cells is obviously higher than that of other groups of NK cells, and the killing capacity of the CAR-NK cells on Her-2 negative breast cancer cells MDA-MB-468 is not obviously different;

2) detection of IFN-gamma and TNF-alpha secretion levels after Co-culture of CAR-NK cells with different cells

And (3) enabling Her-2 positive breast cancer cells BT474 and Her-2 negative breast cancer cells MDA-MB-468 to be in a proportion of E: t is 2.5: 1 co-culturing MockNK cells transfected by an empty vector and different groups of CAR-NK cells for 12h, taking a supernatant, and performing ELISA to detect the concentrations of IFN-gamma and TNF-alpha in the supernatant; the detection result is shown in figure 8, and the secretion levels of IFN-gamma and TNF-alpha after the CAR-NK cells containing DAP10-CD137-CD3zeta intracellular signal domain NK-CAR2 and Her-2 positive breast cancer cells BT474 are cultured together are obviously higher than those of other groups.

The above embodiments are illustrative of the present invention, and are not intended to limit the present invention, and any simple modifications of the present invention are within the scope of the present invention.

Sequence listing

<110> Hangzhou Meizhong disease Gene research institute Co., Ltd

<120> nucleic acid construct for CAR cell therapy, lentiviral vector, cell preparation and application thereof

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 63

<212> DNA

<213> nucleic acid sequence (CD8)

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 2

<211> 21

<212> PRT

<213> polypeptide Sequence (SP)

<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> nucleic acid sequence (Her-2 scFv)

<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> polypeptide sequence (Her-2 scFv)

<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> nucleic acid sequence (CD8H)

<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> polypeptide sequence (CD8H)

<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> nucleic acid sequence (CD28H)

<400> 7

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117

<210> 8

<211> 39

<212> PRT

<213> polypeptide sequence (CD28H)

<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> nucleic acid sequence (IgG4H-CH2-CH3)

<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> polypeptide sequence (IgG4H-CH2-CH3)

<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> nucleic acid sequence (CD8TMD)

<400> 11

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

acc 63

<210> 12

<211> 21

<212> PRT

<213> polypeptide sequence (CD8TMD)

<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> nucleic acid sequence (CD28TMD)

<400> 13

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 14

<211> 27

<212> PRT

<213> polypeptide sequence (CD28TMD)

<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> nucleic acid sequence (NKG2DTMD)

<400> 15

ccattttttt tctgctgctt catcgctgta gccatgggaa tccgtttcat tattatggta 60

aca 63

<210> 16

<211> 21

<212> PRT

<213> polypeptide sequence (NKG2DTMD)

<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> 69

<212> DNA

<213> nucleic acid sequence (DAP10CD)

<400> 17

ctgtgcgcac gcccacgccg cagccccgcc caagatggca aagtctacat caacatgcca 60

ggcaggggc 69

<210> 18

<211> 23

<212> PRT

<213> polypeptide sequence (DAP10CD)

<400> 18

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> 19

<211> 126

<212> DNA

<213> nucleic acid sequence (CD137CD)

<400> 19

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 20

<211> 42

<212> PRT

<213> polypeptide sequence (CD137CD)

<400> 20

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> 21

<211> 123

<212> DNA

<213> nucleic acid sequence (CD28 CD)

<400> 21

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 22

<211> 41

<212> PRT

<213> polypeptide sequence (CD28 CD)

<400> 22

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> 23

<211> 126

<212> DNA

<213> nucleic acid sequence (CD137CD)

<400> 23

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 24

<211> 42

<212> PRT

<213> polypeptide sequence (CD137CD)

<400> 24

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> 25

<211> 359

<212> DNA

<213> nucleic acid sequence (2B4CD)

<400> 25

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 tccaggaaga 240

ggaaccacag cccttccttc aatagcacta tctatgaagt gattggaaag agtcaaccta 300

aagcccagaa ccctgctcga ttgagccgca aagagctgga gaactttgat gtttattcc 359

<210> 26

<211> 120

<212> PRT

<213> polypeptide sequence (2B4CD)

<400> 26

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> 27

<211> 339

<212> DNA

<213> nucleic acid sequence (CD3 ζ SD)

<400> 27

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> 28

<211> 113

<212> PRT

<213> polypeptide sequence (CD3 ζ SD)

<400> 28

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> 29

<211> 534

<212> DNA

<213> nucleic acid sequence (DAP10-CD137-CD3 ζ)

<400> 29

ctgtgcgcac gcccacgccg cagccccgcc caagatggca aagtctacat caacatgcca 60

ggcaggggca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 120

ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 180

ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 240

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 300

gacaagagac gtggccggga ccctgagatg gggggaaagc cgcagagaag gaagaaccct 360

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 420

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 480

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 534

<210> 30

<211> 178

<212> PRT

<213> polypeptide sequence (DAP10-CD137-CD3 ζ)

<400> 30

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 Lys Arg Gly Arg Lys Lys Leu Leu Tyr

20 25 30

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

35 40 45

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Arg

<210> 31

<211> 893

<212> DNA

<213> Artificial sequence (T-CAR)

<400> 31

ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc 60

acgccgccag gccgcaggta caatcgaaaa ccacgacgcc agcgccgcga ccaccaacac 120

cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 180

cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatttttgg gtgctggtgg 240

tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttt attattttct 300

gggtgaggag taagaggagc aggctcctgc acagtgacta catgaacatg actccccgcc 360

gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac ttcgcagcct 420

atcgctccaa acggggcaga aagaaactcc tgtatatatt caaacaacca tttatgagac 480

cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa gaagaagaag 540

gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg taccagcagg 600

gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac gatgttttgg 660

acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg aagaaccctc 720

aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 780

ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 840

cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgc 893

<210> 32

<211> 985

<212> DNA

<213> Artificial sequence (NK-CAR1)

<400> 32

ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc 60

acgccgccag gccgcaggta caatcgaaaa ccacgacgcc agcgccgcga ccaccaacac 120

cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 180

cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatccattt tttttctgct 240

gcttcatcgc tgtagccatg ggaatccgtt tcattattat ggtaacatgg aggagaaaga 300

ggaaggagaa gcagtcagag accagtccca aggaattttt gacaatttac gaagatgtca 360

aggatctgaa aaccaggaga aatcacgagc aggagcagac ttttcctgga ggggggagca 420

ccatctactc tatgatccag tcccagtctt ctgctcccac gtcacaagaa cctgcatata 480

cattatattc attaattcag ccttccagga agtctggtcc aggaagagga accacagccc 540

ttccttcaat agcactatct atgaagtgat tggaaagagt caacctaaag cccagaaccc 600

tgctcgattg agccgcaaag agctggagaa ctttgatgtt tattccagag tgaagttcag 660

caggagcgca gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa 720

tctaggacga agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat 780

ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa 840

agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa 900

ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct 960

tcacatgcag gccctgcccc ctcgc 985

<210> 33

<211> 830

<212> DNA

<213> Artificial sequence (NK-CAR2)

<400> 33

ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc 60

acgccgccag gccgcaggta caatcgaaaa ccacgacgcc agcgccgcga ccaccaacac 120

cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg 180

cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatccattt tttttctgct 240

gcttcatcgc tgtagccatg ggaatccgtt tcattattat ggtaacaata tggagtctgt 300

gcgcacgccc acgccgcagc cccgcccaag atggcaaagt ctacatcaac atgccaggca 360

ggggcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt atgagaccag 420

tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa gaagaaggag 480

gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc 540

agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat gttttggaca 600

agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag aaccctcagg 660

aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt gagattggga 720

tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt ctcagtacag 780

ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 830

<210> 34

<211> 6101

<212> DNA

<213> vector sequence (NK-CAR2)

<400> 34

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct 240

ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 300

gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 360

tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 420

ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 480

cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt 540

ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 600

agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 660

ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 720

ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 780

ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 840

aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa 900

agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag 960

ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 1020

agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 1080

agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac 1140

gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct 1200

gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct 1260

ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg 1320

gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa 1380

taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa 1440

caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa 1500

tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac 1560

aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag 1620

aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc 1680

gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga 1740

aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg 1800

gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 1860

cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aattcaaaat 1920

tttatgtgtc gtgacgcgct agagccacca tggccttacc agtgaccgcc ttgctcctgc 1980

cgctggcctt gctgctccac gccgccaggc cgcaggtaca atcgaaaacc acgacgccag 2040

cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag 2100

aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac ttcgcctgtg 2160

atccattttt tttctgctgc ttcatcgctg tagccatggg aatccgtttc attattatgg 2220

taacaatatg gagtctgtgc gcacgcccac gccgcagccc cgcccaagat ggcaaagtct 2280

acatcaacat gccaggcagg ggcaaacggg gcagaaagaa actcctgtat atattcaaac 2340

aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc tgccgatttc 2400

cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc gcagacgccc 2460

ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg 2520

agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga aagccgcaga 2580

gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg 2640

cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt 2700

accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc 2760

cccctcgcct ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 2820

ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga taagatctgc 2880

tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct 2940

aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt 3000

gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt 3060

ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa 3120

agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat 3180

aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 3240

gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac 3300

tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 3360

aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta 3420

gtgaggaggc ttttttggag gcctagactt ttgcagagac caaattcgta atcatgtcat 3480

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 3540

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 3600

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 3660

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 3720

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3780

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3840

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3900

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3960

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4020

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4080

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4140

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4200

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4260

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 4320

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4380

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4440

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4500

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4560

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4620

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4680

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4740

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4800

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4860

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4920

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4980

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 5040

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 5100

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5160

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5220

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5280

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5340

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5400

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5460

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5520

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5580

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 5640

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg 5700

cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 5760

cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 5820

gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc 5880

atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt 5940

cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 6000

gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt 6060

cccagtcacg acgttgtaaa acgacggcca gtgccaagct g 6101

Claims (10)

1. A nucleic acid construct for CAR cell therapy, characterized in that: comprises a signal peptide, an antigen binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal domain which are sequentially connected in series;

the signal peptide is Human IgKVIII, Human IL-2 or Human insulin;

the antigen binding region is anti-CD19 scFv, anti-Her-2scFv, anti-ROR1 scFv or anti-BCMA scFv;

the extracellular hinge region is a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 with a CH2CH3 region, or IgG4 without a CH2CH3 region;

the transmembrane domain is a transmembrane domain sequence from the α, β or ζ chain, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1 or CTLA of a T cell receptor;

the intracellular domain consists of DAP10CD, CD137CD, and CD3 ζ SD.

2. A nucleic acid construct for CAR cell therapy according to claim 1, wherein: the signal peptide is CD8 SP, and the nucleic acid sequence and the polypeptide sequence of the CD8 SP are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence list.

3. A nucleic acid construct for CAR cell therapy according to claim 1, wherein: the antigen binding region is Her-2scFv, and the nucleic acid sequence and the polypeptide sequence of the Her-2scFv are respectively shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence table.

4. A nucleic acid construct for CAR cell therapy according to claim 1, wherein: the extracellular hinge region is CD8H, CD28H or IgG4H-CH2-CH3, the nucleic acid sequence and the polypeptide sequence of the CD8H are respectively shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28H are respectively shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the IgG4H-CH2-CH3 are respectively shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence table.

5. A nucleic acid construct for CAR cell therapy according to claim 1, wherein: the transmembrane domain is CD8TMD, CD28TMD or NKG2D TMD, the nucleic acid sequence and the polypeptide sequence of the CD8TMD are respectively shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD28TMD are respectively shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the NKG2D TMD are respectively shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence table.

6. A nucleic acid construct for CAR cell therapy according to claim 1, wherein: the nucleic acid sequence and the polypeptide sequence of the DAP10CD are respectively shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence table, the nucleic acid sequence and the polypeptide sequence of the CD137CD are respectively shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence table, and the nucleic acid sequence and the polypeptide sequence of the CD3zeta SD are respectively shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence table.

7. A nucleic acid construct for CAR cell therapy according to claim 6, wherein: the sequence of the intracellular signal domain is DAP10 CD137CD 3zeta SD, and the nucleic acid sequence and the polypeptide sequence of the DAP10 CD137CD CD3zeta SD are respectively shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence table.

8. A lentiviral vector, wherein: the lentiviral vector comprises a gene of the nucleic acid construct for CAR cell therapy of any of claims 1 to 7.

9. A cell preparation, characterized by: the cell preparation is an immune effector cell introduced with the lentiviral vector of claim 8, wherein the immune effector cell is a T cell, an NK cell or a macrophage, and the immune effector cell is derived from an autologous, allogeneic, stem cell differentiation or a specific cell line.

10. A cell preparation according to claim 9 for use in the treatment of cancer.

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