A nucleic acid construct for CAR cell therapy, its lentiviral vector, cell preparation and application

Abstract

The present invention discloses a nucleic acid construct for CAR cell therapy, its lentiviral vector, cell preparation and application. Among them, a nucleic acid construct for CAR cell therapy includes a signal peptide, an antigen-binding region, an extracellular hinge region, a transmembrane domain and an intracellular signaling domain connected in series in sequence, and the intracellular signaling domain is composed of DAP10CD, CD137CD and CD3ζSD. A lentiviral vector contains the gene of the nucleic acid construct for CAR cell therapy. A cell preparation is an immune effector cell into which the aforementioned lentiviral vector is introduced. An application of the cell preparation in cancer treatment. The cell preparation prepared by the present invention has higher activity compared with immune cell preparations constructed with other CAR structures. In particular, NK cells modified with this type of CAR structure have extremely high killing ability against tumor cells expressing the target target (such as breast cancer cells expressing Her-2).

Description

Technical Field

[0001] The present invention relates to the technical field of cellular immunotherapy, in particular to the technical field of nucleic acid constructs for CAR cell therapy, their lentiviral vectors, cell preparations and applications.

Background Art

[0002] Malignant tumors are one of the major diseases endangering human health. Traditional tumor treatment methods, such as surgery, radiotherapy and chemotherapy, have been the main strategies for tumor treatment in recent decades. However, patients are prone to develop resistance to drug and radiotherapy treatments, resulting in a high frequency of tumor recurrence. In recent years, tumor cell therapy has received great attention due to its advantages such as targeting, remarkable effects and almost no side effects, and has gradually become an important means in the comprehensive treatment of tumors. It is known as the green therapy for tumors in the industry and is also the focus and development direction of current basic research and clinical application of tumor treatment.

[0003] The chimeric antigen receptor (CAR) therapy for tumors refers to a new type of precision targeted therapy for treating tumors with immune cells modified with chimeric antigen receptors. In recent years, good results have been achieved in clinical tumor treatment through optimization and improvement. It has the characteristics of precision, rapidity and high efficiency, and has the potential to cure cancer and is very promising. Under normal circumstances, the chimeric antigen receptor cell therapy for tumors uses genetic engineering technology to transfer the chimeric antigen receptor, which is a positioning and navigation device for identifying tumors, into immune cells (such as T cells and NK cells), and correspondingly recombines them into CAR-T cells and CAR-NK cells. CAR-T cells and CAR-NK cells can use the CAR structure they transduce to specifically recognize tumor cells in the body and then be activated. Through the immune effect, a large number of various effector factors are released, thereby efficiently killing tumor cells and achieving the purpose of treating malignant tumors.

[0004] Chimeric antigen receptors are usually composed of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain that contains or does not contain a co-stimulatory domain. With the continuous advancement of scientific and technological research, CARs have now developed to the fourth generation. Among them, the first-generation CARs contain an extracellular single-chain variable fragment scFv, a transmembrane region, and a single intracellular activation signal CD3ζ or FcεRIγ. The first-generation CAR-T cells can only cause transient T cell proliferation and less cytokine secretion, and their anti-tumor activity in vivo is greatly limited, and the reduction of T cell proliferation will eventually lead to T cell apoptosis. The second-generation CARs introduced a co-stimulatory molecule on the basis of the first-generation CARs, which improved the tumor killing effect. In the third-generation CARs, multiple co-stimulatory factors are carried, such as CD28, CD134 (OX40) and CD137 (4-1BB). Costimulatory molecules can activate signaling pathways such as JNK, ERK, and NF-κB in T cells, significantly improving T cells' anti-tumor activity, proliferation activity, survival cycle, and prolonged secretion of cytokines (such as IL-2, TNF-α, and IFN-γ). The fourth-generation CARs add selectable markers and promoters encoding CARs amplification and suicide on the basis of the third-generation CARs.

[0005] Studies have shown that CAR design and intrinsic properties also affect the expansion and persistence of CAR-modified immune cells. Currently, second-generation CARs containing a CD3-ζ signaling domain and a co-stimulatory domain (usually CD28 or 4-1BB) are still the most common nucleic acid constructs in clinical use. In addition, studies have found that when CD28 is used as a co-stimulatory molecule, T cells have a strong killing effect but a short duration, while when 4-1BB is used as a co-stimulatory molecule, T cells have a long duration but limited killing ability.

[0006] Therefore, the existing chimeric antigen receptor immune cell therapy still has many unsatisfactory aspects in the treatment of tumors. For example, a variety of adverse reactions may occur during CAR-T cell therapy, including cytokine release syndrome. Moreover, the clinical manifestations of these toxic and side effects are often complex, varied, and progress rapidly; and each CAR-modified cell product has unique and different effect characteristics due to different production processes or CAR structures, which need to be further fully understood and accurately evaluated. [Summary of the invention]

[0007] The object of the present invention is to solve the problems in the prior art, and to provide a nucleic acid construct for CAR cell therapy containing a specific combination of intracellular signal structures, its lentiviral vector, cell preparation and application. The prepared cell preparation has higher activity compared with immune cell preparations constructed with other CAR structures. In particular, NK cells modified with such CAR structures have extremely high killing ability against tumor cells expressing the target (such as breast cancer cells expressing Her-2).

[0008] To achieve the above object, the present invention is realized through the following technical solutions:

[0009] A nucleic acid construct for CAR cell therapy, comprising a signal peptide, an antigen-binding region, an extracellular hinge region, a transmembrane domain and an intracellular signal domain connected in series in sequence;

[0010] The signal peptide is Human IgKVIII, Human IL-2, Human insulin, etc.;

[0011] The antigen-binding region is anti-CD19 scFv, anti-Her-2scFv, anti-ROR1 scFv, anti-BCMA scFv, etc.;

[0012] The extracellular hinge region is a hinge region sequence from CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 containing the CH2CH3 region or IgG4 without the CH2CH3 region, etc.;

[0013] The transmembrane domain is a transmembrane domain sequence from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1 or CTLA, etc.;

[0014] The intracellular signal domain is composed of DAP10 CD, CD137 CD and CD3ζSD.

[0015] Preferably, the signal peptide is CD8 SP, and the nucleic acid sequence and polypeptide sequence of the CD8 SP are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing respectively.

[0016] Preferably, the antigen-binding region is Her-2scFv, and the nucleic acid sequence and polypeptide sequence of the Her-2scFv are shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing respectively.

[0017] Preferably, the extracellular hinge region is CD8 H, CD28 H or IgG4 H-CH2-CH3. The nucleic acid sequence and polypeptide sequence of CD8 H are shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD28 H are shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of IgG4 H-CH2-CH3 are shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing respectively.

[0018] Preferably, the transmembrane domain is CD8 TMD, CD28 TMD or NKG2D TMD. The nucleic acid sequence and polypeptide sequence of CD8 TMD are shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD28 TMD are shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of NKG2D TMD are shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing respectively.

[0019] Preferably, the nucleic acid sequence and polypeptide sequence of DAP10 CD are shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD137 CD are shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD3ζ SD are shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence listing respectively.

[0020] Preferably, the order of the intracellular signaling domain is DAP10 CD-CD137 CD-CD3ζ SD. The nucleic acid sequence and polypeptide sequence of DAP10 CD-CD137 CD-CD3ζ SD are shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence listing respectively.

[0021] A lentiviral vector, which contains the gene of any one of the nucleic acid constructs for CAR cell therapy as described above.

[0022] A cell preparation, which is an immune effector cell transfected with the above lentiviral vector. The immune effector cell is a T cell, NK cell or macrophage, and the source of the immune effector cell is autologous, allogeneic, stem cell differentiation or a specific cell line.

[0023] Preferably, the NK cells.

[0024] Use of a cell preparation in cancer treatment.

[0025] Advantages of the present invention:

[0026] The present invention constructs a nucleic acid construct composed of a signal peptide, an antigen-binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signal domain in series, and defines that the intracellular signal domain is composed of DAP10 CD, CD137 CD, and CD3ζSD, and then prepares a corresponding cell preparation through lentiviral vector technology. The cell preparation prepared by the present invention has higher activity compared with immune cell preparations constructed with other CAR structures. In particular, NK cells modified with such CAR structures have extremely high killing ability against tumor cells expressing the target target (such as breast cancer cells expressing Her-2).

[0027] The features and advantages of the present invention will be described in detail through examples in combination with the accompanying drawings.

Description of the Drawings

[0028] Figure 1 It is a combined CAR sequence diagram of Examples 1 to 3;

[0029] Figure 2 It is the nucleotide electrophoresis detection result of T-CAR / NK-CAR1 / NK-CAR2 sequences digested with BamHI and XbaI on the pUC57 vector;

[0030] Figure 3 It is the vector map of pCDH-SFFV-Luc-T2A-EGFP;

[0031] Figures 4a to 4l It is a flow cytometry diagram for determining the titer of lentivirus containing CARs with different intracellular structures;

[0032] Figure 5 It is a flow cytometry diagram after purification and amplification of NK cells;

[0033] Figures 6a to 6d It is the flow cytometry analysis result for identifying the transfection efficiency of NK cells transfected with lentivirus of different Her-2-targeted CARs;

[0034] Figures 7a to 7b It is the killing of breast cancer cells in vitro by Her-2 CAR-NK cells with different intracellular structures;

[0035] Figures 8a to 8b It is the secretion levels of IFN-γ (left) and TNF-α (right) of different groups of CAR-NK cells before and after co-culture with different breast cancer cells;

[0036] Figure 9 It is the vector map of NK-CAR2.

[0037] Figure 2 Among them: Lane 1 is the nucleic acid molecular weight standard; Lanes 1-3 correspond to the T-CAR, NK-CAR1, and NK-CAR2 sequence parts respectively; Lane 4 is the pCDH-SFFV-Luc-T2A-EGFP vector part after digestion.

Specific implementation manners

[0038] Examples 1 to 3, design and acquisition of HER-2CAR targeting:

[0039] A nucleic acid construct for CAR cell therapy, characterized in that it comprises a signal peptide, an antigen-binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain connected in series in sequence;

[0040] The signal peptide (Signal peptide, SP) is a short peptide chain (with a length of 5-30 amino acids) responsible for guiding newly synthesized proteins into subcellular organelles with different membrane structures in the cell, and is generally located at the N-terminus of the protein.

[0041] The signal peptide is a signal peptide commonly used for recombinant proteins, such as Human IgKVIII, Human IL-2, or Human insulin, etc. Further, the signal peptide is CD8 SP, and the nucleic acid sequence and polypeptide sequence of the CD8 SP are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing respectively.

[0042] The antigen-binding region can tightly bind to tumor-associated antigens expressed on the surface of tumor cells, determines the targeting of the CAR structure, and is the core structure determining the effect of modified immune cells. The antigen-binding region is a single-chain antibody (single chain fragment variable, scFv) formed by connecting the light chain (VL) and heavy chain (VH) of a monoclonal antibody targeting a specific target antigen and a flexible hinge (linker) located therebetween. The heavy chain or light chain of the single-chain antibody is respectively connected to the signal peptide and the hinge region.

[0043] Among them, the target antigens may include α-folate receptor (ovarian cancer and epithelial cancer), CAIX (renal 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 (various malignancies), EGP-40 (colorectal cancer), EphA2 (glioblastoma), Erb-B2 (breast cancer, prostate cancer, and colon cancer), FBP (ovarian cancer), GD2 (neuroblastoma and melanoma), GD3 (melanoma), HER2 (pancreatic cancer, ovarian cancer, glioblastoma, and osteosarcoma), HMff_MAA (melanoma), IL_13Ra2 (glioma and glioblastoma), KDR (tumor vasculature), κ light chain (B-cell malignancies), L1 (neuroblastoma), MAGE-A1 (melanoma), mesothelin (mesothelioma), MUC1 (breast cancer and ovarian cancer), MUC16 (ovarian cancer), NY-ESO-1 (multiple myeloma), carcinoembryonic antigen (various tumors), PSCA (prostate cancer), PSMA (prostate cancer), ROR1 (B-CLL), TAG-72 (adenocarcinoma), and VEGF-R2 (tumor neovascularization), etc.

[0044] The antigen-binding region is anti-CD19 scFv, anti-Her-2 scFv, anti-ROR1 scFv, or anti-BCMA scFv, etc. Further, the antigen-binding region is Her-2 scFv, and the nucleic acid sequence and polypeptide sequence of the Her-2 scFv are shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing, respectively.

[0045] The 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 position and exposure degree of the target cell antigen epitope. In addition, multiple studies have shown that the activation of CAR-T cells is related to the length of the hinge region. Therefore, adjusting the length of the hinge region can place the CAR-T cells and the target cells at the optimal distance, avoiding the weakening of the CAR signal by large phosphatases during the antigen-antibody binding process. However, in some cases, the antigen epitope may be relatively inaccessible, and a longer hinge region is required to enable the scFv to overcome steric hindrance and effectively bind the antigen. In summary, due to different antigen epitopes, the optimal length of the hinge region is also different, and when targeting neoantigens, the length of the hinge region needs to be adjusted accordingly.

[0046] The extracellular hinge region is the hinge region sequence derived from CD8, CD28, CTLA4, PD-1, NKG2D, IgG1, IgG4 containing CH2CH3 region or IgG4 without CH2CH3 region, etc. Further, the extracellular hinge region is CD8 H, CD28 H or IgG4 H-CH2-CH3. The nucleic acid sequence and polypeptide sequence of CD8 H are shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD28 H are shown as SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of IgG4 H-CH2-CH3 are shown as SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing respectively.

[0047] The transmembrane domain (TMD) can connect the extracellular domain of CAR with the intracellular signal transduction domain and anchor the receptor to the immune cell membrane.

[0048] The transmembrane domain is the transmembrane domain sequence derived from the α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10, DAP12, NKG2A, NKG2D, PD-1 or CTLA, etc. Among them, the CD3ζ transmembrane domain can enable CAR to form a homodimer or a heterodimer with the endogenous TCR, enhancing the activity of CAR-T cells, while the transmembrane domains of CD8α and CD28 can promote the expression of CAR on the cell surface. Further, the transmembrane domain is CD8TMD, CD28 TMD or NKG2D TMD. The nucleic acid sequence and polypeptide sequence of CD8TMD are shown as SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of CD28 TMD are shown as SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing respectively. The nucleic acid sequence and polypeptide sequence of NKG2D TMD are shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing respectively.

[0049] The costimulatory domain (CD) can achieve dual activation of costimulatory molecules and intracellular signals, enabling T cells to continuously proliferate and release cytokines, and enhancing the anti-tumor ability of T cells.

[0050] The intracellular signaling domain is composed of DAP10 CD, CD137 CD, and CD3ζ SD. Further, the nucleic acid sequence and polypeptide sequence of DAP10 CD are shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence listing, respectively; the nucleic acid sequence and polypeptide sequence of CD137 CD are shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence listing, respectively; the nucleic acid sequence and polypeptide sequence of CD3ζ SD are shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence listing, respectively. The order of the intracellular signaling domain is DAP10 CD - CD137 CD - CD3ζ SD, and the nucleic acid sequence and polypeptide sequence of DAP10 CD - CD137 CD - CD3ζ SD are shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence listing, respectively.

[0051] Among them, the nucleic acid sequences and polypeptide sequences involved are shown in Table 1 below:

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Table 1 Sequence Listing

[0065] Sequentially combine and concatenate the above-mentioned various different signal peptides, antigen-binding regions, extracellular hinge regions, transmembrane domains, and intracellular signaling domains to obtain as Figure 1The CAR sequence combinations of Examples 1 to 3 shown, with the specific sequences being SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33 respectively.

[0066] Example 4. Construction of lentiviral vectors:

[0067] Synthesize the CAR sequences of Examples 1 to 3, and clone the synthesized CAR sequences into the pUC57 vector, named pUC57-T-CAR, pUC57-NK-CAR1, and pUC57-NK-CAR2 respectively. Extract the plasmids of pUC57-T-CAR, pUC57-NK-CAR1, and pUC57-NK-CAR2, and perform enzymatic digestion with QuickCut restriction enzymes BamHI and XbaI (Takara). Among them, the enzymatic digestion system is as follows:

[0068] 1 μg of pUC57-T-CAR / pUC57-NK-CAR1 / pUC57-NK-CAR2, 1 μl each of BamHI and XbaI, 2 μl of 10X QuickCut Green Buffer, make up to 20 μl with water, water bath at 37 °C for 15 min. After the enzymatic digestion products are separated by agarose gel electrophoresis, use an agarose gel DNA fragment recovery kit (purchased from Takara) for DNA fragment recovery (the results are shown in Figure 2 , it can be seen from the electrophoresis diagram that lanes 1 to 3 are the bands of T-CAR / NK-CAR1 / NK-CAR2 released after the pUC57-T-CAR / pUC57-NK-CAR1 / pUC57-NK-CAR2 vector is digested by BamHI and XbaI). Then, use the same method to digest the pCDH-SFFV-Luc-T2A-EGFP vector ( Figure 3 ) with BamHI and XbaI, separate and recover the digested vector fragment by agarose gel electrophoresis (the results are shown in Figure 2 , it can be seen from the electrophoresis diagram that lane 4 is the part of the pCDH-SFFV-Luc-T2A-EGFP vector after enzymatic digestion).

[0069] Connect the recovered T-CAR / NK-CAR1 / NK-CAR2 fragments with the digested vector through T4 ligase (purchased from Takara). Among them, the reaction system and conditions are as follows:

[0070] The T-CAR / NK-CAR1 / NK-CAR2 and pCDH-SFFV-Luc-T2A-EGFP vectors digested with BamHI and XbaI were quantified respectively, and then added to the system according to the molar ratio of vector to target band 1:3. 1 μl of 10X T4 DNA Ligase Buffer and 1 μl of ligase were added, and the volume was made up to 10 μl with water. The ligation was carried out at 16 °C for 4 h or overnight. The ligation product was transformed into competent E. coli DH5a bacteria. After overnight culture at 37 °C, single colonies were picked, expanded, and the plasmids of positive clones were extracted using a plasmid extraction kit (purchased from Axygene) according to the kit operation instructions. After digestion and sequencing detection, the correct vectors were named pCDH-T-CAR / pCDH-NK-CAR1 / pCDH-NK-CAR2.

[0071] Example 5. Lentivirus packaging and titer determination:

[0072] 1) Plasmids:

[0073] · pCDH-T-CAR / pCDH-NK-CAR1 / pCDH-NK-CAR2;

[0074] · VSV-G: pMD2.G (Addgene#12259);

[0075] · Rev: pRSV-Rev (Addgene#12253);

[0076] · Gag / Pol: pMDLg / pRRE (Addgene#12251).

[0077] 2) Cell line:

[0078] · Low passage number 293T cells.

[0079] 3) Reagents:

[0080] · 293T medium: high glucose DMEM (containing sodium pyruvate and glutamine) + 10% FBS + GlutaMAX;

[0081] · Virus harvest medium: Take 50 ml of 293T medium, add 0.5 g of BSA (Sigma A9418) and HEPES with a final concentration of 10 - 15 mM, and filter through a 0.22 μm filter;

[0082] · Opti-MEM medium: used for mixing transfection complexes;

[0083] · Transfection reagent: X-tremeGENE HP DNA (or other low-toxicity and high-efficiency reagents).

[0084] 4) Packaging process:

[0085] · On the first day: Seed 293T cells according to Table 2 below;

[0086] · On the second day - morning: Prepare a mixture of Opti-MEM medium, plasmid, and transfection reagent according to Table 2 below. Replace the fresh medium of 293T cells. After the mixture stands at room temperature for 15 - 30 min, add it dropwise to the 293T cell culture system and gently shake well;

[0087]

[0088]

[0089] Table 2 Opti-MEM medium

[0090] · On the second day - afternoon: Gently change the medium to virus harvest medium 6 - 8 h after transfection;

[0091] · On the fourth day: 48 h after transfection, take the cell culture supernatant, centrifuge at 400×g for 4 min, and filter with a 0.45 μm filter; Add the 5×PEG8000 solution by volume, mix once every 20 - 30 min, for a total of 3 - 5 times, place at 4°C for 6 h or overnight; Centrifuge at 4°C, 4000g for 20 min, discard the supernatant, let the tube stand for 1 - 2 min, and aspirate the residual liquid; Add an appropriate amount of virus lysate to dissolve the virus precipitate, divide the virus solution into tubes, and store in a -80°C refrigerator. Take one tube after overnight to further measure the virus titer; Seed 24-well plates with HT-1080, 5 wells, 42000 cells per well. After the cells adhere, perform virus infection; Prepare 10 ml of 8 μg / ml polybrene MEM medium; Prepare 15 μl of virus stock solution + 135 μl of medium; Then perform serial dilutions, take 15 μl + 135 μl, for 4 times; Subsequently, take 50 μl from each group and add it to the wells with 450 μl of medium. The dilution levels are 10 2 、10 3 、10 4 and 10 5 ; 72 h later, digest the cells and perform flow cytometry analysis of GFP%. The results are shown in Figure 4. Calculate according to the following formula to obtain the titer of the packaged lentivirus as:

[0092] TU T-CAR =(8.01×0.01×42000 / 0.5)×10000 = 6.73E7 TU / ml

[0093] TU NK-CAR1 =(2.95×0.01×42000 / 0.5)×100000 = 2.45E8 TU / ml

[0094] TU NK-CAR2 =(8.94×0.01×42000 / 0.5)×10000 = 7.51E7 TU / ml.

[0095] Wherein, titer (TU / mL) = (F × C / V) × D;

[0096] F = GFP positive cell frequency (GFP positive cell percentage / 100);

[0097] C = number of cells per well at transduction (42,000 cells);

[0098] V = transduction volume (mL) (0.5 mL);

[0099] D = lentivirus dilution factor.

[0100] Example 6. Preparation of CAR-NK cells:

[0101] 1) Purification and amplification of NK cells:

[0102] Take 20 ml of fresh anticoagulated blood from healthy volunteers, and isolate peripheral blood mononuclear cells (PBMC) using lymphocyte separation medium (purchased from GE Healthcare); after counting the isolated cells, stimulate and culture them in a 6-well plate coated with CD16 at a density of 2.5X10 6 / well for 72 h, and then transfer them to a common 6-well plate for continued culture and amplification for 72 h; purify the cells using NK magnetic beads (purchased from Miltenyi Biotec), and continue to induce culture with RPMI 1640 medium (purchased from Thermo Scientific) containing 10% FBS + 200 IU / ml IL-2 to obtain purified NK cells, and detect the phenotypic ratios of CD3 and CD56 by flow cytometry. The detection results are as Figure 5 , where the horizontal axis represents CD3, the vertical axis represents CD56, and the cell population represented by CD3 negative and CD56 positive is NK cells, indicating that the proportion of NK cells prepared by this method is greater than 90%;

[0103] 2) Preparation of Her-2-targeted CAR-NK cells:

[0104] After obtaining the purified NK cells, at about 2.5X10 per well 6NK cells were seeded into 24-well plates (BD Biosciences) and mixed with an appropriate amount of virus supernatant in the presence of Protamine sulfate at a final concentration of 8 μg / ml (Sigma-Aldrich) and BX795 at 1.5 μM (Sigma-Aldrich), with the final volume not exceeding 1 ml; cytokines were supplemented, and the plates were centrifuged at 1000·g for 1 h at room temperature; after centrifugation, without removing the virus supernatant, the plates were incubated at 37 °C and 5% CO2 for 4 - 6 h; after incubation, a second centrifugation was performed at 1000·g for 1 h at room temperature, and then 1 ml of fresh NK cell growth medium was removed from the wells; the cells were maintained in medium supplemented with cytokines every day for 2 days to obtain Her-2 CAR-NK cells (hereinafter simply referred to as CAR-NK), and further transfection efficiency was identified; the transfected NK cells were incubated with recombinant human Her-2-Fc protein for 15 min at 4 °C, washed, and then incubated with PE-labeled mouse anti-human anti-IgG1 antibody for 15 min at 4 °C, and further flow cytometry was performed; the results are shown in Figure 6, where the abscissa represents the expression of scFv.

[0105] Example 7. Killing of tumor cells by CAR-NK cells:

[0106] 1) Detection of the killing ability of CAR-NK cells against tumor cells:

[0107] Her-2 positive breast cancer cells BT474 and Her-2 negative breast cancer cells MDA-MB-468 were adjusted to 1X10 6 / ml with medium, labeled with Calcein-AM at a final concentration of 5 μg / ml, incubated at 37 °C for 1 h, washed three times with PBS, and then resuspended with phenol red-free 1640 complete medium, and counted; 10,000 tumor cells per well were adjusted and added to 96-well round bottom plates; Mock NK cells transfected with empty vectors and corresponding different groups of CAR-NK cells were added at E:T ratios of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.03125:1 at 1X10 5 、5X10 4 、2.5X10 4 、1.25X10 4 、0.625X10 4 and 0.03125X10 4; Add another group of tumor cells with 2% Triton X-100 and an untreated group. Centrifuge at 100 g for 5 min, co-culture at 37 °C for 3 h, then centrifuge at 300 g for 5 min. Aspirate 100 μl from each well and transfer it to a 96-well flat bottom plate to detect the OD value; the detection results are shown in Figure 7, indicating that the CAR-NK cells containing the intracellular signaling domain DAP10-CD-CD137-CD-CD3ζSD of NK-CAR2 have significantly higher killing ability against Her-2 positive breast cancer cells than NK cells in other groups, while there is no obvious difference in killing Her-2 negative breast cancer cells MDA-MB-468;

[0108] 2) Detect the secretion levels of IFN-γ and TNF-α after co-culture of CAR-NK cells with different cells

[0109] Co-culture Her-2 positive breast cancer cells BT474 and Her-2 negative breast cancer cells MDA-MB-468 with Mock NK cells transfected with empty vector and different groups of CAR-NK cells at an E:T ratio of 2.5:1 for 12 h, then take the supernatant for ELISA to detect the concentrations of IFN-γ and TNF-α in the supernatant; the detection results are shown in Figure 8, and the secretion levels of IFN-γ and TNF-α after co-culture of CAR-NK cells containing the intracellular signaling domain DAP10-CD137-CD3zeta of NK-CAR2 with Her-2 positive breast cancer cells BT474 are significantly higher than those in other groups.

[0110] The above embodiments are illustrative of the present invention, not limiting of the present invention. Any scheme obtained by simply transforming the present invention belongs to the protection scope of the present invention. Sequence Listing <110> Hangzhou Meizhong Disease Gene Research Institute Co., Ltd. <120> A nucleic acid construct for CAR cell therapy, its lentiviral vector, cell preparation and application <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 (IgG4 H-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 (CD 28CD) <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 (CD28CD) <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 Methionine Isoleucine Glutamine Serine Glutamine Serine Serine Alanine Proline Threonine Serine Glutamine Glutamic Acid Proline Alanine Tyrosine 50 55 60 Threonine Leucine Tyrosine Serine Leucine Isoleucine Glutamine Proline Serine Arginine Lysine Serine Glycine Serine Arginine Lysine 65 70 75 80 Arginine Asparagine Histidine Serine Proline Serine Phenylalanine Asparagine Serine Threonine Isoleucine Tyrosine Glutamic Acid Valine Isoleucine Glycine 85 90 95 Lysine Serine Glutamine Proline Lysine Alanine Glutamine Asparagine Proline Alanine Arginine Leucine Serine Arginine Lysine Glutamic Acid 100 105 110 Leucine Glutamic Acid Asparagine Phenylalanine Aspartic Acid Valine Tyrosine Serine 115 120 <210> 27 <211> 339 <212> DNA <213> Nucleic acid sequence (CD3ζSD) <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 (CD3ζSD) <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-CD3ζ) <400> 29 ctgtgcgcac gcccacgccg cagccccgcc caagatggca aagtctacat caacatgcca 60 Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr Ile Asn Met Pro ggcaggggca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 120 Gly Arg Gly Asn Gly Gln Lys Lys Thr Leu Tyr Tyr Phe Lys Thr Thr Phe Tyr Glu ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 180 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Ala Asp Phe Gln Glu Glu Glu ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 240 Gly Arg Asp Val Asn Glu Ser Glu Val Gln Ala Glu Ala Gln Thr Ala Pro Arg Tyr Gln Gln ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 300 Gly Pro Gln Thr Gln Leu Tyr Asn Glu Leu Asn Leu Arg Asp Glu Glu Glu Tyr Asp Val Leu gacaagagac gtggccggga ccctgagatg gggggaaagc cgcagagaag gaagaaccct 360 Asp Lys Arg Thr Trp Pro Gly Thr Leu Glu Met Gly Gly Lys Ala Gln Arg Lys Glu Asn Pro caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 420 Gln Glu Gly Cys Tyr Asn Glu Leu Gln Lys Asp Lys Met Gly Gly Glu Pro Tyr Ser Glu Ile gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 480 Gly Asp Glu Lys Ala Glu Ala Arg Glu Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Ser Ser acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 534 Thr Ser Thr Lys Asp Thr Tyr Asp Ala Leu His Cys Gln Ala Leu Pro Pro Ser <210> 30 <211> 178 <212> PRT <213> Polypeptide sequence (DAP10 - CD137 - CD3ζ) <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 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 600 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 660 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 660 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 720 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 720 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 780 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 780 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 840 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 840 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa 900 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa 900 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag 960 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag 960 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 1020 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 1020 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 1080 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 1080 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac 1140 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac 1140 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct 1200 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct 1200 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct 1260 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

1. A nucleic acid construct for CAR cell therapy, characterized in that: It includes a signal peptide, an antigen-binding region, an extracellular hinge region, a transmembrane domain, and an intracellular signaling domain connected in series in sequence; The signal peptide is CD8 SP, and the nucleic acid sequence and polypeptide sequence of the CD8 SP are shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing respectively; The antigen-binding region is Her-2 scFv, and the nucleic acid sequence and polypeptide sequence of the Her-2 scFv are shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing respectively; The extracellular hinge region is CD8 H, and the nucleic acid sequence and polypeptide sequence of the CD8 H are shown as SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing respectively; The transmembrane domain is NKG2D TMD, and the nucleic acid sequence and polypeptide sequence of the NKG2D TMD are shown as SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing respectively; The intracellular signaling domain is composed of DAP10 CD, CD137 CD, and CD3ζ SD; The nucleic acid sequence and polypeptide sequence of the DAP10 CD are shown as SEQ ID NO.17 and SEQ ID NO.18 in the sequence listing respectively, the nucleic acid sequence and polypeptide sequence of the CD137 CD are shown as SEQ ID NO.19 and SEQ ID NO.20 in the sequence listing respectively, and the nucleic acid sequence and polypeptide sequence of the CD3ζ SD are shown as SEQ ID NO.27 and SEQ ID NO.28 in the sequence listing respectively; The order of the intracellular signaling domain is DAP10 CD-CD137 CD-CD3ζ SD, and the nucleic acid sequence and polypeptide sequence of the DAP10 CD-CD137 CD-CD3ζ SD are shown as SEQ ID NO.29 and SEQ ID NO.30 in the sequence listing respectively.

2. A lentiviral vector, characterized in that: The lentiviral vector contains the gene of the nucleic acid construct for CAR cell therapy as described in claim 1.

3. A cell preparation, characterized in that: The cell preparation is an immune effector cell into which the lentiviral vector as described in claim 2 is introduced. The immune effector cell is an NK cell, and the source of the immune effector cell is autologous, allogeneic, stem cell differentiation, or a specific cell line.

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