Hypoxia response type synNotch-ODD CAR with dual-target recognition and application of hypoxia response type synNotch-ODD CAR

Abstract

The invention relates to a hypoxia response type synNotch-ODD CAR with dual-target recognition and application thereof, and belongs to the technical field of cell therapy. The invention provides an anti-tumor expression vector. The anti-tumor expression vector comprises nucleic acid sequences of a CD133-synNotchCAR (chimeric antigen receptor) and an EGFR (epidermal growth factor receptor)-hypoxia response element CAR (chimeric antigen receptor). The invention further provides a synNotch-ODD CAR immune cell, and the synNotch-ODD CAR immune cell comprises the expression vector. The polypeptide can be activated and plays a role in killing tumor cells only under the conditions of hypoxia and simultaneous expression of EGFR and CD133 antigens by target cells, and the on-target / off-tube risk caused by activation of metastatic cancer cells or circulating tumor cells of traditional synNotch CAR can be effectively solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and specifically relates to a dual-targeted recognition hypoxia-responsive synNotch-ODD CAR and an application thereof. Background Art

[0002] Chimeric Antigen Receptor-T cell (CAR-T) therapy is a new method of tumor immunotherapy that has developed fastest in recent years. The core of CAR-T technology is to artificially express a chimeric receptor targeting a specific antigen on T cells through genetic engineering technology. The chimeric receptor is composed of a fusion of a specific antigen recognition domain on the cell surface (such as a single-chain fragment antibody scFv) and an intracellular T cell activation signal domain, which gives T cells the ability to specifically recognize and kill tumor cells. Clinical studies have confirmed that CAR-T cells can proliferate, survive for a long time and form immune memory in vivo, thereby obtaining a specific long-term anti-tumor mechanism. CAR-T therapy has a significant therapeutic effect in the treatment of acute leukemia and non-Hodgkin's lymphoma, and has shown good targeting, lethality and persistence in vitro and in clinical trials. At present, a number of CAR-T products have been approved for marketing, which has completely changed the treatment pattern of hematological tumors. Despite this, CAR-T faces huge challenges in the treatment of solid tumors and has developed slowly. Solid tumors account for 90% of all tumors, and there is a greater clinical demand for CAR-T products. Unlike blood tumors, solid tumors lack known tumor-specific antigens. Currently, tumor-associated antigens are usually used in solid tumors, that is, antigens that are highly expressed on tumor cells and also lowly expressed in some normal tissue cells. CAR cells targeting a single antigen may attack normal tissues expressing the antigen at the same time after reinfusion, resulting in an "on-target / off-tumor" effect. As a living cell drug, the potential safety issues of CAR-T technology cannot be ignored. A typical case is that Morgan et al. reported that a patient with colorectal cancer metastasis was treated with third-generation CAR-T cells designed for human epidermal growth factor receptor II (Her2). The patient died 4 days after intravenous injection of CAR-T cells; an autopsy found that the death was caused by low-level expression of Her2 on the surface of lung epithelial cells, and the reinfused CAR-T cells attacked the patient's lungs, resulting in pulmonary toxicity. In addition, this potential safety risk also makes it impossible to improve the efficacy by increasing the dose, limiting the application of CAR cells in the treatment of solid tumors. Therefore, there is an urgent need to develop more efficient and safer CAR cell products or technologies in clinical practice.

[0003] SynNotch CAR is an effective strategy to solve the problem of target specificity. Unlike traditional dual-target AND-gate CAR, SynNotch CAR does not activate immune cells after recognizing the first antigen, but instead releases transcription factors to mediate the expression of the second CAR, which then recognizes and kills target cells. Therefore, synNotch CAR-T cells have almost no responsiveness to cells that express only one target antigen, but have killing activity comparable to that of traditional single-target CAR to target cells that express two antigens. Compared with dual-target CAR, synNotch CAR has advantages over traditional dual-target AND-gate CAR in terms of recognition specificity and activity. However, the prior art Co-opting signalling molecules enables logic-gated control of CAR T cells and Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting discloses that synNotch CAR-T also has a fatal flaw, that is, when treating patients with a large number of metastatic cancer cells or circulating tumor cells, the expression of the second CAR will be activated in the peripheral circulation and spread through the blood circulation, producing on-target / off-tumor toxicity against normal tissue cells expressing the second antigen. Since most patients with solid tumors have metastases and there are a large number of circulating tumor cells in the peripheral circulation, this defect of synNotch CAR-T may endanger the patient's life. Therefore, the existing synNotch CAR needs to be optimized.

[0004] In view of this situation, the present invention provides a novel method for constructing synNotch CAR targeting CD133 and EGFR and its application in the treatment of solid tumors. Among them, the EGFR CAR molecule is stably expressed only under hypoxia, and is rapidly degraded under the normoxic environment of the peripheral circulation, which can effectively avoid the "on-target / off-tumor" toxicity to peripheral tissues. Among them, the tumor stem cell marker targeted by synNotch CAR is CD133, and the second CAR of the induced target targets the expression of a relatively broad spectrum of EGFR. Due to the fusion expression of EGFR CAR and the hypoxia response element ODD, the synNotch CAR immune cells will be rapidly degraded even if they recognize CD133 on peripheral tumor cells under the normoxic state of the peripheral circulation, so that they cannot cause damage to peripheral normal tissue cells. When the synNotch CAR immune cells infiltrate into the hypoxic microenvironment of the solid tumor, the EGFR CAR can be stably expressed after being induced to express, and the immune cells are activated after recognizing the tumor cell EGFR to exert an anti-tumor effect. Summary of the invention

[0005] In view of the defects in the above-mentioned prior art, in order to solve the problem that synNotch CAR-T or CAR-NK products have serious "on-target / off-tumor" effects in patients with tumor metastasis and it is difficult to ensure safety, the purpose of the present invention is to design and provide a method for preparing a hypoxia-responsive synNotch-ODD CAR with dual-targeted recognition and its application in solid tumors. The present invention provides a design scheme of a hypoxia-responsive synNotch-ODD CAR that simultaneously targets EGFR and CD133, and prepares hypoxia-responsive synNotch-ODD CAR immune cells.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides an anti-tumor expression vector comprising nucleic acid sequences of CD133-synNotchCAR and EGFR-hypoxia response element CAR;

[0008] The structure of the CD133-synNotch CAR comprises a CD8α signal peptide, a tag, a first antigen binding domain, a Notch Core, Gal4 (yeast transcription activator protein) and VP64 (tetramer of herpes simplex virus protein 16, i.e., VP16) connected in sequence; after activation of the CD133-synNotch CAR in the present invention, synNotch will be cut to release the Gal4 / VP64 complex in the cell, and the Gal4 / VP64 complex will specifically start the expression of EGFR CAR.

[0009] The structure of the EGFR-hypoxia response element CAR comprises sequentially connected UAS, miniCMV, CD8α signal peptide, second antigen binding domain, tag, CD28 hinge region or CD8α hinge region, CD28 transmembrane region or CD8α transmembrane region, costimulatory domain, signal domain and hypoxia response element;

[0010] The first antigen binding domain is different from the second antigen binding domain;

[0011] Preferably, the first antigen binding domain and the second antigen binding domain are one of anti EGFR scFv or anti CD133 scFv.

[0012] Among them, miniCMV deletes the enhancer sequence of the conventional CMV promoter, so the efficiency of initiating transcription is extremely low, which ensures that EGFR CAR will basically not be expressed in the absence of Gal4 / VP64.

[0013] In the anti-tumor expression vector, the hypoxia response element is ODD, and the nucleic acid sequence is shown in SEQ ID NO.17.

[0014] In the anti-tumor expression vector, the nucleic acid sequence of the CD8α signal peptide is shown as SEQ ID NO.4;

[0015] The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.6;

[0016] The nucleic acid sequence of Notch Core is shown in SEQ ID NO.7;

[0017] The nucleic acid sequence of Gal4 is shown in SEQ ID NO.8;

[0018] The nucleic acid sequence of VP64 is shown in SEQ ID NO.9;

[0019] The nucleic acid sequence of the UAS is shown in SEQ ID NO.10;

[0020] The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.11;

[0021] The nucleic acid sequence of the CD28 hinge region is shown in SEQ ID NO.13;

[0022] The nucleic acid sequence of the CD28 transmembrane domain is shown in SEQ ID NO.14;

[0023] The nucleic acid sequence of the CD28 costimulatory domain is shown in SEQ ID NO.15;

[0024] The nucleic acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO.16.

[0025] In the anti-tumor expression vector, the tag is one of Flag, Myc, HA or PGK;

[0026] The costimulatory domain is one of CD28, 4-1BB, ICOS, CD27 or OX40 costimulatory domains;

[0027] The signal domain is the CD3ζ signal domain or the Fc receptor FcεRIγ.

[0028] The anti-tumor expression vector, the nucleic acid sequence of the CD133-synNotchCAR is shown as SEQ ID NO.1;

[0029] The nucleic acid sequence of the EGFR-hypoxia response element CAR is shown in SEQ ID NO.3.

[0030] In a second aspect, the present invention provides a recombinant virus, wherein the recombinant virus contains any one of the expression vectors described.

[0031] In a third aspect, the present invention provides the use of any one of the expression vectors or the recombinant virus in preparing synNotch-ODD CAR immune cells.

[0032] In a fourth aspect, the present invention provides a synNotch-ODD CAR immune cell, wherein the synNotch-ODDCAR immune cell comprises any one of the expression vectors or the recombinant virus;

[0033] Preferably, the immune cells are NK cells or T cells.

[0034] In a fifth aspect, the present invention provides the use of the synNotch-ODD CAR immune cells in the preparation of drugs for treating anti-tumor diseases.

[0035] In a sixth aspect, the present invention provides an anti-tumor drug, which contains the synNotch-ODD CAR immune cells.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The synNotch-ODD CAR of the present invention is composed of a synNotch CAR targeting CD133 and a hypoxia-responsive CAR molecule targeting EGFR, wherein the CD133 synNotch CAR comprises anti-CD133 scFv, Notch core and Gal4 / VP64; the EGFR CAR comprises anti-EGFR scFv, CD28 hinge / transmembrane region, CD28 co-stimulatory domain, CD3ζ signaling domain and hypoxia-responsive element ODD. Under normoxic conditions, even if CD133 CAR activates the expression of EGFR CAR, the latter will be rapidly degraded and cannot effectively activate immune cells; while under hypoxia, the stability of EGFR CAR protein is improved, and it can effectively kill EGFR-positive tumor cells. Since solid tumors are in a hypoxic state while normal tissues are in a normoxic state, the novel synNotch-ODD CAR-NK cells prepared based on the present invention can be activated and kill tumor cells only when they are hypoxic and the target cells express both EGFR and CD133 antigens. This can effectively solve the on-target / off-tumor risk of traditional synNotch CARs caused by activation of metastatic cancer cells or circulating tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the structure of the synNotch-ODD CAR system of the present invention;

[0039] Figure 2 This is a structural diagram of the synNotch-ODD CAR system of the present invention;

[0040] Figure 3 Schematic diagram of the CD133 overexpression plasmid structure;

[0041] Figure 4 The expression results of synNotch-ODD CAR on NK92MI cells;

[0042] Figure 5 Results of EGFR and CD133 expression in target cells;

[0043] Figure 6The killing of target cells by CAR-NK92MI under normoxic and hypoxic conditions, wherein A is the killing result of target cells by CAR-NK92MI under normoxic conditions, and B is the killing result of target cells by CAR-NK92MI under hypoxic conditions;

[0044] Figure 7 : is the expression of activation marker CD69 after CAR-NK92MI is co-cultured with target cells under normoxia and hypoxia, wherein A is the expression of activation marker CD69 after CAR-NK92MI is co-cultured with target cells under normoxia, B is the statistical graph of A, C is the expression of activation marker CD69 after CAR-NK92MI is co-cultured with target cells under hypoxia, and D is the statistical graph of C;

[0045] Figure 8 : is the expression of EGFR-28z CAR after CAR-NK92MI is co-cultured with target cells under normoxia and hypoxia, wherein A is the expression of EGFR-28z CAR after CAR-NK92MI is co-cultured with target cells under normoxia, B is the statistical graph of A, C is the expression of EGFR-28z CAR after CAR-NK92MI is co-cultured with target cells under hypoxia, and D is the statistical graph of C. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used are all commercially available products.

[0047] Embodiment 1:

[0048] 1. Plasmid structure design and construction

[0049] according to Figure 1The structure shown in the figure inserts the corresponding nucleic acid sequence into the lentiviral packaging backbone plasmid pHR to construct CAR expression plasmids, named CD133-synNotch (nucleic acid sequence as shown in SEQ ID NO.1), EGFR-28z (nucleic acid sequence as shown in SEQ ID NO.2) and EGFR-28z-ODD (nucleic acid sequence as shown in SEQ ID NO.3). Among them, the nucleic acid sequence of CD8α signal peptide (i.e., CD8αSP) is shown in SEQ ID NO.4; the nucleic acid sequence of Flag tag is shown in SEQ ID NO.5; the nucleic acid sequence of anti-CD133scFv is shown in SEQ ID NO.6; the nucleic acid sequence of Notch Core is shown in SEQ ID NO.7; the nucleic acid sequence of Gal4 is shown in SEQ ID NO.8; the nucleic acid sequence of VP64 is shown in SEQ ID NO.9; the nucleic acid sequence of UAS is shown in SEQ ID NO.10; the nucleic acid sequence of antiEGFR scFv is shown in SEQ ID NO.11; the nucleic acid sequence of Myc tag is shown in SEQ ID NO.12; CD28 H / T is split into CD28 Hinge and CD28 TM, and the nucleic acid sequence of CD28 Hinge is shown in SEQ ID NO.13; the nucleic acid sequence of CD28 TM (or CD28 transmembrane domain) is shown in SEQ ID NO.14; the nucleic acid sequence of CD28 (or CD28 co-stimulatory domain) is shown in SEQ ID NO. NO.15; the nucleic acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO.16; the nucleic acid sequence of ODD is shown in SEQ ID NO.17; and the nucleic acid sequence of BFP is shown in SEQ ID NO.18.

[0050] The logic-gated CAR composed of CD133-synNotch and EGFR-28z is named synNotch CAR, and the logic-gated CAR composed of CD133-synNotch and EGFR-28z-ODD is named synNotch-ODD CAR. Figure 2 Shown is a structural schematic diagram of synNotch CAR and synNotch-ODD CAR, in which TF (Transcription Factor) is a complex composed of Gal4 / VP64. Figure 2The structural model diagram of the CAR molecule expressed on the cell membrane of the present invention is shown. The antigen recognition domain anti-CD133 scFv is located outside the cell and is connected to the Notch core domain inside the cell. After activation, Notchcore can cleave and release the transcription factor Gal4 / VP64 connected to it. The complex acts as a transcription factor to enter the cell nucleus and bind to the UAS sequence upstream of CAR to initiate the expression of anti-EGFR CAR. If the anti-EGFR CAR is connected to the hypoxia response element ODD, it can mediate its degradation under normoxia, while it is stably expressed under hypoxia, recognizing the EGFR of the target cell to initiate the killing effect.

[0051] according to Figure 3 The structure shown inserts the CDS sequence of human CD133 (nucleic acid sequence as shown in SEQ ID NO.19) into the lentiviral packaging backbone plasmid pLV3 to construct an antigen expression plasmid named pLV3-CD133.

[0052] 2. Lentivirus packaging and concentration

[0053] 1) HEK293T cells were seeded in 10 cm 2 Incubate the cells in a CO2 incubator overnight in a cell culture dish to allow the cell confluence to reach 70%-90% on the next day;

[0054] 2) Liposome transfection: core plasmid (10 μg), lentiviral packaging plasmid psPAX2 (7.5 μg), and envelope plasmid pMD2.G (2.5 μg) were added to 1 mL of opti-MEM medium at a ratio of 4:3:1, and then 60 μL of PEI transfection reagent was added. After mixing, the mixture was allowed to stand at room temperature for 15 min, and then slowly added to HEK293T cells. The culture dish was gently shaken to mix, and then placed in a 37°C incubator. After 6 hours of culture, fresh complete medium was replaced.

[0055] 3) After 48 hours, the culture medium in the culture dish was collected and filtered through a 0.45 μm filter to obtain the lentiviral supernatant;

[0056] 4) Add 2 mL of 5×PEG8000 to every 8 mL of viral supernatant and shake at 4°C overnight;

[0057] 5) Centrifuge at 4000 g for 30 min at 4°C, remove the supernatant completely, and resuspend the virus precipitate in 100 μL PBS to obtain the concentrated lentivirus.

[0058] 3. Preparation of CAR-NK cells

[0059] 2 × 10 5For NK92MI cells, add concentrated synNotch CAR and synNotch-ODDCAR lentiviruses directly into the wells, add polybrene at a final concentration of 8 μg / mL to enhance infection, replace with fresh culture medium the next day, and detect the CAR positivity rate after another two days.

[0060] 4. Target cell construction

[0061] The expression of EGFR and CD133 in human liver cancer cell line wild-type Huh7 and triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 is as follows: Huh7 EGFR+CD133dim 、MDA-MB-231 EGFR+CD133- and MDA-MB-468 EGFR+CD133dim , requires build MDA-MB-231 EGFR+CD133+ Target cells.

[0062] 2 × 10 were added to each well of a 6-well plate. 5 After MDA-MB-231 cells adhered overnight, 1 mL of unconcentrated pLV3-CD133 lentiviral supernatant was added, and polybrene at a final concentration of 10 μg / mL was added to enhance infection. Fresh culture medium was replaced the next day, and the overexpression of CD133 was detected after another two days.

[0063] 5. Flow cytometry analysis

[0064] 1) Collect infected cells in a 1.5 mL centrifuge tube and collect wild-type cells as a negative control. Centrifuge at 4°C, 200 g for 3 min and remove the culture medium.

[0065] 2) Resuspend the cell pellet in 1 mL of pre-cooled PBS, centrifuge at 200 g for 3 min at 4°C, and remove the supernatant;

[0066] 3) For CAR detection, add 100 μL of diluted anti-Flag antibody working solution to each sample; for target antigen detection, add 100 μL of diluted anti-EGFR or anti-CD133 antibody working solution to each sample, protect from light, and incubate at 4°C for 20 min;

[0067] 4) Add 1 mL of pre-cooled PBS, centrifuge at 4°C, 200 g for 3 min, remove the supernatant, and repeat once;

[0068] 5) After resuspending in 200 μL PBS, flow cytometry was performed to analyze the expression of CD133-synNotch and BFP in CAR-NK92MI cells and EGFR and CD133 in target cells.

[0069] 6. Flow cytometry sorting of CAR-NK92MI and target cells

[0070] 1) Collect approximately 5 × 10 6 The infected CAR-NK92MI and MDA-MB-231 cells were centrifuged at 200 g for 3 min at 4°C, and the culture medium was removed.

[0071] 2) Resuspend the cell pellet in 5 mL of pre-cooled PBS, centrifuge at 200 g for 3 min, and remove the supernatant;

[0072] 3) For CAR-NK92MI sorting, add 500 μL of diluted anti-Flag antibody working solution to each sample; for target cell sorting, add 500 μL of diluted anti-CD133 antibody working solution to each sample and incubate at 4°C for 20 min in a dark environment;

[0073] 4) Add 5 mL of pre-cooled PBS to resuspend, centrifuge at 200 g for 3 min, remove the supernatant, and repeat once;

[0074] 5) Resuspend in 500 μL complete medium and transfer to a 15 mL centrifuge tube and place on ice away from light;

[0075] 6) In the flow cytometer, determine the gate of positive cells based on the negative control and sort out the target cells;

[0076] 7) The sorted cells were centrifuged at 300 g for 5 min, resuspended in complete culture medium containing 3% cyan / chain dual antibody, and cultured in a CO2 incubator to obtain completely positive CAR-NK92MI and MDA-MB-231 EGFR+CD133+ The expression of CAR on the sorted CAR-NK92MI cells is shown in Figure 2 Figure 4 As shown in Figure 2, the sorted CAR-NK92MI cells are all CAR-positive cells. The expression of EGFR and CD133 in wild-type Huh7, MDA-MB-468 and sorted MDA-MB-231 cells is shown in Figure 2. Figure 5 As shown, it can be seen that the sorted MDA-MB-231 cells are all EGFR and CD133 double-positive cells, while Huh7 and MDA-MB-468 are both EGFR-positive and CD133-weakly positive.

[0077] Embodiment 2:

[0078] 1. Construction of in vitro hypoxia model

[0079] The present invention uses 150 μM CoCl2 to treat CAR-NK92MI cells for 24 hours to induce chemical hypoxia.

[0080] 2. Luciferase chemiluminescence assay to determine in vitro tumor killing effect

[0081] 1) The tumor target cells with good growth status were digested and resuspended and counted at 1×10 per well. 4 The cells were seeded in a black opaque 96-well ELISA plate and cultured overnight;

[0082] 2) Remove the target cell culture medium, add 100 μL of different CAR-NK92MI or CAR-T cells according to different effector-target ratios, and culture them in a CO2 incubator for 4-6 hours. Set up 3 replicates for each effector-target ratio in each group, and set up a target cell culture group (negative control group) and a culture medium group (blank group);

[0083] 3) Prepare D-luciferin potassium salt working solution: Dilute 30 mg / mL D-luciferin potassium salt stock solution to 0.15 mg / mL with PBS in a dark place. All subsequent operations must be kept away from light.

[0084] 4) Remove the cell culture medium, add 100 μL of D-luciferin potassium salt working solution to each well using an 8-channel pipette, and incubate at 37°C for 5 min;

[0085] 5) The fluorescence intensity was collected in a microplate reader, the blank group signal was subtracted from the signal in all wells, and then the in vitro killing efficiency was calculated according to the following formula: (1-co-culture group reading / negative control group reading)×100%.

[0086] like Figure 6 The results show the killing effect of synNotch CAR-NK92MI and synNotch-ODD CAR-NK92MI cells on different target cells in the present invention under normoxic or hypoxic conditions. For EGFR and CD133 double-positive target cells MDA-MB-231 and MDA-MB-468, synNotch CAR-NK92MI mediated significant killing under normoxic conditions, while synNotch-ODD CAR-NK92MI had almost no killing function (e.g. Figure 6 A); under hypoxic conditions, synNotch CAR-NK92MI and synNotch-ODD CAR-NK92MI exerted similar killing effects (e.g. Figure 6 B). This result proves that the synNotch-ODD CAR-NK92MI of the present invention has no killing function under normoxic conditions, but has complete killing ability under hypoxic conditions.

[0087] Embodiment 3:

[0088] Flow cytometry detection of CAR-NK92MI activation marker CD69 and EGFR-28z CAR expression:

[0089] 2 × 10 5 CAR-NK92MI was co-cultured with target cells at an effector-target ratio of 1:1. After 24 hours of co-culture, cells were collected and stained for CD56, CD69 and Myc, and then CD56 was detected by flow cytometry. + CD69 + and Myc + proportion.

[0090] Figure 7 The CD69 expression levels of synNotch CAR-NK92MI and synNotch-ODD CAR-NK92MI co-cultured with target cells under normoxic or hypoxic conditions are shown. It can be seen that under normoxic conditions, CD69 is significantly upregulated after synNotch CAR-NK92MI is co-cultured with target cells, while synNotch-ODD CAR-NK92MI has almost no CD69 expression (e.g. Figure 7 AB); however, under hypoxic conditions, the CD69 level of synNotch-ODD CAR-NK92MI was significantly upregulated (e.g. Figure 7 CD). This result also proves that the synNotch-ODD CAR system has an activation mode of hypoxia response.

[0091] Figure 8 The Myc expression levels of synNotch CAR-NK92MI and synNotch-ODD CAR-NK92MI co-cultured with target cells under normoxic or hypoxic conditions are shown. It can be seen that under normoxic conditions, Myc is significantly upregulated after synNotch CAR-NK92MI is co-cultured with target cells, while synNotch-ODD CAR-NK92MI has almost no Myc expression (e.g. Figure 8 AB); however, under hypoxic conditions, the Myc level of synNotch-ODD CAR-NK92MI was significantly upregulated (e.g. Figure 8 CD). This result also proves that the synNotch-ODD CAR system can mediate the stable expression of EGFR-28z CAR when co-cultured with target cells under hypoxic conditions.

[0092] The hypoxia-responsive synNotch-ODD CAR involved in the present invention can activate immune cells only when hypoxia occurs and the target cells express two antigens at the same time, effectively solving the problem of toxicity when the traditional synNotch CAR treats patients with tumor metastasis. Therefore, the hypoxia-responsive synNotch-ODD CAR-NK cells or CAR-T cells constructed based on this structure have better safety than traditional synNotch CAR immune cells.

Claims

1. An anti-tumor expression vector, characterized in that: A nucleic acid sequence comprising CD133-synNotchCAR and EGFR-hypoxia response element CAR; The structure of the CD133-synNotch CAR comprises a CD8α signal peptide, a tag, a first antigen binding domain, a Notch Core, Gal4 and VP64 connected in sequence; The structure of the EGFR-hypoxia response element CAR comprises sequentially connected UAS, miniCMV, CD8α signal peptide, second antigen binding domain, tag, CD28 hinge region or CD8α hinge region, CD28 transmembrane region or CD8α transmembrane region, costimulatory domain, signal domain and hypoxia response element; The first antigen binding domain is different from the second antigen binding domain; Preferably, the first antigen binding domain and the second antigen binding domain are one of anti EGFR scFv and anti CD133 scFv.

2. The anti-tumor expression vector according to claim 1, characterized in that: The hypoxia response element is ODD, and the nucleic acid sequence is shown in SEQ ID NO.

17.

3. The anti-tumor expression vector according to claim 1, characterized in that: The nucleic acid sequence of the CD8α signal peptide is shown in SEQ ID NO.4; The nucleic acid sequence of the anti-CD133 scFv is shown in SEQ ID NO.6; The nucleic acid sequence of Notch Core is shown in SEQ ID NO.7; The nucleic acid sequence of Gal4 is shown in SEQ ID NO.8; The nucleic acid sequence of VP64 is shown in SEQ ID NO.9; The nucleic acid sequence of the UAS is shown in SEQ ID NO.10; The nucleic acid sequence of the anti-EGFR scFv is shown in SEQ ID NO.11; The nucleic acid sequence of the CD28 hinge region is shown in SEQ ID NO.13; The nucleic acid sequence of the CD28 transmembrane domain is shown in SEQ ID NO.14; The nucleic acid sequence of the CD28 costimulatory domain is shown in SEQ ID NO.15; The nucleic acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO.

16.

4. The anti-tumor expression vector according to claim 1, characterized in that: The tag is one of Flag, Myc, HA or PGK; The costimulatory domain is one of CD28, 4-1BB, ICOS, CD27 or OX40 costimulatory domains; The signal domain is the CD3ζ signal domain or the Fc receptor FcεRIγ.

5. The anti-tumor expression vector according to claim 1, characterized in that: The nucleic acid sequence of the CD133-synNotchCAR is shown in SEQ ID NO.1; The nucleic acid sequence of the EGFR-hypoxia response element CAR is shown in SEQ ID NO.

3.

6. A recombinant virus, characterized in that The recombinant virus contains the expression vector according to any one of claims 1 to 5.

7. Use of the expression vector according to any one of claims 1 to 5 or the recombinant virus according to claim 6 in the preparation of synNotch-ODD CAR immune cells.

8. A synNotch-ODD CAR immune cell, characterized in that: The synNotch-ODD CAR immune cell comprises the expression vector according to any one of claims 1 to 5 or the recombinant virus according to claim 6; Preferably, the immune cells are NK cells or T cells.

9. Use of the synNotch-ODD CAR immune cells as described in claim 8 in the preparation of drugs for treating anti-tumor diseases.

10. A drug for anti-tumor, characterized in that: The drug contains the synNotch-ODD CAR immune cell as described in claim 8.

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