CN112921035A - Promoter for regulating and controlling CAR-T specific activation and application thereof - Google Patents

Promoter for regulating and controlling CAR-T specific activation and application thereof Download PDF

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CN112921035A
CN112921035A CN201911236679.3A CN201911236679A CN112921035A CN 112921035 A CN112921035 A CN 112921035A CN 201911236679 A CN201911236679 A CN 201911236679A CN 112921035 A CN112921035 A CN 112921035A
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CN112921035B (en
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张巍
陈军
齐亚男
徐艳敏
赵文旭
黄霞
赵永春
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Chongqing Precision Biotech Co ltd
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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a promoter for regulating and controlling CAR-T specific activation and application thereof. The promoter is regulated by an anoxic environment and is formed by connecting a Hif1a regulating element and a mini promoter, the repetition number of the Hif1 alpha regulating element is 3, and the nucleotide sequence of the Hif1 alpha regulating element is shown in SEQ ID NO:1, and the nucleotide sequence of the promoter is shown as SEQ ID NO:3, respectively. The promoter can strengthen the expression of a target gene in an anoxic environment, can strengthen the activation of CAR-T cells in the anoxic environment, and can improve the killing of the CAR-T cells on tumor target cells.

Description

Promoter for regulating and controlling CAR-T specific activation and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a promoter, a CAR structure, a separated nucleic acid molecule, an expression vector, a CAR-T cell, application of the CAR-T cell in preparation of tumor drugs, and a method for improving killing capacity of the CAR-T cell in an anoxic environment.
Background
CAR-T is known as chimeric antigen receptor T cell immunotherapy and achieves better performance in hematological tumors, but CAR-T is less effective in solid tumors than hematological tumors because CAR-T is less likely to enter solid tumors on the one hand, and because CAR-T cells cannot function normally even if they enter solid tumors because of the tumor microenvironment, both of which affect the efficacy of CAR-T cells in solid tumor therapy; and due to the high heterogeneous degree of the solid tumor, the target of the solid tumor is often expressed in normal tissues, so that the safety problems of off-target risk and the like also exist. Although there are structures currently available to construct fourth generation CARs, to construct dual CARs (to construct CAR-T with dual antigens) or icars with activation inhibition, in the hope of improving the efficacy and safety of CAR-T treatment of solid tumors, these CAR structures still suffer from safety or are more difficult to activate. Therefore, there is a need to construct a CAR structure that specifically initiates activation in the tumor microenvironment.
Acidity and hypoxia are two major physical factors in the tumor microenvironment, most cells in solid tumors are in a hypoxic environment, and hypoxia alters the carbohydrate metabolic pathways of tumor cells, produces a large amount of lactic acid, causes the characteristics of acidity in the tumor microenvironment, and the acidity can reduce tumor cell apoptosis, enhance cell proliferation and growth, and help tumor cell migration. The research of hypoxia in a solid tumor microenvironment is clear, and if a controllable promoter which is activated under the hypoxia condition can be designed, the purpose of specifically activating the expression of downstream proteins in the tumor microenvironment so as to improve the effectiveness and safety of CAR-T cells can be achieved.
Although there is much theoretical research on tumor tissue hypoxia, how to apply hypoxic environments in combination with CAR-T therapy is a new direction, requiring extensive research and effort.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a promoter, and a CAR structure, an expression vector and a CAR-T cell comprising the promoter. The hypoxia-controllable promoter can strengthen the expression of a target gene in an anoxic environment, enhance the expression of target factors (protein, nucleic acid and the like) and improve the curative effect of an anti-tumor medicament.
The invention starts from the hypoxia microenvironment of the tumor, regulates the CAR-T specific activation by constructing the expression of the hypoxia-activated regulatory element specific regulatory protein, and shows that the success of constructing the hypoxia environment by using the in vitro drugs and the subsequent CAR-T killing result are shown in CoCl2The CAR-T under the induced hypoxia condition has stronger killing effect than that of the non-induced CAR-T, and simultaneously, the constructed CAR structure and the CAR-T have the passive targeting characteristic of a hypoxia microenvironment, have stronger activity and lethality in the hypoxia microenvironment, and have safety characteristics in a non-hypoxia microenvironment. The method has important guiding significance for the clinical application of CAR-T and the development of new strategies of tumor combination therapy.
In order to achieve the purpose, the invention adopts the following scheme:
a promoter, wherein the promoter is formed by linking a Hif1a regulatory element with a repeat number of 3 and a mini-promoter, and the Hif1 α regulatory element nucleic acid sequence is shown in SEQ ID NO:1 is shown. The promoter is 3HRE-CMVmini promoter.
Specifically, the Hif1 α regulatory element is a tandem of 3 repeats, which not only means that two nucleic acid sequences are directly linked, i.e., the 3 'end of one nucleic acid sequence is directly linked to the 5' end of the other nucleic acid sequence, but also means that two nucleic acid sequences can be indirectly linked, i.e., other nucleic acid sequences can be linked between the two nucleic acid sequences, as long as the function of the two nucleic acid sequences is not affected.
Further, the promoter is regulated by an anoxic environment, and the mini promoter is selected from any one of a cellular virus promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter, and a synthetic promoter.
Preferably, the mini-promoter is selected for miniCMV.
Further, the structure of the Hif1 alpha regulatory element is optimized to obtain an optimized Hif1 alpha regulatory element, and the nucleotide sequence of the optimized Hif1 alpha regulatory element is shown as SEQ ID NO: 2; the Hif1 α regulatory element can be optimized to fit different expression vectors.
Further, the nucleic acid sequence of the promoter is shown as SEQ ID NO:3, respectively.
Further, the CAR structure comprises a promoter under the regulatory control of a hypoxic environment, the nucleotide sequence of the promoter is set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 sequence is shown.
Specifically, the CAR structure can be a conventional first generation, second generation and third generation CAR structure, and can also be a novel CAR structure such as a modified double CAR and a controllable CAR structure (e.g., FRB/FKBP12 regulation).
Further, the antigen recognition region of the CAR structure is ScFv, and the amino acid sequence of the ScFv is shown as SEQ ID NO.8 or SEQ ID NO.9 or a functional variant thereof.
Further, the amino acid sequence of the hinge region of the CAR structure is shown as SEQ ID NO. 10 or SEQ ID NO. 11 or SEQ ID NO. 12 or a functional variant thereof.
In particular, the hinge region sequence in the CAR structure may be derived from: IgG, CD8, CD7, CD 4; the transmembrane region in the CAR structure may be derived from: CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, and CD 86; intracellular signaling regions in CAR structures may be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD 127.
Further, the amino acid sequence of the transmembrane region of the CAR structure is shown as SEQ ID NO. 13 or SEQ ID NO. 14 or a functional variant thereof.
Further, the amino acid sequence of the intracellular co-stimulatory domain of the CAR structure is shown as SEQ ID NO. 15 or SEQ ID NO. 16, and functional variants thereof.
Further, the amino acid sequence of the intracellular activation signal of the CAR structure is shown as SEQ ID NO 17 or a functional variant thereof.
Further, the amino acid sequence of the CAR structure is as shown in any one of SEQ ID NO 18-21 or a functional variant thereof.
In certain embodiments, the CAR structure comprising the hypoxic promoter carries a truncated EGFRt regulatory tag; in certain embodiments, the CAR comprising the hypoxic promoter is a universal CAR structure; in certain embodiments, the CAR structure comprising the hypoxic promoter carries a suicide gene such as iCasp 9.
In certain embodiments, the antigen recognition region can be a ligand/receptor that recognizes a target antigen; in certain embodiments, the ScFv may be a murine or fully human or human chimeric ScFv, a single domain antibody such as a shark, alpaca or camel antibody, a bispecific antibody, or a combination of target-specific fibronectin type III (FN3) domains designed to recognize a specific target.
In certain embodiments, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thus forming an NKR-CAR. The NKR component may be a transmembrane, hinge or cytoplasmic domain from any of the following natural killer cell receptors: killer immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP 1; natural Cytotoxic Receptors (NCRs), e.g., NKp30, NKp44, NKp 46; the Signaling Lymphocyte Activating Molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD 2F-10; fc receptors (fcrs), e.g., CD16, and CD 64; and Ly49 receptors, e.g., Ly49A, Ly 49C. The NKR-CAR molecule can interact with an adaptor molecule or an intracellular signaling domain (e.g., DAP 12).
Further, a CAR expression vector comprising an anoxic promoter, wherein the nucleotide sequence of the anoxic promoter is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3; the expression vector is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector and a plasmid.
Preferably, the vector is a lentiviral vector.
In certain embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1(HIV-1), human immunodeficiency virus 2(HIV-2), visna-meidi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).
In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR contemplated herein, and a right (3') retroviral LTR.
In certain embodiments, the CAR comprises a hepatitis b virus post-transcriptional regulatory element (HPRE) or woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).
In certain embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.
In certain embodiments, the heterologous promoter is a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or an simian Virus 40(SV40) promoter.
In certain embodiments, the 5'LTR or 3' LTR is a lentiviral LTR.
In certain embodiments, the 3' LTR is a self-inactivating (SIN) LTR.
In certain embodiments, a polynucleotide encoding a CAR encompassed herein comprises an optimized Kozark sequence.
In certain embodiments, the promoter operably linked to a polynucleotide encoding a CAR encompassed herein and the group consisting of: cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF 1-alpha), phosphoglycerate kinase-1 Promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken beta-actin promoter (CAG), polyoma enhancer/herpes simplex thymidine kinase promoter (MC1), beta actin promoter (beta-ACT), simian virus 40 promoter (SV40), and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted (MND) promoter.
In certain embodiments, the CAR-containing vector can comprise a secreted anti-PD-1 ScFv; in certain embodiments, the vector comprising the CAR comprises a PD-1 conjugated transduction peptide (e.g., PD-1-CD28-CD137-CD3 signaling structure); in certain embodiments, the vector comprising the CAR comprises a plurality of CAR combinations, such as 2 CAR combinations targeting different antigens or different recognition sites of the same antigen.
Further, the vector comprises a nucleotide sequence shown in any one of SEQ ID NO 4-7, and specifically, the SEQ ID NO 4-7 comprises a nucleotide sequence encoding a CAR structure and a promoter sequence.
Further, a CAR-T cell comprising the expression vector.
In particular, the "functional variant" is generally meant to include an amino acid sequence that has substantially the same function as it (e.g., may possess the properties of the chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto. In certain embodiments, the variant of the amino acid sequence is substantially the same function as it does.
It is a further object of the present invention to provide an isolated nucleic acid molecule.
In order to achieve the purpose, the invention adopts the following scheme:
the nucleic acid molecule encodes the CAR structure.
It is a further object of the invention to provide a use of such a promoter, CAR structure, expression vector and CAR-T cell.
In order to achieve the purpose, the invention adopts the following scheme:
the application is the application in preparing tumor medicaments, and the tumor is malignant tumor, including acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin lymphoma, prostatic cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, renal cancer, liver cancer, brain cancer and skin cancer.
Further, the tumor is high-expressing one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, CEA, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR. .
In particular, the cells can be used in combination with other agents and/or treatments that enhance the activity of CAR expression.
In particular, the active agent and/or treatment may be surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immune scavengers (immunoablativeagents) such as CAMPATH, anti-CD 3 antibody or other antibody therapy, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine, FK506, rapamycin (rapamycin), mycophenolic acid (mycophenolic acid), steroids (steroids), 90fr 1228, cytokines and radiation.
In certain embodiments, the cell can express other agents, e.g., agents that enhance the activity of the CAR-expressing cell. The active agent may be an active agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 may, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3(CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, GALMII, MHC class II, 9, adenosine, TGFR (TGFR β) and TGFR β. The extracellular domain of the inhibitory molecule may be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.
It is a further object of the present invention to provide a method for increasing the killing ability of CAR-T cells in an anoxic environment. The CAR-T cell can be induced and activated in an anoxic environment and in the presence of a target antigen, the CAR expression capacity and abundance are improved after the target antigen is stimulated in the anoxic environment, the CAR-T effectiveness is enhanced, the CAR expression capacity and abundance are lower than those of conventional CAR-T after the target antigen is stimulated in a non-anoxic environment, and the safety is higher.
In order to achieve the purpose, the invention adopts the following scheme:
the method is to construct an expression vector of the CAR structure and infect T lymphocytes, and then act on target cells to improve the killing capacity of the CAR-T cells in an anoxic environment.
The invention has the beneficial effects that:
1) the promoter induced by the anoxic microenvironment can strengthen the expression of target genes in the anoxic environment;
2) the CAR structure induced and started by the hypoxic microenvironment can be effectively expressed in T lymphocytes, can enhance the activation of CAR-T cells in the hypoxic environment, improves the killing of the CAR-T cells on tumor target cells, and can be used for the targeted therapy of tumors;
3) the chimeric antigen receptor T cell provided by the invention has enhanced activity in an anoxic environment, and can be used for adoptive cell therapy in preparation of medicines for treating malignant hematological diseases and malignant solid tumors.
Drawings
FIG. 1 is a diagram showing the structure of a promoter.
Figure 2 is a hypoxia induction protocol validation.
FIG. 3 is a graph showing the effect of hypoxia inducible drugs on cell proliferation.
FIG. 4 is a CAR positivity test.
FIG. 5 is a measurement of CAR expression intensity after hypoxia induction.
FIG. 6 is a validation of promoter engineered CAR-T efficacy.
FIG. 7 shows a comparison of promoter engineered CAR-T activity in hypoxic and non-hypoxic environments.
FIG. 8 is a comparison of factor secretion following promoter engineering CAR-T activity in hypoxic and non-hypoxic environments.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1 plasmid construction
Synthesizing an anoxic initiation sequence 3HRE nucleic acid sequence shown as SEQ ID NO:1, further constructing a promoter 3HRE-CMVmini promoter-NheI (3H1P) matched with a lentiviral vector, further constructing a promoter 3HRE-CMVmini promoter-NheI nucleic acid sequence shown as SEQ ID NO:3, respectively cutting and recovering fragments by double enzyme digestion, connecting gene fragments, transforming and selecting single clone to obtain a recombinant vector, wherein the vector numbers are respectively 5, 6, 7 and 8; the CAR structure element comprises: ScFv targeting CEA or PSCA with amino acid sequence as SEQ ID NO.8 or SEQ ID NO. 9; a hinge structure having an amino acid sequence as set forth in SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12; a transmembrane structure having an amino acid sequence as set forth in SEQ ID NO. 13 or SEQ ID NO. 14, an intracellular costimulatory domain having an amino acid sequence as set forth in SEQ ID NO. 15 or SEQ ID NO. 16, and an intracellular activation signal having an amino acid sequence as set forth in SEQ ID NO. 17; the obtained CAR modified by the hypoxia promoter has nucleotide sequence shown as SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, and amino acid sequence shown as SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21; FIG. 1 is a diagram showing the structure of a promoter.
Example 2 in vitro hypoxia model validation
CoCl2Induced defectOxygen principle: cobalt dichloride (CoCl)2) Divalent cobalt ion (Co) of (1)2+) Can replace the divalent iron ion of the PHD cofactor because of Co2+The low affinity for oxygen, which makes heme unable to bind oxygen and to be allosteric into an oxidized state, thus forming an anoxic state.
1) And (3) verification of a hypoxia induction scheme:
an in-vitro hypoxic cell model is constructed by using PBMC (peripheral blood mononuclear cell) infected and activated by 3HRE-GFP (high resolution fluorescent protein) virus, and the culture solution is changed after 12-18 h. Using CoCl2Inducing an anoxic environment, culturing until day 4, verifying whether the anoxic model is constructed by green fluorescence detection and WB, and respectively setting a control group (without adding CoCl)2) Experimental group (with CoCl)2) And the expression intensity of GFP is detected by taking a picture of a fluorescence microscope after 24 hours of drug treatment with a positive group (a conventional infected GFP cell not containing a hypoxia regulated promoter). And extracting Protein according to the instruction of M-PER Mammalian Protein Extraction Reagent (Thermo) to detect the expression of HIF-1a in cells by WB.
As a result, as shown in FIG. 2, it was found that CoCl was added2The GFP expression of the induction group (i.e. the microenvironment hypoxia group) is obviously stronger than that of the non-CoCl addition group2The fluorescence intensity of the group is far stronger than that of the positive control group, which indicates that the hypoxia promoter of the PBKL1-3HRE-GFP can normally promote the expression of downstream GFP in a hypoxia environment.
2) Effect of hypoxia-inducible drugs on cell proliferation
Plating 96-well plates with appropriate amount of Hela and PBMC cells in the morning, adhering the cells to the wall for 5h, and then performing CoCl2Adding drugs to final concentrations of 0, 100, 200, 300, 400 and 500uM, adding the incubated CellTiter-Glo One Solution Assay reagent according to the volume of 1:1 after 24 hours of treatment, incubating for 10 minutes at room temperature, and placing the incubated reagent in an enzyme-labeling instrument to detect absorbance. The results are shown in FIG. 3, CoCl2Does not affect the proliferation of the protocell, so that the killing result of the CAR-T cell is not influenced by the externally added CoCl2Influence is only related to whether the hypoxia promoter is included or not.
Example 3 preparation of lentiviruses and infection of T lymphocytes
This example packages lentivirusesThe calcium phosphate method is adopted, and specifically comprises the following steps: the 293T cells are cultured to a better state by using a DMEM medium containing 10% FBS (w/v), the cell confluency is ensured to be 70-80% during transfection, and the cells are replaced by a liquid in advance. The packaging plasmid (RRE: REV:2G) and the expression plasmid are added into a centrifuge tube of 1.5 according to a certain proportion, and 2.5mol/LCaCl is added2And supplementing ddH2O to the total volume of 600ul, uniformly mixing, dropwise adding 2 xHBS into the mixed solution by using a pipette, uniformly mixing, standing at room temperature for 15min, adding into the treated 293T cell culture solution, changing the solution to 10mL of DMEM medium containing 10% FBS after 3-5h, collecting cell supernatant after 48h or 72h, purifying the virus, and measuring the titer.
Separating lymphocytes by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells are activated by anti-CD 3 and CD28 monoclonal antibodies for 24 hours, the activated PBMC is infected according to a certain multiplicity of infection (MOI) and polybrene is added to help virus infection, the cells are cultured overnight, then the cells are changed (10% RPMI 1640+ IL-2+ double antibodies), the cells are cultured continuously in an incubator at 37 ℃, the positive rate of CAR-T is detected on the 12 th day of virus infection, the detection method is flow detection, and the antibodies are: Protein-L-PE, Protein-L can recognize the antibody light chain, and the light chain of ScFv sequence of the CAR antigen recognition region can be recognized by Protein-L, so that the Protein-L can be used to detect the CAR positivity and CAR expression intensity.
The results are shown in FIG. 4: 3H1P promoter modified different structure CAR-T cell, CAR expression is obviously increased under induced hypoxia environment.
The results are shown in FIG. 5: 3H1P promoter-modified CAR-T cells with different structures, wherein a black histogram is the CAR expression strength of the promoter-modified CAR in a non-hypoxic environment; the white histogram is the CAR expression intensity of the promoter-modified CAR in an anaerobic environment, and the CAR expression intensity is enhanced in an induced anaerobic environment.
Example 4 testing of CAR-T effectiveness comprising a hypoxic promoter following hypoxia induction
And respectively taking PSCA positive Hela cells and CEA positive DLD1-CEA cells which are transformed in vitro as target cells to verify the validity function of the CAR-T after promoter transformation. And (2) carrying out anoxic pretreatment on effector cells (CAR-T cells containing promoter modified CAR expression) overnight, collecting the CAR-T cells, paving the CAR-T cells in target cells according to a certain effective target ratio, detecting the killing capacity of different CAR-T on the target cells by using an ACEA xCELLigence RTCA MP instrument, and carrying out experimental steps according to instrument instructions. The ACEA xCELLigence RTCA MP principle is that the resistance index is taken as data to record tumor cells attached to the bottom of a hole every 15 minutes, and the proliferation or death condition of the attached target cells is judged through the resistance index. The result formula of the resistance index analysis is as follows: CAR-T cell killing rate-baseline resistance index-real-time resistance index.
The results are shown in FIG. 6: CAR-T cells of different structure comprising the hypoxic promoter 3H1P were all able to function normally compared to the control group.
FIG. 7 further demonstrates the regulation of CAR-T killing activity by hypoxia-specific promoters engineered in hypoxic and non-hypoxic environments, and the results show that in hypoxic environments, the hypoxia-specific promoters can specifically induce efficient killing of CAR-T at low target ratio of 1:5, whereas conventional CAR-T has very poor killing ability in hypoxic environments, with the percentage killing values as shown in the following table:
Figure BDA0002305077580000111
example 5Elisa detection of IFN-. gamma.detection
Example 4 the collected supernatant was used to detect IFN-. gamma.secretion by ELISA (enzyme-linked immunosorbent assay). IFN-gamma detection is carried out by adopting a BD IFN-gamma kit with the product number of 555142, and the experimental steps are carried out according to the product instruction.
The results are shown in fig. 8, Treat represents the factor secretion situation after target cell killing of the promoter-modified CAR-T cell in an anoxic environment, and unrreatat represents the factor secretion situation after target cell killing of the promoter-modified CAR-T cell in a non-anoxic environment. The result shows that the CAR-T cells with modified promoters can secrete high IFN-gamma only after being activated in the anoxic environment.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Sequence listing
<110> Chongqing accurate Biotechnology Co., Ltd
<120> promoter for regulating and controlling CAR-T specific activation and application thereof
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 119
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
cccacagtgc atacgtgggc tccaacaggt cctcttgtcg agccacagtg catacgtggg 60
ctccaacagg tcctcttgtc gagccacagt gcatacgtgg gctccaacag gtcctcttg 119
<210> 2
<211> 139
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gtcgacgata tcccacagtg catacgtggg ctccaacagg tcctcttgtc gagccacagt 60
gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg ggctccaaca 120
ggtcctcttg tcgagatct 139
<210> 3
<211> 192
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagatctggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 180
tcagatcact ag 192
<210> 4
<211> 2220
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagatctggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 180
tcagatcact aggctagcat ggccctgcca gtgaccgccc tgctgctgcc cctggccctg 240
ctgctgcacg cagcacggcc cgacatccag gatatccagc tgacacagtc ccctagctcc 300
ctgagcgcct ccgtgggcga cagggtgacc atcacatgca gcgcctctag ctccgtgaga 360
ttcatccact ggtaccagca gaagcccggc aaggccccta agaggctgat ctatgatacc 420
tctaagctgg ccagcggagt gccctcccgc ttctctggca gcggctccgg caccgacttt 480
accctgacaa tctctagcct gcagccagag gattttgcca catactattg ccagcagtgg 540
tcctctagcc ccttcacctt tggccagggc acaaaggtgg agatcaaggg atctaccagc 600
ggatccggca agcctggatc tggagagggc agcacaaagg gctccgaggt gcagctggtg 660
gagtccggag gaggactggt gcagccagga ggatctctga ggctgagctg tgcagcctcc 720
ggcttcaaca tcaaggacta ctatatccac tgggtgagac aggcacctgg caagggactg 780
gagtgggtgg catggatcga ccctgagaat ggcgataccg agttcgtgcc aaagtttcag 840
ggcagggcca caatctctgc cgacaccagc aagaacacag cctacctgca gatgaatagc 900
ctgcgcgccg aggataccgc cgtgtactat tgtaagacag gaggattttg gggacagggc 960
accctggtga cagtgtcctc tctcgaggag agcaagtacg gccctccctg ccccccttgc 1020
cctgcccccg agttcctggg cggacccagc gtgttcctgt tcccccccaa gcccaaggac 1080
accctgatga tcagccggac ccccgaggtg acctgtgtgg tggtggacgt gtcccaggag 1140
gaccccgagg tccagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc 1200
aagccccggg aggagcagtt caatagcacc taccgggtgg tgtccgtgct gaccgtgctg 1260
caccaggact ggctgaacgg caaggaatac aagtgtaagg tgtccaacaa gggcctgccc 1320
agcagcatcg agaaaaccat cagcaaggcc aagggccagc ctcgggagcc ccaggtgtac 1380
accctgcccc ctagccaaga ggagatgacc aagaatcagg tgtccctgac ctgcctggtg 1440
aagggcttct accccagcga catcgccgtg gagtgggaga gcaacggcca gcccgagaac 1500
aactacaaga ccaccccccc tgtgctggac agcgacggca gcttcttcct gtacagcagg 1560
ctgaccgtgg acaagagccg gtggcaggag ggcaacgtct ttagctgctc cgtgatgcac 1620
gaggccctgc acaaccacta cacccagaag agcctgtccc tgagcctggg caaggaattc 1680
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 1740
gccttcatca tcttttgggt gaggagcaag cggagcagag gcggccacag cgactacatg 1800
aacatgaccc cccggaggcc tggccccacc cggaagcact accagcccta cgcccctccc 1860
agggacttcg ccgcctaccg gagccgggtg aagttcagcc ggagcgccga cgcccctgcc 1920
taccagcagg gccagagcca gctgtacaac gagctgaacc tgggccggag ggaggagtac 1980
gacgtgctgg acaagcggag aggccgggac cctgagatgg gcggcaagcc ccggagaaag 2040
aaccctcagg agggcctgta taacgaactg cagaaagaca agatggccga ggcctacagc 2100
gagatcggca tgaagggcga gcggcggagg ggcaagggcc acgacggcct gtaccagggc 2160
ctgagcaccg ccaccaagga tacctacgac gccctgcaca tgcaggccct gccccctcgc 2220
<210> 5
<211> 2241
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagatctggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 180
tcagatcact aggctagcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg 240
ctgctccacg ccgccaggcc ggacatccag atgacccaga gcccaagcag cctgagcgcc 300
agcgtgggtg acagagtgac catcacctgt agtaccagtt cgagtgtaag ttacatgcac 360
tggtaccagc agaagccagg taaggctcca aggctgctga tctacagcac atccaacctg 420
gcttctggtg tgccaagcag attcagcggt agcggtagcg gtaccgactt caccttcacc 480
atcagcagcc tccagccaga ggacatcgcc acctactact gccatcagtg gagtagttat 540
cccacgttcg gccaagggac caaggtggaa atcaaaggat ccacttccgg ttcaggaaag 600
cccgggagtg gtgaaggtag cactaaaggc caggtccagc tgcaggagag cggtccaggt 660
cttgtgagac ctagccagac cctgagcctg acctgcaccg tgtctggctt caccatcagc 720
agtggttata gctggcactg ggtgagacag ccacctggac gaggtcttga gtggattgga 780
tacatacagt acagtggtat cactaactac aacccctctc tcaaaagtag agtgacaatg 840
ctggtagaca ccagcaagaa ccagttcagc ctgagactca gcagcgtgac agccgccgac 900
accgcggtct attattgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 960
ggtcaaggca gcacggtcac cgtctcctca ggtgcggccg ccctcgagga gagcaagtac 1020
ggccctccct gccccccttg ccctgccccc gagttcctgg gcggacccag cgtgttcctg 1080
ttccccccca agcccaagga caccctgatg atcagccgga cccccgaggt gacctgtgtg 1140
gtggtggacg tgtcccagga ggaccccgag gtccagttca actggtacgt ggacggcgtg 1200
gaggtgcaca acgccaagac caagccccgg gaggagcagt tcaatagcac ctaccgggtg 1260
gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaggaata caagtgtaag 1320
gtgtccaaca agggcctgcc cagcagcatc gagaaaacca tcagcaaggc caagggccag 1380
cctcgggagc cccaggtgta caccctgccc cctagccaag aggagatgac caagaatcag 1440
gtgtccctga cctgcctggt gaagggcttc taccccagcg acatcgccgt ggagtgggag 1500
agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgacggc 1560
agcttcttcc tgtacagcag gctgaccgtg gacaagagcc ggtggcagga gggcaacgtc 1620
tttagctgct ccgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgtcc 1680
ctgagcctgg gcaaggaatt cttctgggtg ctggtcgtgg tgggtggcgt gctggcctgc 1740
tacagcctgc tggtgacagt ggccttcatc atcttttggg tgaggagcaa gcggagcaga 1800
ggcggccaca gcgactacat gaacatgacc ccccggaggc ctggccccac ccggaagcac 1860
taccagccct acgcccctcc cagggacttc gccgcctacc ggagccgggt gaagttcagc 1920
cggagcgccg acgcccctgc ctaccagcag ggccagagcc agctgtacaa cgagctgaac 1980
ctgggccgga gggaggagta cgacgtgctg gacaagcgga gaggccggga ccctgagatg 2040
ggcggcaagc cccggagaaa gaaccctcag gagggcctgt ataacgaact gcagaaagac 2100
aagatggccg aggcctacag cgagatcggc atgaagggcg agcggcggag gggcaagggc 2160
cacgacggcc tgtaccaggg cctgagcacc gccaccaagg atacctacga cgccctgcac 2220
atgcaggccc tgccccctcg c 2241
<210> 6
<211> 1677
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagatctggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 180
tcagatcact aggctagcat ggccttacca gtgaccgcct tgctcctgcc gctggccttg 240
ctgctccacg ccgccaggcc ggacatccag atgacccaga gcccaagcag cctgagcgcc 300
agcgtgggtg acagagtgac catcacctgt agtaccagtt cgagtgtaag ttacatgcac 360
tggtaccagc agaagccagg taaggctcca aggctgctga tctacagcac atccaacctg 420
gcttctggtg tgccaagcag attcagcggt agcggtagcg gtaccgactt caccttcacc 480
atcagcagcc tccagccaga ggacatcgcc acctactact gccatcagtg gagtagttat 540
cccacgttcg gccaagggac caaggtggaa atcaaaggat ccacttccgg ttcaggaaag 600
cccgggagtg gtgaaggtag cactaaaggc caggtccagc tgcaggagag cggtccaggt 660
cttgtgagac ctagccagac cctgagcctg acctgcaccg tgtctggctt caccatcagc 720
agtggttata gctggcactg ggtgagacag ccacctggac gaggtcttga gtggattgga 780
tacatacagt acagtggtat cactaactac aacccctctc tcaaaagtag agtgacaatg 840
ctggtagaca ccagcaagaa ccagttcagc ctgagactca gcagcgtgac agccgccgac 900
accgcggtct attattgtgc aagagaagac tatgattacc actggtactt cgatgtctgg 960
ggtcaaggca gcacggtcac cgtctcctca ggtgcggccg ccctcgagac cacgacgcca 1020
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 1080
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 1140
gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1200
atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1260
atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1320
gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1380
aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1440
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1500
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1560
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1620
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 1677
<210> 7
<211> 1689
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
gatatcccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 60
gtgggctcca acaggtcctc ttgtcgagcc acagtgcata cgtgggctcc aacaggtcct 120
cttgtcgaga tctggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt 180
gaaccgtcag atcactaggc tagcatggcc ttaccagtga ccgccttgct cctgccgctg 240
gccttgctgc tccacgccgc caggccggac atccagatga cccagagccc aagcagcctg 300
agcgccagcg tgggtgacag agtgaccatc acctgtagta ccagttcgag tgtaagttac 360
atgcactggt accagcagaa gccaggtaag gctccaaggc tgctgatcta cagcacatcc 420
aacctggctt ctggtgtgcc aagcagattc agcggtagcg gtagcggtac cgacttcacc 480
ttcaccatca gcagcctcca gccagaggac atcgccacct actactgcca tcagtggagt 540
agttatccca cgttcggcca agggaccaag gtggaaatca aaggatccac ttccggttca 600
ggaaagcccg ggagtggtga aggtagcact aaaggccagg tccagctgca ggagagcggt 660
ccaggtcttg tgagacctag ccagaccctg agcctgacct gcaccgtgtc tggcttcacc 720
atcagcagtg gttatagctg gcactgggtg agacagccac ctggacgagg tcttgagtgg 780
attggataca tacagtacag tggtatcact aactacaacc cctctctcaa aagtagagtg 840
acaatgctgg tagacaccag caagaaccag ttcagcctga gactcagcag cgtgacagcc 900
gccgacaccg cggtctatta ttgtgcaaga gaagactatg attaccactg gtacttcgat 960
gtctggggtc aaggcagcac ggtcaccgtc tcctcaggtg cggccgccct cgagaagcca 1020
accacaaccc cagcaccaag gccacctaca cctgcaccaa ccatcgcaag ccagccactg 1080
tccctgaggc cagaggcaag acctgcagca ggaggcgccg tgcacacacg gggcctggac 1140
ttcgccgatg aattcatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 1200
ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc 1260
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 1320
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 1380
gcccccgcgt acaagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 1440
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1500
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1560
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1620
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1680
ccccctcgc 1689
<210> 8
<211> 237
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser
100 105 110
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser Glu Val Gln
115 120 125
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
130 135 140
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile
165 170 175
Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg
180 185 190
Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
195 200 205
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly
210 215 220
Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235
<210> 9
<211> 243
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe
85 90 95
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly
100 105 110
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln
115 120 125
Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr
130 135 140
Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp
145 150 155 160
Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln
165 170 175
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
180 185 190
Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser
195 200 205
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr
210 215 220
Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr
225 230 235 240
Val Ser Ser
<210> 10
<211> 228
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 10
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr
65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
100 105 110
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Leu Gly Lys
225
<210> 11
<211> 47
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 11
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 12
<211> 45
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 12
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Arg Pro Ala Ala
20 25 30
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp
35 40 45
<210> 13
<211> 27
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 13
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> 14
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
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 Leu Tyr Cys
20
<210> 15
<211> 41
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 15
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> 16
<211> 44
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 16
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
1 5 10 15
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
20 25 30
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40
<210> 17
<211> 111
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 17
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 18
<211> 644
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 18
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Arg Phe Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Ser Pro Phe Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser
100 105 110
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ser Glu Val Gln
115 120 125
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
130 135 140
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Trp Ile
165 170 175
Asp Pro Glu Asn Gly Asp Thr Glu Phe Val Pro Lys Phe Gln Gly Arg
180 185 190
Ala Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
195 200 205
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Lys Thr Gly
210 215 220
Gly Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys
225 230 235 240
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
450 455 460
Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
465 470 475 480
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
485 490 495
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
500 505 510
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
515 520 525
Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
530 535 540
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
545 550 555 560
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
565 570 575
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
580 585 590
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
595 600 605
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
610 615 620
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
625 630 635 640
Leu Pro Pro Arg
<210> 19
<211> 650
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 19
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe
85 90 95
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly
100 105 110
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln
115 120 125
Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr
130 135 140
Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp
145 150 155 160
Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln
165 170 175
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
180 185 190
Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser
195 200 205
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr
210 215 220
Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr
225 230 235 240
Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
245 250 255
Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
305 310 315 320
Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
340 345 350
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Leu Gly Lys Phe Trp Val Leu Val Val Val Gly Gly
465 470 475 480
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
485 490 495
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
500 505 510
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
515 520 525
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg
530 535 540
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
545 550 555 560
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
565 570 575
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
580 585 590
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
595 600 605
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
610 615 620
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
625 630 635 640
Ala Leu His Met Gln Ala Leu Pro Pro Arg
645 650
<210> 20
<211> 469
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe
85 90 95
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly
100 105 110
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln
115 120 125
Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr
130 135 140
Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp
145 150 155 160
Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln
165 170 175
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
180 185 190
Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser
195 200 205
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr
210 215 220
Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr
225 230 235 240
Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
245 250 255
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
260 265 270
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
275 280 285
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
290 295 300
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys
305 310 315 320
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
325 330 335
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
340 345 350
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
355 360 365
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
370 375 380
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
385 390 395 400
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 21
<211> 467
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 21
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Thr Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile Tyr
35 40 45
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Ile Ala Thr Tyr Tyr Cys His Gln Trp Ser Ser Tyr Pro Thr Phe
85 90 95
Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly
100 105 110
Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln
115 120 125
Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln Thr Leu Ser Leu Thr
130 135 140
Cys Thr Val Ser Gly Phe Thr Ile Ser Ser Gly Tyr Ser Trp His Trp
145 150 155 160
Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile Gly Tyr Ile Gln
165 170 175
Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr
180 185 190
Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser
195 200 205
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Asp Tyr
210 215 220
Asp Tyr His Trp Tyr Phe Asp Val Trp Gly Gln Gly Ser Thr Val Thr
225 230 235 240
Val Ser Ser Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
245 250 255
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Arg
260 265 270
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
290 295 300
Ser Leu Val Ile Thr Leu Tyr Cys Val Lys Arg Gly Arg Lys Lys Leu
305 310 315 320
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
325 330 335
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
340 345 350
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
355 360 365
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
370 375 380
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
385 390 395 400
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
405 410 415
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
420 425 430
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
435 440 445
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
450 455 460
Pro Pro Arg
465

Claims (18)

1. A promoter, wherein the promoter is formed by linking a Hif1a regulatory element with a repeat number of 3 and a mini-promoter, and the Hif1 α regulatory element nucleic acid sequence is shown in SEQ ID NO:1 is shown.
2. The promoter according to claim 1, wherein the promoter is regulated by a hypoxic environment, and the mini-promoter is selected from any one of a cellular viral promoter, a promoter of HSV thymidine kinase, a promoter of simian virus 40, an adenovirus late promoter, and a synthetic promoter.
3. The promoter of claim 1, wherein the Hif1 α regulatory element is optimized in structure to obtain an optimized Hif1 α regulatory element, the nucleotide sequence of which is as shown in SEQ ID NO: 2.
4. the promoter according to any one of claims 1 to 3, wherein the nucleic acid sequence of the promoter is as set forth in SEQ ID NO:3, respectively.
5. A CAR structure comprising a hypoxic promoter, wherein the CAR structure comprises a promoter that is under the regulatory control of a hypoxic environment, the nucleotide sequence of the promoter being as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 sequence is shown.
6. A CAR structure according to claim 5, wherein the antigen recognition region of the CAR structure is an ScFv, the amino acid sequence of which is as shown in SEQ ID No.8 or SEQ ID No.9 or a functional variant thereof.
7. The CAR structure of claim 5, wherein the amino acid sequence of the hinge region of the CAR structure is as set forth in SEQ ID NO 10 or SEQ ID NO 11 or SEQ ID NO 12 or a functional variant thereof.
8. A CAR structure according to claim 5, characterized in that the amino acid sequence of the transmembrane region of the CAR structure is as shown in SEQ ID NO 13 or SEQ ID NO 14 or a functional variant thereof.
9. The CAR structure of claim 5, wherein the intracellular co-stimulatory domain amino acid sequence of the CAR structure is as shown in SEQ ID NO 15 or SEQ ID NO 16 or a functional variant thereof.
10. The CAR structure of claim 5, wherein the intracellular activation signal of the CAR structure has an amino acid sequence as set forth in SEQ ID NO 17 or a functional variant thereof.
11. The CAR structure of any one of claims 5 to 10, wherein the CAR structure has an amino acid sequence as set forth in any one of SEQ ID NOs 18 to 21 or a functional variant thereof.
12. An isolated nucleic acid molecule encoding the CAR structure of any one of claims 5-10.
13. A CAR expression vector comprising a hypoxic promoter, wherein the nucleotide sequence of the hypoxic promoter is as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 is shown in the specification; the expression vector is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector and a plasmid.
14. The CAR expression vector of claim 13, wherein the vector comprises a nucleotide sequence set forth in any one of SEQ ID NOs 4-7.
15. A CAR-T cell comprising the expression vector of claim 13 or 14.
16. A method of increasing the killing ability of CAR-T cells in hypoxic environments, wherein an expression vector for a CAR construct according to claim 13 is constructed and infected with T lymphocytes, which then act on the target cells.
17. Use of the promoter of claim 1 or the CAR structure of claim 5 or the nucleic acid molecule of claim 12 or the expression vector of claim 13 or the CAR-T cell of claim 15 in the preparation of a medicament for the treatment of a tumor.
18. The use of claim 17, wherein the tumor is a malignancy, including acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, non-hodgkin's lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer, and skin cancer; the tumor highly expresses one or more of CD19, CD20, CD123, CD22, BCMA, ROR1, CEA, mesothelin, PSCA, PSMA, c-Met, GPC-3, Her2, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3 TCR.
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EP20769915.8A EP3922721A4 (en) 2019-03-12 2020-02-24 Hypoxia-regulated promoter and application thereof
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WO2024082263A1 (en) * 2022-10-21 2024-04-25 上海鑫湾生物科技有限公司 System for controlling virus replication in response to hypoxic environment and use thereof

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CN116064675A (en) * 2022-07-22 2023-05-05 西安电子科技大学 Recombinant plasmid, recombinant lentiviral vector, stable transgenic cell line, construction method and application thereof
WO2024082263A1 (en) * 2022-10-21 2024-04-25 上海鑫湾生物科技有限公司 System for controlling virus replication in response to hypoxic environment and use thereof

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