CN112921034B - 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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CN112921034B
CN112921034B CN201911236670.2A CN201911236670A CN112921034B CN 112921034 B CN112921034 B CN 112921034B CN 201911236670 A CN201911236670 A CN 201911236670A CN 112921034 B CN112921034 B CN 112921034B
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CN112921034A (en
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张巍
陈军
黄霞
齐亚男
赵永春
徐艳敏
赵文旭
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Chongqing Precision Biotech Co ltd
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Priority to JP2021555018A priority patent/JP7391984B2/en
Priority to EP20769915.8A priority patent/EP3922721A4/en
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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 anoxic environment and is formed by connecting Hif1a regulating elements and mini promoters, the repetition number of the Hif1 alpha regulating elements is 4, and the nucleotide sequence of the Hif1 alpha regulating elements is shown as SEQ ID NO:1, the nucleotide sequence of the promoter is shown as SEQ ID NO: 3. The promoter can strengthen the expression of a target gene in an anoxic environment and strengthen the activation of the CAR-T cells in the anoxic environment, and the killing of the CAR-T cells on tumor target cells is improved.

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, an isolated 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 fully known as chimeric antigen receptor T cell immunotherapy, which achieves better performance in hematological tumors, but CAR-T is less effective in solid tumors than hematological tumors, on the one hand because CAR-T is more difficult to enter into the interior of solid tumors, and on the other hand, CAR-T cells cannot function normally even if entering into the interior of solid tumors because of tumor microenvironment, which all affect the efficacy of CAR-T cells in solid tumor treatment; and because the heterogeneous degree of the solid tumor is high, the target point of the solid tumor is often expressed in normal tissues, and therefore, the safety problems such as off-target risk and the like also exist. While there are structures currently in use to construct fourth generation CARs, to construct dual CARs (to construct CAR-T with dual antigens) or icars with activation inhibition functions in hopes of improving the effectiveness and safety of solid tumor CAR-T treatment, these CAR structures still have the problem of safety or being more difficult to activate. Thus, it is necessary 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 the hypoxic environment, and hypoxia alters the glucose metabolic pathways of tumor cells, producing large amounts of lactic acid, which characterizes acidity in the tumor microenvironment, which can reduce tumor cell apoptosis, enhance cell proliferation and growth, and help tumor cell migration. The study of hypoxia in solid tumor microenvironment is clear, and if the regulatable promoter activated under hypoxia condition can be designed, the aim of specifically activating the expression of downstream protein in tumor microenvironment can be achieved, thereby improving the effectiveness and safety of CAR-T cells.
Although much research is done in theory regarding hypoxia in tumor tissue, how to apply hypoxia-coupled CAR-T therapy is a new direction, requiring comprehensive research and attempts.
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 a hypoxia environment, strengthen the expression of target factors (proteins, nucleic acids and the like) and improve the curative effect of anti-tumor drugs.
The invention starts from the anoxic microenvironment of the tumor itself, regulates and controls the specific activation of the CAR-T by constructing the expression of the regulation element specific regulation protein of the anoxic activation, and shows the success of constructing the anoxic environment and the subsequent CAR-T killing result in the CoCl by in vitro medicaments 2 The CAR-T under the induced hypoxia condition has stronger killing effect than the CAR-T without induction, and meanwhile, the constructed CAR structure and the CAR-T have the passive targeting characteristic of the hypoxia microenvironment, have stronger activity and killing power in the hypoxia microenvironment, and show the safety characteristic in the non-hypoxia microenvironment. The method has important guiding significance for clinical application of the CAR-T and development of new strategies for combined treatment of tumors.
In order to achieve the above purpose, the present invention adopts the following scheme:
a promoter, characterized in that the promoter is formed by connecting a Hif1a regulatory element with the repetition number of 4 and a mini promoter, and the nucleic acid sequence of the Hif1a regulatory element is shown as SEQ ID NO: 1. The promoter is 4HRE-CMVmin promoter.
Specifically, the hif1α regulatory element is a tandem of 4 repeats, where the tandem refers to not only that two nucleic acid sequences are directly connected, i.e., the 3 'end of one nucleic acid sequence is directly connected to the 5' end of the other nucleic acid sequence, but also that two nucleic acid sequences may be indirectly connected, i.e., other nucleic acid sequences may be connected between the two nucleic acid sequences, so long as the expression of the functions of the two nucleic acid sequences is not affected.
Further, the promoter is under the control of an anoxic 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.
Preferably, the mini-promoter selects miniCMV.
Further, the structure of the Hif1 alpha regulating element is optimized to obtain an optimized Hif1 alpha regulating element, and the nucleotide sequence of the optimized Hif1 alpha regulating element is shown as SEQ ID NO:2; the Hif1α regulatory element may be optimized to accommodate different expression vectors.
Further, the nucleic acid sequence of the promoter is shown as SEQ ID NO: 3.
Further, the CAR structure comprises a promoter regulated by the anaerobic environment, and the nucleotide sequence of the promoter is shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3, sequence 3.
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 an improved double CAR structure, a regulatable CAR structure (such as FRB/FKBP12 regulation) and the like.
Further, the antigen recognition region of the CAR structure is an 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 in SEQ ID NO. 10 or SEQ ID NO. 11 or SEQ ID NO. 12 or a functional variant thereof. .
Specifically, the hinge region sequence in the CAR structure may be derived from: igG, CD8, CD7, CD4; the transmembrane region in the CAR structure may be derived from: CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, and CD86; the intracellular signaling region in the CAR structure can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2 3583, CD86 and CD127.
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 as a functional variant thereof.
Further, the amino acid sequence of the intracellular activation signal of the CAR structure is shown in SEQ ID NO. 17 or a functional variant thereof.
Further, the amino acid sequence of the CAR structure is shown in any one of SEQ ID NOs 18-21 or a functional variant thereof.
In certain embodiments, the CAR structure comprising the hypoxia promoter carries a truncated EGFRt regulatory tag; in certain embodiments, the CAR comprising the hypoxia promoter is a universal CAR structure; in certain embodiments, the CAR structure comprising the hypoxia promoter carries a suicide gene such as iCasp9.
In certain embodiments, the antigen recognition region may be a ligand/receptor that recognizes the target antigen; in certain embodiments, the ScFv may be a murine or fully human or a human-murine chimeric ScFv, may be a single domain antibody such as a shark, alpaca or camelbody, may be a bispecific antibody, or may be a combination of artificially designed target-specific fibronectin type III (FN 3) domains that recognize a specific target.
In certain embodiments, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component may be a transmembrane domain, hinge domain or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR DL5B, KIR DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural Cytotoxic Receptors (NCR), e.g., NKp30, NKp44, NKp46; a family of Signaling Lymphocyte Activation Molecules (SLAM) for immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; fc receptors (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g., ly49A, LY C. The NKR-CAR molecule may interact with an adapter 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 or 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-maedi virus (VMV), 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 encompassed herein, and a right (3') retroviral LTR.
In certain embodiments, the CAR comprises a hepatitis b virus posttranscriptional regulatory element (HPRE) or a Woodchuck Posttranscriptional Regulatory Element (WPRE) and an optimized woodchuck posttranscriptional 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 (Rous Sarcoma Virus, RSV) promoter, or a simian virus 40 (SV 40) 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, the polynucleotides encoding the CARs contemplated herein comprise an optimized Kozark sequence.
In certain embodiments, the promoter operably linked to a polynucleotide encoding a CAR encompassed herein and the group consisting of: the cytomegalovirus immediate early gene promoter (CMV), the elongation factor 1 alpha promoter (EF 1-alpha), the phosphoglycerate kinase-1 Promoter (PGK), the ubiquitin-C promoter (UBQ-C), the cytomegalovirus enhancer/chicken beta-actin promoter (CAG), the polyoma enhancer/herpes simplex thymidine kinase promoter (MC 1), the beta actin promoter (beta-ACT), the simian virus 40 promoter (SV 40) and the myeloproliferative sarcoma virus enhancer, the negative control region deleted, the dl587rev primer binding site substituted (MND) promoter.
In certain embodiments, the CAR-comprising vector may comprise a secreted anti-PD-1 ScFv; in certain embodiments, the CAR-comprising vector comprises a PD-1 conjugated transduction peptide (e.g., a PD-1-CD28-CD137-CD3 signal structure); in certain embodiments, the vector comprising the CAR comprises multiple 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 as set forth in any one of SEQ ID NOS.4-7, in particular, SEQ ID NOS.4-7 comprises a nucleotide sequence encoding a CAR structure and a promoter sequence.
Further, CAR-T cells 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 has substantially the same function as it.
It is a second object of the present invention to provide an isolated nucleic acid molecule.
In order to achieve the above purpose, the present invention adopts the following scheme:
the nucleic acid molecule encodes the CAR structure.
It is a further object of the invention to provide the use of said promoter, CAR structure, expression vector and CAR-T cell.
In order to achieve the above purpose, the present invention adopts the following scheme:
the application is the application in preparing tumor medicaments, wherein the tumor is malignant tumor and comprises acute lymphoid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, non-Hodgkin lymphoma, prostate cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, brain cancer and skin cancer.
Further, 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. .
In particular, the cells may be used in combination with other agents and/or treatments that may enhance the activity of CAR expression.
In particular, the active agent and/or treatment may be surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine (cycloporin), azathioprine (azathioprine), methotrexate (methotreate), mycophenolic acid esters (mycophenolate) and FK506, antibodies or other immune scavengers (immunodepleters) such as CAMPATH, anti-CD 3 antibodies or other antibody therapies, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine (cycloporin), FK506, rapamycin (rapamycin), mycophenolic acid (mycophenolic acid), steroids (steroids), FR901228, cytokines and radiation.
In certain embodiments, the cells can express other active agents, e.g., an agent that enhances the activity of a CAR-expressing cell. The active agent may be an agent that blocks an inhibitory molecule. 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 (CD 276), B7-H4 (VTCN 1), HVEM (TNFRSF 14 or CD 270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFR beta) and TGFR beta. The extracellular domain of the inhibitory molecule may be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1CAR.
The third object of the invention is to provide a method for improving the killing capacity of CAR-T cells in an anoxic environment. The CAR-T cells can be induced and activated in the presence of the anoxic environment and the target antigen, the CAR expression capacity and the abundance of the target antigen after stimulation in the anoxic environment are improved, the CAR-T effectiveness is enhanced, the CAR expression capacity and the abundance of the target antigen after stimulation in the non-anoxic environment are lower than those of the conventional CAR-T, and the safety is higher.
In order to achieve the above purpose, the present invention adopts the following scheme:
the method is to construct an expression vector of the CAR structure, 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 hypoxia microenvironment-induced promoter provided by the invention can strengthen the target gene expression in the hypoxia environment;
2) The CAR structure induced and started by the anoxic microenvironment provided by the invention not only can be effectively expressed in T lymphocytes, but also can strengthen the activation of the CAR-T cells in the anoxic environment, improves the killing of the CAR-T cells to tumor target cells, and can be used for targeted treatment 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 preparing adoptive cell therapy in medicines for treating malignant hematopathy and malignant solid tumors.
Drawings
FIG. 1 is a diagram of a structure of a promoter.
Figure 2 is a validation of hypoxia induction protocol.
FIG. 3 is a graph showing the effect of hypoxia-inducible drugs on cell proliferation.
Figure 4 is a CAR positive rate assay.
Figure 5 is a CAR expression intensity assay following hypoxia induction.
FIG. 6 is a graph of the validation of the effectiveness of promoter engineering CAR-T.
FIG. 7 is a comparison of the activity of promoter engineered CAR-T in both anoxic and non-anoxic environments.
FIG. 8 is a comparison of factor secretion after promoter engineering CAR-T activity in both anoxic and non-anoxic environments.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental methods in the preferred embodiments, in which specific conditions are not noted, are generally carried out according to conventional conditions, for example, those described in the guidelines for molecular cloning experiments (third edition, J. Sam Brookfield et al), or according to the manufacturer's recommendations. The examples are presented for better illustration of the present invention, but are not intended to limit the scope of the present invention to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1: plasmid construction
Constructing a promoter 4 HRE-CMVini promoter-NheI (4H 1P) matched with a lentiviral vector, cutting and recovering fragments by double enzyme digestion, connecting gene fragments, transforming and picking monoclone to obtain a recombinant vector, wherein the numbers of the vectors are 1, 2, 3 and 4 respectively, and the nucleic acid sequence is shown as SEQ ID NO. 3. The CAR structural element comprises: scFv of targeting CEA or PSCA with amino acid sequence as SEQ ID NO 8 or SEQ ID NO 9; the amino acid sequence is shown as a hinge structure of SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12; a transmembrane structure with an amino acid sequence shown as SEQ ID NO. 13 or SEQ ID NO. 14, an intracellular co-stimulatory domain with an amino acid sequence shown as SEQ ID NO. 15 or SEQ ID NO. 16, and an intracellular activation signal with an amino acid sequence shown as SEQ ID NO. 17; the nucleotide sequences of the obtained CAR modified by the anoxic promoter are shown as SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, and the amino acid sequences are shown as SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21; FIG. 1 is a diagram of a structure of a promoter. .
Example 2: in vitro hypoxia model validation
CoCl 2 Principle of induced hypoxia: cobalt dichloride (CoCl) 2 ) Of divalent cobalt ions (Co 2+ ) Can replace ferrous ions of PHD cofactor because of Co 2+ The affinity for oxygen is low such that heme cannot bind to oxygen and cannot be altered to an oxidized state, thus forming an anoxic state.
1) Hypoxia induction protocol validation:
infection of living organisms with nHRE-GFP virusThe PBMC is constructed into an in vitro hypoxia cell model, and the culture is carried out for 12-18 hours and then the liquid is changed. By CoCl 2 Inducing anoxic environment, culturing until 4 days, checking whether anoxic model is constructed by green fluorescence detection and WB, and respectively setting control group (without CoCl) 2 ) Experimental group (CoCl added) 2 ) Fluorescence microscopy was performed to measure the expression intensity of GFP after 24 hours of drug treatment compared to the positive group (normal infected GFP cells without hypoxia regulated promoter). And the expression of HIF-1a in the cells was examined by extracting the protein according to the instructions of M-PER Mammalian Protein Extraction Reagent (Thermo).
As a result, as shown in FIG. 2, it was found that CoCl was added 2 The GFP expression in the induction group (i.e. the microenvironment hypoxia group) was significantly stronger than that in the absence of CoCl 2 The fluorescence intensity of the group is far stronger than that of a positive control group, which shows that the hypoxia promoter of PBKL1-nHRE-GFP can normally start the expression of downstream GFP under the hypoxia environment.
2) Hypoxia-inducible drugs affecting cell proliferation
Appropriate amounts of Hela and PBMC were plated in 96-well plates in the morning, and CoCl was performed after 5h of cell attachment 2 Final concentration 0, 100, 200, 300, 400, 500uM is treated by adding the incubated CellTiter-Glo One Solution Assay reagent according to the volume of 1:1 after 24 hours, and the mixture is incubated for 10 minutes at room temperature and then is put into an enzyme-labeled instrument for detecting absorbance. The results are shown in FIG. 3, coCl 2 The addition of (C) does not affect the proliferation of the primordial cells, so the result of killing the CAR-T cells is not subjected to exogenously added CoCl 2 The effect is only relevant to whether or not a hypoxia promoter is included.
Example 3: preparation of lentiviruses and infected T lymphocytes
The embodiment of the packaging lentivirus adopts a calcium phosphate method, which is specifically as follows: 293T cells were cultured to a preferred state with DMEM medium containing 10% FBS (w/v) to ensure a cell confluence of 70-80% at transfection, and the cells were pre-thawed. The packaging plasmid (RRE: REV: 2G) and the expression plasmid were added in a specific ratio to a 1.5 centrifuge tube, and 2.5mol/LCaCl was added 2 Supplement ddH 2 O was added dropwise to the mixture with a pipette to a total volume of 600ul, and 2 XHBS was added dropwiseMixing, 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 virus, and determining titer.
Lymphocyte separation by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was 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 cells are infected according to a certain multiplicity of infection (MOI) and polybrene is added to help virus infection, liquid is changed (10% RPMI 1640+IL-2+ double antibodies) after the PBMC cells are cultured overnight, the culture is continued in a 37 ℃ incubator, the positive rate of CAR-T is detected on the 12 th day of virus infection, and the detection method is flow detection, wherein the antibodies are as follows: protein-L-PE, protein-L can recognize antibody light chain, light chain of ScFv sequence of CAR antigen recognition region can be recognized by Protein-L, so that the positive rate of CAR and the expression intensity of CAR can be detected by utilizing Protein-L.
The results are shown in FIG. 4: the CAR-T cells with different structures and modified by the 4H1P promoter have remarkably increased CAR expression under the induced hypoxia environment.
The results are shown in FIG. 5: different-structure CAR-T cells modified by the 4H1P promoter, wherein a black histogram is the CAR expression intensity of the CAR modified by the promoter in a non-anoxic environment; the white histogram is the CAR expression intensity of the promoter engineered CAR in an anoxic environment, with increased CAR expression intensity in an induced anoxic environment.
Example 4: detection of CAR-T availability comprising hypoxia promoter after hypoxia induction
And respectively taking PSCA positive Hela cells and in-vitro modified CEA positive DLD1-CEA cells as target cells to verify the effective function of the modified CAR-T. Effector cells (CAR-T cells containing the modified CAR expressed by the promoter) are subjected to anoxic pretreatment overnight, then the CAR-T cells are collected and paved in target cells according to a certain effective target ratio, and the killing capacity of different CAR-T cells on the target cells is detected by using a ACEA xCELLigence RTCA MP instrument, and the experimental steps are carried out according to the instrument instruction. ACEA xCELLigence RTCA MP the principle is that tumor cells attached to the bottom of the well are recorded every 15 minutes with the resistance index as data, and proliferation or death of the attached target cells is judged by the resistance index. The formula of the analysis result by using the resistance index is as follows: CAR-T cell killing = baseline resistance index-real-time resistance index.
The results are shown in FIG. 6: CAR-T cells of different structure comprising the hypoxia promoter 4H1P were able to function normally compared to the control group.
FIG. 7 further demonstrates the regulation of CAR-T killing activity by modified hypoxia-specific promoters in both hypoxic and non-hypoxic environments, and shows that the hypoxia-specific promoters can specifically induce efficient killing of CAR-T in hypoxic environments at a low target ratio of 1:5, whereas conventional CAR-T has very poor killing capacity in hypoxic environments, with specific killing percentages as shown in the following table.
Example 5: elisa assay IFN-gamma assay
The supernatant collected in example 4 was assayed for secretion of IFN-. Gamma.and IL-2 by ELISA (enzyme-linked immunosorbent assay). IFN-gamma detection adopts BD IFN-gamma kit detection, the product number is 555142, and the experimental steps are carried out according to the product instruction.
The results are shown in fig. 8, treat represents factor secretion after target cell killing of the promoter-modified CAR-T cells in a hypoxic environment, and un Treat represents factor secretion after target cell killing of the promoter-modified CAR-T cells in a non-hypoxic environment. The results show that higher IFN-gamma secretion is only possible after activation of the promoter engineered CAR-T cells in hypoxic environments.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Sequence listing
<110> Chongqing precision biotechnology Co., ltd
<120> a promoter for controlling CAR-T specific activation and use thereof
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 158
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagccacagt gcatacgtgg gctccaacag gtcctctt 158
<210> 2
<211> 179
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
gtcgacgata tcccacagtg catacgtggg ctccaacagg tcctcttgtc gagccacagt 60
gcatacgtgg gctccaacag gtcctcttgt cgagccacag tgcatacgtg ggctccaaca 120
ggtcctcttg tcgagccaca gtgcatacgt gggctccaac aggtcctctt gtcgagatc 179
<210> 3
<211> 233
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagatctgg taggcgtgta 180
cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcac tag 233
<210> 4
<211> 2261
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagatctgg taggcgtgta 180
cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcac taggctagca 240
tggccctgcc agtgaccgcc ctgctgctgc ccctggccct gctgctgcac gcagcacggc 300
ccgacatcca ggatatccag ctgacacagt cccctagctc cctgagcgcc tccgtgggcg 360
acagggtgac catcacatgc agcgcctcta gctccgtgag attcatccac tggtaccagc 420
agaagcccgg caaggcccct aagaggctga tctatgatac ctctaagctg gccagcggag 480
tgccctcccg cttctctggc agcggctccg gcaccgactt taccctgaca atctctagcc 540
tgcagccaga ggattttgcc acatactatt gccagcagtg gtcctctagc cccttcacct 600
ttggccaggg cacaaaggtg gagatcaagg gatctaccag cggatccggc aagcctggat 660
ctggagaggg cagcacaaag ggctccgagg tgcagctggt ggagtccgga ggaggactgg 720
tgcagccagg aggatctctg aggctgagct gtgcagcctc cggcttcaac atcaaggact 780
actatatcca ctgggtgaga caggcacctg gcaagggact ggagtgggtg gcatggatcg 840
accctgagaa tggcgatacc gagttcgtgc caaagtttca gggcagggcc acaatctctg 900
ccgacaccag caagaacaca gcctacctgc agatgaatag cctgcgcgcc gaggataccg 960
ccgtgtacta ttgtaagaca ggaggatttt ggggacaggg caccctggtg acagtgtcct 1020
ctctcgagga gagcaagtac ggccctccct gccccccttg ccctgccccc gagttcctgg 1080
gcggacccag cgtgttcctg ttccccccca agcccaagga caccctgatg atcagccgga 1140
cccccgaggt gacctgtgtg gtggtggacg tgtcccagga ggaccccgag gtccagttca 1200
actggtacgt ggacggcgtg gaggtgcaca acgccaagac caagccccgg gaggagcagt 1260
tcaatagcac ctaccgggtg gtgtccgtgc tgaccgtgct gcaccaggac tggctgaacg 1320
gcaaggaata caagtgtaag gtgtccaaca agggcctgcc cagcagcatc gagaaaacca 1380
tcagcaaggc caagggccag cctcgggagc cccaggtgta caccctgccc cctagccaag 1440
aggagatgac caagaatcag gtgtccctga cctgcctggt gaagggcttc taccccagcg 1500
acatcgccgt ggagtgggag agcaacggcc agcccgagaa caactacaag accacccccc 1560
ctgtgctgga cagcgacggc agcttcttcc tgtacagcag gctgaccgtg gacaagagcc 1620
ggtggcagga gggcaacgtc tttagctgct ccgtgatgca cgaggccctg cacaaccact 1680
acacccagaa gagcctgtcc ctgagcctgg gcaaggaatt cttctgggtg ctggtcgtgg 1740
tgggtggcgt gctggcctgc tacagcctgc tggtgacagt ggccttcatc atcttttggg 1800
tgaggagcaa gcggagcaga ggcggccaca gcgactacat gaacatgacc ccccggaggc 1860
ctggccccac ccggaagcac taccagccct acgcccctcc cagggacttc gccgcctacc 1920
ggagccgggt gaagttcagc cggagcgccg acgcccctgc ctaccagcag ggccagagcc 1980
agctgtacaa cgagctgaac ctgggccgga gggaggagta cgacgtgctg gacaagcgga 2040
gaggccggga ccctgagatg ggcggcaagc cccggagaaa gaaccctcag gagggcctgt 2100
ataacgaact gcagaaagac aagatggccg aggcctacag cgagatcggc atgaagggcg 2160
agcggcggag gggcaagggc cacgacggcc tgtaccaggg cctgagcacc gccaccaagg 2220
atacctacga cgccctgcac atgcaggccc tgccccctcg c 2261
<210> 5
<211> 2282
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagatctgg taggcgtgta 180
cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcac taggctagca 240
tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac gccgccaggc 300
cggacatcca gatgacccag agcccaagca gcctgagcgc cagcgtgggt gacagagtga 360
ccatcacctg tagtaccagt tcgagtgtaa gttacatgca ctggtaccag cagaagccag 420
gtaaggctcc aaggctgctg atctacagca catccaacct ggcttctggt gtgccaagca 480
gattcagcgg tagcggtagc ggtaccgact tcaccttcac catcagcagc ctccagccag 540
aggacatcgc cacctactac tgccatcagt ggagtagtta tcccacgttc ggccaaggga 600
ccaaggtgga aatcaaagga tccacttccg gttcaggaaa gcccgggagt ggtgaaggta 660
gcactaaagg ccaggtccag ctgcaggaga gcggtccagg tcttgtgaga cctagccaga 720
ccctgagcct gacctgcacc gtgtctggct tcaccatcag cagtggttat agctggcact 780
gggtgagaca gccacctgga cgaggtcttg agtggattgg atacatacag tacagtggta 840
tcactaacta caacccctct ctcaaaagta gagtgacaat gctggtagac accagcaaga 900
accagttcag cctgagactc agcagcgtga cagccgccga caccgcggtc tattattgtg 960
caagagaaga ctatgattac cactggtact tcgatgtctg gggtcaaggc agcacggtca 1020
ccgtctcctc aggtgcggcc gccctcgagg agagcaagta cggccctccc tgcccccctt 1080
gccctgcccc cgagttcctg ggcggaccca gcgtgttcct gttccccccc aagcccaagg 1140
acaccctgat gatcagccgg acccccgagg tgacctgtgt ggtggtggac gtgtcccagg 1200
aggaccccga ggtccagttc aactggtacg tggacggcgt ggaggtgcac aacgccaaga 1260
ccaagccccg ggaggagcag ttcaatagca cctaccgggt ggtgtccgtg ctgaccgtgc 1320
tgcaccagga ctggctgaac ggcaaggaat acaagtgtaa ggtgtccaac aagggcctgc 1380
ccagcagcat cgagaaaacc atcagcaagg ccaagggcca gcctcgggag ccccaggtgt 1440
acaccctgcc ccctagccaa gaggagatga ccaagaatca ggtgtccctg acctgcctgg 1500
tgaagggctt ctaccccagc gacatcgccg tggagtggga gagcaacggc cagcccgaga 1560
acaactacaa gaccaccccc cctgtgctgg acagcgacgg cagcttcttc ctgtacagca 1620
ggctgaccgt ggacaagagc cggtggcagg agggcaacgt ctttagctgc tccgtgatgc 1680
acgaggccct gcacaaccac tacacccaga agagcctgtc cctgagcctg ggcaaggaat 1740
tcttctgggt gctggtcgtg gtgggtggcg tgctggcctg ctacagcctg ctggtgacag 1800
tggccttcat catcttttgg gtgaggagca agcggagcag aggcggccac agcgactaca 1860
tgaacatgac cccccggagg cctggcccca cccggaagca ctaccagccc tacgcccctc 1920
ccagggactt cgccgcctac cggagccggg tgaagttcag ccggagcgcc gacgcccctg 1980
cctaccagca gggccagagc cagctgtaca acgagctgaa cctgggccgg agggaggagt 2040
acgacgtgct ggacaagcgg agaggccggg accctgagat gggcggcaag ccccggagaa 2100
agaaccctca ggagggcctg tataacgaac tgcagaaaga caagatggcc gaggcctaca 2160
gcgagatcgg catgaagggc gagcggcgga ggggcaaggg ccacgacggc ctgtaccagg 2220
gcctgagcac cgccaccaag gatacctacg acgccctgca catgcaggcc ctgccccctc 2280
gc 2282
<210> 6
<211> 1724
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
gatatcccac agtgcatacg tgggctccaa caggtcctct tgtcgagcca cagtgcatac 60
gtgggctcca acaggtcctc ttgtcgagcc acagtgcata cgtgggctcc aacaggtcct 120
cttgtcgagc cacagtgcat acgtgggctc caacaggtcc tcttgtcgag atctggtagg 180
cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcactagg 240
ctagcatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg ctccacgccg 300
ccaggccgga catccagatg acccagagcc caagcagcct gagcgccagc gtgggtgaca 360
gagtgaccat cacctgtagt accagttcga gtgtaagtta catgcactgg taccagcaga 420
agccaggtaa ggctccaagg ctgctgatct acagcacatc caacctggct tctggtgtgc 480
caagcagatt cagcggtagc ggtagcggta ccgacttcac cttcaccatc agcagcctcc 540
agccagagga catcgccacc tactactgcc atcagtggag tagttatccc acgttcggcc 600
aagggaccaa ggtggaaatc aaaggatcca cttccggttc aggaaagccc gggagtggtg 660
aaggtagcac taaaggccag gtccagctgc aggagagcgg tccaggtctt gtgagaccta 720
gccagaccct gagcctgacc tgcaccgtgt ctggcttcac catcagcagt ggttatagct 780
ggcactgggt gagacagcca cctggacgag gtcttgagtg gattggatac atacagtaca 840
gtggtatcac taactacaac ccctctctca aaagtagagt gacaatgctg gtagacacca 900
gcaagaacca gttcagcctg agactcagca gcgtgacagc cgccgacacc gcggtctatt 960
attgtgcaag agaagactat gattaccact ggtacttcga tgtctggggt caaggcagca 1020
cggtcaccgt ctcctcaggt gcggccgccc tcgagaccac gacgccagcg ccgcgaccac 1080
caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc 1140
cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat atctacatct 1200
gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact 1260
gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg agaccagtac 1320
aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa gaaggaggat 1380
gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag cagggccaga 1440
accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt ttggacaaga 1500
gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct caggaaggcc 1560
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt gggatgaaag 1620
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt acagccacca 1680
aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 1724
<210> 7
<211> 1724
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ccacagtgca tacgtgggct ccaacaggtc ctcttgtcga gccacagtgc atacgtgggc 60
tccaacaggt cctcttgtcg agccacagtg catacgtggg ctccaacagg tcctcttgtc 120
gagccacagt gcatacgtgg gctccaacag gtcctcttgt cgagatctgg taggcgtgta 180
cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcac taggctagca 240
tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac gccgccaggc 300
cggacatcca gatgacccag agcccaagca gcctgagcgc cagcgtgggt gacagagtga 360
ccatcacctg tagtaccagt tcgagtgtaa gttacatgca ctggtaccag cagaagccag 420
gtaaggctcc aaggctgctg atctacagca catccaacct ggcttctggt gtgccaagca 480
gattcagcgg tagcggtagc ggtaccgact tcaccttcac catcagcagc ctccagccag 540
aggacatcgc cacctactac tgccatcagt ggagtagtta tcccacgttc ggccaaggga 600
ccaaggtgga aatcaaagga tccacttccg gttcaggaaa gcccgggagt ggtgaaggta 660
gcactaaagg ccaggtccag ctgcaggaga gcggtccagg tcttgtgaga cctagccaga 720
ccctgagcct gacctgcacc gtgtctggct tcaccatcag cagtggttat agctggcact 780
gggtgagaca gccacctgga cgaggtcttg agtggattgg atacatacag tacagtggta 840
tcactaacta caacccctct ctcaaaagta gagtgacaat gctggtagac accagcaaga 900
accagttcag cctgagactc agcagcgtga cagccgccga caccgcggtc tattattgtg 960
caagagaaga ctatgattac cactggtact tcgatgtctg gggtcaaggc agcacggtca 1020
ccgtctcctc aggtgcggcc gccctcgaga agccaaccac aaccccagca ccaaggccac 1080
ctacacctgc accaaccatc gcaagccagc cactgtccct gaggccagag gcaagacctg 1140
cagcaggagg cgccgtgcac acacggggcc tggacttcgc cgatgaattc atctacatct 1200
gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact 1260
gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg agaccagtac 1320
aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa gaaggaggat 1380
gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag cagggccaga 1440
accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt ttggacaaga 1500
gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct caggaaggcc 1560
tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt gggatgaaag 1620
gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt acagccacca 1680
aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 1724
<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> 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 (12)

1. A CAR structure linked to a hypoxia promoter, wherein the promoter has a nucleotide sequence as set forth in SEQ ID NO:3 is shown in the figure; the antigen recognition region of the CAR structure is an ScFv, and the amino acid sequence of the ScFv is shown as SEQ ID NO. 9; the amino acid sequence of the CAR structure is shown as SEQ ID NO. 20 or SEQ ID NO. 21.
2. The CAR structure of claim 1, wherein the amino acid sequence of the hinge region of the CAR structure is set forth in SEQ ID No. 11 or SEQ ID No. 12.
3. The CAR structure of claim 1, wherein the amino acid sequence of the transmembrane region of the CAR structure is set forth in SEQ ID No. 14.
4. The CAR structure of claim 1, wherein the intracellular co-stimulatory domain amino acid sequence of said CAR structure is set forth in SEQ ID No. 16.
5. The CAR structure of claim 1, wherein the amino acid sequence of the intracellular activation signal of the CAR structure is set forth in SEQ ID No. 17.
6. An isolated nucleic acid molecule encoding the CAR structure of any one of claims 1-5.
7. A CAR expression vector comprising an anoxic promoter, wherein the nucleotide sequence of the anoxic promoter is as set forth in SEQ ID NO:3 is shown in the figure; the CAR structure is a CAR structure according to any one of claims 1 to 5; 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.
8. The CAR expression vector of claim 7, wherein the vector comprises a nucleotide sequence set forth in SEQ ID No. 6 or SEQ ID No. 7.
9. A CAR-T cell comprising the expression vector of claim 7 or 8.
10. Use of the CAR expression vector of claim 7 in the preparation of a formulation for improving the killing capacity of CAR-T cells in an anoxic environment.
11. Use of the CAR structure of claim 1 or the nucleic acid molecule of claim 6 or the expression vector of claim 7 or the CAR-T cell of claim 9 in the preparation of a medicament for treating a tumor that highly expresses PSCA.
12. The use according to claim 11, wherein the tumour is cervical cancer.
CN201911236670.2A 2019-03-12 2019-12-05 Promoter for regulating and controlling CAR-T specific activation and application thereof Active CN112921034B (en)

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JP2021555018A JP7391984B2 (en) 2019-03-12 2020-02-24 Hypoxia-regulated promoter and its application
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007038757A2 (en) * 2005-09-28 2007-04-05 Attagene Inc. Methods and compositions for non-invasive assessment of gene expression
CN101200732A (en) * 2007-07-10 2008-06-18 西安交通大学 Hypoxia response elements gene treating plasmid and its constructing method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007038757A2 (en) * 2005-09-28 2007-04-05 Attagene Inc. Methods and compositions for non-invasive assessment of gene expression
CN101200732A (en) * 2007-07-10 2008-06-18 西安交通大学 Hypoxia response elements gene treating plasmid and its constructing method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Abstract A041: Hypoxia-responsive CAR T-cells;Tina Anna Sarén等;《Cancer Immunology Reserch》;20190228;第7卷(第2期);第1页 *
Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells;Christopher Ede等;《ACS Synthetic Biology》;20160301(第5期);第400页左栏和右栏第1段、第401页左栏、图2 *

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