WO2021093831A1 - 一种在活化的t细胞中具有高活性的启动子 - Google Patents

一种在活化的t细胞中具有高活性的启动子 Download PDF

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WO2021093831A1
WO2021093831A1 PCT/CN2020/128526 CN2020128526W WO2021093831A1 WO 2021093831 A1 WO2021093831 A1 WO 2021093831A1 CN 2020128526 W CN2020128526 W CN 2020128526W WO 2021093831 A1 WO2021093831 A1 WO 2021093831A1
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promoter
antibody
vector
nucleic acid
cells
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PCT/CN2020/128526
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French (fr)
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刘韬
方媛
高海霞
钱其军
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上海细胞治疗集团有限公司
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Priority to CA3161286A priority Critical patent/CA3161286A1/en
Priority to US17/776,574 priority patent/US20220372481A1/en
Priority to JP2022528592A priority patent/JP2023502098A/ja
Priority to EP20886705.1A priority patent/EP4060042A4/en
Priority to CN202080078285.4A priority patent/CN114729361B/zh
Publication of WO2021093831A1 publication Critical patent/WO2021093831A1/zh

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    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to a promoter with high activity in activated T cells.
  • a promoter is a component of a gene, usually located upstream of the 5'end of a structural gene, and is a DNA sequence that RNA polymerase recognizes, binds, and starts transcription. Promoters are one of the important factors that affect the efficiency of transgene expression. Choosing high-efficiency promoters is the key to high-efficiency expression of foreign genes.
  • promoters According to the transcription mode of promoters, they can be divided into three categories: constitutive promoters, tissue or organ-specific promoters and inducible promoters.
  • a constitutive promoter means that there is no obvious difference in gene expression in different tissues, organs and developmental stages under the regulation of a constitutive promoter, so it is called a constitutive promoter.
  • Constitutive promoters commonly used in mammals include virus sources: murine or human cytomegalovirus (CMV) promoters (abbreviated as mCMV and hCMV respectively), monkey vacuolar virus SV40 promoters; natural sources of human genome: EF1 ⁇ promoter, pan Ubiquitin (Ubiquitin, Ubi for short), ⁇ -actin promoter, PGK-1 promoter, Rosa26 promoter, HSP70 promoter, GAPDH promoter, eIF4A1 promoter, Egr1 promoter, FerH promoter, SM22 ⁇ promoter, Endothelin-1 promoter and so on.
  • CMV cytomegalovirus
  • mCMV and hCMV monkey vacuolar virus SV40 promoters
  • natural sources of human genome EF1 ⁇
  • the researchers have designed and constructed a series of artificial chimeric promoters, which contain some cis-regulatory elements, mainly including the core sequence of the promoter that can play a stable expression role, and the upstream enhancer or downstream enhancer that can enhance the expression efficiency.
  • Intron the representative is the chimeric promoter CAG (including human CMV enhancer-chicken ⁇ -actin promoter-rabbit ⁇ -globin intron), which is widely used in the expression of foreign genes.
  • Enhancer refers to a DNA sequence that increases the transcription frequency of genes linked to it. Enhancers use promoters to increase transcription of downstream genes. Effective enhancers can be located at the 5'end of the gene, or at the 3'end of the gene, and some can also be located in the intron of the gene. The effect of enhancers is obvious, generally they can increase the frequency of gene transcription by 10-200 times, and some can even be as high as thousands of times.
  • the present invention constructs a promoter combined with CMV enhancer, IFN ⁇ promoter and HTLV (human T cell leukemia virus) long terminal repeat (LTR) sequence.
  • the promoter exhibits a higher ratio in activated immune cells.
  • Existing promoters are more active, but have low or no activity in other non-immune cells.
  • the present invention provides a promoter, which comprises the CMV enhancer, the IFN ⁇ promoter and the long terminal repeat sequence of human T cell leukemia virus sequentially connected from the 5'end to the 3'end.
  • the CMV enhancer is selected from: the CMV enhancer having the nucleotide sequence shown in SEQ ID NO: 8, or the CMV enhancer from human CMV and the nucleotide sequence shown in SEQ ID NO: 8 A CMV enhancer whose sequence has at least 97% sequence identity.
  • the IFN ⁇ promoter is selected from: IFN ⁇ promoter having the nucleotide sequence shown in SEQ ID NO: 4, or human-derived and the nucleotide sequence shown in SEQ ID NO: 4 IFN ⁇ promoter with at least 97% sequence identity.
  • the long terminal repeat sequence of the human T cell leukemia virus is selected from the group consisting of: a long terminal repeat sequence having the nucleotide sequence shown in SEQ ID NO: 3, or a long terminal repeat sequence derived from a human T cell leukemia virus A long terminal repeat sequence having at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3.
  • the present invention also provides a nucleic acid molecule whose base sequence is complementary to that of the promoter.
  • the present invention also provides a nucleic acid construct, which contains the promoter of the present invention and a gene of interest operably linked to the promoter.
  • the nucleic acid construct is an expression cassette.
  • the gene of interest encodes an autocrine antibody, preferably an immune checkpoint antibody, such as PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody , TIGIT antibody and VISTA antibody, more preferably Nanobodies derived from alpaca.
  • an immune checkpoint antibody such as PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody , TIGIT antibody and VISTA antibody, more preferably Nanobodies derived from alpaca.
  • the gene of interest encodes a cytokine.
  • the present invention also provides a vector, which contains the promoter or nucleic acid construct of the present invention.
  • the vector is an expression vector or a cloning vector.
  • the host cell is an immune cell, preferably a T cell, whose genome integrates the nucleic acid construct described in any of the embodiments herein; preferably, the immune cell also expresses CAR or Expression vector containing CAR.
  • promoter of the present invention in increasing the expression of the gene of interest in activated immune cells, or in the preparation of nucleic acid constructs or vectors for enhancing expression in activated immune cells application.
  • Figure 1 pS338B-EGFP plasmid map.
  • Figure 2 pS-IFPT-EGFP plasmid map.
  • Figure 3 pS-IL3en-EGFP plasmid map.
  • Figure 4 pS-uIFP-EGFP plasmid map.
  • Figure 5 pS-uIFPT-EGFP plasmid map.
  • Figure 6 pS-CIFT-EGFP plasmid map.
  • Figure 7 pS-ILFP-EGFP plasmid map.
  • Figure 8 pS-ILPT-EGFP plasmid map.
  • Figure 9 pS-uILP-EGFP plasmid map.
  • Figure 10 pS-uILT-EGFP plasmid map.
  • Figure 11 pS-CILT-EGFP plasmid map.
  • Figure 12 pS-ILFP-EGFP plasmid map.
  • Figure 13 pS-IFen-EGFP plasmid map.
  • Figure 14 pS-uIFen-EGFP plasmid map.
  • Figure 15 pS-CIFen-EGFP plasmid map.
  • Figure 16 pS-LIFen-EGFP plasmid map.
  • Figure 17 pS338B-Fluc plasmid map.
  • Figure 18 pS-IFPT-Fluc plasmid map.
  • Figure 19 pS-CIFT-Fluc plasmid map.
  • Figure 20 pS-ILFP-Fluc plasmid map.
  • Figure 21 pS-ILPT-Fluc plasmid map.
  • Figure 22 pS-CILT-Fluc plasmid map.
  • Figure 23 pS338B- ⁇ PD1 plasmid map.
  • Figure 24 pS-CIFT- ⁇ PD1 plasmid map.
  • Figure 25 Comparison of the effects of IFN- ⁇ gene promoter and IL2 gene promoter on the activity of chimeric gene promoters.
  • Figure 26 Comparison of the effects of TLTR and IFN- ⁇ intron enhancers on the activity of chimeric gene promoters.
  • Figure 27 The effect of different activation methods on the expression of two plasmids eGFP.
  • Figure 28 Test results of dual luciferase reporter system.
  • Figure 29 Expression activity of cytokine gene chimeric promoter in HEK 293T cells.
  • Figure 30 Expression activity of cytokine gene chimeric promoter in CHO cells.
  • Figure 31 Expression activity of cytokine gene chimeric promoter in daughter DC cells.
  • Figure 32 Expression activity of cytokine gene chimeric promoter in Jurkat cells.
  • Figure 33 Expression activity of cytokine gene chimeric promoter in Raji cells.
  • Figure 34 Expression activity of cytokine gene chimeric promoter in Hep G2 cells.
  • Figure 35 Expression activity of cytokine gene chimeric promoter in SKOV3 cells.
  • Figure 36 The chimeric promoter of cytokine genes enhances CAR-T cell secretion of PD1 antibody.
  • the present invention improves the expression intensity of the driving gene by modifying the activity of the promoter, thereby realizing the high-efficiency expression of the exogenous gene in the activated immune cells.
  • immune cells refer to cells involved in or related to immune responses, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes and mast cells.
  • the preferred immune cells of the present invention are peripheral blood mononuclear cells (PBMC), including lymphocytes and monocytes.
  • Lymphocytes include T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes.
  • the cells suitable for the modified promoter of the present invention are immune cells that express high interferon-gamma by themselves.
  • activation refers to the use of corresponding activating reagents to stimulate the immune cells after the immune cells are transferred into the target plasmid or vector to achieve expansion to increase the number of immune cells and activation to enhance the activity of immune cells. the process of.
  • the activation reagent is a conventional activation reagent in the art, such as an anti-CD28 antibody and optionally a corresponding immunogen.
  • the transfer is a chimeric antigen receptor that expresses a certain tumor antigen
  • the activation reagent may also include the tumor antigen or an active fragment thereof.
  • the present invention has no special restrictions on the timing of activation, the concentration of the timing of activation, and the time of activation.
  • the present invention uses an anti-CD28 antibody for activation; in some embodiments, the present invention uses a combination of an anti-CD28 antibody and a tumor antigen for activation.
  • expression cassette refers to the complete elements required to express a gene, including an operably linked promoter and gene coding sequence.
  • Coding sequence refers to the part of the nucleic acid sequence that directly determines the amino acid sequence of its protein product.
  • the boundary of the coding sequence is usually determined by the ribosome binding site immediately upstream of the 5'open reading frame of the mRNA (for prokaryotic cells) and the transcription termination sequence immediately downstream of the 3'open reading frame of the mRNA. Coding sequences can include, but are not limited to DNA, cDNA, and recombinant nucleic acid sequences.
  • operably linked refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences.
  • a promoter is placed at a specific position of the nucleic acid sequence of the gene of interest, for example, the promoter is located at an upstream position of the nucleic acid sequence of the gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thereby ,
  • the promoter region is “operably linked” to the nucleic acid sequence of the gene. "Operable connection” can be achieved by means of gene recombination.
  • the enhancer of the present invention is a CMV enhancer.
  • CMV enhancers known in the art can be used to implement the present invention, including mCMV enhancers from murine cytomegalovirus and hCMV enhancers from human cytomegalovirus, preferably human CMV enhancers.
  • An exemplary CMV enhancer may have the nucleotide sequence shown in SEQ ID NO: 8.
  • the IFN ⁇ promoter of the present invention usually selects its core region sequence or a fragment of the IFN ⁇ promoter containing the core region sequence.
  • IFN ⁇ promoter core region sequence please refer to John C. Chrivia et al., "A model of human cytokine regulation based on transfection of gamma interference on gene fragments directly into isolated peripheral blood T lymphocytes", The Journal of Experiment, August 1990 , Volume 172, Pages 661-664.
  • An exemplary IFN ⁇ promoter is a fragment of the IFN ⁇ promoter having the nucleotide sequence shown in SEQ ID NO:4.
  • the promoter of the present invention uses the nucleotide sequence shown in SEQ ID NO:4.
  • the long terminal repeat sequence of the human T cell leukemia virus suitable for the present invention may have the nucleotide sequence shown in SEQ ID NO: 3.
  • the present invention also includes nucleotide sequences in which one or more base substitutions, deletions and/or additions are made to the nucleotide sequences shown in SEQ ID NOs: 3, 4 and 8, and substitutions, deletions and/or The sequence obtained by adding and modifying still retains the biological functions of SEQ ID NOs: 3, 4, and 8. For example, it can be performed separately or simultaneously at the 5'end and/or 3'end of the nucleotide sequence, and/or within the sequence, for example, no more than 20, such as no more than 15, or no more than 10, or no Substitution, deletion and/or addition of more than 8 or no more than 5 bases.
  • the present invention includes sequences having at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequences shown in SEQ ID NOs: 3, 4, and 8, respectively. Similarly, these sequences The respective biological functions of SEQ ID NOs: 3, 4 and 8 are also retained, and preferably these sequences are derived from human T-cell leukemia virus, human and human cytomegalovirus, respectively. Algorithms well known in the art can be used to determine sequence identity, such as BLAST and BLAST 2.0 algorithms.
  • the promoter sequence of the present invention contains SEQ ID NO: 8, SEQ ID NO: 4, and SEQ ID NO: 3 connected in sequence, or by SEQ ID NO: 8, SEQ ID NO: connected in sequence. 4 is composed of SEQ ID NO: 3.
  • the present invention includes the promoter sequence (that is, the promoter sequence containing SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 3, or the promoter sequence composed of the promoter sequence) having one or more bases. Substitution, deletion and/or addition of modified nucleotide sequences of the base, and the sequence obtained by substitution, deletion and/or addition and modification still retains the biological function of the promoter for high-efficiency expression in activated immune cells.
  • the present invention includes a sequence that has at least 95%, at least 97%, or at least 99% sequence identity with the promoter sequence, and the biological function of the promoter is highly expressed in activated immune cells. It should be understood that substitutions, deletions and/or additions may occur in any one, any two or all three of SEQ ID NO: 8, SEQ ID NO: 4, and SEQ ID NO: 3.
  • Nucleic acid molecules whose base sequence is complementary to the base sequence of the promoter described in any of the embodiments herein are also included in the scope of the present application.
  • the present invention includes nucleic acid constructs containing the sequence of the promoter described herein or its complement, including the promoter sequence with mutation or at least 95% sequence identity or its complement.
  • the nucleic acid construct is an expression cassette containing the enhanced promoter sequence described herein and the coding sequence of the protein of interest.
  • the expression frame usually contains a transcription termination sequence (ie, a transcription terminator), which is a sequence recognized by the host cell to terminate transcription.
  • the transcription termination sequence is operably linked to the 3'end of the coding sequence described herein. Any terminator that is functional in the host cell of choice can be used in the present invention, including but not limited to the SV40polyA transcription termination sequence.
  • the nucleic acid construct is a vector.
  • Vectors generally include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • the vector can be an expression vector, including transient expression vectors, viral expression vectors, and transposable vectors.
  • the vector is preferably a eukaryotic expression vector.
  • Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • the vector can also be a cloning vector to provide the promoter or expression cassette of the present invention.
  • a suitable vector contains at least one origin of replication that functions in the host cell, convenient restriction endonuclease cleavage sites, and one or more selectable markers.
  • Optional restriction sites include but are not limited to Asc I restriction site, Xba I restriction site, Pvu I restriction site, Hind III restriction site, EcoR I restriction site and Sal I restriction site Site.
  • a partial restriction site in the vector is located between the promoter sequence and the transcription termination sequence of the present invention, and is used to cut the vector here and insert the coding sequence of the protein of interest so that the coding sequence is identical to the original The enhanced promoter sequence of the invention and the transcription termination sequence are operably linked.
  • the selectable marker includes either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the cell population infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as kanamycin or neo.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes.
  • the vector is a vector used to integrate the expression cassette of the gene of interest into the genome of the host cell, preferably a transposable vector.
  • the transposable vector is a eukaryotic expression vector containing a transposable element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty. This type of transposable vector contains the 5'inverted terminal repeat (5'ITR) of the corresponding transposon and the 3'inverted terminal repeat (3'ITR) of the corresponding transposon.
  • the transposase can be a transposase from piggybac, sleeping beauty, frog prince, Tn5 or Ty transposal system.
  • sequences of the 5'ITR and 3'ITR in the vector are also correspondingly changed to sequences adapted to the transposable system, which can be easily determined by those skilled in the art .
  • the expression cassette of the present invention is located between the 5'ITR and the 3'ITR.
  • the transposase is a transposase from the piggybac transposition system. Therefore, in these embodiments, the 5'inverted terminal repeat and the 3'inverted terminal repeat of the transposon are the 5'inverted terminal repeat and the 3'inverted terminal repeat of a piggybac transposon, respectively.
  • the transposon 5'inverted terminal repeat sequence is shown in CN 201510638974.7 (the content of which is incorporated herein by reference) SEQ ID NO:1.
  • the 3'inverted terminal repeat sequence of the transposon is shown in SEQ ID NO: 4 of CN 201510638974.7.
  • the piggybac transposase is a transposase containing a c-myc nuclear localization signal coding sequence.
  • the coding sequence of piggybac transposase is shown in SEQ ID NO: 5 of CN 201510638974.7.
  • the promoter of the transposase coding sequence may be various promoters known in the art for controlling the expression of the transposase coding sequence.
  • the CMV promoter is used to control the expression of the transposase coding sequence.
  • the sequence of the CMV promoter may be as shown in CN201510638974.7SEQ ID NO:6.
  • the vector of the present invention uses the pNB328 vector disclosed in CN 201510638974.7 as the backbone, but uses the promoter sequence described herein to replace the EF1 ⁇ promoter originally contained in the vector.
  • the vector of the invention is an empty vector, i.e. does not contain the coding sequence of the protein of interest.
  • empty vectors contain one or more promoter sequences described herein, one or more restriction endonuclease cutting sites and transcription termination sequences in sequence, which are used to digest the coding sequence of the protein of interest by restriction enzyme digestion. It is connected between the promoter sequence and the transcription termination sequence.
  • the vector of the present invention is a vector in which the coding sequence of the protein of interest is inserted between the promoter sequence and the transcription termination sequence described herein, preferably a transposable vector.
  • the promoter sequence described herein, the coding sequence of the protein of interest and the transcription termination sequence are sequentially contained between the 5'ITR and the 3'ITR.
  • the 3'end of the 3'ITR also contains The coding sequence of the transposase and its promoter sequence.
  • the present invention also includes the complementary sequence of each nucleotide sequence described herein.
  • the polynucleotide sequence herein may be in the form of DNA or RNA.
  • nucleotide sequences described herein can usually be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequence disclosed herein, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template to amplify the relevant sequence.
  • a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template to amplify the relevant sequence.
  • synthetic synthesis can be used to synthesize certain nucleotide sequences of the present invention.
  • the protein of interest can be various proteins known in the art, including but not limited to enzymes, antibodies, and other proteins with desired functions, such as cytokines.
  • the protein of interest is a protein known in the art to be expressed in T cells, such as various antibodies with anti-tumor effects, including single-chain antibodies, or chimeric antigen receptors (CAR), etc., and cytokines .
  • Cytokines are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) after stimulation A class of small-molecule proteins with a wide range of biological activities. Cytokines generally regulate cell growth, differentiation and effects by binding to corresponding receptors, and regulate immune responses. Cytokines can have multiple functions such as regulating innate immunity and adaptive immunity, hematogenesis, cell growth, APSC pluripotent cells, and repairing damaged tissues. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines and growth factors.
  • the promoter sequence of the present invention is particularly suitable for driving the gene expression of various types of antibodies, preferably single-chain antibodies, in T cells.
  • the antibody is an autocrine antibody.
  • the antibody is an immune checkpoint antibody, such as PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, VISTA antibody, and the like.
  • the antibody includes a bispecific antibody, such as a bispecific antibody formed by an immune checkpoint antibody and TGF-beta.
  • the antibody is a Nanobody derived from alpaca.
  • the antibody is a PD-1 antibody, and its amino acid sequence may be the amino acid sequence encoded by the sequence shown in SEQ ID NO: 15.
  • Conventional transfection methods can be used to transfer the vector of the present invention into cells of interest. These transfer methods include, but are not limited to: virus transduction, microinjection, particle bombardment, gene gun transformation, and electrotransformation. In certain embodiments, electrotransfection is used to transfect the vector described herein into the cell of interest.
  • the vector of the present application can simultaneously express two or more proteins of interest, such as the antibody and cytokine described in any of the embodiments herein. Therefore, in these embodiments, by transferring such vectors into cells of interest, cells expressing the antibodies and cytokines can be obtained.
  • two or more vectors expressing different proteins of interest can be jointly transferred to the cell of interest to express two or more of them.
  • the protein of interest such as any two or three of antibodies, cytokines, and CAR.
  • the cells of interest can be various T cells known in the art, including but not limited to peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor/regulatory T cells, ⁇ T cells, and cytokines Induced killer cells (CIK), tumor infiltrating lymphocytes (TIL) and other mixed cell populations of T cells.
  • T cells may be derived from PBMCs of patients with B cell malignancies.
  • the T cells are primary cultured T cells.
  • the present invention provides the use of the promoter sequences described herein to drive the expression of exogenous genes (such as single-chain antibody coding sequences) in activated immune cells.
  • the present invention also provides an immune cell, especially a T cell, which contains the promoter sequence or nucleic acid construct or vector described herein, including an antibody for expressing an antibody of interest and/or a vector of interest.
  • Cytokine nucleic acid constructs or vectors Preferably, the genome of the immune cell (especially T cell) integrates the promoter sequence described herein as a promoter to drive the expression of the foreign gene of interest (including the antibody and/or cytokine of interest). Expression box. More preferably, the genome of the immune cell (especially T cell) of the present invention integrates the coding of the immune checkpoint antibody or its bispecific antibody containing the promoter sequence described herein and operably linked to the promoter sequence Sequence expression box.
  • the genome of the immune cell integrates the coding sequence containing the promoter described herein and the cytokine (especially chemokine) operably linked to the promoter Expression box. In some embodiments, the genome of the immune cell (especially T cell) integrates the coding sequence containing the promoter described herein and the cytokine (especially chemokine) operably linked to the promoter An expression cassette containing the promoter described herein and the coding sequence of the immune checkpoint antibody or its bispecific antibody operably linked to the promoter.
  • the immune cell is a CAR T cell, that is, a T cell that expresses a CAR or contains a CAR coding sequence or a vector that expresses a CAR. Therefore, in some embodiments, the CAR T cells provided in this application can express CAR and the antibody of interest at the same time, or express the CAR and the cytokine of interest at the same time, or express the CAR, the antibody of interest and the cell of interest at the same time. factor.
  • the CAR can be any CAR known in the art.
  • the chimeric antigen receptor (CAR) of interest can be directed against one or more of the following antigens: Her2, CD19, CD20, CEA, GD2 (also known as B4GALNT1, ⁇ 1,4-acetyl-galactosamine Base transferase 1), FR (Flavin reductase), PSMA (prostate specific membrane antigen), PMEL (premelanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13R ⁇ 2, MART-1 (also known as Mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family protein, BAGE (B melanoma antigen Family) family protein, GAGE (growth hormone releasing factor) family protein, AFP ( ⁇ -fetoprotein), MUC1 (mucin 1, cell surface related), CD22
  • the CAR-expressing vector and the vector containing other genes of interest (including but not limited to the antibody and/or cytokine of interest described in any of the embodiments herein) operably linked to the promoter of the present invention can be transferred simultaneously or sequentially.
  • CAR T cells whose expression of the gene of interest is controlled by the promoter of the present invention are prepared from the preparation.
  • composition which contains the immune cell of the present invention and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for expressing a protein of interest in a cell of interest, the method comprising transferring a promoter containing the same as described in any of the embodiments herein into the cell of interest.
  • the nucleic acid molecule of the coding sequence of the protein of interest is operably linked, and the cell is cultivated while allowing the expression of the protein of interest.
  • a method for increasing the expression of a gene of interest in activated immune cells the method comprising transferring into the activated immune cells containing the promoter operably linked to the promoter described in any of the embodiments herein.
  • the cell of interest and the protein of interest can be as described in any of the embodiments herein.
  • the cultivation conditions of the cells are well known in the art and can be selected according to different cell types.
  • the method includes constructing a vector containing the promoter and the coding sequence of the protein of interest operably linked to the promoter, and using the vector in a well-known manner (such as electroporation or liposome Transfection) into the cell of interest, and cultivate the cell under conditions suitable for the expression of the gene to produce the protein.
  • this document also provides a cellular immunotherapy, including providing the immune cells described in any of the embodiments herein, and administering a therapeutically effective amount of the immune cells to an individual in need.
  • the individual may be an individual suffering from a disease known in the art that can be treated with proteins expressed by the immune cells, such as antibodies and/or cytokines and/or CARs, such as mammals, especially humans.
  • the immune cells can be prepared by the methods described in any of the embodiments herein to provide the immune cells.
  • the immune cells are autologous cells, that is, immune cells derived from the individual to be treated, which will express the desired antibodies, cytokines and/or cytokines after being treated in vitro by the method described in any of the embodiments herein. CAR is then given back to the individual.
  • the present invention found that when the vector containing the coding sequence of the protein of interest and the promoter of the present invention operably linked to it is transferred into immune cells, the expression level of the protein of interest can be increased several to ten times compared with the control. . Therefore, when the immune cell of the present invention is used as a drug, if the drug of the present invention encounters an activating agent (such as a tumor antigen) in the body, the immune cell of the present invention will be activated and express a large amount of the foreign gene it carries.
  • an activating agent such as a tumor antigen
  • Example 1 Construction of expression vector for human cytokine gene promoter
  • plasmid pS338B-EGFP ( Figure) that contains the chimeric cytokine gene promoters combined with the SV40 enhancer DTS (SEQ ID NO: 1), EF1 ⁇ promoter (SEQ ID NO: 2) and TLTR (SEQ ID NO: 3). 1) As a control plasmid and a modified prototype.
  • IFN- ⁇ gene promoter sequence SEQ ID NO: 4
  • the control vector pS338B-EGFP of the chimeric cytokine gene promoter combined by the promoter and TLTR was digested with Hind III and Pvu I, and the recovered fragment uIFP was ligated to the pS338B-EGFP vector to obtain the SV40 enhancer.
  • IFN- ⁇ promoter and TLTR chimeric cytokine gene promoter vector pS-IFPT-EGFP ( Figure 2).
  • IL3en SEQ ID NO: 5
  • IL3en SEQ ID NO: 5
  • digest the vector pUC57-IL3en with Pvu I and Nhe I double enzymes and recover a fragment containing the IL3 gene enhancer with a size of 266 bp, containing
  • the pS338B-EGFP vector with EF1 ⁇ promoter and TLTR sequence was digested with Xba I and Pvu I
  • the recovered fragment IL3en was ligated to the pS338B-EGFP vector, and the correct clone was the IL3 gene enhancer and EF1 ⁇ promoter.
  • TLTR chimeric cytokine gene promoter vector pS-IL3en-EGFP ( Figure 3).
  • IFNp-f 5’-TCTGCGATCGAAAAGTGCCTTCAAAGAATCC-3’ (SEQ ID NO: 16)
  • the sequence containing the IFN- ⁇ promoter with a length of 256 bp was obtained by PCR amplification, and this fragment was combined with the vector pS-IFPT-EGFP After digestion with Xba I and EcoR I and ligation, after transforming Top10, a single clone is obtained. After the identification is correct with EcoR I and Sal I, it is sent to Jinweizhi for sequencing. The correct clone is the vector containing the IFN- ⁇ gene promoter: pS-uIFP-EGFP ( Figure 4).
  • IFNp-f2 5’-TCTAGAAGGATCTGCGATCGAAAAGTGCCTT-3’ (SEQ ID NO: 18)
  • TLR-r 5’-ATGGTGGCGAATTCGTAGGCGCCGGTCAC-3’ (SEQ ID NO: 19)
  • a 555bp sequence (SEQ ID NO: 7) composed of IFN- ⁇ promoter and TLTR was obtained by PCR amplification, and this fragment was combined with the vector pS- IFPT-EGFP were all digested with Xba I and EcoR I and then ligated. After transforming Top10, a single clone was obtained. After the identification of EcoR I and Sal I was correct, it was sent to Jinweizhi for sequencing. The correct clone is the IFN- ⁇ gene promoter.
  • the vector for the chimeric cytokine gene promoter of TLTR and TLTR pS-uIFPT-EGFP ( Figure 5).
  • a 380bp CMV gene enhancer sequence (SEQ ID NO: 8) was obtained by PCR amplification, and this fragment was combined with the vector pS-IFPT- EGFP were all digested with Xba I and Pvu I and then ligated. After transforming Top10, a single clone was obtained by digesting with EcoR I and Sal I. After the identification was correct, it was sent to Jinweizhi for sequencing. The correct clone is the CMV gene enhancer, IFN- ⁇ gene promoter and TLTR chimeric cytokine gene promoter vector: pS-CIFT-EGFP ( Figure 6).
  • the chimeric cytokine gene promoter vector pS-IFPT-EGFP and the IL3 gene enhancer vector pS-IL3en-EGFP containing the combination of IFN- ⁇ gene promoter and TLTR are both digested with Pvu I and EcoR I After obtaining the corresponding fragments and vectors, ligating and transforming Top10 to obtain a single clone with EcoR I and Sal I double enzyme digestion to obtain the correct clone, which contains the IL3 gene enhancer, IFN- ⁇ gene promoter and TLTR chimeric cells
  • Vector of factor gene promoter pS-ILFP-EGFP ( Figure 7).
  • IL2P human IL-2 gene promoter sequence
  • SEQ ID NO: 9 Entrusted Jinweizhi Company to synthesize the human IL-2 gene promoter sequence (IL2P, SEQ ID NO: 9), digest the vector pUC57-IL2P with Pvu I and Hind III, and recover the 300 bp fragment IL2P, containing the EF1 ⁇ promoter and TLTR
  • the sequence of pS338B-EGFP vector was digested with Hind III and Pvu I
  • the above recovered fragment IL2P was ligated to pS338B-EGFP vector.
  • the clone with the correct ligation is a chimera containing SV40 enhancer, IL2 gene promoter and TLTR Cytokine gene promoter vector: pS-ILPT-EGFP ( Figure 8).
  • IL2p-f 5’-TCTAGAATCTGCGATCGCCCCACCCCC-3’ (SEQ ID NO: 22)
  • IL2p-r 5’-GAATTCCTCGAAGCTTCTTGAACAA-3’ (SEQ ID NO: 23)
  • the IL-2 promoter sequence (SEQ ID NO: 10) with a length of 308 bp was amplified by PCR, and this fragment was used with the vector pS-ILPT-EGFP Xba I and EcoR I were double-enzyme digested and ligated. After transforming Top10, a single clone was obtained. After the EcoR I and Sal I double-enzyme digestion was performed correctly, it was sent to Jinweizhi for sequencing. The correct clone is the vector pS-uILP- containing the IL2 gene promoter. EGFP ( Figure 9).
  • IL2p-f 5’-TCTAGAATCTGCGATCGCCCCACCCCC-3’ (SEQ ID NO: 24)
  • TLR-r 5-CACCATGGTGGCGAATTCGTAGGCGCCGGTC-3' (SEQ ID NO: 25)
  • the IL-2 gene promoter and DTS-EF1 ⁇ sequence (SEQ ID NO: 11) with a length of 598 bp were obtained by PCR amplification, and this fragment was combined with the vector pS- ILPT-EGFP were all digested with Xba I and EcoR I and ligated. After transforming Top10, a single clone was obtained. After the identification was correct with EcoR I and Sal I, it was sent to Jinweizhi for sequencing. The correct clone is the IL2 gene promoter and TLTR chimeric cytokine gene promoter vector: pS-uILT-EGFP ( Figure 10).
  • the vector pS-CIFT-EGFP containing the CMV enhancer sequence and the vector pS-ILPT-EGFP containing the IL2 gene promoter and TLTR sequence were both digested with Pvu I and Xba I to obtain the corresponding fragments and vectors. After connecting and transforming Top10 The obtained single clone was correctly identified by EcoR I and Sal I double enzyme digestion, and the correct clone was obtained: pS-CILT-EGFP ( Figure 11).
  • the vector pS-ILPT-EGFP containing the IL3 gene enhancer sequence and the vector pS-IL3en-EGFP containing the IL2 gene promoter and TLTR sequence were both digested with Pvu I and EcoR I to obtain two corresponding fragments, which were connected to transform Top10 After obtaining a single clone with EcoR I and Sal I double enzyme digestion, the correct clone is obtained, which contains IL3 gene enhancer, IL2 gene promoter and TLTR chimeric cytokine gene promoter vector: pS-ILFP- EGFP ( Figure 12).
  • IFNen-f 5’-GCAGCTGCAGTATTGTTTTAACCTTCTGCTC-3’ (SEQ ID NO: 28)
  • IFNen-r 5’-TGGCGAATTCTAAGGACCTTTTTGAC-3’ (SEQ ID NO: 29)
  • the human IFN- ⁇ gene promoter and enhancer (SEQ ID NO: 13) with a length of 519 bp were obtained by overlapping PCR amplification, and this fragment was combined with the human IFN- ⁇ gene promoter and enhancer (SEQ ID NO: 13).
  • the vector pS-IFPT-EGFP of the SV40 enhancer was digested with Pvu I and EcoR I and ligated. After transforming Top10, a single clone was obtained by digesting with EcoR I and Sal I. After the identification was correct, it was sent to Jinweizhi for sequencing. The correct clone is It is a vector containing SV40 enhancer, IFN- ⁇ gene promoter and IFN- ⁇ enhancer chimeric cytokine gene promoter: pS-IFen-EGFP ( Figure 13).
  • IFNP-f 5’-TCTGCGATCGAAAAGTGCCTTCAAAGAATCC-3’ (SEQ ID NO: 30)
  • IFNen-r 5’-TGGCGAATTCTAAGGACCTTTTTGAC-3’ (SEQ ID NO: 31)
  • the human IFN- ⁇ gene promoter and its enhancer (SEQ ID NO: 14) with a length of 529 bp were amplified by PCR, and this fragment was combined with the vector pS- IFPT-EGFP were all digested with Xba I and EcoR I and then ligated. After transforming Top10, a single clone was obtained. After the identification of EcoR I and Sal I was correct, it was sent to Jinweizhi for sequencing. The correct clone is the IFN- ⁇ gene promoter.
  • the vector for the chimeric cytokine gene promoter of the promoter and the IFN- ⁇ enhancer pS-uIFen-EGFP ( Figure 14).
  • the vector pS-CIFT-EGFP containing the CMV enhancer and IFN- ⁇ gene promoter and the vector pS-IFen-EGFP containing the enhancer in the human IFN- ⁇ gene intron were both digested with Pvu I and Xba I. Obtain the corresponding fragment and vector. After ligating and transforming Top10, the monoclonal is obtained. The EcoR I and Sal I double enzyme digestion is used to identify the correct clone, which contains the CMV enhancer, IFN- ⁇ gene promoter and IFN- ⁇ enhancer.
  • the vector of the chimeric cytokine gene promoter pS-CIFen-EGFP ( Figure 15).
  • the vector pS-CIFen-EGFP containing the IFN- ⁇ gene promoter and the enhancer in the human IFN- ⁇ gene intron and the vector pS-IL3en-EGFP containing the IL3 gene enhancer were both digested with Pvu I and EcoR I. After obtaining the corresponding fragments and vectors, ligating and transforming Top10 to obtain a single clone with EcoR I and Sal I double enzyme digestion, the correct clone is obtained, which contains IL3 gene enhancer, IFN- ⁇ gene promoter and IFN- ⁇ enhancer
  • the vector of the chimeric cytokine gene promoter pS-LIFen-EGFP ( Figure 16).
  • control vector pS338B-EGFP and the vector pS-AD-F-Fluc containing the luciferase reporter gene containing the SV40 enhancer, EF1 ⁇ promoter and TLTR combined into a chimeric cytokine gene promoter are both EcoR I and Xba I
  • the corresponding fragment and vector are obtained.
  • ligation and transformation of Top10 After ligation and transformation of Top10, a single clone is obtained.
  • the double enzyme digestion with EcoR I and Sal I is correctly identified, that is, the correct clone pS338B-Fluc is obtained ( Figure 17).
  • the chimeric cytokine gene promoter vector pS-IFPT-EGFP and the luciferase reporter gene vector pS-AD-F-Fluc containing SV40 enhancer, IFN- ⁇ promoter and TLTR are combined with EcoR I After double digestion with Xba I, the corresponding fragments and vectors were obtained. After ligation and transformation of Top10, the monoclonal antibody was obtained by double digestion with EcoR I and Sal I, and the correct clone pS-IFPT-Fluc was obtained ( Figure 18).
  • the chimeric cytokine gene promoter vector pS-CIFT-EGFP and the luciferase reporter gene vector pS-AD-F-Fluc containing CMV enhancer, IFN- ⁇ promoter and TLTR are combined with EcoR I After double digestion with Xba I, the corresponding fragments and vectors were obtained. After ligation and transformation of Top10, the monoclonal antibody was obtained by double digestion with EcoR I and Sal I, and the correct clone pS-CIFT-Fluc was obtained ( Figure 19).
  • the chimeric cytokine gene promoter vector pS-ILFP-EGFP containing IL3 gene enhancer, IFN- ⁇ gene promoter and TLTR combination and the vector containing luciferase reporter gene pS-AD-F-Fluc are both used After EcoR I and Xba I double enzyme digestion, the corresponding fragment and vector are obtained. After ligation and transformation of Top10, the monoclonal antibody is obtained. The EcoR I and Sal I double enzyme digestion is correct, and the correct clone pS-ILFP-Fluc is obtained ( Figure 20).
  • the vector contains the IL3 gene enhancer, the IL2 gene promoter and the TLTR combination of chimeric cytokine gene promoter pS-ILPT-EGFP and the luciferase reporter gene vector pS-AD-F-Fluc all use EcoR I After double digestion with Xba I, the corresponding fragments and vectors are obtained. After ligation and transformation of Top10, the monoclonal antibody is obtained by double digestion with EcoR I and Sal I, and the correct clone pS-ILPT-Fluc is obtained ( Figure 21).
  • the chimeric cytokine gene promoter vector pS-CILT-EGFP and the luciferase reporter gene vector pS-AD-F-Fluc containing CMV enhancer, IL2 gene promoter and TLTR are combined with EcoR I and After Xba I double enzyme digestion, the corresponding fragment and vector are obtained. After ligation and transformation of Top10, the monoclonal antibody is obtained by double digestion with EcoR I and Sal I, and the correct clone pS-CILT-Fluc is obtained ( Figure 22).
  • the synthetic anti-PD1 antibody sequence ⁇ PD1 was digested with EcoR I and Sal I, and the synthetic anti-PD1 antibody sequence ⁇ PD1 was recovered. After digestion, the above recovered fragment of anti-PD1 antibody sequence ⁇ PD1 is connected to the pS338B-EGFP vector to obtain a vector expressing anti-PD1 antibody, a chimeric cytokine gene promoter composed of DTS, EF1 ⁇ promoter and TLTR : PS338B- ⁇ PD1 ( Figure 23).
  • the synthetic anti-PD1 antibody sequence ⁇ PD1 was digested with EcoR I and Sal I and the synthetic anti-PD1 antibody sequence ⁇ PD1 was recovered, and the vector pS-CIFT-EGFP containing the chimeric cytokine gene promoter composed of the CMV enhancer, IFN- ⁇ promoter and TLTR was combined with EcoR. After double digestion with I and Sal I, the above recovered fragment anti-PD1 antibody sequence ⁇ PD1 is connected to the pS-CIFT-EGFP vector to obtain an expressible anti-PD1 antibody, which is composed of CMV enhancer, IFN- ⁇ promoter and TLTR
  • the vector of the chimeric cytokine gene promoter pS-CIFT- ⁇ PD1 ( Figure 24).
  • Example 2 Detection of the expression of the constructed cytokine gene promoter in T cells
  • PBMC peripheral blood mononuclear cells
  • the plasmid is electroporated into PBMC, placed in a 37°C, 5% CO 2 incubator; 4 hours later, transferred to 5 ⁇ g/ml antiCD28 antibody, or 5 ⁇ g/ml human mesothelin antigen and 5 ⁇ g/ ml antiCD28 antibody (purchased from Novoprotein)-coated culture plate, the medium composition is AIM-V (Gibco), 2% fetal bovine serum (Gibco) and 500IU/ml IL-2 (purchased from Novoprotein); 5 days later The cells were transferred to a culture plate without antigen coating and cultured. The culture medium consisted of AIM-V (Gibco), 2% fetal bovine serum (Gibco) and 200IU/ml IL-2 (purchased from Novoprotein), cultured on the 7th day Perform flow cytometry on cells.
  • Figure 25 is the flow cytometric analysis result of the IFN- ⁇ promoter and IL-2 promoter activity through FlowJo X analysis in the case of the same enhancer. The results showed that the expression intensity of the IFN- ⁇ promoter was stronger than that of the IL-2 promoter, and the enhancer before the promoter sequence enhanced the expression of the promoter. On this basis, the TLTR sequence after the promoter was replaced with the enhancer sequence in the IFN- ⁇ intron, and the effects of the two different sequences after the promoter sequence on the original promoter were compared.
  • Figure 26 is a flow cytometric analysis diagram of the change in the promoter activity of the TLTR sequence after the promoter sequence is changed under the same promoter sequence.
  • Figure 27 compares the effect of pS338B-EGFP and pS-CIFT-EGFP on the promoter activity in the case of using the same promoter sequence and different ways of activating T cells, indicating that compared with the control plasmid pS338B-EGFP, it contains the CMV gene enhancer , IFN- ⁇ gene promoter and TLTR chimeric cytokine gene promoter pS-CIFT-EGFP expression activity is more susceptible to cell activation.
  • the dual luciferase reporter system further detects the expression of the promoter
  • a. Resuspend each group of cells with a volume of 5 ⁇ 10 4 cells in 75uL volume of AIM-V medium and spread them in a 96-well plate; b to a Add an equal volume of 75uL Reagent, mix well and place on a shaker, wait for at least 10 minutes (less than 2 hours) to fully lyse the cells and detect the luminescence intensity of firefly luciferin; c add 75uL Stop& Reagent, mix well and place on a shaker, wait for at least 10 minutes (less than 2 hours) to fully lyse the cells, and detect the luminescence intensity of Renilla fluorescein; d Divide the intensity value of firefly fluorescein detection by the fluorescence intensity of Renilla fluorescein detection Numerical value, you can get the value of signal homogenization, and then reflect the activity of each promoter.
  • Example 3 Detection of expression of constructed cytokine gene promoter in HEK-293T cells
  • A. Cell plating digest HEK-293T in good culture condition with trypsin, count, take 3 ⁇ 10 5 cells and resuspend them in 3mL culture medium, spread them in 6-well plate and culture for 24 hours.
  • the medium composition is DMEM medium. +10% serum;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 4 Detection of the expression of the constructed cytokine gene promoter in CHO cells
  • CHO cell transfection (ViaFect transfection kit): use pS338B-EGFP as the control plasmid
  • A. Cell plating Digest CHO cells in good culture condition with trypsin, count, take 3 ⁇ 10 5 cells and resuspend them in 3mL culture medium, spread them in 6-well plate and culture for 24 hours, culture medium composition is 45% DMEM Base + 45% RPMI-1640 medium + 10% serum + 1% L-glutamine + 1% hypoxanthine-thymidine;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • DC cell transfection (ViaFect transfection kit): use pS338B-EGFP as the control plasmid
  • A. Cell plating Collect the well-cultured DC cells, count them, take 3 ⁇ 10 5 cells and resuspend them in 3mL culture medium, spread them in a 6-well plate and culture for 24 hours, the medium composition is AIM-V;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 6 Detection of the expression of the constructed cytokine gene promoter in the acute T cell leukemia cell line Jurkat cells
  • Jurkat cell transfection use pS338B-EGFP as the control plasmid
  • A. Cell plating Collect the well-cultured Jurkat cells, count them, take 6 ⁇ 10 5 cells and resuspend them in 3mL culture medium, spread them in a 6-well plate and culture for 24 hours.
  • the medium composition is RPMI-1640 medium + 10 %serum;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 7 Detection of the constructed cytokine gene promoter expression in B-cell malignant lymphoma Raji cells
  • A. Cell plating Collect Raji cells in good culture condition, count them, take 6 ⁇ 105 cells and resuspend them in 3mL culture medium, spread them in 6-well plate and culture for 24 hours, the medium composition is RPMI-1640 medium + 10% serum;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 8 Detection of the expression of the constructed cytokine gene promoter in the liver cancer cell line Hep G2
  • Hep G2 cell transfection (ViaFect transfection kit): use pS338B-EGFP as the control plasmid
  • A. Cell plating Collect Hep G2 cells in good culture condition, count them, take 3 ⁇ 10 5 cells and resuspend them in 3mL culture medium, spread them in 6-well plate and culture for 24 hours, the medium composition is RPMI medium + 10% serum;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake for a few times and then stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 9 Detection of expression of constructed cytokine gene promoter in ovarian cancer cell line SKOV3
  • A. Cell plating Collect SKOV3 cells in good culture condition, count them, take 3 ⁇ 105 cells and resuspend them in 3mL culture medium, spread them in 6-well plates and culture for 24 hours.
  • the medium composition is DMEM medium + 10% serum;
  • Fixation Wash the cells with appropriate fluorescence expression intensity after transfection with 1mL PBS buffer solution twice, discard the washing solution and add 1mL 4% paraformaldehyde fixative solution to the cells, and let stand at room temperature for 30 minutes;
  • DAPI staining Wash the permeabilized cells 3 times with 1mL PBS buffer solution for 5 minutes each time, discard the washing solution and add 1mL DAPI dye prepared to the cells, shake it for several times and let stand at room temperature for 4 minutes. Observe under a fluorescence microscope, if the nucleus is completely colored, proceed to step D, if the color is not complete, continue to stand until the nucleus is completely colored;
  • Example 10 Application of chimeric interferon gamma promoter in autocrine anti-PD1 antibody CAR-T cells
  • PBMC peripheral blood mononuclear cells
  • the medium components were AIM-V (Gibco), 2% fetal bovine serum (Gibco) and 200IU/ml IL-2 (purchased from Novoprotein). Take 1 ⁇ 10 4 ovarian cancer SKOV3 cells with high expression of PDL1 and evenly spread them into 96-well plates. After culturing for 24 hours, add 5 ⁇ 10 3 transfected different plasmids to each well, and culture the CAR-T cells on the 10th day. After 24 hours of co-cultivation, the supernatant was taken, centrifuged to collect the supernatant, and the content of PD1 antibody secreted by CAR-T cells in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • A. Antigen plate prepare the PD-1 antigen Human PD-1/PDCD1 Protein (HPLC-verified). For the first use, dissolve the antigen with commercial PBS. Dilute the antigen to 1ug/ml with the coating solution, and coat the enzyme-labeled reaction plate with 100ul/well at 4°C overnight. After overnight, wash with PBST 5 times, 200ul/well, 3 minutes each time, pat dry with absorbent paper;
  • Blocking Add 300ul of blocking solution to each well and incubate for 2 hours in a biochemical incubator at 37°C. Wash 5 times with PBST, 200ul/well, 3 minutes each time, pat dry with absorbent paper;
  • sample and standard zAb 100ul/well, set up multiple wells and control wells, and incubate for 1 hour in a biochemical incubator at 37°C. Dilute the sample and standard with the diluent. The standard starts from 30ng/ml, and sets 7 gradients and 0ng/ml downwards. The sample is diluted according to the actual situation, and the sample in this experiment is set to 40 times and 400 times dilution. Wash 5 times with PBST, 200ul/well, 3 minutes each time, pat dry with absorbent paper;
  • Color development add color development solution TMB, 100ul/hole, avoid light at room temperature for 6min;
  • Stop add 50ul/well of stop solution to stop the reaction. Take computer readings immediately.
  • T cells acquire the ability to autocrine PD1 antibody, and the CMV enhancer-interferon gamma promoter-TLTR chimeric promoter is in The expression activity of the secreted PD1 antibody is significantly better than the DTS-EF1 ⁇ -TLTR chimeric promoter.

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Abstract

提供在活化的T细胞中具有高活性的启动子，其从5'端到3'端包含依次连接的CMV增强子、IFNγ启动子和人T细胞白血病病毒的长末端重复序列。所述启动子表现出在活化的免疫细胞中比现有启动子更强的活性，而在其他非免疫细胞中活性低或无活性。

Description

一种在活化的T细胞中具有高活性的启动子 技术领域
本发明涉及一种在活化的T细胞中具有高活性的启动子。
背景技术
启动子是基因的一个组成部分，通常位于结构基因5’端上游，是RNA聚合酶识别、结合和开始转录的一段DNA序列。启动子是影响转基因表达效率的重要因素之一，选择高效率的启动子是高效率表达外源基因的关键。
根据启动子的转录模式可将其分为3类：组成型启动子、组织或器官特异性启动子和诱导型启动子。
组成型启动子是指在组成型启动子调控下，不同组织器官和发育阶段的基因表达没有明显差异，因而称之为组成型启动子。哺乳动物中常用的组成型启动子包括病毒来源：鼠或人巨细胞病毒(CMV)启动子(分别简称mCMV与hCMV)、猴空泡病毒SV40启动子；人基因组天然来源：EF1α启动子、泛素启动子(Ubiquitin，简称Ubi)、β-actin启动子、PGK-1启动子、Rosa26启动子、HSP70启动子、GAPDH启动子、eIF4A1启动子、Egr1启动子、FerH启动子、SM22α启动子、Endothelin-1启动子等。
在肿瘤免疫治疗中，维持外源基因的高效稳定表达非常重要。然而，一些病毒来源的组成型启动子尽管瞬时表达活性较高(如CMV启动子)，但容易因表观遗传修饰而被关闭表达；而一些人源天然组成型启动子或肿瘤特异性启动子虽然表达稳定，但表达活性相对较弱，难以满足免疫治疗需求。因而，研究人员又设计构建了一系列人工嵌合启动子，它们包含了一些顺式调控元件，主要包括能发挥稳定表达作用的启动子核心序列，以及能增强表达效率的上游增强子或下游内含子，代表者是嵌合启动子CAG(包含人CMV增强子-鸡β-actin启动子-兔β-globin内含子)，广泛应用于外源基因的表达。
增强子指增加同它连锁的基因转录频率的DNA序列，增强子通过启动子来增加下游基因的转录。有效的增强子可以位于基因的5’端，也可位于基因的3’端，有的还可位于基因的内含子中。增强子的效应很明显，一般能使基因转录频率增加10-200倍，有的甚至可以高达上千倍。
发明内容
本发明构建了一种以CMV增强子、IFNγ启动子和HTLV(人T细胞白血病病毒)的长末端重复(LTR)序列组合而成的启动子，该启动子表现出在活化的免疫细胞中比现有启动子更强的活性，而在其他非免疫细胞中活性低或无活性。
因此，本发明提供一种启动子，从5’端到3’端其包含依次连接的CMV增强子、IFNγ启动子和人T细胞白血病病毒的长末端重复序列。
在一个或多个实施方案中，所述CMV增强子选自：具有SEQ ID NO:8所示核苷酸序列的CMV增强子，或来自人CMV的与SEQ ID NO:8所示核苷酸序列具有至少97％序列同一性的CMV增强子。
在一个或多个实施方案中，所述IFNγ启动子选自：具有SEQ ID NO:4所示核苷酸序列的IFNγ启动子，或来自人的与SEQ ID NO:4所示核苷酸序列具有至少97％序列同一性的IFNγ启动子。
在一个或多个实施方案中，所述人T细胞白血病病毒的长末端重复序列选自：具有SEQ ID NO:3所示核苷酸序列的长末端重复序列，或来自人T细胞白血病病毒的与SEQ ID NO:3所示核苷酸序列具有至少97％序列同一性的长末端重复序列。
在一些实施方案中，本发明还提供其碱基序列与所述启动子的碱基序列互补的核酸分子。
本发明还提供一种核酸构建物，其含有本发明所述的启动子和与该启动子操作性连接的感兴趣的基因。
在一个或多个实施方案中，所述核酸构建物是表达框。
在一个或多个实施方案中，所述感兴趣的基因编码自分泌抗体，优选为免 疫检查点抗体，如PD-1抗体、CTLA4抗体、PD-L1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体和VISTA抗体，更优选为来源于羊驼的纳米抗体。
在一个或多个实施方案中，所述感兴趣的基因编码细胞因子。
本发明还提供一种载体，其含有本发明所述的启动子或核酸构建物。
在一个或多个实施方案中，所述载体为表达载体或克隆载体。
还提供的是一种宿主细胞，其含有本文所述的启动子、核酸构建物或载体。
在一个或多个实施方案中，所述宿主细胞为免疫细胞，优选为T细胞，其基因组中整合有本文任一实施方案所述的核酸构建物；优选地，所述免疫细胞还表达CAR或含有CAR的表达载体。
还提供的是本发明所述的启动子在提高感兴趣的基因在活化的免疫细胞中的表达中的应用，或在制备用于在活化的免疫细胞中增强表达的核酸构建物或载体中的应用。
附图说明
图1：pS338B-EGFP质粒图谱。
图2：pS-IFPT-EGFP质粒图谱。
图3：pS-IL3en-EGFP质粒图谱。
图4：pS-uIFP-EGFP质粒图谱。
图5：pS-uIFPT-EGFP质粒图谱。
图6：pS-CIFT-EGFP质粒图谱。
图7：pS-ILFP-EGFP质粒图谱。
图8：pS-ILPT-EGFP质粒图谱。
图9：pS-uILP-EGFP质粒图谱。
图10：pS-uILT-EGFP质粒图谱。
图11：pS-CILT-EGFP质粒图谱。
图12：pS-ILFP-EGFP质粒图谱。
图13：pS-IFen-EGFP质粒图谱。
图14：pS-uIFen-EGFP质粒图谱。
图15：pS-CIFen-EGFP质粒图谱。
图16：pS-LIFen-EGFP质粒图谱。
图17：pS338B-Fluc质粒图谱。
图18：pS-IFPT-Fluc质粒图谱。
图19：pS-CIFT-Fluc质粒图谱。
图20：pS-ILFP-Fluc质粒图谱。
图21：pS-ILPT-Fluc质粒图谱。
图22：pS-CILT-Fluc质粒图谱。
图23：pS338B-αPD1质粒图谱。
图24：pS-CIFT-αPD1质粒图谱。
图25：IFN-γ基因启动子和IL2基因启动子对嵌合基因启动子活性影响的比较。
图26：TLTR和IFN-γ内含子增强子对嵌合基因启动子活性影响的比较。
图27：不同活化方式对两种质粒eGFP表达的影响。
图28：双荧光素酶报告系统检测结果。
图29：细胞因子基因嵌合启动在子HEK 293T细胞中的表达活性。
图30：细胞因子基因嵌合启动在子CHO细胞中的表达活性。
图31：细胞因子基因嵌合启动在子DC细胞中的表达活性。
图32：细胞因子基因嵌合启动在子Jurkat细胞中的表达活性。
图33：细胞因子基因嵌合启动在子Raji细胞中的表达活性。
图34：细胞因子基因嵌合启动在子Hep G2细胞中的表达活性。
图35：细胞因子基因嵌合启动在子SKOV3细胞中的表达活性。
图36：细胞因子基因嵌合启动子增强CAR-T细胞分泌PD1抗体。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本发明通过改造启动子活性来提高其驱动基因表达强度，从而实现外源基 因在活化的免疫细胞内的高效表达。
本发明中，免疫细胞是指参与免疫应答或与免疫应答相关的细胞，包括淋巴细胞、树突状细胞、单核/巨噬细胞、粒细胞和肥大细胞等。本发明优选的免疫细胞为外周血单个核细胞(PBMC)，包括淋巴细胞和单核细胞。淋巴细胞包括T淋巴细胞、B淋巴细胞、K淋巴细胞和NK淋巴细胞。优选的是，适用于本发明改造的启动子的细胞是自身高表达γ干扰素的免疫细胞。
本发明中，“活化”指本领域常规用于免疫细胞转入目的质粒或载体后使用相应的活化试剂对该免疫细胞进行刺激，以达到扩增以增加免疫细胞数量、激活以增强免疫细胞活性的过程。通常，活化试剂为本领域常规的活化试剂，如抗CD28抗体和任选的相应的免疫原。例如，在一些实施方案中，当转入的是表达针对某种肿瘤抗原的嵌合抗原受体时，活化试剂还可包括该肿瘤抗原或其活性片段。本发明对活化的时机、活化时机的浓度和活化的时间等并无特殊限制。在一些实施方案中，本发明使用抗CD28抗体进行活化；在一些实施方案中，本发明使用抗CD28抗体与肿瘤抗原的组合进行活化。
本发明中，术语“表达框”是指表达一个基因所需的完整元件，包括可操作性连接的启动子和基因编码序列。
术语“编码序列”指核酸序列中直接确定其蛋白产物的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。
术语“可操作性连接的”或“可操作性连接”指两个或多个核苷酸区域或核酸序列的功能性的空间排列。例如，在核酸构建物中，启动子被置于感兴趣基因的核酸序列的特定位置，例如启动子位于所述基因核酸序列的上游位置，使得核酸序列的转录受到该启动子区域的引导，从而，启动子区域被“可操作地连接”到该基因的核酸序列上。“可操作性连接”可以通过基因重组的手段实现。
本发明的增强子为CMV增强子。可采用本领域周知的CMV增强子来实施本发明，包括来自鼠巨细胞病毒属的mCMV增强子和来自人巨细胞病毒属 的hCMV增强子，优选为人CMV增强子。示例性的CMV增强子可具有SEQ ID NO:8所示的核苷酸序列。
本发明的IFNγ启动子通常选用其核心区序列或含该核心区序列的IFNγ启动子的片段。IFNγ启动子核心区序列的选取可参考John C.Chrivia等，“A model of human cytokine regulation based on transfection of gamma interferon gene fragments directly into isolated peripheral blood T lymphocytes”，The Journal of Experimental Medicine，1990年8月，第172卷，第661-664页。示例性的IFNγ启动子为具有SEQ ID NO:4所示的核苷酸序列的IFNγ启动子的片段。在优选的实施方案中，本发明的启动子使用SEQ ID NO:4所示的核苷酸序列。
适用于本发明的人T细胞白血病病毒的长末端重复序列可具有SEQ ID NO:3所示的核苷酸序列。
本发明也包括对SEQ ID NO:3、4和8所示的核苷酸序列进行一个或多个碱基的取代、缺失和/或添加修饰的核苷酸序列，且取代、缺失和/或添加修饰所得序列仍然保留SEQ ID NO:3、4和8各自的生物学功能。例如，可分别或同时在所述核苷酸序列的5’端和/或3’端，和/或序列内部进行例如不超过20个，如不超过15个，或不超过10个，或不超过8个，或不超过5个的碱基的取代、缺失和/或添加修饰。在一些实施方案中，本发明包括分别与SEQ ID NO:3、4和8所示的核苷酸序列具有至少95％、至少97％或至少99％序列同一性的序列，同样地，这些序列也保留了SEQ ID NO:3、4和8各自的生物学功能，且优选地这些序列分别来自人T细胞白血病病毒、人以及人巨细胞病毒。可采用本领域周知的算法确定序列同一性，如BLAST和BLAST 2.0算法。
在某些实施方案中，本发明的启动子序列含有依次连接的SEQ ID NO:8、SEQ ID NO:4与SEQ ID NO:3，或由依次连接的SEQ ID NO:8、SEQ ID NO:4与SEQ ID NO:3组成。
本发明包括与此启动子序列(即含有SEQ ID NO:8、SEQ ID NO:4与SEQ ID NO:3的启动子序列，或由其组成的启动子序列)相比具有一个或多个碱基的取代、缺失和/或添加修饰的核苷酸序列，且取代、缺失和/或添加修饰所得序列仍然保留该启动子在活化的免疫细胞内的高效表达的生物学功能。在一些 实施方案中，本发明包括与此启动子序列具有至少95％、至少97％或至少99％序列同一性，并该启动子在活化的免疫细胞内的高效表达的生物学功能的序列。应理解，取代、缺失和/或添加可发生在SEQ ID NO:8、SEQ ID NO:4与SEQ ID NO:3中的任意一条、任意两条或全部三条序列中。
其碱基序列与本文任一实施方案所述的启动子的碱基序列互补的核酸分子也包括在本申请的范围之内。
本发明包括核酸构建物，该核酸构建物含有本文所述的启动子的序列或其互补序列，包括所述具有突变或具有至少95％序列同一性的启动子序列或其互补序列。
在某些实施方案中，该核酸构建物是表达框，含有本文所述的增强的启动子序列和感兴趣蛋白的编码序列。表达框中通常含有转录终止序列(即转录终止子)，转录终止序列是由宿主细胞识别以终止转录的序列。转录终止序列与本文所述的编码序列3’末端可操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明，包括但不限于SV40polyA转录终止序列。
在某些实施方案中，所述核酸构建物是一载体。载体通常包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。载体可以是表达载体，包括瞬时表达载体、病毒表达载体和转座载体。载体优选为真核表达载体。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。载体也可以是克隆载体，用于提供本发明的启动子或表达框。
通常，合适的载体包含在至少一种在宿主细胞中起作用的复制起点、方便的限制性内切酶酶切位点和一个或多个可选择的标记。
可选择的酶切位点包括但不限于Asc I酶切位点、Xba I酶切位点、Pvu I酶切位点、Hind Ⅲ酶切位点、EcoR I酶切位点和Sal I酶切位点。通常，载体中有部分酶切位点位于本发明所述的启动子序列和转录终止序列之间，用于在此处切开载体，插入感兴趣蛋白的编码序列，使得所述编码序列与本发明的增强的启动子序列和转录终止序列可操作性地连接。
可选择的标记包括可选择的标记基因或报道基因中的任一个或两者，以便于从被病毒载体感染的细胞群中鉴定和选择表达细胞。有用的可选择标记基因 包括例如抗生素抗性基因，诸如卡那霉素或neo等。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因。
在某些实施方案中，载体是用于将感兴趣的基因的表达框整合到宿主细胞的基因组中的载体，优选是转座载体。在某些实施方案中，该转座载体是含有选自piggybac、sleeping beauty、frog prince、Tn5或Ty的转座元件的真核表达载体。这类转座载体含有相应转座子的5’反向末端重复序列(5’ITR)和相应转座子的3’反向末端重复序列(3’ITR)。转座酶可以是来自piggybac、sleeping beauty、frog prince、Tn5或Ty转座系统的转座酶。当使用来自不同转座系统的转座酶时，所述载体中的5’ITR和3’ITR的序列也相应改变为与该转座系统适配的序列，这可由本领域技术人员容易地确定。通常，本发明的表达框位于5’ITR和3’ITR之间。
在某些实施方案中，转座酶是来自piggybac转座系统的转座酶。因此，在这些实施方案中，转座子5’反向末端重复序列和3’反向末端重复序列分别为piggybac转座子的5’反向末端重复序列和3’反向末端重复序列。在某些实施方案中，转座子5’反向末端重复序列如CN 201510638974.7(本文将其内容以引用的方式纳入本文)SEQ ID NO:1所示。在某些实施方案中，转座子3’反向末端重复序列如CN 201510638974.7的SEQ ID NO:4所示。在某些实施方案中，piggybac转座酶为含c-myc核定位信号编码序列的转座酶。在某些实施方案中，piggybac转座酶的编码序列如CN 201510638974.7的SEQ ID NO:5所示。
转座酶编码序列的启动子可以是本领域已知的用于控制转座酶编码序列表达的各种启动子。在某些实施方案中，使用CMV启动子控制转座酶编码序列的表达。CMV启动子的序列可如CN 201510638974.7SEQ ID NO:6所示。
在某些实施方案中，本发明的载体以CN 201510638974.7所公开的pNB328载体为骨架，但使用本文所述的启动子序列替换该载体原本含有的EF1α启动子。
在某些实施方案中，本发明的载体是空载体，即不含有感兴趣蛋白的编码 序列。通常，这类空载体依次含有一个或多个本文所述的启动子序列、一个或多个限制性内切酶酶切位点和转录终止序列，用于通过酶切将感兴趣蛋白的编码序列连接到所述启动子序列与转录终止序列之间。在某些实施方案中，本发明的载体是在本文所述的启动子序列和转录终止序列之间插入了感兴趣蛋白的编码序列的载体，优选是转座载体。在这类载体中，在5’ITR和3’ITR之间依次含有本文所述的启动子序列、感兴趣蛋白的编码序列和转录终止序列，优选地，在3’ITR的3’端还含有转座酶的编码序列及其启动子序列。
本发明也包括本文所述各核苷酸序列的互补序列。本文的多核苷酸序列可以是DNA形式或RNA形式。
本文所述的核苷酸序列通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。在某些实施方案中，在合适的情况下，可采用人工合成的方法合成本发明的某些核苷酸序列。
本发明中，感兴趣的蛋白可以是本领域周知的各种蛋白，包括但不限于酶、抗体以及其它具有所需功能的蛋白，如细胞因子。优选的是，感兴趣蛋白是本领域周知的需在T细胞中表达的蛋白，例如具有抗肿瘤作用的各种抗体，包括单链抗体，或嵌合抗原受体(CAR)等，以及细胞因子。
细胞因子是由免疫细胞(如单核细胞、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质。细胞因子一般通过结合相应受体调节细胞生长、分化和效应，调控免疫应答。细胞因子可具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。细胞因子可分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子和生长因子等。
在一些实施方案中，本发明的启动子序列尤其适合用于在T细胞中驱动各类抗体，优选是单链抗体的基因表达。优选地，所述抗体是自分泌抗体。优选 地，所述抗体为免疫检查点抗体，如PD-1抗体、CTLA4抗体、PD-L1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体和VISTA抗体等。在一些实施方案中，所述抗体包括双特异抗体，如免疫检查点抗体与TGF-beta形成的双特异抗体。优选地，所述抗体是来源于羊驼的纳米抗体。在一些实施方案中，所述抗体是PD-1抗体，其氨基酸序列可为SEQ ID NO：15所示序列编码的氨基酸序列。可采用常规的转染方法将本发明的载体转入感兴趣的细胞中，这些转入方法包括但不限于：病毒转导、显微注射、粒子轰击、基因枪转化和电转等。在某些实施方案中，采用电转将本文所述的载体转染入感兴趣的细胞中。在一些实施方案中，本申请的载体可同时表达两种以上感兴趣的蛋白，如同时表达本文任一实施方案所述的抗体和细胞因子。因此，在这些实施方案中，将这类载体转入感兴趣的细胞，即可获得同时表达所述抗体和细胞因子的细胞。或者，当载体仅表达一种感兴趣的蛋白时，可将两种或两种以上分别表达不同的感兴趣蛋白的载体共同转入感兴趣的细胞，以使其表达两种或两种以上的感兴趣的蛋白，如抗体、细胞因子以及CAR中的任意两种或三种。
感兴趣的细胞可以是本领域周知的各种T细胞，包括但不限于外周血T淋巴细胞、细胞毒杀伤T细胞(CTL)、辅助T细胞、抑制/调节性T细胞、γδT细胞以及细胞因子诱导的杀伤细胞(CIK)、肿瘤浸润淋巴细胞(TIL)等混合细胞群体的T细胞。在某些实施方案中，T细胞可来源于B细胞恶性肿瘤患者的PBMC。在某些实施方案中，T细胞为原代培养T细胞。
因此，在某些实施方案中，本发明提供本文所述的启动子序列在驱动外源基因(如单链抗体的编码序列)在活化的免疫细胞内表达中的应用。
在某些实施方案中，本发明还提供一种免疫细胞，尤其是T细胞，其含有本文所述的启动子序列或核酸构建物或载体，包括用于表达感兴趣的抗体和/或感兴趣的细胞因子的核酸构建物或载体。优选地，所述免疫细胞(尤其是T细胞)的基因组中整合了以本文所述的启动子序列作为启动子以驱动感兴趣外源基因(包括感兴趣的抗体和/或细胞因子)表达的表达框。更优选地，本发明的免疫细胞(尤其是T细胞)的基因组中整合了含有本文所述的启动子序列及与该启动子序列可操作性连接的免疫检查点抗体或其双特异抗体的编码序列 的表达框。在一些实施方案中，所述免疫细胞(尤其是T细胞)的基因组中整合了含有本文所述的启动子及与该启动子可操作性连接的细胞因子(尤其是趋化因子)的编码序列的表达框。在一些实施方案中，所述免疫细胞(尤其是T细胞)的基因组中整合了含有本文所述的启动子及与该启动子可操作性连接的细胞因子(尤其是趋化因子)的编码序列的表达框，以及含有本文所述的启动子及与该启动子可操作性连接的免疫检查点抗体或其双特异抗体的编码序列的表达框。
在一些实施方案中，所述免疫细胞是CAR T细胞，即表达CAR或含有CAR编码序列或表达CAR的载体的T细胞。因此，在一些实施方案中，本申请提供的CAR T细胞可同时表达CAR和感兴趣的抗体，或同时表达CAR和感兴趣的细胞因子，或同时表达CAR、感兴趣的抗体以及感兴趣的细胞因子。
CAR可以是本领域周知的任意CAR。本文中，感兴趣的嵌合抗原受体(CAR)可针对如下抗原中的一种或多种：Her2、CD19、CD20、CEA、GD2(又称B4GALNT1，β1,4-乙酰基-氨基半乳糖基转移酶1)、FR(Flavin还原酶)、PSMA(前列腺特异性膜抗原)、PMEL(前黑素小体蛋白)、CA9(碳酸酐酶IX)、CD171/L1-CAM、IL-13Rα2、MART-1(又称粘蛋白-A)、ERBB2、NY-ESO-1(又称CTAG1B，癌/睾丸抗原1B)、MAGE(黑素瘤相关抗原E1)家族蛋白、BAGE(B黑素瘤抗原家族)家族蛋白、GAGE(生长激素释放因子)家族蛋白、AFP(α-胎蛋白)、MUC1(mucin 1，细胞表面相关)、CD22、CD23、CD30、CD33、CD44v7/8、CD70、VEGFR1、VEGFR2、IL-11Rα、EGP-2、EGP-40、FBP、GD3(又称ST8SIA1，ST8α-N-乙酰基-神经酰胺α-2,8-唾液酸转换酶1)、PSCA(前列腺干细胞抗原)、FSA(又称KIAA1109)、PSA(又称KLK3，激肽释放酶相关的肽酶3)、HMGA2、胎儿型乙酰胆碱受体、LeY(又称FUT3)、EpCAM、MSLN(间皮素)、IGFR1、EGFR、EGFRvIII、ERBB3、ERBB4、CA125(又称MUC16，mucin 16，细胞表面相关)、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC(又称SERPINB3)、AFU(又称FUCA1)、EBV-VCA、POA(又称VDR，维生素D(1,25-二氢维生素D3)受体)、β2-MG(β-2-微球蛋白)和PROGRP(GRP胃泌素释放肽)。 应理解的是，除非另有说明，否则本文所述的各种抗原均是本领域周知的抗原，其序列为本领域所周知。
可将表达CAR的载体与含有与本发明启动子操作性连接的其它感兴趣基因(包括但不限于本文任一实施方案所述的感兴趣的抗体和/或细胞因子)的载体同时或先后转入T细胞内，从制备得到由本发明启动子控制其表达的感兴趣基因的CAR T细胞。
还提供的是一种药物组合物，其含有本发明的免疫细胞和药学上可接受的载体。
在一些实施方案中，本发明还提供一种在感兴趣的细胞中表达感兴趣的蛋白的方法，所述方法包括在感兴趣的细胞中转入含有与本文任一实施方案所述的启动子可操作性连接的该感兴趣蛋白的编码序列的核酸分子，在允许所述感兴趣的蛋白表达的情况下培育该细胞。还提供的是提高感兴趣的基因在活化的免疫细胞中的表达的方法，该方法包括在该活化的免疫细胞中转入含有与本文任一实施方案所述的启动子可操作性连接的该感兴趣的基因的载体，以及在适于该感兴趣基因表达的条件下培育该活化的免疫细胞的步骤。感兴趣的细胞和感兴趣的蛋白可如本文任一实施方案所述。细胞的培育条件为本领域所周知，并可根据不同的细胞种类加以选择。在一些实施方案中，所述方法包括构建含有所述启动子和与所述启动子可操作性连接的感兴趣蛋白的编码序列的载体，将该载体以周知的方式(如电转或脂质体转染)的方式转入该感兴趣的细胞内，以及在适合所述基因表达产生所述蛋白的条件下培育该细胞。
在一些实施方案中，本文还提供一种细胞免疫疗法，包括提供本文任一实施方案所述的免疫细胞，并给予需要的个体治疗有效量的所述免疫细胞。该个体可以是患有本领域周知的可用该免疫细胞所表达的蛋白如抗体和/或细胞因子和/或CAR进行治疗的疾病的个体，如哺乳动物，尤其是人。可采用本文任一实施方案所述的方法制备从而提供所述免疫细胞。在一些实施方案中，所述免疫细胞为自体细胞，即为来自于待治疗的个体的免疫细胞，体外经本文任一实施方案所述方法处理后将表达所需的抗体、细胞因子和/或CAR，然后被回输给予该个体。
本发明发现，当将含有感兴趣蛋白的编码序列以及与其操作性连接的本发明启动子的载体转入免疫细胞后，该感兴趣蛋白的表达量相对于对照可提到数倍至十几倍。因此，当将本发明的免疫细胞用作药物时，若本发明的药物在体内遇到活化试剂(如肿瘤抗原)时，本发明的免疫细胞将被活化并大量表达其所携带的外源基因，如各种治疗性分子，如治疗性抗体或嵌合抗原受体等，从而起到治疗作用；当疾病得以治疗而使得活化试剂减少或消除后，该免疫细胞恢复正常。
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：人细胞因子基因启动子表达载体构建
使用含SV40增强子DTS(SEQ ID NO：1)，EF1α启动子(SEQ ID NO：2)和TLTR(SEQ ID NO：3)组合成的嵌合细胞因子基因启动子的质粒pS338B-EGFP(图1)作为对照质粒及改造原型。
1.人IFN-γ基因启动子表达EGFP报告基因载体构建
构建载体pS-IFPT-EGFP
委托金唯智公司合成人IFN-γ基因启动子序列(uIFP，SEQ ID NO：4)，用Pvu I和Hind Ⅲ双酶切载体pUC57-uIFP，回收大小为253bp片段uIFP，含SV40增强子，EF1α启动子和TLTR组合成的嵌合细胞因子基因启动子的对照载体pS338B-EGFP经Hind Ⅲ和Pvu I双酶切后，将上述回收片段uIFP连接到pS338B-EGFP载体上，即得到含SV40增强子、IFN-γ启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-IFPT-EGFP(图2)。
构建载体pS-IL3en-EGFP
委托金唯智公司合成人IL-3基因增强子序列(IL3en,SEQ ID NO：5)，用Pvu I和Nhe I双酶切载体pUC57-IL3en，回收含IL3基因增强子大小为266bp的片段，含EF1α启动子和TLTR序列的pS338B-EGFP载体经Xba I和Pvu I双酶切后，将上述回收片段IL3en连接到pS338B-EGFP载体上，连接正确的克隆即为含IL3基因增强子、EF1α启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-IL3en-EGFP(图3)。
构建载体pS-uIFP-EGFP
委托金唯智公司合成5’端磷酸化修饰的引物：
IFNp-f：5’-TCTGCGATCGAAAAGTGCCTTCAAAGAATCC-3’(SEQ ID NO:16)
IFNp-r：5’-GTTAAAACAATACTGCAGCTGCACCTCCTCTGGCTGC-3’(SEQ ID NO:17)
利用上述合成引物，以载体pS-IFPT-EGFP为模板，通过PCR扩增得到含IFN-γ启动子长度为256bp的序列(SEQ ID NO：6)，将此片段与载体pS-IFPT-EGFP均用Xba I和EcoR I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含IFN-γ基因启动子的载体：pS-uIFP-EGFP(如图4)。
构建载体pS-uIFPT-EGFP
设计引物以从pS-IFPT-EGFP载体上扩增出IFN-γ基因启动子及DTS-EF1α序列，委托金唯智公司合成5’端磷酸化修饰的引物：
IFNp-f2：5’-TCTAGAAGGATCTGCGATCGAAAAGTGCCTT-3’(SEQ ID NO:18)
TLR-r：5’-ATGGTGGCGAATTCGTAGGCGCCGGTCAC-3’(SEQ ID NO:19)
利用上述合成引物，以载体pS-IFPT-EGFP为模板，通过PCR扩增得到由 IFN-γ启动子和TLTR组成的长度为555bp的序列(SEQ ID NO：7)，将此片段与载体pS-IFPT-EGFP均用Xba I和EcoR I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含IFN-γ基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-uIFPT-EGFP(如图5)。
构建载体pS-CIFT-EGFP
设计引物，从pC23-MCS载体上扩增出CMV基因增强子，委托金唯智公司合成5’端磷酸化修饰的引物：
CMVen-f：5’-CACCTCTAGAGACATTGATTATTGACT-3’(SEQ ID NO:20)
CMVen-r：5’-GACTCGATCGCATGGTAATAGCGATG-3’(SEQ ID NO:21)
利用上述合成引物，以含人CMV基因增强子载体pC23-MCS为模板，通过PCR扩增得到长度为380bp的CMV基因增强子序列(SEQ ID NO：8)，将此片段与载体pS-IFPT-EGFP均用Xba I和Pvu I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含CMV基因增强子、IFN-γ基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-CIFT-EGFP(如图6)。
构建载体pS-ILFP-EGFP
将含IFN-γ基因启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-IFPT-EGFP和含IL3基因增强子的载体pS-IL3en-EGFP均用Pvu I和EcoR I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确克隆，其含IL3基因增强子、IFN-γ基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-ILFP-EGFP(如图7)。
2.人IL-2基因启动子表达EGFP报告基因载体构建
构建载体pS-ILPT-EGFP
委托金唯智公司合成人IL-2基因启动子序列(IL2P,SEQ ID NO：9)，用Pvu I和Hind Ⅲ双酶切载体pUC57-IL2P，回收大小为300bp片段IL2P，含EF1α启动子和TLTR序列的pS338B-EGFP载体经Hind Ⅲ和Pvu I双酶切后，将上述回收片段IL2P连接到pS338B-EGFP载体上，连接正确的克隆即为含SV40增强子、IL2基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-ILPT-EGFP(图8)。
构建载体pS-uILP-EGFP
设计引物以从pS-ILPT-EGFP载体上扩增出IL-2基因启动子，委托金唯智公司合成5’端磷酸化修饰的引物：
IL2p-f：5’-TCTAGAATCTGCGATCGCCCCACCCCC-3’(SEQ ID NO:22)
IL2p-r：5’-GAATTCCTCGAAGCTTCTTGAACAA-3’(SEQ ID NO:23)
利用上述合成引物，以载体pS-ILPT-EGFP为模板，通过PCR扩增得到长度为308bp的IL-2启动子序列(SEQ ID NO：10)，将此片段与载体pS-ILPT-EGFP均用Xba I和EcoR I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含IL2基因启动子的载体pS-uILP-EGFP(如图9)。
构建载体pS-uILT-EGFP
设计引物以从pS-ILPT-EGFP载体上扩增出IL-2基因启动子及DTS-EF1α序列，委托金唯智公司合成5’端磷酸化修饰的引物：
IL2p-f：5’-TCTAGAATCTGCGATCGCCCCACCCCC-3’(SEQ ID NO:24)
TLR-r：5-CACCATGGTGGCGAATTCGTAGGCGCCGGTC-3’(SEQ ID NO:25)
利用上述合成引物，以载体pS-ILPT-EGFP为模板，通过PCR扩增得到长度为598bp的IL-2基因启动子及DTS-EF1α序列(SEQ ID NO：11)，将此片段与载体pS-ILPT-EGFP均用Xba I和EcoR I双酶切后连接，转化Top10后得 到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含IL2基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-uILT-EGFP(如图10)。
构建载体pS-CILT-EGFP
将含CMV增强子序列的载体pS-CIFT-EGFP和含IL2基因启动子和TLTR序列的载体pS-ILPT-EGFP均用Pvu I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确克隆：pS-CILT-EGFP(如图11)。
构建载体pS-LILP-EGFP
将含IL3基因增强子序列的载体pS-ILPT-EGFP和含IL2基因启动子和TLTR序列的载体pS-IL3en-EGFP均用Pvu I和EcoR I双酶切后得到两个相应片段，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆，其含IL3基因增强子、IL2基因启动子和TLTR的嵌合细胞因子基因启动子的载体：pS-ILFP-EGFP(如图12)。
3.人IFN-γ基因启动子及其内含子中增强子表达EGFP报告基因载体构建
构建载体pS-IFen-EGFP
委托金唯智公司合成含人IFN-γ基因内含子中增强子序列(uIFen,SEQ ID NO：12)，载体pUC57-uIFen。
设计引物，从pUC57-uIFP及pUC57-uIFen载体上扩增出人IFN-γ基因启动子及增强子，并将其委托金唯智公司合成5’端磷酸化修饰的引物：
IFNP-f：5’-TCTGCGATCGAAAAGTGCCTTCAAAGAATCC-3’(SEQ ID NO:26)
IFNP-r：5’-GTTAAAACAATACTGCAGCTGCACCTCCTCTGGCTGC-3’(SEQ ID NO:27)
IFNen-f：5’-GCAGCTGCAGTATTGTTTTAACCTTCTGCTC-3’(SEQ ID NO:28)
IFNen-r：5’-TGGCGAATTCTAAGGACCTTTTTGAC-3’(SEQ ID NO:29)
利用上述合成引物，以载体pUC57-uIFP及pUC57-uIFNen为模板，通过重叠PCR扩增得到长度为519bp的人IFN-γ基因启动子及增强子(SEQ ID NO：13)，将此片段与含SV40增强子的载体pS-IFPT-EGFP均用Pvu I和EcoR I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含SV40增强子、IFN-γ基因启动子和IFN-γ增强子的嵌合细胞因子基因启动子的载体：pS-IFen-EGFP(如图13)。
构建载体pS-uIFen-EGFP
设计引物，从pS-IFen-EGFP载体上扩增出人IFN-γ基因启动子及增强子，并将其委托金唯智公司合成5’端磷酸化修饰的引物：
IFNP-f：5’-TCTGCGATCGAAAAGTGCCTTCAAAGAATCC-3’(SEQ ID NO:30)
IFNen-r：5’-TGGCGAATTCTAAGGACCTTTTTGAC-3’(SEQ ID NO:31)
利用上述合成引物，以载体pS-IFen-EGFP为模板，通过PCR扩增得到长度为529bp的人IFN-γ基因启动子及其增强子(SEQ ID NO：14)，将此片段与载体pS-IFPT-EGFP均用Xba I和EcoR I双酶切后连接，转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确后，送金唯智测序，正确的克隆即为含IFN-γ基因启动子和IFN-γ增强子的嵌合细胞因子基因启动子的载体：pS-uIFen-EGFP(如图14)。
构建载体pS-CIFen-EGFP载体pS-IFPT-EGFP和
将含CMV增强子和IFN-γ基因启动子的载体pS-CIFT-EGFP和含人IFN-γ基因内含子中增强子的载体pS-IFen-EGFP均用Pvu I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆，其含CMV增强子、IFN-γ基因启动子和IFN-γ增强 子的嵌合细胞因子基因启动子的载体：pS-CIFen-EGFP(如图15)。
构建载体pS-LIFen-EGFP
将含IFN-γ基因启动子和人IFN-γ基因内含子中增强子的载体pS-CIFen-EGFP和含IL3基因增强子的载体pS-IL3en-EGFP均用Pvu I和EcoR I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆，其含IL3基因增强子、IFN-γ基因启动子和IFN-γ增强子的嵌合细胞因子基因启动子的载体：pS-LIFen-EGFP(如图16)。
4.表达Fluc报告基因载体构建
构建载体pS338B-Fluc
将含SV40增强子，EF1α启动子和TLTR组合成的嵌合细胞因子基因启动子的对照载体pS338B-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS338B-Fluc(如图17)。
构建载体pS-IFPT-Fluc
将含SV40增强子，IFN-γ启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-IFPT-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS-IFPT-Fluc(如图18)。
构建载体pS-CIFT-Fluc
将含CMV增强子，IFN-γ启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-CIFT-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS-CIFT-Fluc(如图19)。
构建载体pS-ILFP-Fluc
将含IL3基因增强子，IFN-γ基因启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-ILFP-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS-ILFP-Fluc(如图20)。
构建载体pS-ILPT-Fluc
将载体含IL3基因增强子，IL2基因启动子和TLTR组合成的嵌合细胞因子基因启动子的pS-ILPT-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS-ILPT-Fluc(如图21)。
构建载体pS-CILT-Fluc
将含CMV增强子，IL2基因启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-CILT-EGFP和含荧光素酶报告基因的载体pS-AD-F-Fluc均用EcoR I和Xba I双酶切后得到相应片段和载体，连接转化Top10后得到单克隆用EcoR I和Sal I双酶切鉴定正确，即得到正确的克隆pS-CILT-Fluc(如图22)。
5.表达anti-PD1抗体的基因载体构建
从US 10160806中获取表达人抗PD1抗体(nivolumab)序列(SEQ ID NO：15)，并委托金唯智公司合成序列。
构建载体pS338B-αPD1
用EcoR I和Sal I双酶切并回收合成抗PD1抗体序列αPD1，将含DTS，EF1α启动子和TLTR组合成的嵌合细胞因子基因启动子的对照载体pS338B-EGFP经EcoR I和Sal I双酶切后，将上述回收片段抗PD1抗体序列α PD1连接到pS338B-EGFP载体上，即得到可表达抗PD1抗体，由DTS、EF1α启动子和TLTR组合成的嵌合细胞因子基因启动子的载体：pS338B-αPD1(如图23)。
构建载体pS-CIFT-αPD1
用EcoR I和Sal I双酶切并回收合成抗PD1抗体序列αPD1，将含CMV增强子，IFN-γ启动子和TLTR组合成的嵌合细胞因子基因启动子的载体pS-CIFT-EGFP经EcoR I和Sal I双酶切后，将上述回收片段抗PD1抗体序列αPD1连接到pS-CIFT-EGFP载体上，即得到可表达抗PD1抗体，由CMV增强子、IFN-γ启动子和TLTR组合成的嵌合细胞因子基因启动子的载体：pS-CIFT-αPD1(如图24)。
实施例2：构建的细胞因子基因启动子在T细胞中表达的检测
1.以pS338B-EGFP为对照质粒，通过EGFP表达强度测定启动子表达情况
复苏并重悬5×10 6新鲜分离获得的外周血单个核细胞(PBMC)，使用Lonza 4D-Nucleofector将4μg间皮素CAR质粒pNB338B-MSLN CAR(其构建参见CN201711459160.2或者201711462801.X，不同之处在于，载体骨架由pNB328替换为pNB338B，载体pNB338B的序列及结构参见CN201711476630.6中“pNB338B-E”)及4μg实施例1中得到的细胞因子基因启动子表达EGFP蛋白的报告基因载体(如：pS-uILP-EGFP等)质粒电转到PBMC中，置37℃、5％CO 2培养箱中培养；4小时后转移到用5μg/ml antiCD28抗体，或5μg/ml人间皮素抗原和5μg/ml antiCD28抗体(购自Novoprotein)包被的培养板中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)和500IU/ml IL-2(购自Novoprotein)；5天后将细胞转移至无抗原包被的培养板中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)和200IU/ml IL-2(购自Novoprotein)，培养第7天对细胞进行流式检测。
图25是相同增强子情况下IFN-γ启动子和IL-2启动子活性通过FlowJo Ⅹ 分析得到的流式分析结果。结果显示，IFN-γ启动子表达强度较IL-2启动子强，且启动子序列前的增强子对启动子表达有增强作用。在此基础上，将启动子后TLTR序列替换成IFN-γ内含子中的增强子序列，比较启动子序列后两种不同序列对原启动子的作用。图26是同一启动子序列情况下，改变启动子序列后TLTR序列启动子活性变化的流式分析图。结果显示，改变启动子后TLTR序列对启动子活性有一定程度的影响。图27比较了pS338B-EGFP和pS-CIFT-EGFP在使用相同启动子序列的情况下，不同活化T细胞方式对启动子活性的影响，表明与对照质粒pS338B-EGFP相比，含CMV基因增强子、IFN-γ基因启动子和TLTR的嵌合细胞因子基因启动子pS-CIFT-EGFP表达活性更易受细胞活化影响。
2.以pS338B-Fluc为对照质粒，双荧光素酶报告系统进一步检测启动子表达情况
2.1 T细胞电转：复苏并重悬5×10 6新鲜分离获得的外周血单个核细胞(PBMC)，使用Lonza 4D-Nucleofector将4μg质粒pS-AD-F-Rluc(表达海肾荧光素酶基因载体)及4μg实施例1中得到的细胞因子基因启动子报告基因载体(如：pS-IFPT-Fluc/pS-CIFT-Fluc/pS-ILFP-Fluc/pS-ILPT-Fluc/pS-CILT-Fluc等，表达萤火虫荧光素酶基因载体)质粒电转到PBMC中，置37℃、5％CO 2培养箱中培养；4小时后转移到用5μg/ml anti-CD3抗体和5μg/ml antiCD28抗体(购自Novoprotein)包被的培养板中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)和500IU/ml IL-2(购自Novoprotein)；48小时后用Promega双荧光素酶报告系统检测试剂盒检测细胞双荧光素酶活力，以检测各启动子活性。
2.2双荧光素酶报告系统检测(按试剂盒说明书进行)：a将每组细胞以5×10 4细胞数用75uL体积AIM-V培养基重悬后铺到96孔板中；b向a中加入等体积75uL的
Figure PCTCN2020128526-appb-000001
Reagent，混匀放置于摇床上，等待至少10分钟(少于2小时)，使细胞充分裂解，检测萤火虫荧光素发光强度；c加入75uL的
Figure PCTCN2020128526-appb-000002
Stop&
Figure PCTCN2020128526-appb-000003
Reagent，混匀放置于摇床上，等待至少10 分钟(少于2小时)，使细胞充分裂解，检测海肾荧光素发光强度；d用萤火虫荧光素检测强度数值除以海肾荧光素检测荧光强度数值，即可得到信号均一化的数值，进而反映出各启动子活性。
选取流式细胞术检测荧光强度较高的组别构建双荧光素酶报告系统质粒，转染T细胞后结果如图28所示，其中pS-CIFT-Fluc组萤火虫荧光素检测强度数值除以海参荧光素检测荧光强度数值与对照组质粒pS338B-EGFP组有显著性差异，且比值远超于其他组细胞因子基因启动子及增强子不同的组合荧光素比值，故而CMV增强子与IFN-γ基因启动子及TLTR这一组合启动子活性最高。
实施例3：构建的细胞因子基因启动子在HEK-293T细胞中表达的检测
1.HEK-293T细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：用胰酶消化培养状态良好的HEK-293T，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为DMEM培养基+10％血清；
B.换培液：HEK-293T细胞于6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有HEK-293T细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图29所示，干扰素γ基因启动子和白介素2基因启动子在HEK 293T细胞中表达活性不如对照质粒pS338B-EGFP。
实施例4：构建的细胞因子基因启动子在CHO细胞中表达的检测
1.CHO细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：用胰酶消化培养状态良好的CHO细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为45％DMEM培养基+45％RPMI-1640培养基+10％血清+1％L-谷氨酰胺+1％次黄嘌呤-胸苷；
B.换培液：CHO细胞于6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有CHO细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至 荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图30所示，干扰素γ基因启动子和白介素2基因启动子在CHO细胞中表达活性不如对照质粒pS338B-EGFP。
实施例5：构建的细胞因子基因启动子在树突状细胞(DC)中表达的检测
1.DC细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：收集培养状态良好的DC细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为AIM-V；
B.换培液：DC细胞在6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有DC细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图31所示，干扰素γ基因启动子和白介素2基因启动子在DC细胞中表达活性不如对照质粒pS338B-EGFP。
实施例6：构建的细胞因子基因启动子在急性T细胞白血病细胞株Jurkat细胞中表达的检测
1.Jurkat细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：收集培养状态良好的Jurkat细胞，计数，取6×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为RPMI-1640培养基+10％血清；
B.换培液：Jurkat细胞在6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有Jurkat细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图32所示，干扰素γ基因启动子和白介素2基因启动子在Jurkat细胞中表达活性不如对照质粒pS338B-EGFP。
实施例7：构建的细胞因子基因启动子在B细胞恶性淋巴瘤Raji细胞中表达的检测
1.Raji细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：收集培养状态良好的Raji细胞，计数，取6×105细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为RPMI-1640培养基 +10％血清；
B.换培液：Raji细胞在6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有Raji细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图33所示，干扰素γ基因启动子和白介素2基因启动子在Raji细胞中表达活性不如对照质粒pS338B-EGFP。
实施例8：构建的细胞因子基因启动子在肝癌细胞株Hep G2细胞中表达 的检测
1.Hep G2细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：收集培养状态良好的Hep G2细胞，计数，取3×10 5细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为RPMI培养基+10％血清；
B.换培液：Hep G2细胞在6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有Hep G2细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液 清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照
结果如图34所示，干扰素γ基因启动子和白介素2基因启动子在Hep G2细胞中表达活性不如对照质粒pS338B-EGFP。
实施例9：构建的细胞因子基因启动子在卵巢癌细胞株SKOV3细胞中表达的检测
1.SKOV3细胞转染(ViaFect转染试剂盒)：以pS338B-EGFP为对照质粒
A.细胞铺板：收集培养状态良好的SKOV3细胞，计数，取3×105细胞重悬到3mL培液中，铺到6孔板中培养24小时，培养基成分为DMEM培养基+10％血清；
B.换培液：SKOV3细胞在6孔板培养24h后，弃尽原细胞培养液，加1mL PBS缓冲液清洗细胞，弃尽洗液，向其中加入2mL新培液，并将6孔板重新放回37℃培养箱中培养；
C.配质粒(每孔)：1ug实施例1中构建的表达EGFP的载体(pS-CIFT-EGFP/pS-IFPT-EGFP/pSCILT-EGFP/pSCILT-EGFP/pS338B-EGFP)+1ug表达RFP质粒+6uL转染液(质粒：转染试剂＝1:3)，溶于200uL opt培养基中，涡旋振荡10s，室温孵育5-20min，形成复合物；
D.加转染混合物：迅速将C中配置好的混合物滴加到铺有SKOV3细胞的6孔板中，将6孔板摇晃数次后放回37℃，5％CO 2培养箱中培养24h-48h，至荧光表达强度适宜时进行DAPI染色。
2.DAPI染色
A.固定：将转染后荧光表达强度适宜的细胞用1mL PBS缓冲液清洗2遍，弃尽洗液后向细胞中加1mL 4％多聚甲醛固定液，室温静置30分钟；
B.通透：将固定后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL含0.3％Triton X-100的PBS缓冲液，室温静置30分钟；
C.DAPI染色：将通透后的细胞用1mL PBS缓冲液清洗3次，每次5分钟，弃尽洗液后向细胞中加1mL配制的DAPI染料，轻晃数次后室温静置4min，荧光显微镜下观察，若细胞核已上色完全，进行步骤D，未上色完全，继续静置至细胞核上色完全；
D.荧光显微镜下观察：弃尽DAPI染色液，并向其中加1mL PBS缓冲液清洗细胞，摇晃数次后室温静置5min，重复3次，荧光显微镜拍照。
结果如图35所示，干扰素γ基因启动子和白介素2基因启动子在SKOV3细胞中表达活性不如对照质粒pS338B-EGFP。
实施例10：嵌合干扰素γ基因启动子在自分泌anti-PD1抗体CAR-T细胞中的应用
复苏并重悬5×10 6新鲜分离获得的外周血单个核细胞(PBMC)，使用Lonza4D-Nucleofector将4μg间皮素CAR质粒pNB338B-MSLN CAR及4μg实施例1中得到的细胞因子基因启动子表达PD1抗体的载体(如：pS338B-αPD1等)质粒电转到PBMC中，置37℃、5％CO 2培养箱中培养；4小时后转移到用5μg/ml antiCD28抗体，或5μg/ml人间皮素抗原和5μg/ml antiCD28抗体(购自Novoprotein)包被的培养板中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)和500IU/ml IL-2(购自Novoprotein)；5天后将细胞转移至无抗原包被的培养板中培养，培养基成分为AIM-V(Gibco)、2％胎牛血清(Gibco)和200IU/ml IL-2(购自Novoprotein)。取1×10 4高表达PDL1的卵巢癌SKOV3细胞均匀铺到96孔板中，培养24h后，向每孔中分别加入5×10 3转染不同质粒，培养到第10天的CAR-T细胞，共培养24h后，取上清，离心收集上清，通过酶联免疫吸附剂测定(ELISA)测得上清中CAR-T细胞分泌的PD1抗体含量。
ELISA检测步骤：
A.抗原包板：准备PD-1抗原Human PD-1/PDCD1Protein(HPLC-verified)。第一次使用时，用商业化PBS将抗原溶解。用包被液将抗原稀释至1ug/ml，100ul/孔包被酶标反应板，4℃过夜。过夜后，用PBST清洗5遍，200ul/孔， 每次3分钟，用吸水纸拍干；
B.封闭：每孔加封闭液300ul，37℃生化培养箱孵育2小时。用PBST清洗5遍，200ul/孔，每次3分钟，用吸水纸拍干；
C.加样：加入样品及标准品zAb，100ul/孔，设复孔和对照孔，37℃生化培养箱孵育1小时。用稀释液稀释样品及标准品，其中标准品从30ng/ml开始，向下设置7个梯度及0ng/ml。样品根据实际情况进行稀释，本次实验样本设置40倍、400倍稀释。用PBST清洗5遍，200ul/孔，每次3分钟，用吸水纸拍干；
D.加二抗：用封闭液将IgG HRP 1：20000稀释，100ul/孔，37℃生化培养箱孵育1h。用PBST清洗5遍，200ul/孔，每次3分钟，用吸水纸拍干。
E.显色：加入显色液TMB，100ul/孔，室温避光显色6min；
F.终止：加入终止液50ul/孔，终止反应。立即进行上机读数。
如图36所示，CAR质粒与pS-CIFT-αPD1或pS338B-αPD1质粒共转后，T细胞获得自分泌PD1抗体的能力，且CMV增强子-干扰素γ启动子-TLTR嵌合启动子在分泌PD1抗体中表达活性方面明显优于DTS-EF1α-TLTR嵌合启动子。

Claims (15)

  1. 一种启动子或其碱基序列与该启动子的碱基序列互补的核酸分子，其中，所述启动子从5’端到3’端包含依次连接的CMV增强子、IFNγ启动子和人T细胞白血病病毒的长末端重复序列。
  2. 如权利要求1所述的启动子或其碱基序列与该启动子的碱基序列互补的核酸分子，其特征在于，
    所述CMV增强子选自：具有SEQ ID NO:8所示核苷酸序列的CMV增强子，或来自人CMV的与SEQ ID NO:8所示核苷酸序列具有至少97％序列同一性的CMV增强子；和/或
    所述IFNγ启动子选自：具有SEQ ID NO:4所示核苷酸序列的IFNγ启动子，或来自人的与SEQ ID NO:4所示核苷酸序列具有至少97％序列同一性的IFNγ启动子；和/或
    所述人T细胞白血病病毒的长末端重复序列选自：具有SEQ ID NO:3所示核苷酸序列的长末端重复序列，或来自人T细胞白血病病毒的与SEQ ID NO:3所示核苷酸序列具有至少97％序列同一性的长末端重复序列。
  3. 一种核酸构建物，其特征在于，所述核酸构建物含有权利要求1-2中任一项所述的启动子或其碱基序列与该启动子的碱基序列互补的核酸分子和与该启动子或该核酸分子操作性连接的感兴趣的基因。
  4. 如权利要求3所述的核酸构建物，其特征在于，所述感兴趣的基因编码自分泌抗体和/或细胞因子；优选地，所述自分泌抗体为免疫检查点抗体，如PD-1抗体、CTLA4抗体、PD-L1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体和VISTA抗体，更优选为来源于羊驼的纳米抗体；优选地，所述细胞因子选自白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子和生长因子。
  5. 如权利要求3或4所述的核酸构建物，其特征在于，所述核酸构建物是表达框。
  6. 一种载体，其特征在于，所述载体含有权利要求1-2中任一项所述的启动子或核酸分子，或权利要求3-5中任一项所述的核酸构建物。
  7. 如权利要求6所述的载体，其特征在于，所述载体为表达载体或克隆载体。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求1-2中任一项所述的启动子或核酸分子、权利要求3-5中任一项所示的核酸构建物或权利要求6-7中任一项所示的载体。
  9. 如权利要求8所述的宿主细胞，其特征在于，所述宿主细胞为免疫细胞，优选为T细胞，其基因组中整合有权利要求3-5中任一项所述的核酸构建物；优选地，所述免疫细胞还表达CAR或含有CAR的表达载体。
  10. 如权利要求8所述的宿主细胞，其特征在于，所述宿主细胞为免疫细胞，优选为T细胞，所述免疫细胞含有：权利要求1-2中任一项所述的启动子或核酸分子及与该启动子或核酸分子可操作性连接的细胞因子的编码序列，和/或权利要求1-2中任一项所述的启动子或核酸分子及与该启动子可操作性连接的免疫检查点抗体或其双特异抗体的编码序列。
  11. 如权利要求10所述的宿主细胞，其特征在于，所述免疫细胞的基因组中整合了：含有权利要求1-2中任一项所述的启动子或核酸分子及与该启动子或核酸分子可操作性连接的细胞因子的编码序列的表达框，和/或含有权利要求1-2中任一项所述的启动子或核酸分子及与该启动子或核酸分子可操作性连接的免疫检查点抗体或其双特异抗体的编码序列的表达框。
  12. 如权利要求10或11所述的宿主细胞，其特征在于，所述免疫细胞还表达CAR或含有CAR的表达载体。
  13. 如权利要求10-12中任一项所述的宿主细胞，其特征在于，
    所述免疫检查点抗体选自PD-1抗体、CTLA4抗体、PD-L1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体和VISTA抗体，优选为来源于羊驼的纳米抗体；
    所述细胞因子选自白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子和生长因子。
  14. 权利要求1-2中任一项所述的启动子或核酸分子在提高感兴趣的基因在活化的免疫细胞中的表达中的应用，或在制备用于在活化的免疫细胞中增强表达的核酸构建物或载体中的应用。
  15. 如权利要求14所述的应用，其特征在于，所述感兴趣的基因编码自分泌抗体和/或细胞因子；
    优选地，所述自分泌抗体为免疫检查点抗体，如PD-1抗体、CTLA4抗体、 PD-L1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体和VISTA抗体，更优选为来源于羊驼的纳米抗体；
    优选地，所述细胞因子选自白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子和生长因子。
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