Chimeric promoter having high activity in activated immune cells

Abstract

Provided is a chimeric promoter lacking a CpG motif, which comprises a CMV enhancer, an IFNγ gene core promoter, and an intron, wherein a CG motif in the promoter is subjected to a substitution mutation. The chimeric promoter can improve the expression efficiency of exogenous genes in activated immune cells.

Description

A chimeric promoter with high activity in activated immune cells Technical Field This invention relates to the field of genes, and more particularly to a chimeric promoter that has high activity in activated immune cells. Background Technology Mammalian promoters can be classified into constitutive promoters, tissue-specific promoters, and inducible promoters based on their transcriptional patterns. Commonly used promoters are mainly non-tissue-specific constitutive promoters. These promoters maintain relatively stable expression activity in most cells, but their application is primarily limited to the cellular level. Examples include human or mouse cytomegalovirus promoters (hCMV / mCMV) and Simian virus 40 promoters (SV40). CAG is an artificially constructed chimeric promoter composed of a CMV enhancer, a chicken beta-actin promoter, and a rabbit beta-globin intron. It can be used to drive high-level expression of exogenous genes in mammals. Therefore, constructing a chimeric promoter that specifically and efficiently drives exogenous protein expression in T cells should meet the following conditions: a strong enhancer and a promoter with high expression in T cells. Currently, the main factors affecting the expression of exogenous proteins in mammalian cell expression systems include exogenous gene expression efficiency, copy number and stability of expression plasmids, stability of expression products, and cell culture conditions. Effective transcription initiation is crucial for controlling the expression of exogenous genes in the host, and promoter strength directly influences transcription initiation. However, CpG-induced inflammation often leads to immune-mediated destruction of transgenic cells, inhibiting transgene expression, prolonging inflammation duration, and consequently causing autoimmunity. In mammalian genomes, CpG dinucleotides (PuPuCGPyPy) are typically methylated with cytosine. Methylation of CpG in promoters usually leads to promoter silencing and gene expression shutdown. DNA containing unmethylated CG motifs can activate mouse bone marrow B lymphocytes, stimulating immune defense. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG-containing DNA fragments as foreign DNA and induces an acute inflammatory response accordingly. Summary of the Invention The first aspect of the present invention provides a chimeric promoter comprising a CMV enhancer, an IFN-γ gene core promoter, and an intron. In some embodiments, the chimeric promoter comprises, from the 5' end to the 3' end, a CMV enhancer, an IFN-γ gene core promoter, and an intron connected in sequence. The IFN-γ gene core promoter contains a sequence as shown in SEQ ID NO: 30 or a sequence having at least 90% sequence identity with SEQ ID NO: 30. In some implementations, the CMV enhancer, IFN-γ gene core promoter, and intron are directly linked or operatively linked via a adapter. In some implementations, one or both CG dinucleotide sequences in the IFN-γ gene core promoter undergo substitution mutations. In some implementations, the substitution mutation is an independent mutation of CG into AG, TG, GG, CA, CT, or CC. In some implementations, the CMV enhancers and / or introns do not contain CpG motifs. In some embodiments, the nucleotide sequence of the CMV enhancer is as shown in SEQ ID NO:29, or is selected from a sequence having at least 90% sequence identity with SEQ ID NO:29. In some embodiments, the nucleotide sequence of the IFN-γ gene core promoter is as shown in SEQ ID NO:30, or a sequence that has at least 90% sequence identity but retains promoter function and retains the CG dinucleotide sequence substitution mutation contained in SEQ ID NO:30. In some embodiments, the IFN-γ gene core promoter is as shown in SEQ ID NO:30, and has a mutation at position 62-63, wherein the mutation is selected from any of the following: AG, CA, CC, CT, GG, TG, with CA being preferred. In some embodiments, the IFN-γ gene core promoter is as shown in SEQ ID NO:30, and has a mutation at position 194-195, the mutation being selected from any of the following: AG, CA, CC, CT, GG, TG, preferably CA, CC or GG. In some embodiments, the IFN-γ gene core promoter is as shown in SEQ ID NO:30, and has mutations at a first site and a second site, wherein the first site is position 62-63 of the sequence shown in SEQ ID NO:30, and the second site is position 194-195 of the sequence shown in SEQ ID NO:30. The mutation is selected from any one or more of the following: AG, CA, CC, CT, GG, TG. Preferably, the first site mutation is CA, and the second site mutation is CA, CC, or GG. In some embodiments, the intron is the SI126 intron, preferably, the nucleotide sequence of the intron is as shown in SEQ ID NO:31, or is selected from a sequence having at least 90% sequence identity with SEQ ID NO:31. In some embodiments, the sequence of the chimeric promoter is as shown in any of SEQ ID NO: 32-35. A second aspect of the present invention provides a nucleic acid construct comprising the chimeric promoter described in the first aspect herein, and a gene of interest operatively linked to the promoter. In some implementations, the nucleic acid construct is an expression frame. In some embodiments, the gene-encoding antibody of interest is 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, and more preferably a nanobody derived from alpacas. In some embodiments, the gene of interest encodes a cytokine, preferably selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. In some embodiments, the gene of interest encodes a chimeric antigen receptor, preferably comprising an optional signal peptide sequence, an extracellular target recognition region (antigen-binding domain), a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain. In some embodiments, the CAR, from N-terminus to C-terminus, sequentially comprises a CD8 signal peptide, an anti-MSLN single-domain antibody or an anti-MUC1 single-domain antibody, a CD8 hinge region, a CD28 transmembrane region or a CD8 transmembrane region, a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. In some embodiments, the nucleic acid construct is a vector, preferably a non-viral vector, and more preferably a vector suitable for replication, transcription, and / or expression in animals. Preferably, the animal is a mammal, and more preferably, the animal is a human. In some implementations, the nucleic acid construct is a cloning vector, expression vector, or recombinant vector. The present invention also provides a host cell comprising: (1) Having the expression frame of the chimeric promoter described in the first aspect of this document, or, (2) The nucleic acid constructs described in the second aspect of this article. In some embodiments, the host cell includes an immune effector cell whose genome is integrated with the nucleic acid construct described in any of the embodiments herein; preferably, the immune effector cell also expresses CAR or an expression vector containing CAR. In some embodiments, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; preferably, the immune effector cells are T cells. In some embodiments, the immune effector cells contain: the chimeric promoter as described in any embodiment of the present invention and the coding sequence of a cytokine operably linked to the chimeric promoter. In some embodiments, the immune effector cells contain: the coding sequence of the chimeric promoter as described in any embodiment of the present invention and an immune checkpoint antibody or its bispecific antibody operatively linked to the chimeric promoter. In some embodiments, the genome of the immune effector cells integrates an expression frame containing the coding sequences of a chimeric promoter as described in any embodiment of the present invention and a cytokine operatively linked to the chimeric promoter. In some embodiments, the genome of the immune effector cell integrates an expression cassette containing the coding sequence of the chimeric promoter as described in any embodiment of the present invention and an immune checkpoint antibody or its bispecific antibody operatively linked to the chimeric promoter. In some embodiments, the immune checkpoint antibody is selected from PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody, preferably a nanobody derived from alpaca. In some embodiments, the cytokines are selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. The present invention also provides a kit comprising the chimeric promoter described in the first aspect, the nucleic acid construct described in the second aspect, and / or host cells. The present invention also provides a method for driving the expression of a gene of interest in cells, the method comprising the step of incubating cells under conditions for expression initiated by a chimeric promoter as described in any embodiment herein. In some embodiments, the conditions are conditions suitable for the growth of the cells. In some embodiments, the method includes: (1) introducing the chimeric promoter into the nucleic acid construct described herein; (2) transferring the nucleic acid construct into cells; and (3) incubating the cells under conditions suitable for expression of the nucleic acid construct. In some embodiments, the conditions are conditions suitable for cell growth. In some implementations, the cells are immune effector cells. In some embodiments, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; preferably, the immune effector cells are T cells. The present invention also provides the use of the chimeric promoter described herein in enhancing the expression of a gene of interest in activated immune effector cells, or in the preparation of nucleic acid constructs for enhancing expression in activated immune effector cells. In some implementations, the immune effector cells are activated immune effector cells. In some embodiments, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; preferably, the immune effector cells are T cells. In some embodiments, the gene-encoding antibody of interest is 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, and more preferably a nanobody derived from alpacas. Attached Figure Description Figure 1: pTini-mCIS-dCGFluc plasmid map. Figure 2: Expression efficiency of CpG dinucleotide sequence modification in CMV enhancers and introns in PBMC cells. Figure 3: Expression efficiency of the first point single mutation in the IFN-γ core promoter in PBMC cells using pTini-mCIS-dCGFluc as a template. Figure 4: Expression efficiency of double mutation at the second site of the IFN-γ core promoter in PBMC cells using pTini-mCIS-dCGFluc as a template. Figure 5: pTini-DTS-dCGFluc plasmid map. Figure 6: pTini-JL(DTS)-1444-8E plasmid map. Figure 7: pRL-TK plasmid map. Detailed Implementation Unless otherwise defined, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art. These techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Animal Cell Culture (edited by R.R. Freshney, 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., 1987 edition and its periodically updated editions); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); A Practical Guide to Molecular Cloning (Perbal Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001). This invention uses a combination of transcriptional elements such as CMV enhancer, IFN-γ cytokine gene promoter core and intron to construct a chimeric promoter, which has high expression activity in activated immune cells, especially in activated T cells. The inventors provide a chimeric promoter whose core promoter comprises, from the 5' end to the 3' end, a CMV enhancer, an IFN-γ gene core promoter, and an intron connected sequentially. The CMV enhancer, IFN-γ gene core promoter, and intron can be directly linked or linked via a adapter. In some embodiments, the adapter is an enzyme restriction site that facilitates genetic manipulation. For example, the adapter may contain GTCGAG or GTCGAGAAGGG. In this invention, "activation" refers to a state in which cells have been sufficiently stimulated to induce detectable cell proliferation and / or have been stimulated to exert their effector functions, such as induced cytokine expression and secretion, phagocytosis, cell signaling, antigen processing and presentation, and target cell killing. Immune cells can be activated in vitro by stimulation with appropriate activating agents, or in vivo by stimulation with antigens, growth factors, or cells. Typically, the activating agents are conventional in the art, such as anti-CD28 antibodies and optionally, appropriate immunogens. For example, in some embodiments, when a chimeric antigen receptor expressing a tumor antigen is introduced, the activating agent may also include the tumor antigen or its active fragment. This invention does not impose particular limitations on the timing of activation, the concentration at the time of activation, or the duration of activation. In some embodiments, this invention uses anti-CD28 antibodies for activation; in some embodiments, this invention uses a combination of anti-CD28 antibodies and tumor antigens for activation. The term "coding sequence" refers to the portion of a nucleic acid sequence that directly identifies its protein product. The boundaries of a coding sequence are typically defined by the ribosome-binding site (for prokaryotic cells) immediately upstream of the 5' open reading frame of the mRNA 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. The term "operable link" or "operable connection" refers to a functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. For example, in a nucleic acid construct, a promoter is placed at a specific location on the nucleic acid sequence of a gene of interest, such as an upstream position of the promoter region, so that transcription of the nucleic acid sequence is guided by the promoter region. Thus, the promoter region is "operable linked" to the nucleic acid sequence of the gene. "Operable link" can be achieved through gene recombination. The enhancer of this invention is a CMV enhancer. This invention can be implemented using CMV enhancers known in the art, including mCMV enhancers from the genus *Mice* cytomegalovirus and hCMV enhancers from the genus *Human* cytomegalovirus, preferably human CMV enhancers. An exemplary CMV enhancer may have the nucleotide sequence shown in SEQ ID NO:29. The IFNγ promoter of the present invention is typically selected from its core region sequence or a fragment of an IFNγ promoter containing that core region sequence. An exemplary IFN-γ gene core promoter is a fragment of an IFNγ promoter having the nucleotide sequence shown in SEQ ID NO:30. In preferred embodiments, in some embodiments, the nucleotide sequence of the IFN-γ gene core promoter is as shown in SEQ ID NO:30. In activated immune cells, the IFN-γ gene core promoter, with increased IFN-γ secretion, stimulates the promoter and promotes gene expression; therefore, the chimeric promoter has high activity in activated immune cells. In inactivated immune cells, however, IFN-γ secretion is low, and gene expression levels are low. In some embodiments, one or more CG dinucleotide sequences in the IFN-γ gene core promoter undergo substitution mutations. In some embodiments, the substitution mutation is an independent mutation of each CG to AG, TG, GG, CA, CT, or CC. For example, the IFN-γ gene core promoter is shown in SEQ ID NO:30, and has a mutation at positions 62-63, the mutation being selected from any of the following: AG, CA, CC, CT, GG, TG, preferably CA. As another example, the IFN-γ gene core promoter is shown in SEQ ID NO:30, and has a mutation at positions 194-195, the mutation being selected from any of the following: CA, CT, TG, CC, GG, AG, preferably CA, CC, or GG. In some embodiments, the IFN-γ gene core promoter is as shown in SEQ ID NO:30, and has mutations at positions 62-63 and 194-195, wherein the mutations are selected from any one or more of the following: AG, CA, CC, CT, GG, TG. Preferably, the mutation at position 62-63 is CA, and the mutation at position 194-195 is CA, CC, or GG. In some embodiments, the sequence of the introns suitable for use in this invention is shown in SEQ ID NO:31. This invention also includes nucleotide sequence variants of the nucleotide sequences shown in SEQ ID NO:29, 30, and 31 with one or more base substitutions, deletions, and / or additions (the CG dinucleotide sequence substitution mutations remain unchanged), and the resulting sequences retain the respective biological functions of SEQ ID NO:29, 30, and 31. This invention includes nucleotide sequences with one or more base substitutions, deletions, and / or additions compared to the promoter sequences (i.e., promoter sequences containing SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, or promoter sequences composed thereof) (the CG dinucleotide sequence substitution mutations remain unchanged), and the resulting sequences retain the biological function of efficient expression of the promoter in activated immune cells. For example, substitutions, deletions, and / or additions of, for example, no more than 20, such as no more than 15, or no more than 10, or no more than 8, or no more than 5 bases may be made, either separately or simultaneously, at the 5' end and / or 3' end of the nucleotide sequence, and / or within the sequence. In some embodiments, the invention includes sequences having at least 95%, at least 97%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NO:29, 30, and 31, respectively, and similarly retaining the respective biological functions of SEQ ID NO:29, 30, and 31. Preferably, in variants of the IFN-γ gene core promoter shown in SEQ ID NO:30, the CG dinucleotide sequence substitution mutations at positions 62-63 and / or 194-195 remain unchanged. Sequence identity can be determined using algorithms well known in the art, such as BLAST and BLAST 2.0 algorithms. In some embodiments, the promoter sequence of the present invention contains, or is composed of, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31 connected in sequence. In some embodiments, the promoter sequence of the present invention contains, or is composed of, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31 connected in sequence, and the 62-63 and 194-195 positions shown in SEQ ID NO:30 have mutations, the mutations being selected from any one or more of the following: AG, CA, CC, CT, GG, TG; preferably, the mutation at the 62-63 positions is CA, and the mutation at the 194-195 positions is CA, CC or GG. In some preferred embodiments, the sequence of the chimeric promoter is as shown in any one of SEQ ID NO: 32-35. The promoter shown in any one of SEQ ID NO: 33-35 is a mutated promoter, which not only lacks the CpG motif but also exhibits superior activity compared to the promoter shown in SEQ ID NO: 32. Nucleic acid molecules whose nucleic acid sequences are complementary to the nucleic acid sequences of the promoters described in any embodiment herein are also included within the scope of this application. This invention also includes complementary sequences to the nucleotide sequences described herein. The polynucleotide sequences described herein may be in DNA or RNA form. The present invention also provides a nucleic acid construct containing the sequence of the promoter described herein or its complementary sequence, including the promoter sequence having a mutation or having at least 95% sequence identity or its complementary sequence. The nucleic acid construct may be an expression cassette or vector for DNA or mRNA for RNA. In some embodiments, the nucleic acid construct is an expression cassette containing the enhanced promoter sequence and the coding sequence of the protein of interest as described herein. The expression cassette typically contains a transcription termination sequence (i.e., a transcription terminator), which is a sequence recognized by the host cell to terminate transcription. The transcription termination sequence is operatively linked to the 3' end of the coding sequence as described herein. Any terminator that is functional in a selected host cell can be used in this invention, including but not limited to the SV40polyA transcription termination sequence. In some embodiments, the nucleic acid construct is a vector. Vectors typically include, but are not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. The vector can be an expression vector, including transient expression vectors, viral expression vectors, and transposon vectors. The vector is preferably a eukaryotic expression vector. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. The vector can also be a cloning vector used to provide the promoter or expression cassette of the present invention. Typically, a suitable vector contains at least one origin of replication functioning in the host cell, a convenient restriction endonuclease cleavage site, and one or more optional markers. Optional cleavage sites include, but are not limited to, Asc I, Xba I, Pvu I, HindIII, EcoR I, and Sal I cleavage sites. Typically, a portion of the cleavage site in the vector is located between the promoter sequence and the transcription termination sequence described in this invention, for cleaving the vector at this location to insert the coding sequence of the protein of interest, such that the coding sequence is operatively linked to the enhanced promoter and transcription termination sequences of this invention. Optional markers include any one or both of optional marker genes or reporter genes to facilitate the identification and selection of expressing cells from a cell population infected with the viral vector. Useful optional marker genes include, for example, antibiotic resistance genes, such as kanamycin or neo. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes. An exemplary vector containing the chimeric promoter of this invention is shown in Figure 1. In some embodiments, the vector is a vector for integrating the expression cassette of a gene of interest into the genome of a host cell, preferably a transposon vector. In some embodiments, the transposon vector is a eukaryotic expression vector containing transposon elements selected from piggybac, sleeping beauty, frog prince, PS, ZB, JL, Tn5, or Ty. Such transposon vectors contain a 5' inverted terminal repeat (5'ITR) and a 3' inverted terminal repeat (3'ITR) of the corresponding transposon. The transposase can be a transposon from the piggybac, sleeping beauty, frog prince, Tn5, or Ty transposon system. When using a transposon from a different transposon system, the sequences of the 5'ITR and 3'ITR in the vector are also changed accordingly to sequences adapted to that transposon system, which can be readily determined by those skilled in the art. Typically, the expression cassette of the present invention is located between the 5'ITR and the 3'ITR. In some embodiments, the transposase is a transposase derived from the piggybac transposase system. Therefore, in these embodiments, the 5' inverted terminal repeat (IDR) and 3' inverted terminal repeat (3DR) sequences of the piggybac transposon are respectively the 5' and 3' inverted terminal repeat sequences of the piggybac transposon. In some embodiments, the 5' inverted terminal repeat sequence of the transposon is shown as SEQ ID NO:1 of CN 201510638974.7 (the contents of which are incorporated herein by reference). In some embodiments, the 3' inverted terminal repeat sequence of the transposon is shown as SEQ ID NO:4 of CN 201510638974.7. In some embodiments, the piggybac transposase is a transposase containing a c-myc nuclear localization signal coding sequence. In some embodiments, the coding sequence of the piggybac transposase is shown as SEQ ID NO:5 of CN 201510638974.7. In some embodiments, the transposase is a transposase derived from the JL transposable system, which is the JL transposable subsystem described in any embodiment of CN202310081106.8. The promoter for the transposase coding sequence can be any of the various promoters known in the art for controlling the expression of the transposase coding sequence. In some embodiments, the CMV promoter is used to control the expression of the transposase coding sequence. The sequence of the CMV promoter may be shown as SEQ ID NO:6 in CN 201510638974.7. In some embodiments, the vector of the present invention uses the plasmid disclosed in CN202310072956.1 as the backbone. In some embodiments, the vector of the present invention is an empty vector, i.e., it does not contain the coding sequence of the protein of interest. Typically, such empty vectors sequentially contain one or more chimeric promoter sequences as described herein, one or more restriction endonuclease cleavage sites, and a transcription termination sequence, for linking the coding sequence of the protein of interest between the promoter sequence and the transcription termination sequence by enzyme digestion. In some embodiments, the vector of the present invention is a vector in which the coding sequence of the protein of interest is inserted between the chimeric promoter sequence and the transcription termination sequence as described herein. Expression vectors expressing the chimeric promoter can be constructed using conventional techniques in the art. A typical expression vector contains an expression control sequence that can regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence or its complement described in this invention. Those skilled in the art are familiar with methods for constructing expression vectors containing the chimeric promoter of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to the chimeric promoter in the expression vector to guide mRNA synthesis. Any plasmid and vector can be used, as long as it can replicate and stabilize in a host (especially animal cells). An important characteristic of expression vectors is that they typically contain regulatory sequences, including but not limited to origin of replication, promoter, marker gene, translation control element, ribosome binding site for translation initiation, and transcription terminator. In expression vectors, various nucleic acids and regulatory sequences can be linked together to produce recombinant expression vectors that may include one or more convenient restriction sites that allow the insertion or substitution of nucleotide sequences encoding a polypeptide at such sites. In preparing the expression vectors of the present invention, coding sequences for various different transcriptional activation domains described herein are located in the vector such that the coding sequences are operatively linked for the expression of appropriate regulatory sequences. The expression vector can be any vector (such as a plasmid or virus) capable of readily undergoing recombinant DNA methods and resulting in the expression of the coding sequence of the transcriptional activation domain described herein. The choice of vector generally depends on its compatibility with the host cell in which it is introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication does not depend on chromosome replication, such as a plasmid, extrachromosomal element, miniature chromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome into which it has already been integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids, or transposons, containing the total DNA to be introduced into the host cell genome can be used. The expression vector preferably contains one or more selectable markers that allow for easy selection of cells for transformation, transfection, transduction, etc. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc. Preferably, the expression vector contains elements that allow the vector to integrate into the host cell genome or to replicate autonomously within the cell independently of the genome. This invention provides the application of the chimeric promoter described herein in driving the expression of genes of interest (such as the coding sequence of a single-chain antibody) in activated immune cells. In this invention, the protein of interest can be any protein known in the art, including but not limited to enzymes, antibodies, and other proteins with the desired function, such as cytokines. Preferably, the protein of interest is a protein known in the art that needs to be expressed in T cells, such as various antibodies with anti-tumor activity, including single-chain antibodies, nanobodies, monospecific or multispecific antibodies, cell connectors (TCEs), and cytokines. In some embodiments, the vector of this application can simultaneously express two or more proteins of interest, such as simultaneously expressing the antibody and cytokine described in any embodiment herein. Therefore, in these embodiments, transfecting such a vector into cells of interest can obtain cells that simultaneously express the antibody and cytokine. Alternatively, when the vector expresses only one protein of interest, two or more vectors expressing different proteins of interest can be co-transfected into cells of interest to express two or more proteins of interest, such as antibodies, cytokines, and any two or three of CARs. CAR can be any CAR known in the art. In this paper, the chimeric antigen receptor (CAR) of interest may target one or more of the following antigens: Her2, CD19, CD20, CEA, GD2 (also known as B4GALNT1, β1,4-acetyl-galactosyltransferase 1), FR (Flavin reductase), PSMA (prostate-specific membrane antigen), PMEL (premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, IL-13Rα2, 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 proteins, BAGE (B melanoma antigen family) family proteins, GAGE ​​(growth hormone-releasing factor) family proteins, AFP (alpha-fetoprotein), MUC1 (mucin 1. Cell surface-related), CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, IL-11Rα, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8α-N-acetyl-ceramide α-2,8-sialic acid convertase 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA1109), PSA (also known as KLK3, kallikrein-associated peptidase 3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16. Cell surface-associated antigens, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25-dihydrovitamin D3) receptor), β2-MG (β-2-microglobulin), and PROGRP (GRP gastrin-releasing peptide). It should be understood that, unless otherwise stated, all antigens described herein are antigens well-known in the art, and their sequences are known in the art. The vector expressing CAR and the vector containing other genes of interest operatively linked to the promoter of this invention can be simultaneously or sequentially transferred into T cells to prepare CAR-T cells containing genes of interest whose expression is controlled by the promoter of this invention. The present invention also provides a host cell comprising or genomically integrated with (1) an expression cassette having a chimeric promoter as described in any embodiment herein, or (2) a nucleic acid construct as described in any embodiment herein. The host cell may be used as a recipient of a vector. The host cell may be “transfected” or “transformed,” referring to the process of transfecting or transducing exogenous nucleic acids into the host cell. Transformed cells include primary target cells and their progeny. The terms “engineered” and “recombinant” cells or host cells as used herein often refer to cells in which exogenous nucleic acid sequences, such as vectors, have been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acids. The chimeric promoter sequence described in this invention can be obtained by PCR amplification, recombination or artificial synthesis methods known in the art. For PCR amplification, the primer sequences and templates disclosed herein can be used for amplification. After obtaining the recombinant expression vector, the vector is transformed into a host cell to activate the chimeric promoter and initiate the expression of the exogenous gene. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules; it is the site of recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention includes immune effector cells. Immune effector cells refer to cells that participate in or are related to the immune response, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells. Preferred immune cells in this invention are peripheral blood mononuclear cells (PBMCs), including lymphocytes and monocytes. Lymphocytes include T lymphocytes, B lymphocytes, K lymphocytes, and NK lymphocytes. Preferably, the cells suitable for the promoter modified in this invention are immune cells that overexpress interferon-gamma. In some embodiments, the immune effector cells are selected from one or more of T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Exemplarily, the host cells described in this invention are various T cells known in the art, including but not limited to T cells from a mixed population such as peripheral blood T lymphocytes, cytotoxic T cells (CTLs), helper T cells, suppressor / regulatory T cells, γδ T cells, cytokine-induced killer cells (CIKs), and tumor-infiltrating lymphocytes (TILs). In some embodiments, the T cells may be derived from PBMCs of patients with B-cell malignancies. In some embodiments, the T cells are primary cultured T cells. Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells. After obtaining the expression vectors described herein, they can be transformed into host cells using methods well-known in the art. Conventional transfection methods can be used to transfer the vectors of this invention into cells of interest, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation. For example, when the host is a T cell, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging. In some embodiments, electroporation is used to transfect the vectors described herein into cells of interest. In some embodiments, the present invention also provides an immune cell, particularly a T cell, containing the chimeric promoter sequence or nucleic acid construct or vector described herein, including a nucleic acid construct or vector for expressing an antibody and / or cytokine of interest. Preferably, the genome of the immune cell (particularly the T cell) integrates an expression cassette that uses the chimeric promoter sequence described herein as a promoter to drive the expression of a foreign gene of interest (including an antibody and / or cytokine of interest). In some embodiments, the immune cells are CAR T cells, i.e., T cells expressing CAR or containing a CAR coding sequence or a vector expressing CAR. Therefore, in some embodiments, the CAR T cells provided in this application can simultaneously express CAR and an antibody of interest, or simultaneously express CAR and a cytokine of interest, or simultaneously express CAR, an antibody of interest, and a cytokine of interest. The present invention also provides a kit comprising the chimeric promoter, nucleic acid construct, and / or host cell described in any embodiment herein. This invention also provides a pharmaceutical composition comprising the immune cells of this invention and a pharmaceutically acceptable carrier. This document also provides a cellular immunotherapy comprising providing the immune cells described in any embodiment herein and administering a therapeutically effective amount of the immune cells to a desired individual. 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, particularly humans. The immune cells may be prepared using the methods described in any embodiment herein. In some embodiments, the immune cells are autologous cells, i.e., immune cells derived from the individual to be treated, which are treated in vitro by the methods described in any embodiment herein to express the desired antibodies, cytokines, and / or CARs, and then reinfused to the individual. The present invention also provides a method for driving the expression of a gene of interest in cells (e.g., animal cells), the method comprising the step of incubating the cells under conditions suitable for expression initiated by the chimeric promoter. Typically, the conditions are those suitable for cell growth. Exemplarily, the method comprises: (1) introducing the chimeric promoter into a nucleic acid construct described herein; (2) transferring the nucleic acid construct into a cell; and (3) incubating the cell under conditions suitable for expression of the nucleic acid construct. This invention has discovered that when a vector containing the coding sequence of a protein of interest and the promoter of this invention operatively linked thereto is transferred into immune cells, the immune cells can specifically and efficiently drive gene expression. Partial Specific Implementation Plan Project 1. A chimeric promoter comprising, from the 5' end to the 3' end, a CMV enhancer, an IFN-γ gene core promoter and an intron connected in sequence, wherein the IFN-γ gene core promoter contains a sequence as shown in SEQ ID NO: 30 or a sequence having at least 90% sequence identity with SEQ ID NO: 30. Project 2. The chimeric promoter as described in Project 1, characterized in that one or both CG dinucleotide sequences in the core promoter of the IFN-γ gene undergo a substitution mutation, wherein the substitution mutation is: (1) a mutation at position 62-63 of the sequence shown in SEQ ID NO:30, wherein the mutation is selected from any one of the following: AG, CA, CC, CT, GG, TG, preferably CA; and / or (2) a mutation at position 194-195 of the sequence shown in SEQ ID NO:30, wherein the mutation is selected from any one or more of the following: AG, CA, CC, CT, GG, TG, preferably CA, CC, GG; More preferably, the CG dinucleotide sequence substitution mutation contained in SEQ ID NO:30 is: the 62-63 position of the sequence shown in SEQ ID NO:30 is mutated to CA, and the 194-195 position of the sequence shown in SEQ ID NO:30 is mutated to CA, CC or GG. Project 3. The chimeric promoter as described in Project 1 or 2, characterized in that it further comprises one or more of the following features: The CMV enhancer, IFN-γ gene core promoter, and intron are directly or operatively linked through a adapter. The nucleotide sequence of the CMV enhancer is as shown in SEQ ID NO:29, or is selected from a sequence that has at least 90% sequence identity with SEQ ID NO:29; The intron is the SI126 intron; preferably, the nucleotide sequence of the intron is as shown in SEQ ID NO:31, or is selected from a sequence having at least 90% sequence identity with SEQ ID NO:31. Item 4. The chimeric promoter as described in any one of items 1-3, characterized in that the sequence of the chimeric promoter is as shown in any one of SEQ ID NO: 32-35. Project 5. A nucleic acid construct comprising any one of Projects 1-4, a chimeric promoter, and a gene of interest operatively linked to said chimeric promoter. Preferably, the nucleic acid construct is a cloning vector or an expression vector. Project 6. The nucleic acid construct as described in Project 5, characterized in that the gene of interest encodes an autocrine antibody and / or cytokine; preferably, the autocrine antibody is 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 a nanobody derived from alpacas; preferably, the cytokine is selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. Project 7. A host cell comprising: (1) The chimeric promoter described in any of items 1-4, or, (2) The nucleic acid constructs described in item 5 or 6; Preferably, the host cells include immune effector cells. More preferably, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Item 8. The host cell as described in Item 7, characterized in that the genome of the host cell integrates the nucleic acid constructs described in Item 5 or 6. Preferably, the host cell also expresses CAR or an expression vector containing CAR. Project 9. The use of the chimeric promoter described in any one of Projects 1-4 in enhancing the expression of a gene of interest in activated immune effector cells, or in the preparation of nucleic acid constructs for enhancing expression in activated immune effector cells. Preferably, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Project 10. The application as described in Project 9, characterized in that the gene of interest encodes autocrine antibodies and / or cytokines. Preferably, the immune checkpoint antibody is selected from PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody, and is preferably a nanobody derived from alpaca; Preferably, the cytokines are selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors. The invention will be described below by way of specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Guide (Cold Spring Harbor Laboratory Press, New York, 1989), or as recommended by the manufacturer. Example Example 1: Construction of CpG-Free Mutant Chimeric Promoter Primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the primer sequences are shown in SEQ ID NO:1-12. The DTS sequence is shown in SEQ ID NO:27; the CIT sequence from 5' to 3' consists of a CMV enhancer, an IFN-γ promoter, and a long terminal repeat (LTR) sequence of HTLV (human T-cell leukemia virus) connected sequentially, and the specific structure can be found in CN114729361A; the mCIS sequence from 5' to 3' consists of a CMV enhancer (SEQ ID NO:29), an IFN-γ gene core promoter (SEQ ID NO:30), and an SI126 intron (SEQ ID NO:31) connected sequentially. Taking the construction of plasmid pTini-DTS-dCGFluc as an example, fragments DTS-F1 (as shown in SEQ ID NO:25) and DTS-F2 (as shown in SEQ ID NO:26) were synthesized separately. Fragments DTS-F1 and DTS-F2 were digested with XbaI and BsrGI and then ligated to obtain plasmid pTini-DTS-dCGFluc. The pTini-CIT-dCGFluc plasmid was obtained by constructing fragment CIT-F (as shown in SEQ ID NO:27), digesting it with XbaI and NcoI, and cloning it into the XbaI and NcoI sites of plasmid pTini-DTS-dCGFluc. The constructed fragment mCIS-F (as shown in SEQ ID NO:32) was digested with XbaI and NcoI and cloned into the XbaI and NcoI sites of plasmid pTini-DTS-dCGFluc to obtain plasmid pTini-mCIS-dCGFluc. The spectra of plasmid pTini-mCIS-dCGFluc are shown in Figure 1, and the spectra of plasmid pTini-DTS-dCGFluc are shown in Figure 5. Example 2: 1) PBMC recovery and revitalization Preparation of activation medium: Take AIM-V medium, add 2% fetal bovine serum and 500 IU / ml IL-2. Take a 15ml centrifuge tube, add 9ml of medium to the tube, remove the cryovial from the -80℃ freezer, and thaw it in a 37℃ constant temperature water bath as soon as possible. When a small piece of ice remains, transfer the cell suspension to a centrifuge tube containing medium on a clean bench, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend the cells with an appropriate amount of medium, take 10μL of the cell suspension, and then dilute it with 0.4% trypan blue at a 1:1 ratio. After thorough mixing, perform cell counting. Based on the counting results, for every 1E7 cells, use 30μL of Miltenyi T Cell TransAct. TM To activate the culture, add 3 ml of culture medium to each well, mix well, and incubate at 37°C in a 5% CO2 incubator. 2) PBMC electro-rotation Preparation of culture medium after electroporation: Take AIM-V culture medium and add 2% fetal bovine serum and 100 IU / ml IL-2. Preparation of electroporation mixture: Following the instructions of the Human T Cell Nucleofector transfection kit, 400 μg of pRL-TK plasmid (expressing renilla luciferase) (plasmid map shown in Figure 7) and 2 μg of Fluc plasmid (expressing firefly luciferase) were added to 100 μL of electroporation buffer at a Nucleofector Solution:Supplement ratio of 82:18. Experimental groups are shown in Table 2. pTini-JL(DTS)-1444-8E is a plasmid expressing MSLN CAR; its sequence can be found in Example 1 of CN202111681582.0, specifically MSLN3.

[1444] CAR. Table 2 Prepare 7E6 activated human T cells. Mix the electroporation mixture from the above steps with the cell pellet and add it to the electroporation cuvette. Place the cuvette in a Lonza-4D electroporator and select the program human Tcell stim (EO115) for electroporation. After electroporation, use the micropipette in the kit to aspirate the preheated culture medium and mix it with the electroporation suspension. Quickly transfer the mixture to a 6-well plate containing 3 ml of preheated culture medium, mix well, and incubate at 37°C in a 5% CO2 incubator. 3) MSLN antigen coating 24 hours after electroporation, the MSLN antigen was diluted with PBS to a concentration of 5 μg / ml and added to each well of a 6-well plate using a pipette. The plate was then incubated at 4°C. 4) MSLN antigen stimulation 48 hours after electroporation, the liquid in the 6-well plate coated with MSLN antigen was first discarded. Then, the T cells in the 6-well plate were mixed by pipetting. Half of the cells from each group were seeded into the 6-well plate coated with MSLN antigen. The remaining cells were not stimulated. Both groups of cells were incubated in a 37°C, 5% CO2 incubator. 5) Dual-luciferase expression detection 24 hours after antigen stimulation, T cells from each well were collected into 15 ml centrifuge tubes, centrifuged at 1500 rpm for 3 min, the supernatant was discarded, 1 ml of PBS was added to each tube, the cells were fully resuspended, 10 μL of cell suspension was taken, and then diluted 1:1 with 0.4% trypan blue. After thorough mixing, cell counting was performed. Following the cell count results and the instructions of the Promega dual-luciferase assay kit, add 300-400 μL of 1×Cell lysis buffer to each cell group, shake at room temperature for 10-15 minutes, then centrifuge at 12000g for 2 minutes. Take 20 μL of supernatant from each group and add it to a 96-well microplate. Simultaneously add 100 μL of Luciferase substrate equilibrated to room temperature, mix quickly, and then place the plate in a microplate reader to detect Firefly luciferase reporter gene activity. Record the Firefly luciferase detection data. Then, add 100 μL of freshly prepared Renilla substrate working solution to the microplate, mix quickly, and then place the plate in a microplate reader to detect Renilla luciferase reporter gene activity and record the results. Cell viability was measured as relative luciferase activity, i.e., Firefly luciferase activity / Renilla luciferase activity. The results are shown in Figure 2. Luciferase was still expressed in the absence of MSLN antigen stimulation, but the expression level was significantly lower than after MSLN antigen stimulation. The promoter strength was as follows: mCIS > CIT ≈ DTS. Subsequent designs will be based on modifications to the mCIS promoter. Example 3: The plasmid mutation construction method is the same as in Example 1, except that the primers and templates used are different. The first CpG in the mCIS promoter is mutated to another sequence. Taking pTini-mCIS-dCGFluc-AG as an example, this means mutating the CG sequence to the AG sequence. Referring to Table 1, the corresponding template is amplified using the corresponding primers, and then seamlessly cloned into the corresponding template to obtain the corresponding mutant plasmid. Table 1 The electroporation and detection methods for PBMC cells were the same as in Example 2, except that the plasmid used was a modified plasmid with the first site of the mCIS promoter (positions 62-63 of the sequence shown in SEQ ID NO:30), and the experimental groups were as shown in 3. Table 3 The detection results are shown in Figure 3. Luciferase was still expressed in the absence of MSLN antigen stimulation, but the expression level was significantly lower than after MSLN antigen stimulation. When the CA mutation with the highest promoter activity was used as the comparison object, the significant difference was shown in the figure. Combining the experimental results of the two groups, CA>GG>TG>mCIS>DTS, therefore, the subsequent design will be based on the CA mutant promoter. Example 4 Primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the primer sequences are shown in SEQ ID NO:13-24. Using pTini-mCIS-dCGFluc-CA as a template, plasmids modifying the first (positions 62-63 of the sequence shown in SEQ ID NO:30) and second (positions 194-195 of the sequence shown in SEQ ID NO:30) sites of the mCIS promoter were constructed. The plasmid construction method was the same as in Example 1, except for the primers and template used. Referring to Table 4, the corresponding template was amplified using the corresponding primers, and then seamlessly cloned into the corresponding template to obtain the corresponding mutant plasmid. Table 4 The electroporation and detection methods for PBMC cells were the same as in Example 2, except that the plasmids used were modified plasmids with the first and second sites of the mCIS promoter. The experimental groups are shown in Table 4. Table 4 The test results are shown in Figure 4. Luciferase was still expressed in the absence of MSLN antigen stimulation, but the expression level was significantly lower than after MSLN antigen stimulation. The significant differences are shown in the figure. Among them, the CA-CA, CA-CC, and CA-GG groups showed significant differences from the positive control. Sequence of this article:

Claims

1. A chimeric promoter comprising, from the 5' end to the 3' end, a CMV enhancer, an IFN-γ gene core promoter and an intron connected in sequence, wherein the IFN-γ gene core promoter contains a sequence as shown in SEQ ID NO: 30 or a sequence having at least 90% sequence identity with SEQ ID NO:

30.

2. The chimeric promoter of claim 1, wherein, One or both CG dinucleotide sequences in the core promoter of the IFN-γ gene are subject to substitution mutations, wherein the substitution mutations are: (1) a mutation at position 62-63 of the sequence shown in SEQ ID NO:30, wherein the mutation is selected from any one of the following: AG, CA, CC, CT, GG, TG, preferably CA; and / or (2) a mutation at position 194-195 of the sequence shown in SEQ ID NO:30, wherein the mutation is selected from any one or more of the following: AG, CA, CC, CT, GG, TG, preferably CA, CC, GG; More preferably, the CG dinucleotide sequence substitution mutation contained in SEQ ID NO:30 is: the 62-63 position of the sequence shown in SEQ ID NO:30 is mutated to CA, and the 194-195 position of the sequence shown in SEQ ID NO:30 is mutated to CA, CC or GG.

3. The chimeric promoter of claim 1 or 2, wherein, It also includes one or more of the following features: The CMV enhancer, IFN-γ gene core promoter, and intron are directly or operatively linked through a adapter. The nucleotide sequence of the CMV enhancer is as shown in SEQ ID NO:29, or is selected from a sequence that has at least 90% sequence identity with SEQ ID NO:29; The intron is the SI126 intron; preferably, the nucleotide sequence of the intron is as shown in SEQ ID NO:31, or is selected from a sequence having at least 90% sequence identity with SEQ ID NO:

31.

4. The chimeric promoter of any one of claims 1 to 3, wherein, The sequence of the chimeric promoter is shown in any one of SEQ ID NO: 32-35.

5. A nucleic acid construct comprising a chimeric promoter according to any one of claims 1-4, and a gene of interest operatively linked to said chimeric promoter. Preferably, the nucleic acid construct is a cloning vector or an expression vector.

6. The nucleic acid construct of claim 5, wherein The gene of interest encodes autocrine antibodies and / or cytokines; preferably, the autocrine antibodies are immune checkpoint antibodies, such as PD-1 antibodies, CTLA4 antibodies, PD-L1 antibodies, LAG-3 antibodies, TIM-3 antibodies, TIGIT antibodies, and VISTA antibodies, more preferably, nanobodies derived from alpacas; preferably, the cytokines are selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors.

7. A host cell comprising: (1) The chimeric promoter according to any one of claims 1-4, or, (2) The nucleic acid construct according to claim 5 or 6; Preferably, the host cells include immune effector cells. More preferably, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.

8. The host cell of claim 7, wherein The host cell genome integrates the nucleic acid construct of claim 5 or 6. Preferably, the host cell also expresses CAR or an expression vector containing CAR.

9. The use of the chimeric promoter according to any one of claims 1-4 in enhancing the expression of a gene of interest in activated immune effector cells, or in the preparation of nucleic acid constructs for enhancing expression in activated immune effector cells. Preferably, the immune effector cells are selected from one or more of the following: T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages.

10. Use according to claim 9, wherein The genes of interest encode autosecretory antibodies and / or cytokines. Preferably, the immune checkpoint antibody is selected from PD-1 antibody, CTLA4 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody, and is preferably a nanobody derived from alpaca; Preferably, the cytokines are selected from interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors.

Images