WO2021259334A1 - Self-regulating chimeric antigen receptor and application thereof in tumor immunity - Google Patents

Abstract

A self-regulating chimeric antigen receptor (CAR) and an application thereof in tumor immunity, comprising a tumor antigen-induced gene expression self-regulating nucleic acid construct. A gene expression regulatory sequence can induce the expression of a CAR or an engineered TCR, and is suitable for different tumor targets. When CAR-T or TCR-T is in contact with a tumor, a tumor surface antigen is bound to scFv, a receptor, a ligand, a protein scaffold, or the engineered TCR on a CAR-T surface, so that CAR-T or TCR-T cells are specifically activated, and the expression of more CAR-T cells or the CAR or the engineered TCR on a TCR-T surface is promoted, thereby further enhancing the expansion ability of the CAR-T cells or the TCR-T cells and functioning as effector cells. The approach of locally activating CAR-T or TCR-T by means of a tumor can reduce systemic cytokine release independent of a tumor surface antigen, thereby reducing the toxic and side effects of CAR-T or TCR-T.

Description

Self-regulating chimeric antigen receptor and its application in tumor immunity Technical field

The present disclosure belongs to the field of cellular immunotherapy, and specifically relates to a self-regulating chimeric antigen receptor and its application in tumor immunity.

Background technique

The pathogen antigen stimulates T cells by binding to the T cell receptor (TCR), activates the signal transduction cascade, promotes the proliferation and differentiation of T cells, and finally eliminates the pathogen. After TCR is activated, tyrosine kinase is phosphorylated, thereby activating downstream signal transduction pathways. In response to activation, T cells reorganize their cytoskeleton, changing their metabolism and gene expression.

The three main ways to control gene expression (transcription) through TCR are MAPK (mitogen-activated protein kinase), NF-κB (nuclear factor kappa-B) and calcium pathway (Chan AC). .The role of protein tyrosinekinases and protein tyrosine phospha-tases in T cellantigenreceptorsignaltransduction.AnnuRevImmunol(1994)12:555–92;Brownlie RJ,Zamoyska RT cellreceptor:signalling,bounded,networks Rev Immunol (2013) 13:257–69). The TCR-dependent MAPK pathway first activates Ras, leading to the activation of downstream Erk and the formation of activator protein-1 (AP-1). After AP-1 dimer is combined with AP-1 response element (AP-1-RE), it increases the transcription and expression of genes related to T cell activation. In addition, TCR can transmit signals through the LAT-SPL76 complex, thereby activating PKCθ. Similarly, the costimulatory factor CD28 can also signal through PI3K and PDK1 to activate PKCθ. Activated PKCO causes IKK activation. It further causes phosphorylation of IκBα, leading to ubiquitination and degradation of IκBα, which causes nuclear translocation of NF-κB and combines with NF-κB response elements (NF-κB RE) to cause gene transcription (Suman P, Brian CS. A new look) at TCR signaling to NF-κB. Trends Immunol. 2013 June; 34(6):269–281). Activated T cell nuclear factor (nuclear factor of activated T cell, NFAT) is a type of transcription factor related to the calcium ion signaling pathway. The activated NFAT combines with the NFAT response element (NFAT-RE) to further regulate the development of lymphocytes. Activation and gene expression.

In addition to the above-mentioned main pathways, T cell activation also includes Wnt pathway, Akt pathway and HIF1α pathway. In the classic Wnt pathway, the activation of Wnt requires the participation of Porc (O-palmitoleoyl transferase, O-palmitoleoyl transferase). When activated Wnt binds to Frizzled on the cell membrane, it activates the scaffold protein in the cell matrix (Scaffold, which includes Dvl, GSK-3β, axin, APC, β-catenin, etc.), and then activates the nuclear TCF/ LEF (T cell factor/lymphoid enhancer factor), regulates the transcription of downstream genes. TCF/LEF is a type of transcription factor with two-way regulation function. Its response element is (TCF-RE). It can inhibit gene transcription when combined with Groucho, and it can promote the transcription of downstream target genes when combined with β-Catenin. FoxO transcription factor is the downstream target of PKB/Akt, and its response element is (FoxO RE). When Akt activity decreases, FoxOs is phosphorylated, FoxOs enters the nucleus, and binds to the DNA targeting sequence corresponding to the target gene to perform its transcription function. HIF is a family of heterodimer basic-helix-loop-helix transcription factors, and its hypoxia response element is HRE (hypoxia response element). In the absence of hypoxia, HIF-α is hydroxylated by the factor of inhibiting HIF-1 (FIH) asparaginyl, thereby preventing HIF-α from binding to the co-activator protein p300/CBP. Under hypoxia, the activities of PHD and FIH are restricted by substrates, leading to rapid accumulation of HIF-α, nuclear translocation, and dimerization with HIF-1β. When HIF-1 binds to the DNA consensus sequence (a hypoxia response element (HRE)) in the promoter of the target gene, transactivation occurs.

Common gene transcription regulatory elements in eukaryotic DNA include promoters, enhancers and so on. A promoter is a DNA sequence that RNA polymerase recognizes, binds and starts transcription, and it contains conserved sequences required for RNA polymerase specific binding and transcription initiation. The two most common core promoter elements associated with protein-coding genes are the TATA box and the initiator (Inr), which appear together or separately in most eukaryotic promoters. The TATA box is located 20-30bp upstream of the transcription initiation site (TSS), and serves as a binding site for the general transcription factor TFIID (Mathis DJ, Chambon P. The SV40 early region TATA box is required for accurate in vitro initiation of Nature. .1981 Mar 26; 290(5804):310–315). The conserved sequence of the initiator (Inr) is (YYA+1NT/AYY), which is often found in the core promoter of ubiquitously expressed or "housekeeping" genes, and initiates transcription at multiple discrete sites within a region of about 150 bp, independent Guide accurate transcription initiation (Smale ST, Baltimore D. The "initiator" as a transcription control element. Cell. 1989 Apr 7; 57(1): 103–113). Although TATA box and Inr (Frith MC, Valen E, Krogh A, Hayashizaki Y, Carninci P, Sandelin AA code for transcription initiation in mmammalian genes. Genome Res. 2008Jan; 18() can be found in some complex gene promoters at the same time. 1):1-12), the TATA box mainly initiates the expression of tissue-specific genes such as IL-2 in T cells. In contrast, the initiator (Inr) is used to initiate the expression of ubiquitously expressed or "housekeeping" genes. Therefore, the promoters of immunoglobulin genes and IL-2 genes only contain the TATA box, while the actin promoter only contains the initiator (Inr) element. Many complex genes, such as the MHC (Major Histocompatibility Complex) regulatory region, contain both the TATA box and the initiator (Inr). Enhancers are a kind of conserved specific DNA fragments, which, after binding with transcription factors, enhance the transcription rate of the promoter and regulate gene expression. Response element (RE) is a short conserved DNA sequence that specifically binds transcription factors on enhancers. In response to different environmental stimuli, enhancers can have multiple different response elements and also bind different transcription factors, which together regulate gene expression. In a specific gene, repeated repetition of the same response element can enhance the regulatory effect.

Enhancesome is a functional unit structure composed of enhancers, transcription factors and corresponding cofactors. It can change the structure of local chromosomes, recruit RNA polymerase II to the promoter, and regulate gene Express. IFN-β enhancer is a typical model for studying gene transcription regulation. It is located upstream of the transcription start site of IFN-β gene and can recruit some cofactors that can acetylate histone H1, such as p300/CREB binding protein (CBP) . The acetylated histones can relax the nucleosomes located in the TATA box, exposing the promoter, thereby promoting more transcription factors TFIIB and RNA polymerase II, causing the initiation of transcription. IFN-β enhancer contains 4 positive regulatory domains (Postive regulatory domains, PRDs), interferon regulatory factor (interferon regulatory factor, IRF) combined with positive regulatory domain I (PRDI)) and positive regulatory domain III (PRDⅢ), NF-κB can be combined with positive regulatory region II (PRDⅡ), and ATF-2/c-Jun can be combined with positive regulatory region IV (PRDⅣ). These transcription factors can be combined with their coactivator (Coactivator), CBP, etc., to jointly regulate the transcription and expression of genes (Pan Y, Nussinov. The Role of Response Element Organization in Transcription Factor Seletivity: The IFN-β Enhancesome Example.PLos Comput Biol 7(6):e1002077). Chimeric antigen receptor modified T cell (CAR-T) is a genetically modified T cell that uses gene transduction technology to contain single-chain antibodies that can specifically bind to tumor surface antigens (ScFv), receptors, ligands, protein scaffolds ((including Affibody, DARPin, Monobody (including Centyrin), Anticalin4, etc.)) or other new protein scaffolds and chimeric antigen receptor (CAR) introduction of T cell activation motifs Patient T cells (Alsultan, A., et al. Beyond Antibodies: Development of a Novel Protein Scaffold Based on Human Chaperonin 10. Sci Rep 6, 37348 (2016)), so that these CAR-introduced T cells can directly recognize the surface of cancer cells The antigen is activated to kill cancer cells (Schmitz M, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer. J Biomed Biotechnol, 2010). The chimeric antigen receptor includes an extracellular binding domain, a transmembrane domain (TM), and an intracellular signal domain. Usually the extracellular region contains scFv, receptors, ligands, protein scaffolds (including Affibody, DARPin, Monobody (including Centyrin), Anticalin4, etc.) or other novel protein scaffolds capable of recognizing tumor-associated antigens. The transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signal region adopts cells including immunoreceptor tyrosine activation motif (ITAM) CD3ζ and costimulatory signal molecules CD28, CD137 (4-1BB), CD134, etc. Inner signal area. The intracellular signal region containing only CD3ζ is designed as the first generation CAR-T lymphocytes, in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD3ζ. This kind of CAR T can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively small, and it cannot stimulate long-lasting anti-tumor effects in the body (Zhang T.et al.Chimeric NKG2D-modified T cells inhibition systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Can Res 2007, 67(22): 11029–11036). Subsequent development of the second-generation CAR added a co-stimulatory intracellular signal region such as CD28 or 4-1BB, in which the parts of the chimeric antigen receptor were connected as follows: scFv-TM-CD28-CD3ζ or scFv-TM-4 -1BB-CD3ζ. CD28 or 4-1BB co-stimulation in the intracellular signal area causes the continuous proliferation of T lymphocytes, and can increase the level of IL-2 and IFN-γ secreted by T lymphocytes, and at the same time increase CAR-T in vivo Survival cycle and anti-tumor effect (Dotti G.et al. CD28 costimulation improvement expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121(5): 1822-1826). In the third-generation CAR-T lymphocytes developed in recent years, the parts of its chimeric antigen receptor are connected in the following form: scFv-TM-CD28-4-1BB-CD3ζ or scFv-TM-CD28-CD134-CD3ζ, further Improve the survival cycle of CAR-T in vivo and its anti-tumor effect (Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360-- 3365).

Traditional CAR-T can only target and recognize antigens expressed on the surface of tumor cells, while only about 1% of the antigens of tumor cells are expressed on the cell surface. This means that most of the potential antigens of tumor cells cannot be detected by CAR-T. Recognition. The engineered TCR-T can recognize intracellular proteins. The engineered TCR-T is composed of soluble TCR and the signal part of the CAR. Such a structure binds to the targeted antigen in an MHC Ⅰ restricted manner, and performs the same signal transduction and tumor-killing functions as CAR-T (Walseng, E., et al. al. A TCR-based Chimeric Antigen Receptor. Sci Rep 7,10713 (2017)).

Engineered TCR-T is a TCRα/β heterodimer modified by transduction affinity to improve the specific recognition of intracellular tumor-associated antigen (TAA) (Q Liu, et al. Cancer immunotherapy using T- cell receptor engineered T cell.Ann Blood 2020; 5; 5). TCR-T cell therapy includes the selection of tumor-specific TCR, TCRα/β heterodimer affinity modification, construction of TCRα/β expression vector, T cell transduction, cell reinfusion after TCR-T modification, immune process monitoring, etc. Technology and treatment. Compared with the same type of CAR-T cell therapy technology, TCR-T has a wider choice of antigens, so it can not only expand its applicable tumor range, but also is expected to reduce off-target effects.

Studies have shown that in order to obtain higher anti-tumor activity, the expansion and maintenance of CAR-T cells in the body are two very important factors. In traditional CAR-T cells, the expression of CAR is constitutive. Highly expressed ScFv, self-aggregation, will form basic signaling (Low-level “basal” or “tonic” signal). Studies have found that CAR-T cells expand well in vitro under the effect of tonic signaling, but they are difficult to maintain in vivo (Frigault MJ, et al. Identification of chimeric antigen receptors that mediate constitutive or inducible). proliferation of T cells. Cancer Immunol. Res. 2015; 3: 356–367; Long AH, et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 2015; 21: 581 -590).

Using CRISPR/Cas9 technology to locate CAR on the TCRα gene, or replace the costimulatory factor CD28 with 4-1BB, although it can reduce the basic signal transduction (Eyquem J, et al. Targeting a CAR to the TRAC locus with CRISPR/ Cas9enhances tumor rejection.Nature.2017; 543:113–117), but because of its amplification on γ-retroviral vectors, CAR-T has limited anti-tumor ability in vivo (Diogo G, et al. Tonic 4-1BB) Costimulation in Chimeric Antigen Receptors Impedes T Cell Survival and Is Vector Dependent. Cell Rep. 2017; 21(1):17–26).

Therefore, it is necessary to seek better methods to reduce basic signal transduction, extend the survival time of CAR-T in the body, and provide anti-tumor activity.

Summary of the invention

The present disclosure aims to design a self-regulating CAR-T cell that has low expression of CAR before they are close to tumor antigens and has little cell-based activation. Gene expression self-regulating sequences can induce the expression of CAR or engineered TCR, and are suitable for different tumor targets. Once in contact with the tumor, the tumor surface antigen binds to CAR-T surface scFv, receptor, ligand, protein scaffold or TCR, which will specifically activate CAR-T or TCR-T cells and promote more CAR-T cells or TCRs -The expression of CAR or TCR on the T surface further enhances the expansion ability of CAR-T cells or TCR-T cells and exerts the function of effector cells. This way of activating CAR-T locally through the tumor can effectively reduce the release of systemic cytokines that are not related to tumor surface antigens, reduce the toxic and side effects of CAR-T or TCR-T, and have a powerful tumor-killing effect.

Specifically, the present disclosure proposes the following technical solutions:

In one aspect, the present disclosure provides a tumor antigen-induced gene expression self-regulating nucleotide sequence, the sequence comprising: (1) a promoter sequence, and (2) an enhancer sequence.

In one aspect, the present disclosure provides a nucleic acid construct comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence and a nucleotide sequence encoding a protein that specifically binds to the tumor antigen.

In one aspect, the present disclosure provides the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence and/or nucleic acid construct for preparing genetically modified immune cells directed against tumor-associated antigens.

In one aspect, the present disclosure provides an isolated host cell comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence and/or nucleic acid construct.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence, nucleic acid construct and/or host cell.

In one aspect, the present disclosure provides a use of the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence, nucleic acid construct and/or host cell and/or pharmaceutical composition in the preparation of medicines.

In one aspect, the present disclosure provides a method for preparing CAR-T or TCR-T cells.

In one aspect, the present disclosure provides a method for treating a tumor-associated antigen-related disease in a subject, which comprises administering to the subject a chimeric antigen receptor (CAR) nucleic acid construct, virus, or host cell, and / Or pharmaceutical composition.

The innovative effects of this disclosure include:

The gene expression control sequence formed by the combination of the enhancer of the present disclosure (such as NFAT-RE, NF-κB-RE, AP-1-RE, TCF-RE, HRE, etc.) and a promoter (such as IL-2 TATA box) can Induces the expression of CAR and is suitable for different tumor targets. The combination of the same enhancer and promoter can regulate the expression of different CARs in CAR-T or different engineered TCRs in TCR-T. The combination of different enhancers and IL-2 TATA box can not only regulate gene expression in Jurkat cells, but also play a role in PBMC or mouse spleen cells. Once CAR-T or TCR-T comes into contact with the antigen, it will be quickly activated, thereby regulating and enhancing the expression of its own CAR or engineered TCR. Regulated CAR-T or TCR-T has a powerful tumor killing effect. Compared with CAR-T or TCR-T driven by the EF-1α promoter, this adjustable CAR has a better killing effect in PBMC. . Such an adjustable structure can become another new way to treat tumors, including solid tumors.

Description of the drawings

Figure 1 shows the main structure of the three types of plasmids designed by the present disclosure, in which Figure 1A is the first type of plasmid; Figure 1B is the second type of plasmid; Figure 1C is the third type of plasmid.

Figure 2 shows the regulatory effects of PMA and ionomycin on Jurkat cells transduced with plasmid 112.

Figure 3 shows the regulatory effect of enhancers on Jurkat cells transduced with plasmid 292 (expressing CD19 antigen).

FIG. 4 shows the regulation effect of different stimuli on the PBMC transduced with plasmid 112.

Figure 5 shows the timing of PMA and ionomycin stimulation on PBMC expression of genes.

Figure 6 shows the regulatory effect of the combination of enhancer and IL-2 TATA box in PBMC.

Figure 7 shows the modulatory effects of enhancers on different CARs.

Figure 8 shows the antigen induction effects of different enhancers in PBMC or Jurkat.

Figure 9 shows the flow cytometry (Figure 9A) and data analysis results (Figure 9B) of antigen-stimulated PBMC cells transduced with plasmid 260 and control cells.

Figure 10 shows the regulatory effects of CAR-regulated structures and CAR non-regulated structures on CAR-T cells.

Fig. 11 shows the antigen induction effect of the CAR regulatable structure in retrovirus-transduced Jurkat cells, wherein Fig. 11A is transduced with plasmid 417 and Fig. 11B is transduced with plasmid 440.

Figure 12 shows the killing effect of inducible CAR-T, where Figure 12A shows the CAR after transduction of plasmids 245 and 260 in PBMC; Figure 12B shows the killing of PBMCs transduced with plasmids 245 and 260, respectively The killing rate of tumor cells.

Figure 13 shows the expression of cytokines after PBMC transduced with plasmids 245 and 260 are incubated with target cells.

Figure 14 shows the regulatory effect of the adjustable CAR in mouse spleen cells (SPL).

detailed description

I. Definition

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are all terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.

It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

As used herein, the term "self-regulation" means that once CAR-T or TCR-T cells contact tumor cell surface antigens, CAR-T or TCR-T cells will be activated to produce more CARs or engineered TCRs. It further enhances the expansion ability of T cells and exerts the function of effector cells. This is a process that relies on its own components for regulation.

As used herein, the term "scFv" refers to a fusion protein that includes at least one light chain variable region antibody fragment and at least one heavy chain variable region antibody fragment, wherein the light chain and heavy chain variable regions pass through a short The flexible polypeptide linker is contiguous and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, scFv may have the VL and VH variable regions in any order (for example, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or May include VH-Linker-VL.

As used herein, the term "gene synthesis" refers to the use of recombinant DNA technology or the use of synthetic DNA or amino acid sequence technology available and well-known in the art.

As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response, which may involve the production of antibodies, or the activation of specific immunocompetent cells, or both. The skilled person will understand that any macromolecule, including virtually all proteins or polypeptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. The skilled person will understand any DNA-which includes a nucleotide sequence or part of a nucleotide sequence that encodes a protein that causes an immune response, and therefore encodes the term "antigen" as used herein. In addition, those skilled in the art will understand that the antigen need not be individually encoded by the full-length nucleotide sequence of the gene. It is obvious that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to elicit a desired immune response. In addition, the skilled person will understand that the antigen need not be encoded by a "gene". It is obvious that antigens can be produced, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological body fluids.

As used herein, the term "anti-tumor effect" refers to a biological effect, which can be caused by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or various cancerous disorders. The improvement of physical symptoms is clearly indicated. The "anti-tumor effect" can also be clearly expressed by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure to prevent tumors from occurring in the first place.

As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, brain cancer (such as astrocytoma, meningioma, oligodendroglioma, glioma, etc.), pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, Stomach cancer, bowel cancer, head and neck cancer, thyroid cancer, prostate cancer, Kaposi's sarcoma, etc.

As used herein, examples of the term "inflammatory disease" include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergy, septic shock, pulmonary fibrosis, undifferentiated Spondyloarthropathy, undifferentiated osteoarthropathy, arthritis, inflammatory osteolysis, and chronic inflammation caused by chronic viral and bacterial infections.

As used herein, the term "costimulatory molecule" refers to an associated binding partner on T cells that specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, such as but not limited to proliferation, costimulatory molecules Including but not limited to MHC I molecules, BTLA and Toll ligand receptors.

As used herein, the term "costimulatory signal" refers to a signal that combines with a primary signal, such as TCR/CD3 linkage, leading to T cell proliferation and/or up- or down-regulation of key molecules.

As used herein, the term "expression" is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.

As used herein, the term "nucleic acid construct" refers to a vector that includes a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. The nucleic acid construct includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Nucleic acid constructs include all those known in the art, such as cosmids incorporating recombinant polynucleotides, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus) And adeno-associated virus).

As used herein, the term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in two comparison sequences is occupied by the same base or amino acid monomer subunit, for example, if the position in each of two DNA molecules is occupied by adenine, then the molecules are the same at that position. Source. The percent homology between two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. Take an example to illustrate that the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, when two sequences need to be aligned to give maximum homology, a comparison is made.

Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may include intron(s) in some versions.

"Parenteral" administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection, or injection techniques.

As used herein, the terms "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal or cell thereof that is subject to the methods described herein, whether in vitro or in situ. In some non-limiting embodiments, the patient, subject, or individual is a human.

As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, nucleic acid is a polymer of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides". Monomer nucleotides can be hydrolyzed into nucleosides. Polynucleotides as used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, that is, from a recombinant library or cell genome, using common cloning techniques and PCR And so on clone nucleic acid sequence, and synthetic means.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids, and the maximum number of amino acids in its sequence is not limited. A polypeptide includes any peptide or protein that includes two or more amino acids connected to each other by peptide bonds. As used herein, short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example; and longer chains, which are commonly referred to as proteins in the art, have many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof.

As used herein, the term "promoter" is defined as the initiation of the specific transcription of a polynucleotide sequence, recognized by the synthesis machinery of the cell, or a DNA sequence that guides the synthesis machinery.

As used herein, the term "promoter/regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product operably linked to the promoter/regulatory sequence. In some examples, the sequence may be a core promoter sequence, and in other examples, the sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter/regulatory sequence can be, for example, a sequence that expresses a gene product in a tissue-specific manner.

As used herein, the term "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, allows most cells to produce a gene product under all physiological conditions.

As used herein, the term "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, such that only the corresponding promoter inducer is present in the cell to produce Gene product.

As used herein, "specifically binds" or "immune-specifically binds" with regard to an antibody or an antigen-binding fragment thereof are used interchangeably herein, and refers to an antibody or an antigen-binding fragment passing through the antibody binding site of the antibody and the antigen. The ability of non-covalent interactions with the same antigen to form one or more non-covalent bonds. The antigen may be an isolated antigen or present in tumor cells. Typically, immunospecifically bind (or specific binding) of the antibodies to the antigen is approximately 1 × 10 ⁷ M ^-1 or 1x10 ⁸ M ^-1 or greater affinity constant Ka (1x10 ^-7 M or 1 × 10 ^{- A dissociation constant (Kd) of 8} M or lower) binds the antigen. The affinity constant can be determined by standard kinetic methods of antibody reaction, for example, immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11: 54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of the binding rate are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27 : 335).

As used herein, the viral transduction background value refers to the transduction of the target gene into T lymphocytes through a viral vector, so that the target gene is expressed in the T lymphocytes. The efficiency of viral transduction was tested by flow cytometry. At this time, the expression percentage of fluorescent protein (such as EGFP) or MFI is the viral transduction background value.

As used herein, window or regulatory window refers to the expression percentage of fluorescent protein (e.g. EGFP) or the expression of MFI and unstimulated fluorescent protein (e.g. EGFP) after a cell transduced with a target gene is stimulated by an antigen or other stimulating factor The percentage or the ratio of MFI is the window or adjustment window.

As used herein, the term "therapeutic" means treatment and/or prevention. The therapeutic effect is obtained through the suppression, alleviation or eradication of the disease state.

As used herein, "treating" a disease refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

As used herein, the term "carrier" is a composition of matter, which includes an isolated nucleic acid, and which can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

In some embodiments, an enhancer response element is provided herein, the enhancer response element is selected from NFAT-RE, NF-κB-RE, AP-1-RE, TCF-RE, HRE, and is selected from the following The nucleotide sequence of has at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity: SEQ ID NO: 120-128.

II. Specific implementation

In one aspect, the present disclosure provides a tumor antigen-induced gene expression self-regulating nucleotide sequence, the sequence comprising:

(1) Promoter sequence, and

(2) Enhancer sequence.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of the aforementioned aspect, the promoter is selected from the initiator, the TATA box and/or other core promoter elements, and the other core promoter elements are selected from the following elements : BREu (upstream TFⅡB Recognition Element), MTE (Motif Ten Element), DPE (Downstream Promoter Element), DCE (Downstream Core Element), XCPE1 (X Core Promoter Element 1), etc.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the TATA box is an IL-2 TATA box.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the enhancer is selected from the following response elements: activated T cell nuclear factor response element (NFAT-RE), nuclear factor κB response element (NF-κB) -RE), T-cytokine/lymph enhancement factor response element (TCF-RE), activator protein-1 response element (AP-1-RE), hypoxia-inducible factor response element (HRE), FoxO transcription factor response element (FoxO -RE).

According to any one of the foregoing aspects of the tumor antigen-induced gene expression self-regulating nucleotide sequence, the tumor antigen is a tumor-associated antigen on the surface of tumor cells.

According to the tumor antigen-induced gene expression self-regulating nucleotide according to any one of the foregoing aspects, the enhancer is a single-copy enhancer, a multi-copy enhancer, or a combination of different enhancers.

According to any one of the foregoing aspects of tumor antigen-induced gene expression self-regulating nucleotide sequence, the sequence and the following sequence have at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24. SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID: 128.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the nucleotide sequence of the enhancer single-copy response element is selected from the following sequences: SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID: 128; or at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A sequence of 98%, 99%, 99.5%, or 99.9% identity.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the multiple copies are repeated 2-20 copies; particularly preferably, the multiple copies are repeated 2-9 times or repeated 2-7 Copies; more preferably, the multiple copies are repeated 5-6 copies.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the nucleotide sequence of the enhancer multi-copy response element is selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO :11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; or at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% of the above sequence , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% % Or 99.9% identity sequence.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the single-copy reverse nucleotide sequence is SEQ ID NO: 126 or SEQ ID NO: 127; or is at least 60% with the above sequence. %, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Sequences that are 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the multi-copy reverse nucleotide sequence is SEQ ID NO: 15 or SEQ ID NO: 119; %, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Sequences that are 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the nucleotide sequence of the combined response element of the different enhancer is selected from the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; or at least 60%, 65%, 70% of the above sequence , 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity sequence.

According to the tumor antigen-induced gene expression self-regulating nucleotide sequence of any one of the foregoing aspects, the nucleotide sequence of the promoter is SEQ ID NO: 16; or at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity sequence.

In one aspect, the present disclosure provides a nucleic acid construct comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence and a nucleotide sequence encoding a protein that specifically binds to tumor antigens; preferably, the The tumor antigen is a tumor-associated antigen on the surface of tumor cells.

According to the nucleic acid construct according to any one of the foregoing aspects, the protein capable of specifically binding to tumor antigens is selected from the group consisting of single-chain antibodies (ScFv), receptors, ligands, protein scaffolds and/or other chimeric antigen receptors (CAR ); Preferably, the protein scaffold is selected from Affibody, DARPin, Monobody, Anticalin4; preferably, the monomer is Centyrin; more preferably, the protein that can specifically bind to tumor antigens is a Combined antigen receptor (CAR).

According to the nucleic acid construct of any one of the foregoing aspects, the chimeric antigen receptor (CAR) comprises a tumor-associated antigen binding domain, a transmembrane domain and a signal transduction domain; preferably, the chimeric antigen receptor ( The amino acid sequence of CAR) is SEQ ID NO: 26 or SEQ ID NO: 28; or at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84% of the above sequence , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9 % Identity sequence.

According to the nucleic acid construct of any one of the foregoing aspects, the nucleotide sequence encoding the chimeric antigen receptor (CAR) is SEQ ID NO: 25 or SEQ ID NO: 27; or at least 60%, 65% of the above sequence , 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity sequence.

According to the nucleic acid construct of any one of the foregoing aspects, the nucleotide sequence encoding the chimeric antigen receptor (CAR) is selected from SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109; or at least 60%, 65%, 70%, 75%, 80% of the above sequence , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, 99.5%, or 99.9% identity sequence.

The nucleic acid construct according to any one of the foregoing aspects, which is a cosmid, a plasmid or a viral vector or a non-viral vector.

According to the nucleic acid construct of any one of the foregoing aspects, the viral vector is selected from a lentiviral vector, a retroviral vector, an adenovirus vector, and an adeno-associated virus vector.

According to the nucleic acid construct of any one of the foregoing aspects, the non-viral vector is selected from Sleeping Beauty plasmid transposition system, PiggyBac system or minicircle DNA.

According to the nucleic acid construct of any one of the foregoing aspects, the nucleic acid sequence of the nucleic acid construct is selected from: SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114 , SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; or at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or Sequence with 99.9% identity.

In one aspect, the present disclosure provides the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequences and/or nucleic acid constructs for preparing genetically modified immune cells directed against tumor-associated antigens.

In one aspect, the present disclosure provides an isolated host cell comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence and/or nucleic acid construct; preferably, the host cell is a mammalian cell; Preferably, the host cell is PBMC, T cell, NK cell, NKT cell, macrophage or cell line; preferably, the host cell is a primary cultured T cell; preferably, the host cell is selected from HEK293, HEK293T or Jurkat.

The host cell according to any one of the foregoing aspects also expresses other sequences, the other sequences including cytokine, another CAR, chemokine receptor, siRNA that reduces PD-1 expression, or protein that blocks PD-L1, TCR, or safety switch. Preferably, the cytokine is selected from IL-12, IL-15, IL-21, or type I interferon. Preferably, the chemokine receptor is selected from CCR2, CCR5, and CXCR3. Preferably, the safety switch is selected from iCaspase-9, Truncated EGF.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence, nucleic acid construct and/or host cell, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a use of the aforementioned tumor antigen-induced gene expression self-regulating nucleotide sequence, nucleic acid construct, host cell and/or pharmaceutical composition in the preparation of medicines.

According to the use of any one of the foregoing aspects, the drug is used to diagnose, treat or prevent one or more of cancer, inflammatory disease, and autoimmune disease.

According to the use of any one of the foregoing aspects, the drug is used for the diagnosis, treatment or prevention of tumors, preferably, the tumor is selected from blood cancer and solid tumors; more preferably, the drug is used for diagnosis, treatment or prevention of blood The cancer is selected from one or more of leukemia, lymphoma, and myeloma; more preferably, the solid tumor used by the drug for diagnosis, treatment or prevention is selected from lung cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, and kidney cancer , Bladder cancer, pancreatic cancer, gastric cancer, bowel cancer, prostate cancer, one or more of them.

In one aspect, the present disclosure provides a method for preparing CAR-T or TCR-T cells, the method comprising the following steps:

(1) Construction of a nucleic acid construct expressing a self-inducible CAR or engineered TCR, the nucleic acid construct comprising the tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2 and a nucleotide sequence encoding a tumor antigen The nucleotide sequence of the protein that specifically binds; preferably, the nucleic acid construct is selected from a cosmid, a plasmid, a viral vector or a non-viral vector; preferably, the viral vector is selected from a lentiviral vector, a retroviral vector, Adenovirus vector, adeno-associated virus vector; preferably, the non-viral vector is selected from Sleeping Beauty plasmid transposition system, PiggyBac system or minicircle DNA; preferably, packaging cell line is used to package the virus;

(2) Isolate peripheral blood mononuclear cells to obtain T lymphocytes;

(3) Transducing the nucleic acid construct described in step (1) into the T lymphocytes described in step (2) to obtain T lymphocytes containing the construct; preferably, a viral vector or a non-viral vector Transduction into T lymphocytes;

(4) Expand the culture of the T lymphocytes containing the construct obtained in step (3) to obtain CAR-T or TCR-T cells.

In one aspect, the present disclosure provides a method for treating a tumor-associated antigen-related disease in a subject, which comprises administering the aforementioned nucleic acid construct, host cell and/or pharmaceutical composition to the subject; preferably Preferably, the subject is a mammal; more preferably, the subject is a human.

For the purpose of clear and concise description, the features are described herein as part of the same or separate embodiments. However, it will be understood that the scope of the present disclosure may include a combination of all or some of the features described. Some implementations.

The present disclosure will be further described in detail below in conjunction with specific embodiments, and the examples given are only for clarification of the present disclosure, not for limiting the scope of the present disclosure. The reagents and biological materials described in the following examples can be obtained from commercial sources unless otherwise specified.

The experimental reagents, materials and methods used in the following examples are as follows:

Reagents and materials

1. Reagents and materials for plasmid construction

1.1 The backbone vector of synthetic plasmid 112 is pCDH-CMV-MCS-EF1-Puro (System Bioscience, Cat#: CD510B-1), and the backbone vector of synthetic plasmid 169 is pMSCV PIG (Puro IRES GFP empty vector) (Addgene 21654).

1.2 The PCR primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

1.3 Plasmid 112 was constructed by Suzhou Jinweizhi Biotechnology Co., Ltd.

1.4 High pure dNTPs (High pure dNTPs, Cat#: AD101), Easy Taq DNA polymerase (Easy Taq DNA polymerase, Cat#: AP111), Trans 2K plusII DNA Marker (Cat#: BM121), Trans5α chemically competent cells ( Trans5αChemically competitive cell, Cat#:CD201) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

1.5 Restriction endonucleases SmaI, SalI, ClaI and EcoRV were purchased from Takara.

1.6 Rapid plasmid extraction kit (Cat#:DP105-03), endotoxin-free plasmid small extraction kit (Cat#:DP118-02), ordinary agarose gel DNA recovery kit (Cat#:DP209- 03) All purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

1.7 Homologous recombination enzyme Assembly Mix (Cat#: RN1020) was purchased from Suzhou Hongxun Biotechnology Co., Ltd.

1.8 High-fidelity DNA polymerase Phanta Max super-Fidelity DNA polymerase (Cat#: P505-d1) was purchased from Nanjing Novazan Biotechnology Co., Ltd.

1.9 Tryptone (TRYPTONE, Cat#: LP0042), yeast extract (YEAST EXTRACT, Cat#: LP0021) were all purchased from OXOID.

1.10 Agar Agar was purchased from BIOSHARP.

2. Reagents and materials related to cell culture

2.1 Jurkat, Clone E6-1 cells (human acute T cell leukemia cell line) originated from ATCC TIB-152, HEK293T cells (human embryonic kidney cell line) In ATCC CRL-3216, NIH3T3 cells (mouse fibroblast cell line) are derived from ATCC CRL-1658.

2.2 Medium DMEM(Cat#:11995-065), RPMI Medium1640(Cat#:11875-093), HEPES(Cat#:15630-080), DPBS(Cat#:14190-144), 0.05%Trypsin-EDTA( Cat#:25300-062), Phenol Red (Cat#:25300-062), Dynabeads Human T-Activator CD3/CD28 (Cat#:11161D) were all purchased from Gibco.

2.3 Recombinant human interleukin-2 for injection (Intecom) was purchased from Jiangsu Kingsley Pharmaceutical Co., Ltd.

2.4 Serum ExCell Bio FBS (Cat#: FSP500) was purchased from ExCell Biotechnology (Taicang) Co., Ltd.

2.5 Penicillin-Streptomycin 100×solution (Penicillin-Streptomycin 100×solution, Cat#: SV30010) was purchased from Hyclone.

2.6 Phorbol 12-myristate 13-acetate (Phorbol 12-myristate 13-acetate (PMA), Cat#: P8139-1MG), Dimethyl sulfoxid (DMSO), Cat#: D2650), Phytohaemagglutinin P (PHA-P for short, Cat#: L8754), Concanavalin A (Concanavalin A for short, Con-A, Cat#: L7647-25MG) were all purchased from Sigma -Aldrich.

2.7 Ionomycin Calcium Salt (Cat#: FMS-FZ 208) was purchased from Nanjing Formax Biotechnology Co., Ltd.

2.8 PEI pro Transfection Reagent (Cat#: 115-0015) was purchased from Polyplus.

2.9 Sodium Butyrate (Cat#:HY-B0350A/CS-3924) was purchased from MCE.

2.10 CytoTell ^TM Blue (Cat#: 22251) was purchased from AAT Bioquest.

2.11 Antibiotic puromycin (puromycin, Cat#: ant-pr-5) and blasticidin (Blasticidin, Cat#: ant-bl-05) were purchased from InvivoGen.

2.12 Ficoll-Paque Plus (endotoxin tested <0.12EU/ml, Cat#:17-1440-02) was purchased from GE Healthcare.

2.13 Amicon Ultra-4, PLHK Ultracel-PL ultrafiltration membrane (Cat#: UFC810008) was purchased from Millipore.

2.14 Red blood cell lysate (Cat#: C3702) was purchased from Beyotime.

2.15 Polybrene (Cat#:40804ES76) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

3. Other reagents and materials

3.1 BSA (Albumin Bovine V, Cat#:A8020) was purchased from Solarbio.

3.2 ONE-Glo ^TM Luciferase Assay System (Cat#: E6110) was purchased from Promega.

3.3 Human IL-2ELISA Kit (Cat#:1110202) was purchased from Daktronics Biotechnology Co., Ltd.

Lentivirus packaging, concentration and titer detection

(1) Packaging of lentivirus

On the day before lentivirus packaging, inoculate 9×10 ⁵ /well 293T cells in 2ml DMEM medium in a 6-well plate containing 10% FBS, 100U/ml penicillin, 100μg/ml streptomycin, and 10mM HEPES. On the day of virus packaging, 200 μl of 150 mM NaCl, 1429 ng of target plasmid, 1571 ng of packaging plasmid (RRE:VSVG:REV=1:0.2:1) and 21 μl of PEI were mixed into a transfection solution, vortexed for 8 seconds, and allowed to stand at room temperature for 10 minutes. During this period, replace the medium in the 6-well plate with fresh medium (the medium contains 10% FBS, 100U/ml penicillin, 100μg/ml streptomycin+25uM chloroquine), then suspend and add dropwise The transfection solution was mixed gently and transferred to a 37°C, 5% CO ₂ incubator. 16 hours after transfection, the medium was changed to a fresh medium containing 1 mM sodium butyrate. After 48 hours, collect the cell culture supernatant to obtain a virus liquid containing lentiviral particles.

(2) Concentration of lentivirus

Before concentration, the virus solution obtained in step (1) was centrifuged at 132 g for 5 min to remove cell debris, and the virus supernatant was filtered through a 0.45 μm syringe filter, and then concentrated using an ultrafiltration tube. After the ultrafiltration tube was soaked in 20% ethanol overnight, 4ml of normal saline was added, centrifuged at 3000g for 15min, and the ultrafiltration tube was washed twice with sterile normal saline. Then add 4ml of normal saline and let it stand for 1 min, then remove the normal saline. Then the virus solution was added to the ultrafiltration tube, centrifuged at 3000g for 30 minutes, to obtain the concentrated virus solution, which was stored at -80°C.

(3) Transduction and titer detection of lentivirus

Sequentially add medium (10% FBS+100U/ml penicillin+100μg/ml streptomycin+10mM HEPES+8μg/ml Polybrene), virus solution, Jurkat cell suspension (total cells 1×10 ⁵ ) into a 1.5 In the ml EP tube, keep the total volume of the virus/cell mixture 500μl, and centrifuge at 800g for 30min at room temperature. Then the virus/cell mixture was transferred to a 24-well plate, 37°C, 5% CO ₂ incubator for culture. The next day, the Jurkat cells were collected, centrifuged at 100g for 10 min, and the supernatant was removed, replaced with fresh medium, and the cells were inoculated back to a 24-well plate at 37°C, 5% CO ₂ incubator for 48 hours. Use flow cytometry to detect the transduction efficiency, and calculate the titer of the virus according to the following formula:

Virus titer (TU/ml)=(number of cells×transduction percentage×transduction coefficient×1000)/virus volume (μl)

Notes:

(1) When the transduction percentage is less than 2%, the virus titer cannot be calculated.

(2) 2%≤transduction percentage≤20%, transduction coefficient=1.

(3) Transduction percentage>20%, transduction coefficient=transduction percentage/20%.

Packaging, concentration and titer testing of retrovirus

(1) Packaging of retrovirus

On the day before retrovirus packaging, inoculate 9×10 ⁵ /well packaging cell line D4 or 6# (HEK293 gag-pol stable) in 2ml DMEM medium in a 6-well plate, which contains 10% FBS , 100U/ml penicillin, 100μg/ml streptomycin, 10mM HEPES. On the day of virus packaging, mix 200μl 150mM NaCl, 2.5μg or 2μg target plasmid, 0.5μg packaging plasmid VSVG or 1μg packaging plasmid pCL-Eco (Addgen 12371) and 21μl PEI into a transfection solution, vortex and shake for 8 seconds, and let stand at room temperature 10min. During this period, replace the medium in the 6-well plate with fresh medium (the medium contains 10% FBS, 100U/ml penicillin, 100μg/ml streptomycin+25uM chloroquine), then suspend and add dropwise The transfection solution was mixed gently and transferred to a 37°C, 5% CO ₂ incubator. 16 hours after transfection, the medium was changed to a fresh medium containing 1 mM sodium butyrate. After 48 hours, collect the cell culture supernatant to obtain a virus liquid containing retroviral particles.

(2) Concentration of retrovirus

The method is the same as the lentivirus concentration step (2).

(3) Transduction and titer detection of retrovirus

Take a 1.5ml EP tube under aseptic conditions, and add fresh medium (90% DMEM+10% FBS+100U/ml penicillin+100μg/ml streptomycin+10mM HEPES+8μg/ml Polybrene), virus liquid, NIH3T3 cell suspension (1×10 ⁵ total cells), maintaining a total volume of 500 μl of the virus/cell mixture. After mixing gently, transfer to a 24-well plate. Finally, the 24-well plate was placed in a 37°C, 5% CO ₂ incubator for culture.

After 48h, it was detected by flow cytometry. For example, the calculation of transduction Jurkat and virus titer is the same as that of lentivirus.

Flow cytometry to detect the expression of EGFP on the cell surface

Collect 2×10 ⁵ cells in a 1.5ml EP tube, centrifuge at 500g for 5min, and remove the supernatant. Wash twice with 500 μl DPBS containing 0.1% BSA, each time at 500 g, and centrifuge for 5 min. Finally, 200μl DPBS containing 0.1% BSA was used to resuspend the cells before testing on the machine. Finally, the software of the Beckman Coulter CytoFlex flow cytometer was used to analyze the data.

Antigen (Ag) stimulation test

CytoTell ^TM Blue stain

Take the required Jurkat or hPBMC (the total number of cells does not exceed 1×10 ⁶ ) in a 1.5 ml EP tube, centrifuge at 500 g for 5 min, and remove the supernatant. Resuspend the cells in 1 ml DPBS containing 0.5% FBS, centrifuge again at 500 g for 5 min, and remove the supernatant. Add 500 μl of DPBS containing 0.5% FBS, gently resuspend the cells, then add 1 μl of CytoTell ^TM Blue dye, and incubate for 30 minutes in a 37° C., 5% CO _{2 incubator in the dark.} Centrifuge the cells at 500g for 5min. After removing the supernatant, wash the cells once with DPBS containing 0.5% FBS under the same centrifugation condition at 500g for 5min. Finally, resuspend the cells in fresh medium, take 50μl of cell suspension, and use flow cytometry to detect the CytoTell ^TM Blue staining effect of the cells.

Co-incubation experiment

Inoculate 1×10 ⁵ -2×10 ⁵ CytoTell ^TM Blue stained Jurkat cells or hPBMC expressing CAR in a 24-well plate, and then inoculate Raji B cells expressing CD19 and CD20 antigens. The number of cells inoculated is the same as that of CAR expressing. The number of cells is the same, and the total volume of medium in each well of the 24-well plate is 500 μl. In this experiment, hPBMC or K562 that did not express CD19 and CD20 antigens were used as negative controls. Finally, place the 24-well plate in a 37°C, 5% CO ₂ incubator for culture. Incubate for 24h and 48h, and take 100μl of cell suspension for flow cytometry detection.

The calculation formula of Ag stimulation efficiency is:

Ag stimulated median fluorescence intensity (Median Fluorescence Intensity, MFI) = MFI of EGFP in ^{CytoTell TM} Blue ⁺ EGFP ⁺

Preparation of hPBMC

The steps for preparing hPBMC are as follows:

(1) Pipette 15ml of fresh human peripheral blood into a 50ml centrifuge tube, then add the same volume of DPBS, gently pipette to mix.

(2) Take another 50ml centrifuge tube and add 15ml Ficoll. Then tilt the centrifuge tube at 45°, place the diluted human peripheral blood about 1 cm above the Ficoll liquid level, and slowly add it along the tube wall above the Ficoll.

(3) Centrifuge at 18-20℃, 600g, 30min. Set up the centrifuge, the rise speed is 5, and the fall speed is 0. After centrifugation, it is divided into four layers from the bottom of the tube to the liquid surface, followed by the red blood cell and granulocyte layer, the layered liquid layer, the mononuclear cell layer, and the plasma layer.

(4) Insert the 1ml pipette tip directly into the cloud layer (or suck the upper layer of plasma first), gently suck out the cloud layer, and put it into a new centrifuge tube.

(5) Add 3 times the volume of DPBS larger than PBMC to the new centrifuge tube, centrifuge at 18-20°C, 931g, 30min.

(6) Repeat step (5).

(7) Add 3 times the volume of DPBS larger than PBMC to the centrifuge tube, centrifuge at 18-20°C, 400g, 7min.

(8) After removing the supernatant, add the cryopreservation solution, and divide the cells into cryopreservation tubes, and place them in the program cooling box for cryopreservation.

Cultivation of hPBMC and viral transduction

Day 0 (D0): Take out a tube of frozen hPBMC from liquid nitrogen and quickly melt it in a 37°C water bath. Gently add hPBMC dropwise to 3ml of pre-warmed complete medium (90%RPMI 1640+10%FBS+100U/ml penicillin+100μg/ml streptomycin+10mM HEPES), and centrifuge at 500g for 5min. After removing the supernatant, resuspend the cells in 3ml medium and take 10μl of the cell suspension for counting. According to the counting results, 1×10 ⁵ cells were seeded in a 96-well plate (round bottom), and a certain volume of culture medium was added to make the total volume 200 μl. Then add CD3/CD28 Dynabeads according to the ratio of hPBMC:Dynabeads=1:0.8. The Dynabeads need to be washed once in accordance with the steps in the product instructions before use. Finally, 100IU/ml human interleukin-2 was added, and after gently mixing, the 96-well plate was placed in a 37°C, 5% CO ₂ incubator for 24 hours.

Day 1 (D1): Gently pipette 110 μl of supernatant (do not touch the cells), and discard. Take another 500μl EP tube, add virus, 100ng/ml Protamine Sulfate, 20mM HEPES, and mix gently (the amount of protamine sulfate and HEPES added is calculated based on the total medium in the 96-well plate) . The prepared virus suspension was added dropwise to hPBMC, and centrifuged at 800g at 32°C for 2h. After the end, the plate was placed in a 37°C, 5% CO ₂ incubator to continue culturing.

Day 2 (D2): Take a 500μl EP tube, add virus, 100ng/ml protamine sulfate, and mix gently (the amount of protamine sulfate added is calculated based on the volume of the virus). Add the prepared virus suspension dropwise to hPBMC, centrifuge at 800g at 32°C for 2h, add 100μl of medium (90%RPMI1640+10%FBS+100IU/ml human interleukin-2+100U/ml penicillin+100μg/ml Streptomycin), place the plate in a 37°C, 5% CO ₂ incubator for culture.

Day 3 (D3): Change the medium: remove 100 μl of supernatant (do not touch the cells), and then add 100 μl of complete medium.

Day 4 (D4): Transfer the cells from the 96-well plate to the 24-well plate for culture, and add 250 μl of complete medium.

Day 5 (D5): Change the medium: remove 220 μl of supernatant, and then add 650 μl of complete medium.

Day 6 (D6): Transfer the cells in 24 wells once to two, and add 500 μl of complete medium to each well.

Day 7 (D7): Inoculate the cells in two 24-wells into a T25 culture flask and maintain the cell density at 5×10 ⁵ -1×10 ⁶ cells/ml. From now on, cells can be maintained in T25, counted every other day, and replaced with fresh medium. The cell seeding density is 5×10 ⁵ -1×10 ⁶ cells/ml.

Obtaining, culturing and virus transduction of mouse spleen cells

Day 0 (D0): Place the mouse spleen in a 10cm dish, add 5ml DMEM medium, gently grind the mouse spleen with 2 glass slides (rough side), and pass the cell suspension through a 70-mesh screen Filter and collect in a 50ml centrifuge tube. Then rinse the 10cm dish with 5ml DMEM medium, and filter and collect it in the same way. Let stand at room temperature for 1 min. Transfer the supernatant to a new 15ml centrifuge tube and centrifuge at 500g for 5min. After removing the supernatant, add 1.5ml of red blood cell lysate, leave it at room temperature for 6 minutes, and add 10ml of DPBS to stop the reaction. After centrifugation at 500g for 5min, resuspend in pre-warmed 5ml complete medium (90%RPMI 1640+10%FBS+100U/ml penicillin+100μg/ml streptomycin+10mM HEPES+100IU/ml IL2+50μMβ-mercaptoethanol) Take 10μl of cells and count them. According to the experimental requirements, take the corresponding number of spleen cells and centrifuge them in a 15ml centrifuge tube at 500g for 5min. Then resuspend the cells in culture medium, and inoculate the cells in a 24-well plate with 3×10 ⁶ cells in each well and 1 ml of culture medium. Finally, add 0.1μg/ml CD3 and 0.1μg/ml CD28 antibodies to each well, mix gently, and incubate in a 37°C, 5% CO ₂ incubator.

Day 1 (D1): Gently pipette 600μl of cell supernatant into a 1.5ml EP tube, centrifuge at 500g for 5min. After removing the supernatant, add 100μl of complete medium to resuspend the cells, and then add virus, 2μg/ml Lipo2000 (ThermoFisher), 1.6μg/ml Polybrene. Lipo2000 and Polybrene are calculated based on a total volume of 500μl. After mixing, add dropwise to a 24-well plate, centrifuge at 930g at 37°C for 1.5h. After centrifugation, add 500μl of complete medium, 37°C, 5% CO ₂ incubator to continue culturing.

Day 2 (D2): Remove 500μl of cell supernatant, taking care not to touch the cells. After gently mixing the cells, take 10μl and count. According to the cell count results, the cells in the 24-well plate were transferred to T25 culture flasks for culture. The inoculation cell density was 2×10 ⁵ cells/ml, and the total volume was 6 ml. From this time on, the cells can be maintained in T25 and the cell density is maintained at 4×10 ⁵ -1×10 ⁶ cells/ml. Because the cells grow rapidly, it is necessary to count and replace with fresh medium every day.

Example 1 Construction of Nucleic Acid Encoding Chimeric Antigen Receptor (CAR)

1. For CD20

CD20 CAR was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and its nucleotide sequence is: SEQ ID NO: 25, and its amino acid sequence is: SEQ ID NO: 26.

2. For CD19

CD19 CAR was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and its nucleotide sequence is: SEQ ID NO: 27, and its amino acid sequence is: SEQ ID NO: 28.

Example 2 Structural design of inducible CAR

In order to study the induction of chimeric antigen receptors (CAR) by enhancers, this example uses response elements combined with transcription factors related to T cell receptor (TCR) gene transcription regulation as enhancers, and designed a single connection or multiple Copy the linked response elements to regulate gene expression. The response elements include AP-1-RE (MAPK pathway), NF-κB-RE (NF-κB pathway), NAFT-RE (calcium pathway), FoxO-RE (PKB/Akt pathway), TCF-RE (Wnt Pathway), HRE (HIF1a pathway). Among them, the nucleotide sequence of a single copy of NF-κB-RE is SEQ ID NO: 120 or 121; the nucleotide sequence of a single copy of NAFT-RE is SEQ ID NO: 122; the nucleotide sequence of a single copy of TCF-RE It is SEQ ID NO: 123; the nucleotide sequence of HRE single copy is SEQ ID NO: 124; the nucleotide sequence of AP-1-RE single copy is SEQ ID NO: 125; NF-κB reverse single copy nucleus The nucleotide sequence of the nucleotide sequence is SEQ ID NO: 126; the nucleotide sequence of the reverse single copy of NAFT-RE is SEQ ID NO: 127; the nucleotide sequence of the single copy of FoxO-RE is SEQ ID NO: 128.

In this example, the following three types of plasmids are designed: The first type of plasmid contains enhancer, IL-2 TATA promoter (SEQ ID NO: 16) and EGFP (enhancer-IL-2 TATA promoter-EGFP), and its main structure As shown in Figure 1A, plasmids 112, 241, 242, 243-1, 243-5, 244, 262, 293, 417, 445, 446, 459, and 460 are involved. The second type of plasmid contains enhancer, IL-2 TATA promoter, CAR, mouse IL-7 and EGFP (enhancer-IL-2 promoter-CAR-F2A-IL-7-F2A-EGFP), the main structure is as follows As shown in Figure 1B, plasmids 228, 259, 260, 261, 263, 280, 281, 292, 294, 407, 410, 437, 438-13, 438-18, 439, 440, 448 are involved. The third type of plasmid contains EF1α enhancer, EF1α promoter, CAR, murine IL-7 and EGFP (EF1α enhancer-EF1α promoter-CAR-F2A-IL-7-F2A-EGFP), and its main structure is shown in Figure 1C As shown, plasmid 245 is involved.

1. Plasmids 026, 056, 099, 112, 245, and 169 are all synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and their nucleotide sequences are: SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 118.

2. Construction of plasmids 241, 242, 243-1, 243-5, 244, 262, 293, 445, 446, 459, 460

The enhancers NFAT-RE (SEQ ID NO: 1), NF-κB-RE (SEQ ID NO: 2 or 3), TCF-RE (SEQ ID NO: 4 or 5), FoxO-RE (SEQ ID NO: 2 or 3), TCF-RE (SEQ ID NO: 4 or 5) were synthesized by conventional molecular biology PCR. RE (SEQ ID NO: 6), HRE (SEQ ID NO: 7), AP-1-RE (SEQ ID NO: 8), IFN-βenhancesome-RE (SEQ ID NO: 21, 22, 23 or 24), The primer sequences are shown in the table below. Then according to the requirements of the homologous recombination instructions, the enhancer was homologously recombined into the plasmid 112ClaI and EcoRV restriction sites. Unless otherwise specified, the molecular cloning techniques involved in this disclosure are all conventional techniques of molecular biology.

Table 1

Plasmid	Primer sequence (SEQ ID NO:)
241	24, 35
242	36, 37
243-1	38, 39, 40, 41, 42, 43
243-5	38, 39, 40, 41, 42, 43
244	44, 45, 46, 47
262	48, 49, 50, 51
293	52, 53
445	54, 55
446	56, 57
459	58, 59, 60, 61
460	61, 62, 63, 64

3. Construction of plasmids 228, 259, 260, 261, 263, 280, 281, 292, 294, 407, 410, 437, 438-13, 438-18, 439, 440, 448

Use conventional molecular biology methods to PCR and recover enhancer and promoter fragments. The specific templates and primers used are as follows. Then according to the requirements of the homologous recombination instructions, the enhancer and promoter fragments were homologously recombined to the restriction site of the corresponding plasmid. Unless otherwise specified, the molecular cloning techniques involved in this disclosure are all conventional techniques of molecular biology.

Table 2

Plasmid	PCR template	Primer (SEQ ID NO:)	Restriction digestion plasmid	Restriction endonuclease
228	112	65, 66	245	Spel and 8amHI
259	241	66, 67	245	Spel and BamHI
260	241	68, 69	228	EcoRV
261	228	68, 69	259	EcoRV
263	262	66, 70	245	Spel and 8amHI
280	262	68, 71	228	EcoRV
281	112	68, 72	263	EcoRV
292	026	73, 74	260	Xhol and 8amHI
294	293	65, 70	245	Spel and BamHI
407	/	75, 76	259	EcoRV
410	/	77, 78	259	EcoRV
437	/	77, 78	410	EcoRV
438-13	263	79, 80	263	Spel
438-18	263	79, 80	263	Spel
439	/	81, 82	294	EcoRV
448	446	60, 68	245	Spel and BamHI
408	/	83, 84, 85, 86	169	Xhol and SalI
417	112	87, 88	408	SalI
440	410	89, 90, 91, 92	417	HindIII/BamHI

Example 3 PMA and ionomycin regulate the expression of EGFP in plasmid 112

When culturing cells in vitro, PMA and ionomycin can usually be used to stimulate the cells to study the activation mechanism of T cells. PMA is an activator of protein kinase C (PKC). PKC can activate the phosphorylation of many downstream protein kinases to form a cascade reaction and induce the expression of many proteins through NFAT. In normal cell physiology, PKC can be activated by the combined action of ^{DAG (diacylglycerol) and Ca 2+.} Ionomycin is a transport agent of Ca ^2+, the intracellular Ca ²⁺ can be transported to the cytoplasm, thus contributing to T-cell activation. Therefore, PMA and ionomycin can synergistically activate T cells, and can detect the expression of genes related to TCR induction.

Jurkat cells are an acute T cell leukemia cell line, which retains the signal transduction function of T cells and has a wide range of applications in cell biology research on T cell functions. In order to simulate the TCR activation process, the NFAT enhancer and IL-2 TATA box were combined, and EGFP was used as the reporter gene. They were constructed into a lentiviral vector, and then HEK293T cells were used to package the virus, and the plasmid 112 was transduced to Test its regulatory effect in Jurkat cells.

Jurkat was stimulated with PMA and ionomycin. The experimental method is as follows:

Collect 1×10 ⁵ -2×10 ⁵ Jurkat cells in a 1.5 ml EP tube and centrifuge at 500 g for 5 min, and remove the supernatant. Resuspend the cells in 200 μl of fresh medium containing the corresponding concentrations of PMA and ionomycin. The total volume of PMA and ionomycin should not exceed 2μl. DMSO was added to the control group, and the volume of DMSO added was consistent with the total volume of PMA and ionomycin. Finally, the cells were seeded in a 96-well plate and cultured in a 37°C, 5% CO ₂ incubator. After 24 hours, the cells were collected, and flow cytometry was used to detect the expression of EGFP on the cell surface.

Different concentrations of PMA and ionomycin were used to stimulate Jurkat cells, and flow cytometry was used to observe the percentage of EGFP cells or the average fluorescence intensity (MFI) after 24 hours, reflecting the effect of gene regulation. It can be seen from Figure 2 that the expression of EGFP in the unstimulated control group was 16.5%. After 10ng/ml PMA was stimulated with 0.03μM, 1μM, and 2.2μM ionomycin, the expression of EGFP gradually increased. The EGFP expression after stimulation with 2.2μM ionomycin was the highest, which was 51.4%. Similarly, after stimulation with different concentrations of PMA/Iono, the MFI of EGFP also gradually increased, and the trend was the same as that of the percentage of EGFP. When the concentration of PMA is 10ng/ml and the concentration of ionomycin reaches 2.2μM, the cell viability is lower after 24h stimulation. Therefore, in subsequent experiments, the fixed PMA concentration is 10ng/ml and the concentration of ionomycin is 1μM. This experiment can be used to evaluate the induction efficiency of different enhancers.

Example 4 PMA and ionomycin test different enhancers' regulatory effects in Jurkat

1. Regulating role of enhancer in RE-IL-2 TATA box-EGFP structure

This example first selected some enhancers related to T cell activation, such as AP-1-RE (MAPK pathway), NF-κB-RE (NF-κB pathway), NFAT-RE (Ca ⁺ pathway), TCF- RE (Wnt pathway), FoxO-RE (Akt pathway), HRE (HIF1α pathway), combine them with IL-2 TATA box and EGFP through homologous recombination, and then transduce them into Jurkat cells through lentivirus , And stimulated with PMA/Iono. After 24h, flow cytometry was used to detect the expression of EGFP in each combination. The expression of EGFP can reflect the ability of different enhancers to induce gene expression.

As shown in Table 3: All RE-IL-2 TATA box-EGFP structures (plasmid 112, 241, 242, 243-1, 243-5, 244, 262, 293) can be stimulated by PMA/Iono, Jurkat cells Both the expression percentage of EGFP and MFI increased significantly, the former has a window of 1.3-8.6 times, and the latter has a window of 1.1-5.2 times. Under such experimental conditions, when the basic expression is not stimulated by PMA/Iono, the structure with higher expression of EGFP has a smaller induction percentage, such as plasmid 262. The percentage is only up-regulated from 58.5% to 76.5%, but MFI is lower than 6,200 increased to 11877. In addition, the regulatory window of the MFI of plasmid 241 and plasmid 293 is also larger. Therefore, both the percentage of EGFP and the change in MFI can be used to evaluate the effect of regulation.

table 3

2. Regulating role of enhancer in RE-IL-2 TATA box-CAR-F2A-IL-7-F2A-EGFP structure

In order to simulate the final TCR response, in this example, the above-mentioned different enhancers and the IL-2 TATA box were homologously recombined into the vector expressing CD20 CAR, and the structure is RE-IL-2 TATA box-CAR-F2A-IL -7-F2A-EGFP (plasmid 228, 259, 263, 294).

It can be seen from Table 4 that under PMA/Iono stimulation, the expression of EGFP in Jurkat cells increased significantly, with a window ranging from 2.6 times to 18.8 times. Similarly, the MFI of EGFP also increased, from 1.2 times to 1.8 times the window.

Table 4

3. Regulatory effects of different RE combinations

In order to further study the regulatory effects of different enhancers, this example also combined different REs to obtain the following plasmid: 260 (including enhancer NFAT-RE×NF-κB-RE, and its nucleotide sequence is as SEQ ID NO: 17), 261 (including the enhancer NF-κB-RE×NFAT-RE, and its nucleotide sequence is shown in SEQ ID NO: 18), 280 (including the enhancer NFAT-RE×HRE-RE , Its nucleotide sequence is shown in SEQ ID NO: 19), 281 (including the enhancer HRE-RE×NFAT-RE, and its nucleotide sequence is shown in SEQ ID NO: 20), 445 (including the enhancer IFN -βenhancesome(1)-RE, whose nucleotide sequence is shown in SEQ ID NO: 21) and plasmid 446 (including enhancer IFN-βenhancesome(2)-RE, whose nucleotide sequence is shown in SEQ ID NO: 22) Show).

It can be seen from Table 5 that under the same stimulation conditions, the adjustment windows of NFAT-RE×HRE (280) and HRE×NFAT-RE (281) are 3.3 times and 3.6 times, respectively. The regulatory effect of the combination of NFAT-RE and NF-κB-RE is more obvious, and the EGFP expression percentage window is 12.7 times (260) and 18.5 times (261), respectively. Plasmid 445 and plasmid 446 have smaller regulatory windows, which may be related to high background values.

table 5

4. Regulatory effects of multi-copy enhancers NF-κB-RE, HRE, and AP-1-RE

As mentioned above, the response element is a highly conserved DNA sequence with independent or multiple copies of enhancers. This example also uses enhancers NF-κB-RE, HRE and AP-1-RE as examples to discuss different copy numbers. The regulatory role of the enhancer. The NF-κB-RE, HRE and AP-1-RE used in the previous article are all 3 repeats, but here we use the PCR method to synthesize 3, 4 and 11 NF-κB-REs, 6 With 9 HRE repeats and 6 AP-1-RE repeats, and through homologous recombination, the following plasmids were obtained: 410 (6 NF-κB-RE, SEQ ID NO: 9), 407 (7 NF-κB-RE, SEQ ID NO: 10), 437 (11 NF-κB-RE, SEQ ID NO: 11), 438-18 (6 HRE, SEQ ID NO: 12), 438-13 ( Nine HREs, SEQ ID NO: 13) and 439 (6 AP-1-REs, SEQ ID NO: 14). Similarly, HEK293T was used to package the viruses, and they were transduced into Jurkat cells via lentivirus, and then stimulated with PMA/Iono for 24h, and finally, the expression of EGFP was detected by flow cytometry.

As shown in Table 6, increasing the number of NF-κB-RE, the adjustment window for the percentage of EGFP expression is larger than the three repeat fragments, and the plasmid 410 has the largest adjustment window for the percentage of EGFP expression, reaching 122.7. Similarly, increasing the number of AP-1-REs will increase the regulatory window for the percentage of EGFP expression, that is, the window of plasmid 439 is 46.3, which is higher than that of plasmid 294 (the regulatory window is 18.8). Different from NF-κB-RE and AP-1-RE, the repeat fragments of HRE are increased, and the EGFP percentage regulation window is not higher than the regulation window of 3 HREs, that is, the EGFP percentage regulation window of plasmids 438-13 and 438-18 They are 1.6 and 2.1 respectively, both lower than or close to plasmid 263 (regulation window is 2.6).

Table 6

5. Regulatory effects of enhancers on different CARs

All the above structures are based on CD20 CAR. In order to prove that the structure of the inducible CAR can be applied to other different targets, this example also uses plasmid 260 as the vector backbone, replacing CD20scFv with CD19scFv. First, use the plasmid 026 containing CD19 CAR as a template, design primers, and obtain CD19scFv by PCR. After the DNA fragment of CD19scFv was recovered, homologous recombination was used to homologate it to the BamHI/XhoI site of plasmid 260, and finally plasmid 292 containing CD19CAR was obtained. It was also transduced into Jurkat cells using the method of lentivirus, PMA/Iono was stimulated for 24h, and finally the EGFP expression was detected by flow cytometry.

As shown in Table 5 and Figure 3, for Jurkat cells transduced with plasmid 292, when PMA/Iono is not added, EGFP expression is 8.1%, and after PMA/Iono is added, EGFP expression rises to 72.6%, and the window is 9.0 times This shows that the combination of an adjustable enhancer and IL-2 TATA box is also suitable for CD19 CAR.

Example 5 Regulation of Plasmid 112 in PBMC

Human peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMC) are cells with mononuclear nuclei in peripheral blood, mainly including lymphocytes (T cells and B cells), monocytes, phagocytes, dendritic cells and others A small number of cell types are an important cell component of the body's immune response function. PBMC is a research material often needed by scholars in the fields of immunology, antibody drug development, infectious diseases, hematological malignancies, vaccine development, and transplantation immunity. In CAR-T therapy, the final prepared CAR is also transduced into human PBMC through viruses and other methods.

In the in vitro experiment, this example uses Ficoll reagent to separate PBMC to obtain T cells. Then use HEK293T cells to package the virus of plasmid 112, and use an ultrafiltration tube to concentrate the virus. Finally, the plasmid 112 was transduced into PBMC by way of virus, and the gene regulation of the combination of enhancer NFAT and IL-2TATA box in PBMC was further tested. effect.

For this reason, a multi-factor stimulation experiment was carried out in this example, and the experiment method is as follows:

Collect 1.5×10 ⁵ PBMC cells in a 1.5 ml EP tube, centrifuge at 500 g for 5 min, and remove the supernatant. Add 200μl of fresh medium containing CD3/CD28 Dynabeads (PBMC:Beads=1:0.8), 10ng/ml PMA, 1μM ionomycin, 2μg/ml PHA-P, 10μg/ml PHA-P, 20μg /ml PHA-P, 1μg/ml Con-A, 2μg/ml Con-A or 5μg/ml Con-A. After resuspending the cells, they were seeded in a 96-well plate and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Collect cells and use flow cytometry for detection.

As shown in Figure 4, PBMC transduced with plasmid 112, when not stimulated, only 1.3% of the cells in the control group expressed EGFP. After 24h stimulation with PMA/Iono, the expression of EGFP was as high as 62.6%, with a window of 48 times . In addition to PBMC can be stimulated by PMA/Iono, CD3/CD28 Dynabeads (activating TCR/costimulatory factor CD28), PHA-P and Con A (promoting mitosis) can all activate T cells, thereby promoting the expression of EGFP in PBMC. After CD3/CD28 Dynabeads stimulation, EGFP expression increased significantly, reaching 40%, and the regulation window was 31 times. PHA-P and Con A stimulate the expression of plasmid 112 genes in a dose-dependent manner, and the 20μg/ml PHA-P and 5μg/ml Con A induction windows reached the maximum, 18.3% and 34.2%, respectively, but their maximum regulation The windows are lower than the induction windows of PMA/Iono and CD3/CD28. And when PHA-P and ConA were used at concentrations greater than or equal to 20μg/ml and 5μg/ml, the viability of PBMC cells decreased.

According to the review, CD3/CD28 Dynabeads, PHA-P, Con A and PMA/Iono all activate T cells. Once T cells are activated, the enhancer NFAT and IL-2 TATA box can regulate the transcription and expression of the downstream gene EGFP. In addition, under the action of these stimulating factors, the windows for plasmid 112 to regulate gene expression are different. PMA/Iono has the best effect, with a window of 48 times. Therefore, the follow-up PBMC experiment also uses PMA/Iono as a stimulating factor to activate T cells.

Example 6 Selection of PMA and ionomycin stimulation time

PMA/Iono stimulation was used to test the time node changes of plasmid 112 gene regulation in PBMC. Firstly, the plasmid 112 was transduced into PBMC through the concentrated lentivirus, and then PBMC was stimulated with PMA/Iono, and the PMA and ionomycin stimulation time exploration experiment was carried out. The experimental method is as follows:

Inoculate 1.5×10 ⁵ hPBMC cells in a 96-well plate, the total volume of the medium is 200 μl, a total of 6 wells, respectively labeled AF. Add 1μl DMSO to well A, and add 10ng/ml PMA+1μM ionomycin (total volume 1μl) to the remaining wells. Place the 96-well plate in a 37°C, 5% CO ₂ incubator for 1h, 2h, 3h, and 6h. Take one well of the cells in a 1.5ml EP tube and centrifuge at 500g for 5min. After removing the supernatant, Wash once with 500μl DPBS and centrifuge at 500g for 5min. The supernatant was removed, and the cells were resuspended in 200 μl of fresh medium, and then inoculated in 96 wells to continue culturing. After 24h (calculated by adding PMA and ionomycin from the beginning), all the cells were collected, and the expression of EGFP on the cell surface was detected by flow cytometry.

1h, 2h, 3h, 6h after stimulation, wash off PMA/Iono with Dulbecco’s phosphate buffered saline (DPBS), and then add fresh complete medium to continue culturing PBMC. Counting from the beginning of adding PMA/Iono, culture for a total of 24h. Finally, together with the cells stimulated for 24h, flow cytometry was used to detect the expression of EGFP. As shown in Figure 5, the expression of EGFP in unstimulated PBMC was 1.6%. When stimulated with PMA/Iono for 1 h, the expression of EGFP increased to 36.8%. When the stimulation was extended for another 1 hour, the expression of EGFP continued to rise to 48%, almost reaching a plateau. As the stimulation time was extended again, the expression of EGFP increased slowly, reaching a maximum of 56% at 24h. This shows that PMA/Iono can quickly stimulate the activation of T cells within 1-2 hours, thereby promoting NFAT and IL-2 TATA boxes to regulate downstream EGFP expression. Different from the change of EGFP percentage, after PMA/Iono stimulation 24h, EGFP MFI increased from 2477 to 74065, reaching the maximum value, which was 30 times that of the control group. As mentioned in the previous article, PMA/Iono can rapidly activate T cells within 1-2 hours, thereby promoting NFAT and IL-2 TATA boxes to up-regulate the expression of EGFP. However, the MFI of EGFP is still low at this time, which indicates that the amount of EGFP expressed by each cell is still low, and the maximum value can only be reached when the induction time reaches more than 24 hours. Therefore, this disclosure will use PMA/Iono to stimulate PBMC for 24 hours or longer for subsequent experiments.

Example 7 Regulatory effects of the combination of enhancer and IL-2 TATA box in PBMC

1. Regulatory effects of the combination of different enhancers and IL-2 TATA box

In this example, three different enhancers and IL-2 TATA box combinations were selected. They are plasmid 260 (NFAT-RE×NF-κB-RE), plasmid 261 (NF-κB-RE×NFAT-RE) and Plasmid 280 (NFAT-RE×HRE-RE) was transduced into PBMC through concentrated lentivirus, and then stimulated with PMA/Iono for 24h, and finally the expression of EGFP was detected by flow cytometry.

As shown in Figure 6, after transduction of plasmids 260, 261, and 280 in PBMC, they were 21.8%, 3.5%, and 6.8% without PMA/Iono stimulation, respectively. After PMA/Iono stimulation for 24 hours, the expression level of EGFP increased significantly, 77.8%, 43.7%, and 43.89%, and the change of MFI of EGFP was the same as the percentage change. This shows that these three combinations can all be in PBMC. Stimulated by PMA/Iono.

2. The regulatory effect of the combination of other enhancers and IL-2 TATA box

In this embodiment, the same method is used to test the regulation effect of the combination of other enhancers and IL-2 TATA box in PBMC. First, the plasmids 228 (NFAT-RE), 259 (NF-κB-RE), 281 (HRE×NFAT-RE), 294 (AP-1-RE), 407 (NF-κB-RE×7), 410 ( NF-κB-RE×6), 437 (11 NF-κB-RE), 438-13 (9 HRE), 438-18 (6 HRE), and 448 (IFN-βenhancesome(2)), pass slowly The virus was transduced into PBMC, and then stimulated with PMA/Iono for 24h, and finally the expression of EGFP was detected by flow cytometry. As shown in Table 7, among these combinations, the EGFP percentage adjustment window of plasmid 439 is the largest at 12 times, the adjustment window of plasmid 281 is 10.2 times, the adjustment window of plasmid 228 is 8 times, and the adjustment window of plasmid 448 is 7.6 times. The regulatory windows of plasmids 259 and 407 are both close to 2 times, and the regulatory windows of plasmids 437, 438-13 and 438-18 are the smallest.

Table 7

3. Regulatory effects of enhancers on different CARs

The foregoing proves in Jurkat cells that the regulatory structure designed in the present disclosure is suitable for different CARs. In order to further prove, the same use of lentivirus was used to transduce plasmid 292 (CD19 CAR) and plasmid 260 (CD20 CAR) expressing CD19 and CD20 CAR respectively into PBMC. These two plasmids have NFAT-RE×NF-κB-RE As part of the IL-2 TATA box, PMA/Iono was stimulated for 24 hours, and the expression of EGFP was detected by flow cytometry. As shown in Table 7 and Figure 7, PBMC transduced with plasmid 260 and plasmid 292 after PMA/Iono stimulation, EGFP expression increased significantly, and the regulatory window was 3.7 times and 5.7 times, respectively. This shows that CD19 CAR can also be regulated in PBMC.

Example 8 Antigen induction effect of different enhancers in PBMC or Jurkat

To stimulate self-regulated CAR-T in vitro, Raij B cells are used as target cells.

Raji B cells are human lymphoma cells. Raji B cells are pre-labeled with PE-labeled anti-human CD20 flow cytometry antibody. As shown in Figure 8, almost 100% of Raji B cells express CD20 antigen on their surface, so Raji B cells can be used as Ideal target cells for in vitro antigen induction experiments.

The antigen induction experiment requires that CAR-T cells are incubated with target cells expressing antigen to induce CAR expression. In order to distinguish Raji B from CAR-transduced T cells, dyes are used to stain CAR-T cells. At present, there are many kinds of dyes on the market for staining living cells, such as the widely used CFSE dye. However, the adjustable CAR-T structure designed in the present disclosure uses EGFP to reflect the expression of CAR, due to CFSE and its fluorescein The excitation spectrum and emission spectrum of the analog are almost the same as EGFP, so CAR-T cells cannot be labeled with CFSE and its fluorescein analog dye. In this embodiment, the living cell dye CytoTell ^TM Blue is used to label CAR-T cells. CytoTell ^TM Blue is a blue fluorescent dye that can uniformly stain cells. It is less cytotoxic and can be applied to EGFP-transduced cells.

1. Regulating role of CAR adjustable structure and CAR non-adjustable structure in PBMC

Plasmid 260 was transduced into PBMC by lentivirus to obtain CAR-T cells transduced with plasmid 260. The plasmid 260 has a CAR adjustable structure, namely NFAT-RE×NF-κB-RE and IL-2 TATA box The combination.

In order to ensure the authenticity and reliability of the experiment, plasmid 245 was transduced into PBMC through lentivirus simultaneously to obtain CAR-T cells transduced with plasmid 245. Plasmid 245 contains a constitutive promoter EF1α that regulates CAR expression. This constitutive promoter EF1α The CAR-T cell transduced with plasmid 245 is a CAR-T cell that does not have an adjustable structure for constitutive expression. CAR-T cells transduced with plasmid 245 can be used as control cells for CAR-T cells transduced with plasmid 260.

Empty PBMCs that did not express CD20 antigen were used as control cells.

The 260 T cells transduced with CytoTell ^TM Blue were respectively stained and incubated with Raji B cells at a ratio of 1:1. As a control, T cells stained with 260 were incubated with empty PBMCs that did not express CD20 antigen. Cells were collected at 24-48h, and the expression of EGFP in ^{Cyto Tell TM Blue positive cell population was detected by flow cytometry.}

Figures 9A and 9B show the flow cytometry and data analysis results of antigen stimulation. When CAR-T cells transduced with plasmid 260 were incubated with empty PBMCs that did not express CD20 antigen, the expression percentage of EGFP was 18.6% (=(9.36/(9.36+40.91))×100). After Raji B cells were stimulated for 24 hours, the expression of EGFP increased to 42.9% (=(32.58/(32.58+43.29))×100). Using this method, compare the stimulation effects of antigen stimulation and the other two in vitro stimulation methods (CD3/CD28 and PMA/Iono).

As shown in Figure 10, after CAR-T cells transduced with plasmid 260 are stimulated by CD3/CD28 or PMA/Iono, the percentage window of EGFP expression is close to 4 times, and after co-incubation with Raji B cells, the EGFP regulatory window is 3 times. The CAR-T transduced with plasmid 260 was incubated with PBMC or culture medium that did not express CD20 antigen, and the expression of EGFP was similar, which indicated that PBMC that did not express CD20 antigen had no stimulating effect on CAR-T. The CAR-T cell transduced with plasmid 245 was used as a control. When it was cultured in medium, the expression of EGFP was 3.3%. After co-incubation with Raji B cells, the expression of EGFP was 3.7%. This indicates that the expression of plasmid 245 is Structure has no regulatory effect in PBMC.

2. Regulatory effects of different enhancers

Using the same method to test other combinations, as shown in Table 8, CARs with different enhancer and IL-2 TATA box combinations can be activated after recognizing tumor antigens, and then regulate their own EGFP expression, with a window of 1.4 times to 8.5 times . Plasmid 228, that is, NFAT-RE, has the strongest regulatory effect, with a window of 8.5 times, while plasmid 259, that is, NF-κB-RE has the smallest regulatory window, which is 1.4 times. This may be related to the high background in PBMC after its transduction. Value related.

Table 8

3. Regulatory effects of enhancers on different CARs

As mentioned above, after replacing CD20 ScFv with CD19 ScFv, the regulated CAR can still be activated by PMA/Iono stimulation. This article uses the same method to test its antigen stimulation effect. Raji B is a lymphoblast-like cell line cultured in vitro. In addition to high expression of CD20 protein on the cell surface, it also highly expresses CD19 protein. Therefore, it can be used as a target cell for CD19 CAR-T. This example is based on the combination of NFAT-RE×NF-κB-RE and IL-2 TATA box to explore the antigen induction of CD19 CAR. Similarly, first use the concentrated lentivirus to transduce the CD19 CAR-expressing plasmid 292 into PBMC, and then ^{stain Cyto Tell TM} Blue to transduce the plasmid 292 PBMC, and compare it with Raji B cells or empty PBMC cells respectively. Incubate at a ratio of 1:1, and detect the expression of EGFP for 24-48h. As shown in Table 8, before antigen stimulation, EGFP expression was 0.6%, and after co-incubation with RajiB cells, EGFP expression rose to 1.8%, and the adjustment window was 3 times. This shows that the adjustable CAR designed in this example The structure can be applied to different antigens. Once activated by the antigen, T cells are activated to promote the high expression of their own CAR.

4. Antigen induction in Jurkat cells transduced by retrovirus

The combinations tested above are all constructed on lentiviral vectors. In addition, this example uses retroviral vectors to test the regulatory effects of these combinations. Through the method of homologous recombination, the combination of NFAT-RE (SEQ ID NO: 15) and IL-2 TATA box was constructed on the retroviral vector-plasmid 169, and plasmid 417 was obtained. And the retrovirus was packaged by the cell line D4, then the plasmid 417 was transduced into Jurkat cells by the retrovirus, and finally stimulated with PMA/Iono. As shown in Figure 11A), after PMA/Iono stimulation, the expression of EGFP in Jurkat cells transduced with plasmid 417 increased from 0.28% to 2.1%, with a window of 7.5 times. Similarly, after PMA/Iono stimulation, the MFI of EGFP also increases, which shows that the regulatory structure designed in this embodiment is also applicable to retroviral vectors.

In addition, the present disclosure also constructs the structure of the enhancer response element on 410 and the CAR through the method of homologous recombination on the retroviral vector-plasmid 169, to obtain plasmid 440. Then the retrovirus was packaged with the cell line D4, and then the plasmid 440 was transduced into Jurkat cells by the retrovirus, and finally stimulated with PMA/Iono. As shown in Figure 11B), after stimulating with PMA/Iono transduced with 440, the expression of EGFP in Jurkat cells transduced with plasmid 440 increased from 0.01% to 3.3%, with a window of 330 times. Similarly, after PMA/Iono stimulation, the MFI of EGFP also increases, which shows that the regulatory structure designed in this embodiment can also regulate the expression of CAR on the retroviral vector.

In summary, the adjustable CAR that combines different enhancers and IL-2 TATA boxes designed in this embodiment is suitable for different tumor targets. Once CAR-T comes into contact with the antigen, it will be quickly activated to regulate the expression of its own CAR. The unregulated promoter EF1α, even after being activated by an antigen, still cannot regulate the expression of its own CAR. Such an adjustable structure can become another new way to treat tumors, including solid tumors.

Example 9 Killing effect of inducible CAR-T

In this embodiment, the ONE-Glo ^TM Luciferase Assay System is used to detect the tumor-killing effect of the adjustable CAR-T, and the specific method is as follows:

First, the plasmid 056 was transduced into K562 cells by lentivirus, and the CD20 expression of K562 cells was detected by flow cytometry 72 hours later. Then the cell suspension is prepared, that is, 100 K562/056 polyclonal cells are added to 10ml of medium containing 8μg/ml puromycin, and after mixing, they are inoculated in a 96-well plate with 100μl of medium per well. After 10-15 days, select monoclonal cells, expand the culture, and finally detect the expression of CD20 expressed by K562 cells by flow cytometry, and then K562/056 cell line can be obtained. Then the K562/056/099 cell line was constructed on the basis of the K562/056 cell line. Similarly, lentivirus was used to transduce plasmid 099 into K562/056 cell line. After 72 hours, the expression of EGFP on the cell surface was detected by flow cytometry. The method for preparing cell suspension is the same as the construction of K562/056 cell line. After 10-15 days, select monoclonal cells and expand the culture, and finally detect the expression of EGFP on the cell surface by flow cytometry. The K562/056/099 cell line can be obtained.

Inoculate 2×10 ⁴ effector cells (Effector), that is, PBMC cells expressing CAR in a 384-well plate, and then inoculate target cells expressing the corresponding antigen and Luciferase at the same time according to the experimental requirements. The total culture system is 80 μl, and the control group is no The CAR-expressing effector cells are incubated with the same target cells. Place the 384-well plate in a 37°C, 5% CO ₂ incubator for culture. After 24 hours, take out 30μl of supernatant from each well, then add 20μl ONE-Glo ^TM Assay buffer (configured according to the instructions) into the 384-well plate, leave it at room temperature for 3 minutes, and use a microplate reader to detect the reading of luciferase. Calculate the kill rate according to the following formula:

Killing rate (%) = ((control group-experimental group)/control group)×100%

First, construct K562/056/099 cells, a stable cell line expressing both human CD20 antigen and firefly luciferase in K562 cells, and then transduce plasmid 260 into PBMC by lentivirus, as a control, synchronously transduce了 Plasmid 245. Then the expression of CAR in PBMC was detected by flow cytometry. As shown in FIG. 12A, plasmid 245 and plasmid 260 were transduced into PBMC, and their CAR expression was 11% on the 15th and 18th days, and then the 15th and 18th day PBMC were used for the killing experiment. The CAR-expressing PBMC and the target cell K562/056/099 (Target) were incubated at a ratio of 1:1, 3:1, or 10:1. After 24 hours, the tumor killing was detected using Promega's Luciferase detection kit. As shown in Figure 12B, when the effector cell PBMC (Effector) and the target cell K562/056/099 are incubated at a ratio of 1:1, the PBMC transduced with plasmid 245 and plasmid 260 can both kill the tumor, with a killing ratio of 50 respectively. % And 62%. With the increase in the number of PBMC, the better the killing effect. When effector cell PBMC: target cell K562/056/099 = 10:1, the killing rate of PBMC transduced with plasmid 245 is 92.5%, while that of plasmid 260 is 99.5%. Plasmid 245 and plasmid 260 are CAR in PBMC The expression of both is 11%, but the killing effect of plasmid 260 is better than that of plasmid 245, which shows that the killing effect of the adjustable CAR with the NFAT-RENF-κB-RE-IL-2 TATA box structure is better than the traditional non-adjustable CAR is good.

Cytokine is a low molecular weight soluble protein produced by immunogens, mitogens or other factors that stimulate cells, such as interleukin 2 (IL-2) and interferon IFN-γ, which can regulate innate immunity and adaptability. Immune response, promotion of hematopoiesis, and stimulation of cell activation, proliferation and differentiation. IL-2 is a cytokine of the chemokine family, which is mainly ^{synthesized by T cells (especially CD4 +} T cells) after being stimulated by antigens or mitotic sources, and plays an important role in the body's immune response and anti-viral infection. IFN-γ belongs to type II interferon. It is an important immune regulatory factor in the body. It can promote the processing and presentation of MHC class I and II antigens. It can also increase the cytotoxic T cells by up-regulating the presentation of MHC class I antigens ( The sensitivity of Cytotoxic lymphocyte (CTL) to pathogens allows CTL to eliminate pathogens more effectively. In this example, the expression of IL2 and IFN-γ in the supernatants of plasmid 245 and plasmid 260 incubated with K562/056/099, respectively, was detected by enzyme-linked immunolabeling (ELISA), and the expression of these two cytokines reflected T cells The activation of CTL and the killing of tumor by CTL. As shown in Figure 13, after plasmid 245 and plasmid 260 were incubated with target cells K562/056/099, the expression of IL-2 and IFN-γ increased. Plasmid 245 was incubated with target cells, and the expression of IL-2 was 2.7-3.9 ng/ml, and the expression of IFN-γ was 2.3-6.8 ng/ml. The expression of the corresponding plasmid 260IL-2 is 5.9ng/ml-9ng/ml, and the expression of IFN-γ is 6.3-7.3ng/ml. After plasmid 260 and target cells were co-incubated, the expressions of IL-2 and IFN-γ were higher than those of plasmid 245. This indicates that the adjustable CAR designed in this example activates T cells and has higher tumor-killing activity than non-adjustable CAR.

In summary, the adjustable CAR designed in this example has a powerful tumor-killing effect, and compared to the CAR expression driven by the EF-1α promoter (that is, the CAR non-regulated promoter), this adjustable CAR CAR has better killing effect in PBMC.

Example 10 Regulatory effect of adjustable CAR in mouse spleen cells (SPL)

This example investigates the regulation of the combination of different enhancer response elements and promoters in mouse spleen cells (SPL). Firstly, plasmid 417 (containing the NFAF enhancer, whose nucleotide sequence is shown in SEQ ID NO: 15) and plasmid 440 (containing the NF-κB enhancer, whose nucleotide sequence is shown in SEQ ID NO: : 119), and the retrovirus was packaged by cell line 6#, and then the plasmids 417 and 440 were transduced into SPL by the retrovirus. Finally, PMA/Iono were used to stimulate the mouse spleen cells (SPL) transduced with these two plasmids respectively. It can be seen from Fig. 14 that when the SPL transduced with 417 and 440 is not stimulated by PMA/Iono, the expression percentage of EGFP is 0.3%, and after stimulation, the expression of EGFP both increases to 13.1% and 0.6%, respectively. Prove the regulatory effect of the adjustable CAR structure in mouse spleen cells (SPL).

Claims (10)

1. A tumor antigen-induced gene expression self-regulating nucleotide sequence, the sequence comprising:

   (1) Promoter sequence, and

   (2) Enhancer sequence,

   Preferably, the promoter is selected from an initiator, a TATA box and/or other core promoter elements, and the other core promoter elements are selected from the following elements: BREu (upstream TFIIB Recognition Element), MTE (Motif Ten Element) , DPE (Downstream Promoter Element), DCE (Downstream Core Element), XCPE1 (X Core Promoter Element 1), etc.;

   Preferably, the TATA box is an IL-2 TATA box;

   Preferably, the enhancer is selected from the following response elements: activated T cell nuclear factor response element (NFAT-RE), nuclear factor κB response element (NF-κB-RE), T cell factor/lymph enhancer factor response element (TCF- RE), Activin-1 Response Element (AP-1-RE), Hypoxia Inducible Factor Response Element (HRE), FoxO Transcription Factor Response Element (FoxO-RE);

   Preferably, the tumor antigen is a tumor-associated antigen on the surface of tumor cells.
2. The tumor antigen-induced gene expression self-regulating nucleotide sequence according to claim 1, wherein the enhancer is a single-copy enhancer, a multi-copy enhancer, or a combination of different enhancers;

   Preferably, the sequence and the following sequence have at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity: SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID: 128;

   Preferably, the nucleotide sequence of the single copy of the enhancer is selected from the following sequences: SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID: 128;

   Preferably, the multiple copies are repeated 2-20 times; especially preferably, the multiple copies are repeated 2-9 times or repeated 2-7 times; more preferably, the multiple copies are repeated 5-6 times. Copy

   Preferably, the nucleotide sequence of the enhancer multi-copy response element is selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14;

   Preferably, the reverse nucleotide sequence of the single copy of the enhancer is SEQ ID NO: 126 or SEQ ID NO: 127;

   Preferably, the reverse nucleotide sequence of the multiple copies of the enhancer is SEQ ID NO: 15 or SEQ ID NO: 119;

   Preferably, the nucleotide sequence of the combined response element of the different enhancers is selected from the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24;

   Preferably, the nucleotide sequence of the promoter is SEQ ID NO: 16.
3. A nucleic acid construct comprising the tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2 and a nucleotide sequence encoding a protein capable of specifically binding to tumor antigens; preferably, said Tumor antigens are tumor-associated antigens on the surface of tumor cells;

   Preferably, the protein that can specifically bind to tumor antigens is selected from the group consisting of single-chain antibodies (ScFv), receptors, ligands, protein scaffolds and/or other chimeric antigen receptors (CAR); preferably, the The protein scaffold is selected from Affibody, DARPin, Monobody, Anticalin4; preferably, the monomer is Centyrin; more preferably, the protein that can specifically bind to tumor antigens is a chimeric antigen receptor (CAR) ；

   Preferably, the chimeric antigen receptor (CAR) comprises a tumor-associated antigen binding domain, a transmembrane domain and a signal transduction domain; preferably, the amino acid sequence of the chimeric antigen receptor (CAR) is SEQ ID NO: 26 or SEQ ID NO: 28;

   Preferably, the nucleotide sequence encoding the chimeric antigen receptor (CAR) has at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity: SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO :98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106 , SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109;

   Preferably, the nucleotide sequence encoding the chimeric antigen receptor (CAR) is SEQ ID NO: 25 or SEQ ID NO: 27;

   Preferably, the nucleotide sequence encoding the chimeric antigen receptor (CAR) is selected from SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97 , SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109.
4. The nucleic acid construct of claim 3, which is a cosmid, a plasmid, a viral vector or a non-viral vector;

   Preferably, the viral vector is selected from a lentiviral vector, a retroviral vector, an adenovirus vector, and an adeno-associated virus vector;

   Preferably, the non-viral vector is selected from Sleeping Beauty plasmid transposition system, PiggyBac system or minicircle DNA;

   Preferably, the nucleic acid sequence of the nucleic acid construct is selected from: SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117; or, the nucleic acid sequence of the nucleic acid construct has at least 60%, 65%, 70%, 75%, 80%, 81%, 82% of the above-mentioned nucleotide sequence. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity.
5. The tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2 and/or the nucleic acid construct of claim 3 or 4 are used to prepare genetically modified immune cells against tumor-associated antigens.
6. An isolated host cell comprising the tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2 and/or the nucleic acid construct of claim 3 or 4; preferably, the host cell is Mammalian cells; more preferably, the host cells are PBMC, T cells, NK cells, NKT cells, macrophages or cell lines; preferably, the host cells are selected from HEK293, HEK293-T or Jurkat;

   Preferably, the host cell also expresses other sequences, and the other sequences include cytokine, another CAR, chemokine receptor, siRNA that reduces PD-1 expression, or protein that blocks PD-L1, TCR, or Safety switch;

   Preferably, the cytokine is selected from IL-12, IL-15, IL-21, or type I interferon;

   Preferably, the chemokine receptor is selected from CCR2, CCR5, CXCR3;

   Preferably, the safety switch is selected from iCaspase-9, Truncated EGFR.
7. A pharmaceutical composition comprising the tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2, the nucleic acid construct of claim 3 or 4, and/or the host cell of claim 6, And a pharmaceutically acceptable carrier.
8. The tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2, the nucleic acid construct of claim 3 or 4, the host cell of claim 6 and/or the nucleotide sequence of claim 7 The use of the pharmaceutical composition in the preparation of medicines;

   Preferably, the medicine is used to diagnose, treat or prevent one or more of cancer, inflammatory disease, and autoimmune disease;

   Preferably, the drug is used to diagnose, treat or prevent tumors. Preferably, the tumors are selected from blood cancers and solid tumors; more preferably, the blood cancers used for the diagnosis, treatment or prevention of the drugs are selected from leukemia, One or more of lymphoma and myeloma; more preferably, the solid tumor used by the drug for diagnosis, treatment or prevention is selected from lung cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, pancreas One or more of cancer, stomach cancer, bowel cancer, and prostate cancer.
9. A method for preparing CAR-T or TCR-T cells, the method comprising the following steps:

   (1) Construction of a nucleic acid construct expressing a self-inducible CAR or engineered TCR, the nucleic acid construct comprising the tumor antigen-induced gene expression self-regulating nucleotide sequence of claim 1 or 2 and a nucleotide sequence encoding a tumor antigen The nucleotide sequence of the protein that specifically binds; preferably, the nucleic acid construct is selected from a cosmid, a plasmid, a viral vector or a non-viral vector; preferably, the viral vector is selected from a lentiviral vector, a retroviral vector, Adenovirus vector, adeno-associated virus vector; preferably, the non-viral vector is selected from Sleeping Beauty plasmid transposition system, PiggyBac system or minicircle DNA; preferably, packaging cell line is used to package the virus;

   (2) Isolate peripheral blood mononuclear cells to obtain T lymphocytes;

   (3) Transducing the nucleic acid construct described in step (1) into the T lymphocytes described in step (2) to obtain T lymphocytes containing the construct; preferably; a viral vector or a non-viral vector Transduction into T lymphocytes;

   (4) Expand the culture of the T lymphocytes containing the construct obtained in step (3) to obtain CAR-T or TCR-T cells.
10. A method for treating a tumor-associated antigen-related disease in a subject, which comprises administering to the subject the nucleic acid construct according to claim 3 or 4, the host cell according to claim 6, and/or The pharmaceutical composition of claim 7; preferably, the subject is a mammal; more preferably, the subject is a human.

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