WO2021259334A1 - Récepteur d'antigène chimère auto-régulateur et son application dans l'immunité tumorale - Google Patents

Récepteur d'antigène chimère auto-régulateur et son application dans l'immunité tumorale Download PDF

Info

Publication number
WO2021259334A1
WO2021259334A1 PCT/CN2021/101900 CN2021101900W WO2021259334A1 WO 2021259334 A1 WO2021259334 A1 WO 2021259334A1 CN 2021101900 W CN2021101900 W CN 2021101900W WO 2021259334 A1 WO2021259334 A1 WO 2021259334A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cells
car
tumor
nucleotide sequence
Prior art date
Application number
PCT/CN2021/101900
Other languages
English (en)
Chinese (zh)
Inventor
周高潮
李凤霞
卫郦娜
鲁满
刘士超
Original Assignee
南京博望医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京博望医药科技有限公司 filed Critical 南京博望医药科技有限公司
Publication of WO2021259334A1 publication Critical patent/WO2021259334A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464424—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • 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.
  • 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.
  • TCR T cell receptor
  • tyrosine kinase is phosphorylated, thereby activating downstream signal transduction pathways.
  • T cells reorganize their cytoskeleton, changing their metabolism and gene expression.
  • TCR TCR-dependent MAPK pathway
  • AP-1 activator protein-1
  • AP-1 dimer 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.
  • TCR can transmit signals through the LAT-SPL76 complex, thereby activating PKC ⁇ .
  • the costimulatory factor CD28 can also signal through PI3K and PDK1 to activate PKC ⁇ .
  • Activated PKCO causes IKK activation. It further causes phosphorylation of I ⁇ B ⁇ , leading to ubiquitination and degradation of I ⁇ B ⁇ , which causes nuclear translocation of NF- ⁇ B and combines with NF- ⁇ B response elements (NF- ⁇ B RE) to cause gene transcription (Suman P, Brian CS. A new look) at TCR signaling to NF- ⁇ B.
  • Activated T cell nuclear factor 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.
  • T cell activation also includes Wnt pathway, Akt pathway and HIF1 ⁇ pathway.
  • Wnt pathway In the classic Wnt pathway, the activation of Wnt requires the participation of Porc (O-palmitoleoyl transferase, O-palmitoleoyl transferase).
  • Wnt binds to Frizzled on the cell membrane, it activates the scaffold protein in the cell matrix (Scaffold, which includes Dvl, GSK-3 ⁇ , axin, APC, ⁇ -catenin, etc.), and then activates the nuclear TCF/ LEF (T cell factor/lymphoid enhancer factor), regulates the transcription of downstream genes.
  • Scaffold which includes Dvl, GSK-3 ⁇ , axin, APC, ⁇ -catenin, etc.
  • TCF/ LEF T cell factor/lymphoid enhancer factor
  • 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 ⁇ -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).
  • HIF- ⁇ is hydroxylated by the factor of inhibiting HIF-1 (FIH) asparaginyl, thereby preventing HIF- ⁇ from binding to the co-activator protein p300/CBP.
  • FIH HIF-1
  • the activities of PHD and FIH are restricted by substrates, leading to rapid accumulation of HIF- ⁇ , nuclear translocation, and dimerization with HIF-1 ⁇ .
  • HIF-1 binds to the DNA consensus sequence (a hypoxia response element (HRE)) in the promoter of the target gene, transactivation occurs.
  • HRE hypoxia response element
  • TATA box 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.
  • TFS transcription initiation site
  • TFIID general transcription factor
  • the SV40 early region TATA box is required for accurate in vitro initiation of Nature.
  • 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).
  • 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.
  • the TATA box mainly initiates the expression of tissue-specific genes such as IL-2 in T cells.
  • 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.
  • RE Response element
  • 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- ⁇ enhancer is a typical model for studying gene transcription regulation. It is located upstream of the transcription start site of IFN- ⁇ 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.
  • CBP p300/CREB binding protein
  • IFN- ⁇ 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- ⁇ B can be combined with positive regulatory region II (PRDII), and ATF-2/c-Jun can be combined with positive regulatory region IV (PRDIV).
  • PRDs Postive regulatory domains, PRDs
  • IRF interferon regulatory factor
  • PRDI positive regulatory domain I
  • PRDIII positive regulatory domain III
  • NF- ⁇ B can be combined with positive regulatory region II (PRDII)
  • 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- ⁇ Enhance
  • Chimeric antigen receptor modified T cell 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.
  • 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.
  • TM transmembrane domain
  • 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) CD3 ⁇ and costimulatory signal molecules CD28, CD137 (4-1BB), CD134, etc. Inner signal area.
  • the intracellular signal region containing only CD3 ⁇ 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-CD3 ⁇ .
  • 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).
  • 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- ⁇ 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.
  • 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 TCR ⁇ / ⁇ 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, TCR ⁇ / ⁇ heterodimer affinity modification, construction of TCR ⁇ / ⁇ 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.
  • CAR-T cells in order to obtain higher anti-tumor activity, the expansion and maintenance of CAR-T cells in the body are two very important factors.
  • CAR-T cells 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).
  • 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).
  • CRISPR/Cas9 technology to locate CAR on the TCR ⁇ 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 ⁇ -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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides a method for preparing CAR-T or TCR-T cells.
  • 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.
  • a chimeric antigen receptor (CAR) nucleic acid construct e.g., a chimeric antigen receptor (CAR) nucleic acid construct, virus, or host cell, and / Or pharmaceutical composition.
  • CAR chimeric antigen receptor
  • the gene expression control sequence formed by the combination of the enhancer of the present disclosure (such as NFAT-RE, NF- ⁇ B-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.
  • CAR-T or TCR-T 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-1 ⁇ 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any macromolecule including virtually all proteins or polypeptides, can be used as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • 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.
  • 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.
  • 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.
  • brain cancer such as astrocytoma, meningioma, oligodendroglioma, glioma, etc.
  • pancreatic cancer ovarian cancer
  • kidney cancer bladder cancer
  • pancreatic cancer Stomach cancer
  • bowel cancer bowel cancer
  • head and neck cancer thyroid cancer
  • prostate cancer Kaposi's sarcoma
  • inflammatory disease examples 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.
  • COPD chronic obstructive pulmonary disease
  • 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.
  • 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.
  • expression is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.
  • 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).
  • homologous refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules.
  • 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.
  • 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.
  • 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.
  • the patient, subject, or individual is a human.
  • 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.
  • peptide 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.
  • 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.
  • 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.
  • promoter/regulatory sequence refers to a nucleic acid sequence required for the expression of a gene product operably linked to the promoter/regulatory sequence.
  • 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.
  • 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.
  • 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.
  • immunospecifically 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 antigen may be an isolated antigen or present in tumor cells.
  • 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).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • 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.
  • the expression percentage of fluorescent protein (such as EGFP) or MFI is the viral transduction background value.
  • 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.
  • the term "therapeutic" means treatment and/or prevention.
  • the therapeutic effect is obtained through the suppression, alleviation or eradication of the disease state.
  • treating refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
  • 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.
  • 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.
  • an enhancer response element is provided herein, the enhancer response element is selected from NFAT-RE, NF- ⁇ B-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.
  • the present disclosure provides a tumor antigen-induced gene expression self-regulating nucleotide sequence, the sequence comprising:
  • 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.
  • the TATA box is an IL-2 TATA box.
  • the enhancer is selected from the following response elements: activated T cell nuclear factor response element (NFAT-RE), nuclear factor ⁇ B response element (NF- ⁇ B) -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).
  • NFAT-RE activated T cell nuclear factor response element
  • NF- ⁇ B response element nuclear factor ⁇ B response element
  • TCF-RE T-cytokine/lymph enhancement factor response element
  • AP-1-RE activator protein-1 response element
  • HRE hypoxia-inducible factor response element
  • FoxO transcription factor response element FoxO transcription factor response element
  • the tumor antigen is a tumor-associated antigen on the surface of tumor cells.
  • the enhancer is a single-copy enhancer, a multi-copy enhancer, or a combination of different enhancers.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 );
  • 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).
  • the chimeric antigen receptor 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.
  • 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.
  • 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
  • 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.
  • the viral vector is selected from a lentiviral vector, a retroviral vector, an adenovirus vector, and an adeno-associated virus vector.
  • the non-viral vector is selected from Sleeping Beauty plasmid transposition system, PiggyBac system or minicircle DNA.
  • 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.
  • 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.
  • 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 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.
  • the cytokine is selected from IL-12, IL-15, IL-21, or type I interferon.
  • the chemokine receptor is selected from CCR2, CCR5, and CXCR3.
  • the safety switch is selected from iCaspase-9, Truncated EGF.
  • the present disclosure provides a pharmaceutical composition
  • 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.
  • 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.
  • the drug is used to diagnose, treat or prevent one or more of cancer, inflammatory disease, and autoimmune disease.
  • the drug is used for the diagnosis, treatment or prevention of tumors
  • 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.
  • the present disclosure provides a method for preparing CAR-T or TCR-T cells, the method comprising the following steps:
  • 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;
  • step (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;
  • 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.
  • 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).
  • PCR primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.
  • Plasmid 112 was constructed by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • 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
  • Trans5 ⁇ chemically competent cells Trans5 ⁇ Chemically competitive cell, Cat#:CD201
  • Homologous recombination enzyme Assembly Mix (Cat#: RN1020) was purchased from Suzhou Hongxun Biotechnology Co., Ltd.
  • Tryptone TRYPTONE, Cat#: LP0042
  • yeast extract YEAST EXTRACT, Cat#: LP0021
  • Agar Agar was purchased from BIOSHARP.
  • 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.
  • Penicillin-Streptomycin 100 ⁇ solution (Penicillin-Streptomycin 100 ⁇ solution, Cat#: SV30010) was purchased from Hyclone.
  • 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.
  • CytoTell TM Blue (Cat#: 22251) was purchased from AAT Bioquest.
  • Antibiotic puromycin (puromycin, Cat#: ant-pr-5) and blasticidin (Blasticidin, Cat#: ant-bl-05) were purchased from InvivoGen.
  • Ficoll-Paque Plus (endotoxin tested ⁇ 0.12EU/ml, Cat#:17-1440-02) was purchased from GE Healthcare.
  • Red blood cell lysate (Cat#: C3702) was purchased from Beyotime.
  • the medium in the 6-well plate contains 10% FBS, 100U/ml penicillin, 100 ⁇ g/ml streptomycin+25uM chloroquine
  • the medium contains 10% FBS, 100U/ml penicillin, 100 ⁇ g/ml streptomycin+25uM chloroquine
  • 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.
  • 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 ⁇ m 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.
  • 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.
  • virus packaging mix 200 ⁇ l 150mM NaCl, 2.5 ⁇ g or 2 ⁇ g target plasmid, 0.5 ⁇ g packaging plasmid VSVG or 1 ⁇ g packaging plasmid pCL-Eco (Addgen 12371) and 21 ⁇ l PEI into a transfection solution, vortex and shake for 8 seconds, and let stand at room temperature 10min.
  • the medium in the 6-well plate contains 10% FBS, 100U/ml penicillin, 100 ⁇ g/ml streptomycin+25uM chloroquine
  • the medium contains 10% FBS, 100U/ml penicillin, 100 ⁇ g/ml streptomycin+25uM chloroquine
  • 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.
  • the method is the same as the lentivirus concentration step (2).
  • transduction Jurkat and virus titer is the same as that of lentivirus.
  • MFI Median Fluorescence Intensity
  • the steps for preparing hPBMC are as follows:
  • Day 0 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+100 ⁇ g/ml streptomycin+10mM HEPES), and centrifuge at 500g for 5min. After removing the supernatant, resuspend the cells in 3ml medium and take 10 ⁇ l 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 ⁇ l.
  • pre-warmed complete medium 90%RPMI 1640+10%FBS+100U/ml penicillin+100 ⁇ g/ml streptomycin+10mM HEPES
  • the Dynabeads need to be washed once in accordance with the steps in the product instructions before use.
  • 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 Gently pipette 110 ⁇ l of supernatant (do not touch the cells), and discard. Take another 500 ⁇ l 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 Take a 500 ⁇ l 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).
  • Day 3 Change the medium: remove 100 ⁇ l of supernatant (do not touch the cells), and then add 100 ⁇ l of complete medium.
  • Day 4 Transfer the cells from the 96-well plate to the 24-well plate for culture, and add 250 ⁇ l of complete medium.
  • Day 5 Change the medium: remove 220 ⁇ l of supernatant, and then add 650 ⁇ l of complete medium.
  • Day 6 Transfer the cells in 24 wells once to two, and add 500 ⁇ l of complete medium to each well.
  • Day 7 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.
  • Day 0 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.
  • 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
  • Day 1 Gently pipette 600 ⁇ l of cell supernatant into a 1.5ml EP tube, centrifuge at 500g for 5min. After removing the supernatant, add 100 ⁇ l of complete medium to resuspend the cells, and then add virus, 2 ⁇ g/ml Lipo2000 (ThermoFisher), 1.6 ⁇ g/ml Polybrene. Lipo2000 and Polybrene are calculated based on a total volume of 500 ⁇ l. After mixing, add dropwise to a 24-well plate, centrifuge at 930g at 37°C for 1.5h. After centrifugation, add 500 ⁇ l of complete medium, 37°C, 5% CO 2 incubator to continue culturing.
  • D1 Gently pipette 600 ⁇ l of cell supernatant into a 1.5ml EP tube, centrifuge at 500g for 5min. After removing the supernatant, add 100 ⁇ l of complete medium to resuspend the cells, and then add virus, 2 ⁇ g/ml Lipo2000 (
  • Day 2 (D2): Remove 500 ⁇ l of cell supernatant, taking care not to touch the cells. After gently mixing the cells, take 10 ⁇ l 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.
  • 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.
  • 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.
  • 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- ⁇ B-RE (NF- ⁇ B pathway), NAFT-RE (calcium pathway), FoxO-RE (PKB/Akt pathway), TCF-RE (Wnt Pathway), HRE (HIF1a pathway).
  • the nucleotide sequence of a single copy of NF- ⁇ B-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- ⁇ B 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.
  • 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 EF1 ⁇ enhancer, EF1 ⁇ promoter, CAR, murine IL-7 and EGFP (EF1 ⁇ enhancer-EF1 ⁇ promoter-CAR-F2A-IL-7-F2A-EGFP), and its main structure is shown in Figure 1C As shown, plasmid 245 is involved.
  • 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.
  • the enhancers NFAT-RE (SEQ ID NO: 1), NF- ⁇ B-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- ⁇ enhancesome-RE SEQ ID NO: 21, 22, 23 or 24
  • the enhancer was homologously recombined into the plasmid 112ClaI and EcoRV restriction sites.
  • the molecular cloning techniques involved in this disclosure are all conventional techniques of molecular biology.
  • 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 protein kinase C
  • PKC can activate the phosphorylation of many downstream protein kinases to form a cascade reaction and induce the expression of many proteins through NFAT.
  • DAG diacylglycerol
  • Ca 2+ 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.
  • 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.
  • This example first selected some enhancers related to T cell activation, such as AP-1-RE (MAPK pathway), NF- ⁇ B-RE (NF- ⁇ B pathway), NFAT-RE (Ca + pathway), TCF- RE (Wnt pathway), FoxO-RE (Akt pathway), HRE (HIF1 ⁇ 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.
  • enhancers related to T cell activation such as AP-1-RE (MAPK pathway), NF- ⁇ B-RE (NF- ⁇ B pathway), NFAT-RE (Ca + pathway), TCF- RE (Wnt pathway), FoxO-RE (Akt pathway), HRE (HIF1 ⁇ pathway)
  • 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).
  • this example also combined different REs to obtain the following plasmid: 260 (including enhancer NFAT-RE ⁇ NF- ⁇ B-RE, and its nucleotide sequence is as SEQ ID NO: 17), 261 (including the enhancer NF- ⁇ B-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 - ⁇ enhancesome(1)-RE, whose nucleotide sequence is shown in SEQ ID NO: 21) and plasmid 446 (including enhancer IFN- ⁇ enhancesome(2)-RE, whose nucleotide sequence is shown in SEQ ID NO: 22)
  • the response element is a highly conserved DNA sequence with independent or multiple copies of enhancers.
  • This example also uses enhancers NF- ⁇ B-RE, HRE and AP-1-RE as examples to discuss different copy numbers.
  • the regulatory role of the enhancer is a highly conserved DNA sequence with independent or multiple copies of enhancers.
  • NF- ⁇ B-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- ⁇ B-REs, 6 With 9 HRE repeats and 6 AP-1-RE repeats, and through homologous recombination, the following plasmids were obtained: 410 (6 NF- ⁇ B-RE, SEQ ID NO: 9), 407 (7 NF- ⁇ B-RE, SEQ ID NO: 10), 437 (11 NF- ⁇ B-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).
  • 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.
  • 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.
  • 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).
  • 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).
  • this example also uses plasmid 260 as the vector backbone, replacing CD20scFv with CD19scFv.
  • plasmid 026 containing CD19 CAR as a template, design primers, and obtain CD19scFv by PCR.
  • 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.
  • PBMC Human peripheral blood mononuclear cells
  • T cells and B cells lymphocytes
  • monocytes monocytes
  • phagocytes dendritic cells
  • 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 researchers in the fields of immunology, antibody drug development, infectious diseases, hematological malignancies, vaccine development, and transplantation immunity.
  • CAR-T therapy the final prepared CAR is also transduced into human PBMC through viruses and other methods.
  • 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.
  • PBMC transduced with plasmid 112 when not stimulated, only 1.3% of the cells in the control group expressed EGFP.
  • the expression of EGFP was as high as 62.6%, with a window of 48 times .
  • CD3/CD28 Dynabeads activating TCR/costimulatory factor CD28
  • PHA-P and Con A promoting mitosis
  • 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 20 ⁇ g/ml PHA-P and 5 ⁇ g/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 20 ⁇ g/ml and 5 ⁇ g/ml, the viability of PBMC cells decreased.
  • CD3/CD28 Dynabeads, PHA-P, Con A and PMA/Iono all activate T cells.
  • the enhancer NFAT and IL-2 TATA box can regulate the transcription and expression of the downstream gene EGFP.
  • 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.
  • 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 ⁇ l, a total of 6 wells, respectively labeled AF.
  • Add 1 ⁇ l DMSO to well A and add 10ng/ml PMA+1 ⁇ M ionomycin (total volume 1 ⁇ l) to the remaining wells.
  • the supernatant was removed, and the cells were resuspended in 200 ⁇ l 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.
  • plasmid 260 NFAT-RE ⁇ NF- ⁇ B-RE
  • plasmid 261 NF- ⁇ B-RE ⁇ NFAT-RE
  • 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.
  • the same method is used to test the regulation effect of the combination of other enhancers and IL-2 TATA box in PBMC.
  • the plasmids 228 NFAT-RE
  • 259 NF- ⁇ B-RE
  • 281 HRE ⁇ NFAT-RE
  • 294 AP-1-RE
  • 407 NF- ⁇ B-RE ⁇ 7
  • 410 NF- ⁇ B-RE ⁇ 6
  • 437 11 NF- ⁇ B-RE
  • 438-13 9 HRE
  • 438-18 6 HRE
  • 448 IFN- ⁇ enhancesome(2)
  • 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
  • the adjustment window of plasmid 448 is 7.6 times.
  • the regulatory windows of plasmids 259 and 407 are both close to 2 times
  • the regulatory windows of plasmids 437, 438-13 and 438-18 are the smallest.
  • 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.
  • 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.
  • dyes are used to stain CAR-T cells.
  • 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.
  • 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.
  • 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- ⁇ B-RE and IL-2 TATA box The combination.
  • Plasmid 245 was transduced into PBMC through lentivirus simultaneously to obtain CAR-T cells transduced with plasmid 245.
  • Plasmid 245 contains a constitutive promoter EF1 ⁇ that regulates CAR expression. This constitutive promoter EF1 ⁇
  • 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.
  • the 260 T cells transduced with CytoTell TM Blue were respectively stained and incubated with Raji B cells at a ratio of 1:1.
  • 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.
  • this method compare the stimulation effects of antigen stimulation and the other two in vitro stimulation methods (CD3/CD28 and 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.
  • 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- ⁇ B-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.
  • 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.
  • 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- ⁇ B-RE and IL-2 TATA box to explore the antigen induction of CD19 CAR.
  • NFAT-RE SEQ ID NO: 15
  • IL-2 TATA box was constructed on the retroviral vector-plasmid 169, and plasmid 417 was obtained.
  • 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.
  • 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.
  • 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.
  • the expression of EGFP in Jurkat cells transduced with plasmid 440 increased from 0.01% to 3.3%, with a window of 330 times.
  • 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.
  • 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.
  • Such an adjustable structure can become another new way to treat tumors, including solid tumors.
  • 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:
  • 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 8 ⁇ g/ml puromycin, and after mixing, they are inoculated in a 96-well plate with 100 ⁇ l 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.
  • 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 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 ⁇ l, 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.
  • K562/056/099 cells 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 ⁇ 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 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.
  • 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- ⁇ B-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- ⁇ , 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- ⁇ belongs to type II interferon. It is an important immune regulatory factor in the body.
  • CTL Cytotoxic lymphocyte
  • the adjustable CAR designed in this example has a powerful tumor-killing effect, and compared to the CAR expression driven by the EF-1 ⁇ promoter (that is, the CAR non-regulated promoter), this adjustable CAR CAR has better killing effect in PBMC.
  • This example investigates the regulation of the combination of different enhancer response elements and promoters in mouse spleen cells (SPL).
  • SPL mouse spleen cells
  • plasmid 417 containing the NFAF enhancer, whose nucleotide sequence is shown in SEQ ID NO: 15
  • plasmid 440 containing the NF- ⁇ B enhancer, whose nucleotide sequence is shown in SEQ ID NO: : 119
  • the retrovirus was packaged by cell line 6#, and then the plasmids 417 and 440 were transduced into SPL by the retrovirus.
  • PMA/Iono were used to stimulate the mouse spleen cells (SPL) transduced with these two plasmids respectively. It can be seen from Fig.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Récepteur d'antigène chimère (CAR) auto-régulateur et application de celui-ci dans l'immunité tumorale, comprenant une construction d'acide nucléique auto-régulatrice à expression génique induite par un antigène tumoral. Une séquence régulatrice d'expression génique peut induire l'expression d'un CAR ou d'un TCR modifié, et est appropriée pour différentes cibles tumorales. Lorsque qu'un CAR-T ou TCR-T est en contact avec une tumeur, un antigène de surface tumorale est lié à scFv, un récepteur, un ligand, un échafaudage protéique, ou le TCR modifié sur une surface de CAR-T, de telle sorte que les cellules CAR-T ou TCR-T sont spécifiquement activées, et l'expression de plusieurs cellules CAR-T ou CAR ou TCR modifié sur une surface TCR-T est favorisée, ce qui permet de renforcer l'amélioration de la capacité d'expansion des cellules CAR-T ou TCR-T et leur fonctionnement comme cellules effectrices. L'approche d'activation locale de CAR-T ou de TCR-T au moyen d'une tumeur permet de réduire la libération systémique de cytokine indépendante d'un antigène de surface tumorale, ce qui permet de réduire les effets toxiques et secondaires de CAR-T ou TCR-T.
PCT/CN2021/101900 2020-06-24 2021-06-23 Récepteur d'antigène chimère auto-régulateur et son application dans l'immunité tumorale WO2021259334A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010592743 2020-06-24
CN202010592743.8 2020-06-24

Publications (1)

Publication Number Publication Date
WO2021259334A1 true WO2021259334A1 (fr) 2021-12-30

Family

ID=79282006

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/101900 WO2021259334A1 (fr) 2020-06-24 2021-06-23 Récepteur d'antigène chimère auto-régulateur et son application dans l'immunité tumorale

Country Status (1)

Country Link
WO (1) WO2021259334A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111315402A (zh) * 2017-08-15 2020-06-19 艾达普特免疫有限公司 T细胞修饰
WO2023010038A1 (fr) * 2021-07-29 2023-02-02 Seattle Children's Hospital (dba Seattle Children's Research Institute) Éléments d'acide nucléique de synthèse pour améliorer l'efficacité des lymphocytes de type car-t

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040214757A1 (en) * 1986-01-09 2004-10-28 David Baltimore Nuclear factors associated with transcriptional regulation
CN110913871A (zh) * 2017-05-17 2020-03-24 西雅图儿童医院（Dba西雅图儿童研究所） 生成哺乳动物t细胞活化诱导型合成启动子(syn+pro)以改善t细胞疗法
WO2020118634A1 (fr) * 2018-12-13 2020-06-18 科济生物医药（上海）有限公司 Cellules effectrices immunitaires ciblant gpc3 et leur utilisation
CN111315402A (zh) * 2017-08-15 2020-06-19 艾达普特免疫有限公司 T细胞修饰

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040214757A1 (en) * 1986-01-09 2004-10-28 David Baltimore Nuclear factors associated with transcriptional regulation
CN110913871A (zh) * 2017-05-17 2020-03-24 西雅图儿童医院（Dba西雅图儿童研究所） 生成哺乳动物t细胞活化诱导型合成启动子(syn+pro)以改善t细胞疗法
CN111315402A (zh) * 2017-08-15 2020-06-19 艾达普特免疫有限公司 T细胞修饰
WO2020118634A1 (fr) * 2018-12-13 2020-06-18 科济生物医药（上海）有限公司 Cellules effectrices immunitaires ciblant gpc3 et leur utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KATHARINA ZIMMERMANN, JOHANNES KUEHLE, ANNA CHRISTINA DRAGON, MELANIE GALLA, CHRISTINA KLOTH, LOREEN SOPHIE RUDEK, I. EROL SANDALC: "Design and Characterization of an "All-in-One" Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines", CANCERS, vol. 12, no. 2, pages 375, XP055727985, DOI: 10.3390/cancers12020375 *
KULEMZIN SERGEY V., MATVIENKO DARIA A., SABIROV ARTUR H., SOKRATYAN ARPINE M., CHERNIKOVA DARIA S., BELOVEZHETS TATYANA N., CHIKAE: "Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-cell lines", BMC MEDICAL GENOMICS, vol. 12, no. S2, 1 March 2019 (2019-03-01), pages 20 - 25, XP055833898, DOI: 10.1186/s12920-019-0489-4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111315402A (zh) * 2017-08-15 2020-06-19 艾达普特免疫有限公司 T细胞修饰
CN111315402B (zh) * 2017-08-15 2024-05-07 艾达普特免疫有限公司 T细胞修饰
WO2023010038A1 (fr) * 2021-07-29 2023-02-02 Seattle Children's Hospital (dba Seattle Children's Research Institute) Éléments d'acide nucléique de synthèse pour améliorer l'efficacité des lymphocytes de type car-t

Similar Documents

Publication Publication Date Title
JP7008350B2 (ja) Ｃａｒ発現ベクター及びｃａｒ発現ｔ細胞
AU2016238963B2 (en) Method and compositions for cellular immunotherapy
RU2716716C2 (ru) Композиции и способы для бустинга эффективности адоптивной клеточной иммунотерапии
JP6971986B2 (ja) 免疫療法の抗腫瘍活性を高めるための間葉系幹細胞
JP2021516256A (ja) Ｃｄ１９に基づくキメラ抗原受容体及びその利用
US20210155702A1 (en) Antibody-modified chimeric antigen receptor modified t cell and uses thereof
WO2022007804A1 (fr) Lymphocyte t et son utilisation
TW202016295A (zh) Car表現t細胞及car表現載體
JP7475088B2 (ja) ヒトメソセリンを特異的に認識する細胞表面分子、ｉｌ－７、及びｃｃｌ１９を発現する免疫担当細胞
WO2022007795A1 (fr) Récepteur antigénique chimérique et son utilisation
CN112204133B (zh) Car nk细胞
JP2021525101A (ja) 改変された抗ｃｄ１９ ｃａｒ−ｔ細胞
WO2021259334A1 (fr) Récepteur d'antigène chimère auto-régulateur et son application dans l'immunité tumorale
US20230000918A1 (en) Chimeric antigen receptors, compositions and applications thereof
WO2021136538A1 (fr) Cellule cart améliorée favorisant l'infiltration dans des tumeurs solides, procédé de préparation associé et médicament cellulaire correspondant
JP2022522786A (ja) 構成的に活性なキメラサイトカイン受容体
JP2021517450A (ja) Ｓｓｅａ４に結合するキメラ抗原受容体及びその使用
JP2023524873A (ja) 方法
JP2021509290A (ja) 双方向活性化共刺激分子受容体及びその用途
CN111378046B (zh) 一种免疫效应细胞转换受体
CN111983218A (zh) 一种用于检测活细胞-活细胞表面受体-配体相互作用的试剂盒
US11359012B1 (en) Specific chimeric antigen receptor cells targeting human CLDN18A2, preparation method and application thereof
WO2021093818A1 (fr) Dimère de protéine tmem59 ou récepteur d'expression chimérique améliorant la fonction des lymphocytes t
US11364267B1 (en) Bi-specific targeting human NKG2DL and CLDN18A2 chimeric antigen receptor cells, preparation method and application thereof
WO2024066026A1 (fr) RÉCEPTEUR ANTIGÉNIQUE CHIMÉRIQUE OPTIMISÉ CIBLANT IL13Rα2 ET SON UTILISATION

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21828756

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21828756

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 21828756

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 04.07.2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21828756

Country of ref document: EP

Kind code of ref document: A1