Recombinant promoter system for specifically inducing gene expression after T cell activation

Abstract

The invention provides a recombinant promoter system for specifically inducing gene expression after T cell activation. The invention provides a novel T cell activation inducible recombinant promoter constructed by modifying and optimizing GM-CSF, IFNg and IL2 promoters, when CAR-T cells are activated, expression of downstream genes of the CAR-T cells can be efficiently induced, and the activity of the novel T cell activation inducible recombinant promoter is superior to that of a traditional NFAT response type promoter constructed on the basis of a T cell activation nuclear factor binding sequence. The invention also provides a CAR-T cell containing the T cell activation inducible recombinant promoter and application of the CAR-T cell.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and in particular to a recombinant promoter system for specifically inducing gene expression after T cell activation. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) immunotherapy is an adoptive T cell therapy that uses genetic engineering technology to make T cells express receptors and co-stimulatory signal molecules that can specifically recognize antigens, then amplify them in vitro and then infuse them back into the patient's body, so that they do not rely on major histocompatibility complex presentation to recognize tumor-associated antigens and kill tumor cells. In recent years, CAR-T cell therapy has made great achievements in the treatment of hematological tumors. Currently, many CAR-T therapies have been approved for the treatment of hematological tumors. Among them, CAR-T cell therapy targeting CD19 can achieve a remission rate of more than 90% in refractory and relapsed acute lymphoblastic leukemia; CAR-T cell therapy targeting BCMA has an overall remission rate (ORR) of up to 96% in the treatment of multiple myeloma, providing hope for the use of CAR-T therapy to cure tumors.

[0003] Although CAR-T therapy has achieved great success in the clinical treatment of hematological tumors, its clinical treatment effect in solid tumors is very limited and still faces many difficulties and challenges: antigen heterogeneity is common in solid tumors, leading to antigen escape; CAR-T cells cannot effectively infiltrate solid tumor tissues; CAR-T cells have poor persistence and are easily exhausted; the immunosuppressive microenvironment in solid tumors inhibits CAR-T function; and off-target effects cause strong toxic side effects. Therefore, many strategies have been tried to transform and optimize CAR-T to have optimal activity and low toxicity to enhance its efficacy in solid tumors.

[0004] In addition to optimizing the chimeric antigen receptor itself, a more effective strategy is to allow CAR-T to express relevant functional proteins and enhance its killing ability by "arming" CAR-T. For example, making it express immunoregulatory factors (such as cytokines, immune checkpoint inhibitory molecules, bacterial virulence factor proteins, etc.) can effectively improve the persistence of CAR-T and activate the immune system. T cells that overexpress γ-chain cytokines (such as IL2, IL7, IL15 and IL21) or inflammatory cytokines (such as IL12, IL18, and IL23) after engineering modification show better anti-tumor activity. However, the continuous expression of immunoregulatory factors may cause serious toxic side effects, thus preventing its advantages from being brought into play. For example, CAR-T overexpressing IL2 or IL18 was found to cause serious toxic side effects in preclinical studies, limiting its clinical translational application. Therefore, strictly controlling the expression of these load proteins and making them secreted only in tumor lesions can maximize the functional advantages of load proteins and reduce their effects on normal tissues at the same time.

[0005] Therefore, there is a need in the art to develop methods for enabling T cells to express immunomodulatory factors with high efficiency and high specificity. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant promoter expression system for specifically inducing gene expression after T cell activation.

[0007] Another object of the present invention is to provide T cells containing the recombinant promoter expression system of the present invention and applications thereof.

[0008] In a first aspect of the present invention, a genetically modified promoter element is provided, wherein the promoter element is a T cell activation-inducible promoter, and the promoter element has a fragment derived from a promoter selected from the group consisting of a GM-CSF promoter, an IFNg promoter, an IL2 promoter, or a combination thereof.

[0009] In another preferred embodiment, the T cell activation-inducible promoter means that the target gene regulated by the promoter is not expressed or expressed at a low level when the T cells are not activated, and is highly expressed when the T cells are activated.

[0010] In another preferred embodiment, the promoter element is induced by a T cell activation pathway selected from the group consisting of:

[0011] a) Induction by ligand binding to the T cell receptor (TCR);

[0012] b) induced by ligand binding of chimeric antigen receptor (CAR);

[0013] c) Activation through antibodies against immune cell surface markers (including anti-CD3 and anti-CD28);

[0014] d) Activation by compound activators (such as PMA, inomycin).

[0015] In another preferred embodiment, under the condition of T cell activation, the transcription efficiency of the promoter element is increased by ≥50%, preferably ≥100%, and more preferably ≥150% compared with the nuclear factor of activation of T cells (NFAT) promoter.

[0016] In another preferred embodiment, the promoter element has a structure as shown in the following formula (I):

[0017] Z0-Z1-Z2(I)

[0018] In the formula, “-” is a bond or a nucleotide linking sequence;

[0019] Z0 is none, a fragment derived from a minimal promoter, or a fragment derived from another promoter;

[0020] Z1 is a fragment derived from a promoter selected from the group consisting of GM-CSF promoter, IFNg promoter, IL2 promoter, or a combination thereof;

[0021] Z2 is none, a fragment derived from a minimal promoter, or a fragment derived from another promoter.

[0022] In another preferred embodiment, Z0 is nothing; and Z2 is a fragment derived from a minimal promoter.

[0023] In another preferred embodiment, Z2 is nothing; and Z0 is a fragment derived from a minimal promoter.

[0024] In another preferred embodiment, the Z1 is a GM-CSF promoter fragment.

[0025] In another preferred embodiment, the nucleotide sequence length of the fragment is 50-800 bp, preferably 150-700 bp, and more preferably 200-400 bp.

[0026] In another preferred embodiment, the Z1 is derived from the nucleic acid segment from position p1 to position q1 of the human GM-CSF promoter, wherein

[0027] p1 is any integer from -800 to -100, preferably from -400 to -150;

[0028] q1 is any integer from -50 to 50, preferably from -40 to +40, and more preferably from -36 to +36.

[0029] In another preferred embodiment, the Z1 is a nucleic acid segment from position -626 (preferably position -336) to position +36 (preferably position +35) of the human GM-CSF promoter, or a truncated fragment thereof.

[0030] In another preferred embodiment, the Z1 comprises the nucleic acid segment from -217 to -1 of the GM-CSF promoter.

[0031] In another preferred embodiment, the promoter element comprises:

[0032] (a) a nucleotide sequence as shown in any one of SEQ ID NOs: 11-17;

[0033] (b) a nucleotide sequence having a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the sequence shown in any one of SEQ ID NOs: 11-17, and having substantially the function of the polynucleotide shown in any one of SEQ ID NOs: 11-17; or

[0034] (c) A polynucleotide having one or more nucleotides, preferably 1-30, more preferably 1-6 nucleotides, truncated at the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs: 11-17, and having substantially the function of the polynucleotide shown in any one of SEQ ID NOs: 11-17.

[0035] In another preferred embodiment, the Z1 is an IL-2 promoter fragment.

[0036] In another preferred embodiment, the nucleotide sequence length of the fragment is 200-600 bp, preferably 300-500 bp, and more preferably 350-400 bp.

[0037] In another preferred embodiment, the Z1 is derived from the nucleic acid segment from position p2 to position q2 of the human IL-2 promoter, wherein

[0038] p2 is any integer from -500 to -200, preferably from -400 to -300;

[0039] q2 is any integer from 0 to +100, preferably from +30 to +70, and more preferably from +40 to +60.

[0040] In another preferred embodiment, the Z1 comprises the nucleic acid segment from -326 to +47 of the IL-2 promoter.

[0041] In another preferred embodiment, the promoter element comprises:

[0042] (a) the nucleotide sequence shown in SEQ ID NO: 18;

[0043] (b) a nucleotide sequence having a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the sequence shown in SEQ ID NO: 18, and having substantially the function of the polynucleotide shown in SEQ ID NO: 18; or

[0044] (c) a polynucleotide having one or more nucleotides, preferably 1-30, more preferably 1-6 nucleotides, truncated at the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO: 18, and having substantially the function of the polynucleotide shown in SEQ ID NO: 18.

[0045] In another preferred embodiment, the Z1 is an IFNg promoter fragment.

[0046] In another preferred embodiment, the nucleotide sequence length of the fragment is 100-1000 bp, preferably 300-800 bp, and more preferably 500-700 bp.

[0047] In another preferred embodiment, the Z1 is derived from the nucleic acid segment from position p3 to position q3 of the IFNg promoter, wherein

[0048] p3 is any integer from -800 to -100, preferably from -600 to -400;

[0049] q3 is any integer from +20 to +120, preferably from +40 to +80, and more preferably from +50 to +70.

[0050] In another preferred embodiment, the Z1 is the nucleic acid segment from -538 to +64 of the IFNg promoter, or a truncated fragment thereof.

[0051] In another preferred embodiment, the Z1 comprises the nucleic acid segment from -108 to +64 of the IFNg promoter.

[0052] In another preferred embodiment, the promoter element comprises:

[0053] (a) the nucleotide sequence shown in SEQ ID NO: 19 or 20;

[0054] (b) a nucleotide sequence having a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the sequence shown in SEQ ID NO: 19 or 20, and having substantially the function of the polynucleotide shown in SEQ ID NO: 19 or 20; or

[0055] (c) a polynucleotide having one or more nucleotides, preferably 1-30, more preferably 1-6 nucleotides, truncated at the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO: 19 or 20, and having substantially the function of the polynucleotide shown in SEQ ID NO: 19 or 20.

[0056] In another preferred embodiment, the minimal promoter is a constitutive promoter or an inducible promoter.

[0057] In another preferred embodiment, the minimal promoter is selected from the following group: any minimal promoter such as minimal CMV promoter, minimal IL2 promoter, synthetic minimal promoter, etc.

[0058] In another preferred embodiment, the nucleotide sequence of the fragment derived from the minimal promoter is shown in SEQ ID NO:21.

[0059] In another preferred embodiment, the nucleotide sequence of the promoter element is:

[0060] (a) a nucleotide sequence as shown in any one of SEQ ID NOs: 22-29;

[0061] (b) a nucleotide sequence having a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the sequence shown in any one of SEQ ID NOs: 22-29, and having substantially the function of the polynucleotide shown in any one of SEQ ID NOs: 22-29; or

[0062] (c) A polynucleotide having one or more nucleotides, preferably 1-30, more preferably 1-6 nucleotides, truncated at the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs: 22-29, and having substantially the function of the polynucleotide shown in any one of SEQ ID NOs: 22-29.

[0063] In another preferred embodiment, the nucleotide sequence of the promoter element is shown in any one of SEQ ID NOs: 11-20, 22-29.

[0064] In the second aspect of the present invention, an expression cassette is provided, wherein the expression cassette has the following elements: the promoter element described in the first aspect of the present invention, and an ORF sequence of a target gene operably linked to the promoter element.

[0065] In another preferred embodiment, the target gene encodes an immunomodulatory factor protein.

[0066] In another preferred embodiment, the immunomodulatory factor is selected from the following group: cytokines, chemokines, antibodies, chimeric antigen receptors, or a combination thereof.

[0067] In another preferred embodiment, the cytokines include but are not limited to cytokines selected from the following group: IL-2, IL-15, IL-12, IL-18, IL-21, IL-7, IL9, IL23, INF-α, IFN-β, IFN-γ, or a combination thereof.

[0068] In another preferred embodiment, the chemokine includes but is not limited to cytokines selected from the following group: CCL20, CCL1, CCL17, CCL22, CCL27, CCL28, CXCL9, CXLC10, or a combination thereof.

[0069] In another preferred example, the antibody and / or chimeric antigen receptor may target an antigen selected from the following group: PD1, 4-1BB, OX40, CD40, CTLA-4, CSF1R, FAP, or a combination thereof, but not limited thereto.

[0070] In another preferred embodiment, the immunomodulatory factor is selected from neutrophil-activating protein (NAP) and MMPs (Matrix metalloproteinases).

[0071] In another preferred embodiment, the immunomodulatory factor is mouse IL2 or human IL2.

[0072] In another preferred embodiment, the expression cassette further comprises one or more elements selected from the following group: a poly(A) element, an enhancer, a transport element, or a gene targeting element.

[0073] In the third aspect of the present invention, an expression cassette for co-expressing a chimeric antigen receptor and an immunomodulatory factor is provided, wherein the expression cassette has a promoter element as described in the first aspect of the present invention, and the promoter element regulates the expression of the immunomodulatory factor.

[0074] In another preferred embodiment, the chimeric antigen receptor and the immunomodulatory factor are expressed in the same direction or in opposite directions.

[0075] In another preferred embodiment, the expression cassette has a structure shown in the following formula II from the 5' end to the 3' end:

[0076] CAR-PA2-IF-Prom(II)

[0077] In the formula,

[0078] Each "-" is independently a bond or a connecting nucleic acid sequence;

[0079] CAR is a chimeric antigen receptor coding sequence;

[0080] PA2 is a bidirectional polyA sequence;

[0081] IF is the immunomodulatory factor coding sequence in reverse;

[0082] Prom is a promoter element as described in the first aspect of the present invention in reverse orientation.

[0083] In another preferred embodiment, the bidirectional PolyA sequence is shown as SEQ ID NO:30.

[0084] In another preferred embodiment, the immunomodulatory factor is selected from the following group: cytokines, chemokines, antibodies, chimeric antigen receptors, or a combination thereof.

[0085] In another preferred embodiment, the cytokines include but are not limited to cytokines selected from the following group: IL-2, IL-15, IL-12, IL-18, IL-21, IL-7, IL9, IL23, INF-α, IFN-β, IFN-γ, or a combination thereof.

[0086] In another preferred embodiment, the chemokine includes but is not limited to cytokines selected from the following group: CCL20, CCL1, CCL17, CCL22, CCL27, CCL28, CXCL9, CXLC10, or a combination thereof.

[0087] In another preferred example, the antibody and / or chimeric antigen receptor may target an antigen selected from the following group: PD1, 4-1BB, OX40, CD40, CTLA-4, CSF1R, FAP, or a combination thereof, but not limited thereto.

[0088] In another preferred embodiment, the immunomodulatory factors are selected from NAP and MMPs.

[0089] In another preferred embodiment, the immunomodulatory factor is mouse IL2 or human IL2.

[0090] In another preferred example, the amino acid sequence of the immunomodulatory factor is shown in SEQ ID NO:10.

[0091] In another preferred embodiment, the encoding nucleotide sequence of the immunomodulatory factor is shown in SEQ ID NO:9.

[0092] In another preferred embodiment, the chimeric antigen receptor has a structure as shown in the following formula III:

[0093] LF-EB-H-TM-C-CD3ζ(III)

[0094] In the formula,

[0095] Each "-" is independently a bond or a connecting nucleic acid sequence;

[0096] L is none or a signal peptide coding sequence;

[0097] F is the None or label sequence;

[0098] EB is the extracellular binding domain coding sequence;

[0099] H is no or hinge region;

[0100] TM is the transmembrane domain;

[0101] C is no or co-stimulatory signal molecule;

[0102] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0103] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, SECTM1 or a combination thereof.

[0104] In another preferred embodiment, the extracellular binding domain is selected from the group consisting of scFv, ligand, or a combination thereof.

[0105] In another preferred example, the extracellular binding domain specifically binds to B7-H3.

[0106] In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown in SEQ ID NO:2.

[0107] In another preferred embodiment, the encoding nucleotide sequence of the extracellular binding domain is shown in SEQ ID NO:1.

[0108] In another preferred embodiment, the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, antibody Fc or a combination thereof.

[0109] In another preferred embodiment, the H is the hinge region derived from CD8α.

[0110] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following groups: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or a combination thereof.

[0111] In another preferred embodiment, the TM is a transmembrane region derived from CD8α.

[0112] In another preferred embodiment, the amino acid sequences of the hinge region and the transmembrane region are as shown in SEQ ID NO:4.

[0113] In another preferred example, the encoding nucleotide sequences of the hinge region and transmembrane region are as shown in SEQ ID NO:3.

[0114] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, HVEM, or a combination thereof.

[0115] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from CD28.

[0116] In another preferred example, the amino acid sequence of C is shown in SEQ ID NO:6.

[0117] In another preferred example, the encoding nucleotide sequence of C is shown in SEQ ID NO:5.

[0118] In another preferred example, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3ζ is shown in SEQ ID NO:8.

[0119] In another preferred example, the encoding nucleotide sequence of the cytoplasmic signal transduction sequence derived from CD3ζ is shown as SEQ ID NO:7.

[0120] In the fourth aspect of the present invention, a vector is provided, wherein the vector contains the expression cassette described in the second aspect of the present invention, or the expression cassette for co-expressing CAR and an immunomodulatory factor described in the third aspect of the present invention.

[0121] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0122] In another preferred embodiment, the vector is a retroviral vector.

[0123] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the fourth aspect of the present invention, or the exogenous promoter element described in the first aspect of the present invention is integrated into its chromosome, or the expression cassette described in the second aspect of the present invention is integrated into its chromosome.

[0124] In another preferred embodiment, the chromosome of the host cell has one or more (such as 1-50, preferably 2-6) copies of the promoter element described in the first aspect of the present invention, or the expression cassette described in the second aspect of the present invention.

[0125] In the sixth aspect of the present invention, an engineered immune cell is provided, wherein the immune cell contains the vector described in the fourth aspect of the present invention, or its chromosome is integrated with the expression cassette described in the second aspect of the present invention or the expression cassette for co-expressing CAR and immunomodulatory factors described in the third aspect of the present invention.

[0126] In another preferred embodiment, the engineered immune cells are selected from the following group: T cells, CAR-T cells, TCR-T cells, NK cells, γδT cells, NKT cells, macrophages, dendritic cells, neutrophils, or a combination thereof.

[0127] In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells), T cell receptor gene-modified T cells (TCR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0128] In the seventh aspect of the present invention, a method for preparing an engineered immune cell as described in the sixth aspect of the present invention is provided, comprising the following steps: transducing the expression cassette as described in the second aspect of the present invention, or the expression cassette for co-expressing CAR and immunomodulatory factors as described in the third aspect of the present invention, or the vector as described in the fourth aspect of the present invention into an immune cell, thereby obtaining the engineered immune cell.

[0129] In the eighth aspect of the present invention, a pharmaceutical composition is provided, which contains the vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, and / or the engineered immune cell as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0130] In another preferred embodiment, the preparation is a liquid preparation.

[0131] In another preferred embodiment, the preparation is in the form of an injection.

[0132] In another preferred embodiment, the concentration of the engineered immune cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0133] In the ninth aspect of the present invention, there is provided a use of the vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, or the engineered immune cell as described in the sixth aspect of the present invention for preparing a drug or preparation for preventing and / or treating a disease.

[0134] In another preferred embodiment, the disease is a tumor.

[0135] In another preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0136] In another preferred embodiment, the solid tumor is selected from the following group: breast cancer, lung cancer, pancreatic cancer, osteosarcoma, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, glioma, prostate cancer, nasopharyngeal cancer, head and neck cancer, and biliary tract cancer.

[0137] In another preferred embodiment, the blood tumor is selected from the following group: acute leukemia, chronic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0138] In another preferred embodiment, the disease is a disease with high expression of B7-H3.

[0139] In the tenth aspect of the present invention, a method for treating a disease is provided, comprising administering an effective amount of the host cells as described in the fifth aspect of the present invention, or the engineered immune cells as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention to a subject in need of treatment.

[0140] In another preferred embodiment, the disease is a tumor.

[0141] In another preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0142] In another preferred embodiment, the solid tumor is selected from the following group: breast cancer, lung cancer, pancreatic cancer, osteosarcoma, gastric cancer, colorectal cancer, hepatobiliary cancer, bladder cancer, non-small cell lung cancer, ovarian cancer, cervical cancer, esophageal cancer, glioma, prostate cancer, nasopharyngeal cancer, head and neck cancer, and biliary tract cancer.

[0143] In another preferred embodiment, the blood tumor is selected from the following group: acute leukemia, chronic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0144] In another preferred embodiment, the disease is a disease with high expression of B7-H3.

[0145] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells (autologous cells) derived from the subject.

[0146] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0147] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0148] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0149] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0151] Figure 1 A schematic diagram of the molecular structure of the B7H3.28ζCAR molecule and different versions of the T cell activation induced promoter (TAIP) inducible gene expression system co-expression vector (B7H3.28ζ.TAIP.mIL2) is shown. Among them, aB7H3: anti-B7H3 antibody scFV, CD8: hinge region and transmembrane domain of CD8α, 28ζ: intracellular co-stimulatory domain of CD28 and intracellular domain of CD3ζ, Flag: Flag protein, PA2: bidirectional PolyA sequence, mIL2: mouse IL2 gene, pGM-CSF: GM-CSF promoter (GM-CSF promoter), pIFN: IFN promoter (IFN promoter), pIL2: IL2 promoter (IL2 promoter), minCMV: CMV minimal promoter (minimalCMV promoter), 6*NFAT: 6 times NFAT binding sequence repeat sequence, minIL2: IL2 minimal promoter (minimal IL2promoter), pGM-CSF (+36to-217) represents the GM-CSF promoter +36 to -217 nucleic acid segment, and so on.

[0152] Figure 2 The expression efficiency of different versions of B7H3.28ζ.TAIP.mIL2 vector in T cells is shown. Figure 2 A is a representative flow cytometry graph of the infection rate of B7H3.28ζ.TAIP.mIL2 in T cells detected by anti-Flag antibody on the 4th day after B7H3.28ζ.TAIP.mIL2 infected T cells, where NT (Non-transduced T) is the non-transduced T cell control group. This figure represents at least 6 independent experiments with similar results. Figure 2 B is a statistical chart of the infection rate of B7H3.28ζ.TAIP.mIL2 in T cells. Figure 2C is a statistical graph of the expression intensity of B7H3.28ζ.TAIP.mIL2 in T cells.

[0153] Figure 3 The results show the effect of different versions of TAIP on the expression of genes induced by T cell activation signals. Figure 3 A is the ELISA test of mIL2 secretion in the supernatant on the 7th day after T cells were infected with B7H3.28ζ.TAIP.mIL2 and stimulated with B7H3-FC-coated antigen for 24 hours. Figure 3 B is the relative expression of mIL2 induced by different TAIPs after T cell activation. The expression of mIL2 was first calibrated with the CAR expression intensity and then compared with the induced expression of the NFAT promoter group.

[0154] Figure 4 The results show the detection of induction activity of different segments of pGM-CSF and the minCMV recombinant promoter. Figure 4 A shows the secretion of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after 24 hours of T cell activation (ELISA determination). Figure 4 B is the relative expression level of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after T cell activation. The expression level of mIL2 was first calibrated with the CAR expression intensity and then compared with the expression level induced by the NFAT promoter group.

[0155] Figure 5 The results show the detection of induction activity of different segments of pGM-CSF and the minCMV recombinant promoter. Figure 5 A shows the secretion of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after 24 hours of T cell activation (ELISA determination). Figure 5 B is the relative expression level of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after T cell activation. The expression level of mIL2 was first calibrated with the CAR expression intensity and then compared with the expression level induced by the NFAT promoter group. DETAILED DESCRIPTION

[0156] After extensive and in-depth research, the inventors have developed for the first time a new type of T cell activation inducible recombinant promoter constructed by GM-CSF, IFNg and IL2 promoter transformation and optimization. The recombinant promoter of the present invention can efficiently induce the expression of its downstream genes when CAR-T cells are activated, and its activity is better than the traditional NFAT-responsive promoter constructed based on NFAT (nuclear factor of T cell activation) binding sequence. This type of T cell activation inducible recombinant promoter can be used to induce CAR-T cells to secrete and express cytokines, antibody-like proteins and other immunomodulatory molecules in tumors to promote the activity and persistence of CAR-T cells, thereby promoting the application of CAR-T cells in solid tumors. On this basis, the present invention has been completed.

[0157] the term

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. As used herein, the term "about" when used in reference to a specific recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0159] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not occur.

[0160] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0161] "Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides to host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells (such as Escherichia coli).

[0162] "Gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification to produce the RNA or protein product of the gene.

[0163] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-stranded and double-stranded molecules interchangeably. Unless otherwise indicated, the polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands that are known or predictable to constitute the double-stranded form.

[0164] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0165] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0166] Promoter

[0167] The term "promoter" as used herein refers to a nucleic acid sequence that accurately and effectively initiates gene transcription, which guides the transcription of the gene nucleic acid sequence into mRNA, and is usually present upstream (5' end) of the target gene coding sequence. The promoter or promoter region generally provides recognition sites for RNA polymerase and other factors necessary for the correct initiation of transcription.

[0168] As used herein, the term "constitutive promoter" refers to a promoter under which the expression of a structural gene is generally constant at a certain level, and there is no obvious difference in the expression level in different tissues and parts.

[0169] As used herein, the term "inducible promoter" refers to a promoter that can significantly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals.

[0170] As used herein, the term "T cell activation-inducible promoter" refers to a type of promoter that can be induced to activate and thus initiate the expression of genes driven by the promoter when T cells are activated under the stimulation of certain specific physical or chemical signals.

[0171] As used herein, when used for immune cells (such as T cells), the term "activation" or "activation" refers to the stimulation of immune cells by corresponding stimulatory factors to achieve amplification to increase the number of immune cells and activation to enhance the activity of immune cells. The stimulatory factor of immune cells can be any factor that can amplify or enhance the activity of immune cells. In some embodiments, the stimulatory factor can also be an antibody targeting immune cell surface markers such as CD3 and CD28, or a compound activator such as PMA and inomycin. In some embodiments, when immune cells express a T cell receptor (TCR) or a chimeric antigen receptor (CAR) for a specific antigen, the stimulatory factor of the immune cell may also include the specific antigen or its binding epitope fragment, which causes TCR and CAR activation by contacting with T cells. For example, for T cells expressing anti-B7-H3 chimeric antigen receptors, the stimulatory factor may be B7-H3 expressed by tumor cells.

[0172] As used herein, the term "minimal promoter" refers to a short DNA segment that is not active by itself, but can mediate strong transcription when combined with other transcriptional regulatory elements. The characteristic of enhancing transcription is common to minimal promoters, and therefore, the promoter element of the present invention can include any minimal promoter. The minimal promoter sequence can be derived from a variety of different sources, including prokaryotic and eukaryotic genes. In one embodiment, the promoter element of the present invention includes the miniCMV promoter, which is a minimal promoter derived from human cytomegalovirus.

[0173] The promoter of the present invention has a fragment derived from the GM-CSF promoter, the IFNg promoter, or the IL2 promoter. The GM-CSF promoter is described in Gene ID: 1437; the IFNg promoter is described in Gene ID: 3458; and the IL2 promoter is described in Gene ID: 3558. In the present invention, the base numbering of the promoter fragment is calculated according to the conventional method in the art, that is, the base numbering is calculated with the transcription start site as the origin (+1 position), the upstream of the transcription start site is recorded as a negative number, and the downstream of the transcription start site is recorded as a positive number. For example, "the nucleic acid segment from -626 to +36 of the GM-CSF promoter" refers to the fragment of the GM-CSF promoter from 626 upstream to 36 downstream of the transcription start site.

[0174] In the promoter of the present invention, the fragment derived from the GM-CSF promoter, IFNg promoter, or IL2 promoter and the minimal promoter (or other promoter fragment) can be directly connected or connected through an additional nucleotide sequence. In one embodiment, in order to facilitate the insertion of the derived promoter fragment and the minimal promoter into the vector separately during the construction process, the additional nucleotide sequence used can be a restriction site, such as GCGGCCGC.

[0175] The promoter fragment used to construct the promoter element of the present invention can be derived from any animal, such as mouse, rat, rabbit, primate (such as human) and the like.

[0176] After testing, the present invention screened a series of T cell activation inducible promoters. Although the present invention specifically provides promoter elements as shown in SEQ ID NO.11-20, 22-29, this is only to provide exemplary embodiments to illustrate the parent promoter fragments and minimal promoters that the promoter elements of the present invention can contain. For example, the present invention provides test results of promoter elements containing GM-CSF promoter +36 to -217 and promoter elements containing GM-CSF promoter +36 to -626. Those skilled in the art should understand that other promoter elements between the two can achieve the effects of the present invention, such as promoter elements containing GM-CSF promoter +36 to -300 (or -400, -500). Therefore, the present invention includes these exemplary promoter elements and their derivative promoter elements.

[0177] The recombinant promoter expression system of the present invention can be used to specifically induce gene expression after T cell activation, and can be used to optimize, transform, and enhance the anti-tumor function of gene-edited immune cells or transform the tumor immune microenvironment.

[0178] Expression cassette

[0179] The present invention provides an expression cassette, which has the promoter element described in the first aspect of the present invention and an ORF sequence of a target gene.

[0180] The promoter of the present invention can be used to express any target gene. In one embodiment, the promoter of the present invention is connected downstream to the coding sequence of the immunomodulatory factor for regulating the expression of the immunomodulatory factor. As used herein, the term "immunomodulatory factor" refers to a protein or small molecule polypeptide that can transmit information between cells and has immunomodulatory and effector functions. The promoter of the present invention can be used to express immunomodulatory factors selected from the group consisting of cytokines, chemokines, antibodies, chimeric antigen receptors, but not limited thereto. In one embodiment, the expressed cytokines may be: IL-2, IL-15, IL-12, IL-18, IL-21, IL-7, INF-α, IFN-β, IFN-γ, but not limited thereto. In one embodiment, the expressed chemokines may be: CCL20, CCL1, CCL17, CCL22, CCL27, CCL28, CXCL9, CXLC10, but not limited thereto. In one embodiment, the expressed antibody and / or chimeric antigen receptor targets an antigen selected from the group consisting of PD1, 4-1BB, OX40, CD40, CTLA-4, CSF1R, FAP, but not limited thereto. In one embodiment, the expressed immunomodulatory factor may also be NAP (neutrophil-activating protein) or MMPs (Matrix metalloproteinases).

[0181] In one embodiment, the promoter of the present invention is used to regulate the expression of cytokines. The promoter of the present invention does not express or only expresses a very small amount of target protein in a non-activated T cell state, and can efficiently respond to T cell activation signals and induce a large amount of target protein expression when the T cells are activated. This performance can effectively improve the activity and persistence of T cells and reduce the generation of side effects such as cytokine storms. Therefore, the promoter of the present invention is suitable for CAR-T cells targeting tumors.

[0182] Chimeric Antigen Receptor Immune Cells (CAR-Immune Cells)

[0183] In the present invention, a chimeric antigen receptor immune cell is provided, which simultaneously expresses a chimeric antigen receptor and an immunomodulatory factor.

[0184] In the CAR-immune cells of the present invention, the coding sequences of the chimeric antigen receptor and the immunomodulatory factor can be located in the same vector or in different vectors, wherein the expression of the immunomodulatory factor is regulated by the promoter element of the present invention, thereby expressing the immunomodulatory factor under the condition that the immune cell is activated by the tumor antigen. In one embodiment, the CAR-immune cell contains an expression cassette for co-expressing the chimeric antigen receptor and the immunomodulatory factor as described in the third aspect of the present invention.

[0185] It should be understood that the promoter or immunomodulatory factor expression cassette of the present invention can be used in immune cells expressing any chimeric antigen receptor, and these chimeric antigen receptors can target any known antigen (such as tumor antigen). Therefore, although the present invention provides an example of a chimeric antigen receptor targeting B7-H3, the content of the present invention should not be limited thereto.

[0186] The chimeric antigen receptor immune cell of the present invention can be any immune cell, for example, it can be a CAR-T cell, or it can be a CAR-NK cell, or a CAR-macrophage. Preferably, the chimeric antigen receptor immune cell of the present invention is a CAR-T cell.

[0187] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the present invention" all refer to the CAR-T cells described in the sixth aspect of the present invention.

[0188] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, once the CAR gene construction targeting a certain tumor marker is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0189] As used herein, the terms "CAR-NK cell", "CAR-NK", and "CAR-NK cell of the present invention" all refer to the CAR-NK cell described in the sixth aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors.

[0190] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxic effects.

[0191] Compared with CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0192] Carrier

[0193] The present invention also provides a vector comprising an expression cassette of the present invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentivirus vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0194] In brief summary, the promoter of the present invention is usually operably connected to the target nucleic acid sequence and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0195] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0196] The expression cassette of the present invention can be cloned into many types of vectors. For example, the expression cassette can be cloned into the following vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0197] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0198] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0199] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0200] Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. Typically, reporter genes are genes that are not present in or expressed by a receptor organism or tissue, and that encode a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). In one embodiment of the invention, reporter genes are genes encoding Flag tag proteins. Suitable expression systems are well known and can be prepared or commercially available using known techniques. Typically, constructs with a minimum of 5 flanking regions showing the highest level of reporter gene expression are identified as promoters. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0201] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0202] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0203] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0204] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0205] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0206] In a preferred embodiment of the present invention, the vector is a retroviral vector.

[0207] preparation

[0208] The present invention provides a preparation, which contains the vector described in the fourth aspect of the present invention, or the host cell described in the fifth aspect of the present invention, or the engineered immune cell described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0209] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0210] Therapeutic applications

[0211] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a vector encoding the expression cassette of the present invention. The transduced T cells can target tumor cell markers (e.g., B7-H3), and after activating the T cells, induce the expression of immune regulatory factors, causing sustained T cell activation and immune response, thereby significantly improving its efficiency in killing tumor cells.

[0212] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-cell of the present invention to the mammal.

[0213] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0214] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells expressing B7-H3 CARs cause a specific immune response against B7-H3 cells.

[0215] Although the data disclosed herein specifically discloses a retroviral vector comprising a combination of an anti-B7-H3 scFv, hinge and transmembrane regions, a CD28 co-stimulatory domain, and a CD3ζ signaling domain, the present invention should be construed to include any number of variations to each of the construct components.

[0216] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors. Cancer includes non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) and solid tumors. The types of cancer treated with the CAR of the present invention include but are not limited to cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas. Also included are adult tumors / cancers and childhood tumors / cancers.

[0217] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0218] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0219] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0220] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0221] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0222] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0223] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0224] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0225] When an "effective amount", "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6The T cell composition can also be administered at multiple doses of 1 cell / kg body weight (including all integer values ​​within those ranges). The cells can be administered by using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0226] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0227] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment modalities (e.g., before, simultaneously or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for specific tumor patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0228] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10The CAR-T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0229] The main advantages of the present invention include:

[0230] 1) The present invention provides a series of novel T cell activation-inducible recombinant promoters constructed by optimizing and modifying GM-CSF, IFNg and IL2 promoters, which can efficiently induce the expression of their downstream genes when CAR-T cells are activated, and their activity is superior to that of traditional NFAT-responsive promoters constructed based on NFAT (nuclear factor of T cell activation) binding sequences.

[0231] 2) The present invention provides CAR-T cells containing the T cell activation-inducible recombinant promoter, which can induce CAR-T cells to secrete and express cytokines, antibody-like proteins and other immunoregulatory molecules in tumors to promote the activity and persistence of CAR-T cells, thereby promoting the application of CAR-T cells in solid tumors.

[0232] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0233] Example 1 Construction of different versions of B7H3.28ζ.TAIP.mIL2 plasmid

[0234] Different segments of GM-CSF, IFNg and IL2 promoters were selected to construct T cell activation induced promoter (TAIP), and mIL2 gene was inserted into it as an indicator gene. Then TAIP-mIL2 was reversely cloned into SFG.B7H3.28 ζ In the expression vector, a series of B7H3.28 ζ .TAIP.mIL2 vector (such as Figure 1 In the GM-CSF promoter and minCMV minimal promoter recombinant promoter, multiple versions of the recombinant promoter were constructed in which minCMV was located at different positions downstream of the GM-CSF promoter (pGM-CSF) (as shown in FIG. Figure 1) to enhance the induction activity of pGM-CSF. Among them, SFG.B7H3.28z.CAR was used as a negative control vector, and B7H3.28ζ.NFAT.mIL2 was used as a reference vector.

[0235] The construction method is as follows: the variable region (VL and VH) fragment sequences of the light chain and heavy chain of the monoclonal antibody 376.96 against B7H3 were synthesized by gene sequence synthesis, and cloned into the retroviral vector SFG.CAR.CD28ζ after digestion with Mlu1 and Nco1 to construct the SFG.B7H3.CD28ζ vector; the different segments of the GM-CSF, IFNg and IL2 promoters were amplified from the genome of human cells by PCR, and the mIL2 gene was amplified from the genome of activated mouse T cells, and then the two were connected by fusion and reversely cloned into the SFG.B7H3.CD28ζ vector. In the plasmid with added minCMV, minCMV was introduced into the downstream of the GM-CSF promoter segment in the form of primers, and then cloned into the SFG.B7H3.CD28ζ vector by fusion with the mIL2 gene.

[0236] Example 2 Preparation of retrovirus expressing B7H3.28ζ.TAIP.mIL2

[0237] 6×10 5 293T cells were seeded in a 6-well cell culture dish. After 18 hours, 0.75 μg of different versions of B7H3.28ζ.TAIP.mIL2 plasmids were mixed with 0.75 μg of PEQ plasmid and 0.5 μg of RDF plasmid, respectively. The plasmid mixture was then mixed with GeneJuice transfection reagent (Merck Millipore), allowed to stand for 10 minutes, and then transfected into 293T cells. 48 hours after transfection, the culture supernatant containing the virus was collected and filtered with a 0.45 μm filter, and then frozen at -80°C for use.

[0238] Example 3 Preparation and in vitro expansion of CAR-T cells

[0239] PBMC lymphocytes were isolated from the peripheral blood of healthy volunteers and then added to 24-well plates coated with CD3 and CD28 antibodies at 1×10 6 cells per well. After 24 hours, IL7 and IL15 (5 ng / mL) were added. After two days of stimulation, T cells were collected and counted for later use. The stimulated T cells were infected with the above virus. The specific infection steps were as follows: 1 mL of virus supernatant was added to a 24-well plate coated with fibronectin and centrifuged at 2000 g for 1.5 hours. After centrifugation, the supernatant was discarded and 3×10 5After stimulation, the T cells were centrifuged at 1000 g for 10 min. The cells were cultured in a 37°C cell culture incubator and fresh culture medium was replaced after 72 hours. Thereafter, the culture medium was replaced every two days. After 4 days of infection, CAR-T cells were collected and stained with anti-Flag antibody to detect the CAR positive rate by flow cytometry. After 7 days of infection, 3×10 5 Cells were collected and CAR-T cells were used for B7H3-FC antigen stimulation experiment.

[0240] Example 4 Flow cytometry

[0241] After 4 days of virus infection, 1×10 6 CAR-T cells were stained by adding 0.5ug Flag-APC (Biolegend) antibody and incubating at 4°C for 15 minutes. The cells were washed with PBS and detected by BD FACSCanto II flow cytometer. At least 10,000 live cells were collected for each sample, and the collected results were analyzed by Flowjo 10 software.

[0242] Results: B7H3.28ζ-TAIP-mIL2 can be efficiently expressed in T cells (such as Figure 2 shown). Figure 2 B and Figure 2 C shows that the positive rate and expression level of the chimeric antigen receptor were not significantly affected, and basically met the requirements for use.

[0243] Example 5 B7H3-FC antigen stimulation experiment

[0244] B7H3-FC protein (ACROBiosystem) was diluted in PBS to a final concentration of 1ug / ml, and then 500ul was added to a non-tissue culture 24-well plate and placed at 4°C for overnight coating. On the second day, the B7H3-FC dilution was discarded, 1mL of serum-containing culture medium was added, and incubated at room temperature for 10min, and then 1×10 6 CAR-T cells, and the same number of CAR-T cells were plated in a 24-well plate not coated with B7H3-FC as an unstimulated control group. After incubation at 37°C for 24 hours, 1 ml of supernatant was collected for ELISA detection. The mouse IL2 ELISA kit (Mabtech) was used to detect the secretion of mIL2 in the supernatant of B7H3.28ζ.TAIP.mIL2-infected T cells after B7H3-FC stimulation.

[0245] Results: Figure 3As shown, after 24 hours of antigen stimulation, a series of inducible promoters constructed can respond to the activation of T cells and induce the expression and secretion of mIL2. Except for the pIFN (+64 to -108) promoter, the induction activity of the other inducible promoters is higher than that of the NFAT promoter. Among them, the recombinant promoter composed of the GM-CSF-derived promoter and mimCMV (pGM-CSF (-1 to -336).minCMV, pGM-CSF (-35 to -336).minCMV and pGM-CSF (-1 to 217).minCMV) can efficiently induce the expression of mIL2. The quantitative results are shown in Table 1 below:

[0246] Table 1 mIL2 expression levels

[0247]

[0248]

[0249] The results showed that when minCMV was located at -35 bp downstream of pGM-CSF, the promoter of pGM-CSF (-35 to 336).minCMV was stronger than that of pGM-CSF (-1 to 336).minCMV ( Figure 3 The induction activity of pGM-CSF(-35to 217).minCMV was then compared with that of pGM-CSF(-1to 217).minCMV. The results showed that the induction activity of pGM-CSF(-35to 217).minCMV was slightly better than that of pGM-CSF(-1to 217).minCMV (as shown). Figure 4 shown).

[0250] Example 6 Promoter truncation test

[0251] The GM-CSF promoter segment was further truncated to test whether the shorter GM-CSF promoter still has strong T cell activation induction activity. Figure 5 As shown in Figure 2, the further truncated pGM-CSF(-35to-179).minCMV still has the same induction activity as pGM-CSF(-35to-217).minCMV. However, when the promoter segment is reduced to about 100 bp, such as the pGM-CSF(-35to106).minCMV promoter, its induction activity is significantly lower than that of pGM-CSF(-35to 217).minCMV, but still higher than that of the NFAT binding sequence 6 repeat synthetic promoter ( Figure 5 ).

[0252] sequence:

[0253] 1. 376.96 scFv DNA sequence:

[0254] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAATTGGAGCCAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGAACTGCTGTAGCCTGGTATCAACAGAAACCAGGCCAGTCTCCTAAACTACTAATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATGGTACTCCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGCGGAGGGGGCTCTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGAAGCCTCTAGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAGCCATTAGTGGAGGTGGTAGGTACACCTACTATCCAGACAGTATGAAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAATTTCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGACACTATGATGGTTATCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:1)

[0255] 2. 376.96 scFv amino acid sequence:

[0256] DIVMTQSHKFMSTSIGARVSITCKASQDVRTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYGTPPWTFGGGTKLEIKGGGGSGGGGSGGGG SEVQLVESGGGLVKPGGSLKLSCEASRFTFSSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRFTISRDNAKNFLYLQMSSLRSEDTAMYYCARHYDGYLDYWGQGTTLTVSS(SEQ ID NO:2)

[0257] 3.CD8α hinge region and transmembrane region DNA sequence:

[0258] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 3)

[0259] 4.Amino acid sequence of CD8α hinge region and transmembrane region:

[0260] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:4)

[0261] 5. DNA sequence of CD28 intracellular co-stimulatory domain:

[0262] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGG CCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 5)

[0263] 6.Amino acid sequence of CD28 intracellular co-stimulatory domain:

[0264] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:6)

[0265] 7. DNA sequence of the CD3ζ intracellular domain:

[0266] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:7)

[0267] 8. Amino acid sequence of the CD3ζ intracellular domain:

[0268] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:8)

[0269] 9. Mouse IL2 (mIL2) CDS DNA sequence:

[0270] ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAATAA(SEQ ID NO:9)

[0271] 10. Amino acid sequence of mouse IL2 (mIL2) protein:

[0272] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRME NYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVK LKGSDNTFECQFDDESATVVDFLRRWIAFCQSI ISTSPQ*(SEQ ID NO:10)

[0273] 11. pGM-CSF(+36to-336) promoter DNA sequence

[0274] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGA(SEQ ID NO:11)

[0275] 12. pGM-CSF (-1 to -336) promoter DNA sequence

[0276] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCC(SEQ ID NO:12)

[0277] 13. pGM-CSF (-35 to -336) promoter DNA sequence

[0278] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGGGGCTGTCGGTTCTTGG AAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGATTCCACAGTCAGGTAGTCCCCGCCTCCCTGGCATTTGTGGTCACCATCATTTCCT(SEQ ID NO:13)

[0279] 14.pGM-CSF(+36to-217)

[0280] CAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGGCTGATAAGGGCCAGGAGATTCCACAG TTCAGGTAGTTCCCCCGCCTCCCTGGCATTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAGGCTAAAGTTCTCTGGAGGA(SEQ ID NO:14)

[0281] 15.pGM-CSF(-1to-217)

[0282] CAGGAAGGCGGGTGGGTGGCTGTCGGTTCTTGGAAAGGTTCATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGGCTGATAAGG GCCAGGAGATTCCCAGTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCC(SEQID NO:15)

[0283] 16.pGM-CSF(-35to-217)

[0284] CAGGAAGGCGGGTGGGTGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCC TGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGGCTGATAAGGGCCAGGAGATTCCACAGTT CAGGTAGTTCCCCCGCCTCCCTGGCATTTTGGGTCACCATTCATCACTTAQ: IDCATTQ:6

[0285] 17.pGM-CSF(+36to-626)

[0286] GCTTGCTGAGAGTGGCTGCAGTCTCGCTGCTGGATGTGCACATGGTGGTCATTCCCTCTGCTCACAGGGGCAGGGGTCCCCCCTTACTGGACTGAGGTTGCCCCCTGCTCCAGGTCCTGGGTGGGAGCCCATGTGAACTGTCAGTGGGGCAGGTCTGTGAGAGCTCCCCTCACACTCAAGTCTCTCACAGTGGCCAGAGAAGAGGAAGGCTGGAGTCAGAATGAGGCACCAGGGCGGGCATAGCCTGCCCAAAGGCCCCTGGGATTACAGGCAGGATGGGGAGCCCTATCTAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGA(SEQ ID NO:17)

[0287] 18. pIL2(+47 to -326) promoter DNA sequence

[0288] TTTTCTGAGTTACTTTTGTATCCCCACCCCCTTAAAGAAAGGAGGAAAAACTGTTTCATACAGAAGGCGTTAATTGCATGAATTAGAGCTATCACCTAAGTGTGGGCTAATGTAACAAAGAGGGATTTCACCTACATCCATTCAGTCAGTCTTTGGGGGTTTAAAGAAATTCCAAAGAGTCATCAGAAGAGGAAAAATGAAGGTAATGTTTTTTCAGACAGGTAAAGTCTTTGAAAATATGTGTAATATGTAAAACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCACA(SEQ ID NO:18)

[0289] 19. pIFN(+64 to -538) promoter DNA sequence

[0290] TAGAGCAATTTGAAACTTGTGGTAGATATTTTACTAACCAACTCTGATGAAGGACTTCCTCACCAAATTGTTCTTTTAACCGCATTCTTTCCTTGCTTTCTGGTCATTTGCAAGAAAAATTTTAAAAGGCTGCCCCTTTGTAAAGGTTTGAGAGGCCCTAGAATTTCGTTTTTCACTTGTTCCCAACCACAAGCAAATGATCAATGTGCTTTGTGAATGAAGAGTCAACATTTTACCAGGGCGAAGTGGGGAGGTACAAAAAAATTTCCAGTCCTTGAATGGTGTGAAGTAAAAGTGCCTTCAAAGAATCCCACCAGAATGGCACAGGTGGGCATAATGGGTCTGTCTCATCGTCAAAGGACCCAAGGAGTCTAAAGGAAACTCTAACTACAACACCCAAATGCCACAAAACCTTAGTTATTAATACAAACTATCATCCCTGCCTATCTGTCACCATCTCATCTTAAAAAACTTGTGAAAATACGTAATCCTCAGGAGACTTCAATTAGGTATAAATACCAGCAGCCAGAGGAGGTGCAGCACATTGTTCTGATCATCTGAAGATCAGCTATTAGAAGAGAAAGATCAGTTAAGTCCTTTGG(SEQ ID NO:19)

[0291] 20. pIFN(+64 to -108) promoter DNA sequence

[0292] CTATCATCCCTGCCTATCTGTCACCATCTCATCTTAAAAAACTTGTGAAAATACGTAATCCTC AGGAGACTTCAATTAGGTATAAATACCAGCAGCCAGAGGAGGTGCAGCACATTGTTCTGATCATCTG AAGATCAGCTATTAGAAGAGAAAGATCAGTTAAGTCCTTTGG(SEQ ID NO:20)

[0293] 21. minCMV (CMV minimal promoter) DNA sequence

[0294] CCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:21)

[0295] 22. pGM-CSF(+36 to -336). minCMV promoter DNA sequence

[0296] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAA AAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGAGCGGCCGCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:22)

[0297] 23. pGM-CSF(-1 to -336). minCMV promoter DNA sequence

[0298] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCGCGGCCGCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:23)

[0299] 24. pGM-CSF(-35 to -336). minCMV promoter DNA sequence

[0300] TAAGTGTCTCCCACGCCCCACCCCAGCCATTCCAGGCCAGGAAGTCCAAACTGTGCCCCTCAGAGGGAGGGGGCAGCCTCAGGCCCATTCAGACTGCCCAGGGAGGGCTGGAGAGCCCTCAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTGCGGCCGCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:24)

[0301] 25. pGM-CSF(-1 to -217). minCMV promoter DNA sequence

[0302] CAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCGCGGCCGCcCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:25)

[0303] 26. pGM-CSF(-35 to -217). minCMV promoter DNA sequence

[0304] CAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTGCGGCCGCcCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:26)

[0305] 27. pGM-CSF(-35 to -179). minCMV promoter DNA sequence

[0306] GTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTGCGGCCGCcCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:27)

[0307] 28. pGM-CSF(-35 to -106). minCMV promoter DNA sequence

[0308] GCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAA TCATTTCCTGCGGCCGCcCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:28)

[0309] 29. pGM-CSF(+36 to -217). minCMV promoter DNA sequence:

[0310] CAGGAAGGCGGGTGGGTGGGCTGTCGGTTCTTGGAAAGGTTCATTAATGAAAACCCCCAAGCCTGACCACCTAGGGAAAAGGCTCACCGTTCCCATGTGTGGCTGATAAGGGCCAGGAGATTCCACAGTTCAGGTAGTTCCCCCGCCTCCCTGGCATTTTGTGGTCACCATTAATCATTTCCTCTGTGTATTTAAGAGCTCTTTTGCCAGTGAGCCCAGTACACAGAGAGAAAGGCTAAAGTTCTCTGGAGGAGCGGCCGCcCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA(SEQ ID NO:29)

[0311] 30. 6*NFAT. minIL2 promoter DNA sequence

[0312] Ggaggaaaaactgtttcatacagaaggcgtcaattaggaggaaaaactgtttcatacagaaggcgtcaattaggaggaaaaactgtttcatacagaaggcgtcaattggtcccatcgaattaggaggaaaaactgtttcatacagaaggcgtcaattaggaggaaaaactgtttcata cagaaggcgtcaattaggaggaaaaactgtttcatacagaaggcgtcaattggtcccgggacattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctga(SEQ ID NO:30)

[0313] 31.PA2 bidirectional PolyA DNA sequence

[0314] AATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTG(SEQ ID NO:31)

[0315] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A genetically modified promoter element, characterized in that The promoter element is a T cell activation-inducible promoter, and the promoter element has a fragment derived from a promoter selected from the group consisting of a GM-CSF promoter, an IFNg promoter, an IL2 promoter, or a combination thereof.

2. The promoter element according to claim 1, characterized in that The promoter element has a structure as shown in the following formula (I): Z0-Z1-Z2(I) In the formula, "-" is a bond or a nucleotide connection sequence; Z0 is none, a fragment derived from a minimal promoter, or a fragment derived from another promoter; Z1 is a fragment derived from a promoter selected from the group consisting of GM-CSF promoter, IFNg promoter, IL2 promoter, or a combination thereof; Z2 is none, a fragment derived from a minimal promoter, or a fragment derived from another promoter.

3. The promoter element according to claim 2, characterized in that (1) Z1 is derived from the nucleic acid segment from position p1 to position q1 of the human GM-CSF promoter, wherein p1 is any integer from -800 to -100, preferably from -400 to -150; q1 is any integer from -50 to 50, preferably from -40 to +40, more preferably from -36 to +36; or (2) Z1 is derived from the nucleic acid segment from position p2 to position q2 of the human IL-2 promoter, wherein p2 is any integer from -500 to -200, preferably from -400 to -300; q2 is any integer from 0 to +100, preferably from +30 to +70, more preferably from +40 to +60; or (3) Z1 is derived from the nucleic acid segment from position p3 to position q3 of the IFNg promoter, wherein p3 is any integer from -800 to -100, preferably from -600 to -400; q3 is any integer from +20 to +120, preferably from +40 to +80, and more preferably from +50 to +70.

4. The promoter element according to claim 2, characterized in that The minimal promoter is selected from the group consisting of a minimal CMV promoter, a minimal IL2 promoter, a synthetic minimal promoter, or a combination thereof.

5. The promoter element according to claim 1, characterized in that The nucleotide sequence of the promoter element is: (a) a nucleotide sequence as shown in any one of SEQ ID NOs: 11-20, 22-29; (b) a nucleotide sequence having a homology of ≥90% (preferably ≥95%, more preferably ≥98%) to the sequence shown in any one of SEQ ID NOs: 11-20, 22-29, and substantially having the function of the polynucleotide shown in any one of SEQ ID NOs: 11-20, 22-29; or (c) A polynucleotide having one or more nucleotides, preferably 1-30, more preferably 1-6 nucleotides, truncated at the 5' end and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs: 11-20, 22-29, and having substantially the function of the polynucleotide shown in any one of SEQ ID NOs: 11-20, 22-29.

6. An expression cassette, characterized in that The expression cassette comprises the promoter element according to claim 1 and an ORF sequence of a target gene operably linked to the promoter element.

7. An expression cassette for co-expressing a chimeric antigen receptor (CAR) and an immunomodulatory factor, characterized in that: The expression cassette has the promoter element of claim 1, and the promoter element regulates the expression of the immunomodulatory factor.

8. The expression cassette for co-expressing CAR and an immunomodulatory factor according to claim 7, characterized in that: The expression cassette has a structure shown in the following formula II from the 5' end to the 3' end: CAR-PA2-IF-Prom(II) In the formula, Each "-" is independently a bond or a connecting nucleic acid sequence; CAR is a chimeric antigen receptor coding sequence; PA2 is a bidirectional polyA sequence; IF is the immunomodulatory factor coding sequence in reverse; Prom is the promoter element of claim 1 in reverse orientation.

9. A carrier, characterized in that The vector contains the expression cassette of claim 6, or the expression cassette for co-expressing CAR and an immunomodulatory factor of claim 7.

10. An engineered immune cell, characterized in that: The immune cell contains the vector according to claim 9, or its chromosome is integrated with the expression cassette according to claim 6 or the expression cassette for co-expressing CAR and immune regulatory factors according to claim 7.

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