Recombinant promoter system specifically inducing gene expression after t cell activation

Abstract

Provided is a recombinant promoter system specifically inducing gene expression after T cell activation. Also provided is a new T cell activation-induced recombinant promoter constructed by modifying and optimizing GM-CSF, IFNg and IL2 promoters. When CAR-T cells are activated, the recombinant promoter can efficiently induce the expression of downstream genes, with the activity thereof being superior to that of a traditional NFAT-responsive promoter constructed based on a binding sequence of a nuclear factor of activated T cells. Further provided are a CAR-T cell comprising the T cell activation-induced recombinant promoter and the use thereof.

Description

A recombinant promoter system for specifically inducing gene expression after T cell activation 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 to engineer T cells to express receptors and co-stimulatory molecules that specifically recognize antigens. These cells are then expanded in vitro and reinfused back into the patient, enabling them to recognize tumor-associated antigens and kill tumor cells independently of major histocompatibility complex presentation. In recent years, CAR-T cell therapy has achieved significant success in the treatment of hematologic malignancies. Currently, several CAR-T therapies have been approved for the treatment of hematologic malignancies. CAR-T cell therapy targeting CD19 has achieved a remission rate of over 90% in refractory, relapsed acute lymphoblastic leukemia. CAR-T cell therapy targeting BCMA has achieved an overall response rate (ORR) of up to 96% in the treatment of multiple myeloma, offering hope for the potential of CAR-T therapy to cure cancer.

[0003] While CAR-T therapy has achieved tremendous success in the clinical treatment of hematologic malignancies, its efficacy in solid tumors is very limited and still faces numerous difficulties and challenges: antigen heterogeneity is prevalent 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 can lead to strong toxic side effects. Therefore, numerous strategies have been explored to modify and optimize CAR-T to achieve optimal activity while minimizing toxicity, thereby enhancing its efficacy in solid tumors.

[0004] In addition to optimizing the chimeric antigen receptor itself, a more effective strategy is to load CAR-T cells with relevant functional proteins, thereby enhancing their anti-tumor potential by "arming" them. For example, loading them with immunomodulatory factors (such as cytokines, immune checkpoint inhibitors, and bacterial virulence factor proteins) can effectively improve CAR-T persistence and activate the immune system. T cells engineered to overexpress γ-chain cytokines (such as IL2, IL7, IL15, and IL21) or inflammatory cytokines (such as IL12, IL18, and IL23) have shown enhanced anti-tumor activity. However, persistent expression of immunomodulatory factors can cause severe toxic side effects, potentially limiting their potential benefits. For example, CAR-T cells overexpressing IL2 or IL18 have been found to cause severe toxic side effects in preclinical studies, limiting their clinical translation. Therefore, strictly controlling the expression of these cargo proteins and ensuring their secretion only within tumor lesions can maximize their functional benefits while minimizing their impact on normal tissues.

[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.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a recombinant promoter expression system that specifically induces gene expression after T cell activation.

[0008] Another object of the present invention is to provide T cells containing the recombinant promoter expression system of the present invention and applications thereof.

[0009] 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.

[0010] 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.

[0011] In another preferred embodiment, the promoter element is induced by a T cell activation pathway selected from the group consisting of:

[0012] a) Induction by ligand binding to the T cell receptor (TCR);

[0013] b) induced by ligand binding of chimeric antigen receptor (CAR);

[0014] c) Activation through antibodies against immune cell surface markers (including anti-CD3 and anti-CD28);

[0015] d) Activation by compound activators (such as PMA, inomycin).

[0016] In another preferred embodiment, under the conditions 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.

[0017] In another preferred embodiment, the promoter element has a structure as shown in the following formula (I): Z0-Z1-Z2 (I)

[0018] Wherein, “-” represents 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 absent; 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, 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) the nucleotide sequence 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 or substantially having 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' and / or 3' end of the polynucleotide represented by any one of SEQ ID NOs: 11-17, and having or substantially having the function of the polynucleotide represented by 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, 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 of SEQ ID NO: 18, and substantially having the function of the polynucleotide of 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' and / or 3' end of the polynucleotide shown in SEQ ID NO: 18, and substantially having 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, 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 of SEQ ID NO: 19 or 20, and substantially having the function of the polynucleotide of 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' and / or 3' end of the polynucleotide shown in SEQ ID NO: 19 or 20, and substantially having 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: minimal CMV promoter, minimal IL2 promoter, synthetic minimal promoter and other minimal promoters.

[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) the nucleotide sequence 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 substantially having 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' and / or 3' end of the polynucleotide shown in any one of SEQ ID NOs: 22-29, and substantially having the functions 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 a second aspect of the present invention, an expression cassette is provided. The expression cassette comprises the following elements: the promoter element according to the first aspect of the present invention, and an ORF sequence of a gene of interest 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 group consisting of 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 can 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, characterized in that the expression cassette has the 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: CAR-PA2-IF-Prom (II)

[0076] Where,

[0077] Each "-" is independently a bond or a connecting nucleic acid sequence;

[0078] CAR is the chimeric antigen receptor coding sequence;

[0079] PA2 is a bidirectional polyA sequence;

[0080] IF is the immunomodulatory factor coding sequence in reverse;

[0081] Prom is a promoter element as described in the first aspect of the present invention in reverse orientation.

[0082] In another preferred embodiment, the bidirectional PolyA sequence is shown as SEQ ID NO: 30.

[0083] In another preferred embodiment, the immunomodulatory factor is selected from the group consisting of cytokines, chemokines, antibodies, chimeric antigen receptors, or a combination thereof.

[0084] 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.

[0085] 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.

[0086] In another preferred example, the antibody and / or chimeric antigen receptor can 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.

[0087] In another preferred embodiment, the immunomodulatory factors are selected from NAP and MMPs.

[0088] In another preferred embodiment, the immunomodulatory factor is mouse IL2 or human IL2.

[0089] In another preferred embodiment, the amino acid sequence of the immunomodulatory factor is shown in SEQ ID NO: 10.

[0090] In another preferred embodiment, the nucleotide sequence encoding the immunomodulatory factor is shown in SEQ ID NO:9.

[0091] In another preferred embodiment, the chimeric antigen receptor has a structure shown in the following formula III: LF-EB-H-TM-C-CD3ζ (III)

[0092] Where,

[0093] Each "-" is independently a bond or a connecting nucleic acid sequence;

[0094] L is none or a signal peptide coding sequence;

[0095] F is the None or label sequence;

[0096] EB is the extracellular binding domain coding sequence;

[0097] H is the absence or hinge region;

[0098] TM is the transmembrane domain;

[0099] C is no or costimulatory signaling molecule;

[0100] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0101] 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.

[0102] In another preferred embodiment, the extracellular binding domain is selected from the group consisting of scFv, ligand, or a combination thereof.

[0103] In another preferred embodiment, the extracellular binding domain specifically binds to B7-H3.

[0104] In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown in SEQ ID NO: 2.

[0105] In another preferred embodiment, the nucleotide sequence encoding the extracellular binding domain is shown in SEQ ID NO: 1.

[0106] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, antibody Fc, or a combination thereof.

[0107] In another preferred embodiment, the H is a hinge region derived from CD8α.

[0108] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, HVEM, or a combination thereof.

[0109] In another preferred embodiment, the TM is a transmembrane region derived from CD8α.

[0110] In another preferred embodiment, the amino acid sequences of the hinge region and transmembrane region are as shown in SEQ ID NO:4.

[0111] In another preferred embodiment, the nucleotide sequences encoding the hinge region and transmembrane region are shown in SEQ ID NO: 3.

[0112] In another preferred embodiment, the 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.

[0113] In another preferred embodiment, the C is a costimulatory signal molecule derived from CD28.

[0114] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO: 6.

[0115] In another preferred embodiment, the coding nucleotide sequence of C is shown in SEQ ID NO:5.

[0116] In another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3ζ is shown in SEQ ID NO:8.

[0117] In another preferred embodiment, the nucleotide sequence encoding the cytoplasmic signaling sequence derived from CD3ζ is shown in SEQ ID NO:7.

[0118] In the fourth aspect of the present invention, a vector is provided, which contains 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.

[0119] 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.

[0120] In another preferred embodiment, the vector is a retroviral vector.

[0121] 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.

[0122] 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.

[0123] In the sixth aspect of the present invention, an engineered immune cell is provided, which 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.

[0124] In another preferred embodiment, the engineered immune cells are selected from the group consisting of T cells, CAR-T cells, TCR-T cells, NK cells, γδT cells, NKT cells, macrophages, dendritic cells, neutrophils, or a combination thereof.

[0125] 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).

[0126] In the seventh aspect of the present invention, a method for preparing the engineered immune cells 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 the immune cells, thereby obtaining the engineered immune cells.

[0127] In the eighth aspect of the present invention, a pharmaceutical composition is provided, which contains the vector described in the fourth aspect of the present invention, the host cell described in the fifth aspect of the present invention, and / or the engineered immune cell described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0128] In another preferred embodiment, the preparation is a liquid preparation.

[0129] In another preferred embodiment, the preparation is in the form of an injection.

[0130] 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.

[0131] 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.

[0132] In another preferred embodiment, the disease is a tumor.

[0133] In another preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0134] 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.

[0135] In another preferred embodiment, the blood tumor is selected from the group consisting of acute leukemia, chronic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0136] In another preferred embodiment, the disease is a disease with high expression of B7-H3.

[0137] 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.

[0138] In another preferred embodiment, the disease is a tumor.

[0139] In another preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0140] 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.

[0141] In another preferred embodiment, the blood tumor is selected from the group consisting of acute leukemia, chronic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0142] In another preferred embodiment, the disease is a disease with high expression of B7-H3.

[0143] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0144] 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).

[0145] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0146] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0147] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] 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.

[0149] Figure 1 shows 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). 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 (minimal CMV promoter), 6*NFAT: 6 NFAT binding sequence repeats, minIL2: IL2 minimal promoter (minimal IL2 promoter), pGM-CSF (+36 to -217) represents the GM-CSF promoter +36 to -217 nucleic acid segment, and so on.

[0150] Figure 2 shows the expression efficiency of different versions of the B7H3.28ζ.TAIP.mIL2 vector in T cells. Figure 2A is a representative flow cytometry plot of the infection rate of B7H3.28ζ.TAIP.mIL2 in T cells using an anti-Flag antibody on day 4 after infection. NT (Non-transduced T) represents the untransduced T cell control group. This figure is representative of at least six independent experiments with similar results. Figure 2B is a statistical graph 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.

[0151] Figure 3 shows the expression of genes induced by T cell activation signals by different versions of TAIP. Figure 3A shows the secretion of mIL2 in the supernatant measured by ELISA 7 days after infection of T cells with B7H3.28ζ.TAIP.mIL2 and 24 hours of stimulation with B7H3-FC-coated antigen. Figure 3B shows the relative expression of mIL2 induced by different TAIPs upon T cell activation. mIL2 expression was first calibrated to CAR expression intensity and then compared to the expression induced by the NFAT promoter group.

[0152] Figure 4 shows the detection of induction activity of different segments of pGM-CSF and the minCMV recombinant promoter. Figure 4A shows the amount of mIL2 secretion induced by different pGM-CSF-minCMV recombinant promoters 24 hours after T cell activation (ELISA assay). Figure 4B shows the relative expression levels of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after T cell activation. The expression levels of mIL2 were first calibrated with CAR expression intensity and then compared with the expression levels induced by the NFAT promoter group.

[0153] Figure 5 shows the detection of induction activity of different segments of pGM-CSF and the minCMV recombinant promoter. Figure 5A shows the amount of mIL2 secretion induced by different pGM-CSF-minCMV recombinant promoters 24 hours after T cell activation (ELISA assay). Figure 5B shows the relative expression levels of mIL2 induced by different pGM-CSF-minCMV recombinant promoters after T cell activation. The expression levels of mIL2 were first calibrated with CAR expression intensity and then compared with the expression levels induced by the NFAT promoter group. DETAILED DESCRIPTION

[0154] After extensive and in-depth research, the inventors have developed for the first time a novel T cell activation inducible recombinant promoter constructed by optimizing the transformation of GM-CSF, IFNg and IL2 promoters. 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 the 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.

[0155] the term

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" 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 all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0157] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not.

[0158] As used herein, the term "comprising" or "including" may be open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0159] "Transduction", "transfection", "transformation" or other terms used in this article refer to the process of transferring exogenous polynucleotides into 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).

[0160] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification to produce the RNA or protein product of a gene.

[0161] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs thereof. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. As used herein, the term polynucleotide refers interchangeably to single-stranded and double-stranded molecules. Unless otherwise indicated, a polynucleotide in any embodiment described herein includes a double-stranded form and two complementary single strands that are known or predicted to constitute a double-stranded form.

[0162] 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.

[0163] 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.

[0164] promoter

[0165] As used herein, the term "promoter" refers to a nucleic acid sequence that effectively and efficiently initiates gene transcription, directing the transcription of a gene's nucleic acid sequence into mRNA. It is typically found upstream (5') of the target gene's coding sequence. A promoter or promoter region generally provides recognition sites for RNA polymerase and other factors necessary for the proper initiation of transcription.

[0166] 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 locations.

[0167] 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.

[0168] As used herein, the term "T cell activation-inducible promoter" refers to a type of promoter that can be induced to activate and thereby initiate the expression of genes driven by the promoter when T cells are activated under the stimulation of certain specific physical or chemical signals.

[0169] 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 stimulating factors to achieve amplification to increase the number of immune cells and activation to enhance the activity of immune cells. The stimulating factor of immune cells can be any factor that can cause immune cells to amplify or enhance their activity. In some embodiments, the stimulating factor can also be an antibody targeting immune cell surface markers such as CD3 and CD28, or a compound activator such as PMA or inomycin. In some embodiments, when immune cells express a T cell receptor (TCR) or a chimeric antigen receptor (CAR) for a specific antigen, the stimulating factor of the immune cell may also include the specific antigen or its binding epitope fragment, which causes TCR and CAR activation by contact with T cells. For example, for T cells expressing anti-B7-H3 chimeric antigen receptors, the stimulating factor can be B7-H3 expressed by tumor cells.

[0170] As used herein, the term "minimal promoter" refers to a short DNA segment that is inactive on its own but can mediate strong transcription when combined with other transcriptional regulatory elements. The property of enhancing transcription is common to minimal promoters, and therefore, the promoter element of the present invention can comprise 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 comprises the miniCMV promoter, which is a minimal promoter derived from human cytomegalovirus.

[0171] The promoters of the present invention include fragments 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 promoter fragments is calculated according to conventional methods in the art, i.e., the base numbering is calculated with the transcription start site as the origin (position +1), with positions upstream of the transcription start site being designated as negative numbers and positions downstream of the transcription start site being designated as positive numbers. For example, the "nucleic acid segment from position -626 to +36 of the GM-CSF promoter" refers to the fragment of the GM-CSF promoter from position 626 upstream to position 36 downstream of the transcription start site.

[0172] In the promoters 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 linked or linked via an additional nucleotide sequence. In one embodiment, to facilitate the insertion of the derived promoter fragment and the minimal promoter into a vector separately during construction, the additional nucleotide sequence used can be a restriction enzyme cleavage site, e.g., GCGGCCGC.

[0173] 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), etc.

[0174] The present invention has been tested and screened for a series of T cell activation-inducible promoters. While the present invention specifically provides promoter elements such as SEQ ID NOs. 11-20 and 22-29, this is merely an exemplary embodiment to illustrate the parent promoter fragments and minimal promoters that may be included in the promoter elements of the present invention. For example, the present invention provides test results for a promoter element comprising positions +36 to -217 of the GM-CSF promoter and a promoter element comprising positions +36 to -626 of the GM-CSF promoter. Those skilled in the art will appreciate that other promoter elements between these positions can achieve the effects of the present invention, such as a promoter element comprising positions +36 to -300 (or -400, -500) of the GM-CSF promoter. Therefore, the present invention encompasses these exemplary promoter elements and their derivatives.

[0175] 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.

[0176] Expression cassette

[0177] The present invention provides an expression cassette comprising the promoter element described in the first aspect of the present invention and an ORF sequence of a target gene.

[0178] 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 an 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 following group: cytokines, chemokines, antibodies, chimeric antigen receptors, but not limited thereto. In one embodiment, the cytokines expressed can 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 chemokines expressed can 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, but not limited to, PD1, 4-1BB, OX40, CD40, CTLA-4, CSF1R, and FAP. In one embodiment, the expressed immunomodulatory factor may also be NAP (neutrophil-activating protein) or MMPs (Matrix metalloproteinases).

[0179] 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 expresses only a very small amount of target protein in the non-activated state of T cells, and can efficiently respond to T cell activation signals and induce large amounts of target protein expression when T cells are activated. This performance can effectively enhance 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.

[0180] Chimeric antigen receptor immune cells (CAR-immune cells)

[0181] The present invention provides a chimeric antigen receptor immune cell that simultaneously expresses a chimeric antigen receptor and an immunomodulatory factor.

[0182] 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 the expression cassette for co-expressing the chimeric antigen receptor and the immunomodulatory factor as described in the third aspect of the present invention.

[0183] 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 a 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.

[0184] 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.

[0185] 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.

[0186] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, once the CAR gene construction for 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.

[0187] 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 of the sixth aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors.

[0188] Natural killer (NK) cells are a major type of immune effector cell that protects 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 possess enhanced anti-tumor cytotoxicity.

[0189] 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 storm; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0190] carrier

[0191] The present invention also provides vectors comprising the expression cassettes of the present invention. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral 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.

[0192] In brief summary, the promoter of the present invention is generally operably linked to the target nucleic acid sequence and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0193] 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, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0194] The expression cassette of the present invention can be cloned into many types of vectors. For example, the expression cassette can be cloned into 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.

[0195] 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. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism and work in the origin of replication, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0196] 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 the 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.

[0197] In order to evaluate the expression of the CAR polypeptide or its portion, 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 that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can 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 to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0198] 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 recipient organisms or tissues, and that encode polypeptides whose expression is clearly indicated by some easily detectable properties, such as enzymatic activity. After DNA has been introduced into recipient cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes can 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 using known techniques or commercially available. Typically, constructs with at least 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be connected to reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0199] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily 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.

[0200] 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.

[0201] 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 method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0202] 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).

[0203] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, may form 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.

[0204] In a preferred embodiment of the present invention, the vector is a retroviral vector.

[0205] preparation

[0206] The present invention provides a preparation comprising 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.

[0207] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or 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.

[0208] Therapeutic applications

[0209] The present invention encompasses therapeutic applications of cells (e.g., T cells) transduced with vectors encoding the expression cassettes of the present invention. Transduced T cells can target tumor cell markers (e.g., B7-H3). Upon activation, these cells induce the expression of immune regulatory factors, leading to sustained T cell activation and immune responses, thereby significantly enhancing their tumor cell-killing efficiency.

[0210] 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 step of administering a CAR cell of the present invention to the mammal.

[0211] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach significantly reduces the risk of graft-versus-host disease, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0212] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain for an extended period of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein 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 CAR elicit a specific immune response against B7-H3 cells.

[0213] Although the data disclosed herein specifically disclose retroviral vectors comprising a combination of an anti-B7-H3 scFv, hinge and transmembrane regions, a CD28 co-stimulatory domain, and a CD3ζ signaling domain, the invention should be construed to encompass any number of variations in each of the construct's components.

[0214] Treatable cancers include tumors that are not vascularized or substantially not yet 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 lymphoma, benign and malignant tumors and malignant tumors, such as sarcoma, cancer and melanoma. Also included are adult tumors / cancers and childhood tumors / cancers.

[0215] 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, granulocytic-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 lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0216] The CAR-modified T cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0217] 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.

[0218] In vitro 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 a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a 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.

[0219] 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.

[0220] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of CAR-modified T cells of the present invention to a subject in need thereof.

[0221] The CAR-modified T cells of the present invention can 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. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0222] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to 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 appropriate dosages can be determined by clinical trials.

[0223] When an "effective amount," "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise 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 a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0224] 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.

[0225] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of relevant treatment modalities, 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 present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. 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 before or after surgery.

[0226] 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. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The CAR-T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0227] The main advantages of the present invention include:

[0228] 1) The present invention provides a series of novel T cell activation-inducible recombinant promoters constructed by modifying and optimizing GM-CSF, IFNg and IL2 promoters. When CAR-T cells are activated, they can efficiently induce the expression of their downstream genes, and their activity is superior to traditional NFAT-responsive promoters constructed based on NFAT (nuclear factor of T cell activation) binding sequences.

[0229] 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 various immunomodulatory molecules such as cytokines and antibody-like proteins in tumors to promote the activity and persistence of CAR-T cells, thereby promoting the application of CAR-T cells in solid tumors.

[0230] 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, for which specific conditions are not specified, are generally 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 according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0231] Example 1 Construction of different versions of B7H3.28ζ.TAIP.mIL2 plasmids

[0232] 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. TAIP-mIL2 was then reverse cloned into SFG.B7H3.28 ζ In the expression vector, a series of B7H3.28 ζ .TAIP.mIL2 vector (as shown in Figure 1). In the recombinant promoter of GM-CSF promoter and minCMV minimal promoter, multiple versions of recombinant promoters with minCMV located at different positions downstream of GM-CSF promoter (pGM-CSF) were constructed (as shown in Figure 1) to enhance the induction activity of pGM-CSF. Among them, SFG.B7H3.28z.CAR was used as a negative control vector, B7H3.28 ζ .NFAT.mIL2 served as a reference vector.

[0233] The construction method is as follows: The variable region (VL and VH) fragments of the light and heavy chains of the anti-B7H3 monoclonal antibody 376.96 were synthesized by gene sequence synthesis, digested with Mlu1 and Nco1, and cloned into the retroviral vector SFG.CAR.CD28ζ to construct the SFG.B7H3.CD28ζ vector. Different segments of the GM-CSF, IFNg, and IL2 promoters were amplified from the human cell genome by PCR, and the mIL2 gene was amplified from the genome of activated mouse T cells. The two were then linked by fusion and reverse cloned into the SFG.B7H3.CD28ζ vector. In the plasmid containing minCMV, the minCMV was introduced downstream of the GM-CSF promoter segment using primers and then cloned into the SFG.B7H3.CD28ζ vector by fusion with the mIL2 gene.

[0234] Example 2 Preparation of Retrovirus Expressing B7H3.28ζ.TAIP.mIL2

[0235] 6×10 5 293T cells were seeded in 6-well plates. 18 hours later, 0.75 μg of different versions of the B7H3.28ζ.TAIP.mIL2 plasmid were mixed with 0.75 μg of the PEQ plasmid and 0.5 μg of the RDF plasmid. The plasmid mixture was then mixed with GeneJuice transfection reagent (Merck Millipore), allowed to stand for 10 minutes, and then transfected into the 293T cells. 48 hours after transfection, the culture supernatant containing the virus was collected, filtered through a 0.45 μm filter, and frozen at -80°C until use.

[0236] Example 3 Preparation and in vitro expansion of CAR-T cells

[0237] 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 a density of 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 2000g for 1.5 hours. After centrifugation, the supernatant was discarded and 3×10 5 After stimulation, the T cells were centrifuged at 1000 g for 10 minutes. 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 CAR positivity by flow cytometry. After 7 days of infection, 3×105 Cells were collected and CAR-T cells were used for B7H3-FC antigen stimulation experiments.

[0238] Example 4 Flow cytometry

[0239] After 4 days of virus infection, 1×10 6 CAR-T cells were stained by adding 0.5 μg of 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 living cells were collected for each sample, and the collected results were analyzed using Flowjo 10 software.

[0240] Results: B7H3.28ζ-TAIP-mIL2 was efficiently expressed in T cells (as shown in Figure 2). Figures 2B and 2C show that the positive rate and expression level of the chimeric antigen receptor were not significantly affected, basically meeting the requirements for use.

[0241] Example 5 B7H3-FC antigen stimulation experiment

[0242] B7H3-FC protein (ACROBiosystem) was diluted in PBS to a final concentration of 1 μg / ml. 500 μl was then 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, 1 mL of serum-containing culture medium was added, and the plate was incubated at room temperature for 10 minutes. 1×10 6 CAR-T cells were plated in 24-well plates without B7H3-FC coating. The same number of CAR-T cells were plated in a 24-well plate without B7H3-FC coating as an unstimulated control group. After incubation at 37°C for 24 hours, 1 ml of supernatant was collected for ELISA. A mouse IL2 ELISA kit (Mabtech) was used to detect the amount of mIL2 secreted in the supernatant of B7H3.28ζ.TAIP.mIL2-infected T cells after B7H3-FC stimulation.

[0243] Results: As shown in Figure 3, after 24 hours of antigen stimulation, a series of constructed inducible promoters all responded to T cell activation and induced the expression and secretion of mIL2. With the exception of the pIFN (+64 to -108) promoter, the inducible activity of the remaining inducible promoters was higher than that of the NFAT promoter. Among them, the recombinant promoters 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) were highly effective in inducing the expression of mIL2. Quantitative results are shown in Table 1 below:

[0244] Table 1 mIL2 expression levels

[0245] The results showed that when minCMV was located -35 bp downstream of pGM-CSF, the pGM-CSF(-35 to 336).minCMV promoter was stronger than the pGM-CSF(-1 to 336).minCMV promoter (as shown in Figure 3). Subsequently, the induction activity of pGM-CSF(-35 to 217).minCMV and pGM-CSF(-1 to 217).minCMV was compared, and the results showed that the induction activity of pGM-CSF(-35 to 217).minCMV was slightly better than that of pGM-CSF(-1 to 217).minCMV (as shown in Figure 4).

[0246] Example 6 Promoter truncation test

[0247] The GM-CSF promoter segment was further truncated to test whether shorter GM-CSF promoters still exhibited potent T cell activation-inducing activity. As shown in Figure 5 , the further truncated pGM-CSF(-35 to -179).minCMV still exhibited comparable induction activity to that of pGM-CSF(-35 to -217).minCMV. However, when the promoter segment was shortened to approximately 100 bp, for example, the pGM-CSF(-35 to 106).minCMV promoter, its induction activity was significantly lower than that of the pGM-CSF(-35 to 217).minCMV promoter, but still higher than that of the promoter synthesized with six repeats of the NFAT binding sequence (Figure 5).

[0248] sequence:

[0249] 1. 376.96 scFv DNA sequence:

[0250] 2. 376.96 scFv amino acid sequence:

[0251] 3.CD8α hinge region and transmembrane region DNA sequence:

[0252] 4.Amino acid sequences of CD8α hinge and transmembrane regions:

[0253] 5. DNA sequence of CD28 intracellular costimulatory domain:

[0254] 6.Amino acid sequence of the CD28 intracellular costimulatory domain:

[0255]

[0256] 7. DNA sequence of CD3ζ intracellular domain:

[0257] 8. Amino acid sequence of CD3ζ intracellular domain:

[0258] 9. Mouse IL2 (mIL2) CDS DNA sequence:

[0259] 10. Mouse IL2 (mIL2) protein amino acid sequence:

[0260] 11.pGM-CSF (+36 to -336) promoter DNA sequence

[0261] 12.pGM-CSF (-1 to -336) promoter DNA sequence

[0262] 13.pGM-CSF (-35 to -336) promoter DNA sequence

[0263] 14.pGM-CSF (+36 to -217) promoter DNA sequence

[0264] 15.pGM-CSF (-1 to -217) promoter DNA sequence

[0265] 16.pGM-CSF (-35 to -217) promoter DNA sequence

[0266] 17.pGM-CSF (+36 to -626) promoter DNA sequence

[0267] 18.pIL2 (+47 to -326) promoter DNA sequence

[0268] 19.pIFN (+64 to -538) promoter DNA sequence

[0269] 20.pIFN (+64 to -108) promoter DNA sequence

[0270] 21. minCMV (CMV minimal promoter) DNA sequence

[0271] 22.pGM-CSF (+36 to -336). minCMV promoter DNA sequence

[0272] 23.pGM-CSF (-1 to -336). minCMV promoter DNA sequence

[0273] 24.pGM-CSF (-35 to -336). minCMV promoter DNA sequence

[0274] 25.pGM-CSF (-1 to -217). minCMV promoter DNA sequence

[0275] 26.pGM-CSF (-35 to -217). minCMV promoter DNA sequence

[0276] 27.pGM-CSF (-35 to -179). minCMV promoter DNA sequence

[0277] 28.pGM-CSF (-35 to -106). minCMV promoter DNA sequence

[0278] 29.pGM-CSF (+36 to -217). minCMV promoter DNA sequence:

[0279] 30.6*NFAT.minIL2 promoter DNA sequence

[0280] 31.PA2 bidirectional PolyA DNA sequence

[0281] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 having or 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 or substantially having 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.

11. The engineered immune cell according to claim 10, characterized in that The immune cells are T cells.

12. A method for preparing engineered immune cells, characterized in that: The method comprises the following steps: transducing the expression cassette as described in claim 6, or the expression cassette for co-expressing CAR and immunomodulatory factors as described in claim 7, or the vector as described in claim 9 into immune cells, thereby obtaining the engineered immune cells.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the vector according to claim 9 and / or the engineered immune cell according to claim 10, and a pharmaceutically acceptable carrier, diluent or excipient.

14. The vector according to claim 9 or the engineered immune cell according to claim 10, which is used for preparing a drug or preparation for preventing and / or treating a disease.

15. The vector or engineered immune cell used as claimed in claim 14, characterized in that: The disease is a tumor with high expression of B7-H3.

16. A method for treating a disease, comprising administering an effective amount of the engineered immune cell of claim 10 or the pharmaceutical composition of claim 13 to a subject in need of treatment.

Images