Differential gene expression regulation system in response to hypoxia environment and use thereof

Abstract

Provided are a differential gene expression regulation system in response to a hypoxia environment and a use thereof. The gene expression regulation system comprises: a hypoxia-sensitive promoter, a target gene expression cassette, and a polynucleotide encoding a truncated oxygen-dependent degradation domain, which can be effectively used in CAR-T immune cell therapy. Since a CAR structure comprises a promoter specifically activated under hypoxia conditions, a CAR functional sequence, and the truncated oxygen-dependent degradation domain, the target killing activity of transduced CAR-T cells in a hypoxia environment is enhanced, thereby achieving good safety while ensuring the effective treatment of CAR-T, and having broad application values and prospects.

Description

A differential gene expression regulation system in response to hypoxic environment and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a differential gene expression regulation system in response to hypoxic environment and application thereof in treating hypoxic diseases, such as tumors. BACKGROUND

[0002] During the development of tumors, due to abnormal vascular formation and insufficient blood supply and excessive proliferation of tumor cells, tumor cells and stromal cells often have difficulty in obtaining nutrients and oxygen, resulting in the existence of transient or permanent hypoxic regions in the assembly of most solid tumors (Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006; 441: 437-443.). Hypoxia is a common feature of various solid tumors, and the development of new technologies for treating solid tumors against hypoxic microenvironment helps to enhance the targeting and specificity of tumor treatment and reduce damage to normal tissues.

[0003] The HIF (hypoxia-inducible factor) family is rapidly responsive to changes in the oxygen content of cells, and is formed by an oxygen-unstable alpha subunit (HIF1alpha, HIF2alpha) and a constitutively stable beta subunit HIF1beta to form a heterodimer, which enters the nucleus to bind to the hypoxia-responsive elements (HREs) of the target gene DNA, thereby regulating the gene expression pattern of cells, resulting in increased glycolysis and decreased mitochondrial activity, reduced oxygen consumption, and ultimately adapted to the hypoxic microenvironment.

[0004] The two proline residues (HIF1-a: P402 / P564, HIF2-a: P405 / P531) in the oxygen-dependent degradation domain (ODD) contained in the oxygen-labile alpha subunit of HIF (HIF1a, HIF2a) are hydroxylated by the protein of proline hydroxylase domain (PHDs) under normoxia, which leads to ubiquitination and proteasome degradation of the alpha subunit. Studies have shown that the fusion of the ODD domain of HIF-1a to the target protein can make it degrade under normoxic environment, thereby reducing the protein level, while under hypoxic environment, the protein is stable and enriched. However, the target protein coupled with ODD under normoxia is still at a high level in the cell, and the protein level is still low under hypoxic conditions, so that the target protein has little difference in different environments of normoxia and hypoxia, which has certain application limitations (Juillerat A, Marechal A, Filhol JM, et al. An oxygen sensitive self-decision making engineered CAR T-cell [J]. Sci Rep. 2017, 7: 39833; Liao Q, He H, Mao Y, et al. Engineering T cells with hypoxia-inducible chimeric antigen receptor (HiCAR) for selective tumor killing. Biomark Res. 2020, 8(1): 56). Therefore, it is necessary to further develop more safe and effective hypoxia-sensitive target gene expression regulation technology.

[0005] The microenvironment of solid tumors is relatively complex, such as tumor suppressive microenvironment, tumor heterogeneity, etc., which limits the application of CAR-T therapy. At present, CAR-T has not yet achieved the case of significant efficacy similar to hematological tumors in the treatment of solid tumors. Moreover, solid tumors are generally derived from somatic mutations, making it difficult to find tumor-specific targets, and solid tumor CAR-T has much greater off-target risk than hematological tumor CAR-T. Therefore, it is necessary to construct a CAR structure that is specifically activated in the tumor microenvironment. Due to the rapid growth of tumor tissue and insufficient blood supply, hypoxia becomes a distinct feature in the microenvironment of solid tumors. By designing a regulatable promoter that is specifically activated under hypoxic conditions, the purpose of specifically activating downstream protein expression in the tumor microenvironment is achieved, thereby significantly improving the effectiveness and safety of CAR-T cells.

[0006] CD276 (B7-H3) is a member of the immune-regulatory ligand B7 family that modulates T lymphocyte activation and differentiation, which affects immune responses and cancer progression through immune and non-immune pathways. CD276 can inhibit T cell proliferation and down-regulate cytokine production (Vigdorovich V, Ramagopal UA, et al. Structure and T cell inhibition properties of B7 family member, B7-H3. Structure. 2013; 21(5): 707-17.). CD276 inhibits the activity of T cells, NK cells, macrophages, neutrophils, dendritic cells by promoting the production of IL-10 and TGF-β1, and inhibits the secretion of IFN-γ, IL-2, perforin, granzyme B, thereby forming a tumor immunosuppressive microenvironment (Long C, Li G, Zhang C, et al. B7-H3 as a Target for CAR-T Cell Therapy in Skull Base Chordoma. Front Oncol. 2021; 11: 659662.). In addition, it is reported that in addition to regulating the immune microenvironment, CD276 can activate signaling pathways such as ERK, PI3K and Stat3 in cancer cells, which can lead to accelerated cell proliferation and tumor growth (Ding M, Liao H, Zhou N, et al. B7-H3-Induced Signaling in Lung Adenocarcinoma Cell Lines with Divergent Epidermal Growth Factor Receptor Mutation Patterns. Biomed Res Int. 2020; 2020: 8824805.). The expression level of CD276 in most normal tissues is usually relatively low. In contrast, in malignant tumor tissues, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, biliary tract cancer, oral squamous cell carcinoma, endometrial cancer, squamous cell carcinoma, gastric cancer, glioma, melanoma and adrenal gland malignant tumor, etc., the expression level of CD276 is significantly up-regulated. In addition, it has been found that higher CD276 expression levels are associated with poorer prognosis in cancer patients (Getu, A. A., Tigabu, A., Zhou, M., et al. New frontiers in immune checkpoint B7-H3 (CD276) research and drug development. Mol Cancer. 2023, 22, 43.).These characteristics make CD276 a promising target for cancer therapy.

[0007] SUMMARY

[0008] Therefore, the present application aims to provide a differential gene expression regulation system in response to hypoxic environment and its application in the treatment of hypoxic diseases such as solid tumors.

[0009] The differential gene expression regulation system in response to hypoxic environment provided by the present application is composed of a hypoxic response element HRE that can bind with a hypoxia-inducible factor (HIF) complex, an activity-limited promoter, a target gene expression frame, and a truncated oxygen-dependent degradation domain. The system has the following advantages: (1) the promoter is mainly induced to express under hypoxic environment, and has low activity under normoxic environment, which reduces the leakage expression of the target functional molecules, especially the chimeric antigen receptor (CAR), in the normal tissue environment, and enhances the biosafety of the expression regulation system; (2) the protein encoded by the target gene is coupled with the truncated oxygen-dependent degradation domain, which further enhances the hypoxic specificity of the system without significantly increasing the influence of the target protein molecular structure on its activity function, and maximally reduces the leakage expression of the target gene.

[0010] In a first aspect, the present application provides a gene expression regulation system in response to hypoxic environment, comprising: a hypoxia-sensitive promoter, a target gene expression frame, and a polynucleotide encoding a truncated oxygen-dependent degradation domain. The hypoxia-sensitive promoter is a regulatable promoter that upregulates RNA transcription in response to hypoxia to upregulate gene expression, and the truncated oxygen-dependent degradation domain is used to regulate the degradation of the target protein. The system has obvious hypoxic expression specificity and can specifically express the target protein in a hypoxic environment, which can greatly improve the biosafety of the immune cell treatment method when used for precise targeted therapy of tumors.

[0011] According to some embodiments of the present application, the hypoxia-sensitive promoter is a promoter connected with a hypoxic response element (HRE), preferably a truncated promoter connected with multiple, preferably at least 3, more preferably 4 or 5, hypoxic response elements in series. The truncated promoter provides the minimum nucleotide sequence required to initiate transcription of the target gene. The nucleotide sequence of the truncated promoter connected with 4 hypoxic response elements in series is shown in SEQ ID NO: 1 or a functional variant thereof; the nucleotide sequence of the truncated promoter connected with 5 hypoxic response elements in series is shown in SEQ ID NO: 2 or a functional variant thereof.

[0012] According to some embodiments of the present application, the promoter is selected from one or more of the group consisting of human cytomegalovirus promoter, the promoter of HSV thymidine kinase, the promoter of simian virus 40, the adenovirus late promoter, and synthetic promoters, preferably human cytomegalovirus promoter truncated CMVd2 and / or CMVmini.

[0013] According to some embodiments of the present application, the truncated oxygen-dependent degradation domain is a core oxygen degradation peptide segment of the oxygen-dependent degradation (ODD) domain of HIF-1a of different lengths, for example, a peptide segment of 18 residues (ODD18) or 22 residues (ODD22) cut from the oxygen-dependent degradation (ODD) domain of HIF-1a. For example, the sequence of the polynucleotide encoding the truncated oxygen-dependent degradation domain is shown in SEQ ID NO: 3 (ODD18) or a functional variant thereof, or SEQ ID NO: 5 (ODD22) or a functional variant thereof, respectively, and the corresponding amino acid sequence is shown in SEQ ID NO: 4 or a functional variant thereof, or SEQ ID NO: 6 or a functional variant thereof, respectively. The present application optimizes the combination of these DNA elements of different lengths to effectively induce the expression of the target gene in a hypoxia-specific manner.

[0014] According to some embodiments of the present application, the adjacent hypoxia response elements in the tandem hypoxia response elements are separated by a random spacer nucleotide segment, preferably a random spacer nucleotide segment of 15-30 bp. By introducing a random spacer nucleotide segment of, for example, 15-30 bp to separate the tandem HREs, appropriate physical space can be provided for the binding of HIF protein to HRE, promoting the effective binding of transcription factors or regulatory factors on the promoter, increasing the activity of the promoter in a hypoxic environment, and reducing the DNA genetic instability caused by the repetitive sequences formed by directly tandem HREs.

[0015] According to some embodiments of the present application, the target gene comprises a polynucleotide encoding one or more anti-tumor effector molecules selected from the group consisting of cytokines, chemokines, antibodies, immune checkpoint blockers, T cell receptors, and cytotoxic molecules, etc.

[0016] Preferably, the cytokines and chemokines are selected from one or more of the group consisting of GM-CSF, IFN-a / b / g, IL-2, IL-3, IL-7, IL-12, IL-15, IL-21, IL-33, IL-35, IL-37, CCL4, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, MIP-1a, and MIP-1b.

[0017] Preferably, the cytotoxic molecule is selected from one or more of TNF-a, T cell engager, bispecific T cell engager protein (BiTE), chimeric antigen receptor, apoptosis gene, pyroptosis gene, and toxin.

[0018] Preferably, the antibody is selected from one or more of agonistic antibodies, such as anti-CD28 antibody, anti-4-1BB antibody, anti-ICOS antibody, anti-GITR antibody, anti-OX40 antibody, and anti-CD27 antibody; and blocking antibodies, such as one or more of CTLA-4 antibody, PD-1 antibody, PD-L1 antibody, LAG-3 antibody, and Tim3 antibody.

[0019] According to some preferred embodiments of the present application, the gene of interest comprises a polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR that binds to a tumor antigen. The structure of the CAR can be a conventional first, second, or third generation CAR structure, or a novel CAR structure such as a modified dual CAR, a regulatable CAR structure (e.g., FRB / FKBP12 regulation), etc.

[0020] The antigen target of the chimeric antigen receptor is selected from one or more of AXL, EGFR, MHC, CD24, CD47, FAP, CD147, HER-2, CD55, CD59, ROR1, ROR2, CD73, CD133, CD44v6, CD44v7, CD44v8, CD126, CD171, CEA, EpCAM, TAG72, IL-13R a, EGFRvIII, GD2, GD3, FR a, PSCA, PSMA, GPC3, CAIX, Claudin 18.2, VEGFR2, PD-L1, PD-L2, MSLN, MUC1, c-Met, FOLR1, B7-H3 (CD276), and Trop2.

[0021] Further, the hinge region of the CAR can be derived from IgG, CD8, CD7, and CD4; the transmembrane region of the CAR can be derived from CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, and CD86; and the intracellular signaling region of the CAR can be derived from CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86, and CD127.

[0022] Preferably, the gene-of-interest expression cassette comprises a polynucleotide encoding a chimeric antigen receptor targeting CD276. Further preferably, the CAR targeting CD276 comprises an anti-CD276 single-chain antibody, a CD8 hinge region, a CD8 transmembrane region, and a CD137 (4-1BB) and CD3ξ dual stimulatory signaling region, i.e., the specific structure of the CAR is CD276 ScFv-CD8 hinge region-CD8 transmembrane region-CD137-CD3ξ, the amino acid sequence of which is shown in SEQ ID NO: 7 or a functional variant thereof. Thus, the present application provides a hypoxia-inducible promoter-driven CAR targeting CD276 and immune cells comprising the same, the structure of the CAR targeting CD276 not only can effectively enhance the expression of the CAR molecule under hypoxic environment, improve the killing ability of CAR-T cells against tumor target cells, but also has very low leakage expression under normoxia, which can greatly enhance the precise targeted therapy of CAR-T cells on tumors. Accordingly, the present application also provides a nucleic acid molecule encoding a hypoxia-inducible promoter-driven CAR targeting CD276, the nucleotide sequence of which is shown in SEQ ID NO: 8 or a functional variant thereof.

[0023] According to some preferred embodiments of the present application, the gene expression control system is a hypoxia-inducible promoter-driven CAR targeting CD276 expression control system, which comprises a hypoxia-sensitive promoter, a CAR expression cassette targeting CD276, and a polynucleotide encoding a truncated oxygen-dependent degradation region. Further preferably, the nucleotide sequence of the gene expression control system is shown in SEQ ID NO: 8 or a functional variant thereof.

[0024] According to some embodiments of the present application, the gene-of-interest can comprise a polynucleotide encoding a plurality of CARs, for example, a polynucleotide encoding two CARs targeting different antigens or different recognition sites of the same antigen.

[0025] According to some embodiments of the present application, the gene-of-interest can comprise a polynucleotide encoding a CAR and other anti-tumor effector molecules, for example, a combination of a CAR and one or more factors selected from the group consisting of GM-CSF, IFN-α / β / γ, IL-2, IL-3, IL-7, IL-12, IL-15, IL-21, IL-33, IL-35, IL-37, CCL4, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CCL3, and CCL4.

[0026] According to some embodiments of the application, the gene of interest can comprise a polynucleotide encoding one or more components of a CAR and a Natural Killer Cell Receptor (NKR), thereby forming a NKR-CAR. The NKR component can be a transmembrane domain, a hinge domain, and a cytoplasmic domain of any one or more of the Natural Killer Cell Receptors selected from the group consisting of Killer-cell immunoglobulin-like receptors (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; Natural Cytotoxicity Receptors (NCR), such as NKp30, NKp44, NKp46; Signaling Lymphocyte Activation Molecules (SLAM) family of immune cell receptors, such as CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; Fc receptors (FcR), such as CD16 and CD64; and Ly49 receptors, such as LY49A and LY49C. The NKR-CAR molecule can interact with an adaptor molecule or an intracellular signaling domain, such as DAP12.

[0027] According to some embodiments of the application, the polynucleotide encoding the CAR further comprises a polynucleotide encoding a tag peptide, such as a Flag tag peptide, a His tag peptide, a Myc tag peptide, a HA tag peptide, or a combination thereof. The presence of these tags is for the convenience of detection and does not contribute to the function of the gene of interest per se.

[0028] According to some embodiments of the application, the polynucleotide encoding the CAR can be operably linked to one or more promoters selected from the group consisting of cytomegalovirus immediate early gene promoter (CMV), elongation factor 1 alpha promoter (EF1-alpha), phosphoglycerate kinase-1 promoter (PGK), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / herpes simplex thymidine kinase promoter (MCI), beta actin promoter (beta-ACT), simian virus 40 promoter (SV40), myeloproliferative sarcoma virus enhancer, and a negative control region-deleted, dl587rev primer binding site-substituted (MND) promoter.

[0029] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a gene expression control system responsive to hypoxic environment according to the first aspect of the present application.

[0030] According to some embodiments of the present application, the isolated nucleic acid molecule is a nucleic acid molecule encoding a target CD276 chimeric antigen receptor under the control of a hypoxia-inducible promoter.

[0031] Preferably, the target CD276 chimeric antigen receptor comprises an anti-CD276 single chain antibody, a CD8 hinge region, a CD8 transmembrane region, and a CD137 (4-1BB) and CD3 zeta dual costimulatory signaling region.

[0032] Further preferably, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 8 or a functional variant thereof.

[0033] In a third aspect, the present application provides a vector comprising the isolated nucleic acid molecule according to the second aspect of the present application.

[0034] According to some embodiments of the present application, the vector is selected from one or more of the following: DNA, RNA, plasmid, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia virus vector, herpes simplex virus vector, forest encephalitis virus vector, polio virus vector, Newcastle disease virus vector, and transposon, preferably the vector is a lentiviral vector.

[0035] According to some specific embodiments of the present application, the vector is selected from one or more of the following: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus, caprine arthritis- encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0036] In a fourth aspect, the present application provides a host cell comprising the vector according to the third aspect of the present application, or the isolated nucleic acid molecule according to the second aspect of the present application integrated (exogenously) into the chromosome of the host cell or delivered non-integratively into the host cell for expression of a gene of interest.

[0037] According to some embodiments of the present application, the host cell is an isolated human cell, such as one or more of an isolated embryonic stem cell, an umbilical cord blood-derived stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose stem cell, a T cell, an NK cell, an NKT cell, and a macrophage.

[0038] According to some embodiments of the present application, the host cell is a genetically engineered human cell, such as a genetically engineered immune cell, including one or more of genetically engineered T cells, NK cells, NKT cells, and macrophages, preferably genetically engineered T cells. Alternatively, the genetically engineered immune cell is selected from one or more of chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), chimeric antigen receptor NKT cells (CAR-NKT cells), chimeric antigen receptor macrophages (CAR-M cells), and T cell receptor T cells (TCR-T cells). The cell) and T cell receptor T cells (TCR-T cells).

[0039] Preferably, the genetically engineered immune cell is a chimeric antigen receptor T cell (CAR-T cell). Wherein the CAR-T cell is introduced with the isolated nucleic acid molecule according to the second aspect of the present application by transfection or transduction with the vector according to the third aspect of the present application, thereby enabling the expression of chimeric antigen receptor under hypoxic environment.

[0040] According to some embodiments of the present application, the introduction includes the simultaneous or sequential introduction of one or more vectors to co-express the CAR and other molecules as a fusion protein. For example, the other molecules are inhibitory molecules in their native state, but can be engineered to enhance the activity of the cell by coupling with CAR, including PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR and TGFR beta. The extracellular domain of the inhibitory molecule can be fused to the transmembrane domain and intracellular signaling domain to form, for example, a PD1 CAR.

[0041] In a fifth aspect, the present application provides a pharmaceutical composition for treating hypoxic diseases, ischemic diseases or cancer, comprising the differential gene expression regulation system according to the first aspect of the present application, the isolated nucleic acid molecule according to the second aspect of the present application, the vector according to the third aspect of the present application, or the host cell according to the fourth aspect of the present application, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0042] According to some embodiments of the present application, the cancer is a solid tumor, for example one or more solid tumors selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, head and neck cancer, bladder cancer, and skin cancer.

[0043] In a sixth aspect, the present application provides use of the differential gene expression regulation system according to the first aspect of the present application, the isolated nucleic acid molecule according to the second aspect of the present application, the vector according to the third aspect of the present application, or the host cell according to the fourth aspect of the present application in the preparation of a medicament for treating a hypoxic disease, an ischemic disease, or a cancer.

[0044] According to some embodiments of the present application, the cancer is a solid tumor, for example one or more solid tumors selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, head and neck cancer, bladder cancer, and skin cancer.

[0045] In a seventh aspect, the present application provides a method for treating a hypoxic disease, an ischemic disease, or a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the differential gene expression regulation system according to the first aspect of the present application, the isolated nucleic acid molecule according to the second aspect of the present application, the vector according to the third aspect of the present application, or the host cell according to the fourth aspect of the present application.

[0046] According to some embodiments of the present application, the cancer is a solid tumor, for example one or more solid tumors selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, head and neck cancer, bladder cancer, and skin cancer.

[0047] In the present application, the term "functional variant" of a sequence generally refers to a variant having substantially the same function (e.g., having the function of a chimeric antigen receptor) as the protein, peptide, gene, or nucleic acid molecule, and having at least 85% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to its amino acid sequence or nucleotide sequence. In certain embodiments, the functional variant of the protein, peptide, gene, or nucleic acid molecule has the same function as it.

[0048] As to a gene or nucleic acid molecule, functional variants of its sequence include:

[0049] (1) a nucleotide sequence complementary to the indicated nucleotide sequence;

[0050] (2) a nucleotide sequence that hybridizes under stringent hybridization conditions to the indicated nucleotide sequence;

[0051] (3) a nucleotide sequence having at least 80% homology, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, to the indicated nucleotide sequence; or

[0052] (4) a nucleotide sequence obtained by deletion, substitution or insertion of one or more, for example 1, 2, 3, 4 or 5, nucleotides in the indicated nucleotide sequence.

[0053] In the case of a protein or peptide, functional variants of the sequence thereof include:

[0054] (1) an amino acid sequence having at least 80% homology, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, to the indicated amino acid sequence; or

[0055] (2) an amino acid sequence obtained by deletion, substitution or insertion of one or more, for example 1, 2, 3, 4 or 5, amino acids in the indicated amino acid sequence.

[0056] The differential gene expression regulation system responding to hypoxic environment provided by the application adopts a hypoxia microenvironment-induced promoter, and can strengthen the expression of factors such as target genes and proteins under a hypoxic environment. The use of the system can specifically activate CAR-T cells in a hypoxic microenvironment, effectively clear tumors in the body, reduce the risk of CAR-T cells attacking normal tissues off-target, and has high safety.

[0057] The CAR structure targeting CD276 and the CAR-T cell or immune cell provided by the application can effectively kill tumors, and achieve a good balance between effectiveness and safety in CAR-T treatment, and is a promising cellular immunotherapy product.

[0058] The CAR-T cell provided by the application has enhanced activity under a hypoxic environment, and can be used for adoptive cell therapy in tumor treatment, and has important guiding significance for the clinical application of CAR-T cells and the development of new strategies for combined treatment of tumors.

[0059] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limitation of the length, they will not be repeated one by one here.

[0060] Brief Description of the Drawings

[0061] Hereinafter, the embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0062] Figure 1 is a structural schematic diagram of a system for differentially regulating the expression of a target gene in response to a hypoxic environment (i.e., hypoxia-specific induction of expression) according to the present application.

[0063] Figure 2 is a structural schematic diagram of a plasmid for hypoxia-specific induction of GFP expression and a control plasmid in Example 1; wherein the plasmid for hypoxia-specific induction of GFP expression mediates hypoxia-specific expression of GFP via HRE, and the control plasmid overexpresses via the EF1a promoter.

[0064] Figure 3 is a flow cytometry analysis of the expression of a plasmid for hypoxia-regulated GFP expression under different oxygen concentrations in 293T cells in Example 1; wherein the GFP plasmid that is completely mediated by HRE and odd18 for hypoxia-specific expression of GFP shows obvious hypoxia specificity, while the plasmid that is only mediated by HRE for hypoxia expression fails to show significant hypoxia specificity.

[0065] Figure 4 is a flow cytometry analysis of the expression of CMVd2 promoter-mediated GFP under different oxygen concentrations in T cells in Example 2; wherein in human T cells, the hypoxia-inducible system of 4*HRE-CMVd2 combined with different oxygen instability domains can effectively mediate hypoxia-specific expression of GFP.

[0066] Figure 5 is a flow cytometry analysis of the expression of CMVmini promoter-mediated GFP under different oxygen concentrations in T cells in Example 2; wherein in human T cells, 4*HRE-CMVmini combined with different oxygen instability domains can effectively mediate hypoxia-specific expression of GFP.

[0067] Figure 6 is a flow cytometry analysis of the expression of GFP mediated by unseparated direct concatenation of 5*HRE and CMVd2 promoter and different oxygen instability domains under different oxygen concentrations in T cells in Example 3; wherein the unseparated 5*HRE system can partially mediate hypoxia-specific expression of GFP.

[0068] Figure 7 is a flow cytometry analysis of the expression of GFP mediated by unseparated direct concatenation of 5*HRE and CMVmini promoter and different oxygen instability domains under different oxygen concentrations in T cells in Example 3.

[0069] Figure 8 is the sequencing results of HRE sequences in different series in Example 4, showing that the separated series of HRE repeat sequences have better genetic stability than the non-separated ones.

[0070] Figure 9 is the flow cytometry analysis of the hypoxia-specific expression of CD276-41BB CAR-T cells containing different hypoxia environment-induced gene expression regulation systems in Example 5; wherein the separated HRE combination system mediates the excellent hypoxia-specific expression of CD276-CAR-T.

[0071] Figure 10 is the detection results of the killing of tumor cell line NCI-H292 cells by CD276-41BB CAR-T cells containing different hypoxia environment-responsive gene expression regulation systems in Example 5 under different oxygen environments, showing that the CD276-CAR-T cells with hypoxia-specific expression have the ability to specifically kill NCI-H292 cells under hypoxic conditions.

[0072] Figure 11 is the detection results of the killing of tumor cell line SK-OV-3 cells by CD276-41BB CAR-T cells containing different hypoxia environment-responsive gene expression regulation systems in Example 5 under different oxygen environments, showing that the CD276-CAR-T cells with hypoxia-specific expression have the ability to specifically kill SK-OV-3 cells under hypoxic conditions.

[0073] Figure 12 is the safety evaluation of CD276-41BB CAR-T cells with hypoxia-regulated expression in a tumor-bearing immunodeficient mouse model in Example 6.

[0074] Figure 13 is the therapeutic effect of CD276-41BB CAR-T cells with hypoxia-regulated expression on two tumors in a tumor-bearing immunodeficient mouse model in Example 6.

[0075] Best mode for carrying out the invention

[0076] The present application will be further described in conjunction with specific examples, and the advantages and features of the present application will become more apparent from the following description. The following examples are only used to illustrate the present application, but not to limit the scope of the present application. Therefore, non-essential improvements and adjustments to the embodiments of the present application made by those skilled in the art based on the content of the present application still fall within the scope of the present application.

[0077] The structural schematic diagram of the differential gene expression regulation system responding to hypoxic environment of the application is shown in Figure 1, which comprises tandem HREs and truncated promoters (for example, Mini Promoter), target genes (GOI) and truncated oxygen-dependent degradation domains ODD18 or ODD22 separated by random sequences. Different combinations can be selected according to the specific expression requirements of the target gene, such as different numbers of tandem HREs, different strengths of promoters and different oxygen-dependent degradation domains, to achieve optimal hypoxic-specific expression control.

[0078] The GFP used in the embodiments of the application is a conventional eGFP, for example, the nucleotide sequence thereof is shown in SEQ ID NO: 9, and the amino acid sequence thereof is shown in SEQ ID NO: 10.

[0079] The amino acid sequence of the CD276-41Z CAR structure in the embodiments of the application is shown in SEQ ID NO: 7, and the nucleotide sequence thereof is shown in SEQ ID NO: 30. The 41BB in the embodiments of the application is CD137, and Z is CD3ξ, and the sequences thereof are conventional sequences.

[0080] In the embodiments of the application, the method for verifying whether the hypoxic model is constructed is to use the recombinant plasmid to transfect 293T cells or to use lentivirus to infect activated T cell constructs to construct an in vitro hypoxic cell model. The cells are divided into two parts and placed in culture boxes with different oxygen concentrations (21% O2 normoxia and 1% O2 hypoxia) for a certain period of time. Then, the expression of CAR is detected by flow cytometry analysis instrument to detect the GFP fluorescence signal or flag tag antibody.

[0081] The lentivirus packaging method in the embodiments of the application comprises:

[0082] ① HEK293T cell processing: 24h before transfection, collect HEK293T cells in the logarithmic growth phase, inoculate them in a 10cm cell culture dish (6-8x10 6(1) Cells were grown in 10 mL of complete DMEM medium and cultured at 37°C with 5% CO2 for 18–24 h until the cell density reached 70–90%, at which point plasmid transfection could be performed. ② HEK293T cell transfection: 1 mL of basal DMEM medium was added to a 15 mL centrifuge tube. A transfection mixture was prepared at a mass ratio of lentiviral expression plasmid: packaging plasmid: envelope plasmid = 1:3:1, with a total plasmid content of 15 μg / dish. 30 μL of TurboFect transfection reagent was added at a plasmid (μg) : transfection reagent (μL) ratio of 1:2. After incubation at room temperature for 15–20 min, the mixture was added to a culture dish containing HEK293T cells. The cells were cultured at 37°C with 5% CO2 for 48 h. The viral supernatant was then collected, centrifuged at 1000 × g at 4°C for 10 min, and the precipitate at the bottom of the tube was discarded. ③ Lentiviral Concentration: The collected viral supernatant was further filtered through a 0.45 μm filter. Lenti-X lentivirus concentration reagent (1 / 3 volume of viral supernatant) was added, and the mixture was inverted several times. The mixture was incubated overnight at 4°C. Centrifuged at 2000×g, 4°C for 45 min. A white precipitate was observed at the bottom of the centrifuge tube; this was the concentrated viral particles. The supernatant was carefully discarded. The white precipitate was resuspended in 1 / 50 to 1 / 100 volume of blank RPMI 1640 medium, aliquoted, and stored at -80°C for later use. ④ Lentiviral Titer Determination: Jurkat T cells were prepared according to a 1×10⁻⁶ titer... 5 Cells / well were seeded onto 96-well U-plates, and the collected lentivirus concentrate was serially diluted 10-fold. 100 μL of the virus dilution was added to each well, and protamine sulfate was added to adjust the concentration to 10 μg / mL. The cells were centrifuged at 1000×g and 32℃ for 90 min, and incubated overnight. The medium was then replaced with fresh RPMI 1640 complete medium, and the cells were cultured for another 48 h. The proportion of fluorescently positive cells was detected by flow cytometry. The viral titer was calculated using the following formula:

[0083] Viral titer (TU / mL) = 1 × 10 5 ×Proportion of fluorescent positive cells / 100 × 1000 × Corresponding dilution factor.

[0084] In embodiments of the present invention, the method for preparing lentivirus-mediated genetically engineered T cells includes adding concentrated lentivirus vectors to a layer of 1×10⁻⁶ cells at an MOI of 3. 6 In 48-well plates containing pre-activated peripheral blood mononuclear cells, protamine sulfate, an infection-promoting reagent, was added to a working concentration of 10 μg / mL. The cells were centrifuged at 1000 × g and 32°C for 90 min for infection. After overnight culture, the medium was replaced with fresh T-cell growth medium for continued culture. Fresh T-cell growth medium was added every 2–3 days, and the cell density was adjusted to 0.5–2 × 10⁶ cells / well. 6The activated T cells were removed from the culture medium 6-7 days after the infection, and the genetically engineered T cells were continuously cultured and expanded until the cells were rested (9-14 days after the removal of the activated T cells). Then, the subsequent functional experiments were performed.

[0085] In the embodiments of the present application, the method for detecting the killing ability of different CAR-T cells on target cells is a luciferase-based cytotoxicity assay. First, 1 x 10 4 SK-OV-3-Luc (human ovarian cancer cells modified with a firefly luciferase gene) or NCI-H292-Luc (human lung cancer cells modified with a firefly luciferase gene) were inoculated in a 96-well flat-bottom black plate at 100 μL of culture medium per well and incubated in a 37°C, 5% CO2 cell incubator for 18 h. The next day, genetically engineered CAR-T cells and non-transduced T cells cultured at the same time were added to the wells containing the target cells at an effector cell: target cell ratio of 1:1, 2:1, and 4:1, respectively, and incubated for 20 h. After the incubation, the luciferase activity of the target cells was detected using a microplate luminometer.

[0086] The formula for calculating the cell killing rate is as follows:

[0087] Cell killing rate (%) = (luciferase activity of the non-transduced T cell group - luciferase activity of the experimental group) / luciferase activity of the non-transduced T cell group x 100

[0088] In the embodiments of the present application, the in vivo verification uses NCG mice, which are one of the most complete mouse models of immune system defects to date and are very suitable for long-term transplantation and efficacy evaluation. The tumor-forming target cells used in the in vivo verification are human lung cancer cell line NCI-H292 and human esophageal squamous cell carcinoma cell line KYSE-150 cells, which are used to construct a human solid tumor-bearing mouse model.

[0089] The experimental methods used in the following examples are conventional experimental methods in the art unless otherwise specified. The experimental materials used in the following examples were purchased from biochemical reagent sales companies unless otherwise specified.

[0090] Example 1: Construction of a GFP-expressing recombinant plasmid with a hypoxia-responsive gene expression regulation system and expression test in 293T cells

[0091] (1) Construction of pXW-HRE-GFP-odd18

[0092] ​The hypoxic promoter sequence 4 / 5HRE-CMVd2 Promoter was synthesized by a truncated CMV promoter CMVd2 promoter (as shown in SEQ ID NO: 31) and four or five HRE elements, and the nucleotide sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Then, the lentiviral expression vector, 4 / 5HRE-CMVd2 Promoter, GFP protein coding sequence and coding sequence of the truncated oxygen-dependent degradation domain ODD18 were linked, transformed and single colonies were picked to construct the hypoxia-specific expression plasmids pXW-4HRE-CMVd2-GFP-odd18 (SEQ ID NO: 11) and pXW-5HRE-CMVd2-GFP-odd18 (SEQ ID NO: 13) as shown in FIG. 2 (the amino acid sequence of GFP-odd18 is shown in SEQ ID NO: 12). As a comparison, the GFP expression recombinant plasmids pXW-4HRE-CMVd2-GFP (SEQ ID NO: 15) and pXW-5HRE-CMVd2-GFP (SEQ ID NO: 16) carrying the conventional overexpression promoter EF1a and not containing the truncated oxygen-dependent degradation domain ODD18 were constructed, respectively, as well as the pXW-EF1a-GFP (SEQ ID NO: 14).

[0093] (2) Verification of hypoxia model in 293T cells

[0094] The 293T cells transfected with the five plasmids pXW-EF1a-GFP, pXW-4HRE-CMVd2-GFP, pXW-5HRE-CMVd2-GFP, pXW-4HRE-CMVd2-GFP-odd18 and pXW-5HRE-CMVd2-GFP-odd18 cultured under hypoxic conditions were set as experimental groups, and those cultured under normoxic conditions were set as control groups, and the GFP positive rate and fluorescence expression intensity were detected after 24 hours. The results are shown in FIG. 3, and it can be found that the overexpression system of the conventional EF1a promoter is limited in expression under the hypoxic condition of the cells (FIG. 3A), while the hypoxia-sensitive expression system of the application has good hypoxia induction characteristics, and the expression amount of GFP is significantly increased under the physiological hypoxia condition (1% O2) compared with that under the normoxic condition (21% O2) (FIG. 3D, 3E). When GFP does not carry the oxygen-dependent degradation region ODD18 and ODD22, the leaky expression under normoxia is obviously increased, and the hypoxia induction specificity is poor (FIG. 3B, 3C).

[0095] Example 2: Verification of hypoxia-regulated GFP expression system in human T cells

[0096] To further verify the specificity and stability of the gene expression regulation system responding to hypoxic environment, the human T cells were infected with the lentivirus packaged with the GFP plasmid having different expression systems, and the expression of GFP was detected under hypoxic and normoxic culture conditions. The genetically engineered T cells of the experimental group and the control group were respectively placed under normoxic conditions (21% O2) or hypoxic conditions (1% O2) for 24h, and after the culture was completed, the cells were collected and washed once with FACS buffer (1x PBS containing 2% FBS), centrifuged at 500xg for 5min, and then resuspended with 300μL FACS buffer, and the expression of GFP was detected using a flow cytometer.

[0097] To test the effect of different combinations on the gene expression regulation system responding to hypoxic environment, the combinations of 4HRE and CMVd2 promoter and different oxygen-dependent degradation domains were first analyzed: 4HRE-CMVd2-GFP, 4HRE-CMVd2-GFP-ODD18, 4HRE-CMVd2-GFP-ODD22 (SEQ ID NO: 17 and SEQ ID NO: 18), 4HRE-CMVd2-GFP-ODD full (SEQ ID NO: 19 and SEQ ID NO: 20). The results are shown in Figure 4. When no oxygen-dependent degradation region is carried, the target gene GFP has higher expression fluorescence intensity under hypoxic conditions, indicating that the HRE-mediated hypoxic induction system can effectively overcome the disadvantage of poor host cell state under hypoxic conditions. However, from the cell positive rate, the hypoxic specificity is poor, and there is a high positive rate under normoxic and hypoxic conditions (Figure 4A). The hypoxic regulation expression system of the present application has very significant hypoxic specificity expression in human T cells, very low positive rate under normoxic conditions and higher positive rate under hypoxic conditions, and can maintain a good fluorescence intensity (background leakage under normoxic conditions <5%, efficient induction of expression of GFP under hypoxic conditions, and the positive rate increases to about 60% or more (Figures 4B-C). Although the ODD domain of the full-length HIF1a protein (ODD full) can also effectively mediate specific expression under hypoxic conditions, due to the overbulky ODD full-length structure, the expression amount of the target gene is affected (the fluorescence intensity is significantly lower than that of the truncated oxygen-dependent degradation domain ODD18 or ODD22 (Figure 4D).

[0098] Next, the CMVd2 promoter was changed to CMVmini promoter (as shown in SEQ ID NO: 21), and the combinations of 4 HREs with CMVmini promoter and different oxygen-dependent degradation domains were analyzed: 4HRE-CMVmini-GFP, 4HRE-CMVmini-GFP-ODD18, 4HRE-CMVmini-GFP-ODD22, 4HRE-CMVmini-GFP-ODD full. The results are shown in Figure 5, the overall trend of the ability of hypoxia-specific induction of GFP mediated by 4 HREs in tandem combined with CMVmini promoter and different oxygen-dependent degradation domains in human T cells is consistent with the CMVd2 promoter in Figure 4, and the expression system carrying the truncated oxygen-dependent degradation domain has good hypoxia expression specificity (Figure 5B, C). Although the proportion of positive cells activated by expression under hypoxic conditions is similar, the fluorescence intensity is lower than that mediated by the CMVd2 promoter, indicating that the activity of the promoter has an important influence on the expression ability of the hypoxia induction system.

[0099] Example 3: Verification of non-spaced tandem HRE-mediated hypoxia-inducible expression system in human T cells

[0100] To compare the difference between the spaced tandem HRE elements of the present application and the non-spaced tandem HRE elements, the 4*HRE elements in the GFP plasmid used in Example 2 were replaced with non-spaced 5 HREs (5tandem-HRE, as shown in SEQ ID NO: 22), and combinations of the non-spaced 5 HREs with the CMVd2 promoter and different oxygen-dependent degradation domains were constructed: 5tandem-HRE-CMVd2-GFP, 5tandem-HRE-CMVd2-GFP-ODD18, 5tandem-HRE-CMVd2-GFP-ODD22, 5tandem-HRE-CMVd2-GFP-ODD full; and combinations of the non-spaced 5 HREs with the CMVmini promoter and different oxygen-dependent degradation domains: 5tandem-HRE-CMVmini-GFP, 5tandem-HRE-CMVmini-GFP-ODD18, 5tandem-HRE-CMVmini-GFP-ODD22, 5tandem-HRE-CMVmini-GFP-ODD full. After hypoxia induction of the corresponding genetically engineered T cells, the GFP expression was detected, and the results are shown in Figures 6 and 7. The hypoxia-specific inducible expression system constructed by the non-spaced 5 HREs (5tandem-HRE) directly linked together in combination with the CMVd2 or CMVmini promoter and different oxygen-dependent degradation domains also showed certain specific expression under hypoxic conditions, but the effect was obviously inferior to that of the spaced tandem HRE system of the present application (the positive rate decreased from about 60% to about 30% under hypoxic induction, and the background leakage positive rate under normoxia also increased slightly), indicating that the non-spaced 5tandem-HRE elements, although more in number than the 4*HRE of the present application, failed to fully exert the activation ability for expression under hypoxic conditions.

[0101] Example 4: Verification of genetic stability of tandem HRE repeat sequences

[0102] It is known that genomic DNA repeat sequences have genetic instability, and there is a possibility of mutation during DNA replication or repair. The cultured and expanded genetically engineered T cells (5tandem-HRE-CMVd2-GFP and 4*HRE-CMVd2-GFP) were each taken at 5x10 5One, 2000xg centrifugation for 5min, after the end of the supernatant, using conventional genomic DNA extraction method to extract genomic DNA as a template, according to the HRE element upstream and downstream sequence of lentiviral vector plasmid sequence design specific primers, genomic DNA PCR amplification. PCR amplification product purification and TA cloning after sanger sequencing. The sequencing results are shown in Figure 8, a part of the unseparated direct tandem HRE T cell genomic DNA clone sample exists HRE element repeat sequence partial deletion, 5 tandem HRE elements will lose 1-2, genetic stability is poor. While the separated HRE repeat sequence of the application has good genetic stability, all cloned sequencing has not found fragment loss.

[0103] Example 5: Expression test of CD276 CAR containing hypoxia environment responsive gene expression control system in human T cells

[0104] The lentiviral expression plasmid of example 1, the lentiviral vector of example 2 and the genetic engineering T cell preparation method were used to construct different hypoxia inducible expression systems, including unseparated 5*tandem-HRE and separated 4*HRE and 5*HRE CMVd2-CD276 CAR-T, respectively 5tandem-HRE-CMVd2-CD276-41Z-odd18 (as shown in SEQ ID NO: 23), 4HRE-CMVd2-CD276-41Z-odd18 (as shown in SEQ ID NO: 8), 4HRE-CMVmini-CD276-41Z-odd18 (as shown in SEQ ID NO: 24), 5HRE-CMVd2-CD276-41Z-odd18 (as shown in SEQ ID NO: 25), 5HRE-CMVmini-CD276-41Z-odd18 (as shown in SEQ ID NO: 26) CAR-T cells. The amino acid sequence of the CD276-41Z CAR of the above hypoxia inducible expression system is shown in SEQ ID NO: 27. The above five kinds of genetically engineered T cells were placed in normoxic conditions (21% O2) or hypoxic conditions (1% O2) for 24h, and then the cells were collected and detected for CD276 CAR molecule expression using flow cytometry antibody PE-anti-DYKDDDDK.

[0105] As shown in FIG. 9, the CD276 CAR-T cells controlled by the gene expression control system responding to hypoxic environment in different combinations specifically highly express CAR molecules under hypoxic environment, while the expression under normoxic condition is weak. Among them, the unseparated tandem HRE system (5*tandem HRE) has weak expression activation ability for the target gene, and has more leakage expression under normoxic condition (FIG. 9A), while the CD276 CAR mediated by the gene expression control system responding to hypoxic environment of the application has good hypoxic-specific expression (compared with normoxic condition, the CAR positive rate under hypoxic condition is 10-20 times higher, and the average fluorescence intensity is also 2-3 times higher, FIGS. 9B-9E).

[0106] Example 6: In vitro killing ability detection of CD276 CAR-T cells containing the gene expression control system responding to hypoxic environment.

[0107] The CAR-T cells in Example 5 were detected for their killing ability in vitro on cultured tumor cell lines. The killing of NCI-H292 and SK-OV-3 cells by CD276 CAR-T cells was performed by luciferase-based cytotoxicity assay, and the evaluation results are shown in FIGS. 10 and 11. Among them, the CD276 CAR-T cells with the 4*HRE and 5*HRE mediated gene expression control system of the application showed high efficient specific killing ability under hypoxic condition, while the unseparated 5tandem-HRE hypoxic induction system as a control also had partial hypoxic expression specificity, but had higher leakage killing ability under normoxic condition, and the killing ability under hypoxic condition was also significantly weaker than that of the application. Under different effector cell: target cell ratios, the killing ability of the 4*HRE induction expression system was slightly smaller than that of the 5*HRE system, but the latter also brought slightly higher leakage under normoxic condition. In addition, the activity of the promoter also affected the killing of CAR-T cells, and the killing activity of CD276 CAR-T cells mediated by CMVd2 promoter was slightly stronger than that of CAR-T cells mediated by CMVmini promoter.

[0108] Example 7: Detection of the ability of CD276 CAR-T cells containing the gene expression control system responding to hypoxic environment to treat tumors in immunodeficient mice

[0109] To detect the therapeutic effect of the CD276 CAR-T cells containing the gene expression control system responding to the hypoxic environment of the present application on tumors in vitro, traditional EF1a overexpression promoter driven EF1a-Her2-41Z CAR-T cells (nucleotide sequence as shown in SEQ ID NO: 28, amino acid sequence as shown in SEQ ID NO: 29) and hypoxia specific 4HRE-CMVd2-CD276-41Z-odd18 CAR-T cells were constructed for the reinfusion treatment of immunodeficient mice.

[0110] After the purchased 8-week-old female NCG mice were adaptively fed in a sterile isolator for 1 week, they were partially shaved on the back. The shaving can use depilatory cream or animal shaver, so that the skin at the tumor cell inoculation site is exposed. 5x10 6 NCI-H292 and KYSE-150 cells were inoculated subcutaneously on the left and right sides of the back of NCG mice, respectively, with 125 μL of tumor cell suspension injected on each side. The tumor formation and health status of the mice were observed every 2-3 days after tumor cell inoculation, and the baseline tumor volume was measured with a vernier caliper after tumor formation and subsequent experiments. The formula for calculating the tumor volume is as follows:

[0111] Tumor volume = (long diameter x short diameter 2 ) / 2.

[0112] According to the tumor volume, the mice were randomly divided into Control (PBS) group, EF1a-Her2 CAR-T and 4HRE-CD276-41Z-odd18 CAR-T experimental groups. Ten days after tumor inoculation, the prepared CAR-T cells or the same volume of PBS solution were injected intravenously, and each tumor-bearing mouse was reinfused with 5x10 6 CAR-T cells. After tumor cell inoculation, the body weight change and tumor size of the mice were measured every 2-3 days. Each group of mice was reinfused with the same amount of CAR-T cells or PBS intravenously again on the 18th day.

[0113] As shown in FIGS. 12 and 13, compared with the traditional EF1a promoter overexpressed Her2 target CAR-T cells, the hypoxia-sensitive CD276 CAR-T cells of the present application can effectively inhibit the growth of KYSE-150 (esophageal squamous cell carcinoma) and NCI-H292 (lung cancer) cells, and significantly prolong the survival of mice. The untreated mice (PBS) all died within 30 days, the traditional Her2 CAR-T treated mice survived for a maximum of 42 days, while the hypoxia-sensitive CD276 CAR-T cell treated mice were still mostly alive at 50 days (4 / 5). In terms of body weight, the hypoxia-sensitive CD276 CAR-T cell treated mice were also very stable, except for the 2nd mouse that died prematurely (FIG. 12D); while the mice in the traditional Her2 CAR-T group generally lost weight, and the mouse that gained weight may have been due to rapid tumor growth (FIG. 12D). From the growth curves of the two tumors, the hypoxia-sensitive CD276 CAR-T cells can effectively control the growth of esophageal squamous cell carcinoma (KYSE-150) and lung cancer cells (NCI-H292), especially for the treatment of NCI-H292, which effectively shrunk the tumor, while the traditional EF1a-Her2 CAR-T cells could not control the growth of the two tumors (FIG. 13B, D).

[0114] Finally, it should be noted that although the present application has been disclosed above with preferred embodiments, the above embodiments are only used to illustrate the technical solutions of the present application and not to limit it. Any modification, equivalent change and modification of the above embodiments by any person skilled in the art according to the technical essence of the present application to obtain equivalent embodiments of equivalent changes, without departing from the purpose and scope of the technical solutions of the present application, should be covered in the scope of the claims of the present application.

Claims

1. A gene expression regulation system responsive to hypoxic environment, comprising: a hypoxia-sensitive promoter, a gene-of-interest expression cassette, and a polynucleotide encoding a truncated oxygen-dependent degradation domain.

2. The gene expression regulation system of claim 1, wherein the hypoxia-sensitive promoter is a promoter linked to hypoxia response elements (HREs), preferably a truncated promoter linked to multiple, preferably at least 3, more preferably 4 or 5, hypoxia response elements in tandem; preferably, the nucleotide sequence of the truncated promoter linked to 4 hypoxia response elements in tandem is set forth in SEQ ID NO: 1 or a functional variant thereof; the nucleotide sequence of the truncated promoter linked to 5 hypoxia response elements in tandem is set forth in SEQ ID NO: 2 or a functional variant thereof; preferably, the promoter is selected from one or more of human cytomegalovirus promoter, the promoter of HSV thymidine kinase, the promoter of simian virus 40, adenovirus late promoter, and synthetic promoters, preferably human cytomegalovirus promoter truncated CMVd2 and / or CMVmini.

3. The gene expression regulation system of claim 1 or 2, wherein the truncated oxygen-dependent degradation domain is a peptide segment of 18 residues (ODD18) or 22 residues (ODD22) truncated from the oxygen-dependent degradation (ODD) domain of HIF-1a; preferably, the sequence of the polynucleotide encoding the truncated oxygen-dependent degradation domain is set forth in SEQ ID NO: 3 (ODD18) or a functional variant thereof, or SEQ ID NO: 5 (ODD22) or a functional variant thereof, respectively, and the corresponding amino acid sequence is set forth in SEQ ID NO: 4 or a functional variant thereof, or SEQ ID NO: 6 or a functional variant thereof, respectively.

4. The gene expression regulation system of any one of claims 1 to 3, wherein adjacent hypoxia response elements in the tandem hypoxia response elements are separated by a random spacer nucleotide segment, preferably a random spacer nucleotide segment of 15-30 bp.

5. The gene expression regulation system of any one of claims 1 to 4, wherein the gene-of-interest comprises a polynucleotide encoding one or more anti-tumor effector molecules selected from the group consisting of cytokines, chemokines, antibodies, immune checkpoint blockers, T cell receptors, and cytotoxic molecules; preferably, the cytokines and chemokines are selected from one or more of GM-CSF, IFN-a / b / g, IL-2, IL-3, IL-7, IL-12, IL-15, IL-21, IL-33, IL-35, IL-37, CCL4, CCL20, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, MIP-1a, and MIP-1b. ​ Preferably, the cytotoxic molecule is selected from one or more of TNF-a, T cell engager, bispecific T cell engager protein (BiTE), chimeric antigen receptor, apoptosis gene, pyroptosis gene, and toxin; Preferably, the antibody is selected from one or more of an agonistic antibody, such as an anti-CD28 antibody, an anti-4-1BB antibody, an anti-ICOS antibody, an anti-GITR antibody, an anti-OX40 antibody, and an anti-CD27 antibody; and a blocking antibody, such as one or more of a CTLA-4 antibody, a PD-1 antibody, a PD-L1 antibody, a LAG-3 antibody, and a Tim3 antibody; Further preferably, the gene of interest comprises a polynucleotide encoding a chimeric antigen receptor, such as a polynucleotide encoding a chimeric antigen receptor that binds to a tumor antigen; Further preferably, the antigen target of the chimeric antigen receptor is selected from one or more of AXL, EGFR, MHC, CD24, CD47, FAP, CD147, HER-2, CD55, CD59, ROR1, ROR2, CD73, CD133, CD44v6, CD44v7, CD44v8, CD126, CD171, CEA, EpCAM, TAG72, IL-13R a, EGFRvIII, GD2, GD3, FR a, PSCA, PSMA, GPC3, CAIX, Claudin 18.2, VEGFR2, PD-L1, PD-L2, MSLN, MUC1, c-Met, FOLR1, B7-H3 (CD276), and Trop2; Preferably, the gene of interest expression cassette comprises a polynucleotide encoding a chimeric antigen receptor targeting CD276; further preferably, the chimeric antigen receptor targeting CD276 comprises an anti-CD276 single chain antibody, a CD8 hinge region, a CD8 transmembrane region, and CD137 (4-1BB) and CD3 zeta costimulatory signaling regions; more preferably, the amino acid sequence of the chimeric antigen receptor targeting CD276 is set forth in SEQ ID NO: 7 or a functional variant thereof.

6. An isolated nucleic acid molecule comprising the gene expression control system responsive to hypoxic environment according to any one of claims 1 to 5; Preferably, the isolated nucleic acid molecule is a nucleic acid molecule encoding a hypoxia-inducible promoter-targeted CD276 chimeric antigen receptor; Further preferably, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO: 8 or a functional variant thereof.

7. A vector comprising the isolated nucleic acid molecule according to claim 6; Preferably, the vector is selected from one or more of DNA, RNA, plasmid, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia viral vector, herpes simplex viral vector, forest brain encephalitis viral vector, polio viral vector, Newcastle disease viral vector, and transposon; more preferably, the vector is a lentiviral vector; Preferably, the vector is selected from one or more of DNA, RNA, plasmid, retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia viral vector, herpes simplex viral vector, forest brain encephalitis viral vector, polio viral vector, Newcastle disease viral vector, and transposon; more preferably, the vector is a lentiviral vector; Preferably, the vector is selected from one or more of the following: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV) virus, caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

8. A host cell comprising the vector of claim 7, or the isolated nucleic acid molecule of claim 6 integrated (exogenously) into the chromosome of the host cell or delivered non-integratively into the host cell for expression of a gene of interest; Preferably, the host cell is one or more of an isolated human cell, such as an isolated embryonic stem cell, an umbilical cord blood-derived stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose stem cell, a T cell, an NK cell, an NKT cell and a macrophage. Preferably, the host cell is a genetically engineered human cell, such as a genetically engineered immune cell, including one or more of genetically engineered T cells, NK cells, NKT cells, and macrophages, preferably genetically engineered T cells; or the genetically engineered immune cell is selected from one or more of chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), chimeric antigen receptor NKT cells (CAR-NKT cells), chimeric antigen receptor macrophages (CAR-macrophages), and chimeric antigen receptor B cells (CAR-B cells). Preferably, the genetically engineered immune cell is a chimeric antigen receptor T cell (CAR-T cell).

9. A pharmaceutical composition for treating a hypoxic disease, an ischemic disease or a cancer, comprising the differential gene expression modulation system of any one of claims 1 to 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, or the host cell of claim 8, and optionally a pharmaceutically acceptable carrier, diluent or excipient; Preferably, the cancer is a solid tumor, such as one or more solid tumors selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestinal cancer, large intestinal cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, head and neck cancer, bladder cancer and skin cancer.

10. Use of the differential gene expression modulation system of any one of claims 1 to 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, or the host cell of claim 8 in the manufacture of a medicament for treating a hypoxic disease, an ischemic disease or a cancer; Preferably, the cancer is a solid tumor, such as one or more solid tumors selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestinal cancer, large intestinal cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, head and neck cancer, bladder cancer and skin cancer. ​

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