Il-33 modified chimeric antigen receptor and uses thereof

Abstract

The application relates to the technical field of immunotherapy, in particular to an IL-33 modified chimeric antigen receptor and application thereof. The application discloses a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence; the extracellular domain comprises an antigen recognition region and a hinge region; and the chimeric antigen receptor is modified by IL-33. The IL-33 modified chimeric antigen receptor provided by the application can significantly increase the anti-tumor function of T cells.

Description

Technical Field

[0001] This specification relates to the field of immunotherapy technology, and in particular to an IL-33 modified chimeric antigen receptor and its applications. Background Technology

[0002] In recent years, chimeric antigen receptor T cell (CAR-T) therapy has received considerable attention and achieved significant breakthroughs as an adoptive immunotherapy. CAR-T cell therapy involves modifying a patient's peripheral blood T cells in vitro using genetic engineering methods, endowing these T cells with the ability to target and recognize tumor cell surface antigens. After in vitro expansion and culture, these cells are then reinfused into the patient to treat tumors. Although CAR-T technology has made progress in treating hematologic malignancies such as lymphoma and leukemia, many challenges remain in treating solid tumors.

[0003] The key feature of CAR-T cell therapy is its ability to target any antigen on the selected cell surface, generating an immune response against tumor cells in a manner independent of the major histocompatibility complex (MHC), thereby eliminating the tumor. However, the application of CAR-T technology in solid tumors is limited due to the lack of effective targets and the immunosuppressive effects of the tumor microenvironment. Therefore, improving the efficacy of CAR-T therapy for solid tumors is currently a major research focus. Summary of the Invention

[0004] To address the aforementioned technical challenges, this application constructs CAR-T cells targeting the tumor antigen CD133, establishes a preliminary technical platform for CAR-T cell preparation, and conducts a series of preclinical studies. Based on CD133-CAR-T cell therapy technology, this application further constructs a functionally enhanced IL-33.CD133-CAR-T cell that continuously expresses the cytokine IL-33 for the treatment of solid tumors.

[0005] This application provides a chimeric antigen receptor comprising: an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence; the extracellular domain comprising an antigen recognition region and a hinge region; and the chimeric antigen receptor being modified with IL-33.

[0006] This application also provides a polynucleotide sequence selected from: (1) a polynucleotide sequence encoding the chimeric antigen receptor described above; and (2) a complementary sequence to the polynucleotide sequence described in (1).

[0007] This application also provides a nucleic acid construct containing the above-described polynucleotide sequence.

[0008] This application also provides a lentiviral vector system containing the above-mentioned nucleic acid constructs and lentiviral vector auxiliary components.

[0009] This application also provides a cell containing the above-described polynucleotide sequence, or containing the above-described nucleic acid construct, or infected with the above-described lentiviral vector system.

[0010] This application also provides the application of the above-mentioned chimeric antigen receptor, polynucleotide sequence, nucleic acid construct, and lentiviral vector system in the preparation of products for any one or more of the following purposes: (1) preparing T cells; (2) enhancing the anti-tumor function of T cells.

[0011] This application also provides the application of the aforementioned chimeric antigen receptor, polynucleotide sequence, nucleic acid construct, lentiviral vector system, or T cell in the preparation of tumor therapeutic products. Preferably, the tumor is a tumor expressing CD133. More preferably, the tumor is selected from liver cancer, adrenocortical carcinoma, urothelial carcinoma of the bladder, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colon cancer, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, and hepatocellular carcinoma. The tumor is selected from one or more of the following: cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, or retinoblastoma. More preferably, the tumor is selected from one or more of liver cancer, pancreatic cancer, or colon cancer. Even more preferably, the tumor is liver cancer.

[0012] The IL-33-modified chimeric antigen receptor proposed in this specification offers beneficial effects including, but not limited to: The treatment of malignant solid tumors such as liver cancer, pancreatic cancer, and colon cancer remains a challenge in cancer therapy. Traditional treatment methods, including surgery, radiotherapy, and chemotherapy, still have limitations. Many patients with metastatic or recurrent diseases still face poor prognoses, and survival rates have not significantly improved. This invention first constructs CAR-T cells targeting the tumor antigen CD133, initially establishing a technical platform for CAR-T cell preparation, and conducting a series of preclinical studies. Based on CD133-CAR-T cell therapy technology, this invention constructs a functionally enhanced IL-33.CD133-CAR-T cell that continuously expresses the cytokine IL-33. Through a series of in vitro experiments, it is confirmed that IL-33.CD133-CAR-T cells have a stronger tumor-killing effect compared to CD133-CAR-T cells. Furthermore, it is demonstrated that IL-33.CD133-CAR-T cells are a safe, specific, and effective CAR-T cell type with significant clinical application value. Attached Figure Description

[0013] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0014] Figure 1 This is a schematic diagram of the structure of IL-33.CD133-CAR-T according to some embodiments of this application.

[0015] Figure 2 This is a schematic diagram of the entire expression frame structure of a CD133-CAR-T expressing IL-33, constructed according to some embodiments of this application.

[0016] Figure 3 Based on the RT-PCR results shown in some embodiments of this application, the target gene is expressed at a high level in CD133-CAR-T and IL-33.CD133-CAR-T.

[0017] Figure 4 According to flow cytometry results shown in some embodiments of this application, CD133 is highly expressed in human hepatocellular carcinoma cells Hep3B.

[0018] Figure 5A and 5B Based on the in vitro killing experiment results shown in some embodiments of this application, the results show that the antitumor effect of IL-33.CD133-CAR-T is significantly better than that of CD133-CAR-T. Detailed Implementation

[0019] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0020] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0021] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0022] Interleukin-33 (IL-33) is a bifunctional cytokine from the IL-1 family, highly expressed in endothelial cells, epithelial cells, and fibroblasts, and particularly highly expressed in hepatic stellate cells. As an alarm protein, IL-33 participates in maintaining tissue homeostasis, signaling through inhibition of the IL-1 receptor-associated ST2 receptor and inducing Th2 immune responses. Upregulation of IL-33 expression in the tumor microenvironment significantly increases the infiltration of CD8+ in tumor tissue. + The ratio of T cells to NK cells is increased, and their effector function is enhanced. Downregulation of IL-33 expression is crucial for tumor immune evasion and is an important mechanism leading to tumor "immune neglect." Previous studies have confirmed that the IL-33 receptor ST2 is expressed on CD8. + This finding was observed on T cells, Th1 cells, NK cells, and NKT cells, indicating that IL-33 / ST2 plays an important role in type I antitumor immune responses.

[0023] This application provides a chimeric antigen receptor comprising: an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence; the extracellular domain comprising an antigen recognition region and a hinge region; and the chimeric antigen receptor being modified with IL-33.

[0024] The term "antigen recognition region" refers to one or more extracellular regions of a chimeric antigen receptor that are specific to a particular antigen.

[0025] The term "transmembrane domain" refers to any three-dimensional protein structure that is thermodynamically stable within a membrane. This three-dimensional protein structure can be a single α-helix, a transmembrane β-barrel, a β-helix of bacitracin A, or any other structure. The length of a transmembrane helix is ​​typically about 20 amino acids. Generally, a transmembrane domain refers to a single transmembrane α-helix of a transmembrane protein, also known as an integrative protein. The primary function of a transmembrane domain is to anchor CARs to the T cell membrane, and there is evidence that transmembrane domains may also be involved in CAR cellular function. In some embodiments, the transmembrane domain can be a native protein, such as CD4, CD8α, CD3ζ, or CD28. In some embodiments, preferably, the transmembrane domain is CD28.

[0026] In some embodiments, the chimeric antigen receptor may include an antigen recognition region, a hinge region, a co-stimulatory signal transduction region, an intracellular CD3ζ segment, and IL-33 connected in sequence. In some embodiments, the IL-33 may be the full-length IL-33 or a fragment thereof.

[0027] The term "hinge" refers to a variable-length amino acid sequence typically encoded between two or more domains in a polypeptide construct. The hinge extends from the transmembrane domain to the extracellular domain of the binding unit. The hinge functions to provide flexibility to overcome spatial barriers and helps increase length to allow the antigen-binding domain to access the target epitope. The chosen hinge affects CAR function because differences in hinge region length and composition influence flexibility, CAR expression, signal transduction, epitope recognition, and activation output strength.

[0028] In some embodiments, the co-stimulatory signal transduction region may be selected from one or more of the CD27 fragment, CD28 fragment, CD134 fragment, 4-1BB fragment, OX40 fragment, and ICOS fragment.

[0029] The costimulatory signaling region refers to a portion of the CAR containing the intracellular domain of the costimulatory molecule. The costimulatory molecule is a cell surface molecule, in addition to an antigen receptor or its ligand, that is essential for an effective lymphocyte response to an antigen. In some embodiments, amino acid residues or functional derivatives thereof from the cytoplasmic domain of CD3ζ can functionally deliver the initial signal necessary for T cell activation. Various costimulatory domains have been reported to confer different properties. For example, the 4-1BB (CD137) costimulatory domain has shown enhanced persistence in in vivo xenograft models (Milone et al., Mol Ther 2009; 17:1453-1464; Song et al., Cancer Res 2011; 71:4617-4627).

[0030] The term "CD28" refers to protein differentiation group 28, which is one of the proteins expressed on T cells that provides the co-stimulatory signals required for T cell activation and survival.

[0031] In some embodiments, the chimeric antigen receptor includes an antigen recognition region, a hinge region, a CD28 fragment, a 4-1BB fragment, an intracellular CD3ζ segment, and IL-33 connected in sequence.

[0032] In some embodiments, the amino acid sequence of the CD28 fragment is as shown in SEQ ID NO: 13, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 13.

[0033] The term "sequence" in this article should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the amino acid sequence, unless a more specific interpretation is required in this article.

[0034] The "homology" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Homology" also indicates the percentage of identical or conserved amino acid substitutions. Methods for evaluating the degree of homology between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence homology is typically measured using sequence analysis software. For instance, homology can be determined using the BLAST program in the NCBI database.

[0035] In some embodiments, the amino acid sequence of the 4-1BB fragment is as shown in SEQ ID NO: 14, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 14.

[0036] In some embodiments, the amino acid sequence of the CD3ζ intracellular segment is as shown in SEQ ID NO: 15, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 15.

[0037] In some embodiments, the CD3ζ intracellular segment and the IL-33 fragment are connected by a linker fragment, preferably T2A.

[0038] In some embodiments, the amino acid sequence of the IL-33 fragment is as shown in SEQ ID NO: 20, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 20.

[0039] In some embodiments, the antigen recognition region is selected from single-chain antibodies against tumor surface antigens. In some embodiments, preferably, the single-chain antibody is selected from single-chain antibodies against CD133.

[0040] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Single-chain antibody fragments (scFvs) are antibodies composed of variable regions of the antibody heavy and light chains linked by a short peptide (linker) of 15–20 amino acids. scFvs endow T cells with the ability to specifically recognize and bind to target antigens, exhibiting significantly increased affinity for target antigens compared to unmodified native TCR-T cells. CAR-T cells' recognition and binding of target antigens via scFvs is independent of MHC antigen presentation, preventing tumor cells from escaping through MHC molecule regulation and endowing CAR-T cells with the ability to recognize non-peptide antigens.

[0041] In some embodiments, the transmembrane domain is selected from one or more transmembrane regions of CD28, CD4, CD8α, OX40, or H2-Kb.

[0042] In some embodiments, the hinge region is selected from one or more of the CD28 hinge region, CD8α hinge region, CD4 hinge region, and immunoglobulin IgG4 hinge region.

[0043] In some embodiments, the amino acid sequence of the hinge region of immunoglobulin IgG4 is as shown in SEQ ID NO: 16, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 16.

[0044] In some embodiments, the antigen recognition region of the chimeric antigen receptor is selected from a single-chain antibody against CD133, and the hinge region is selected from the CD28 hinge region and the immunoglobulin IgG4 hinge region; in some embodiments, the transmembrane domain is selected from the CD28 transmembrane region; in some embodiments, the intracellular domain includes the intracellular segment of CD28, the 4-1BB intracellular segment, the CD3ζ intracellular segment, and the IL-33 fragment connected in sequence.

[0045] In some embodiments, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 21, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 21.

[0046] This application also provides a polynucleotide sequence selected from: (1) a polynucleotide sequence encoding the chimeric antigen receptor described above; and (2) a complementary sequence to the polynucleotide sequence described in (1).

[0047] In some embodiments, the polynucleotide sequence is as shown in SEQ ID NO: 19, or has more than 80% homology with the amino acid sequence shown in SEQ ID NO: 19.

[0048] This application also provides a nucleic acid construct containing the above-described polynucleotide sequence.

[0049] In some embodiments, the nucleic acid construct is a vector; in some embodiments, preferably, the nucleic acid construct is a lentiviral vector containing a replication start site, a 3'LTR, and a 5'LTR.

[0050] As used herein, "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. A vector can deliver fragments of nucleic acids, or individual polynucleotides, into a host cell. It may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. Vectors according to this application may be in circular or linear (linearized) form and also include vector fragments. The term "vector" also includes artificial chromosomes or similar individual polynucleotides that allow the transfer of exogenous nucleic acid fragments.

[0051] The term "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in that they can infect non-dividing cells; they can deliver genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0052] The term "lentiviral vector" refers to a vector containing one or more nucleic acid sequences derived from at least a portion of the lentiviral genome. Lentiviral vectors may contain non-coding sequences of one or more proteins derived from lentiviruses (such as HIV-1).

[0053] In some embodiments, the nucleic acid construct can be used to transform host cells. The nucleic acid construct further includes one or more regulatory sequences operatively linked to the aforementioned polynucleotide sequence. The coding sequence of the CAR described in this application can be manipulated in various ways to ensure the expression of the protein. The nucleic acid construct can be manipulated according to the expression vector or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0054] In some embodiments, the polynucleotide sequence encoding the CAR of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phage particles, phage derivatives, animal viruses, and granules. Further, in some embodiments, the vector can be an expression vector. In some embodiments, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers.

[0055] Methods for introducing genes into cells and expressing genes into cells are known in the art. Vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0056] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and others. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0057] This application also provides a lentiviral vector system containing the aforementioned nucleic acid constructs and lentiviral vector auxiliary components. In some embodiments, the lentiviral auxiliary components may include lentiviral packaging plasmids and cell lines. In some embodiments, the lentiviral vector system can be prepared by viral packaging with the assistance of lentiviral packaging plasmids and cell lines. The method for constructing the lentiviral vector system is a commonly used method in the art.

[0058] This application also provides a T cell containing the above-described polynucleotide sequence, or containing the above-described nucleic acid construct, or infected with the above-described lentiviral vector system. In some embodiments, the T cell may be derived from a human or other mammal.

[0059] In some embodiments, T cells may be CD4 + T cells, CD8 + T cells, naive T (TN) cells, effector T (TEFF) cells, memory T cells, stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, regulatory T (Treg) cells, helper T cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells are any one or more of the following:

[0060] This application also provides the application of the above-mentioned chimeric antigen receptor, polynucleotide sequence, nucleic acid construct, and lentiviral vector system in the preparation of products for any one or more of the following purposes: (1) preparing T cells; (2) enhancing the anti-tumor function of T cells.

[0061] In some embodiments, the application further includes enhancing the killing effect of T cells on tumor cells. In some embodiments, the application further includes enhancing the killing effect of T cells on CD133-expressing tumor cells. In some embodiments, the tumor is selected from one or more of the following: liver cancer, adrenocortical carcinoma, urothelial carcinoma of the bladder, breast cancer, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, bile duct carcinoma, colon cancer, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, or retinoblastoma. In some embodiments, preferably, the tumor is selected from one or more of liver cancer, pancreatic cancer, or colon cancer. In some embodiments, more preferably, the tumor is liver cancer.

[0062] This application also provides the application of the aforementioned chimeric antigen receptor, polynucleotide sequence, nucleic acid construct, lentiviral vector system, or T cell in the preparation of tumor therapeutic products. Preferably, the tumor is a tumor expressing CD133. More preferably, the tumor is selected from liver cancer, adrenocortical carcinoma, urothelial carcinoma of the bladder, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colon cancer, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, and hepatocellular carcinoma. The tumor is selected from one or more of the following: cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, or retinoblastoma. More preferably, the tumor is selected from one or more of liver cancer, pancreatic cancer, or colon cancer. Even more preferably, the tumor is liver cancer.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0064] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0065] Example 1: Construction of a chimeric antigen receptor expression vector targeting CD133 and a chimeric antigen receptor expression vector expressing interleukin-33 targeting CD133

[0066] In this invention, the chimeric antigen receptor targeting CD133 can be simply referred to as CD133-CAR.

[0067] In this invention, the chimeric antigen receptor that expresses interleukin-33 (IL-33) and targets CD133 can be simply referred to as IL-33.CD133-CAR.

[0068] In this invention, such as Figure 2As shown, CD133-CAR is composed of a sequentially linked peptide that binds CD133, a CD28 transmembrane region (CD28-TM), an intracellular 4-1BB, and an intracellular CD3ζ fusion.

[0069] The peptide that binds to CD133 is an anti-CD133 single-chain antibody (scFv). This single-chain antibody includes a light chain variable region (V... L ) and heavy chain variable region (V H ).

[0070] The light chain variable region (V) L The encoding nucleotide sequence of ) is shown in SEQ ID NO: 1, specifically as follows:

[0071] GACATTGTTCTCTCCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTATATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0072] The heavy chain variable region (V) H The encoding nucleotide sequence of ) is shown in SEQ ID NO: 2, specifically as follows:

[0073] CTCGAGGTGAAGCTGGTGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATAACCTTCACAGACTATTCAATGCACTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCA TCATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTACCGATTACGGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0074] The encoding nucleotide sequence of the anti-CD133 single-chain antibody (scFv) is shown in SEQ ID NO: 3, specifically:

[0075] GACATTGTTCTCTCCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTATATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAATCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGGGTGGTTCCTCTAGATCTTCCCTCGAGGTGAAGCTGGTGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTC AATGCACTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCATCATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTACCGATTACGGGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0076] The coding nucleotide sequence of the CD28 transmembrane domain (CD28-TM) is shown in SEQ ID NO: 4, specifically as follows:

[0077] ATGTTCTGGGTGCTGGTGGTGGTGGGCGGGGTGCTGGCCTGCTACAGCCTGCTGGTGACAGTGGCCTTCATCATCTTTTGGGTG

[0078] The coding nucleotide sequence of intracellular 4-1BB is shown in SEQ ID NO: 5, specifically as follows:

[0079] CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0080] The nucleotide sequence encoding intracellular CD3ζ is shown in SEQ ID NO: 6, specifically:

[0081] CGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAA GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG

[0082] A linker fragment of immunoglobulin IgG4 hinge region between the CD133 peptide and the CD28 transmembrane region (CD28-TM) is used as the linker fragment. The encoding nucleotide sequence of the immunoglobulin IgG4 hinge region fragment is shown in SEQ ID NO: 7, specifically:

[0083] GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCGAAGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC AGGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCCAAAGCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAA GAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCTCCCTCCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTG CCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGT GGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG

[0084] like Figure 2 As shown, T2A-Tag BFP was linked to the C-terminus of CD3ζ intracellularly to facilitate the recognition of CD133-CAR.

[0085] Sequencing results showed that the full-length gene sequence of CD133-CAR was correct and consistent with expectations.

[0086] Specifically, the encoding nucleotide sequence of CD133-CAR is shown in SEQ ID NO: 8, and is as follows:

[0087]

[0088] Using existing technology, a high-quality plasmid of the recombinant expression vector was transformed into host cells to induce expression, and CD133-CAR was isolated and purified from the expression product. N / C-terminal sequence analysis of the obtained CD133-CAR showed that the expressed CD133-CAR had accurate reading frames and was consistent with the theoretical N / C-terminal amino acid sequence.

[0089] Therefore, the amino acid sequence of CD133-CAR is shown in SEQ ID NO: 9, specifically as follows:

[0090] METDTLLLWVLLLWVPGSTGDDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGGSSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRWRALEGGGEGRGSLLTCGDVEENPGPRMSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN

[0091] The amino acid sequence of the anti-CD133 single-chain antibody (scFv) is shown in SEQ ID NO: 10, specifically:

[0092] DIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGGGGSS RSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSS

[0093] The amino acid sequence of the light chain variable region of the anti-CD133 single-chain antibody (scFv) is shown in SEQ ID NO: 11, specifically:

[0094] DIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK

[0095] The amino acid sequence of the heavy chain variable region of the anti-CD133 single-chain antibody (scFv) is shown in SEQ ID NO: 12, specifically:

[0096] LEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSYADDF KGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSS

[0097] The amino acid sequence of the CD28 transmembrane region (CD28-TM) is shown in SEQ ID NO: 13, specifically:

[0098] MFWVLVVVGGVLACYSLLVTVAFIIFWV

[0099] The amino acid sequence of intracellular 4-1BB is shown in SEQ ID NO: 14, specifically:

[0100] RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0101] The intracellular CD3ζ amino acid sequence is shown in SEQ ID NO: 15, specifically:

[0102] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0103] A linker fragment of the immunoglobulin IgG4 hinge region between the CD133 peptide and the CD28 transmembrane region (CD28-TM) is used as the linker fragment. The amino acid sequence of the immunoglobulin IgG4 hinge region fragment is shown in SEQ ID NO: 16, specifically:

[0104] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0105] like Figure 1 As shown, IL-33.CD133-CAR is a T2A-IL-33 linked to the C-terminus of CD3ζ in the intracellular cell of CD133-CAR.

[0106] The sequence of the polynucleotide encoding T2A is shown in SEQ ID NO: 17, specifically:

[0107] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0108] The encoding polynucleotide sequence of human secreted IL-33 is shown in SEQ ID NO.18, specifically:

[0109] AGTATCACAGGAATTTCACCTATTACAGAGTATCTTGCTTCTCTAAGCACATACAATGATCAATCCATTACTTTTGCTTTGGAGGATGAAAGTTATGAGATATATGTTGAAGACTTGAAAAAAGATGAAAAGAAA GATAAGGTGTTACTGAGTTACTATGAGTCTCAACACCCCTCAAATGAATCAGGTGACGGTGTTGATGGTAAGATGTTAATGGTAACCCTGAGTCCTACAAAAGACTTCTGGTTGCATGCCAACAAGGAACACT CTGTGGAGCTCCATAAGTGTGAAAAACCACTGCCAGACCAGGCCTTCTTTGTCCTTCATAATATGCACTCCAACTGTGTTTCATTTGAATGCAAGACTGATCCTGGAGTGTTTATAGGTGTAAAGGATAATCATCTTGCTTCTGATTAAAGTAGACTCTTCTGAGAATTTGTGTACTGAAAATATCTTGTTTAAGCTCTCTGAAACTTAG

[0110] The commissioned company synthesized the entire expression frame of IL-33.CD133-CAR, as follows: Figure 2 As shown, the lentiviral vector (inserted by Shanghai Jikai Company) was correctly sequenced, and the plasmid was extracted and purified using Qiagen's plasmid purification kit to obtain a high-quality plasmid of the recombinant expression vector.

[0111] Sequencing confirmed that the full-length coding gene of IL-33.CD133-CAR was correct and consistent with expectations. Specifically, the coding nucleotide sequence of IL-33.CD133-CAR is shown in SEQ ID NO: 19, and is as follows:

[0112]

[0113] Using existing technology, high-quality plasmids of recombinant expression vectors were transfected into host cells to induce expression. CD133-CAR and IL-33 were then isolated and purified from the expression products. N / C-terminal sequence analysis of the obtained CD133-CAR and IL-33 showed that the expressed CD133-CAR and IL-33 had correct reading frames and were consistent with the theoretical N / C-terminal amino acid sequences.

[0114] The amino acid sequence of CD133-CAR is shown in SEQ ID NO: 9.

[0115] The amino acid sequence of IL-33 is shown in SEQ ID NO: 20, specifically:

[0116] SITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET

[0117] The amino acid sequence of IL-33.CD133-CAR is shown in SEQ ID NO: 21, specifically:

[0118] METDTLLLWVLLLWVPGSTGDDIVLSQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELKSSGGGGSGGGG GGSSRSSLEVKLVESGPELKKPGETVKISCKASGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPSYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCATDYGDYFDYWGQGTTLTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVT VAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRWRALEGGGEGRGSLLTCGDV EENPGPRMALPVTALLLPLALLLHAARPSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET

[0119] Example 2: Preparation of lentiviruses expressing chimeric antigen receptors targeting CD133 and lentiviruses expressing IL-33 targeting CD133.

[0120] I. Preparation of Lentiviral Viruses

[0121] 1. Objective: To introduce a method for producing and concentrating lentiviruses for subsequent mammalian cell transduction.

[0122] 2. Equipment and materials 3.

[0124] Table 1

[0125]

[0126] * The complete DMEM medium is DMEM medium supplemented with 10% heat-inactivated FBS, and also supplemented with 100 U / ml penicillin-streptomycin. § If heat-inactivated FBS is unavailable, conventional FBS can be placed in a 56°C water bath for 30 minutes to completely thaw and inactivate the FBS.

[0127] 4. Steps

[0128] Note: Traditional virus preparation requires eight 10cm culture dishes. This number can be adjusted as needed. In this process, three types of viruses are prepared simultaneously, each requiring eight 10cm culture dishes.

[0129] 4.1 Preparing HEK 293T:

[0130] 4.1.1. When preparing lentivirus, passage and seed HEK 293T cells in T-150 culture flasks 3 days in advance. The HEK 293T cell culture method is as follows (Note: Minimize the time cells spend in concentrated trypsin and outside the incubator):

[0131] 4.1.1.1. Absorb the culture medium from the culture flask and discard it.

[0132] 4.1.1.2. Add 2 ml of 0.25% trypsin to each T-150 culture flask and tilt the flask to ensure that the trypsin covers the entire bottom of the flask.

[0133] 4.1.1.3. Incubate at 37℃ for 2-3 minutes.

[0134] 4.1.1.4. Check if the cells have detached from the bottom of the flask; the culture flask can be tilted to promote cell digestion and detachment.

[0135] 4.1.1.5. After the cells have been digested and detached, add 10 ml of DMEM complete culture medium to each culture flask and mix thoroughly with a pipette to ensure that the cells are completely suspended in the solution.

[0136] 4.1.1.6. Transfer 10 μl of cell suspension to a 600 μl centrifuge tube, add 90 μl of culture medium to dilute and mix well.

[0137] 4.1.1.7. Take 15 μl of the cell suspension diluted 1:10 above and mix it with 15 μl of trypan blue. Count the cells and calculate the cell density.

[0138] 4.1.1.8. Using 5×10 6 Cells were seeded into T-150 culture flasks at a density of 45-60 × 10⁶ cells / 20 ml of culture medium after 3 days (the cell count in each T-150 culture flask reached 45-60 × 10⁶ cells / 20 ml of culture medium). 6 ).

[0139] 4.1.1.9. Place the culture flask horizontally in the CO2 incubator.

[0140] 4.1.2. After culturing for 72 hours, observe cell growth. If the cell density reaches 80-90% and the cells are in good condition (bright cells, tightly attached to the bottom of the flask, with smooth cell edges), then they can be used for subsequent lentivirus preparation.

[0141] 4.2. Day 1 of lentivirus preparation:

[0142] Note: CalPhos transfection can only begin after the cells have adhered to the bottom of the culture dish, a process that takes approximately 4-6 hours. Therefore, cells should be seeded into 10cm culture dishes as early as possible on day 1 of lentivirus preparation. Note: Alternatively, cells can be seeded at a rate of 3.5 × 10⁻⁶ cells / day 24 hours before transfection. 6 Inoculate at a density of 9 individuals per 10 cm culture dish.

[0143] 4.2.1. To prepare a lentivirus, eight 10cm cell culture dishes are required, and each dish should be labeled with the date, name, and plasmid ID number.

[0144] 4.2.2. As described in 3.1.1.1-3.1.1.7, HEK 293T cells were digested with trypsin, digestion was terminated with DMEM complete medium, and the cells were counted.

[0145] 4.2.3. Determine the number of cells required for lentivirus preparation: 7 × 10⁻⁶ 6 1 cell / plate, 8 plates / virus.

[0146] 4.2.4. The actual number of cells collected should be 3.5 × 10⁻⁶ more than the required number. 6 To prevent losses (for example, preparing two types of viruses requires an actual charge of 171.5 × 10⁻⁶ units). 6 (8 cells per culture dish for each virus).

[0147] 4.2.5. Add an appropriate amount of culture medium to bring the final concentration to 7 × 10⁻⁶. 6 1 cell / 9ml

[0148] 4.2.6. Divide 9 ml of cell suspension into each labeled 10 cm culture dish.

[0149] 4.2.7. Place each culture dish flat on the biosafety cabinet and gently move it back and forth in all directions to ensure that the cells are evenly distributed on the bottom of the dish.

[0150] 4.2.8. Place the culture dish back into the incubator. Do not stack multiple culture flasks in the incubator. Due to varying cell growth stages, it takes approximately 3-6 hours to reach 80% cell density. After this, CalPhos transfection can begin.

[0151] 4.2.9. Calculate the required reagent volume according to Table 2 below.

[0152] Table 2

[0153]

[0154] 4.2.10. Add sterile water (from the CalPhos transfection kit), plasmid DNA, and 2M CaCl2 solution (from the CalPhos transfection kit) to a 15ml centrifuge tube in the following order and mix thoroughly.

[0155] 4.2.11. Add an appropriate amount of 2X HBS to a 50ml centrifuge tube according to an average of 500μl of 2X HBS per reaction system, and increase the volume appropriately to avoid loss (for example, a total of 4100μl of HBS can be added to 8 culture dish reaction systems).

[0156] 4.2.12. Using a 1ml pipette, add an equal volume of DNA / CaCl2 mixture dropwise to the HBS, gently shaking the HBS tube as you add the mixture.

[0157] 4.2.13. Incubate the mixture at room temperature for 20 minutes. When the incubation is about to end, remove the 293T cells that were spread out in the morning.

[0158] 4.2.14. Mix the HBS / CaCl2 / DNA solution by pipetting up and down with a 1ml pipette.

[0159] 4.2.15. Add 1 ml of solution dropwise to each petri dish.

[0160] 4.2.16. Repeat step 3.2.7 and mix thoroughly.

[0161] 4.2.17. After processing all the petri dishes, place them in an incubator overnight for no more than 18 hours before proceeding to the next experiment.

[0162] 4.3. Day 2 of lentivirus preparation (CalPhos component removed)

[0163] 4.3.1. Prepare a 0.6M sodium butyrate solution as follows:

[0164] 4.3.1.1. Dissolve 3.3027 g of sodium butyrate (Sigma, catalog number B5887-5G) in 50 ml of H2O. Sterilize using a 0.22 μm vacuum filter. Store at room temperature.

[0165] 4.3.2. Remove CalPhos components (which are toxic to cells) and change the culture medium within 18 hours after transfection.

[0166] 4.3.2.1. Prepare the culture medium for Day 2: Add 2.45 ml of 0.6 M sodium butyrate to 245 ml of DMEM complete medium. (Note: There are 24 culture dishes in this experiment. Add 10 ml of culture medium to each dish. Adjustments can be made as needed.)

[0167] 4.3.2.2. Preheat the above culture medium and magnesium- and calcium-free 1X PBS to 37°C.

[0168] 4.3.2.3. To avoid cells staying outside the incubator for too long, two culture dishes can be removed at once and processed sequentially.

[0169] 4.3.2.4. Mark a line in the center of the petri dish to distinguish the sites where culture medium is removed and added, in order to reduce damage to cells in other parts of the body.

[0170] 4.3.2.5. Use a 10ml pipette and pipette to slowly and gently remove the culture medium.

[0171] 4.3.2.6. Rotate the culture dish to the opposite side where the culture medium was removed. Using a 10ml pipette, slowly add 5ml of the preheated PBS along the top edge of the culture dish, allowing the culture medium to flow slowly down the bottom of the dish.

[0172] 4.3.2.7. Move the culture dish back and forth and side to side (as shown in step 3.2.7) to rinse thoroughly. Then rotate the culture dish to remove the PBS wash solution at the location where the culture medium was removed.

[0173] 4.3.2.8. Slowly add 10 ml of DMEM complete culture medium containing sodium butyrate prepared in step 3.3.2.1.

[0174] 4.3.2.9. Place the petri dish back into the incubator.

[0175] 4.3.2.10. Process the remaining petri dishes according to steps 3.3.2.3-3.3.2.9.

[0176] 4.4. Day 3 of lentivirus production (first-generation collection and PEG treatment)

[0177] 4.4.1. Collect the viral supernatant (change the solution after 24 hours)

[0178] 4.5. Virus Resuspension

[0179] 4.5.8.1. Add 50 μl of serum-free 1640 to each plate of collected virus. Do not mix with a pipette.

[0180] 4.5.8.2. Seal the clean tube with sealing film and vibrate it on a shaker for several seconds to completely resuspend it.

[0181] 4.5.8.3. Let it stand at 4°C for 30 min to 1 h, and mark each virus on its V-bottom cryovial.

[0182] 4.5.8.4. Shake the virus suspension on a shaker until there are no obvious clumps. (The solution will be viscous even if there is no obvious precipitation after ultracentrifugation).

[0183] 4.5.8.5. Add resuspensions of the same virus to the same tube.

[0184] 4.5.8.6. Place the empty ultraclean tube into a 50ml centrifuge tube and shake for 15 seconds to transfer the viral supernatant to the bottom of the tube.

[0185] 4.5.8.7. Transfer the supernatant from 3.4.4.5 to a main collection tube.

[0186] 4.5.8.8. Shake the supernatant to make it uniform.

[0187] 4.5.8.9. Transfer one portion of the supernatant to a V-bottom cryovial (approximately 25-50 μl per portion).

[0188] 4.5.9. Store each virus sample at -80°C.

[0189] II. Lentiviral titration

[0190] 1. Objective: To introduce a method for evaluating lentivirus transduction efficiency.

[0191] 2. Equipment and materials

[0192] Table 3

[0193]

[0194] * The complete DMEM medium is DMEM medium supplemented with 10% heat-inactivated FBS, and also supplemented with 100 U / ml penicillin-streptomycin.§ If heat-inactivated FBS is unavailable, conventional FBS can be placed in a 56°C water bath for 30 minutes to completely thaw and inactivate the FBS.

[0195] 3. Steps

[0196] Day 1

[0197] Note: Use the maximum viral load to transfect a few additional wells for long-term culture and cryopreservation.

[0198] Note: Cells transduced with empty vector should be used as a negative control.

[0199] 1) Add 0.1 × 10⁻⁶ mg of RPMI complete medium to each well. 6 One Jurkat cell was seeded into a 24-well plate.

[0200] 2) Add polybrene (4 mg / ml) to each well at a ratio of 1:1000 (e.g., add 0.5 μl of 4 mg / ml polybrene to 0.5 ml of culture medium).

[0201] 3) Reviving the virus

[0202] 4) Dilute the concentrated virus 1:1000 with RPMI complete medium.

[0203] 5) Add the virus to each well at the following concentrations.

[0204] a. Add 50 μl of virus diluted 1:1000 (final virus volume: 5 × 10⁻⁶). -5 ml)

[0205] b. Add 0.5 μl of undiluted virus (final virus volume: 5 × 10⁻⁶) -4 ml)

[0206] c. Add 1 μl of undiluted virus (final virus volume: 1 × 10⁻⁶) -3 ml)

[0207] d. Add 5 μl of undiluted virus (final virus volume: 5 × 10⁻⁶) -3 ml)

[0208] 6) Incubate overnight at 37°C.

[0209] Day 2

[0210] Add 0.5 ml of fresh RPMI complete medium to each well to dilute the polybrene.

[0211] Day 3

[0212] 1) Collect samples and use flow cytometry to analyze gene expression.

[0213] 2) Transfect several additional wells with the highest viral load for long-term culture and cryopreservation.

[0214] Titer calculation:

[0215]

[0216] Note: The linear range for titer calculation is 15%-45% of the vector expression rate.

[0217] Ultimately, calculations showed that the titers of the chimeric antigen receptor-expressing lentivirus targeting CD133 and the chimeric antigen receptor-expressing lentivirus expressing IL-33 targeting CD133 met the requirements.

[0218] Example 3 - In vitro culture, genetic modification and expansion of T cells

[0219] Human T lymphocytes were transduced using the chimeric antigen receptor-expressing lentivirus targeting CD133 and the IL-33-expressing chimeric antigen receptor-expressing lentivirus targeting CD133 prepared in Example 2.

[0220] 1. Human T lymphocytes obtained by magnetic bead sorting were seeded into 3 wells of a 24-well plate, 5 million cells per well, with 1 ml of RPMI.

[0221] 2. Add 1 μg / ml OKT3 and IL-2 diluted 1:1000 to each well to stimulate cells.

[0222] 3. Incubate at 37℃ for 24 hours.

[0223] 4. Add 6 μl of polybrene (4 mg / ml) to 6 ml of virus supernatant and mix well.

[0224] 5. Add 2 ml of virus / polyglobulin mixture to each well.

[0225] 6. Centrifuge at 1800 rpm for 50 minutes.

[0226] 7. Aspirate 2 / 3 of the supernatant from each well; and add 2 ml of virus / polyglobulin mixture and 1:1000 diluted IL-2 to each well.

[0227] 8. Incubate overnight at 37°C.

[0228] 9. Aspirate 2 / 3 of the supernatant; and add 2 ml of fresh RPMI and 1:1000 diluted IL-2 to each well.

[0229] 10. Incubate overnight at 37°C.

[0230] 11. Collect samples and analyze the expression of the target gene using Real-Time One-Step RT-PCR. The specific steps are as follows:

[0231] (1) Primer design.

[0232] The amplification primers used are a sequence from the CAR structure, as follows:

[0233] Table 4

[0234]

[0235] (2) RNA extraction

[0236] RNA was extracted using TRIzol reagent, and its purity and concentration were determined using Thermo Nano-drop.

[0237] (3) Real-Time One-Step RT-PCR Detection

[0238] Table 5 PCR reaction system

[0239]

[0240]

[0241] Table 6 PCR Reaction Procedure

[0242]

[0243] After the reaction is complete, pass through 2 (-ΔΔCt) The method was used to process and analyze the relative gene expression levels among the groups. For example... Figure 3 The PCR results showed that the target gene was highly expressed in CD133-CAR-T and IL-33.CD133-CAR-T.

[0244] Example 4 - In vitro assay to verify the antitumor effects of CD133-CAR-T and IL-33 CD133-CAR-T cells

[0245] The CD133-CAR-T cells and IL-33.CD133-CAR-T cells constructed in Example 3 were selected, and the human liver cancer cell line Hep3B cells were used as target cells. Co-culture killing experiments were conducted with T cells at different E / T ratios (0.5:1, 1:1, 2:1, 5:1, 10:1) to explore the anti-tumor effect of IL-33.CD133-CAR-T in vitro.

[0246] Figure 4Flow cytometry results showed that CD133 was expressed in Hep3B, SW1990, Panc-1, SW480, RKO and LOVO cells, with high expression in human hepatocellular carcinoma cells Hep3B.

[0247] Figure 5A In vitro killing experiments were conducted by co-culturing IL-33.CD133-CAR-T with Hep3B cells, and the results showed that the anti-tumor effect of IL-33.CD133-CAR-T was significantly better than that of CD133-CAR-T.

[0248] Figure 5B In vitro killing experiments were conducted by co-culturing IL-33.CD133-CAR-T with Hep3B, SW1990, Panc-1, SW480, RKO and LOVO cells, respectively. The results showed that the antitumor effect of IL-33.CD133-CAR-T on Hep3B, SW1990, Panc-1 and LOVO cells was significantly better than that on CD133-CAR-T.

[0249] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0250] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0251] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0252] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes: The extracellular domain, transmembrane domain, and intracellular domain are connected sequentially. The extracellular domain includes an antigen recognition region and a hinge region; The chimeric antigen receptor is modified with IL-33; The antigen recognition region of the chimeric antigen receptor is selected from a single-chain antibody against CD133, and the hinge region is selected from the CD28 hinge region and the immunoglobulin IgG4 hinge region. The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO:

21.

2. A polynucleotide, said polynucleotide being selected from the polynucleotide sequence encoding the chimeric antigen receptor of claim 1.

3. The polynucleotide as described in claim 2, characterized in that, The polynucleotide sequence is shown in SEQ ID NO:

19.

4. A nucleic acid construct comprising the polynucleotide sequence of claim 2 or 3.

5. The nucleic acid construct as described in claim 4, characterized in that, The nucleic acid construct is a vector.

6. The nucleic acid construct according to claim 4, characterized in that, The nucleic acid construct is a lentiviral vector containing a replication start site, a 3'LTR, and a 5'LTR.

7. A lentiviral vector system, wherein the lentiviral vector system comprises the nucleic acid construct and lentiviral vector auxiliary components as described in any one of claims 4 to 6.

8. A T cell, characterized in that, The cell contains the polynucleotide sequence of claim 2 or 3, or the nucleic acid construct of any one of claims 4 to 6, or is infected with the lentiviral vector system of claim 7.

9. The use of the chimeric antigen receptor of claim 1, the polynucleotide sequence of claim 2 or 3, or the nucleic acid construct containing any one of claims 4 to 6, or the lentiviral vector system of claim 7 in the preparation of T cells.

10. The use of the chimeric antigen receptor of claim 1, the polynucleotide sequence of claim 2 or 3, or the nucleic acid construct containing any one of claims 4 to 6, or the lentiviral vector system of claim 7, or the T cell of claim 8 in the preparation of liver cancer treatment products.

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