WO2023161845A1

WO2023161845A1 - Gpc3-targeting chimeric antigen receptor and use thereof

Info

Publication number: WO2023161845A1
Application number: PCT/IB2023/051678
Authority: WO
Inventors: 许元剑; 郭志刚
Original assignee: 南京蓝盾生物科技有限公司
Priority date: 2022-02-23
Filing date: 2023-02-23
Publication date: 2023-08-31

Abstract

Provided in the invention are a GPC3-targeting chimeric antigen receptor (CAR) construct, and a CAR-T cell expressing the CAR construct. The antigen binding domain of the CAR construct comprises an antibody heavy chain variable region as shown in SEQ ID NO: 3, and an antibody light chain variable region as shown in SEQ ID NO: 7. The GPC3 CAR-T cell of the present invention can efficiently target GPC3, and an excellent anti-tumor effect can be obtained at a low dose.

Description

A Chimeric Antigen Receptor Targeting GPC3 and Its Application Technical Field The present invention relates to the field of cellular immunotherapy, in particular, to a chimeric antigen receptor targeting Glypican 3 (GPC3) and its application. Background technique

The concept of CAR-T (Chimeric Antigen Receptor T cell) was first proposed by Israeli scientist Zelig Eshhar in a PNAS article published in 1989. It consists of antigen-binding region, chain region, transmembrane region and intracellular signal. It has gone through several generations of structure development and optimization. In 2017, the world's first CART product, Kymriah, was approved by the FDA for use in acute lymphoblastic leukemia (ALL) in children and young adults. So far, a number of CAR-T products targeting CD19 or BCMA have been launched in many countries or regions, and all of them have achieved amazing therapeutic effects; however, these products are all aimed at hematological tumors. No CAR-T product has been approved for marketing, and the clinical results of CAR-T in most solid tumors are not as prominent as in hematological tumors. Therefore, there is an urgent need in this field to develop CAR-T products that can effectively treat solid tumors. Glypican 3 (GPC3) protein is a heparan sulfate glycoprotein on the surface of the cell membrane, expressed in some tissues during infancy, especially the liver and kidney, and is a cell related to organ formation. Extracellular matrix protein; The expression of GPC3 is hardly observed in adult tissues, but it is highly expressed in various cancer tissues such as hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. Therefore, GPC3 is a good target for tumor therapy and can be used in the research and development of tumor-targeted therapy drugs.

The first aspect of the present invention provides a chimeric antigen receptor (CAR) construct targeting GPC3, the antigen-binding domain of the chimeric antigen receptor comprises a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region includes the following CDRs:

CDR1 shown in SEQ ID NO: 19,

CDR2 shown in SEQ ID NO: 20, and

The CDR3 shown in SEQ ID NO: 21; and the light chain variable region includes the following complementarity determining region CDR:

CDR1' shown in SEQ ID NO:22,

CDR2 shown in SEQ ID NO: 23 ^? , and

(GPC3). In another preferred example, the heavy chain variable region and the light chain variable region of the antigen-binding domain are derived from murine, human, or humanized antibodies.

In another preferred example, said H is a hairpin chain region derived from CD8. In another preferred example, the amino acid sequence of H is shown in SEQ ID NO: 9. In another preferred example, the TM is a transmembrane region of a protein selected from the group consisting of ICOS, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, GD2, CD33, CD37, CD64, CD80,

The second aspect of the present invention provides an isolated nucleic acid molecule encoding the chimeric antigen receptor (CAR) construct described in the first aspect of the present invention. In another preferred example, the nucleic acid molecule is shown in SEQ ID NO: 18. In another aspect of the present invention, a nucleotide sequence encoding a chimeric antigen receptor is provided, the main part of the chimeric antigen receptor includes an antigen binding domain, a transmembrane domain and a costimulatory signal transduction region . The GPC3-specific antibody coding gene is connected to the modified cloning site region of the CAR backbone containing the transmembrane structural gene and costimulatory signal gene to obtain the CAR gene. The antigen-binding domain can specifically bind the tumor-specific antigen GPC3, and activate the NK cell through the transmembrane domain and costimulatory signal transduction region. In another preferred example, the antigen-binding domain is an antibody that specifically binds GPC3 or an antigen-binding fragment thereof, and the antigen-binding fragment is Fab or scFv o In another preferred example, the original binding domain It is scFv, the nucleic acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the nucleic acid sequence of its light chain variable region is shown in SEQ ID NO: 8. The third aspect of the present invention provides a vector containing the nucleic acid molecule described in the second aspect of the present invention. In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), retroviral vector, transposon, or a combination thereof . In another preferred example, the vector is selected from the group consisting of plasmids and viral vectors. In another preferred embodiment, the vector is in the form of virus particles. In another preferred example, the vector is a lentiviral vector. The fourth aspect of the present invention provides a host cell containing the vector according to the third aspect of the present invention, or an exogenous nucleic acid molecule as described in the second aspect of the present invention integrated in the chromosome, Or express the CAR construct as described in the first aspect of the present invention. In another preferred example, the host cells include eukaryotic cells and prokaryotic cells. In another preferred example, the host cells include Escherichia coli.

The sixth aspect of the present invention provides a preparation comprising the CAR construct according to the first aspect of the present invention, the isolated nucleic acid molecule according to the second aspect of the present invention, the third aspect of the present invention The carrier or the immune cell according to the fifth aspect of the present invention, and a pharmaceutically acceptable carrier. In another preferred example, the formulation is a liquid formulation. In another preferred example, the dosage form of the preparation is injection. In another preferred example, the concentration of immune cells (GPC3 CAR-T cells) in the preparation is IX

1O ³ -1 X 1 O ⁸ cells/ml, preferably 1 X 10 ⁴ -5 X l (f cells/ml. In another preferred embodiment, the preparation may include a buffer such as neutral buffered saline , sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg eg, aluminum hydroxide); and preservatives. The formulations of the invention are preferably formulated for intravenous administration. In another preferred example, the preparation further includes a second anti-tumor active ingredient, preferably a second antibody or a chemotherapeutic agent. In another preferred example, the chemotherapeutic agent is selected from the group consisting of docetaxel, calipers, or a combination thereof. In another preferred example, the preparation is a pharmaceutical composition. In another preferred example, when the preparation is administered, the effect-to-target ratio of GPC3 CAR-T cells to tumor cells is (0.1-20): 1, preferably, the effect-to-target ratio is (1-4): 1. The seventh aspect of the present invention provides the CAR construct according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, the fifth aspect of the present invention The use of the immune cells according to the aspect, or the preparation according to the sixth aspect of the present invention is used to prepare a drug for preventing and/or treating tumor or cancer. In another preferred example, the tumor or cancer highly (over) expresses GPC3. In another preferred example, the tumor or cancer is a solid tumor. In another preferred example, the solid tumor is selected from the group consisting of hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, lung squamous cell carcinoma, breast cancer, renal carcinoma, cholangiocarcinoma or a combination thereof. In another preferred example, the tumor or cancer is hepatocellular carcinoma. In another preferred example, the tumor or cancer is a liver cell that highly (over)expresses GPC3. The eighth aspect of the present invention provides a method for preparing the engineered immune cells according to the fifth aspect of the present invention, the method comprising the following steps:

(a) providing immune cells to be engineered; and

(b) Transducing the nucleic acid molecule according to claim 2 or the vector according to claim 3 into the immune cells, thereby obtaining the engineered immune cells. In another preferred example, the engineered immune cells are CART cells or CAR-NK cells. In another preferred example, the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells. The ninth aspect of the present invention provides a method for treating tumor or cancer, comprising administering to a subject in need an effective amount of the immune cells described in the fifth aspect of the present invention, or the preparation described in the sixth aspect of the present invention . In another preferred example, the tumor or cancer highly (over) expresses GPC3. In another preferred example, the tumor or cancer is a solid tumor. In another preferred example, the solid tumor is selected from the group consisting of hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, lung squamous cell carcinoma, breast cancer, kidney cancer, cholangiocarcinoma or a combination thereof In another preferred example, the tumor or cancer is hepatocellular carcinoma. It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a schematic diagram of the structure of the GPC3-targeting chimeric antigen receptor GC52BB& of the present invention. Figure 2 shows the construction and detection of HuH7-Luc>HepG2-Luc cells. Figure 3 shows the detection of GC52BB C positive rate. Figure 4 shows that GC52BB V kills HuH7-luc cells in vitro; the left panel shows the killing ability of GC52BB E on HuH7Tuc cells when E/T (effect-to-target ratio) is 4; Target ratio) is 1, the killing ability of GC52BB V on HuH7-luc cells. Figure 5 shows that GC52BB C kills HepG2-luc cells in vitro; the left picture shows the killing ability of GC52BB to HepG2Tuc cells when E/T (effect-to-target ratio) is 4; Target ratio) is 1, GC52BB has the ability to kill HepG2-luc cells. Figure 6 shows the anti-tumor effect of GC52BB V in vivo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS After extensive and in-depth research, the present inventors unexpectedly discovered for the first time a chimeric antigen receptor that targets and recognizes GPC3, its encoding nucleotides, and GPC3 CAR-T cells expressing the CAR and its preparation method and applications. The CART cells of the present invention are specific to GPC3, and can specifically recognize and kill tumors, especially solid tumors (such as hepatocellular carcinoma) that highly express or overexpress GPC3, and have more efficient tumor killing activity, at low doses (approximately It is only 1/5 of the usual dose in the prior art to achieve a good anti-tumor effect, and its properties are stable, and it can be mass-produced and prepared. On this basis, the present invention has been accomplished. In order to better understand the present invention, certain terms are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, "antigen-binding domain" and "single-chain antibody fragment" all refer to a Fab fragment, Fab' fragment, F(ab ⁷ ) ₂ fragment, or a single Fv fragment with antigen-binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming antigenic Combine the desired structure. The antigen-binding domain is usually scFv (single-chain variable fragment) _o The size of scFv is generally 1/6 of a complete antibody. A single chain antibody is preferably a sequence of one amino acid chain encoded by one nucleotide chain. As a preferred mode of the present invention, the scFv comprises an antibody that specifically recognizes GPC3, preferably a humanized single-chain antibody. For the hinge region and transmembrane region (transmembrane domain), CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some instances, transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with the receptor complex. interactions with other members. In the present invention, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins, derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F (ab) 2 , as well as single-chain antibodies and humanized antibodies, etc. (Harlow et al., 1999, In : Using Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies : A Laboratory Manual , Cold Spring Harbor , New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426). The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenically determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies and multispecific antibodies formed from antibody fragments. Unless otherwise specified, "encoding nucleotides" or "encoding nucleic acids" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein may include introns. The term "lentivirus" refers to a genus of the Retroviridae family that is capable of efficiently infecting aperiodic and post-mitotic cells; they can transfer significant amounts of genetic information into the host cell's DNA, so that they are the most suitable vectors for gene delivery. one of the effective methods. The term "promoter" is defined as a DNA sequence that is recognized by or directs the synthetic machinery of a cell, required to initiate specific transcription of a polynucleotide sequence. The term "specifically binds" refers to recognition of a specific antigen but substantially no recognition or binding of other molecules in the sample. The term "vector" is a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include the facilitation of transfer of nucleic acid into Cellular non-plasmid and non-viral compounds such as, for example, polylysine compounds, liposomes and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. The term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. As used herein, "comprising" is synonymous with "including", "comprising" or "characterized by" and is inclusive or open-ended and does not exclude additional unstated elements or method steps. Any expression of the term "comprising" herein, especially when describing the methods, uses or products of the present invention, should be understood as including consisting essentially of the components or elements or steps and consisting of the components or elements or steps consisting of those products, methods and uses. The invention exemplified herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. The terms and expressions which have been employed herein are used as terms of description rather than limitation, and in the use of such terms and expressions it is not intended to exclude any equivalents of the features shown and described or parts thereof, but various modifications are recognized It is possible within the scope of the claimed invention. Accordingly, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art and that such modifications and variations are considered to be defined within the scope of the invention. The English names appearing in this article are not case-sensitive, and they have the same meaning; for example, scFv, ScFv, SCFV, etc. have the same meaning; GPC3 CAR-T and Anti GPC3-CAR T have the same meaning, and both represent the anti-GPC3 molecule CAR-T cells. Chimeric Antigen Receptor (CAR)

The design of CARs has gone through the following process: The first-generation CAR has only one intracellular signaling component CD3 & or Fc γ RI molecule, because there is only one activation domain in the cell, so it can only cause transient T cell proliferation and less The secretion of cytokines does not provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so good clinical efficacy has not been achieved. The second-generation CARs introduce a co-stimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, and the persistence of CAR-T cells and the ability to protect tumor cells are further enhanced. lethality. On the basis of the second-generation CARs, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs. The chimeric antigen receptor (CAR) of the present invention is a second-generation CAR, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes target-specific binding elements (also known as antigen-binding structures area). The intracellular domain includes the co-stimulatory signaling domain and the chain portion. A co-stimulatory signaling region refers to a portion of an intracellular domain that includes co-stimulatory molecules. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigens. A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids. In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting GPC3. When the CAR of the present invention is expressed in T cells, it can perform antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing tumor cells not to grow, being induced to die, or otherwise affected, and resulting in a reduction or elimination of the patient's tumor burden. The antigen binding domain is preferably fused to an intracellular domain from one or more of a co-stimulatory molecule and a chain. The nucleic acid sequence of the vector encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology, directly isolated from cells and tissues containing the gene. Alternatively, the gene of interest can be produced synthetically. The present invention also provides a vector into which an expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an advantage over vectors derived from oncogenic retroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity. Briefly summarized, the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration in eukaryotic cells. A typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence. The expression constructs of the invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the present invention provides gene therapy vectors. The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology in this It is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and US Patent No. 6,326,193). A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used. Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically these are located in the 30-110 bp region upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible in order to preserve promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act cooperatively or independently to initiate transcription. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α) _. However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse Mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Lu Steiner's sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. In order to evaluate the expression of the CAR polypeptide or a part thereof, the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene, so as to seek transfected or infected cell populations from viral vectors Identification and selection of expressing cells. In other aspects, selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Flanking both selectable marker and reporter gene

is well known in the field. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) _{. o} A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, eg human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, U.S. Patent Nos. 5,350,674 and 5,585,362. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems. plastid. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane vesicle). Where a non-viral delivery system is used, an exemplary delivery vehicle is liposomes. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides into liposomes, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, Or otherwise associated with lipids. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles or have a "col lapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic cores and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. In a preferred embodiment of the present invention, the vector is a lentiviral vector. Therapeutic Applications The present invention includes therapeutic applications of cells (eg T cells) transduced with a lentiviral vector (LV) encoding a CAR of the present invention. The transduced T cells can target the tumor cell marker GPC3, activate T cells cooperatively, and cause T cell immune responses, thereby significantly improving their killing efficiency against tumor cells. Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CART cells of the present invention to the mammal. In one embodiment, the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterogeneous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. In this way, the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers expressing the antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. In one embodiment, CAR-T cells of the invention can undergo robust in vivo NK cell expansion for extended amounts of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR. For example, anti-GPC3 CART cells elicit a specific immune response against GPC3-positive cells. Although the data disclosed herein specifically discloses lentiviral vectors comprising anti-GPC3 scFv, CD8 chain and CD8 transmembrane and intracellular regions, 4-1BB intracellular region and CD3 & signaling domain, the present invention should be considered Interpretation is to include any number of variations to each of the construct components. Treatable cancers include non-solid tumors (such as hematological tumors, eg leukemias and lymphomas) or solid tumors, especially solid tumors. The types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma and melanoma. Also includes adult tumors/cancers and childhood tumors/cancers. Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic , monocytic and erythroleukemia), chronic leukemias such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulin leukemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, and kidney cancer. In a preferred embodiment, the treatable cancer is a GPC3-positive tumor, such as hepatocellular carcinoma. The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human. For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into the mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and/or iii) cryopreserving the cells. Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient. Alternatively, cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient. In addition to the use of cell-based vaccines for ex vivo immunization, the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient. The present invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention. The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical composition of the present invention may include the target cell population as described herein combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives. The compositions of the invention are preferably formulated for intravenous administration. The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials. When an "immunologically effective amount", "antitumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of a composition of the invention to be administered can be determined by a physician, taking into account the patient's Individual differences in (subjects') age, weight, tumor size, degree of infection or metastasis, and disease. It may generally be noted that: Pharmaceutical compositions comprising T cells described herein may be dosed at 1.4 to 1.9 cells/kg body weight, preferably at doses of 10, to ¹⁰¹³ cells/kg body weight (including those within the range All integer values) apply. T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly. Administration of the composition to a subject can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, a T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by iv injection. Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection. In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (eg, previously , concurrently or subsequently) to the patient in the form of treatment including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, arabinocytosomes (also known ARA-C) or natalizumab in MS patients or erfatizumab in psoriasis or other treatments in PML. In a further embodiment, the T cells of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressants, such as cyclosporine, thiamin, methotrein, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery. Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 x 1.6 to 1 x 10 "modified T cells (for example, CAR-T cells) of the present invention can be administered to Patients. According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention can be administered to a subject with any effective dose. Preferably, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention It can be administered in multiple doses, for example, from about 2 to about 20 doses, more preferably from about 4 to 0 doses.In a particularly preferred embodiment, during the administration, the frequency of administration about once every three weeks will be The pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention is administered to the subject, such as injection, infusion or orally. In a particularly preferred embodiment, the administration is by injection into the tumor-bearing site. It should be understood that the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be formulated in any suitable manner for administration by any suitable route. The dosage unit of the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention is administered to a subject on a routine basis. For example, dosage units may be administered more than once daily, weekly, monthly, etc. The dosage unit may be administered on a twice/week basis, that is, twice a week, for example, once every three days. The instructions related to the pharmaceutical product contained in the pharmaceutical product of the present invention may contain the following contents: indications (eg, glioblastoma), dosage (eg, as exemplified above), and possible side effects, etc. . Sequences of the invention

GC52BB V sequence: signal peptide SP amino acid:

MALPVTALLLPLALLLLHAARPGS (SEQ ID NO: 1) signal peptide SP nucleic acid:

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCCCTGCTGCTCCACGCCGCTAGACCCGGAA

GC (SEQ ID NO: 2) heavy chain VH amino acid:

QIQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIDPKTGGTAYNQKFKD

KAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYSYAYWGQGTLVTVSA (SEQ ID NO: 3) heavy chain VH nucleic acid:

CAGATCCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCTGGCGCTTCCGTGACACTGTCC

TGTAAGGCCAGCGGCTACACATTCACCGATTACGAGATGCACTGGGTGAAGCAGACACCCGTGCACGGCCTGGAGTGGATCGGCGCTATCGACCCTAAGACAGGCGGCACCGCCTACAATCAGAAGTTCAAGGATAAGGCCATCCTGACCGCCGACAAGAGCTCCTCCACCGCCTACATGGAGCTGAGGTCCCTGACCTCCGAGGATTCCGCCGTGT ACTACTGTACAAGATACTACAGCTACGCCTACTGGGGCCAGGGCACACTGG

TGACAGTGTCCGCC (SEQ ID NO: 4) linker (Linker) amino acid:

GGGGSGGGGSGGGGS (SEQ ID NO: 5) linker (Linker) nucleic acid:

GGCGGAGGCGGAAGCGGAGGAGGAGGAAGCGGCGGAGGCGGTAGC (SEQ ID NO: 6) Light chain VL amino acids:

DVVMTQTPLSLPVSLGDQAS ISCRSSQSPVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDR

FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIK (SEQ ID NO: 7) light chain VL nucleic acid: GATGTGGTGATGACCCAGACACCTCTGAGCCTGCCTGTGTCCCTGGGCGATCAGGCCTCCATCTCCTGCAGATCCAGCCAGAGCCCCGTGCACAGCAATGGCAATACATACCTGCACTGGTACCTGCAGA AGCCTGGCCAGAGCCCTAAGCTGCTGATCTACAAGGTGAGCAACAGATTCAGCGGCGTGCCCGACAGATTCTCCGGCAGCGGCTCCGGC ACAGACTTCACACTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCAGCCAGAGCACCCACGTGCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGA TCAAG (SEQ ID NO: 8) Hinge amino acid in the hairpin chain region:

SGTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 9) Hinge nucleic acid of chain region:

TCCGGCACAACCACCCCTGCCCCCAGACCCCCTACCCCAGCTCCTACAATCGCCAGCCAGCCCCTGAGCCTCAGGCCTGAGGCCTGCAGGCCCGCTGCTGGAGGAGCTGTGCACACCAGGGGCCTGGACT TCGCCTGTGACATCTAC (SEQ ID NO: 10) Transmembrane region TM amino acids:

IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 11) Transmembrane Region TM Nucleic Acid:

ATCTGGGCCCCCCTGGCCGGCACCTGTGGAGTTCTGCTGCTGTCCCTGGTGATCACACTGTAC TGC (SEQ ID NO: 12) co-stimulatory domain 4-1BB amino acids:

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 13) costimulatory domain 4-IBB nucleic acid:

AGATTCTCCGTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATG AGGCCTGTGCAGACAACACAGGAGGAGGATGGCTGTTCCTGCAGATTCCCCGAGGAGGAGGAGGGCG GCTGTGAGCTG (SEQ ID NO: 14) Intracellular signal CD3 V amino acid:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSE I GMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (SEQ ID NO: 15) intracellular signal CD3, nucleic acid:

AGAGTGAAGTTCAGCAGATCCGCCGATGCCCCCGCCTACCAGCAGGGACAGAATCAGCTGTAC AACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGGGGCAGGGACCCTGAGATGGGCGGCAAGCCCAGGAGGAAGAACCCCCAGGAGGGCCTGTACAACGAACTGCAGAAGGACAA GATGGCCGAGG CCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACATACGACGCCCTGCACATGCAGGCCCTGCCTCCCAGATGA (SEQ ID NO: 16)

Amino acid sequence of GC52BB V CAR:

MALPVTALLLPLALLLLHAARPGSQIQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPV HGLEWIGAIDPKTGGTAYNQKFKDKAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYSYAYWGQGTLVTV

SAGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSPVHSNGNTYLHWYLQKPGQSPKLLI

YKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEIKSGTTTPAPPRPP

TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKR

GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG

RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR (SEQ ID NO: 17)

GC52BB, the nucleic acid sequence of CAR:

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCCCTGCTGCTCCACGCCGCTAGACCCGGAA

GCCAGATCCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCTGGCGCTTCCGTGACACTGTCCTG

TAAGGCCAGCGGCTACACATTCACCGATTACGAGATGCACTGGGTGAAGCAGACACCCGTGCACGGC

CTGGAGTGGATCGGCGCTATCGACCCTAAGACAGGCGGCACCGCCTACAATCAGAAGTTCAAGGATA

AGGCCATCCTGACCGCCGACAAGAGCTCCTCCACCGCCTACATGGAGCTGAGGTCCCTGACCTCCGA

GGATTCCGCCGTGTACTACTGTACAAGATACTACAGCTACGCCTACTGGGGCCAGGGCACACTGGTG

ACAGTGTCCGCCGGCGGAGGCGGAAGCGGAGGAGGAGGAAGCGGCGGAGGCGGTAGCGATGTGGTGA

TGACCCAGACACCTCTGAGCCTGCCTGTGTCCCTGGGCGATCAGGCCTCCATCTCCTGCAGATCCAG

CCAGAGCCCCGTGCACAGCAATGGCAATACATACCTGCACTGGTACCTGCAGAAGCCTGGCCAGAGC

CCTAAGCTGCTGATCTACAAGGTGAGCAACAGATTCAGCGGCGTGCCCGACAGATTCTCCGGCAGCG

GCTCCGGCACAGACTTCACACTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTG

CAGCCAGAGCACCCACGTGCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGTCCGGCACA

ACCACCCCTGCCCCCAGACCCCCTACCCCAGCTCCTACAATCGCCAGCCAGCCCCTGAGCCTCAGGC

CTGAGGCCTGCAGGCCCGCTGCTGGAGGAGCTGTGCACACCAGGGGCCTGGACTTCGCCTGTGACAT

CTACATCTGGGCCCCCCTGGCCGGCACCTGTGGAGTTCTGCTGCTGTCCCTGGTGATCACACTGTAC

TGCAGATTCTCCGTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGA

GGCCTGTGCAGACAACACAGGAGGAGGATGGCTGTTCCTGCAGATTCCCCGAGGAGGAGGAGGGCGG

CTGTGAGCTGAGAGTGAAGTTCAGCAGATCCGCCGATGCCCCCGCCTACCAGCAGGGACAGAATCAG

CTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGGGGCAGGG

ACCCTGAGATGGGCGGCAAGCCCAGGAGGAAGAACCCCCAGGAGGGCCTGTACAACGAACTGCAGAA

GGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCAC

GACGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACATACGACGCCCTGCACATGCAGGCCC

TGCCTCCCAGATGA (SEQ ID NO: 18)

(2) The CAR-T cells of the present invention can efficiently target GPC3, and when the dose is 1 X 10 ⁶ /mouse or lower, CAR-T can have a good tumor inhibitory effect; while the current solid tumor CAR-T The dosage of T is generally 5 X 10&/mouse or above to achieve a good tumor inhibitory effect;

(3) A good therapeutic effect can be achieved with a relatively low dose, which provides better possibility and convenience for later-stage technology and industrialization, and also makes CAR-T products safer, and less likely to occur due to a large number of cell regeneration. Toxic and side effects caused by infusion. The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., Molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 2 HuH7-Luc, HepG2-Luc cell construction and detection By querying the luciferase gene (GenBank: ACF93193.1) sequence, it was synthesized in CRO company and constructed into the PLVX-Puro vector, and the constructed vector was named PLVX-Luc -Puro _o Construct a stable transfected cell line by lentivirus transfection, that is, use the pGPol, pVSVG packaging plasmid and the target plasmid PLVX-Luc-Puro to produce high-titer lentivirus on the company's own lentivirus packaging platform, and then Infect the target cells HuH7 and _HepG2 respectively according to MOI = 1. After several rounds of Purmycin pressurized screening, the luciferase signal value of the qualified cell line should be more than 100 times that of the original cells not transfected with lentivirus. The qualified cells They were named HuH7-Luc and HepG2-Luc respectively; the results of luciferase signal detection are shown in Figure 2.

GC76BB E, 4 ug pGPol and 2 ug pVSVG plasmids, pipette up and down to mix thoroughly, add 18 uL PEL, immediately pipette up and down to mix, and let stand at room temperature for 15 minutes;

(4) Add the above-mentioned DNA/PEI complex dropwise into a 15cm petri dish, shake the petri dish gently, and mix well. Place the culture dish in a 37°C, 5% CO incubator, and after culturing for 6 to 8 hours, remove the medium containing the transfection reagent and replace it with fresh complete medium;

(5) After 48 hours of continuous culture, collect the medium supernatant containing the virus in the culture dish, filter it with a 0.45 μm filter membrane, and then centrifuge at 20000g for 2 hours under 4P conditions; after the centrifugation, carefully remove the liquid in the centrifuge tube Aspirate, add 400 uL of PBS buffer to resuspend the pellet, aliquot the virus into 1.5ml centrifuge tubes, each tube is 100 H 1 , and store the virus at -80°C. Example 4 Sorting and activation of T cells Follow the steps below to sort and activate T cells:

(1) Take 1 frozen PBMC cell and resuscitate in a 37°C water bath; add the resuscitated PBMC to a 15ml centrifuge tube that has been pre-added with AlOml preheated medium; then centrifuge at 500g for 5 minutes;

(2) After resuspending cells with sorting buffer, perform cell counting;

(3) Centrifuge at 500g for 5 minutes, and adjust the cell density to 1.0*10 ⁷ /ml;

(4) According to the cells of 1.0*10', add 20 Li 1 volume of sorting magnetic beads, and add sorting magnetic beads;

(5) Incubate for 15 minutes at 4°C;

(6) Add the incubated cells into the sorting column to perform the sorting operation;

(7) Collect the effluent in the sorting column, and centrifuge at 500g for 5 minutes;

(8) resuspend the cells in X-vivo 15 medium (containing 300U/mL IL-2), count, and adjust the cell density to 1.0^lOVml;

(9) According to the ratio of 1.0*10 ⁶ cells to 10 11 1 activated magnetic beads, add activated magnetic beads, and then place the cells in a 37°C. 5% CO? incubator for 48 hours. Thus, activated T cells are obtained.

And obtain CAR-T cells. Example 6 Detection of positive rate of CAR-T cells

The detection method of CAR-T cell positive rate is as follows:

(1) Centrifuge at 500g for 5 minutes, and collect the CAR-T cells infected with lentivirus on the 7th day in Example 5;

(2) Resuspend CAR-T cells in 500 uL PBS, centrifuge at 500g for 5 minutes to wash the cells once;

(3) Cells were resuspended in 1 mL of PBS, and 2011 L of resuspended cells were counted on a cell counter;

(4) According to the cell counting results, 5*107 reaction cells were taken to detect the positive rate of CART;

(5) Add the detection antibody at a concentration of 2 μg/ml, the reaction volume is 100uL, and then incubate at 4°C for 30 minutes;

(6) After the incubation, centrifuge at 500g for 5 minutes;

(7) Resuspend the cells in 500 uL PBS, centrifuge at 500g for 5 minutes to wash the cells once;

(8) Repeat step 7;

(9) Cells were resuspended in 200 uL PBS for testing on the machine. The results are shown in Figure 3, the positive rate of CAR-T cells was 68.79%. Example 7 CAR-T cells kill target cells

(1) Collect logarithmic growth phase target cells (HuH7Tuc or HepG2-luc), adjust the target cell density to 2*107ml, take a new 96-well plate, and inoculate the target cells according to the amount of 50UL/well. To the unused wells around the 96-well plate, 100 PL medium was added to each well to prevent water evaporation in the middle experimental wells.

(2) Collect the above-prepared CAR-T cells by centrifugation, use X-vivo 15 medium (containing 300U/mL IL-2); then add 50U 1 CAR-T cells to each well according to the designed effect-to-target ratio (E/T). T cells to 96-well plate. Positive control BM (CAR-T constructed by scFv of GPC3 antibody GC33) was set; UN-T was T cells not transduced with lentivirus, and all cells were prepared through the same operation.

(3) Place the well plate in a 5% CO ₂ 37°C incubator and incubate for 14-18 hours.

(4) After the incubation, take the well plate out of the incubator, add 50u 1 One-Gio luciferase detection substrate, centrifuge the 96-well plate at room temperature for 3 minutes at 300g, gently take the plate out, and read it on a microplate reader.

(5) Calculate the killing efficiency of CAR-T cells on target cells by using the microplate reader data; Calculation formula: killing % =1-(sample reading value-Min)/(Max-Min).

The results of GC52BB V killing HuH7-luc cells and HepG2-luc cells in vitro are shown in Figure 4 and Figure 5 . The results showed that for HuH7-luc cells, when the in vitro killing activity of GC52BBV was 4 when the E/T (effect-to-target ratio) was 4, GC52BBC could kill nearly 100% of tumor cells, while the BM positive control CAR-T had only 60% killing effect; Even when the E/T (effect-to-target ratio) is 1, GC52BBV has a killing ability of nearly 40%. At this time, the BM positive control CAR-T has almost no killing effect (Figure 4); for HepG2-luc cells, When the killing activity of GC52BBC in vitro was E/T (effect-to-target ratio) of 4, both GC52BB £ and BM control CAR-T could It can kill nearly 100% of tumor cells, but when the E/T (effect-to-target ratio) is 1, GC52BB E can still have 80% killing ability, and at this time, the killing effect of BM positive control CAR-T has dropped to 50% following (Figure 5).

The dose was 1x107 mice, and the Mock T group only set up a high-dose 3x10° group; the day of grouping was defined as D1 day, and the drug was administered on the day of grouping. Experimental observation and data collection: After administration, the effect of tumor on the normal behavior of animals was routinely monitored weekly. The specific content includes the activity of experimental animals, food intake and drinking conditions, weight gain or loss, eyes, coat and other abnormal conditions. Measure and record tumor size and mouse body weight. The calculation method of tumor volume is: tumor volume (mm ³ ) = 0.5 X (tumor long diameter X tumor short diameter?). The results are shown in Figure 6, the CART cells of the present invention have an excellent tumor inhibitory effect at a lower dosage (1x10°/mouse). All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

22 claims

White transmembrane region: ICOS, CD28, CD3 eps i lon. CD45, CD4, CD5, CD8, CD9, CD16, GD2, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

7. The CAR construct according to claim 4, wherein said C is a co-stimulatory signal molecule of a protein selected from the group consisting of ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dapl 0, CDS, ICAMT, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

8. The CAR construct according to claim 4, wherein the amino acid sequence of the CAR is as shown in SEQ ID NO: 17.

9. An isolated nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the chimeric antigen receptor (CAR) construct of claim 1.

10. a carrier, is characterized in that, described carrier contains nucleic acid molecule described in claim 9.

11. A host cell, characterized in that, the host cell contains the vector according to claim 10, or the exogenous nucleic acid molecule according to claim 9 is integrated into the chromosome, or expresses the vector according to claim 1 The CAR construct.

12. An engineered immune cell, characterized in that, the immune cell expresses the CAR construct according to claim 1.

13. A preparation, characterized in that, the preparation comprises the CAR construct as claimed in claim 1, the isolated nucleic acid molecule as claimed in claim 9, the carrier as claimed in claim 10 or the vector as claimed in claim 12 The immune cells, and a pharmaceutically acceptable carrier.

14. The CAR construct as claimed in claim 1, the nucleic acid molecule as claimed in claim 9, the carrier as claimed in claim 10, the immune cell as claimed in claim 12, or the The use of the preparation is used for preparing a medicine for preventing and/or treating tumor or cancer.

15. A method for treating tumor or cancer, comprising administering an effective amount of the immune cell according to claim 12, or the preparation according to claim 13 to a subject in need.