Chimeric antigen receptor and uses thereof

Abstract

A chimeric antigen receptor and its application are provided. The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence; the extracellular domain recognizes and binds to antigens; the intracellular domain includes an activation domain, or includes a co-stimulatory domain and an activation domain; relative to the parental intracellular domain, lysine residues in the co-stimulatory domain and / or the activation domain are partially or completely replaced with glycine. Some of the provided chimeric antigen receptors have prolonged half-lives and exhibit strong killing activity against tumors or cancers even at low target-to-cell ratios, which helps to improve the anti-tumor activity of CAR-T therapy against solid tumors and has broad clinical application prospects.

Description

Chimeric antigen receptor and its application

[0001] This application claims the benefit of Chinese Patent Application No. 2023115312166, filed on November 16, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to a chimeric antigen receptor and applications thereof. Background Art

[0003] Tumor immunotherapy is a treatment approach that controls and eliminates tumors by reinitiating and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. Tumor immunotherapy encompasses treatments such as monoclonal antibody-based immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapies, and small molecule inhibitors. In recent years, tumor immunotherapy has rapidly advanced, becoming the fourth most popular cancer treatment technology after surgery, radiotherapy, and chemotherapy. While immune checkpoint inhibitors, such as anti-PD-1 monoclonal antibodies, have been approved for multiple cancer indications and continue to challenge current standard treatment options, cell therapy has taken a significant lead in the field of hematological malignancies. In tumor immunotherapy, cell therapy involves the use of immune cells, induced by cytokines, to generate large numbers of highly active anti-tumor cells ex vivo. These cells are then adoptively transferred into the patient to restore the patient's immune system and attack tumor cells. Currently, several CAR-T therapy-based drugs have been approved for hematological malignancies, establishing the importance of cell therapy in the treatment of various hematological malignancies. Summary of the Invention

[0004] A first aspect of the present invention provides a chimeric antigen receptor, comprising an extracellular domain, a transmembrane domain, and an intracellular domain connected in sequence;

[0005] The extracellular domain recognizes and binds to the antigen;

[0006] The intracellular domain includes an activation domain, or includes a co-stimulatory domain and an activation domain;

[0007] Relative to the parent intracellular domain, the lysine residues in the costimulatory domain and / or activation domain are partially or completely replaced by glycine residues.

[0008] In some embodiments of the present invention, the number of the costimulatory domain is at least one.

[0009] In some embodiments of the present invention, relative to the parent intracellular domain, some or all of the lysine residues in the activation domain are replaced by glycine residues.

[0010] In some embodiments of the present invention, relative to the parent intracellular domain, the lysine residues in at least one of the costimulatory domain and the activation domain are partially or completely replaced by glycine residues.

[0011] In some embodiments of the invention, at least one, at least two, or at least three lysine residues in at least one of the costimulatory domain and the activation domain are replaced with glycines relative to the parent intracellular domain.

[0012] In some embodiments of the invention, at least one lysine in at least one of the costimulatory domains and activation domains is replaced with glycine relative to the parent intracellular domain. In some embodiments of the invention, at least two lysines in at least one of the costimulatory domains and activation domains are replaced with glycines relative to the parent intracellular domain. In some embodiments of the invention, at least three lysines in at least one of the costimulatory domains and activation domains are replaced with glycines relative to the parent intracellular domain.

[0013] In some embodiments of the invention, one lysine in a costimulatory domain is replaced with glycine relative to the parent intracellular domain. In some embodiments of the invention, two lysines in a costimulatory domain are replaced with glycines relative to the parent intracellular domain. In some embodiments of the invention, three lysines in a costimulatory domain are replaced with glycines relative to the parent intracellular domain.

[0014] In some embodiments of the invention, one lysine residue in an activation domain is replaced with a glycine residue relative to the parent intracellular domain. In some embodiments of the invention, two lysine residues in an activation domain are replaced with glycines relative to the parent intracellular domain. In some embodiments of the invention, three lysine residues in an activation domain are replaced with glycines relative to the parent intracellular domain.

[0015] In some embodiments of the present invention, the parent intracellular domain consists of a native sequence. In some embodiments of the present invention, the parent intracellular domain comprises a costimulatory domain extracted from a native sequence, and an activation domain extracted from a native sequence. In some embodiments of the present invention, the costimulatory domain of the parent intracellular domain is a native sequence. In some embodiments of the present invention, the activation domain of the parent intracellular domain is a native sequence.

[0016] In other embodiments of the present invention, relative to the parent intracellular domain, the lysine residues in at least one of the costimulatory domain and the activation domain are partially or completely replaced by glycine residues.

[0017] In some embodiments of the present invention, the activation domain comprises a functional signaling domain selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10 and / or DAP12.

[0018] In some specific embodiments of the present invention, the activation domain is a functional signaling domain of CD3ζ.

[0019] In some examples of the present invention, the activation domain comprises the amino acid sequence shown in SEQ ID NO:16.

[0020] In some embodiments of the present invention, the costimulatory domain is selected from the costimulatory domains of the following proteins: 4-1BB, CD27, CD28, OX-40 and / or ICOS.

[0021] In some specific embodiments of the present invention, the costimulatory domain is selected from CD28 and / or 4-1BB.

[0022] In some embodiments of the present invention, the costimulatory structure comprises the amino acid sequence shown in SEQ ID NO: 12 and / or SEQ ID NO: 14.

[0023] In some embodiments of the present invention, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and / or CD154.

[0024] In some specific embodiments of the present invention, the transmembrane domain is the transmembrane domain of CD8.

[0025] In some examples of the present invention, the transmembrane domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:10.

[0026] In some embodiments of the present invention, the extracellular domain is an antibody or antigen-binding fragment, such as scFv, single domain antibody or F(ab)'.

[0027] In some embodiments of the present invention, the chimeric antigen receptor further comprises a hinge region, which connects the extracellular domain and the transmembrane domain.

[0028] In some embodiments of the present invention, the hinge region comprises the hinge region of CD8α or CD28.

[0029] In some specific embodiments of the present invention, the hinge region comprises the hinge region of CD8α.

[0030] In some examples of the present invention, the hinge region comprises the amino acid sequence shown in SEQ ID NO:8.

[0031] In some embodiments of the present invention, the chimeric antigen receptor further comprises a linker signal peptide.

[0032] In some specific embodiments of the present invention, the connecting signal peptide is the signal peptide of CD8α

[0033] In some examples of the present invention, the linker signal peptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0034] In some embodiments of the present invention, the extracellular domain targets a tumor antigen.

[0035] In some embodiments of the present invention, the tumor antigen is selected from GPC3, CLDN18.2, GCC, EGFRvIII, ROR1, CLDN6, MSLN, ALPP, MUC1, LGR5, HER2, OR2H1, DLL-3, C-MET, glyco-cMET, glyco-LAMP1, CD123, CD33, CLL-1, CD70, CD38, FLT3 and GRP78.

[0036] The second aspect of the present invention provides a polypeptide comprising the chimeric antigen receptor according to the first aspect.

[0037] In some embodiments of the present invention, the polypeptide is further linked to other functional molecules.

[0038] In some preferred embodiments of the present invention, the other functional molecules are selected from one or more of the following: signal peptides, protein tags, other antigen binding molecules and cytokines.

[0039] The third aspect of the present invention provides an isolated nucleic acid encoding the chimeric antigen receptor according to the first aspect or the polypeptide according to the second aspect.

[0040] In some specific embodiments of the present invention, the nucleic acid sequence encoding the transmembrane domain of the chimeric antigen receptor is shown in SEQ ID NO:9.

[0041] In some specific embodiments of the present invention, the nucleic acid sequence encoding the activation domain of the chimeric antigen receptor is shown in SEQ ID NO:15.

[0042] In some specific embodiments of the present invention, the nucleic acid sequence encoding the costimulatory domain of the chimeric antigen receptor is shown in SEQ ID NO:11 and / or SEQ ID NO:13.

[0043] In some specific embodiments of the present invention, the nucleic acid sequence encoding the hinge region of the chimeric antigen receptor is shown in SEQ ID NO:7.

[0044] In some specific embodiments of the present invention, the nucleic acid sequence encoding the signal peptide of the chimeric antigen receptor is shown as SEQ ID NO: 3.

[0045] The fourth aspect of the present invention provides a vector comprising the nucleic acid as described in the third aspect.

[0046] In some embodiments of the present invention, the vector is a plasmid, cosmid, phage or viral vector.

[0047] In some specific embodiments of the present invention, the backbone of the viral vector is pHAGE.

[0048] The fifth aspect of the present invention provides a gene expression cassette, comprising a promoter and the nucleic acid according to the third aspect.

[0049] The sixth aspect of the present invention provides an immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor as described in the first aspect or the polypeptide as described in the second aspect.

[0050] In some embodiments of the present invention, the immune effector cells are selected from one or more of T cells, NK cells, NKT cells, monocytes, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells.

[0051] In some specific embodiments of the present invention, the immune effector cells are T cells, NK cells and NKT cells; for example, T cells.

[0052] The seventh aspect of the present invention provides a method for preparing immune effector cells, comprising the step of introducing the vector described in the fourth aspect or the gene expression cassette described in the fifth aspect into parent immune effector cells, thereby obtaining immune effector cells.

[0053] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises the chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the vector as described in the fourth aspect, the gene expression cassette as described in the fifth aspect and / or the immune effector cell as described in the sixth aspect.

[0054] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0055] In the present invention, the pharmaceutical composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid such as glycine; antioxidant; chelating agent such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative.

[0056] The ninth aspect of the present invention provides the use of the chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the vector as described in the fourth aspect, the gene expression cassette as described in the fifth aspect, the immune effector cell as described in the sixth aspect and / or the pharmaceutical composition as described in the eighth aspect in the preparation of a drug for diagnosing, preventing and / or treating tumors or cancer; the tumors include solid tumors and non-solid tumors.

[0057] In some embodiments of the present invention, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0058] The tenth aspect of the present invention provides a method for diagnosing, preventing and / or treating tumors or cancers in a patient in need thereof, the method comprising administering to a patient in need thereof an effective amount of the chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the vector as described in the fourth aspect, the gene expression cassette as described in the fifth aspect, the immune effector cell as described in the sixth aspect and / or the pharmaceutical composition as described in the eighth aspect; the tumor includes solid tumors and non-solid tumors.

[0059] The pharmaceutical compositions of the present invention can be administered in a manner suitable for diagnosing, preventing, and / or treating tumors or cancers. The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease. Administration can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection, or intraperitoneally.

[0060] In some embodiments of the present invention, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0061] The eleventh aspect of the present invention provides a composition for diagnosing, preventing and / or treating tumors or cancer, the composition comprising the chimeric antigen receptor as described in the first aspect, the polypeptide as described in the second aspect, the nucleic acid as described in the third aspect, the vector as described in the fourth aspect, the gene expression cassette as described in the fifth aspect, the immune effector cell as described in the sixth aspect and / or the pharmaceutical composition as described in the eighth aspect; the tumor includes solid tumors and non-solid tumors.

[0062] In some embodiments of the present invention, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0063] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0064] The reagents and raw materials used in the present invention are commercially available.

[0065] In some embodiments of the present invention, the chimeric antigen receptor has an extended half-life and has a strong killing activity against tumors at a low efficiency target ratio, which helps to improve the anti-tumor activity of CAR-T against solid tumors and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 is a diagram showing the expression of CAR on the surface of CAR-T cell membrane detected by flow cytometry in Example 3.

[0067] Figure 2 is a diagram showing the killing effect of CAR-T-1, CAR-T-2, CAR-T-3, CAR-T-4 and CAR-T-5 cells on target cells in Example 4.

[0068] FIG3 is an in vivo imaging diagram of tumor-bearing mice in Example 5.

[0069] Figure 4 is a diagram showing the killing effect of CAR-T-GC33-1 and CAR-T-1-B1-1 cells on target cells in Example 7.

[0070] Figure 5 is a diagram showing the killing effect of CAR-T-GC33-2 and CAR-T-1-B1-2 cells on target cells in Example 9. DETAILED DESCRIPTION

[0071] Definition of terms

[0072] The term "chimeric antigen receptor" or "CAR (Chimeric Antigen Receptor)" used herein generally refers to a fusion protein comprising an extracellular domain and an intracellular domain that can bind to an antigen. CAR is a core component of a chimeric antigen receptor immune effector cell, which may include an antigen (e.g., tumor-associated antigen (TAA)) binding domain, a transmembrane domain, and an intracellular domain. In the present application, the chimeric antigen receptor can be combined with an immune effector cell receptor activation intracellular domain based on the antigen (e.g., GPC3) specificity of an antibody. Genetically modified immune effector cells expressing CAR can specifically identify and eliminate malignant cells expressing target antigens.

[0073] The term "extracellular domain" as used herein generally refers to a domain that can specifically bind to an antigen, including but not limited to a single-chain antibody, an antibody or its antigen-binding fragment, a tethered ligand or an extracellular domain of a co-receptor. For example, the extracellular domain may comprise an antibody or its antigen-binding fragment that can specifically bind to an antigen expressed on a cell. The terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" used in this application are used interchangeably, and provide a domain or fragment of a CAR with the ability to specifically bind to a target antigen (e.g., GPC3). The extracellular domain can be of natural origin, synthetic origin, semi-synthetic origin or recombinant origin. The "specific binding" generally refers to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to an antigen (which can be an epitope) is an antibody that binds to the antigen with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other antigens. As used herein, the term "epitope" and its grammatical alternatives may refer to a portion of an antigen that is recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope may be a tumor epitope or pathogen epitope recognized by a chimeric antigen receptor. Multiple epitopes within an antigen may also be recognized. Epitopes may also mutate.

[0074] The term "antibody" is used in the broadest sense herein and refers to a polypeptide or combination of polypeptides comprising sufficient sequence from the variable region of the heavy chain of an immunoglobulin and / or sufficient sequence from the variable region of the light chain of an immunoglobulin to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit desired antigen-binding activity. In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only complete antibodies, but also antigen-binding fragments of antibodies. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for CDR grafting, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0075] The term "antigen-binding fragment" herein does not have the entire structure of a complete antibody, but only comprises a partial or partial variant of a complete antibody, wherein the partial or partial variant has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody, and single-domain antibody. The terms "single domain antibody (sdAb)", "VHH domain" and "nanobody" herein have the same meaning and are used interchangeably, referring to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, a heavy chain antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region. It should be noted that nanobodies can be used to form other forms of antibodies, such as antibodies that can contain VH-CH2-CH3 from N-terminus to C-terminus, or contain VH-CH1-CH2-CH3; and can form homodimers, such as heavy chain dimer antibodies without light chains.

[0076] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0077] The term "transmembrane domain" as used herein refers to a structure that can anchor CAR to the plasma membrane of a cell, which is connected to the intracellular domain and plays a role in transmitting signals. The natural transmembrane portion of common cell adhesion molecules can be used for the "transmembrane domain" of CAR. For example, the natural transmembrane portion of CD8α or the natural transmembrane portion of CD28 can be used as the "transmembrane domain" in CAR.

[0078] As used herein, the term "activation domain" refers to a molecule on an immune effector cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0079] As used herein, the term "costimulatory domain" refers to a cognate binding partner on an immune effector cell that specifically binds a costimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, but not limited to, proliferation.

[0080] As used herein, the term "targeting" refers to specific binding, specifically to a non-random binding reaction between two molecules, such as an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 The antigen is bound with an affinity (KD) of M or less.

[0081] As used herein, the term "tumor" or "cancer" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0082] The term "tumor antigen" as used herein refers to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). TAA or TSA can be expressed on blood cancer cells. TAA or TSA can be expressed on solid tumor cells. The tumor antigen is selected from one or more of the following: CD19, CD133, CD123, CD20, CD22, CD30, CD33, CD171, CD80 / 86, CA125, C-met, L1CAM, EC, DLL3, CD99, GRP78, 5T4, CD138, CS-1 (also known as CD2 subclass 1, CRACC, SLAMF7, CD319 or 19A24), Glycan-3 (GPC3), Claudin 18.2 (Claudin 18.2), Glycan-4 (GPC4), Glycan-5 (GPC6), Glycan-7 (GPC8), Glycan-9 (GPC9), Glycan-10 (GPC11), Glycan-11 (GPC12), Glycan-10 (GPC13), Glycan-10 (GPC14), Glycan-10 (GPC15), Glycan-10 (GPC16), Glycan-10 (GPC17), Glycan-10 (GPC18), Glycan-10 (GPC19), Glycan-10 (GPC11), Glycan-10 (GPC11), Glycan-10 (GPC15), Glycan-10 (GPC16), Glycan-10 (GPC17), Glycan-10 (GPC19), Glycan-10 (GPC1 ... 18.2), guanylate cyclase C (GCC / GUCY2C), mesothelin (MSLN), epidermal growth factor receptor (EGFR), prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP), tyrosine protein kinase receptor UFO (AXL), death receptor 5 (DR5), NKG2D ligand, prostate stem cell antigen (PSCA), macrophage stimulating protein receptor (MST1R), inhibitory leukocyte immunoglobulin-like receptor (LILRB4), C-type lectin-like molecule-1 (CLL-1 or CLECL1), epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn Ag, GalNAcα-Ser / Thr), glyco-cMET, glyco-LAMP1, receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3);Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, B7H3 (CD276), B7-H4, KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen-4 (SSEA-4), folate receptor alpha (FR-α), receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface-associated mucin 1 (MUC1), cell surface-associated Mucin-16 (MUC16), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), mutant elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (e.g., proteasome, megalin) subunit type β 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1;Sialyl Lewis adhesion molecule (sLe), transglutaminase 5 (TGS5), high molecular weight-melanoma associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tight junction protein 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), globoH The hexose moiety of glycoceramide (GloboH), breast differentiation antigen (NY-BR-1), urothelial differentiation-specific glycoprotein (uroplakin) 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor β3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative open reading frame protein (TARP), Wilm tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation variant gene 6 on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, p53, p53 mutant, prostate-specific protein (prostein), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1);Rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), melanoma inhibitor of apoptosis protein (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytic leukemia virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), squamous cell carcinoma antigen 3 recognized by T cells (SART3), paired box protein Pax-5 (PAX5), pre-acrosomal protein binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, mutant heat shock protein 70-2 (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of the IgA receptor (FCAR or CD890), leukocyte immunoglobulin-like receptor subfamily A, member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12, member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), lymphocyte antigen 75 (LY75), placental alkaline phosphatase (ALPP), Fc receptor-like 5 (FCRL5) and / or immunoglobulin lambda-like polypeptide 1, leucine-rich repeat G-protein-coupled receptor 5 (LGR5), and olfactory receptor OR2H.

[0083] As used herein, the term "solid tumor" refers to a tumor selected from the group consisting of liver cancer, stomach cancer, lung cancer, breast cancer, colon cancer, renal cell carcinoma, non-small cell lung cancer, small intestine cancer, esophageal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, liposarcoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, neurilemmoma, malignant fibrous histiocytoma, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, spinal vertebral tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, and squamous cell carcinoma.

[0084] As used herein, the term "non-solid tumor" refers to a tumor selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, T-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, B lymphocytic neoplasm, and Waldenstrom macroglobulinemia.

[0085] As used herein, the term "Glypican-3 (GPC3)" is a heparan sulfate (HS) glycoprotein that belongs to the heparan sulfate proteoglycan family and is anchored to the cell membrane surface via phosphatidylinositol (GPI). "GPC3" herein includes mature or immature full-length wild-type GPC3 proteins or mutants thereof (e.g., point mutations, insertion mutations, or deletion mutations), splice variants, orthologs, and fragments of the aforementioned GPC3. Exemplarily, "GPC3" herein can be derived from mammals, e.g., humans, primates, such as monkeys (e.g., rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats.

[0086] The term "hinge region" as used herein can be considered as a part for providing flexibility to the extracellular antigen binding region, which is generally used to maintain further stable expression and activity of the chimeric antigen receptor in immune effector cells. The hinge region can be derived from the hinge region of CD8α or CD28 extracellular domain or IgG.

[0087] As used herein, the term "linker signal peptide" refers to a polypeptide attached to the N-terminus of a chimeric antigen receptor that may affect protein secretion efficiency. The linker signal peptide can be derived from a cell adhesion molecule, such as the signal peptide of CD8, or an immunoglobulin heavy chain signal peptide of an immune effector cell, such as a T cell or NK cell.

[0088] As used herein, the term "polypeptide" refers to a compound composed of amino acid residues covalently linked by peptide bonds. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0089] As used herein, the term "isolated" refers to a substance obtained artificially from its natural state. If a substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been separated from its natural environment, or both. For example, a highly purified polynucleotide or polypeptide isolated from a naturally occurring unisolated polynucleotide or polypeptide in a living animal is considered "isolated." The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0090] The term "coding" as used herein refers to the intrinsic properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) as a template for synthesizing other polymers and macromolecules with a definite nucleotide sequence (e.g., rRNA, tRNA and mRNA) or definite amino acid sequence in a biological process, and the resulting biological properties. Therefore, if transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and non-coding strand (as a template for transcription of a gene or cDNA) can be referred to as coded protein or other products of the gene or cDNA.

[0091] As used herein, the term "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. A "vector" may contain "regulatory elements" to regulate the expression of a gene or sequence.

[0092] As used herein, the term "regulatory element" includes promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoters or 35S enhanced promoters), internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). In some cases, regulatory elements include those that direct the constitutive expression of a nucleotide sequence in many types of cells and those that direct the nucleotide sequence to be expressed only in certain cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific.

[0093] The term "immune effector cells" as used herein refers to immune cells that can participate in the clearance of foreign antigens and perform effector functions in immune responses, and can be used in cell therapy as autologous cells or allogeneic cells for cell immunotherapy.

[0094] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0095] The present invention is further illustrated by way of examples below, but the invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or according to the product specifications. All reagents and raw materials used in the present invention are commercially available.

[0096] Example 1

[0097] This example prepares mutant CAR-T (CAR-T-1) cells, in which all lysine K sites in the intracellular segment of CAR-T-1 are mutated to glycine G, including the following steps:

[0098] 1) Construction of CAR plasmid. This example uses a third-generation CAR targeting GPC3, with a mutant CAR (CAR-1) encoding sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2, comprising the following parts in sequence: CD8α signal peptide (encoding sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4), hGPC3 scFv (YP7, encoding sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6), CD8α hinge region (encoding sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8), CD8α transmembrane region (encoding sequence SEQ ID NO: 9, amino acid sequence SEQ ID NO: 10), mutant CD28 costimulatory domain (encoding sequence SEQ ID NO: 11, amino acid sequence SEQ ID NO: 12), mutant 4-1BB costimulatory domain (encoding sequence SEQ ID NO: 13, amino acid sequence SEQ ID NO: 14), and mutant CD3ζ intracellular domain (encoding sequence SEQ ID NO: 15, amino acid sequence SEQ ID NO: 16). The above CAR-1 coding sequence was cloned into the pHAGE-EF1αL-eGFP vector (Addgene, catalog number 126686) and sequenced for verification.

[0099] 2) Construction of CAR lentiviral vector. This example uses a second-generation lentiviral packaging system. A plasmid mixture was prepared in the following ratio: psPAX2 (Addgene, Catalog No. 12260): pMD2.G (Addgene, Catalog No. 12259): CAR plasmid = 3:1:4. After mixing, the mixture was added dropwise to a 293T cell culture dish. The virus was collected at 48 hours and 72 hours, and concentrated by ultrafiltration and centrifugation to obtain the CAR lentiviral vector.

[0100] 3) Preparation of CAR-T-1 cells. PBMCs from healthy human donors were sorted using magnetic beads (Stemcell, Catalog No. 17951) to obtain T cells. T cells were activated using TransAct (Miltenyi Biotec, Catalog No. 130-111-160). One day later, the CAR lentiviral vector from step 2) was added for infection to obtain CAR-T-1 cells.

[0101] Example 2

[0102] This example prepares mutant CAR-T (CAR-T-2) cells. This example differs from Example 1 in that only all lysine K sites of CD3ζ in the CAR are mutated to glycine G. The amino acid sequence of CAR in CAR-T-2 is SEQ ID NO: 17.

[0103] Comparative Example 1

[0104] This comparative example prepares unmutated CAR-T (CAR-T-NT) cells, mutant CAR-T-3 (all lysine K sites in the intracellular segment of CAR are mutated to arginine R) cells, mutant CAR-T-4 (all lysine K sites in the intracellular segment of CAR are mutated to histidine H) cells, and mutant CAR-T-5 (all lysine K sites in the intracellular segment of CAR are mutated to glutamate E) cells. The only difference from Example 1 is that the CAR coding sequence is different, so that all lysine K sites in the intracellular segment of CAR are not mutated or mutated to corresponding amino acids. The CAR amino acid sequences in unmutated CAR-T, mutant CAR-T-3, mutant CAR-T-4, and mutant CAR-T-5 are SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0105] In this comparative example, mock cells were prepared, i.e., wild-type T cells not infected with lentivirus. T cells were obtained by magnetic bead sorting (Stemcell, Catalog No. 17951) of PBMCs from healthy human donors and activated using TransAct (Miltenyi Biotec, Catalog No. 130-111-160).

[0106] Example 3: Detection of the degradation rate of CAR on the surface of T cells

[0107] In this example, the degradation levels of CAR mediated by antigen stimulation of GPC3 CAR-T cells, namely CAR-T-NT cells, CAR-T-1 cells, CAR-T-2 cells, CAR-T-3 cells, CAR-T-4 cells and CAR-T-5, were detected in vitro.

[0108] GPC3 CAR-T cells were co-cultured with GPC3-positive target cells Hep-G2 at an effector-target ratio of 1:4 in a 24-well cell culture plate. Three replicate wells were set up for each CAR-T cell type, and T cells in the culture supernatant were collected at 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. Flow cytometry was used to detect the expression of CAR on the surface of the CAR-T cell membrane.

[0109] The results are shown in Figure 1. The values ​​shown on the horizontal axis are relative CAR expression levels (CAR expression at 0 h is 1). After CAR-T cells interact with target cells, the expression of CAR on the surface of CAR-T-NT cells rapidly decreases. The expression of CAR on the surface of CAR-T-1 cells, CAR-T-2 cells, CAR-T-3 cells, CAR-T-4 cells, and CAR-T-5 cells increases briefly and then decreases. Compared with the CAR-T-NT group, the expression of CAR on the surface of CAR-T-1 cells, CAR-T-3 cells, CAR-T-4 cells, and CAR-T-5 cells with lysine mutations in the CAR intracellular domain decreases less. Among them, the CAR expression of CAR-T-1 cells with all lysine K sites in the CAR intracellular domain mutated to glycine G decreases the least. At 96 h of co-culture, the relative average expression of CAR on the surface is 66.19%, while that of the CAR-T-NT group is only 29.47%. At the same time, the experiment found that the expression level of CAR on the surface of CAR-T-2 cells in which only the lysine K site in the CD3ζ region was mutated to glycine G was also significantly lower than that in the CAR-T-NT group, indicating that only the mutation of the CD3ζ lysine K site of CAR to glycine G can still significantly slow down the downregulation of CAR expression.

[0110] Example 4: Detection of T cell cytotoxicity in vitro

[0111] This example detects the in vitro tumor cell killing activity of Mock cells, CAR-T-NT cells, CAR-T-1 cells, CAR-T-2 cells, CAR-T-3 cells, CAR-T-4 cells, and CAR-T-5 cells.

[0112] T cells were seeded with luciferase-expressing Hep-G2 cells at three different effector-target ratios (1:2, 1:1, and 2:1) in 96-well U-shaped cell culture plates. After 16 hours of incubation, luciferase substrate was added and the cells were read on a microplate reader to measure in vitro cytotoxicity. Negative controls were used, with Hep-G2 cells expressing luciferase and no T cells inoculated. The average value of the negative control group was designated as a, and the value of the experimental wells was designated as b. The killing percentage (%) of the experimental wells = (1-b / a) × 100%.

[0113] The results are shown in Figure 2. There was no significant difference in the killing effect of CAR-T cells on target cells at a high-efficiency target ratio (2:1) among all groups. However, at a low-efficiency target ratio (1:2), the killing rate of CAR-T-NT was only 45.58%. Correspondingly, the killing rates of CAR-T-1 were 80.17%, CAR-T-2 were 79.08%, CAR-T-3 were 68.72%, CAR-T-4 were 54.87%, and CAR-T-5 were 68.94%. The results showed that at a low target ratio (1:2), compared with CAR-T-NT cells without mutation, mutation of all lysine K sites in the intracellular domain of CAR to glycine G, arginine R, histidine H, and glutamate E can enhance the tumor cell killing activity of CAR-T cells. The tumor cell killing activity of CAR with all lysine K sites in the intracellular domain mutated to glycine G was the most improved. In addition, the experiment found that the tumor cell killing activity of the CAR-T-2 cell group in which only the lysine K site in the CD3ζ region was mutated to glycine G was also significantly stronger than that of the CAR-T-NT group at a low target ratio (1:2), indicating that only mutating the CD3ζ lysine K site of CAR to glycine G can still significantly enhance the tumor cell killing activity of CAR-T.

[0114] Example 5: Detection of the anti-tumor effect of T cells in tumor-bearing mice

[0115] This example detects the anti-tumor effects of CAR-T-NT cells, CAR-T-1 cells, CAR-T-2 cells, CAR-T-3 cells, CAR-T-4 cells, and CAR-T-5 cells in a tumor mouse model.

[0116] A peritoneal liver cancer model was established in severely immunodeficient N-BDG mice using luciferase-containing Hep-G2 cells. Six days after modeling, the mice were randomly divided into three groups based on in vivo imaging: mock, CAR-T-NT, CAR-T-1, CAR-T-2, CAR-T-3, CAR-T-4, and CAR-T-5, with five mice in each group. Seven days after modeling, the mice were administered: the mock group was injected with wild-type T cells not infected with lentivirus, while the other experimental groups were injected with CAR-T cells (the CAR-T cell injection dose was calculated based on the number of positive CAR-T cells, and the same dose of positive CAR-T cells was injected). The day of drug administration was designated as Day 0. In vivo imaging was performed every week, and body weight changes were recorded every three days to observe tumor growth and mouse health indicators.

[0117] The results are shown in Figure 3. Compared with the mock group, tumors in the CAR-T-NT, CAR-T-1, CAR-T-2, CAR-T-3, CAR-T-4, and CAR-T-5 groups all showed some degree of tumor suppression. Compared with unmutated CAR-T-NT cells, mutations of all lysine K sites in the CAR intracellular domain to glycine G, arginine R, histidine H, and glutamic acid E enhanced the tumor cell killing activity of CAR-T cells and further improved the tumor suppression effect. Among them, mutations of all lysine K sites in the CAR intracellular domain to glycine G had the greatest tumor suppression effect, achieving complete tumor clearance in two CAR-T-1 mice. Furthermore, the experiment found that the tumor suppression effect of the CAR-T-2 cell group, in which only the lysine K site in the CD3ζ region was mutated to glycine G, was significantly stronger than that of the CAR-T-NT group, indicating that mutation of only the CD3ζ lysine K site in the CAR to glycine G can still significantly enhance the tumor suppression effect of CAR-T.

[0118] Example 6

[0119] In this example, mutant CAR-T (CAR-T-GC33-1, CAR-T-1-B1-1) cells were prepared. The difference from the CAR-T-1 in Example 1 was that the antibody YP7 in the CAR was changed to GC33 (amino acid sequence as shown in SEQ ID NO: 22) or 1-B1 (amino acid sequence as shown in SEQ ID NO: 23) to obtain CAR-T-GC33-1 cells and CAR-T-1-B1-1 cells.

[0120] In this example, unmutated CAR-T (CAR-T-GC33-NT, CAR-T-1-B1-NT) cells were prepared. The difference from the CAR-T-NT cells in Comparative Example 1 was that the antibody YP7 in CAR was changed to GC33 (amino acid sequence as shown in SEQ ID NO: 22) or 1-B1 (amino acid sequence as shown in SEQ ID NO: 23) to obtain CAR-T-GC33-NT cells and CAR-T-1-B1-NT cells.

[0121] Example 7

[0122] This example detects the in vitro tumor cell killing activity of CAR-T-GC33-1 cells, CAR-T-1-B1-1 cells, CAR-T-GC33-NT cells, and CAR-T-1-B1-NT cells in Example 6.

[0123] The CAR-T cells were seeded in a 96-well U-shaped cell culture plate at an effector-target ratio of 1:2 with luciferase-expressing Hep-G2 cells. After 16 hours of culture, luciferase substrate was added and the cells were read on a microplate reader to detect in vitro cytotoxicity. A negative control group was used with luciferase-expressing Hep-G2 cells and no T cells. The average value of the negative control group was designated as a, and the value of the experimental well was designated as b. The killing percentage of the experimental well (killing%) = (1-b / a) × 100%.

[0124] The results are shown in Figure 4. At an effector-target ratio of 1:2, the killing ratio of CAR-T-GC33-1 cells was much higher than that of CAR-T-GC33-NT cells, and the killing ratio of CAR-T-1-B1-1 cells was much higher than that of CAR-T-1-B1-NT cells, indicating that compared with CAR-T cells in which the CAR intracellular domain CD28 costimulatory domain, 4-1BB costimulatory domain, and CD3ζ intracellular domain were not mutated, mutations of the CAR intracellular domain CD28 costimulatory domain, 4-1BB costimulatory domain, and CD3ζ intracellular domain to glycine G can greatly enhance the tumor cell killing activity.

[0125] Example 8

[0126] In this example, mutant CAR-T (CAR-T-GC33-2, CAR-T-1-B1-2) cells were prepared. The difference from CAR-T-2 in Example 2 was that the antibody YP7 in CAR was changed to GC33 (amino acid sequence as shown in SEQ ID NO: 22) or 1-B1 (amino acid sequence as shown in SEQ ID NO: 23) to obtain CAR-T-GC33-2 cells and CAR-T-1-B1-2 cells.

[0127] In this example, unmutated CAR-T (CAR-T-GC33-NT, CAR-T-1-B1-NT) cells were prepared. The difference from the CAR-T-NT cells in Comparative Example 1 was that the antibody YP7 in CAR was changed to GC33 (amino acid sequence as shown in SEQ ID NO: 22) or 1-B1 (amino acid sequence as shown in SEQ ID NO: 23) to obtain CAR-T-GC33-NT cells and CAR-T-1-B1-NT cells.

[0128] Example 9

[0129] This example detects the in vitro tumor cell killing activity of Example 8 CAR-T-GC33-2 cells, CAR-T-1-B1-2 cells, Example 6 CAR-T-GC33-NT cells, and CAR-T-1-B1-NT cells.

[0130] The CAR-T cells were seeded in a 96-well U-shaped cell culture plate at an effector-target ratio of 1:2 with luciferase-expressing Hep-G2 cells. After 16 hours of culture, luciferase substrate was added and the cells were read on a microplate reader to detect in vitro cytotoxicity. A negative control group was used with luciferase-expressing Hep-G2 cells and no T cells. The average value of the negative control group was designated as a, and the value of the experimental well was designated as b. The killing percentage of the experimental well (killing%) = (1-b / a) × 100%.

[0131] The results are shown in Figure 5. At an effector-target ratio of 1:2, the killing ratio of CAR-T-GC33-2 cells was much higher than that of CAR-T-GC33-NT cells, and the killing ratio of CAR-T-1-B1-2 cells was much higher than that of CAR-T-1-B1-NT cells, indicating that compared with CAR-T cells in which the CD3ζ region of the CAR intracellular domain has not been mutated, mutating the lysine K site in the CD3ζ region of the CAR intracellular domain to glycine G can greatly enhance the tumor cell killing activity.

Claims

1. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain connected in sequence; The extracellular domain recognizes and binds to an antigen; The intracellular domain includes an activation domain, or includes a co-stimulatory domain and an activation domain; Relative to the parent intracellular domain, the lysine in the co-stimulatory domain and / or the activation domain is partially or completely replaced by glycine.

2. The chimeric antigen receptor according to claim 1, wherein The number of the co-stimulatory domains is at least one; Preferably: Relative to the parent intracellular domain, the lysine in the activation domain is partially or completely replaced by glycine; or, relative to the parent intracellular domain, the lysine in at least one of the co-stimulatory domain and the activation domain is partially or completely replaced by glycine.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The activation domain comprises a functional signaling domain selected from the following proteins: CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10 and / or DAP12; preferably CD3ζ; And / or, the costimulatory domain is selected from the costimulatory domains of the following proteins: 4-1BB, CD27, CD28, OX-40 and / or ICOS; preferably CD28 and / or 4-1BB; And / or, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and / or CD154; preferably CD8; And / or, the extracellular domain is an antibody or antigen-binding fragment, such as scFv, single domain antibody or F(ab)'.

4. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that The chimeric antigen receptor further comprises a hinge region, wherein the hinge region connects the extracellular domain and the transmembrane domain; preferably, the hinge region comprises the hinge region of CD8α or CD28, for example, CD8α; And / or, the chimeric antigen receptor further comprises a linker signal peptide, preferably a signal peptide of CD8α; and / or, the extracellular domain targets a tumor antigen; Preferably, the tumor antigen is selected from GPC3, CLDN18.2, GCC, EGFRvIII, ROR1, CLDN6, MSLN, ALPP, MUC1, LGR5, HER2, OR2H1, DLL-3, C-MET, glyco-cMET, glyco-LAMP1, CD123, CD33, CLL-1, CD70, CD38, FLT3 and GRP78.

5. The chimeric antigen receptor according to any one of claims 1 to 4, characterized in that The activation domain is a functional signal transduction domain of CD3ζ, and the activation domain preferably comprises an amino acid sequence as shown in SEQ ID NO: 16; and / or, the costimulatory domain is the costimulatory domain of CD28, and the costimulatory domain preferably comprises the amino acid sequence shown in SEQ ID NO: 12 and / or SEQ ID NO: 14; And / or, the transmembrane domain is the transmembrane domain of CD8, and the transmembrane domain preferably comprises the amino acid sequence shown in SEQ ID NO: 10; and / or, the hinge region is the hinge region of CD8α, preferably comprising the amino acid sequence shown in SEQ ID NO: 8; And / or, the connection signal peptide is a signal peptide of CD8α, preferably comprising an amino acid sequence as shown in SEQ ID NO:

4.

6. A polypeptide, characterized in that The polypeptide comprises the chimeric antigen receptor according to any one of claims 1 to 5; Preferably, the polypeptide is also connected to other functional molecules; More preferably, the other functional molecules are selected from one or more of the following: signal peptides, protein tags, other antigen binding molecules and cytokines.

7. An isolated nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor according to any one of claims 1 to 5 or the polypeptide according to claim 6; Preferably, the nucleic acid sequence encoding the transmembrane domain of the chimeric antigen receptor is as shown in SEQ ID NO:9; and / or, the nucleic acid sequence encoding the activation domain of the chimeric antigen receptor is as shown in SEQ ID NO:15; and / or, the nucleic acid sequence encoding the co-stimulatory domain of the chimeric antigen receptor is as shown in SEQ ID NO:11 and / or SEQ ID NO:13; and / or, the nucleic acid sequence encoding the hinge region of the chimeric antigen receptor is as shown in SEQ ID NO:7; and / or, the nucleic acid sequence encoding the signal peptide of the chimeric antigen receptor is as shown in SEQ ID NO:

3.

8. A carrier, characterized in that The vector comprises the nucleic acid of claim 7; Preferably, the vector is a plasmid, cosmid, phage or viral vector; More preferably, the backbone of the viral vector is pHAGE.

9. A gene expression cassette, characterized in that: The gene expression cassette comprises a promoter and the nucleic acid according to claim 7.

10. An immune effector cell, characterized in that The immune effector cell expresses the chimeric antigen receptor according to any one of claims 1 to 5 or comprises the polypeptide according to claim 6; Preferably, the immune effector cells are selected from one or more of T cells, NK cells, NKT cells, monocytes, mast cells, macrophages, dendritic cells, CIK cells and stem cell-derived immune effector cells; More preferably, the immune effector cells are T cells, NK cells or NKT cells; for example, T cells.

11. A method for preparing immune effector cells, characterized in that: The preparation method comprises the step of introducing the vector according to claim 8 or the gene expression cassette according to claim 9 into parent immune effector cells, thereby obtaining immune effector cells.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 1 to 5, the polypeptide according to claim 6, the nucleic acid according to claim 7, the vector according to claim 8, the gene expression cassette according to claim 9 and / or the immune effector cell according to claim 10; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

13. Use of the chimeric antigen receptor according to any one of claims 1 to 5, the polypeptide according to claim 6, the nucleic acid according to claim 7, the vector according to claim 8, the gene expression cassette according to claim 9, the immune effector cell according to claim 10 and / or the pharmaceutical composition according to claim 12 in the preparation of a drug for diagnosing, preventing and / or treating tumors or cancer; The tumors include solid tumors and non-solid tumors; Preferably, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, schwannoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

14. A method for diagnosing, preventing and / or treating a tumor or cancer in a patient in need thereof, characterized in that: The method comprises administering to a patient in need thereof an effective amount of a chimeric antigen receptor according to any one of claims 1 to 5, a polypeptide according to claim 6, a nucleic acid according to claim 7, a vector according to claim 8, a gene expression cassette according to claim 9, an immune effector cell according to claim 10, and / or a pharmaceutical composition according to claim 12; The tumors include solid tumors and non-solid tumors; Preferably, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, schwannoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

15. A composition for diagnosing, preventing and / or treating tumors or cancers, characterized in that: The composition comprises the chimeric antigen receptor according to any one of claims 1 to 5, the polypeptide according to claim 6, the nucleic acid according to claim 7, the vector according to claim 6, the gene expression cassette according to claim 9, the immune effector cell according to claim 10 and / or the pharmaceutical composition according to claim 12; The tumors include solid tumors and non-solid tumors; Preferably, the solid tumor is selected from liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, adrenal cancer, schwannoma, malignant fibrous histiocytoma and esophageal cancer; the non-solid tumor is selected from B lymphocytic neoplasm, Hodgkin's lymphoma, chronic myeloid leukemia and acute myeloid leukemia.