Engineered immune cells and uses thereof

Abstract

The invention provides an engineered immune cell and application thereof, and relates to the technical field of biology. The cell membrane of the engineered immune cell expresses the chimeric antigen receptor; the antibody or the antigen binding fragment of the antibody specifically binding to CD47 is expressed by the overexpression cytokine of the antibody or the antigen binding fragment of the antibody. The engineered immune cell can efficiently activate macrophages, and the tumor treatment effect is improved.

Description

[0001] Priority Statement

[0002] This application claims the priority of a Chinese patent application with the application number 202311839395.X, the filing date of December 27, 2023, and the invention title of "Engineered Immune Cells and Their Applications", the entire content of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and particularly to an engineered immune cell and its application. Background Art

[0004] Currently, CAR-modified immune cells have achieved great breakthroughs in hematological malignancies, but have limited efficacy against solid tumors. Solid tumors have an immunosuppressive tumor microenvironment, and immunosuppressive molecules and cells in the tumor microenvironment highly limit the intratumoral proliferation and anti-tumor activity of CAR-modified immune cells. Tumor-associated macrophages are a common immunosuppressive myeloid cell subset in solid tumors. A large number of studies have shown that this group of cells can promote tumor growth and metastasis and inhibit T cell immune responses. Inducing and activating these cells into pro-inflammatory macrophages can promote their antigen presentation, secretion of inflammatory cytokines, and ultimately promote T cell immune responses. Therefore, how to further improve CAR-modified immune cells to promote the activation of tumor-associated macrophages by CAR-modified immune cells in the tumor, thereby inducing a high-intensity intratumoral immune response to eliminate solid tumors, is a problem to be solved.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide an engineered immune cell that can efficiently activate macrophages to improve the tumor treatment effect.

[0007] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0008] In the first aspect, an engineered immune cell is provided, the cell membrane of which expresses a chimeric antigen receptor; and it overexpresses cytokines; and expresses an antibody or an antigen-binding fragment thereof that specifically binds to CD47;

[0009] The chimeric antigen receptor contains a signal transduction domain;

[0010] The antibody or antigen-binding fragment thereof that specifically binds to CD47 contains complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and / or complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region;

[0011] The HCDR1, HCDR2, and HCDR3 comprise amino acid sequences identical to the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1, and the LCDR1, LCDR2, and LCDR3 comprise amino acid sequences identical to the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.2;

[0012] In an alternative embodiment, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of the variable region is defined by any one system of Kabat, Chothia, IMGT, ABM, or Contact or a combination of multiple definition systems.

[0013] In a second aspect, a polynucleotide is also provided, which contains a nucleotide sequence encoding the chimeric antigen receptor in the first aspect, a nucleotide sequence encoding the cytokine in the first aspect, and a nucleotide sequence encoding an antibody specifically binding to CD47 or an antigen-binding fragment thereof in the first aspect.

[0014] In a third aspect, a biomaterial is also provided, and the biomaterial includes any one of the following (i) to (ii):

[0015] (i) A transgenic immune cell, characterized in that the genome of the transgenic immune cell contains a nucleotide sequence encoding the chimeric antigen receptor in the first aspect, a nucleotide sequence encoding the cytokine in the first aspect, and a nucleotide sequence encoding an antibody specifically binding to CD47 or an antigen-binding fragment thereof in the first aspect;

[0016] In an alternative embodiment, the polynucleotide according to claim 11 or 12 is integrated into the genome of the transgenic immune cell;

[0017] In an alternative embodiment, the transgenic immune cell includes a T cell, a natural killer cell, a monocyte, a macrophage, an NKT cell, a dendritic cell, a granulocyte, a B cell, a lymphocyte, a leukocyte, or a peripheral blood mononuclear cell;

[0018] In an alternative embodiment, the transgenic immune cell includes a T cell;

[0019] (ii) A recombinant vector, characterized in that the recombinant vector contains the polynucleotide described in the second aspect;

[0020] In an alternative embodiment, the recombinant vector is a lentiviral vector.

[0021] Fourthly, there is provided a method for preparing the engineered immune cells of the first aspect, the preparation method comprising introducing into immune cells a polynucleotide encoding the first aspect, a polynucleotide encoding the cytokine of the first aspect, and a polynucleotide encoding the antibody specifically binding to CD47 or an antigen-binding fragment thereof of the first aspect.

[0022] Fifthly, there is also provided the use of the immune cells of the first aspect, or the polynucleotides of the second aspect, or the biological materials of the third aspect, or the preparation method of the fourth aspect in the preparation of anti-tumor drugs.

[0023] Sixthly, there is also provided a cellular pharmaceutical composition, which comprises the engineered immune cells of the first aspect, or the transgenic immune cells of the third aspect.

[0024] Seventhly, there is also provided a method for treating tumors, the treatment method comprising administering to a subject in need of treatment the engineered immune cells of the first aspect, or the transgenic immune cells of the third aspect, or the cellular pharmaceutical composition of the sixth aspect. Description of the Drawings

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 Schematic diagram of the plasmid constructed for Example 1. From top to bottom, they are plasmids expressing only GPC3 CAR-T, plasmids expressing GPC3 CAR-T with IL-12, plasmids expressing GPC3 CAR-T with CD47, and plasmids expressing GPC3 CAR-T with IL-12-aCD47.

[0027] Figure 2 Results of the CAR positive rate detected by CytoFlex flow cytometer in Example 2.

[0028] Figure 3 IL-12 concentration in the culture supernatant of CAR-T cells detected by ELISA on the 7th day in Example 3.

[0029] Figure 4 Secretion results of IFN-γ of different CAR-T cells in Example 4.

[0030] Figure 5 Secretion results of granzyme B of different CAR-T cells in Example 4.

[0031] Figure 6 For the detection results of the memory phenotypes (CD45RA + CD62L + double-positive cells and CD45RA + CD27 + double-positive cells) of CAR-T cells detected by the flow cytometer in Example 5;

[0032] Figure 7 For the detection results of the macrophage activation phenotypes detected by the flow cytometer in Example 6;

[0033] Figure 8 For the bright-field microscope photos of the macrophage-hepatocellular carcinoma cell co-cultures treated with different CAR-T activation supernatants in Example 6;

[0034] Figure 9 For the flow cytometry quantitative detection results of the macrophage-hepatocellular carcinoma cell co-cultures treated with different CAR-T activation supernatants in Example 6;

[0035] Figure 10 For the cell number statistical results of the flow cytometry quantitative detection of the macrophage-hepatocellular carcinoma cell co-cultures treated with different CAR-T activation supernatants in Example 6. Detailed implementation manners

[0036] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0037] The articles "a / an" and "the" in the present invention include plural references unless the context clearly indicates otherwise. For example, "an antibody" refers to one antibody or more than one antibody.

[0038] In the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0039] In the present invention, the term "CD47", also known as integrin-associated protein (IAP), is a 50 kDa membrane protein with an amino-terminal immunoglobulin domain and a carboxy-terminal multiple transmembrane region. It interacts with a variety of ligands, including but not limited to signal regulatory protein alpha (SIRPα), SIRPγ, integrin, and thrombospondin 1 (TSP1). SIRPα is mainly expressed on myeloid cells, including macrophages, bone marrow dendritic cells (DCs), granulocytes, mast cells, and their precursors, including hematopoietic stem cells. The CD47 / SIRPα interaction transmits a "don't eat me" signal, preventing autophagy.

[0040] The molecules mentioned in the present invention, such as but not limited to CD47, GPC3, GUCY2C, CD19, MSLN, MUC16, MICA, HER2, CD70, FSHR, BAFFR, EPHA1, EGFRVIII, CEA, IL-12, IL-21, IL-15, and IL-7, etc., unless otherwise specified, the terms include molecules naturally expressed by cells or expressed by gene-transfected cells. The terms also include artificially modified molecules, and the artificial modification includes but is not limited to polypeptides or proteins after mutation, truncation, or fusion with other domains.

[0041] In the present invention, the term "antibody" includes any immunoglobulin that can bind to a specific antigen. The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, full-length antibodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Generally, a natural intact antibody consists of two heavy (H) chains and two light (L) chains. Antibodies can be classified into five main classes or isotypes, namely IgA, IgD, IgE, IgG, and IgM, depending on whether they contain α, δ, ε, γ, and μ heavy chains, respectively. Several main antibody classes can also be further divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain), etc. Each heavy chain consists of a variable region (VH) and first, second, third, and fourth (optionally) constant regions (CH1, CH2, CH3, CH4, respectively). Mammalian light chains can be divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region (CL). The variable regions of the light and heavy chains determine antigen binding. The variable region of each chain usually contains three hypervariable regions, called "complementary determining regions (CDRs)". Among them, the light chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy chain CDRs include HCDR1, HCDR2, and HCDR3. The variable regions (VH and VL) each consist of three complementary determining regions connected by four framework regions (FRs). Usually, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the CDRs and FRs numbered as follows in the following combination arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0042] In the present invention, the term "antigen-binding fragment" refers to a substance that contains the CDRs of an antibody, lacks some amino acids present in the full-length chain, but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include but are not limited to Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), and scFv dimers (bivalent bifunctional antibodies). The above antigen-binding fragments can bind to the same antigen as the parental antibody.

[0043] In the present invention, the term "Fab" of an antibody refers to a part of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and the first constant region of a single heavy chain bound together by a disulfide bond. The "Fab' fragment" refers to a Fab fragment containing a part of the hinge region. "F(ab')2" refers to a dimer of Fab'. The "Fv fragment" is composed of the variable region of a single light chain and / or the variable region of a single heavy chain bound together. The "single-chain Fv antibody" or "scFv" refers to an antibody fragment formed by directly connecting the variable region of the light chain and the variable region of the heavy chain or by connecting them through a peptide linker sequence. The "minimal antibody recognition unit" refers to a structure containing only a single CDR in the variable region. Although the minimal recognition unit has a small molecular weight and low affinity, it has the ability to bind to an antigen.

[0044] The CDR boundaries of the antibody or its antigen-binding fragment in the present invention can be defined or identified according to the IMGT, Kabat, Chothia, AbM, Contact definition methods, and the CDRs defined in other acceptable ways in the art also fall within the protection scope of the present invention (Kaas, Q et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains. Dev. Comp. Immunol. 29, 185-203, (2005); R.M. MacCallum et al.,. Antibody–antigen interactions: contact analysis and binding site topography J. Mol. Biol. (1996); Martin, A.C.R. Protein sequence and structure analysis of antibody variable domains (Book chapter). In Antibody engineering lab manual Eds. Duebel, S. and Kontermann, R. (2001); Marie-Paule Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Developmental and Comparative Immunology 27 (2003) 55–77).

[0045] In the present invention, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs typically have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of amino acids but act in a manner similar to naturally occurring amino acids.

[0046] In the present invention, the term "percentage of identity" refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Alignment of the percentage of amino acid sequence identity can be carried out in various ways in the art, such as by software well-known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment of the full length of the comparison sequences.

[0047] In the present invention, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, for example, the reaction between an antibody and an antigen. In some specific embodiments, for example, it is determined according to the fluorescence-activated cell sorting technique.

[0048] In the present invention, the term "overexpression" refers to an increase in the expression level of a polypeptide or protein of interest compared to the corresponding natural cells. The polypeptide or protein with increased expression level can be expressed endogenously and / or exogenously. For example, introducing a vector containing the gene encoding the polypeptide or protein of interest enables the independent expression of the encoding gene of the polypeptide or protein in the cells, aiming to increase the expression level. Additionally, for example, increasing the copy number of the gene encoding the polypeptide or protein of interest in the cell genome, or altering the regulatory elements controlling its expression can achieve an increase in the expression level of the polypeptide or protein of interest. "Overexpression" can be achieved by methods known and conventional in the art, such as introducing exogenous genes, or performing gene editing on the cells through gene editing. The present invention does not limit this.

[0049] The "chimeric antigen receptor" in the present invention, CAR, is an artificial receptor that mimics the function of TCR and is composed of an extracellular domain, a transmembrane domain, and an intracellular signaling domain connected in sequence. When the antigen (receptor) on the surface of tumor cells binds to the antibody (ligand) of the chimeric antigen receptor, signals can be transmitted to the intracellular through the transmembrane region. The intracellular signaling domain then converts the signals into activation signals to activate effector cells. The effector cells kill tumor cells by secreting perforin or producing cytokines. At the same time, the effector cells themselves also undergo amplification, further expanding the immune killing effect.

[0050] In an alternative embodiment, the structure of an exemplary CAR is as follows:

[0051] The extracellular domain generally consists of a single-chain antibody (single-chain variable fragment, scFv) of a monoclonal antibody responsible for recognizing and binding the antigen and a hinge region (Hinge) that serves as a linker. The single-chain antibody is the antigen-binding domain of CAR, and this domain determines the specificity and function of the immune cells expressing CAR. The extracellular domain also contains a hinge region, which is the extracellular structural region of CAR that connects the single-chain variable fragment and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells. The hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8α / CD28. The type and length of the hinge region have an important impact on the functional activity of CAR. The transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8, CD28, and CD3ζ or their derivatives. The selection of the transmembrane domain affects the activation degree of the CAR structure in cell function. The intracellular domain contains a signal transduction domain, or is composed of a co-stimulatory domain and a signal transduction domain together. The co-stimulatory domain usually comes from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The signal transduction domain is usually the T cell receptor TCR / CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain.

[0052] The CAR of the present invention (including its functional parts and functional variants) can be obtained by methods known in the art, for example, it can be prepared by any suitable method for preparing polypeptides or proteins.

[0053] In the present invention, the term "engineered immune cell" refers to an immune cell expressing a CAR or an immune cell modified with a CAR, wherein the immune cells include but are not limited to T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, natural killer T cells (NKT cells), dendritic cells, granulocytes, B cells, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells.

[0054] In the present invention, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, and nucleic acid molecules include ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include but are not limited to single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemical, or biochemical modifications, unnatural, or derived nucleobases. When a polynucleotide encodes a protein or polypeptide, the encoding may optionally be for the sense strand or the antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" can be used interchangeably.

[0055] In an alternative embodiment, the nucleic acid molecule is RNA or DNA, the nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. When a nucleic acid molecule is placed in a functional relationship with another nucleic acid sequence, the nucleic acid molecule is "operably linked". For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. When incorporated into a vector, DNA is preferably used.

[0056] In the present invention, the term "vector" refers to a vehicle into which a genetic element (such as the aforementioned nucleic acid molecule) can be operably inserted and which enables the genetic element to be expressed, for example, to produce a protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries can be expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses, etc. The vector can contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication. The vector can also include components that assist its entry into the cell, including but not limited to, virus particles, liposomes or protein coats. The vector can be an expression vector or a cloning vector. In some embodiments, the vector (such as an expression vector) provided by the present invention contains the nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof according to the present invention, at least one promoter (such as SV40, CMV, EF1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0057] In the present invention, the terms "purified" or "isolated" in relation to a polypeptide or nucleic acid mean that the polypeptide or nucleic acid is not in its natural medium or natural form. Thus, the term "isolated" includes a polypeptide or nucleic acid removed from its original environment, for example, if it is naturally occurring, from its natural environment. In relation to a nucleic acid, the terms isolated or purified indicate, for example, that the nucleic acid is not in its natural genomic context (such as in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

[0058] In the present invention, the term "pharmaceutical composition" is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.

[0059] In the present invention, a "pharmaceutically acceptable carrier" can include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc.

[0060] In the present invention, the term "effective amount" refers to a therapeutic amount, an amount sufficient to reduce or ameliorate the severity and / or duration of a disorder and / or one or more of its symptoms; prevent the progression of a disease; cause regression of a disease; prevent recurrence, development, or progression of one or more symptoms associated with a disease; detect a disease; or enhance or improve the prophylactic or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). The therapeutically effective dose of the antibody or antigen-binding fragment thereof described in the present invention depends on a variety of factors well known in the art, such as body weight, age, medical history, current treatment, the health status of the subject and the potential for cross-infection, allergies, hypersensitivity, and side effects, as well as the route of administration and the degree of tumor development. A person skilled in the art (e.g., a doctor or veterinarian) can proportionally reduce or increase the dose according to these or other conditions or requirements.

[0061] In a first aspect, there is provided an engineered immune cell, the engineered immune cell membrane expressing a chimeric antigen receptor; and overexpressing a cytokine; and expressing an antibody or an antigen-binding fragment thereof that specifically binds CD47.

[0062] The antibody or antigen-binding fragment thereof that specifically binds CD47 comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and / or complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region;

[0063] The HCDR1, HCDR2, and HCDR3 comprise amino acid sequences identical to the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1, and HCDR1, HCDR2, and HCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, or the IMGT definition, as shown in Table 1.

[0064] The LCDR1, LCDR2, and LCDR3 comprise amino acid sequences identical to the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO.2. LCDR1, LCDR2, and LCDR3 are determined according to the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, or the IMGT definition, as shown in Table 1.

[0065] Table 1

[0066]

[0067]

[0068] In an alternative embodiment, the HCDR1 comprises the amino acid residues SN, and may include, for example, but not limited to, any of the sequences shown in SEQ ID NOs. 13 to 17; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 18, and may include, for example, but not limited to, any of the sequences shown in SEQ ID NOs. 18 to 22; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 23, and may include, for example, but not limited to, any of the sequences shown in SEQ ID NOs. 24 to 26. The LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 27, and may include, for example, but not limited to, any of the sequences shown in SEQ ID NOs. 28 to 30; the LCDR2 comprises the amino acid residues WA, and may include, for example, but not limited to, the sequences shown in SEQ ID NO. 31 or 32; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 33, and may include, for example, but not limited to, the sequences shown in SEQ ID NO. 33 or 34.

[0069] In an alternative embodiment, the above antigen-binding fragment contains a heavy-chain framework region and / or a light-chain framework region, and the above heavy-chain framework region and / or light-chain framework region is derived from at least one of a murine antibody, a primate antibody or a mutant thereof.

[0070] In an alternative embodiment, the three CDRs of the above antigen-binding fragment are separated by flanking portions called framework regions (FR, the light-chain FR contains LFR1, LFR2, LFR3 and LFR4, and the heavy-chain FR contains HFR1, HFR2, HFR3 and HFR4).

[0071] In an alternative embodiment, 1, 2, 3 or 4 of HFR1, HFR2, HFR3 and HFR4 are selected from the framework regions of the heavy-chain variable region having an amino acid sequence such as SEQ ID NO. 1. The CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition or IMGT definition in Table 1, and the FR regions in the corresponding definition are obtained according to the structure of the heavy-chain variable region. Taking the IMGT definition as an example, HFR1, HFR2, HFR3 and HFR4 respectively contain the amino acid sequences shown in SEQ ID NOs. 35, 36, 37 and 38.

[0072] In an alternative embodiment, 1, 2, 3, or 4 of LFR1, LFR2, LFR3, and LFR4 are selected from the framework regions of the light chain variable region having the amino acid sequence shown in SEQ ID NO.2. Its CDR regions are determined according to the Kabat definition, Chothia definition, AbM definition, Contact definition, or IMGT definition in Table 1, and the FR regions of the corresponding definition methods are obtained according to the structure of the light chain variable region. Taking the IMGT definition as an example, LFR1, LFR2, LFR3, and LFR4 respectively contain the amino acid sequences shown in SEQ ID NO.39, 40, 41, and 42.

[0073] In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment that specifically binds CD47 is as shown in SEQ ID NO.1, or an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0074] In an alternative embodiment, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment that specifically binds CD47 is as shown in SEQ ID NO.2, or an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence shown in SEQ ID NO.2.

[0075] In an alternative embodiment, the antibody or its antigen-binding fragment that specifically binds CD47 includes scFv.

[0076] In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the scFv is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2.

[0077] In an alternative embodiment, the heavy chain variable region and the light chain variable region in the scFv that specifically binds CD47 are linked by a flexible linker peptide.

[0078] In an alternative embodiment, the scFv has the following structure from the N-terminus to the C-terminus: VL-Linker-VH or VH-linker-VL; the VL is the light chain variable region, the VH is the heavy chain variable region, and the linker is a flexible linker peptide.

[0079] The amino acid sequence of the exemplary linker peptide is (GGGGS)n, where n represents the number of tandem repeats of the amino acid sequence GGGGS, n = 1 to 10, n is a positive integer, and its amino acid sequence is SEQ ID NO.43. In an alternative embodiment, the amino acid sequence of the linker peptide is GGGGSGGGGSGGGGS (SEQ ID NO.44), i.e., n = 3.

[0080] In an alternative embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO.3.

[0081] In an alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds CD47 comprises a partial or complete sequence of the constant region, and the constant region is derived from at least one of murine antibodies, primate antibodies or mutants thereof;

[0082] In an alternative embodiment, the heavy chain constant region contains a partial or complete constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0083] In an alternative embodiment, the light chain constant region is a κ or λ chain.

[0084] In an alternative embodiment, the species from which the constant region is derived includes one or a combination of more than one of mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey and human.

[0085] The engineered immune cell provided in the first aspect has a chimeric antigen receptor expressed on the cell membrane, and the chimeric antigen receptor contains a signal transduction domain.

[0086] The signaling domain is located within the intracellular signaling domain and is responsible for activating at least one effector function of the cell expressing the CAR. The intracellular signaling domain transduces effector function signals and directs the cell (e.g., immune cell) to perform its specific function, such as harming and / or destroying target cells. Examples of intracellular signaling domains include, but are not limited to, fragments or domains from one or more molecules or receptors, including but not limited to CD3ζ, TCR, CD3γ, CD3δ, CD3ε, CD86, FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP 10, DAP 12, CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-l, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds to CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1Id, ITGAE, CD103, ITGAL, CD11 a, LFA-l, ITGAM, CD lib, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD 96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and any derivatives, variants or fragments thereof, any synthetic sequences of co-stimulatory molecules with the same functional capabilities, and any combination thereof.

[0087] In an alternative embodiment, the signaling domain of the chimeric antigen receptor is selected from CD3ζ.

[0088] In an alternative embodiment, the chimeric antigen receptor further comprises a co-stimulatory domain and a transmembrane domain.

[0089] The co-stimulatory domain is located within the intracellular signaling domain and contributes to CAR-T cell expansion, function, persistence, and anti-tumor activity. Examples of co-stimulatory domains include, but are not limited to, CD3, CD4, CD8, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, co-stimulatory molecules that specifically bind to CD83 or any fragment thereof.

[0090] The transmembrane domain connects the extracellular domain of the CAR to the intracellular signal transduction domain, and the transmembrane domain can be any sequence derived from natural molecules, artificial sequences, or combinations thereof that facilitate the insertion of the CAR into the cell membrane. Examples of transmembrane domains include, but are not limited to, those derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), transmembrane domains of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9, or any derivatives, variants, or fragments thereof, any synthetic sequence having the same function, and any combination thereof.

[0091] Alternatively, the transmembrane domain can be synthetic and can contain hydrophobic residues such as leucine and valine. In an exemplary embodiment, one or both ends of the synthetic transmembrane domain are a triplet of phenylalanine, tryptophan, and valine.

[0092] In alternative embodiments, the extracellular domain of the chimeric antigen receptor contains an antigen-binding domain that directs the chimeric antigen receptor-modified immune cells to target specific targets. The antigen-binding domain generally includes a single-chain antibody (scFv). Sites specifically bound by the antigen-binding domain of the chimeric antigen receptor include, but are not limited to, at least one of GPC3 (glypican 3), GUCY2C (guanylate cyclase C), CD19, MSLN (mesothelin), MUC16 (also known as CA-125, a highly glycosylated multi-domain type I transmembrane protein), MICA (class I human major histocompatibility complex-related protein A), HER2 (human epidermal growth factor receptor 2), CD70 (tumor necrosis factor receptor superfamily TNFSF factor, type II transmembrane protein), FSHR (follicle-stimulating hormone receptor), BAFFR (B cell-activating factor receptor), EGFRVIII (epidermal growth factor receptor type III mutant), and CEA (carcinoembryonic antigen).

[0093] In alternative embodiments, the antigen-binding domain of the chimeric antigen receptor specifically binds GPC3.

[0094] In alternative embodiments, the antigen-binding domain of the chimeric antigen receptor includes scFv.

[0095] In alternative embodiments, the amino acid sequence of the heavy-chain variable region of the scFv is as shown in SEQ ID NO.45, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO.46.

[0096] In alternative embodiments, the heavy-chain variable region and the light-chain variable region in the scFv that specifically binds GPC3 are linked by a flexible linker peptide;

[0097] In alternative embodiments, the scFv has the following structure from the N-terminus to the C-terminus: VL-Linker-VH or VH-linker-VL; the VL is the light-chain variable region, the VH is the heavy-chain variable region, and the linker is a flexible linker peptide.

[0098] The amino acid sequence of an exemplary linker peptide is (GGGGS)n (SEQ ID NO.43), where n represents the tandem repeat number of the amino acid sequence GGGGS, n = 1 to 10, n is a positive integer, and its amino acid sequence is SEQ ID NO.43. In alternative embodiments, the amino acid sequence of the linker peptide is GGGGSGGGGSGGGGS (SEQ ID NO.44), i.e., n = 3.

[0099] In an alternative embodiment, the amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO.44.

[0100] In an alternative embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO.5.

[0101] In an alternative embodiment, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain.

[0102] In an alternative embodiment, the chimeric antigen receptor comprises [scFv]-[CD28TM]-[4-1BB]-[CD3ζ] from the N-terminus to the C-terminus;

[0103] [scFv] is a single-chain antibody targeting an antigen; [CD28TM] represents a membrane domain, and the transmembrane domain is from CD28; [4-1BB] is a co-stimulatory domain, which is 4-1BB; [CD3ζ] is a signal transduction domain, which is CD3ζ.

[0104] In an alternative embodiment, [scFv] is a single-chain antibody targeting GPC3, and the amino acid sequence is as shown in SEQ ID NO.5.

[0105] The engineered immune cells provided in the first aspect also overexpress cytokines.

[0106] In an alternative embodiment, the cytokine is selected from at least one of interleukin, interferon, colony-stimulating factor, chemokine, and growth factor.

[0107] In an alternative embodiment, the interleukin includes but is not limited to at least one of IL-12, IL-21, IL-15, and IL-7.

[0108] In an alternative embodiment, the interleukin is selected from IL-12. In an alternative embodiment, the interleukin is selected from wild-type IL-12 or mutant IL-12. IL-12 is a key cytokine bridging innate immunity and adaptive immunity. Research reports that IL-12 can promote the differentiation of mouse M1 macrophages by activating T cells and NK cells to produce a large amount of IFN-γ, improve the solid tumor microenvironment, and ultimately promote the intratumoral proliferation and accumulation of CAR-T cells, synergistically enhancing the anti-tumor activity. Integrating IL-12 and CD47 antibody in immune cells can theoretically synergistically activate tumor-associated macrophages and promote a strong and persistent anti-tumor immune response. An exemplary amino acid sequence of IL-12 is as shown in SEQ ID NO.7.

[0109] In alternative embodiments, the interferon includes, but is not limited to, at least one of IFN-γ, IFN-α, and IFN-β.

[0110] In alternative embodiments, the colony-stimulating factor includes, but is not limited to, at least one of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF).

[0111] In alternative embodiments, the chemokine includes, but is not limited to, at least one of CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CXCL1, CXCL2, CXCL9, CXCL10, CCL21, and CXCL12.

[0112] In alternative embodiments, the growth factor includes, but is not limited to, at least one of epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, and platelet-derived growth factor.

[0113] In alternative embodiments, in the engineered immune cells, the immune cells include T cells, natural killer cells, monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, leukocytes, or peripheral blood mononuclear cells.

[0114] In alternative embodiments, the immune cells are selected from T cells.

[0115] In alternative embodiments, the sources of the immune cells may include allogeneic and autologous sources.

[0116] In alternative embodiments, the immune cells can be differentiated from stem cells or induced pluripotent stem cells (iPSCs).

[0117] In alternative embodiments, the immune cells can be isolated from umbilical cord blood, peripheral blood, human embryonic stem cells, or iPSCs.

[0118] In alternative embodiments, the immune cells are primary cells, such as T cells or NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0119] In alternative embodiments, the immune cells are human-derived cells.

[0120] The engineered immune cells provided in the first aspect above have the following beneficial effects:

[0121] The engineered immune cells provided in the first aspect described above express a chimeric antigen receptor and express an antibody or an antigen-binding fragment thereof that specifically binds to CD47. CD47 is an important immune checkpoint molecule of myeloid cells. Blocking CD47 with an antibody can promote the phagocytosis of macrophages and the antigen presentation of dendritic cells. Therefore, blocking CD47 can also activate tumor-associated macrophages; the engineered immune cells also overexpress cytokines to promote a strong and persistent anti-tumor immune response. The engineered immune cells provided by the present invention show significant activity in promoting macrophage activation.

[0122] In a second aspect, there is also provided a polynucleotide, which contains a nucleotide sequence encoding the chimeric antigen receptor in the first aspect, a nucleotide sequence encoding the cytokine in the first aspect, and a nucleotide sequence encoding the antibody or an antigen-binding fragment thereof that specifically binds to CD47 in the first aspect.

[0123] In an optional embodiment, the polynucleotide is an isolated nucleic acid molecule.

[0124] In an optional embodiment, the antibody or an antigen-binding fragment thereof that specifically binds to CD47 is an scFv, and the nucleotide sequence includes the sequence shown in SEQ ID NO.4.

[0125] In an optional embodiment, the antigen-binding domain of the chimeric antigen receptor specifically binds to GPC3, and its antigen-binding structure domain is an scFv, and the nucleotide sequence includes the sequence shown in SEQ ID NO.6.

[0126] In an optional embodiment, the cytokine is selected from IL-12, and the nucleotide sequence includes the sequence shown in SEQ ID NO.8.

[0127] In an optional embodiment, the nucleotide sequence encoding the chimeric antigen receptor, the nucleotide sequence encoding the cytokine, and the nucleotide sequence encoding the antibody or an antigen-binding fragment thereof that specifically binds to CD47 are connected by a cleavable linker.

[0128] In an optional embodiment, the cleavable linker includes, but is not limited to, an F2A linker (foot-and-mouth disease virus (FMDV) 2A peptide), a P2A linker (porcine teschovirus-1 2A peptide), a T2A linker (Plutella xylostella virus 2A peptide), or an E2A linker (equine rhinitis A virus 2A peptide).

[0129] In an optional embodiment, the cleavable linker includes a P2A linker, and the amino acid sequence of the P2A linker is as shown in SEQ ID NO.9 (ATNFSLLKQAGDVEENPGP).

[0130] In an alternative embodiment, the nucleotide sequence encoding the P2A linker comprises the sequence shown in SEQ ID NO.10 (gccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggcca).

[0131] In an alternative embodiment, the cleavable linker comprises an F2A linker, and the amino acid sequence of the F2A linker is shown in SEQ ID NO.11 (KIVAPVKQTLNFDLLKLAGDVESNPGP).

[0132] In an alternative embodiment, the nucleotide sequence encoding the F2A linker comprises the sequence shown in SEQ ID NO.12 (AAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTA GAAAGCAATCCAGGTCCA).

[0133] In an alternative embodiment, the polynucleotide comprises [GPC3-CAR]-[P2A]-[IL-12]-[F2A]-[CD47] from the 5'-end to the 3'-end;

[0134] wherein, [GPC3-CAR] is the nucleotide sequence encoding the chimeric antigen receptor targeting GPC3, [P2A] is the nucleotide sequence encoding the P2A linker, [IL-12] is the nucleotide sequence encoding the cytokine IL-12, [F2A] is the nucleotide sequence encoding the F2A linker, and [CD47] is the nucleotide sequence encoding the antibody specifically binding to CD47 or its antigen-binding fragment.

[0135] In an alternative embodiment, [CD47] is the scFv encoding the antibody specifically binding to CD47.

[0136] In a third aspect, a transgenic immune cell is also provided. The genome of the transgenic immune cell contains the nucleotide sequence encoding the chimeric antigen receptor in the first aspect, the nucleotide sequence encoding the cytokine in the first aspect, and the nucleotide sequence encoding the antibody specifically binding to CD47 or its antigen-binding fragment in the first aspect.

[0137] In an alternative embodiment, the polynucleotide of the second aspect is integrated into the genome of the transgenic immune cell.

[0138] In an alternative embodiment, the transgenic immune cell includes T cells, natural killer cells, monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, white blood cells or peripheral blood mononuclear cells.

[0139] In an alternative embodiment, the genetically modified immune cells include T cells.

[0140] In an alternative embodiment, the sources of the genetically modified immune cells may include allogeneic and autologous sources.

[0141] In an alternative embodiment, the genetically modified immune cells can be differentiated from stem cells or induced pluripotent stem cells (iPSCs).

[0142] In an alternative embodiment, the genetically modified immune cells can be isolated from umbilical cord blood, peripheral blood, human embryonic stem cells or iPSCs.

[0143] In an alternative embodiment, the genetically modified immune cells are primary cells, such as T cells or NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0144] In an alternative embodiment, the immune cells are human-derived cells.

[0145] In a fourth aspect, a recombinant vector is also provided, which contains the polynucleotide of the second aspect. In an alternative embodiment, the recombinant vector is a lentiviral vector.

[0146] In a fifth aspect, a method for preparing the engineered immune cells of the first aspect is provided, and the preparation method includes introducing the polynucleotide encoding the first aspect, the polynucleotide encoding the cytokine of the first aspect, and the polynucleotide encoding the antibody or its antigen-binding fragment specifically binding to CD47 of the first aspect into immune cells.

[0147] In an alternative embodiment, the introduction includes expressing the introduced polynucleotide independently of the genome or integrating it into the genome.

[0148] In an alternative embodiment, some or all of the polynucleotide encoding the first aspect, the polynucleotide encoding the cytokine of the first aspect, and the polynucleotide encoding the antibody or its antigen-binding fragment specifically binding to CD47 of the first aspect are expressed independently of the genome; and / or some or all are integrated into the genome of the immune cells.

[0149] In an alternative embodiment, at least two of the polynucleotide encoding the first aspect, the polynucleotide encoding the cytokine of the first aspect, and the polynucleotide encoding the antibody or its antigen-binding fragment specifically binding to CD47 of the first aspect are carried by two different independent vectors respectively.

[0150] In an alternative embodiment, the polynucleotide encoding the first aspect, the polynucleotide encoding the cytokine of the first aspect, and the polynucleotide encoding the antibody or antigen-binding fragment thereof that specifically binds CD47 of the first aspect are carried by the same vector.

[0151] In an alternative embodiment, the preparation method includes introducing the polynucleotide of the second aspect into immune cells. The introduction can be carried out by conventional techniques well-known to those skilled in the art. Exemplary introduction methods include, but are not limited to: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, or liposome packaging, etc.

[0152] In the sixth aspect, there is also provided the use of the immune cells of the first aspect, or the polynucleotide of the second aspect, or the transgenic immune cells of the third aspect, or the recombinant vector of the fourth aspect, or the preparation method of the fifth aspect in the preparation of anti-tumor drugs.

[0153] In the seventh aspect, there is also provided a cell drug composition, which comprises the engineered immune cells of the first aspect, or the transgenic immune cells of the third aspect.

[0154] In an alternative embodiment, the cell drug composition is used for treating tumors.

[0155] In an alternative embodiment, the drug composition further comprises a pharmaceutically acceptable carrier and / or excipient. The acceptable carrier and pharmaceutically acceptable excipients can be any carriers and / or excipients known and conventional in the art. Examples of carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.; examples of excipients include, but are not limited to, fillers, disintegrants, preservatives, solubilizers, and emulsifiers, etc.

[0156] In an alternative embodiment, the drug composition further comprises one or several drug active ingredients having other therapeutic effects, including but not limited to a combination of one or several of chemotherapeutic agents, anti-cancer drugs, radiotherapy agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

[0157] In the eighth aspect, there is also provided a method for treating tumors, which comprises administering to a subject in need of treatment the engineered immune cells of the first aspect, or the transgenic immune cells of the third aspect, or the cell drug composition of the seventh aspect.

[0158] In alternative embodiments, the dosage (administered dosage) of any of the above may vary during the course of treatment. For example, in some embodiments, the initial administered dosage may be higher than subsequent administered dosages. In some embodiments, the administered dosage is adjusted during the course of treatment based on the response of the subject being administered.

[0159] In alternative embodiments, when administering any of the above, it may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a hormone therapeutic agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine).

[0160] In alternative embodiments, the tumors involved in the fifth, sixth, seventh, and eighth aspects include, but are not limited to, liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor, neuroblastoma, mesothelioma, pancreatic cancer, cholangiocarcinoma, colon cancer, thymic cancer, esophageal cancer, endometrial cancer, prostate cancer, head and neck cancer, non-Hodgkin lymphoma, bladder cancer, glioblastoma, cervical cancer, chondrosarcoma, thyroid cancer, kidney cancer, osteosarcoma, bladder cancer, meningioma, multiple squamous cell carcinoma, small cell lung cancer, colorectal cancer, medulloblastoma, acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or diffuse large B-cell lymphoma (DLBCL).

[0161] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0162] Example 1 Construction of expression vectors and lentiviruses

[0163] 1. Plasmid construction: The schematic diagram of the plasmid structure is as Figure 1 shown, specifically as follows:

[0164] Plasmid expressing only GPC3 CAR-T (GPC3-T):

[0165] GC33 ScFv-CD28 TM-4-1BB-CD3ζ;

[0166] Plasmid expressing GPC3 CAR-T with IL-12 (GPC3-IL12-T):

[0167] GC33 ScFv-CD28 TM-4-1BB-CD3ζ-P2A-rh IL-12;

[0168] Plasmid of GPC3 CAR-T expressing aCD47 (GPC3-aCD47-T, where aCD47 represents scFv specifically binding to CD47):

[0169] GC33 ScFv-CD28 TM-4-1BB-CD3ζ-P2A-CD47 ScFv;

[0170] Plasmid of GPC3 CAR-T expressing IL-12-aCD47 (GPC3-IL12-aCD47-T):

[0171] GC33 ScFv-CD28 TM-4-1BB-CD3ζ-P2A-rh IL-12-F2A-CD47 ScFv. The above nucleotide sequences were synthesized by gene synthesis. According to the restriction enzyme cleavage sites of the lentiviral vector, the nucleotide fragments were constructed onto the lentiviral vector, primers were designed, and the correctness of the vector construction was verified by the sequencing results.

[0172] In the above plasmids, the nucleotide sequence of GC33 ScFv is as shown in SEQ ID NO.6, and the amino acid sequence is as shown in SEQ ID NO.5; the nucleotide sequence of IL-12 is as shown in SEQ ID NO.8, and the amino acid sequence is as shown in SEQ ID NO.7; the nucleotide sequence of CD47 ScFv is as shown in SEQ ID NO.4, and the amino acid sequence is as shown in SEQ ID NO.3;

[0173] Nucleotide sequence of GC33 ScFv:

[0174] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCTCCTACGTTCGGATCGGGGACCAAGCTGGAAATAAAAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTAAAATGGATTGGAGCTCTTGATCCTAAAACTGGTGATACTGCCTACAGTCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGATTCTACTCCTATACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO.6);

[0175] Amino acid sequence of GC33 ScFv (its structural schematic diagram is VL-linker-VH, the amino acid sequence of VL of GC33 is shown in bold, the amino acid sequence of the flexible linker peptide is shown underlined, and the amino acid sequence of VH of GC33 is shown in italics):

[0176] MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIK GGGGSGGGGS GGGGS QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSA(SEQ ID NO.5);

[0177] Nucleotide sequence of IL-12:

[0178]

[0179] Amino acid sequence of IL-12:

[0180] MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO.7);

[0181] Nucleotide sequence of CD47 ScFv:

[0182] GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGAGGGCCACCATGAACTGCAAGAGCAGCCAGAGCCTGCTGAACACCAGGACCAGGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCAGGGAGAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCAAGCAGAGCTACAACCTGAGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGTGGCGGGTCAGGCGGTGGAGGTTCTGGCGGAGGCGGAAGCCAGGTGCAGCTGGTGCAGCCCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGCAGCAGCTTCACCAGCAACTGGATGAACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCATGATCCACCCCAGCGACAGCGAGACCAGGCTGAACCAGAAGTTCAAGGACAGGGCCACCCTGACCGTGGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCCCAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCACCACCGTGGTGGACGCCTTCGCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO.4);

[0183] Amino acid sequence of CD47 ScFv (its structural schematic diagram is VL-linker-VH, the amino acid sequence of VL of GC33 (SEQ ID NO.2) is shown in bold, the amino acid sequence of the flexible linker peptide is shown underlined, and the amino acid sequence of VH of CD47 ScFv (SEQ ID NO.1) is shown in italics):

[0184] DIVMTQSPDSLAVSLGERATMNCKSSQSLLNTRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLRTFGGGTKLEIK GGGGSGGGGSGGGGS QVQLVQPGAEVKKPGASVKLSCKASGSSFTSNWMNWVRQAPGQGLEWIGMIHPSDSETRLNQKFKDRATLTVDKSTSTAYMELSSPRSEDTAVYYCARGTTVVDAFAYWGQGTTVTVSS(SEQ ID NO.3).

[0185] 2. Lentivirus packaging: 293T cells were seeded at a density of 5×10 6 cells / 100 mm 2 culture dish. The cell status was observed the next day. Three generations of lentiviral packaging vectors were co-transfected into 293T cells by PEI transfection method. The medium was changed 16 hours after transfection. DMEM medium containing 10% fetal bovine serum was added at 10 ml / 100 mm 2 culture dish. The virus supernatant was collected 48 and 72 hours after transfection, centrifuged at 3000 rpm, 4 °C for 10 minutes to remove cell debris, and then ultrafiltered and concentrated for 30 minutes at 3000 rpm, 4 °C using an Amicon Ultra-15 centrifugal filtration device (100 kDa, Merck Millipore UFC901096). The concentrated virus was aliquoted appropriately and stored at -80 °C. The virus titer was detected by flow cytometry using PE-Labeled Human GPC3 (Acro, GP3-HP2E3).

[0186] Example 2 Preparation of CAR-T cells

[0187] 1. The reagents are shown in the following table:

[0188] Table 2

[0189]

[0190]

[0191] 2. Operation steps:

[0192] After the normal donor peripheral blood mononuclear cells PBMCs stored in liquid nitrogen were resuscitated in a 37 °C water bath, they were resuspended with complete T cell medium (95% X-VIVO, 5% AB serum, 300 IU / mL IL-2), counted, and seeded at 2.0×10 7Resuspend at a density of 1.0×10 cells / mL. Then add anti-human CD3 / 28 T cell positive selection magnetic beads at a magnetic bead:cell ratio of 1:1 (the magnetic beads can both positively select and stimulate T cells), incubate at room temperature for 30 minutes with gentle mixing, place in a magnetic stand and let stand for 5 minutes, remove the supernatant to obtain the T cell count, and culture at a density of 1.0×10 6 cells / mL for 24 h. Adjust the T cell density to 4.0×10 6 cells / mL (the culture medium is X-VIVO), add 10 μg / mL of transfection enhancer, and transfect T cells at a lentivirus ratio of MOI = 5. After co-culturing T cells and lentivirus for 4 hours, supplement the culture medium (X-VIVO 99% + AB serum 1%) until the cell density is 1.0×10 6 cells / mL. After 16 - 18 h, centrifuge to remove the remaining virus, and then dilute the T cells with complete T cell medium to 5.0×10 5 cells / mL, and continue to culture in a 37°C, 5% CO₂ incubator. Supplement a certain volume of complete T cell medium every 2 days until the cell density reaches 5.0×10 5 cells / mL. On the 4th day after lentivirus infection, take appropriate amounts of WT-T cells and CAR-T cells in each group, remove the magnetic beads, stain with PE-GPC3, and detect the CAR positive rate of the test cells using a CytoFlex flow cytometer. The results are as Figure 2 shown. The percentage of CAR expression in T cells transduced with lentivirus all exceeded 30%.

[0193] Example 3 Detection of IL-12 Secretion by CAR-T Cells

[0194] On the 7th day of CAR-T cell culture, take the supernatant and detect the IL-12 concentration by ELISA (Human IL-12p70 Precoated ELISA Kit, Dayou, 1111203). The results show that the concentration of IL-12 in the supernatants of GPC3-IL12-T and GPC3-IL12-aCD47-T cells was significantly higher than that in the WT-T and GPC3-T groups ( Figure 3 ).

[0195] Example 4 Detection of Secretion Levels of IFN-γ and Granzyme B by CAR-T Cells

[0196] Co-culture CAR-T cells with huh7 human hepatocellular carcinoma cells at an E:T ratio of 1:1 for 48 h, collect the supernatant, dilute it 10-fold, and detect it according to the operation steps of the IFN-γ and IL-2 detection kits (IFN-γ detection kit, Invitrogen, 88-7316-88; IL-2 detection kit, Invitrogen, 88-7025-88). The results show that overexpression of IL-12 can significantly increase the IFN-γ secretion level of CAR-T cells after activation by target cells, but does not affect the secretion level of Granzyme B ( Figure 4 and Figure 5 ).

[0197] Example 5 Detection of the memory cell differentiation phenotype of CAR-T

[0198] On the 7th day of CAR-T cell culture, take 2×10 5 cells and detect the distribution of T cell memory and differentiation subsets by flow cytometry. The results show that the proportions of CD8 + CD45RA + CD62L + double-positive cells and CD45RA + CD27 + double-positive cells in the CAR-T cells of the IL-12 overexpression group (GPC3-IL12-T, GPC3-IL12aCD47-T) are both significantly increased, suggesting that overexpression of IL-12 promotes the maintenance of the memory cell subset of CD8 + CAR-T cells ( Figure 6 ).

[0199] Example 6 IL-12 and CD47 synergistically promote macrophage activation and tumor killing ability

[0200] Culture non-T cells in human peripheral blood-derived monocytes (PBMC) in X-vivo medium containing 10 ng / mL GM-CSF for 7 days, aspirate the suspended cells to obtain adherent human primary macrophages. Digest these cells with trypsin, resuspend them in 250 μL of X-vivo medium and add them to a 48-well plate (2×10 5 cells / well), and add 1×10 5Huh7 liver cancer cells, and then 250 μL of the activation supernatant (same as Example 4) of the co-culture of the collected GPC3-T, GPC3-IL12-T, and GPC3-IL12-aCD47-T cells with the target cells Huh7 was added. After 48 hours, observation was performed under a microscope, and all cells were digested with trypsin for flow cytometry analysis. The results showed that the supernatants of IL-12 and aCD47 overexpressing CAR-T cells could significantly up-regulate the expression levels of CD86 and MHC-II molecules in human macrophages, suggesting that IL-12 and aCD47 secreted by CAR-T cells could synergistically promote macrophage activation( Figure 7 ).

[0201] It was observed under a microscope that large patches of clear areas appeared in the co-culture of macrophages and tumors treated with the supernatants of IL-12 and aCD47 overexpressing CAR-T cells, suggesting a significant reduction in tumor cells( Figure 8 ).

[0202] Flow cytometry detection found that the supernatants of IL-12 and aCD47 overexpressing CAR-T cells could significantly promote the increase in the proportion of macrophages in the co-culture. At the same time, the proportion of tumor cells decreased significantly, suggesting that macrophages treated with the supernatants of IL-12 and aCD47 overexpressing CAR-T cells significantly inhibited the expansion of tumor cells, thus proving that IL-12 and aCD47 overexpressing CAR-T cells could inhibit and eliminate tumor cells by highly activating macrophages( Figure 9 and Figure 10 ).

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An engineered immune cell, characterized in that: Its cell membrane expresses a chimeric antigen receptor; it overexpresses cytokines; and it expresses an antibody or an antigen-binding fragment thereof that specifically binds to CD47; The chimeric antigen receptor contains a signaling domain; The antibody or antigen-binding fragment thereof that specifically binds to CD47 contains the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and / or the complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region; The HCDR1, HCDR2 and HCDR3 include amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO.1, and the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO.2; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 of the variable region is defined by any one system or a combination of multiple definition systems of Kabat, Chothia, IMGT, ABM or Contact.

2. The engineered immune cell according to claim 1, characterized in that The HCDR1 includes the amino acid residue SN; the HCDR2 includes the amino acid sequence shown in SEQ ID NO.18; the HCDR3 includes the amino acid sequence shown in SEQ ID NO.23; The LCDR1 comprises the amino acid sequence shown in SEQ ID NO.27; the LCDR2 comprises the amino acid residue WA; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.33; Optionally, according to the IMGT definition: the HCDR1 includes the amino acid sequence shown as SEQ ID NO.17; the HCDR2 includes the amino acid sequence shown as SEQ ID NO.22; the HCDR3 includes the amino acid sequence shown as SEQ ID NO.26; the LCDR1 includes the amino acid sequence shown as SEQ ID NO.30; the LCDR2 includes the amino acid residue WA; the LCDR3 includes the amino acid sequence shown as SEQ ID NO.

34.

3. The engineered immune cell according to claim 1 or 2, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to CD47 is as shown in SEQ ID NO.1, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.1; And / or, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof that specifically binds to CD47 is as shown in SEQ ID NO.2, or an amino acid sequence that has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO.

2.

4. The engineered immune cell according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof that specifically binds to CD47 includes scFv; Optionally, the amino acid sequence of the heavy chain variable region of the scFv is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; Optionally, the heavy chain variable region and the light chain variable region in the scFv that specifically binds to CD47 are connected by a flexible linker peptide; Optionally, the scFv has the following structure from N-terminus to C-terminus: VL-Linker-VH or VH-linker-VL; the VL is the light chain variable region, the VH is the heavy chain variable region, and the linker is a flexible connecting peptide; Optionally, the amino acid sequence of the flexible connecting peptide is (GGGGS)n, n=1-10, n is a positive integer, and its amino acid sequence is SEQ ID NO.43; Optionally, the amino acid sequence of the flexible connecting peptide is shown in SEQ ID NO.44; Optionally, the amino acid sequence of the scFv is shown in SEQ ID NO.

3.

5. The engineered immune cell according to any one of claims 1 to 4, characterized in that: The signaling domain of the chimeric antigen receptor is selected from CD3ζ, TCR, CD3γ, CD3δ, CD3ε, CD86, FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS; Optionally, the signaling domain of the chimeric antigen receptor is selected from CD3ζ.

6. The engineered immune cell according to any one of claims 1 to 5, characterized in that: The chimeric antigen receptor also contains a transmembrane domain and a co-stimulatory domain; Optionally, the chimeric antigen receptor includes, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, a co-stimulatory domain and a signal transduction domain; Optionally, the transmembrane domain is selected from the transmembrane domains of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9, or derivatives thereof; Optionally, the costimulatory domain is selected from the costimulatory molecule of CD3, CD4, CD8, TCR, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or any fragment thereof; Optionally, the chimeric antigen receptor comprises [scFv]-[CD28TM]-[4-1BB]-[CD3ζ] from N-terminus to C-terminus; [scFv] is a single-chain antibody targeting an antigen; [CD28TM] represents a transmembrane domain, which is derived from the transmembrane domain of CD28; [4-1BB] is a co-stimulatory domain, which is a 4-1BB molecule; and [CD3ζ] is a signaling domain, which is a CD3ζ molecule.

7. The engineered immune cell according to any one of claims 1 to 6, characterized in that: The antigen binding domain of the chimeric antigen receptor specifically binds to at least one of GPC3, GUCY2C, CD19, MSLN, MUC16, MICA, HER2, CD70, FSHR, BAFFR, EPHA1, EGFRVIII and CEA.

8. The engineered immune cell according to claim 7, characterized in that The antigen binding domain of the chimeric antigen receptor specifically binds to GPC3; Optionally, the antigen binding domain comprises a scFv; Optionally, the amino acid sequence of the heavy chain variable region of the scFv is shown in SEQ ID NO.45, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.46; Optionally, the heavy chain variable region and the light chain variable region in the scFv that specifically binds to GPC3 are connected by a flexible connecting peptide; Optionally, the scFv has the following structure from N-terminus to C-terminus: VL-Linker-VH or VH-linker-VL; the VL is the light chain variable region, the VH is the heavy chain variable region, and the linker is a flexible connecting peptide; Optionally, the amino acid sequence of the flexible connecting peptide is (GGGGS)n, n=1-10, n is a positive integer, and its amino acid sequence is SEQ ID NO.43; Optionally, the amino acid sequence of the flexible connecting peptide is shown in SEQ ID NO.44; Optionally, the amino acid sequence of the scFv is shown in SEQ ID NO.

5.

9. The engineered immune cell according to any one of claims 1 to 8, characterized in that: The cytokine is selected from at least one of interleukins, interferons, colony stimulating factors, chemokines and growth factors; Optionally, the cytokine is selected from interleukins; Optionally, the interleukin includes at least one of IL-12, IL-21, IL-15 and IL-7; Optionally, the interleukin is selected from IL-12; Optionally, the interleukin is selected from wild-type IL-12 or mutant IL-12; Optionally, the amino acid sequence of interleukin IL-12 is shown in SEQ ID NO.

7.

10. The engineered immune cell according to any one of claims 1 to 9, characterized in that: The immune cells include T cells, natural killer cells, monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, leukocytes or peripheral blood mononuclear cells; Optionally, the immune cells are selected from T cells.

11. A polynucleotide, characterized in that A nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 10, a nucleotide sequence encoding the cytokine according to any one of claims 1 to 10, and a nucleotide sequence encoding the antibody or antigen-binding fragment thereof that specifically binds to CD47 according to any one of claims 1 to 10; Optionally, the antibody or antigen-binding fragment thereof that specifically binds to CD47 is a scFv, and the nucleotide sequence includes the sequence shown in SEQ ID NO.4; Optionally, the antigen binding domain of the chimeric antigen receptor specifically binds to GPC3, and its antigen binding domain is scFv, and the nucleotide sequence includes the sequence shown in SEQ ID NO.6; Optionally, the cytokine is selected from IL-12, and the nucleotide sequence includes the sequence shown in SEQ ID NO.

8.

12. The polynucleotide according to claim 11, characterized in that The nucleotide sequence encoding the chimeric antigen receptor, the nucleotide sequence encoding the cytokine and the nucleotide sequence encoding the antibody or antigen-binding fragment thereof that specifically binds to CD47 are connected by a cleavable linker; Optionally, the cleavable linker comprises at least one of a P2A linker, a F2A linker, a T2A linker and an E2A linker; Optionally, the cleavable linker comprises a P2A linker, and the amino acid sequence of the P2A linker is shown in SEQ ID NO.9; Optionally, the nucleotide sequence encoding the P2A linker includes the sequence shown in SEQ ID NO.10; Optionally, the cleavable linker comprises a F2A linker, and the amino acid sequence of the F2A linker is shown in SEQ ID NO.11; Optionally, the nucleotide sequence encoding the F2A linker includes the sequence shown in SEQ ID NO.

12. Optionally, the polynucleotide includes [GPC3-CAR]-[P2A]-[IL-12]-[F2A]-[CD47] from the 5' end to the 3' end; Among them, [GPC3-CAR] is a nucleotide sequence encoding a chimeric antigen receptor targeting GPC3, [P2A] is a nucleotide sequence encoding a P2A linker, [IL-12] is a nucleotide sequence encoding a cytokine IL-12, [F2A] is a nucleotide sequence encoding an F2A linker, and [CD47] is a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that specifically binds to CD47.

13. A biomaterial, characterized in that: The biological material includes any one of the following (i) to (ii): (i) A transgenic immune cell, characterized in that the genome of the transgenic immune cell contains a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 10, a nucleotide sequence encoding the cytokine according to any one of claims 1 to 10, and a nucleotide sequence encoding the antibody or antigen-binding fragment thereof that specifically binds to CD47 according to any one of claims 1 to 10; Optionally, the genome of the transgenic immune cell is integrated with the polynucleotide of claim 11 or 12; Optionally, the transgenic immune cells include T cells, natural killer cells, monocytes, macrophages, NKT cells, dendritic cells, granulocytes, B cells, lymphocytes, leukocytes or peripheral blood mononuclear cells; Optionally, the transgenic immune cells include T cells; (ii) a recombinant vector, characterized in that the recombinant vector contains the polynucleotide according to claim 11 or 12; Optionally, the recombinant vector is a lentiviral vector.

14. The method for preparing engineered immune cells according to any one of claims 1 to 10, characterized in that: The method comprises introducing a polynucleotide encoding the chimeric antigen receptor according to any one of claims 1 to 10, a polynucleotide encoding the cytokine according to any one of claims 1 to 10, and a polynucleotide encoding the antibody or antigen-binding fragment thereof expressing specific binding to CD47 according to any one of claims 1 to 10 into an immune cell; Optionally, the method comprises introducing the polynucleotide of claim 11 or 12 into an immune cell.

15. Use of the engineered immune cell according to any one of claims 1 to 10, or the polynucleotide according to claim 11 or 12, or the biomaterial according to claim 13, or the preparation method according to claim 14 in the preparation of tumor drugs.

16. A cell pharmaceutical composition, characterized in that: It comprises the engineered immune cell according to any one of claims 1 to 10, or the transgenic immune cell according to claim 13.

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