Fusion gene containing genes encoding chimeric antigen receptors and chimeric switch receptors and uses thereof

Abstract

The present invention provides a fusion gene containing a gene encoding a chimeric antigen receptor and a chimeric switch receptor, and uses thereof. Specifically, the present invention discloses a fusion protein having the amino acid sequence SEQ ID No. 1, and a nucleic acid molecule (SEQ ID No. 3) containing a gene encoding a chimeric antigen receptor and a chimeric switch receptor. By designing a fusion gene containing a gene encoding a chimeric antigen receptor and a chimeric switch receptor, the suppression of anti-tumor T cell responses due to PD-L1 activation is converted into an activation signal via CD27, conferring the ability of CAR-T cells to resist the immunosuppressive tumor microenvironment and enhancing the in vivo persistence of CAR-T cells. By promoting the secretion of cytokines IFN-γ and IFN-α2, the peri-tumor microenvironment is regulated, relieving its immunosuppressive properties and utilizing the body's own immune system to kill tumor cells, thereby improving the anti-tumor effect of CAR-T cells.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a fusion gene comprising a gene encoding a chimeric antigen receptor and a chimeric switch receptor (CSR), and uses thereof. [Background technology]

[0002] Chimeric antigen receptor (CAR) technology uses genetic engineering to modify immune cells to express exogenous antitumor genes, enabling immune cells such as lymphocytes to recognize tumor cell surface antigens and specifically recognize and kill tumor cells. In recent years, CAR-T cell therapy has shown promising therapeutic effects in hematological malignancies, including B-cell malignancies. However, its significant success in hematological malignancies has yet to be replicated in solid tumors. Unlike hematological malignancies, CAR-T cells must overcome the inherent heterogeneity of tumor cells and the complexity of the suppressive tumor microenvironment to penetrate solid tumors. The immunosuppressive nature and structural complexity of the tumor microenvironment present major obstacles to the widespread use of T-cell immunotherapy in solid tumors.

[0003] Specific recognition of tumor antigens is only the first step toward successful CAR-T therapy. To survive, tumors create an immunosuppressive microenvironment that is unfavorable to CAR-T therapy. Tumor cell glycolysis creates a hypoxic, acidic, and nutrient-poor environment. While glycolysis and oxidative phosphorylation are necessary for CAR-T function, tumor cell consumption and depletion of glucose reduces the effector function of CAR-T cells. In an inflammatory environment, tumor cells typically upregulate inhibitory ligands such as PD-L1 and Galectin-9. The tumor microenvironment also harbors numerous suppressive immune and stromal cells, including tumor-associated fibroblasts (CAFs), myeloid suppressor cells (MDSCs), tumor-associated macrophages (TAMS), tumor-associated neutrophils (TANS), mast cells, and regulatory T cells (Tregs). These cells, as well as tumor cells, secrete VEGF and TGF-β, which cause tumor vascular malformations, promote the polarization of immune cells such as TAMS toward anti-inflammatory forms, and contribute to epithelial-mesenchymal transition (EMT). They also produce reactive oxygen species (ROS), lactic acid, indoleamine 2,3-dioxygenase (Ido), prostaglandin e2 (PGE2), soluble fatty acids, and adenosine, creating a suppressive immune microenvironment. Recent studies have shown that the immunosuppressive effects of the tumor microenvironment become stronger after CAR-T cell therapy.

[0004] Therefore, CAR-T cell therapy faces a serious challenge: how to modify CAR-T cells appropriately to overcome the immunosuppressive tumor microenvironment and improve clinical efficacy. The development of novel CAR-T cells with potent antitumor effects has important theoretical and practical significance in tumor immune cell therapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the prevention or treatment of tumors (such as tumors expressing GPC3 antigen) and / or the control of the immunosuppressive effect of the tumor microenvironment. The technical problem to be solved is not limited to the technical subject matter described above, and those skilled in the art will be able to clearly understand other technical subject matter not described herein through the following explanation.

[0006] To solve the above technical problems, the present invention provides a nucleic acid molecule, which may contain a gene encoding a chimeric antigen receptor (CAR) and a gene encoding a chimeric switch receptor (CSR).

[0007] Furthermore, in the above nucleic acid molecule, the chimeric switch receptor may comprise the extracellular domain of the TGFβ type II receptor (the extracellular domain of the transforming growth factor β type II receptor), μPD-1, and the transmembrane and cytoplasmic domains of CD27. μPD-1 may be the extracellular domain of a mutation-optimized PD-1, and its amino acid sequence may be positions 187 to 332 of SEQ ID No. 1.

[0008] Furthermore, in the above nucleic acid molecule, the gene encoding the chimeric switch receptor may be any one of the following: B1) a nucleic acid molecule encoding the fusion protein; B2) a DNA molecule whose coding sequence is SEQ ID No. 2; B3) a DNA molecule having the nucleotide sequence SEQ ID No. 2; The fusion protein may be any one of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) A protein having 80% or more identity and the same function as the protein shown in A1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1; A3) A fusion protein having the same function as A1) or A2) tagged at the N-terminus and / or C-terminus.

[0009] The fusion protein may be a Chimeric Switch Receptor (CSR).

[0010] The fusion protein and the gene encoding it can be used to improve the ability of CAR cells to resist the immunosuppressive tumor microenvironment, increase the in vivo persistence of CAR cells, and / or inhibit the PD-1 and TGFβ signaling pathways and enhance the CD27 signaling pathway.

[0011] The gene encoding the fusion protein can be used to prepare a nucleic acid molecule according to the present invention or a CAR cell comprising a nucleic acid molecule according to the present invention.

[0012] Tags described herein include, but are not limited to, GST (glutathione S-transferase) tagged proteins, His6 tagged proteins (His-tag), MBP (maltose binding protein) tagged proteins, Flag tagged proteins, SUMO tagged proteins, HA tagged proteins, Myc tagged proteins, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag tagged proteins.

[0013] Those skilled in the art can easily mutate the nucleotide sequence encoding the fusion protein of the present invention using conventional methods such as directed evolution and point mutation. These artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the fusion protein of the present invention are derived from and equivalent to the nucleotide sequence of the present invention, as long as they encode the fusion protein and have the function of the fusion protein.

[0014] 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0015] As used herein, "identity" refers to the identity of an amino acid sequence or a nucleotide sequence. Amino acid sequence identity can be measured using an online homology search site, such as the BLAST page on the NCBI homepage. For example, in the advanced BLAST 2.1, the program blastp is used, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as the matrix, and the gap existence cost, per residue gap cost, and lambda ratio are set to 11, 1, and 0.85 (default values), respectively. The percent identity can be calculated by searching for amino acid sequence identity.

[0016] As used herein, 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0017] Furthermore, the nucleic acid molecule may further comprise an IFNα gene (α interferon gene) and / or a P2A gene.

[0018] The nucleotide sequence of the IFNα gene may be from positions 1552 to 2118 of SEQ ID No.3.

[0019] The nucleotide sequence of the P2A gene may be from positions 1474 to 1551 of SEQ ID No. 3.

[0020] Furthermore, in the above nucleic acid molecule, the gene encoding the chimeric antigen receptor may be a gene encoding a chimeric antigen receptor that targets GPC3.

[0021] GPC3 is glypican-3.

[0022] Furthermore, the gene encoding the chimeric antigen receptor that targets GPC3 may be any one of the following: C1) a nucleic acid molecule encoding a protein having the amino acid sequence SEQ ID No. 4; C2) a DNA molecule whose coding sequence is positions 1 to 1473 of SEQ ID No. 3; C3) A DNA molecule having the nucleotide sequence of positions 1 to 1473 of SEQ ID No. 3.

[0023] Furthermore, the nucleic acid molecule may be any one of the following: D1) a DNA molecule having the nucleotide sequence SEQ ID No. 3; D2) A DNA molecule having 70% or more identity and function with the DNA molecule shown in D1), in which the nucleotide sequence shown in SEQ ID No. 3 has been modified and / or one or more nucleotides have been substituted and / or deleted and / or added.

[0024] 70% or greater identity may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0025] The present invention further provides a biomaterial, which may be any one of the following: E1) an expression cassette comprising a nucleic acid molecule; E2) a recombinant vector comprising a nucleic acid molecule or an expression cassette according to E1; E3) A recombinant microorganism comprising a nucleic acid molecule, or an expression cassette according to E1), or a recombinant microorganism comprising a recombinant vector according to E2); E4) A recombinant cell comprising a nucleic acid molecule, or an expression cassette according to E1), or a recombinant cell comprising a recombinant vector according to E2); E5) a gene encoding a chimeric switch receptor; E6) chimeric switch receptor, E7) Fusion proteins.

[0026] In the above biomaterial, the cells in E4) may be T cells, NK cells, γδT cells, NKT cells, macrophages or stem cells.

[0027] The recombinant cell expresses the nucleic acid molecule.

[0028] Furthermore, the cells in E4) may be T cells.

[0029] Additionally, the cells in E4) may be human T cells.

[0030] As used herein, a vector refers to a vector capable of introducing foreign DNA or a target gene into a host cell, amplifying it, and expressing it. The vector may be a cloning vector or an expression vector, and includes, but is not limited to, a plasmid, a phage (such as a lambda phage or an M13 filamentous phage), a cosmid, or a viral vector (such as a baculovirus vector, a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, or a herpes virus (such as a herpes simplex virus)). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a retroviral vector MP71.

[0031] The microorganisms described herein may be bacteria, yeast, algae, or fungi. The bacteria may be, but are not limited to, Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc. The yeast may be, but is not limited to, Saccharomyces sp., Pichia sp., Yarrowia sp., Candida sp., Schizosaccharomyces sp., Hansenula sp., Kluyreromyces sp., etc. The algae may be, but are not limited to, Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc. The fungi may be, but are not limited to, Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., etc. In one or more embodiments of the present invention, the microorganism is Escherichia coli DH5α.

[0032] As used herein, a cell (host cell) refers to a cell that can be used to introduce a vector, including, but not limited to, a mammalian immune cell or a stem cell that can differentiate into an immune cell. Host cells may include, but are not limited to, T cells, NK cells, γδ T cells, NKT cells, macrophages, stem cells, etc. In one or more embodiments of the present invention, the cell is a human T cell.

[0033] As used herein, a recombinant vector refers to a recombinant vector DNA molecule constructed by ligating an exogenous target gene to a vector in vitro, and can be constructed by any suitable method, so long as the constructed recombinant vector delivers the exogenous target gene to a recipient cell and confers the exogenous target gene with the ability to replicate, integrate, amplify, and / or express in the recipient cell. In one or more embodiments of the present invention, the recombinant vector is the recombinant vector pUC57-GPC3 CAR-IC and / or the recombinant retroviral vector MP71-GPC3 CAR-IC.

[0034] The recombinant vector pUC57-GPC3 CAR-IC is a recombinant vector obtained by cloning the GPC3 CAR-IC gene, whose nucleotide sequence is SEQ ID No. 3, into the pUC57 vector. The recombinant vector pUC57-GPC3 CAR-IC contains the DNA molecule shown in SEQ ID No. 3.

[0035] The recombinant retroviral vector MP71-GPC3 CAR-IC is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with the DNA fragment shown in SEQ ID No. 3 in the Sequence Listing, without changing any other nucleotide sequence of the retroviral vector MP71. The recombinant retroviral vector MP71-GPC3 CAR-IC contains the DNA molecule shown in SEQ ID No. 3.

[0036] As used herein, a recombinant microorganism refers to a microorganism whose function has been altered by manipulating or modifying the genes of a target microorganism. Examples of such a microorganism include a recombinant microorganism obtained by introducing an exogenous target gene or a recombinant vector into a target microorganism, or a recombinant microorganism obtained by directly editing an endogenous gene of a target microorganism. In one or more embodiments of the present invention, the recombinant microorganism is a recombinant bacterium containing the DNA molecule shown in SEQ ID No. 3, which was obtained by introducing the recombinant retroviral vector MP71-GPC3 CAR-IC into Escherichia coli DH5α.

[0037] As used herein, recombinant cells refer to cells whose function has been altered by manipulating or modifying the genes of a target cell. Examples include recombinant cells obtained by introducing an exogenous target gene or a recombinant vector into a target cell. Furthermore, recombinant cells described herein include, but are not limited to, recombinant cells (e.g., CAR-T cells, CAR-NK cells, CAR-macrophages (CAR-M cells), CAR-iPSCs, and CAR-PSCs) obtained by stably expressing a CAR gene in recipient cells (e.g., T cells, NK cells, γδT cells, NKT cells, macrophages, or stem cells). In one or more embodiments of the present invention, the recombinant cells are GPC3 CAR-IC T cells.

[0038] GPC3 CAR-IC T cells are recombinant cells obtained by packaging the recombinant retroviral vector MP71-GPC3 CAR-IC in packaging cells to obtain a recombinant retrovirus, and then introducing the recombinant retrovirus into T cells. The packaging cells may be Phoenix Ecotropic (ECO) cells or PG13 cells.

[0039] GPC3 CAR-IC T cells contain the DNA molecule shown in SEQ ID No. 3 and express the GPC3 CAR-IC gene (SEQ ID No. 3).

[0040] The present invention further provides any of the following uses of the nucleic acid molecules and / or biomaterials described herein: F1) Use in the manufacture of a medicament for the prevention or treatment of tumors; F2) Use in the manufacture of a pharmaceutical for the prevention or treatment of tumors expressing the GPC3 antigen; F3) Modulation of the immunosuppressive effect of the tumor microenvironment or use in the manufacture of a product for modulating the immunosuppressive effect of the tumor microenvironment; F4) Use in the prevention or treatment of tumors; F5) Use in the prevention or treatment of tumors expressing GPC3 antigen; F6) Use in the prevention or treatment of liver cancer, melanoma, Wilms' tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma, or glioma.

[0041] Furthermore, the modulation in F3) may be a decrease or inhibition.

[0042] In the above-mentioned use, the tumor expressing the GPC3 antigen may be, but is not limited to, liver cancer, melanoma, Wilms' tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma, or glioma.

[0043] Furthermore, the liver cancer may be hepatocellular carcinoma (HCC).

[0044] Additionally, the squamous cell carcinoma may be lung squamous cell carcinoma.

[0045] Additionally, the melanoma may be a malignant melanoma.

[0046] Additionally, the glioma may be a glioblastoma.

[0047] Furthermore, the tumor expressing the GPC3 antigen may be a tumor that highly expresses GPC3.

[0048] Furthermore, F1) may be for use in the manufacture of a medicament for inhibiting and / or killing tumors.

[0049] Furthermore, F2) may be used in the manufacture of a medicament for inhibiting and / or killing tumors expressing the GPC3 antigen.

[0050] The inhibition may be an inhibition of tumor proliferation and / or growth.

[0051] The present invention further provides a pharmaceutical composition, the active ingredient of which may be a CAR cell (such as a CAR-T cell, a CAR-NK cell, a CAR-macrophage, a CAR-iPSC, or a CAR-PSC) containing or expressing any one of the nucleic acid molecules of the present invention.

[0052] Additionally, the pharmaceutical composition comprises GPC3 CAR-IC T cells as described herein.

[0053] Additionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, which may be, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorption carriers, surfactants, or lubricants.

[0054] The pharmaceutical composition has an antitumor effect and can be used for the prevention or treatment of tumors.

[0055] Furthermore, the pharmaceutical composition can be used to prevent or treat tumors in mammals.

[0056] The mammal may be, but is not limited to, any one of a human, mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, and horse.

[0057] The tumors described herein may be solid tumors (such as pancreatic cancer, lung cancer, kidney cancer, liver cancer, or brain glioma).

[0058] Furthermore, the active ingredient of the pharmaceutical composition may be an immune effector cell expressing the GPC3 CAR-IC gene (SEQ ID No. 3).

[0059] Furthermore, the active ingredient of the pharmaceutical composition may be GPC3 CAR-IC T cells.

[0060] The present invention further provides a method for preventing or treating tumors, which may comprise administering to a patient suffering from a tumor disease a pharmaceutical comprising a recombinant cell as described in E4) of the present specification.

[0061] In the above method, the recombinant cell may be a GPC3 CAR-IC T cell, and the GPC3 CAR-IC T cell may comprise the DNA molecule shown in SEQ ID No. 3.

[0062] In the above method, the tumor may be a tumor that expresses the GPC3 antigen.

[0063] In the above method, the tumor expressing the GPC3 antigen may be, but is not limited to, liver cancer, melanoma, Wilms' tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma, or glioma.

[0064] In the above method, the liver cancer may be hepatocellular carcinoma.

[0065] In the above method, the squamous cell carcinoma may be lung squamous cell carcinoma.

[0066] In the above method, the melanoma may be malignant melanoma.

[0067] In the above method, the glioma may be a glioblastoma.

[0068] The present invention further provides a method for preparing GPC3 CAR-IC T cells, the method comprising: (1) constructing and synthesizing the GPC3 CAR-IC gene (SEQ ID No. 3); (2) cloning the GPC3 CAR-IC gene into the retroviral vector MP71 to obtain the recombinant retroviral vector MP71-GPC3 CAR-IC; (3) introducing the recombinant retroviral vector MP71-GPC3 CAR-IC into packaging cells and packaging it to obtain a recombinant retrovirus; (4) transfecting T cells with the recombinant retrovirus to obtain GPC3 CAR-IC T cells.

[0069] In the above method, the packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0070] The present invention further provides the use of the fusion protein and / or nucleic acid molecule in the preparation of GPC3 CAR-IC T cells.

[0071] The nucleic acid molecules described herein may be a fusion gene comprising a gene encoding a chimeric antigen receptor and a chimeric switch receptor.

[0072] The nucleic acid molecules described herein are capable of expressing a chimeric antigen receptor, an IFNα, and a fusion protein, which may be any one of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) A protein having 80% or more identity and the same function as the protein shown in A1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1; A3) A fusion protein having the same function as A1) or A2) tagged at the N-terminus and / or C-terminus.

[0073] Furthermore, the target of the chimeric antigen receptor may be GPC3.

[0074] Additionally, the chimeric antigen receptor may be any one of the following: F1) a protein whose amino acid sequence is SEQ ID No. 4; F2) A protein having 80% or more identity and the same function as the protein shown in F1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 4. F3) A fusion protein with the same function as F1) or F2) tagged at the N-terminus and / or C-terminus.

[0075] Additionally, the IFNα may be any one of the following: G1) a protein having the amino acid sequence SEQ ID No. 5; G2) A protein having 80% or more identity and the same function as the protein shown in G1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 5; G3) A fusion protein with the same function as G1) or G2) tagged at the N-terminus and / or C-terminus.

[0076] Additionally, the nucleic acid molecules described herein are capable of expressing a chimeric antigen receptor having the amino acid sequence of SEQ ID No. 4, an IFNα having the amino acid sequence of SEQ ID No. 5, and a fusion protein (chimeric switch receptor) having the amino acid sequence of SEQ ID No. 1.

[0077] The nucleic acid molecule described herein may be the GPC3 CAR-IC gene, the nucleotide sequence of which may be SEQ ID No. 3, and the GPC3 CAR-IC gene may consist, from N- to C-terminus, of a gene encoding a chimeric antigen receptor, a P2A gene, an IFNα gene, a promoter element gene, and a gene encoding a chimeric switch receptor. A structural diagram of the gene is shown in Figure 1 (the IFNα gene, the promoter element gene, and the gene encoding the chimeric switch receptor can be collectively referred to as the IC gene).

[0078] The nucleotide sequence of the IC gene (IFNα gene+promoter element gene+gene encoding the chimeric switch receptor) may be from positions 1552 to 3882 of SEQ ID No.3.

[0079] The gene encoding the chimeric antigen receptor may be a gene encoding, from the N-terminus to the C-terminus, Signal (signal peptide), GPC3 scFv (tumor antigen-binding region, i.e., single-chain antibody region), Hinge (hinge region), CD8 (transmembrane region), 4-1BB (intracellular signal region), and CD3ζ (intracellular signal region). The nucleotide sequence of the gene encoding the chimeric antigen receptor may be positions 1 to 1473 of SEQ ID No. 3, and the amino acid sequence encoding the chimeric antigen receptor (CAR) may be SEQ ID No. 4.

[0080] Among the genes encoding the chimeric antigen receptor, the nucleotide sequence of the gene encoding Signal (signal peptide) may be the sequence of positions 1 to 63 of SEQ ID No. 3, the nucleotide sequence of the gene encoding GPC3 scFv may be the sequence of positions 64 to 789 of SEQ ID No. 3, the nucleotide sequence of the gene encoding Hinge (hinge region) + CD8 (transmembrane region) may be the sequence of positions 790 to 996 of SEQ ID No. 3, the nucleotide sequence of the gene encoding 4-1BB (intracellular signal region) may be the sequence of positions 997 to 1137 of SEQ ID No. 3, and the nucleotide sequence of the gene encoding CD3ζ (intracellular signal region) may be the sequence of positions 1138 to 1473 of SEQ ID No. 3.

[0081] The nucleotide sequence of the P2A gene may be from positions 1474 to 1551 of SEQ ID No. 3. The P2A peptide encoded by the P2A gene may be a short viral peptide, commonly referred to as a "self-cleaving" peptide. The "self-cleaving" function of the P2A peptide allows it to generate multiple proteins from a single transcript, i.e., to form two proteins, an upstream product and a downstream product, by self-cleavage.

[0082] The nucleotide sequence of the IFNα gene may be positions 1552 to 2118 of SEQ ID No. 3, and the amino acid sequence of IFNα (α interferon) encoded by the IFNα gene may be SEQ ID No. 5.

[0083] The nucleotide sequence of the promoter element gene may be from positions 2119 to 2661 of SEQ ID No. 3, where positions 2136 to 2635 of SEQ ID No. 3 is the promoter.

[0084] The gene encoding the chimeric switch receptor may be a gene encoding, from the N-terminus to the C-terminus, the TRII extracellular domain (the extracellular domain of the TGFβ type II receptor), a linker, μPD-1, and the transmembrane and cytoplasmic domains of CD27. Furthermore, the gene encoding the transmembrane and cytoplasmic domains of CD27 may be oCD27 obtained by codon optimization, which is suitable for expression in human cells if the encoded amino acid sequence remains unchanged. The nucleotide sequence of the gene encoding the chimeric switch receptor may be positions 2662 to 3882 of SEQ ID NO. 3 (i.e., the nucleotide sequence shown in SEQ ID NO. 2), and the amino acid sequence encoding the chimeric switch receptor (CSR) may be SEQ ID NO. 1.

[0085] Among the genes encoding the chimeric switch receptor, the nucleotide sequence of the gene encoding the TRII extracellular domain (extracellular domain of the TGFβ type II receptor) may be at positions 2662 to 3159 of SEQ ID No. 3, the nucleotide sequence of the gene encoding the Linker may be at positions 3160 to 3219 of SEQ ID No. 3, the nucleotide sequence of the gene encoding μPD-1 may be at positions 3220 to 3657 of SEQ ID No. 3, and the nucleotide sequence of the gene encoding the transmembrane and cytoplasmic domains of CD27 (i.e., oCD27) may be at positions 3658 to 3882 of SEQ ID No. 3.

[0086] The chimeric antigen receptor described herein can specifically kill GPC3-positive tumor cells. GPC3 (Glypican-3), a member of the heparan sulfate proteoglycan family, binds to the cell surface via a glycosylphosphatidylinositol anchor on the cell membrane and is specifically highly expressed in many liver cancer tissues. It can be used to target liver cancer cells and induce tumor killing, making it an ideal target for tumor therapy.

[0087] IFNα (alpha interferon) described herein has a broad range of anti-tumor effects, directly controlling tumor cell proliferation, apoptosis, and migration, inhibiting tumor angiogenesis and metastasis, and possessing immunomodulatory functions, such as upregulating the expression of MHCI, tumor antigens, and PD-L1 on tumor cells; activating innate immune cells such as NK cells, DCs, and γδT cells (particularly NK cell proliferation, migration, and function); regulating adaptive immune cells such as T cells and B cells (particularly T cell proliferation, migration, function, and survival); suppressing immune suppressive cells such as tumor activators (TAMs), Tregs, and myeloid suppressor cells (MDSCs); and upregulating the expression of PD-L1 on tumor cells, thereby enhancing anti-tumor immune activity.

[0088] The chimeric switch receptor described herein inhibits the PD-1 and TGFβ signaling pathways and enhances the CD27 signaling pathway, thereby conferring resistance to the immunosuppressive tumor microenvironment and enhancing in vivo persistence of CAR-T cells. Because tumor cells typically express PD-L1 on their surface, which activates inhibitory PD-1 on T cells and evades or inhibits anti-tumor T cell responses, the chimeric switch receptor converts this inhibitory effect into an activating signal via CD27, thereby targeting PD-1 and advantageously attracting CAR-T cells to tumor sites expressing PD-L1.

[0089] In the present invention, the GPC3 CAR-IC gene having the nucleotide sequence of SEQ ID No. 3 was constructed, but the present invention is not limited to this specific sequence. Those skilled in the art can replace the P2A gene and / or promoter element gene in the GPC3 CAR-IC gene with, for example, but not limited to, F2A (VKQTLNFDLLKLAGCVESNPG, SEQ ID No. 6), T2A (EGRGSLLTCGDVEENPG, SEQ ID No. 7), or E2A (QCTNYALLKLAGDVESNPG, SEQ ID No. 8), which have a "self-cleavage" function similar to that of the P2A gene. The promoter element gene can be any promoter known in the art. As long as the constructed nucleic acid molecules are capable of expressing a chimeric antigen receptor having the amino acid sequence of SEQ ID No. 4, an IFNα having the amino acid sequence of SEQ ID No. 5, and a fusion protein (chimeric switch receptor) having the amino acid sequence of SEQ ID No. 1, and have the same functions as the nucleic acid molecules described in the present invention, they are all considered to be equivalent to the nucleic acid molecules of the present invention, and these equivalent nucleic acid molecules do not deviate from the scope of protection of the present invention.

[0090] After extensive research, the inventors of this application designed a fusion gene containing genes encoding a chimeric antigen receptor and a chimeric switch receptor. They then designed and constructed a GPC3-targeting GPC3 CAR-IC gene (SEQ ID No. 3), packaged it, and retrovirally infected it to obtain GPC3 CAR-IC T cells. The GPC3 CAR-IC T cells constructed according to this invention express two artificial receptors. One is a chimeric antigen receptor (CAR), which recognizes tumor-associated antigens and specifically kills GPC3-positive tumor cells, targeting liver cancer cells and inducing tumor killing. The other is a chimeric switch receptor (CSR), which converts immunosuppressive signals in the tumor microenvironment into activation signals in CAR-T cells. The extracellular domain of TRII binds to TGF-β, which is secreted in large amounts in HCC, blocking its inhibitory pathway and activating the CD27 signaling pathway in CAR-T cells, stimulating CAR-T cell growth, promoting CAR-T cell survival and migration, and inhibiting the tumor microenvironment. Furthermore, μPD-1 in CSR binds to PD-L1 expressed in HCC, blocking the PD-1 signaling pathway on T cells and suppressing T cell exhaustion. It also activates the CD27 signaling pathway in CAR-T cells, enhancing tumor killing. Furthermore, the GPC3 CAR-IC T cells constructed according to the present invention also express IFNα. IFNα is used for broad-spectrum antitumor activity, reducing antigen heterogeneity and tumor cell escape, enhancing immune cell infiltration, immunomodulation, improving the tumor microenvironment, and resisting HBV / HCV infection.

[0091] Experimental results showed that, compared with control cells that do not express the IC gene (IFNα gene + promoter element gene + gene encoding a chimeric switch receptor), the GPC3 CAR-IC T cells constructed according to the present invention secreted higher levels of IFN-γ and, upon antigen stimulation, secreted high levels of IFN-α2, exhibited stronger specific cytotoxicity and killing activity against GPC3-positive target cells, and the secreted IFN-α2 was able to induce NK cells to secrete the functional effector molecule IFN-γ. In vivo antitumor experiments using a HepG2 subcutaneous tumor-bearing NSG mouse model showed that administration of GPC3-IC CAR-T cells significantly increased the secretion of IFN-α2 and IFN-γ in mouse serum. This indicates that the GPC3 CAR-IC T cells of the present invention have a more rapid and superior tumor-suppressing effect and stronger in vivo tumor-killing activity.

[0092] The present invention constructs CAR-T cells by designing a fusion gene containing genes encoding a chimeric antigen receptor and a chimeric switch receptor. This gene converts the suppression of anti-tumor T cell responses caused by PD-L1 and TGFβ activation into an activation signal mediated by CD27, endowing CAR-T cells with the ability to resist the immunosuppressive tumor microenvironment, enhancing their in vivo persistence, promoting CAR-T cell proliferation, extending their in vivo survival time, and promoting cytokine secretion. This promotes the secretion of cytokines IFN-γ and IFN-α2, thereby regulating the peri-tumor microenvironment, relieving immunosuppression, and utilizing the body's own immune system to kill tumor cells. This improves the anti-tumor effect of CAR-T cells, and is of great significance and broad application in CAR-T treatment of tumors (such as hepatocellular carcinoma). [Brief explanation of the drawings]

[0093] [Figure 1] 1 is a schematic diagram of the structure of the GPC3 CAR-IC gene. [Figure 2] 1 shows the results of detecting CAR expression in Example 2. [Figure 3]3A and 3B show the results of detecting the cell function of IFN-γ (FIG. 3A) and IFN-α2 (FIG. 3B) secretion by CAR-IC cells in Example 3. [Figure 4] 1 shows the results of detecting the cytotoxicity (CD107a degranulation) function of CAR-IC cells in Example 4. [Figure 5] 5 shows the results of detecting the cytotoxicity of CAR-T cells in Example 5. Figure 5A shows the killing effect on target cells HepG2, and Figure 5B shows the killing effect on negative control cells U87 MG. [Figure 6] 10 shows in vitro detection of the NK cell-inducing effect of IFN-α2 secreted by co-culturing GPC3 CAR-IC T cells with target cells in Example 6. [Figure 7] 10 shows the evaluation of in vivo antitumor effects and animal survival in the HepG2-NSG tumor transplant model in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention will be described in detail below with reference to specific embodiments, but the examples shown are for the purpose of illustrating the present invention and do not limit the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, but do not limit the present invention in any way.

[0095] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0096] The pUC57 vector used in the following examples is a product of Beijing Jingke Biotechnology Co., Ltd.

[0097] The retroviral vector MP71 used in the following examples is described in the following literature: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes [J]. Human Gene Therapy, 2003, 14(12):1155-1168. The public can obtain this biological material from the applicant. This biological material will be used only to repeat the experiments of the present invention and cannot be used for any other purpose.

[0098] In the examples below, human peripheral blood mononuclear cells (PBMCs) were obtained from the venous blood of healthy volunteers.

[0099] The HepG2 cells in the following examples are a product of the Cell Bank of the Chinese Academy of Sciences, product number SCSP-510.

[0100] The U87 MG cells in the following examples are a product of the Cell Bank of the Chinese Academy of Sciences, product number TCHu138.

[0101] The NSG mice (6-7 weeks old, weighing 18-25 g) used in the following examples were purchased from Baiosaito Jiangsu Gene Biotechnology Co., Ltd.

[0102] The RPMI-1640 medium used in the following examples is a product of Sigma with product number R8758.

[0103] The RPMI-1640 medium containing 10% fetal bovine serum (FBS) (10% FBS RPMI-1640 medium) used in the following examples was prepared using RPMI-1640 medium (R8758) as a solvent and fetal bovine serum as a solute, and is also called complete medium.

[0104] Example 1: Construction of CAR-IC cells The CAR-IC cells constructed in this example target GPC3 and express two artificial receptors: a chimeric antigen receptor (CAR) that recognizes tumor-associated antigens, and a chimeric switch receptor (CSR) that converts immunosuppressive signals in the tumor microenvironment into activation signals in CAR-T cells. This stimulates CAR-T cell proliferation, promotes CAR-T cell survival and migration, and inhibits the tumor microenvironment. The specific construction method is as follows.

[0105] 1. Design of the GPC3 CAR-IC gene The GPC3 CAR-IC gene consists of, from the N-terminus to the C-terminus, a gene encoding a chimeric antigen receptor, a P2A gene, an IFNα gene, a promoter element gene, and a gene encoding a chimeric switch receptor. A structural diagram is shown in Figure 1 (the IFNα gene, promoter element gene, and gene encoding the chimeric switch receptor are collectively referred to as the IC gene).

[0106] The nucleotide sequence of the IC gene (IFNα gene + promoter element gene + gene encoding the chimeric switch receptor) is from positions 1552 to 3882 of SEQ ID No. 3.

[0107] The gene encoding the chimeric antigen receptor encodes, from the N-terminus to the C-terminus, Signal (signal peptide), GPC3 scFv (tumor antigen-binding region, i.e., single-chain antibody region), Hinge (hinge region), CD8 (transmembrane region), 4-1BB (intracellular signal region), and CD3ζ (intracellular signal region). The nucleotide sequence of the gene encoding the chimeric antigen receptor is positions 1 to 1473 of SEQ ID NO. 3, and the amino acid sequence encoding the chimeric antigen receptor (CAR) is SEQ ID NO. 4.

[0108] Among the genes encoding chimeric antigen receptors, the nucleotide sequence of the gene encoding Signal (signal peptide) is at positions 1 to 63 of SEQ ID No. 3, the nucleotide sequence of the gene encoding GPC3 scFv is at positions 64 to 789 of SEQ ID No. 3, the nucleotide sequence of the gene encoding Hinge (hinge region) + CD8 (transmembrane region) is at positions 790 to 996 of SEQ ID No. 3, the nucleotide sequence of the gene encoding 4-1BB (intracellular signal region) is at positions 997 to 1137 of SEQ ID No. 3, and the nucleotide sequence of the gene encoding CD3ζ (intracellular signal region) is at positions 1138 to 1473 of SEQ ID No. 3.

[0109] The nucleotide sequence of the P2A gene is located at positions 1474-1551 of SEQ ID NO. 3. The P2A peptide encoded by the P2A gene is a short viral peptide commonly referred to as a "self-cleaving" peptide. The "self-cleaving" function of the P2A peptide allows it to generate multiple proteins from a single transcript, i.e., it can self-cleave to form two proteins, an upstream product and a downstream product.

[0110] The nucleotide sequence of the IFNα gene is from positions 1552 to 2118 of SEQ ID No. 3, and the amino acid sequence of IFNα (α interferon) encoded by the IFNα gene is SEQ ID No. 5.

[0111] The nucleotide sequence of the promoter element gene is from position 2119 to position 2661 of SEQ ID No. 3, where position 2136 to position 2635 of SEQ ID No. 3 is the promoter.

[0112] The gene encoding the chimeric switch receptor encodes, from the N-terminus to the C-terminus, the TRII extracellular domain (the extracellular domain of the TGFβ type II receptor), a linker, μPD-1, and the transmembrane and cytoplasmic domains of CD27. Furthermore, the gene encoding the transmembrane and cytoplasmic domains of CD27 is oCD27, which was obtained by codon optimization and is suitable for expression in human cells when the encoded amino acid sequence remains unchanged. μPD-1 is the extracellular domain of PD-1 optimized by mutation. The nucleotide sequence of the gene encoding the chimeric switch receptor is positions 2662 to 3882 of SEQ ID No. 3 (i.e., the nucleotide sequence shown in SEQ ID No. 2), and the amino acid sequence encoding the chimeric switch receptor (CSR) is SEQ ID No. 1.

[0113] Among the genes encoding the chimeric switch receptors, the nucleotide sequence of the gene encoding the TRII extracellular domain (extracellular domain of the TGFβ type II receptor) is at positions 2662 to 3159 of SEQ ID No. 3, the nucleotide sequence of the gene encoding the Linker is at positions 3160 to 3219 of SEQ ID No. 3, the nucleotide sequence of the gene encoding μPD-1 is at positions 3220 to 3657 of SEQ ID No. 3, and the nucleotide sequence of the gene encoding the transmembrane and cytoplasmic domains of CD27 (i.e., oCD27) is at positions 3658 to 3882 of SEQ ID No. 3.

[0114] Chimeric antigen receptors (CARs) can specifically kill GPC3-positive tumor cells. GPC3 (Glypican-3), a member of the heparan sulfate proteoglycan family, binds to the cell surface via a glycosylphosphatidylinositol anchor on the cell membrane and is highly expressed specifically in many liver cancer tissues. This makes it an ideal target for tumor therapy, as it can be used to target liver cancer cells and induce tumor killing.

[0115] IFNα (α interferon) has a wide range of antitumor effects, directly controlling tumor cell proliferation, apoptosis, and migration, inhibiting tumor angiogenesis and metastasis, and possessing immunomodulatory functions, such as upregulating the expression of MHCI, tumor antigens, and PD-L1 on tumor cells, activating innate immune cells such as NK cells, DCs, and γδT cells (especially NK cell proliferation, migration, and function), regulating adaptive immune cells such as T cells and B cells (especially T cell proliferation, migration, function, and survival), suppressing immune suppressive cells such as TAMs, Tregs, and myeloid suppressor cells (MDSCs), and upregulating PD-L1 expression on tumor cells, thereby enhancing antitumor immune activity.

[0116] The chimeric switch receptor, which is composed of a TRII extracellular domain, μPD-1, and the transmembrane and cytoplasmic domains of CD27, inhibits the PD-1 and TGFβ signaling pathways and enhances the CD27 signaling pathway, thereby conferring resistance to the immunosuppressive tumor microenvironment and enhancing in vivo persistence of CAR-T cells. Because tumor cells typically express PD-L1 on their surface, which activates inhibitory PD-1 on T cells and evades or inhibits anti-tumor T cell responses, the chimeric switch receptor converts this inhibitory effect into an activating signal via CD27, thereby targeting PD-1 and advantageously attracting CAR-T cells to tumor sites expressing PD-L1.

[0117] 2. Synthesis of GPC3 CAR-IC gene The full-length sequence of the gene encoding the transmembrane and cytoplasmic domains of wild-type human CD27 is designated nCD27, and codon optimization yields oCD27, which is suitable for human cell expression when the encoded amino acid sequence remains unchanged. The nucleotide sequence of the gene encoding oCD27 is shown at positions 3658 to 3882 of SEQ ID No. 3.

[0118] The GPC3 CAR-IC gene was constructed according to the design in Step 1 (see Figure 1), synthesized by Jingke Biotechnology Co., Ltd., and cloned into the pUC57 vector to obtain the recombinant vector pUC57-GPC3 CAR-IC, which was sequenced and confirmed.

[0119] The nucleotide sequence of the GPC3 CAR-IC gene is shown in SEQ ID NO. 3, and it expresses a chimeric antigen receptor whose amino acid sequence is SEQ ID NO. 4, IFNα whose amino acid sequence is SEQ ID NO. 5, and a fusion protein (chimeric switch receptor) whose amino acid sequence is SEQ ID NO. 1.

[0120] 3. Construction of CAR-IC cells The GPC3 CAR-IC gene constructed in step 2 was introduced into T cells for stable expression, and T cells expressing the GPC3 CAR-IC gene (SEQ ID No. 3) were obtained and named GPC3 CAR-IC T cells. The specific procedure is as follows.

[0121] 3-1. Construction of recombinant retroviral vectors (1) The recombinant vector pUC57-GPC3 CAR-IC was double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragment was excised from the gel and recovered. (2) Retroviral vector MP71 was double digested with NotI and EcoRI, and the large vector fragment was excised from the gel and recovered. (3) The target gene fragment and the large vector fragment were ligated using T4 ligase (NEB) to obtain the recombinant retroviral vector MP71-GPC3 CAR-IC carrying the GPC3 CAR-IC gene. (4) The recombinant retroviral vector MP71-GPC3 CAR-IC was transformed into competent Escherichia coli DH5α, and the plasmid was extracted and purified using a Qiagen plasmid purification kit to obtain the MP71-GPC3 CAR-IC plasmid. (5) According to the above method, the MP71-GPC3 CAR plasmid without IC gene was constructed as a control.

[0122] The recombinant retroviral vector MP71-GPC3 CAR-IC (i.e., MP71-GPC3 CAR-IC plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment whose nucleotide sequence is SEQ ID NO. 3 in the Sequence Listing, without changing any other nucleotide sequence of the retroviral vector MP71.

[0123] The recombinant retroviral vector MP71-GPC3 CAR (i.e., MP71-GPC3 CAR plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment (a gene encoding a chimeric antigen receptor) whose nucleotide sequence is shown at positions 1 to 1473 of SEQ ID No. 3 in the Sequence Listing, without changing any other nucleotide sequences of the retroviral vector MP71.

[0124] 3-2. Retrovirus packaging The MP71-GPC3 CAR-IC and MP71-GPC3 CAR plasmids prepared in step 3-1 were then introduced into packaging cells for packaging, completing viral assembly and yielding retroviruses. The specific steps for viral packaging are as follows:

[0125] a) Day 1: Phoenix Ecotropic (ECO) cells should be within 20 passages and not overcrowded. Cell density: 0.6 x 10 6 10 mL of DMEM medium was added to a 10 cm dish, the cells were mixed well, and the cells were cultured at 37°C overnight. b) Day 2: Transfection was performed when ECO cells reached approximately 90% confluency (usually 14-18 h after seeding). 12.5 μg of plasmid, 250 μL of 1.25 M CaCl2, and 1 mL of HO were prepared for a total volume of 1.25 mL. In a separate tube, an equal volume of 2x HBS was added to the plasmid complex, and the mixture was vortexed for 20 seconds while adding the plasmid complex. The mixture was gently poured along the side of the ECO dish and incubated at 37°C for 4 hours. The medium was removed, washed once with PBS, and fresh prewarmed medium was added. c) Day 4: 48 hours after transfection, the supernatant was collected and filtered through a 0.45 μm filter to obtain a retroviral solution, which was then aliquoted and stored at −80°C. d) 1.2 mL of 15 μg / mL Retronectin coating solution was added to each well of an NTC 6-well plate and incubated at 4°C overnight. e) The blocking solution was carefully removed, and 2 mL / well of PBS was added to wash the wells. 5 mL of the above virus solution was added to each well, and the wells were centrifuged at 32°C and 2000 x g for 2 hours. The unbound virus supernatant was then aspirated and discarded. f) PG13 cells in the logarithmic growth phase were rinsed once with 10 mL of PBS, and 1 mL of 0.25% recombinant trypsin was added and allowed to stand at room temperature for 2 to 3 minutes. g) Digestion was stopped by adding 5 ml of complete medium containing 10% FBS and centrifuging at 1500 rpm for 5 minutes. h) Discard the supernatant and culture in complete medium at a cell density of 0.5 x 10 5 The final cell number was adjusted to 1.5 × 10 cells / mL and added to the virus-coated NTC 6-well plate at 3 mL / well. 5 The number of cells / well was 1. i) The mixture was centrifuged at 1000 rpm for 1 minute and cultured at 37°C and 5% CO2 for 48 hours. j) After 1 to 2 passages, the cells were transferred to a T175 culture flask and cultured in DEME medium containing 12% FBS for 2 days. k) The medium was replaced with fresh DEME medium containing 12% FBS, and after culturing for 48 hours, the supernatant was collected and filtered through a 0.45 μm filter to obtain a retrovirus solution, which was then aliquoted and stored at -80°C.

[0126] 3-3. Retroviral infection of human T cells a) Thaw cryopreserved healthy human peripheral blood PBMCs and culture at a cell density of 1 × 10 using RPMI-1640 medium containing 10% fetal bovine serum (FBS). 6 ~2×10 6 Adjusted to cells / mL. b) PBMCs were collected using Ficoll separation solution (Tianjin Jingni Ougai Yang), and CD3-positive T cells were isolated using magnetic beads. Clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added at a ratio of magnetic beads:CD3-positive cells = 3:1 to activate the T cells. c) Two days after T cell activation, RetroNectin (TAKARA) diluted in PBS to a final concentration of 15 μg / mL was coated onto a non-tissue culture-treated plate. 1.2 mL of RetroNectin was added to each well of a 6-well plate. The plate was then stored overnight at 4°C in the dark. d) After 2 days of T cell activation culture, the coated 6-well plate was removed, the coating solution was aspirated and discarded, and the plate was washed once with PBS. e) 5-6 mL of the retrovirus solution prepared in step 3-2 was added to each well, and the mixture was centrifuged at 2000 × g at 32 °C for 2 hours. 3 mL of fresh complete medium containing hIL-2 (500 U / mL) was added to each well, and the mixture was cultured for 1 day. f) After cell infection, the cell density was monitored daily and the T cell density was maintained at approximately 5 × 10 5 To maintain the T cell culture medium at 100 U / mL to promote cell proliferation, T cell culture medium containing 100 U / mL IL-2 was added at the appropriate time. g) CAR-IC cells and control CAR T cells infected with the retrovirus prepared in step 3-2 were obtained and designated GPC3 CAR-IC T cells (i.e., T cells expressing the GPC3 CAR-IC gene whose nucleotide sequence is SEQ ID No. 3) and control GPC3 CAR T cells (i.e., T cells expressing the DNA molecule whose nucleotide sequence is set forth in 1 to 1473 of SEQ ID No. 1), respectively.

[0127] Example 2: Detection of CAR expression in CAR-IC cells The GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, as well as CTR T cells (i.e., T cells not transfected with a virus, as a control), were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C, and the day marked as D0 was recorded. CAR expression was detected by culturing until D8.

[0128] 1. Detection of GPC3 CAR expression: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. The cells were resuspended in 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) in each well of a 96-well round-bottom plate and centrifuged at 1500 rpm for 5 min. (2) 60 μL of the prepared premix of fluorescently labeled antibody and fluorescently labeled recombinant protein (see Table 1 for the preparation of the premix, where the fluorescently labeled antibody is a fluorescently labeled anti-human CD3 / CD4 / CD8 antibody and the fluorescently labeled recombinant protein (rp) is an FITC-labeled GPC3 recombinant protein) was added to each well, resuspended, mixed, and incubated at 4°C for 30 minutes. TIFF2025538568000001.tif72170(3) 200 μL of FACS buffer was added to each well and centrifuged at 1500 rpm for 5 minutes. (4) The supernatant was discarded, and the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II) to analyze the proportion of GPC3 CAR in T cells.

[0129] 2. Detection of interferon α2 (IFNα2) expression: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. The cells were resuspended in 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) in each well of a 96-well round-bottom plate and centrifuged at 1500 rpm for 5 min. (2) 60 μL of the prepared premix of fluorescently labeled antibody and fluorescently labeled recombinant protein (see Table 1 for the preparation of the premix, where the fluorescently labeled antibody is a fluorescently labeled anti-human CD3 / CD4 / CD8 antibody and the fluorescently labeled recombinant protein (rp) is an FITC-labeled GPC3 recombinant protein) was added to each well, resuspended, mixed, and incubated at 4°C for 30 minutes. (3) 200 μL of FACS buffer was added to each well, and the plate was centrifuged at 1500 rpm for 5 minutes. (4) The supernatant was discarded, and 150 μL of Cytofix / Cytoperm (BD, product number 55472) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 15 minutes. (5) After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, 200 μL of Perm / Wash buffer (BD, product number 554723) was added to each well, the cells were resuspended and mixed, and the cells were centrifuged at 1500 rpm for 5 minutes to wash twice. (6) 20 μL of the prepared anti-human IFN-α2 was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 20 minutes. (7) 200 μL of 0.5% BSA was added to each well, and the plate was centrifuged at 1500 rpm for 5 minutes to wash twice. (8) 20 μL of the prepared SA-APC was added to each well, resuspended, mixed, and incubated at 4°C for 10 minutes. (9) 200 μL of 0.5% BSA was added to each well and centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II) to analyze the proportion of IFNα2 in T cells.

[0130] 3. Detection of μPD-1 / TRII expression: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. The cells were resuspended in 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) in each well of a 96-well round-bottom plate and centrifuged at 1500 rpm for 5 min. (2) 60 μL of the prepared premix of fluorescently labeled antibody and fluorescently labeled recombinant protein (see Table 1 for the preparation of the premix, where the fluorescently labeled antibody is a fluorescently labeled anti-human CD3 / CD4 / CD8 antibody and the fluorescently labeled recombinant protein (rp) is an FITC-labeled GPC3 recombinant protein) was added to each well, resuspended, mixed, and incubated at 4°C for 30 minutes. (3) 200 μL of FACS buffer was added to each well, and the plate was centrifuged at 1500 rpm for 5 minutes. (4) The supernatant was discarded, and the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II) and the proportion of μPD-1 / TRII in T cells was analyzed.

[0131] The detection results are shown in Figure 2. CAR (i.e., GPC3 CAR), IFNα2, μPD-1, and TRII were simultaneously expressed in CAR-IC cells (i.e., GPC3 CAR-IC T cells prepared in Example 1), indicating that the expression of CAR molecules in CAR-IC cells is an expected design.

[0132] Example 3: Cellular function detection of IFN-γ and IFN-α2 secretion by CAR-IC cells The test cells were the GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells not transfected with a virus).

[0133] The test cells were cultured until D10 and plated in a 24-well TC plate at 1 × 10 6 HepG2 cells were added at a test cell:target cell ratio of 1:1, and the cells were co-cultured in a 37°C incubator, and the result was recorded as D0.

[0134] The detection group and control group are as follows: CTR T+Medium: 1×10 61 mL of medium (10% FBS RPMI-1640 medium) was added to cells / mL / well of T cells that had not been transfected with a virus (CTR T cells). CTR T+HepG2: 1×10 6 cells / mL / well of virus-untransfected T cells (CTR T cells) with 1 × 10 6 1 mL of HepG2 cells / mL was added. GPC3 CAR T+Medium: 1 x 10 6 1 mL of medium (10% FBS RPMI-1640 medium) was added to cells / mL / well of conventional GPC3-targeting CAR T cells (GPC3 CAR T cells). GPC3 CAR T+HepG2: 1×10 6 cells / mL / well of conventional GPC3-targeted CAR T cells (GPC3 CAR T cells) plus 1 × 10 6 1 mL of HepG2 cells / mL was added. GPC3 CAR-IC T+Medium: 1 x 10 6 1 mL of medium (10% FBS RPMI-1640 medium) was added to cells / mL / well of GPC3-targeting CAR-IC T cells (GPC3 CAR-IC T cells). GPC3 CAR-IC T+HepG2: 1×10 6 1 × 10 cells / mL / well of GPC3-targeted CAR-IC T cells (GPC3 CAR-IC T cells) 6 1 mL of HepG2 cells / mL was added.

[0135] The co-cultured cells were harvested for three consecutive days (D1, D2, D3) and secreted IFN-γ and IFN-α2 were detected.

[0136] The specific detection procedure is as follows. The cell density of the co-cultured cells cultured on D1, D2, and D3 was 2 × 10 6100 μL of the solution was added to a 96-well U-bottom plate, and Brefeldin A (Med Chem Express, HY-16592) was added to each well to a final concentration of 5 μg / mL, followed by incubation in a 37°C incubator for 5 to 6 hours. 2. After incubation, flow cytometry staining was performed. The procedure is as follows: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) was added to each well to resuspend the cells. The cells were then centrifuged at 1500 rpm for 5 min. This step was repeated twice. (2) 60 μL of the prepared premix of fluorescently labeled antibody and fluorescently labeled recombinant protein (see Table 1 for the preparation of the premix, where the fluorescently labeled antibody is a fluorescently labeled anti-human CD3 / CD4 / CD8 antibody and the fluorescently labeled recombinant protein (rp) is a FITC-labeled GPC3 recombinant protein) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 10 minutes. (3) 200 μL of FACS buffer was added to each well, and the plate was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. (4) 150 μL of Cytofix / Cytoperm (BD, product number 55472) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 15 minutes. (5) After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, 200 μL of Perm / Wash buffer (BD, product number 554723) was added to each well, the cells were resuspended and mixed, and the cells were centrifuged at 1500 rpm for 5 minutes to wash twice. (6) 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, product number 506510) or anti-human IFN-α2 (Biolegend, product number 537204) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 20 minutes. (7) 200 μL of Perm buffer was added to each well and centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II) to analyze the proportions of functional effector molecules IFN-γ and IFN-α2 in GPC3 CAR-IC T cells and control cells.

[0137] The detection results are shown in Figure 3. After co-culture with target cells (HepG2 cells), both GPC3 CAR T cells and GPC3 CAR-IC T cells were able to specifically secrete the T cell effector molecule IFN-γ. Compared with GPC3 CAR T cells, GPC3 CAR-IC T cells secreted higher levels of IFN-γ. Furthermore, in the absence of antigen stimulation, GPC3 CAR-IC T cells secreted low levels of IFN-α2, but upon antigen stimulation, GPC3 CAR-IC T cells were able to secrete high levels of IFN-α2.

[0138] Example 4: Detection of cytotoxic (CD107a degranulation) function of CAR-IC cells The degranulation ability of CAR-IC cells, i.e., the expression of CD107a, was detected. The test cells were the GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells not transfected with a virus).

[0139] 1) The test cells were cultured until D10, and the cell density was increased to 2 × 10 6 / mL, and 100 μL was added to a 96-well U-bottom plate. 2) 2 × 10 cells per well were added as CAR antigen-specific stimulation (+HepG2) or negative control U87 MG cells (+U87 MG). 5 100 μL of HepG2 cells were added at a test cell:target cell ratio of 1:1, and 100 μL of Medium (10% FBS RPMI-1640 medium) was added to each well as a negative control (+Medium). 3) 1 μL of APC-labeled anti-human CD107a antibody (Biolegend, product number 328620) was added to each well, and the plate was incubated at 37° C. for 1 hour. 4) 10 μL of Monensin Solution (Invitrogen, product number 00-4505-51) diluted 1:50 was added to each well and incubated for 3 hours. 5) After incubation, flow cytometry staining was performed, and the procedure was as follows: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) was added to each well, resuspended, mixed, and centrifuged at 1500 rpm for 5 min. This step was repeated twice. (2) 60 μL of the prepared premix of fluorescently labeled antibody and fluorescently labeled recombinant protein (see Table 1 for the preparation of the premix, where the fluorescently labeled antibody is a fluorescently labeled anti-human CD3 / CD4 / CD8 antibody and the fluorescently labeled recombinant protein (rp) is a FITC-labeled GPC3 recombinant protein) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 10 minutes. (3) After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended in 400 μL of FACS buffer, and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II), and the proportion of the cell degranulation molecule CD107a in T cells was analyzed.

[0140] The detection results are shown in Figure 4. After co-culture with target cells, both GPC3 CAR T cells and GPC3 CAR-IC T cells could specifically enhance CD107a degranulation. Furthermore, compared with GPC3 CAR-T cells, GPC3 CAR-IC T cells had stronger specific cytotoxicity (CD107a).

[0141] Example 5: Detection of CAR-T cell cytotoxicity The test cells were the GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells not transfected with a virus).

[0142] 1) The test cells were cultured until D10, and the cell density was increased to 2 × 10 5 The solution was adjusted to 100 μg / mL and 100 μL was added to a 96-well U-bottom plate. D-luciferin sodium salt (Yeasen Biotechnology, product number 40901ES08, stock concentration 100 mg / mL) was added to a final concentration of 100 μg / mL in triplicate wells. 2) Different effector / target ratios (1:1, 1:3, 1:9, 1:27) were used to culture HepG2 target cells or negative control U87 MG cells (at a cell density of 2 × 10 target cells at a 1:1 ratio). 4 Cells) were added to the cells and cultured at 37°C for 16 hours. 3) Fluorescence values ​​were measured using a TECAN spark microplate reader, and the specific killing activity (cytotoxicity) of CAR-T cells was calculated by taking the average of three replicate wells. Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin) Eexp: RLU value when effector cells and target cells were co-cultured. Emin: RLU value of spontaneous death of effector cells in the absence of cells. Tmax: RLU value for spontaneous death of target cells in the absence of effector cells. Tmin: RLU value under the condition of maximum killing rate.

[0143] The detection results are shown in Figure 5. Both GPC3 CAR T cells and GPC3 CAR-IC T cells were able to specifically kill GPC3-positive target cells (HepG2 cells) but showed no killing effect on GPC3-negative target cells (U87 MG cells). The control CTR T cells showed no killing effect on GPC3-positive or GPC3-negative target cells. Furthermore, compared with GPC3 CAR-T cells, GPC3 CAR-IC T cells had a stronger killing effect on GPC3-positive target cells (HepG2 cells).

[0144] Example 6: In vitro detection of the NK cell-inducing effect of IFN-α2 secreted by co-culture of GPC3 CAR-IC T cells with target cells The test cells were the GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells not transfected with a virus). The detection procedure was as follows.

[0145] 1) The test cells were cultured until D10, and the cell density was increased to 2 × 10 6 / mL and 1 mL was added to a 96-well U-bottom plate. 2) 2 x 10 cells per well 6 / mL HepG2 cells were added at a test cell:target cell ratio of 1:1 and cultured for 3 days. 3) The supernatant was collected by centrifugation and incubated with PBMC cells. 5 / well, 0.1mL / well; supernatant: 0.1mL / well. 4) After overnight incubation, Brefeldin A (Med Chem Express, HY-16592) was added to each well to a working concentration of 5 μg / mL and incubated at 37°C for 4 hours. 5) After incubation, flow cytometry staining was performed, and the procedure was as follows: (1) The cells were centrifuged (1500 rpm x 5 min) and the supernatant was discarded. 200 μL of FACS buffer (1x PBS containing 0.1% NaN3 and 2% FBS) was added to each well to resuspend the cells, and the cells were centrifuged at 1500 rpm for 5 min. (2) 40 μL of the prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD56) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 10 minutes. (3) 200 μL of FACS buffer was added to each well, and the plate was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. (4) 150 μL of Cytofix / Cytoperm (BD, product number 55472) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 15 minutes. (5) After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, 200 μL of Perm / Wash buffer (BD, product number 554723) was added to each well, the cells were resuspended and mixed, and the cells were centrifuged at 1500 rpm for 5 minutes to wash twice. (6) 40 μL of the prepared fluorescent antibody (fluorescently labeled anti-human IFNγ / Granzyme B) was added to each well, resuspended, mixed, and incubated in the dark at room temperature for 20 minutes. (7) 200 μL of Perm buffer was added to each well and centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube. The cells were read using a flow cytometer (BD Canto-II) and the proportions of the effector molecules IFN-γ and Granzyme B in NK cells were analyzed.

[0146] The detection results are shown in Figure 6. IFN-α2 secreted by GPC3 CAR-IC T cells can induce NK cells to secrete the functional effector molecule IFN-γ.

[0147] Example 7: Evaluation of the in vivo antitumor effect and animal survival of GPC3 CAR-IC T cells in the transplanted tumor HepG2-NSG model The in vivo antitumor effect and animal survival of GPC3 CAR-IC T cells were evaluated using a HepG2 subcutaneous tumor-bearing NSG mouse model (HepG2-NSG model). The specific procedures were as follows.

[0148] 1) Inject 1 x 10 IgG into the dorsal surface of the right lower limb of NSG mice (6-7 weeks old). 7 HepG2 cells were inoculated subcutaneously at 1 cell / mouse. 2) On day 12, the tumors in the mice were 80-100 mm 3 At the time of tumor growth, GPC3 CAR-IC T cells were injected and recorded as D0. During this period, tumor-bearing mice were randomly divided into three groups (5 mice / group), as follows: Control group (CTR T): CTR T cells (3 × 10 6 cells / animal). GPC3 CAR T group: GPC3 CAR-T cells (3×10 6 cells / animal). GPC3 CAR-IC T group: GPC3-IC CAR-T cells (3×10 6 cells / animal). 3) After administration, tumor size was measured with a vernier caliper on D7, D14, D21, D28, and D35. 4) Blood was collected from the submandibular region of the mice one day before administration (D-1) and on D4, D7, D12, D21, and D28 after administration, and serum was collected. IFN-γ and IFN-α2 secreted from GPC3 CAR-IC T cells and control cells in the mice were detected using a multiple cytokine assay.

[0149] The detection results are shown in Figure 7. Compared with GPC3 CAR-T cells, GPC3 CAR-IC T cells exhibited a more rapid and superior tumor suppression effect at D7 / D14. High levels of IFN-α2 secretion were observed in the serum of mice adoptively transferred with GPC3 CAR-IC T cells. Higher levels of IFN-γ secretion were observed in the serum of mice adoptively transferred with GPC3 CAR-IC T cells compared with mice adoptively transferred with GPC3 CAR-T cells. This indicates that GPC3 CAR-IC T cells have stronger in vivo antitumor activity.

[0150] The present invention has been described in detail above. Those skilled in the art can practice the present invention to a broader extent under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation. Although the present invention has been described with specific examples, it is understood that the present invention can be further improved. In short, based on the principles of the present invention, the present application is intended to cover any changes, uses, or improvements to the present invention that depart from the scope disclosed herein, including improvements made using conventional techniques known in the art. Some basic features may be applied within the scope of the following appended claims. [Industrial Applicability]

[0151] After extensive research, the inventors of the present application have designed a fusion gene containing genes encoding a chimeric antigen receptor and a chimeric switch receptor, constructed a GPC3-targeting GPC3 CAR-IC gene (SEQ ID No. 3), packaged it, and infected it with a retrovirus to obtain GPC3 CAR-IC T cells. The GPC3 CAR-IC T cells constructed according to the present invention specifically kill GPC3-positive tumor cells, exhibiting excellent tumor-killing effects, more rapid and superior tumor suppression effects, and stronger in vivo antitumor activity. They are also suitable for broad-spectrum antitumor activity, reducing antigen heterogeneity and tumor cell escape, enhancing immune cell infiltration, immune regulation, improving the tumor microenvironment, and resisting HBV / HCV infection. These properties are of great significance and have broad application value in CAR-T treatment of tumors (e.g., hepatocellular carcinoma).

Claims

1. A nucleic acid molecule comprising a gene encoding a chimeric antigen receptor and a gene encoding a chimeric switch receptor.

2. The nucleic acid molecule of claim 1, wherein the chimeric switch receptor comprises the extracellular domain of a TGFβ type II receptor, μPD-1, and the transmembrane and cytoplasmic domains of CD27, and the amino acid sequence of μPD-1 is positions 187 to 332 of SEQ ID No.

1.

3. The nucleic acid molecule of claim 2, wherein the gene encoding the chimeric switch receptor is any one of the following: B1) a nucleic acid molecule encoding the fusion protein; B2) a DNA molecule whose coding sequence is SEQ ID No. 2; B3) a DNA molecule having the nucleotide sequence SEQ ID No. 2; The fusion protein is one of the following: A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) A protein having 80% or more identity and the same function as the protein shown in A1), in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1; A3) A fusion protein having the same function as A1) or A2) tagged at the N-terminus and / or C-terminus.

4. The nucleic acid molecule according to any one of claims 1 to 3, further comprising an IFNα gene and / or a P2A gene.

5. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene encoding the chimeric antigen receptor is a gene encoding a chimeric antigen receptor that targets GPC3.

6. The nucleic acid molecule according to claim 5, wherein the gene encoding the chimeric antigen receptor that targets GPC3 is any one of the following: C1) a nucleic acid molecule encoding a protein having the amino acid sequence SEQ ID No. 4; C2) a DNA molecule whose coding sequence is positions 1 to 1473 of SEQ ID No. 3; C3) A DNA molecule having the nucleotide sequence of positions 1 to 1473 of SEQ ID No.

3.

7. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule is one of the following: D1) a DNA molecule having the nucleotide sequence SEQ ID No. 3; D2) A DNA molecule having 70% or more identity and the same function as the DNA molecule shown in D1), in which the nucleotide sequence shown in SEQ ID No. 3 has been modified and / or one or more nucleotides have been substituted and / or deleted and / or added.

8. A biomaterial, characterized in that it is any one of the following: E1) An expression cassette comprising a nucleic acid molecule according to any one of claims 1 to 7, E2) A recombinant vector comprising a nucleic acid molecule according to any one of claims 1 to 7 or an expression cassette according to E1. E3) A recombinant microorganism comprising a nucleic acid molecule according to any one of claims 1 to 7, or an expression cassette according to E1), or a recombinant microorganism comprising a recombinant vector according to E2). E4) A recombinant cell comprising a nucleic acid molecule according to any one of claims 1 to 7, or a recombinant cell comprising an expression cassette according to E1), or a recombinant cell comprising a recombinant vector according to E2). E5) A gene encoding the chimeric switch receptor according to claim 2 or 3; E6) The chimeric switch receptor of claim 2. E7) The fusion protein of claim 3.

9. The biomaterial according to claim 8, characterized in that the cells in step E4) are T cells, NK cells, γδT cells, NKT cells, macrophages or stem cells.

10. Use of a nucleic acid molecule according to any one of claims 1 to 7 and / or a biomaterial according to claim 8 or 9 in any one of the following: F1) Use in the manufacture of a medicament for the prevention or treatment of tumors; F2) Use in the manufacture of a medicament for the prevention or treatment of tumors expressing the GPC3 antigen; F3) Modulation of the immunosuppressive effect of the tumor microenvironment or use in the manufacture of a product for modulating the immunosuppressive effect of the tumor microenvironment; F4) Use in the prevention or treatment of tumors; F5) Use in the prevention or treatment of tumors expressing the GPC3 antigen; F6) Use in the prevention or treatment of liver cancer, melanoma, Wilms' tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma or glioma.

11. A pharmaceutical composition comprising, as an active ingredient, a CAR cell containing or expressing the nucleic acid molecule according to any one of claims 1 to 7.

12. A method for preventing or treating tumors, comprising administering a pharmaceutical comprising the recombinant cell of claim 8 to a patient suffering from a tumor disease.

13. The method of claim 12, wherein the recombinant cell is a GPC3 CAR-IC T cell, and the GPC3 CAR-IC T cell comprises a DNA molecule represented by SEQ ID No.

3.

14. 14. The method according to claim 12 or 13, wherein the tumor is a tumor that expresses the GPC3 antigen.

15. The method according to claim 14, wherein the tumor expressing the GPC3 antigen is liver cancer, melanoma, Wilms' tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma, or glioma.

16. 16. The method of claim 15, wherein the liver cancer is hepatocellular carcinoma.

17. 16. The method of claim 15, wherein the squamous cell carcinoma is lung squamous cell carcinoma.

18. 16. The method of claim 15, wherein the melanoma is malignant melanoma.

19. 16. The method of claim 15, wherein the glioma is a glioblastoma.

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