Fusion gene containing chimeric antigen receptor and chimeric switch receptor encoding gene and its application

Abstract

The present invention discloses a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene and its application. Specifically disclosed is a fusion protein whose amino acid sequence is SEQ ID No.1 and a nucleic acid molecule (SEQ ID No.3) comprising a chimeric antigen receptor and a chimeric switch receptor encoding gene. The present invention converts the PD-L1 activated inhibitory anti-tumor T cell response into a CD27-mediated activation signal by designing a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene, giving CAR-T cells the ability to resist an immunosuppressive tumor microenvironment and making them more persistent in vivo, regulating the microenvironment near the tumor by promoting the secretion of cytokines IFN-γ and IFN-α2, relieving its immunosuppression, mobilizing the body's own immunity to participate in the killing of tumor cells, and improving the anti-tumor effect of CAR-T cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor (CSR) encoding gene and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) technology uses genetic engineering technology to modify immune cells to express exogenous anti-tumor genes, so that immune cells such as lymphocytes have the ability to recognize tumor cell surface antigens and specifically recognize and kill tumor cells. In recent years, CAR-T cell therapy has achieved encouraging therapeutic effects in hematological tumors such as B cells. However, its great success in hematological tumors has not yet been replicated in solid tumors. Solid tumors are different from hematological tumors. When entering solid tumors, CAR-T cells need to overcome the inherent heterogeneity of tumor cells and the complexity of the inhibitory tumor microenvironment. The immunosuppressive nature of the tumor microenvironment and the complexity of its structure are the main obstacles to the widespread application of T cell immunotherapy in solid tumors.

[0003] Specific recognition of tumor antigens is only the first step to the success of CAR-T. In order to survive, the tumor creates an immunosuppressive microenvironment for itself, which is unfriendly to CAR-T. Glycolysis of tumor cells makes the environment hypoxic, acidic, and nutrient-depleted. The execution of CAR-T function also requires glycolysis and oxidative phosphorylation, but sugars are depleted by tumor cells, and the effector function of CAR-T cells is impaired. In an inflammatory environment, tumor cells usually upregulate inhibitory ligands such as PD-L1 and Galectin-9. In addition, the tumor microenvironment also has a large number of inhibitory immune cells and stromal cells, such as tumor-associated fibroblasts (CAFs), myeloid suppressor cells (MDSCs), tumor-associated macrophages (TAMS), tumor-associated neutrophils (TANS), mast cells, and regulatory T cells (Tregs). VEGF, TGF-β, etc. secreted by these cells and tumor cells can cause tumor vascular malformations, promote anti-inflammatory polarization of immune cells such as TAMS, and participate in epithelial-mesenchymal transition (EMT). They can also produce reactive oxygen species (ROS) and molecules such as lactate, indoleamine 2,3-dioxygenase (Ido), prostaglandin e2 (PGE2), soluble fatty acids and adenosine, forming an inhibitory immune microenvironment. Recent studies have shown that the immunosuppressive effect of the tumor microenvironment becomes stronger after CAR-T cell therapy.

[0004] In view of this, how to perform corresponding structural modification on CAR-T cells to overcome the immunosuppressive tumor microenvironment and thus improve the clinical treatment effect is still a severe challenge facing CAR-T cell therapy. Researching and developing new CAR-T cells with powerful anti-tumor effects has important theoretical significance and application value for immune cell therapy of tumors. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to prevent or treat tumors (such as tumors expressing GPC3 antigen) and / or regulate the immunosuppressive effect of the tumor microenvironment. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

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

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

[0008] Furthermore, in the above nucleic acid molecule, the gene encoding the chimeric switch receptor may be any of the following:

[0009] B1) a nucleic acid molecule encoding a fusion protein;

[0010] B2) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0011] B3) a DNA molecule whose nucleotide sequence is SEQ ID No. 2;

[0012] The fusion protein may be any of the following:

[0013] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0014] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0015] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0016] The fusion protein may be a chimeric switch receptor (CSR).

[0017] The fusion protein and its encoding gene can be used to improve the ability of CAR cells to resist immunosuppressive tumor microenvironment, make CAR cells have stronger in vivo persistence and / or inhibit PD-1 and TGFβ signaling pathways to enhance the effect of CD27 signaling pathway.

[0018] The gene encoding the fusion protein can be used to prepare the nucleic acid molecule of the present invention or a CAR cell containing the nucleic acid molecule of the present invention.

[0019] The tags described in this article include but are not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0020] Those skilled in the art can easily mutate the nucleotide sequence encoding the fusion protein of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the fusion protein of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the fusion protein and have the function of the fusion protein.

[0021] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0022] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0023] Herein, the 80% or greater 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.

[0024] Furthermore, in the above nucleic acid molecule, the nucleic acid molecule may also include IFNα gene (α interferon gene) and / or P2A gene.

[0025] The nucleotide sequence of the IFNα gene may be positions 1552-2118 of SEQ ID No.3.

[0026] The nucleotide sequence of the P2A gene may be positions 1474-1551 of SEQ ID No.3.

[0027] Furthermore, in the nucleic acid molecule, the gene encoding the chimeric antigen receptor may be a gene encoding a chimeric antigen receptor targeting GPC3.

[0028] The GPC3 is Glypican-3.

[0029] Furthermore, the gene encoding the chimeric antigen receptor targeting GPC3 may be any of the following:

[0030] C1) a nucleic acid molecule encoding a protein having an amino acid sequence of SEQ ID No. 4;

[0031] C2) the coding sequence is a DNA molecule having positions 1 to 1473 of SEQ ID No. 3;

[0032] C3) The nucleotide sequence is the DNA molecule of positions 1-1473 of SEQ ID No.3.

[0033] Furthermore, the nucleic acid molecule may be any of the following:

[0034] D1) a DNA molecule having a nucleotide sequence of SEQ ID No. 3;

[0035] D2) The nucleotide sequence shown in SEQ ID No. 3 is modified and / or one or more nucleotides are substituted and / or deleted and / or added to obtain a DNA molecule that has more than 70% identity with the DNA molecule shown in D1) and has the same function.

[0036] The greater than 70% 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.

[0037] The present invention also provides a biomaterial, which may be any of the following:

[0038] E1) an expression cassette containing the nucleic acid molecule;

[0039] E2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in E1);

[0040] E3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in E1), or a recombinant microorganism containing the recombinant vector described in E2);

[0041] E4) a recombinant cell containing the nucleic acid molecule, or a recombinant cell containing the expression cassette described in E1), or a recombinant cell containing the recombinant vector described in E2);

[0042] E5) a gene encoding the chimeric switch receptor;

[0043] E6) the chimeric switch receptor;

[0044] E7) the fusion protein.

[0045] In the above-mentioned biological materials, the cells in E4) may be T cells, NK cells, γδT cells, NKT cells, macrophages or stem cells.

[0046] The recombinant cell expresses the nucleic acid molecule.

[0047] Furthermore, the cell in E4) may be a T cell.

[0048] Furthermore, the cells in E4) may be human T cells.

[0049] The vector described herein refers to a vector that can carry exogenous DNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus)). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a retrovirus vector MP71.

[0050] The microorganism described herein may be bacteria, yeast, algae or fungi. Among them, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto. The yeast may be from Saccharomyces sp., Pichia sp., Yarrowia sp., Candida sp., Schizosaccharomyces sp., Hansenula sp., Kluyreromyces sp., etc., but not limited thereto. The algae may be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but not limited thereto. The fungi may be from Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., Cephalosporium sp., etc., but not limited thereto. In one or more embodiments of the present invention, the microorganism is Escherichia coli DH5α.

[0051] The cells (host cells) described herein refer to cells that can be used to introduce vectors, including but not limited to: mammalian immune cells or stem cells with the ability to differentiate into immune cells. The host cells may include but are not limited to T cells, NK cells, γδT cells, NKT cells, macrophages or stem cells. In one or more embodiments of the present invention, the cells are human T cells.

[0052] The recombinant vector described herein refers to a recombinant vector DNA molecule constructed by connecting an exogenous target gene to a vector in vitro, and can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with replication, integration, amplification and / or expression capabilities in the recipient cell. In one or more embodiments of the present invention, the recombinant vector is a recombinant vector pUC57-GPC3 CAR-IC and / or a recombinant retroviral vector MP71-GPC3 CAR-IC.

[0053] The recombinant vector pUC57-GPC3 CAR-IC is a recombinant vector obtained by cloning the GPC3 CAR-IC gene with the nucleotide sequence of 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.

[0054] 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 of SEQ ID No.3 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged. The recombinant retroviral vector MP71-GPC3CAR-IC contains the DNA molecule shown in SEQ ID No.3.

[0055] The recombinant microorganism described herein refers to a microorganism whose function has changed by manipulating and modifying the genes of the target microorganism. For example, a recombinant microorganism obtained by introducing an exogenous target gene or a recombinant vector into the target microorganism, or a recombinant microorganism obtained by directly editing the endogenous gene of the target microorganism. In one or more embodiments of the present invention, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant retroviral vector MP71-GPC3 CAR-IC into Escherichia coli DH5α, which contains the DNA molecule shown in SEQ ID No.3.

[0056] The recombinant cell described herein refers to the manipulation and modification of the genes of the target cells to obtain cells with changed functions. Such as recombinant cells obtained by introducing an exogenous target gene or a recombinant vector into a target cell. Further, the recombinant cells described herein include recombinant cells obtained by stably expressing the CAR gene in receptor cells (such as T cells, NK cells, γδT cells, NKT cells, macrophages or stem cells), such as CAR-T cells, CAR-NK cells, CAR-macrophages (CAR-M cells), CAR-iPSC or CAR-PSC, but not limited thereto. In one or more embodiments of the present invention, the recombinant cell is a GPC3 CAR-IC T cell.

[0057] The GPC3 CAR-IC T cells are recombinant cells obtained by packaging the recombinant retroviral vector MP71-GPC3 CAR-IC with packaging cells to obtain recombinant retroviruses, and then introducing the recombinant retroviruses into T cells. The packaging cells can be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0058] The 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).

[0059] The present invention also provides any of the following uses of the nucleic acid molecules described herein, and / or the biological materials:

[0060] F1) Use in the preparation of drugs for preventing or treating tumors;

[0061] F2) Use in the preparation of a medicament for preventing or treating a tumor expressing the GPC3 antigen;

[0062] F3) Use in regulating the immunosuppressive effect of tumor microenvironment or preparing a product for regulating the immunosuppressive effect of tumor microenvironment;

[0063] F4) Application in the prevention or treatment of tumors;

[0064] F5) Use in preventing or treating tumors expressing GPC3 antigen;

[0065] F6) Use in the prevention or treatment of liver cancer, melanoma, Wilm's tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma or glioma.

[0066] Furthermore, the regulation in F3) may be reduction or inhibition.

[0067] In the above application, the tumor expressing the GPC3 antigen may be liver cancer, melanoma, Wilm's tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma or glioma, but is not limited thereto.

[0068] Furthermore, the liver cancer may be hepatocellular carcinoma (HCC).

[0069] Furthermore, the squamous cell carcinoma may be lung squamous cell carcinoma.

[0070] Furthermore, the melanoma may be malignant melanoma.

[0071] Furthermore, the glioma may be a glioblastoma.

[0072] Furthermore, the tumor expressing the GPC3 antigen may be a tumor with high expression of GPC3.

[0073] Furthermore, the F1) can be used in the preparation of drugs for inhibiting and / or killing tumors.

[0074] Furthermore, the F2) can be used in the preparation of a drug for inhibiting and / or killing a tumor expressing the GPC3 antigen.

[0075] The inhibition may be inhibition of tumor proliferation and / or growth.

[0076] The present invention also provides a pharmaceutical composition, the active ingredient of which may be a CAR cell (such as a CAR-T cell, CAR-NK cell, CAR-macrophage, CAR-iPSC or CAR-PSC) containing or expressing any of the nucleic acid molecules described in the present invention.

[0077] Furthermore, the pharmaceutical composition comprises the GPC3 CAR-IC T cells described herein.

[0078] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers may be diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants but are not limited thereto.

[0079] The pharmaceutical composition has an anti-tumor effect and can be used to prevent or treat tumors.

[0080] Furthermore, the pharmaceutical composition can be used to prevent or treat mammalian tumors.

[0081] The mammal may be any one of humans, mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0082] The tumor described herein may be a solid tumor (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, or brain glioma).

[0083] Furthermore, the active ingredient of the pharmaceutical composition may be immune effector cells expressing the GPC3 CAR-IC gene (SEQ ID No. 3).

[0084] Furthermore, the active ingredient of the pharmaceutical composition may be the GPC3 CAR-IC T cells.

[0085] The present invention also provides a method for preventing or treating tumors, which method comprises administering a drug containing the recombinant cell described in E4) herein to a patient suffering from a tumor disease.

[0086] In the above method, the recombinant cell may be a GPC3 CAR-IC T cell, and the GPC3 CAR-IC T cell may contain the DNA molecule shown in SEQ ID No.3.

[0087] In the above method, the tumor may be a tumor expressing GPC3 antigen.

[0088] In the above method, the tumor expressing GPC3 antigen may be liver cancer, melanoma, Wilm's tumor, non-small cell lung cancer, ovarian clear cell carcinoma, squamous cell carcinoma, renal cell carcinoma, prostate cancer, colorectal cancer, hepatoblastoma or glioma, but is not limited thereto.

[0089] In the above method, the liver cancer may be hepatocellular carcinoma.

[0090] In the above method, the squamous cell carcinoma may be lung squamous cell carcinoma.

[0091] In the above method, the melanoma may be malignant melanoma.

[0092] In the above method, the glioma may be a glioblastoma.

[0093] The present invention also provides a method for preparing the GPC3 CAR-IC T cells, the method comprising:

[0094] (1) constructing and synthesizing the GPC3 CAR-IC gene (SEQ ID No. 3);

[0095] (2) cloning the GPC3 CAR-IC gene into the retroviral vector MP71 to obtain a recombinant retroviral vector MP71-GPC3 CAR-IC;

[0096] (3) introducing the recombinant retroviral vector MP71-GPC3 CAR-IC into packaging cells for packaging to obtain a recombinant retrovirus;

[0097] (4) Transfecting T cells with the recombinant retrovirus to obtain the GPC3 CAR-IC T cells.

[0098] In the above method, the packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0099] The present invention also provides the use of the fusion protein and / or the nucleic acid molecule in preparing GPC3 CAR-IC T cells.

[0100] The nucleic acid molecule described herein may be a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene.

[0101] The nucleic acid molecules described herein can express chimeric antigen receptors, IFNα and fusion proteins, and the fusion protein can be any of the following:

[0102] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0103] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0104] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0105] Furthermore, the target of the chimeric antigen receptor may be GPC3.

[0106] Furthermore, the chimeric antigen receptor may be any of the following:

[0107] F1) a protein whose amino acid sequence is SEQ ID No. 4;

[0108] F2) a protein having more than 80% identity with the protein shown in F1) and having the same function as the protein shown in F1) obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 4;

[0109] F3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of F1) or F2).

[0110] Furthermore, the IFNα may be any of the following:

[0111] G1) a protein whose amino acid sequence is SEQ ID No. 5;

[0112] G2) a protein having more than 80% identity with the protein shown in G1) and having the same function as the protein shown in G1) obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.5;

[0113] G3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of G1) or G2).

[0114] Furthermore, the nucleic acid molecule described herein can express a chimeric antigen receptor having an amino acid sequence of SEQ ID No.4, an IFNα having an amino acid sequence of SEQ ID No.5, and a fusion protein (chimeric switch receptor) having an amino acid sequence of SEQ ID No.1.

[0115] The nucleic acid molecule described herein may be a GPC3 CAR-IC gene, the nucleotide sequence of the GPC3 CAR-IC gene may be SEQ ID No. 3, and the GPC3 CAR-IC gene may be a chimeric antigen receptor encoding gene, a P2A gene, an IFNα gene, a promoter element gene, and a chimeric switch receptor encoding gene from the N-terminus to the C-terminus. The structural diagram is shown in Figure 1 As shown (the IFNα gene, the promoter element gene, and the coding gene of the chimeric switch receptor can be collectively referred to as the IC gene). Among them:

[0116] The nucleotide sequence of the IC gene (IFNα gene+promoter element gene+chimeric switch receptor encoding gene) may be positions 1552-3882 of SEQ ID No.3.

[0117] The coding genes of the chimeric antigen receptor may be 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), CD3ζ (intracellular signal region) coding genes from N-terminus to C-terminus. The nucleotide sequence of the coding gene of the chimeric antigen receptor may be positions 1-1473 of SEQ ID No.3, and the amino acid sequence encoding the chimeric antigen receptor (CAR) may be SEQ ID No.4.

[0118] Among the coding genes of the chimeric antigen receptor, the nucleotide sequence of the coding gene of Signal (signal peptide) may be positions 1-63 of SEQ ID No.3, the nucleotide sequence of the coding gene of GPC3 scFv may be positions 64-789 of SEQ ID No.3, the nucleotide sequence of the coding gene of Hinge (hinge region) + CD8 (transmembrane region) may be positions 790-996 of SEQ ID No.3, the nucleotide sequence of the coding gene of 4-1BB (intracellular signal region) may be positions 997-1137 of SEQ ID No.3, and the nucleotide sequence of the coding gene of CD3ζ (intracellular signal region) may be positions 1138-1473 of SEQ ID No.3.

[0119] The nucleotide sequence of the P2A gene may be positions 1474-1551 of SEQ ID No. 3. The P2A peptide encoded by it may be a short peptide derived from a virus, generally referred to as a "self-cleaving" peptide. The "self-cleavage" function of the P2A peptide can enable a transcription product to produce multiple proteins, that is, to form two proteins, an upstream product and a downstream product, through self-cleavage.

[0120] The nucleotide sequence of the IFNα gene may be positions 1552-2118 of SEQ ID No.3, and the amino acid sequence of the IFNα (interferon α) encoded by it may be SEQ ID No.5.

[0121] The nucleotide sequence of the promoter element gene may be positions 2119-2661 of SEQ ID No. 3, wherein positions 2136-2635 of SEQ ID No. 3 are the promoter.

[0122] The coding genes of the chimeric switch receptor can be the coding genes of the TRII extracellular region (TGFβ receptor type II extracellular region), Linker (linker), μPD-1, CD27 transmembrane region and cytoplasmic region from N-terminus to C-terminus. Furthermore, the coding genes of the transmembrane region and cytoplasmic region of CD27 can be oCD27 obtained by codon optimization, which is more suitable for human cell expression when the encoded amino acid sequence remains unchanged. The nucleotide sequence of the coding gene of the chimeric switch receptor can be 2662-3882 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) can be SEQ ID No.1.

[0123] Among the coding genes of the chimeric switch receptor, the nucleotide sequence of the coding gene of the TRII extracellular region (TGFβ receptor type II extracellular region) may be positions 2662-3159 of SEQ ID No.3, the nucleotide sequence of the coding gene of the Linker (connector) may be positions 3160-3219 of SEQ ID No.3, the nucleotide sequence of the coding gene of μPD-1 may be positions 3220-3657 of SEQ ID No.3, and the nucleotide sequence of the coding gene of the transmembrane region and cytoplasmic region of CD27 (i.e., oCD27) may be positions 3658-3882 of SEQ ID No.3.

[0124] The chimeric antigen receptor described herein can specifically kill GPC3 + Tumor cells. GPC3, the full name of which is Glypican-3, is a member of the heparan sulfate proteoglycan family. It is connected to the cell surface through the glycosylphosphatidylinositol anchor on the cell membrane. It is highly expressed in most liver cancer tissues and can be used to target liver cancer cells to initiate tumor killing. It is an ideal target for tumor treatment.

[0125] The IFNα (α interferon) described in this article has a broad spectrum of anti-tumor effects, directly regulates the proliferation, apoptosis and migration of tumor cells; inhibits the angiogenesis and metastasis of tumors; has immunomodulatory functions, and can enhance anti-tumor immune activity in the following ways: upregulating the expression of MHC I, tumor antigens and PD-L1 in tumor cells; activating innate immune cells such as NK, DC and γδT cells, especially the expansion, migration and function of NK cells; regulating adaptive immune cells such as T cells and B cells, especially the expansion, migration, function and survival of T cells; negatively regulating immunosuppressive cells such as TAM, Treg and myeloid suppressor cells MDSCs; upregulating the expression of PD-L1 in tumor cells.

[0126] The chimeric switch receptor described in this article can inhibit the PD-1 and TGFβ signaling pathways to achieve the effect of enhancing the CD27 signaling pathway, giving CAR-T cells the ability to resist the immunosuppressive tumor microenvironment and enabling them to have stronger in vivo persistence; since tumor cells usually express PD-L1 on their surface to activate the inhibitory PD-1 on T cells to circumvent / inhibit anti-tumor T cell responses, the chimeric switch receptor converts the above-mentioned inhibitory effect into a CD27-mediated activation signal, and at the same time, PD-1 targeting is beneficial for CAR-T cells to aggregate to tumor sites expressing PD-L1.

[0127] Although the present invention constructs a GPC3 CAR-IC gene with a nucleotide sequence of SEQ ID No. 3, the present invention is not limited to this specific sequence. Those skilled in the art may replace the P2A gene and / or the promoter element gene in the GPC3 CAR-IC gene, for example, using F2A (VKQTLNFDLLKLAGCVESNPG), T2A (EGRGSLLTCGDVEENPG), E2A (QCTNYALLKLAGDVESNPG) and the like having the same "self-splicing" function as the P2A gene, but not limited thereto. The promoter element gene may use a promoter known in the art. As long as the constructed nucleic acid molecule can express a chimeric antigen receptor with an amino acid sequence of SEQ ID No. 4, an IFNα with an amino acid sequence of SEQ ID No. 5, and a fusion protein (chimeric switch receptor) with an amino acid sequence of SEQ ID No. 1, and has the same function as the nucleic acid molecule described in the present invention, they are all regarded as nucleic acid molecules equivalent to the nucleic acid molecule of the present invention, and these equivalent nucleic acid molecules do not depart from the protection scope of the present invention.

[0128] After extensive and in-depth research, the inventors of this application designed a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene, and designed and constructed a GPC3 CAR-IC gene (SEQ ID No. 3) targeting GPC3, which was packaged and infected by retrovirus to obtain GPC3 CAR-IC T cells. The GPC3 CAR-IC T cells constructed by the present invention express two artificial receptors, one of which is a chimeric antigen receptor (CAR), which is used to recognize tumor-associated antigens and specifically kill GPC3. + Tumor cells, targeting liver cancer cells to initiate tumor killing. The other is a chimeric switch receptor (CSR), which is used to convert immunosuppressive signals in the tumor microenvironment into activation signals in CAR-T cells, in which the TRII extracellular region can bind to TGFβ secreted in large quantities in HCC, block its inhibitory pathway, and activate the CD27 signaling pathway in CAR-T cells, providing growth stimulation for CAR-T cells, enhancing the survival and migration of CAR-T cells, and inhibiting the tumor microenvironment; μPD-1 in CSR can also bind to PD-L1 expressed in HCC, blocking the PD-1 signaling pathway on T cells, inhibiting T cell exhaustion, and activating the CD27 signaling pathway in CAR-T cells to enhance the tumor killing effect. In addition, the GPC3 CAR-IC T cells constructed by the present invention also express IFNα, which is used for broad-spectrum anti-tumor, reducing antigen heterogeneity and tumor cell escape, enhancing immune cell infiltration, immunomodulation, improving tumor microenvironment, and resisting HBV / HCV infection.

[0129] The experimental results show that, compared with the control cells that do not express IC genes (IFNα gene + promoter element gene + chimeric switch receptor encoding gene), the GPC3 CAR-IC T cells constructed by the present invention have higher IFN-γ secretion levels, can secrete high levels of IFN-α2 under antigen stimulation, have stronger specific cytotoxicity, and can kill GPC3 + The target cells have a stronger effect, and the IFN-α2 secreted by them can induce NK cells to secrete the functional effector molecule IFN-γ. The results of the in vivo tumor killing experiment of the HepG2 subcutaneous tumor-bearing NSG mouse model showed that after administration (GPC3-IC CAR-T cells), the secretion of IFN-α2 and IFN-γ in the mouse serum increased significantly, indicating that the GPC3 CAR-IC T cell tumor inhibition effect of the present invention is faster and better, and has stronger in vivo tumor killing activity.

[0130] The present invention constructs CAR-T cells by designing a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene, converting the inhibitory anti-tumor T cell response activated by PD-L1 and TGFβ into a CD27-mediated activation signal, giving CAR-T cells the ability to resist the immunosuppressive tumor microenvironment, and making them more persistent in vivo, promoting the expansion of CAR-T cells, prolonging the survival time in vivo, and promoting cytokine secretion, and achieving the microenvironment near the tumor by promoting the secretion of cytokines IFN-γ and IFN-α2, relieving its immunosuppression, and participating in the killing of tumor cells by mobilizing the body's own immunity. The anti-tumor effect of CAR-T cells is improved, and the CAR-T treatment of tumors (such as hepatocellular carcinoma) has important significance and wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 Schematic diagram of the GPC3 CAR-IC gene structure.

[0132] Figure 2 This is the CAR expression detection result in Example 2.

[0133] Figure 3 The CAR-IC cells in Example 3 secrete IFN-γ ( Figure 3 A) and IFN-α2 ( Figure 3 (B) Cell function test results.

[0134] Figure 4 This is the result of the cytotoxic (degranulation CD107a) function test of the CAR-IC cells in Example 4.

[0135] Figure 5 This is the CAR-T cell cytotoxicity test result in Example 5. Figure 5A in the middle is the killing effect on the target cell HepG2. Figure 5 B in the middle shows the killing effect on the negative control cells U87 MG.

[0136] Figure 6 This is the in vitro detection of the induction effect of IFN-α2 secreted by GPC3 CAR-IC T cells co-cultured with target cells on NK cells in Example 6.

[0137] Figure 7 The in vivo tumoricidal effect and animal survival of the transplanted tumor HepG2-NSG model in Example 7 were evaluated. DETAILED DESCRIPTION

[0138] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0139] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0140] The pUC57 vector in the following examples is a product of Beijing Qingke Biotechnology Co., Ltd.

[0141] The retroviral vector MP71 in the following examples is described in the following document: 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 the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0142] The human peripheral blood mononuclear cells (PBMC) in the following examples were derived from the venous blood of healthy volunteers.

[0143] The HepG2 cells in the following examples are products of the cell bank of the Chinese Academy of Sciences, with the catalog number SCSP-510.

[0144] The U87 MG cells in the following examples are products of the cell bank of the Chinese Academy of Sciences, with the catalog number being TCHu138.

[0145] The NSG mice (6-7 weeks old, weighing 18-25 g) in the following examples were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0146] The RPMI-1640 culture medium in the following examples is a product of Sigma, with the product number R8758.

[0147] In the following examples, the RPMI-1640 medium containing 10% fetal bovine serum (FBS) (10% FBS RPMI-1640 medium) is prepared with RPMI-1640 medium (R8758) as solvent and fetal bovine serum as solute, and is also called complete medium.

[0148] Example 1. Construction of CAR-IC cells

[0149] The CAR-IC cells constructed in this embodiment target GPC3 and express two artificial receptors, one is a chimeric antigen receptor (CAR) for the recognition of tumor-associated antigens, and the other is a chimeric switch receptor (CSR) for converting immunosuppressive signals in the tumor microenvironment into activation signals in CAR-T cells, thereby providing growth stimulation for CAR-T cells, enhancing the survival and migration of CAR-T cells, and inhibiting the tumor microenvironment. The specific construction method is as follows:

[0150] 1. GPC3 CAR-IC gene design

[0151] The GPC3 CAR-IC gene is composed of the chimeric antigen receptor coding gene, P2A gene, IFNα gene, promoter element gene, and chimeric switch receptor coding gene from N-terminus to C-terminus. Figure 1 As shown (the IFNα gene, the promoter element gene, and the coding gene of the chimeric switch receptor are collectively referred to as the IC gene). Among them:

[0152] The nucleotide sequence of the IC gene (IFNα gene+promoter element gene+chimeric switch receptor encoding gene) is positions 1552-3882 of SEQ ID No.3.

[0153] The coding genes of the chimeric antigen receptor are 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) from N-terminal to C-terminal. The nucleotide sequence of the coding gene of the chimeric antigen receptor is 1-1473 of SEQ ID No.3, and the amino acid sequence encoding the chimeric antigen receptor (CAR) is SEQ ID No.4.

[0154] Among the genes encoding the chimeric antigen receptor, the nucleotide sequence of the gene encoding Signal (signal peptide) is positions 1-63 of SEQ ID No. 3, the nucleotide sequence of the gene encoding GPC3 scFv is positions 64-789 of SEQ ID No. 3, the nucleotide sequence of the gene encoding Hinge (hinge region) + CD8 (transmembrane region) is positions 790-996 of SEQ ID No. 3, the nucleotide sequence of the gene encoding 4-1BB (intracellular signal region) is positions 997-1137 of SEQ ID No. 3, and the nucleotide sequence of the gene encoding CD3ζ (intracellular signal region) is positions 1138-1473 of SEQ ID No. 3.

[0155] The nucleotide sequence of the P2A gene is 1474-1551 of SEQ ID No. 3. The P2A peptide it encodes is a short peptide derived from a virus, commonly known as a "self-cleaving" peptide. The "self-cleavage" function of the P2A peptide can produce multiple proteins from one transcription product, that is, two proteins, an upstream product and a downstream product, are formed through self-cleavage.

[0156] The nucleotide sequence of the IFNα gene is positions 1552-2118 of SEQ ID No.3, and the amino acid sequence of the IFNα (interferon α) encoded by it is SEQ ID No.5.

[0157] The nucleotide sequence of the promoter element gene is positions 2119-2661 of SEQ ID No. 3, wherein positions 2136-2635 of SEQ ID No. 3 are the promoter.

[0158] The coding genes of the chimeric switch receptor are, from N-terminus to C-terminus, the coding genes of the TRII extracellular region (TGFβ receptor type II extracellular region), Linker, μPD-1, CD27 transmembrane region and cytoplasmic region. Furthermore, the coding genes of the CD27 transmembrane region and cytoplasmic region are oCD27 obtained by codon optimization, which is more suitable for human cell expression when the encoded amino acid sequence remains unchanged. μPD-1 is the extracellular region of mutation-optimized PD-1. The nucleotide sequence of the coding gene of the chimeric switch receptor is 2662-3882 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.

[0159] Among the coding genes of the chimeric switch receptor, the nucleotide sequence of the coding gene of the TRII extracellular region (TGFβ receptor type II extracellular region) is positions 2662-3159 of SEQ ID No.3, the nucleotide sequence of the coding gene of the Linker is positions 3160-3219 of SEQ ID No.3, the nucleotide sequence of the coding gene of μPD-1 is positions 3220-3657 of SEQ ID No.3, and the nucleotide sequence of the coding gene of the CD27 transmembrane region and cytoplasmic region (i.e., oCD27) is positions 3658-3882 of SEQ ID No.3.

[0160] Chimeric antigen receptor can specifically kill GPC3 + Tumor cells. GPC3, the full name of which is Glypican-3, is a member of the heparan sulfate proteoglycan family. It is connected to the cell surface through the glycosylphosphatidylinositol anchor on the cell membrane. It is highly expressed in most liver cancer tissues and can be used to target liver cancer cells to initiate tumor killing. It is an ideal target for tumor treatment.

[0161] IFNα (α-interferon) has a broad spectrum of anti-tumor effects, directly regulates the proliferation, apoptosis and migration of tumor cells; inhibits the formation and metastasis of tumor blood vessels; has immunomodulatory functions, and can enhance anti-tumor immune activity in the following ways: upregulating the expression of MHC I, tumor antigens and PD-L1 in tumor cells; activating innate immune cells such as NK, DC and γδT cells, especially the expansion, migration and function of NK cells; regulating adaptive immune cells such as T cells and B cells, especially the expansion, migration, function and survival of T cells; negatively regulating immunosuppressive cells such as TAM, Treg and myeloid suppressor cells MDSCs; upregulating the expression of PD-L1 in tumor cells.

[0162] The chimeric switch receptor is composed of a TRII-type extracellular region, μPD-1 and CD27 transmembrane regions and cytoplasmic regions. It can inhibit the PD-1 and TGFβ signaling pathways to achieve the effect of enhancing the CD27 signaling pathway, giving CAR-T cells the ability to resist the immunosuppressive tumor microenvironment and making them more persistent in vivo. Since tumor cells usually express PD-L1 on their surface to activate the inhibitory PD-1 on T cells to evade / inhibit anti-tumor T cell responses, the chimeric switch receptor converts the above-mentioned inhibitory effect into a CD27-mediated activation signal, and at the same time, PD-1 targeting is beneficial for CAR-T cells to aggregate to tumor sites expressing PD-L1.

[0163] 2. GPC3 CAR-IC gene synthesis

[0164] The full-length sequence of the gene encoding the transmembrane region and cytoplasmic region of wild-type human CD27 is called nCD27, and oCD27 is obtained after codon optimization to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence. The nucleotide sequence of the gene encoding oCD27 is shown in positions 3658-3882 of SEQ ID No.3.

[0165] According to the design in step 1 (see Figure 1 ) The GPC3 CAR-IC gene was constructed and synthesized by Qingke Biotechnology Co., Ltd., cloned into the pUC57 vector to obtain the recombinant vector pUC57-GPC3 CAR-IC, and sequenced.

[0166] The nucleotide sequence of the GPC3 CAR-IC gene is shown in SEQ ID No. 3, and it expresses a chimeric antigen receptor with an amino acid sequence of SEQ ID No. 4, an IFNα with an amino acid sequence of SEQ ID No. 5, and a fusion protein (chimeric switch receptor) with an amino acid sequence of SEQ ID No. 1.

[0167] 3. CAR-IC cell construction

[0168] The GPC3 CAR-IC gene constructed in step 2 is transferred into T cells for stable expression, and T cells expressing the GPC3 CAR-IC gene (SEQ ID No. 3) are obtained, which are named GPC3 CAR-IC T cells. The specific steps are as follows:

[0169] 3-1. Construction of recombinant retroviral vector

[0170] (1) The recombinant vector pUC57-GPC3 CAR-IC was double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragment was recovered by gel excision.

[0171] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel excision.

[0172] (3) Use T4 ligase (NEB) to connect the above-mentioned target gene fragment and the large vector fragment to obtain the recombinant retroviral vector MP71-GPC3 CAR-IC carrying the GPC3CAR-IC gene.

[0173] (4) The recombinant retroviral vector MP71-GPC3 CAR-IC was transformed into competent Escherichia coli DH5α, and the plasmid was extracted and purified using the plasmid purification kit of Qiagen Company to obtain the MP71-GPC3 CAR-IC plasmid.

[0174] (5) According to the above method, MP71-GPC3 CAR plasmid without IC gene was constructed as a control.

[0175] 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 list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0176] 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 (coding gene for the chimeric antigen receptor) having a nucleotide sequence shown at positions 1-1473 of SEQ ID No. 3 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0177] 3-2. Retroviral packaging

[0178] The MP71-GPC3 CAR-IC and MP71-GPC3 CAR plasmids prepared in step 3-1 are respectively introduced into packaging cells for packaging to complete virus assembly and obtain retrovirus. The specific steps of virus packaging are as follows:

[0179] a) Day 1: Phoenix Ecotropic (ECO) cells should be less than 20 generations and not overgrown. 6 / ml cell density, add 10mL DMEM medium to a 10cm dish, mix the cells thoroughly, and culture at 37℃ overnight;

[0180] b) Day 2: When the confluency of ECO cells reaches about 90%, transfection is performed (usually about 14-18 hours after plating); prepare 12.5μg of plasmid, 250μL of 1.25M CaCl2, 1mL of H2O, and a total volume of 1.25mL; add 2×HBS equal to the volume of plasmid complex to another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture along the edge to the ECO dish, culture at 37℃ for 4 hours, remove the culture medium, wash once with PBS, and re-add preheated fresh culture medium;

[0181] c) Day 4: 48 hours after transfection, collect the supernatant, filter with a 0.45 μm filter to obtain the retroviral solution, and store it in aliquots at -80°C;

[0182] d) Add 1.2 mL of 15 ug / mL Retronectin coating solution to each well of a 6-well NTC plate and incubate at 4°C overnight;

[0183] e) Carefully remove the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 32°C and 2000×g for 2 h, and discard the unbound virus supernatant;

[0184] f) Take logarithmic phase PG13 cells and rinse them once with 10 mL PBS, add 1 mL 0.25% recombinant trypsin, and let stand at room temperature for 2-3 minutes;

[0185] g) Add 5 ml of complete medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min;

[0186] h) Discard the supernatant and adjust the cell density to 0.5×10 5 cell / mL, and add 3 mL / well to the NTC 6-well plate coated with the above virus to make the final cell number 1.5×10 5 cell / well;

[0187] i) centrifugation at 1000 rpm for 1 min and incubation at 37°C, 5% CO2 for 48 h;

[0188] j) After 1-2 generations of subculture, the cells were transferred to T175 culture flasks and cultured in DEME medium containing 12% FBS for 2 days;

[0189] k) Replace with new DEME medium containing 12% FBS and continue culturing for 48 hours, then collect the supernatant, filter with a 0.45 μm filter to obtain a retrovirus solution, and store it in aliquots at -80°C.

[0190] 3-3. Retrovirus infection of human T cells

[0191] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1×10 in RPMI-1640 medium containing 10% fetal bovine serum (FBS). 6 -2×10 6 cells / mL.

[0192] b) PBMCs were collected using Ficoll separation medium (Tianjin Haoyang), and CD3 + T cells, by magnetic beads: CD3 + Clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added to activate T cells at a ratio of 3:1.

[0193] c) On the second day after T cell activation, RetroNectin (TAKARA) diluted with PBS to a final concentration of 15 μg / mL was coated on non-tissue-treated culture plates, 1.2 mL per well of a 6-well plate. Protect from light and store at 4°C overnight for use.

[0194] d) After two days of T cell activation culture, the coated 6-well plate was taken out, the coating solution was discarded, and PBS was added to wash the plate once.

[0195] e) Add 5-6 mL of the retrovirus solution prepared in step 3-2 to each well, centrifuge at 32°C, 2000×g for 2 h. Add 3 mL of fresh complete medium containing hIL-2 (500 U / mL) to each well, and continue culturing for 1 day.

[0196] f) After cell infection, the cell density was observed every day, and T cell culture medium containing 100 U / mL IL-2 was added in time to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0197] g) CAR-IC cells and control CAR T cells infected with the retrovirus prepared in step 3-2 are obtained, and are named GPC3 CAR-IC T cells (i.e., T cells expressing the GPC3 CAR-IC gene with a nucleotide sequence of SEQ ID No. 3) and GPC3 CAR T cells as a control (i.e., T cells expressing the DNA molecule shown in positions 1-1473 of SEQ ID No. 1).

[0198] Example 2. Detection of CAR expression in CAR-IC cells

[0199] 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 without virus transfection, as a control) were cultured at 37° C. in RPMI-1640 medium containing 10% fetal bovine serum (FBS), and recorded as D0 day, and CAR expression was detected until D8 day.

[0200] 1. GPC3 CAR expression detection:

[0201] (1) After centrifugation (1500 rpm × 5 min), the supernatant was discarded, and 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) was added to each well of a 96-well round-bottom plate to resuspend the cells and centrifuge at 1500 rpm for 5 min;

[0202] (2) Add 60 μL of the prepared fluorescent-labeled antibody and fluorescent-labeled recombinant protein premix to each well (see Table 1 for the preparation of the premix, where the fluorescent-labeled antibody is a fluorescent-labeled anti-human CD3 / CD4 / CD8 antibody, and the fluorescent-labeled recombinant protein (rp) is a FITC-labeled GPC3 recombinant protein), resuspend and mix, and incubate at 4°C for 30 min;

[0203] Table 1. Preparation of premix

[0204] Markers Fluorescent labeling Part Number brand Dilution Dosage (μL) CD3 PE 344806 Biolegend 1:100 0.6 CD4 APCCy7 317450 Biolegend 1:200 0.3 CD8a PY7 301012 Biolegend 1:200 0.3 GPC3 recombinant protein FITC GP3-HF258 Acrobiosystems 1:50 1.2

[0205] (3) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0206] (4) Discard the supernatant, resuspend the cells in 400 μL FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of GPC3 CAR in T cells.

[0207] 2. Interferon α2 (IFNα2) expression detection:

[0208] (1) After centrifugation (1500 rpm × 5 min), the supernatant was discarded, and 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) was added to each well of a 96-well round-bottom plate to resuspend the cells and centrifuge at 1500 rpm for 5 min;

[0209] (2) Add 60 μL of the prepared fluorescent-labeled antibody and fluorescent-labeled recombinant protein premix to each well (see Table 1 for the preparation of the premix, where the fluorescent-labeled antibody is a fluorescent-labeled anti-human CD3 / CD4 / CD8 antibody, and the fluorescent-labeled recombinant protein (rp) is a FITC-labeled GPC3 recombinant protein), resuspend and mix, and incubate at 4°C for 30 min;

[0210] (3) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0211] (4) Discard the supernatant and add 150 μL Cytofix / Cytoperm (BD, Catalog No. 55472) to each well to resuspend and mix, and incubate at room temperature for 15 min in the dark.

[0212] (5) After centrifugation at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Catalog No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0213] (6) Add 20 μL of prepared anti-human IFN-α2 to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0214] (7) Add 200 μL of 0.5% BSA to each well, centrifuge at 1500 rpm for 5 min, and wash twice;

[0215] (8) Add 20 μL of prepared SA-APC to each well, resuspend and mix, and incubate at 4°C for 10 min;

[0216] (9) Add 200 μL of 0.5% BSA to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL FACS buffer and transfer them to a flow cytometer (BD Canto-II) to read the cells and analyze the percentage of IFNα2 in T cells.

[0217] 3. μPD-1 / TRⅡ expression detection:

[0218] (1) After centrifugation (1500 rpm × 5 min), the supernatant was discarded, and 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) was added to each well of a 96-well round-bottom plate to resuspend the cells and centrifuge at 1500 rpm for 5 min;

[0219] (2) Add 60 μL of the prepared fluorescent-labeled antibody and fluorescent-labeled recombinant protein premix to each well (see Table 1 for the preparation of the premix, where the fluorescent-labeled antibody is a fluorescent-labeled anti-human CD3 / CD4 / CD8 antibody, and the fluorescent-labeled recombinant protein (rp) is a FITC-labeled GPC3 recombinant protein), suspend and mix well, and incubate at 4°C for 30 min;

[0220] (3) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0221] (4) Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer them to a flow cytometer, read the cells using a flow cytometer (BD Canto-II), and analyze the percentage of μPD-1 / TRⅡ in T cells.

[0222] Test results such as Figure 2 As shown, CAR (i.e., GPC3 CAR), IFNα2, μPD-1, and TRⅡ are simultaneously expressed in CAR-IC cells (i.e., the GPC3 CAR-IC T cells prepared in Example 1), indicating that the expression of CAR molecules in CAR-IC cells meets the expected design.

[0223] Example 3. Cell function detection of CAR-IC cells secreting IFN-γ and IFN-α2

[0224] The test cells were GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (ie, T cells without virus transfection).

[0225] The above test cells were cultured until D10, and 1×10 6 The cells were added into a 24-well TC plate, and HepG2 cells were added at a ratio of test cells: target cells = 1:1, and co-cultured in a 37°C incubator, and recorded as D0 day.

[0226] The test groups and controls are as follows:

[0227] CTR T+Medium: 1 mL per well, total 1×10 6 Add 1 mL of Medium (10% FBS RPMI-1640 medium) to each cell of virus-free T cells (CTR T cells);

[0228] CTR T+HepG2: 1 mL per well, 1×10 6 Add 1 mL of 1×10 T cells without virus transfection (CTR T cells) 6 HepG2 cells;

[0229] GPC3 CAR T+Medium: 1 mL per well, 1×10 6 Add 1 mL of Medium (10% FBS RPMI-1640 medium) to each conventional GPC3-targeted CAR T cell (GPC3 CAR T cell);

[0230] GPC3 CAR T+HepG2: 1 mL per well, 1×10 61×10 CAR T cells targeting GPC3 were added to 1 mL of conventional GPC3 CAR T cells (GPC3 CAR T cells). 6 HepG2 cells;

[0231] GPC3 CAR-IC T+Medium: 1 mL per well, 1×10 6 Add 1 mL of Medium (10% FBS RPMI-1640 medium) to each GPC3-targeting CAR-IC T cell (GPC3CAR-IC T cell);

[0232] GPC3 CAR-IC T+HepG2: 1 mL per well, 1×10 6 1×10 CAR-IC T cells targeting GPC3 (GPC3 CAR-IC T cells) were added in 1 mL. 6 HepG2 cells.

[0233] The co-cultured cells were collected for three consecutive days (D1, D2, and D3) to detect the secreted IFN-γ and IFN-α2.

[0234] The specific detection steps are as follows:

[0235] 1. The co-cultured cells on D1, D2, and D3 were cultured as above and the cell density was adjusted to 2×10 6 / mL, take 100 μL and add it to 96-well U-bottom plate, add Brefeldin A (Med Chem Express, HY-16592) with a final concentration of 5 μg / mL to each well, and incubate in a 37°C incubator for 5 to 6 hours.

[0236] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0237] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0238] (2) Add 60 μL of prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD4 / CD8 / GPC3(rp)) to each well, resuspend and mix, and incubate at room temperature for 10 min in a dark environment;

[0239] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0240] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Cat. No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in a dark place.

[0241] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Catalog No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0242] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Catalog No. 506510) or anti-human IFN-α2 (Biolegend, Catalog No. 537204) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0243] (7) Add 200 μL of Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells with 400 μL of FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of functional effector molecules IFN-γ and IFN-α2 in GPC3 CAR-IC T cells and control cells.

[0244] Test results such as Figure 3 As shown in the figure, after co-culture with target cells (HepG2 cells), both GPC3 CAR T cells and GPC3CAR-IC T cells can specifically secrete the T cell effector molecule IFN-γ; compared with GPC3 CAR T cells, GPC3 CAR-IC T cells secrete higher levels of IFN-γ. In addition, GPC3 CAR-IC T cells can secrete low levels of IFN-α2 without antigen stimulation; under antigen stimulation, GPC3 CAR-IC T cells can secrete high levels of IFN-α2.

[0245] Example 4. Detection of cytotoxicity (degranulation CD107a) function of CAR-IC cells

[0246] The degranulation ability of CAR-IC cells, i.e., the expression of CD107a, was detected. The test cells were GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells without virus transfection).

[0247] 1) The above test cells were cultured until D10 and the cell density was adjusted to 2×10 6 / mL, take 100μL and add it into a 96-well U-bottom plate;

[0248] 2) Add 100 μL of 2×10 5 HepG2 cells were used as CAR antigen-specific stimulation (+HepG2) or negative control U87 MG cells (+U87 MG), and 100 μL Medium (10% FBS RPMI-1640 medium) was added to each well as a negative control (+Medium);

[0249] 3) Add 1 μL of APC-labeled anti-human CD107a antibody (Biolegend, Cat. No. 328620) to each well and incubate at 37°C for 1 h;

[0250] 4) Add 10 μL of 1:50 diluted Monensin Solution (Invitrogen, Cat. No. 00-4505-51) to each well and continue incubation for 3 h;

[0251] 5) After incubation, flow cytometry staining is performed. The steps are as follows:

[0252] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and repeat this step twice;

[0253] (2) Add 60 μL of prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD4 / CD8 / GPC3(rp)) to each well, resuspend and mix, and incubate at room temperature for 10 min in a dark environment;

[0254] (3) Centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) and analyze the percentage of cell degranulation molecule CD107a in T cells.

[0255] Test results such as Figure 4 As shown in the figure, after co-culture with target cells, both GPC3 CAR T cells and GPC3 CAR-IC T cells can specifically upregulate CD107a degranulation. In addition, compared with GPC3 CAR-T cells, GPC3 CAR-IC T cells have stronger specific cytotoxic effect (CD107a).

[0256] Example 5. Detection of CAR-T cell cytotoxicity

[0257] The test cells were GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (ie, T cells without virus transfection).

[0258] 1) The above test cells were cultured until D10 and the cell density was adjusted to 2×10 5 / mL, take 100 μL and add it to a 96-well U-bottom plate; add D-firefly luciferin sodium salt (Yeasen Biotechnology, product number 40901ES08, storage concentration is 100 mg / mL) at a final concentration of 100 μg / mL, and repeat for 3 wells;

[0259] 2) Target HepG2 cells or negative control U87 MG cells were added at different effector-target ratios (1:1, 1:3, 1:9, 1:27) (the cell density of target cells under 1:1 condition was 2×10 4 cells) and cultured at 37°C for 16 h.

[0260] 3) Measure the fluorescence value with TECAN spark microplate reader, and take the average value of three replicate wells to calculate the specific killing activity (Cytotoxicity) of CAR-T cells:

[0261] Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0262] Eexp: RLU value when effector cells and target cells are co-cultured;

[0263] Emin: RLU value of spontaneous death of effector cells in the absence of cells;

[0264] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0265] Tmin: RLU value under the condition of maximum killing rate.

[0266] Test results such as Figure 5 As shown in Figure 2, both GPC3 CAR T cells and GPC3 CAR-IC T cells can specifically kill GPC3 + Target cells (HepG2 cells), GPC3 - The target cells (U87 MG cells) had no killing effect; the control CTR T cells had no killing effect on GPC3 + Target cells and GPC3 - In addition, compared with GPC3 CAR-T cells, GPC3 CAR-IC T cells killed GPC3 +The effect on target cells (HepG2 cells) was stronger.

[0267] Example 6: In vitro detection of the induction effect of IFN-α2 secreted by GPC3 CAR-IC T cells co-cultured with target cells on NK cells

[0268] The test cells are the GPC3 CAR-IC T cells and GPC3 CAR T cells prepared in Example 1, and CTR T cells (i.e., T cells without virus transfection). The detection steps are as follows:

[0269] 1) The above test cells were cultured until D10 and the cell density was adjusted to 2×10 6 / mL, take 1mL and add it into a 96-well U-bottom plate;

[0270] 2) Add 1 mL of 2 × 10 6 HepG2 cells were co-cultured for 3 days;

[0271] 3) Collect the supernatant by centrifugation and incubate with PBMC cells, where PBMC: 2×10 5 / well, 0.1mL / well; supernatant: 0.1mL / well;

[0272] 4) After incubation overnight, add Brefeldin A (Med Chem Express, HY-16592) to each well to a working concentration of 5 μg / mL and incubate at 37°C for 4 h;

[0273] 5) After incubation, flow cytometry staining is performed. The steps are as follows:

[0274] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant, add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well, resuspend, and centrifuge at 1500 rpm for 5 min;

[0275] (2) Add 40 μL of prepared fluorescent antibody (fluorescently labeled anti-human CD3 / CD56) to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark;

[0276] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0277] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Catalog No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in a dark place.

[0278] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Catalog No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0279] (6) Add 40 μL of prepared fluorescent antibody (fluorescently labeled anti-human IFNγ / Granzyme B) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0280] (7) Add 200 μL of Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL of FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of effector molecules IFN-γ and Granzyme B in NK cells.

[0281] Test results such as Figure 6 As shown, IFN-α2 secreted by GPC3 CAR-IC T cells can induce NK cells to secrete the functional effector molecule IFN-γ.

[0282] Example 7: Evaluation of the in vivo tumoricidal effect of GPC3 CAR-IC T cells and animal survival in the transplanted tumor HepG2-NSG model

[0283] The HepG2 subcutaneous tumor-bearing NSG mouse model (HepG2-NSG model) was used to evaluate the in vivo tumoricidal effect of GPC3 CAR-IC T cells and animal survival. The specific steps are as follows:

[0284] 1) NSG mice (6-7 weeks old) were subcutaneously inoculated with HepG2 cells at 1×10 7 Cells / piece.

[0285] 2) On day 12, the mouse tumor grew to 80-100 mm 3 GPC3 CAR-IC T cells were then injected, which was recorded as D0. During this period, the tumor-bearing mice were randomly divided into 3 groups, 5 mice / group, and the groups were as follows:

[0286] Control group (CTR T): injected with CTR T cells, 3×10 6 Cells / piece;

[0287] GPC3 CAR T group: injected with GPC3 CAR-T cells, 3×10 6 Cells / piece;

[0288] GPC3 CAR-IC T group: injected with GPC3-IC CAR-T cells, 3×106 Cells / piece.

[0289] 3) The tumor size was measured with a vernier caliper on D7, D14, D21, D28, and D35 after administration.

[0290] 4) Blood was collected from the submandibular area of ​​the mice 1 day before administration (D-1) and on D4, D7, D12, D21, and D28 after administration, and the serum multiple cytokine method was used to detect the IFN-γ and IFN-α2 secreted by GPC3 CAR-IC T cells and control cells in the mice.

[0291] Test results such as Figure 7 As shown, compared with GPC3 CAR-T cells, the tumor suppression effect of GPC3 CAR-IC T cells on D7 / D14 was faster and better. There was a high level of IFN-α2 secretion in the serum of mice adoptively transferred with GPC3 CAR-IC T cells. Compared with the adoptively transferred GPC3 CAR-T mice, there was a higher level of IFN-γ secretion in the serum of GPC3 CAR-IC T mice. This shows that GPC3 CAR-IC T cells have stronger tumoricidal activity in vivo.

[0292] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A nucleic acid molecule, characterized in that The nucleic acid molecule is composed of a chimeric antigen receptor coding gene, a P2A gene, an IFNα gene, a promoter element gene and a chimeric switch receptor coding gene from N-terminus to C-terminus, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.4, the amino acid sequence of the IFNα is shown in SEQ ID No.5, and the amino acid sequence of the chimeric switch receptor is shown in SEQ ID No.

1.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleotide sequence of the P2A gene is shown in positions 1474-1551 of SEQ ID No. 3, and the nucleotide sequence of the promoter element gene is shown in positions 2119-2661 of SEQ ID No.

3.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.

3.

4. Biomaterial, characterized in that The biological material is any of the following: E1) An expression cassette containing the nucleic acid molecule according to any one of claims 1 to 3; E2) a recombinant vector containing the nucleic acid molecule according to any one of claims 1 to 3, or a recombinant vector containing the expression cassette according to E1); E3) a recombinant microorganism containing the nucleic acid molecule according to any one of claims 1 to 3, or a recombinant microorganism containing the expression cassette according to E1), or a recombinant microorganism containing the recombinant vector according to E2); E4) A recombinant cell containing the nucleic acid molecule according to any one of claims 1 to 3, or a recombinant cell containing the expression cassette according to E1), or a recombinant cell containing the recombinant vector according to E2).

5. The biomaterial according to claim 4, characterized in that The cells in E4) are T cells, NK cells, macrophages or stem cells.

6. The biomaterial according to claim 4, characterized in that E4) The cells are γδT cells or NKT cells.

7. Any of the following uses of the nucleic acid molecule according to any one of claims 1 to 3, and / or the biomaterial according to any one of claims 4 to 6: F1) Use in the preparation of a drug for preventing or treating liver cancer; F2) Application in the preparation of products for regulating the immunosuppressive effect of liver cancer microenvironment.

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