Irradiated CAR-T cell and its preparation method and application

Abstract

The present invention discloses an irradiated CAR-T cell and a preparation method and application thereof. Specifically disclosed is an irradiated GPC3CAR-IC cell, which can be prepared according to a method comprising the following steps: irradiating the GPC3CAR-IC cell with ionizing radiation to obtain an irradiated GPC3CAR-IC cell, which contains a nucleic acid molecule shown in SEQ ID No.3. The irradiated GPC3CAR-IC cell of the present invention achieves the immunosuppressive effect of suppressing the tumor microenvironment by modifying the CAR gene at the gene level, improves the anti-tumor effect of the GPC3CAR-IC cell, and the irradiated GPC3CAR-IC cell of the present invention can reduce the risk of GVHD without gene editing, has the characteristics of safety, generality and low cost, can realize large-scale industrial production, and has a wide range of clinical application value for CAR-T treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of cell immunotherapy, and specifically relates to an irradiated CAR-T cell and a preparation method and application thereof. Background Art

[0002] Adoptive cell therapy (ACT) refers to a therapy that directly or indirectly kills tumor cells by infusing autologous or allogeneic specific or nonspecific immune cells that have been expanded in vitro into the body. Adoptive cell immunotherapy includes lymphokine activated killer cell (LAK cell) immunotherapy, tumor infiltrating lymphocyte (TIL) immunotherapy, cytokine induced killer cell (CIK cell) immunotherapy, cytotoxic T cell (CTL) immunotherapy, T cell receptor gene engineered T cell (TCR-T) immunotherapy and chimeric antigen receptor T cell (CAR-T) immunotherapy. Studies have shown that CAR-T cells are specific, efficient and sustained in attacking tumors, especially in the treatment of hematological tumors. In recent years, CAR-T cell therapy has achieved encouraging results in blood system tumors such as B cells, but the difficulty in industrializing this therapy remains a bottleneck hindering its development.

[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] At present, conventional autologous CAR-T or allogeneic CAR-T treatment still faces the following problems: (1) Therapeutic window and cell preparation: The therapeutic window and cell production time of conventional autologous CAR-T are relatively long, which makes it not suitable for the treatment of rapidly progressing invasive tumors; the cell source of autologous CAR-T is limited by the number of T cells in the patient's own immune system and their killing ability, resulting in high and low treatment effects; each batch of autologous CAR-T cells is produced in a small batch size and is only suitable for use in one patient, which is not easy to mass produce; autologous CAR-T cells need to be produced according to the specific situation of each patient, the process is complicated, and it is not easy to unify quality control. (2) Safety issues: During conventional autologous CAR-T or allogeneic CAR-T treatment, patients may face cytokine release syndrome (CRS) and CAR-T cell-related encephalopathy syndrome (CRES); even if allogeneic CAR-T knocks out the relevant HLA I and TCR through CRISPR gene editing, graft versus host disease (GVHD) may still occur; at the same time, allogeneic CAR-T uses CRISPR technology to edit the genes of T cells during the preparation process, which poses related gene editing risks. In addition, not every patient who is suitable for CAR-T treatment can successfully prepare cells. For those patients who have undergone multiple chemotherapy, the proliferation ability of their own T cells is greatly weakened, and nearly 1 / 4 of patients fail to successfully prepare CAR-T cells. Therefore, how to produce efficient and safe universal CAR-T cells is a huge challenge.

[0005] In view of the above aspects, it is particularly urgent to establish a universal CAR-T technology that can overcome the immunosuppressive tumor microenvironment and is safe, efficient, and low-cost. It will be a new direction for future development. It can realize the industrialization of CAR-T cell therapy, widely and conveniently provide it to suitable patients, and greatly reduce the production cost of cell products and the treatment cost of patients. Summary of the invention

[0006] One of the purposes of the present invention is to provide an irradiated GPC3 CAR-IC cell for preventing or treating tumors expressing GPC3 antigen, which can resist the immunosuppressive effect of the tumor microenvironment, reduce the risk of GVHD (Graft versus host disease), and can be efficiently produced, safe, universal and / or low-cost. The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0007] To achieve the above-mentioned object, the present invention first provides irradiated CAR-T cells, which can be prepared according to a method comprising the following steps: irradiating in vitro CAR-T cells with ionizing rays to obtain irradiated CAR-T cells, wherein the ionizing rays are selected from one or more of α rays, β rays, γ rays, X-rays, electron beams, proton beams, heavy ion beams, and neutron beams, and the in vitro CAR-T cells may be GPC3 CAR-IC cells, and the GPC3 CAR-IC cells contain nucleic acid molecules, and the nucleic acid molecules may include a gene encoding a chimeric antigen receptor and a gene encoding a chimeric switch receptor, and the chimeric switch receptor may include a TGFβ receptor type II extracellular region (transforming growth factor β type II receptor extracellular region), μPD-1 and a CD27 transmembrane region and a cytoplasmic region, and the amino acid sequence of the μPD-1 (mutation-optimized PD-1 extracellular region) may be positions 187-332 of SEQ ID No. 1, and the gene encoding the chimeric antigen receptor may be a gene encoding a chimeric antigen receptor targeting GPC3.

[0008] Furthermore, the irradiated CAR-T cells have a reduced proliferation activity compared to the GPC3 CAR-IC cells, while maintaining the effects of specific secretion of T cell effector molecule IFN-γ, cytotoxicity, and specific killing of GPC3 + The effects on tumor cells, the effects on specific secretion of T cell effector molecule IFNα2, the effects on inducing NK cells to secrete functional effector molecule IFN-γ and / or the in vivo tumor inhibition effects.

[0009] The irradiated CAR-T cells (including irradiated GPC3 CAR-IC cells) are cells that have CAR elements introduced into T cells of healthy individuals, and then the cells are irradiated to a certain extent to inhibit excessive proliferation in the body after cell reinfusion, thereby reducing the GVHD reaction, but without affecting the killing activity of CAR-T cells against tumor cells.

[0010] The irradiated CAR-T cells may be irradiated GPC3 CAR-IC cells.

[0011] Furthermore, the encoding gene of the chimeric switch receptor may be any of the following:

[0012] A1) DNA molecule encoding fusion protein;

[0013] A2) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0014] A3) a DNA molecule whose nucleotide sequence is SEQ ID No. 2;

[0015] The fusion protein may be any of the following:

[0016] B1) a protein whose amino acid sequence is SEQ ID No. 1;

[0017] B2) a protein having more than 80% identity with the protein shown in B1) and having the same function as the protein shown in B1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1;

[0018] B3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2).

[0019] The fusion protein may be a chimeric switch receptor (CSR).

[0020] 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.

[0021] 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.

[0022] The tags described in this article include but are not limited to: GST (glutathione sulfhydryltransferase) tag protein, His 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.

[0023] 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.

[0024] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0025] 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.

[0026] 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.

[0027] Furthermore, the nucleic acid molecule may also include IFNα gene (interferon α gene) and / or P2A gene.

[0028] The nucleotide sequence of the IFNα gene may be positions 1552-2118 of SEQ ID No.3.

[0029] The nucleotide sequence of the P2A gene may be positions 1474-1551 of SEQ ID No.3.

[0030] Furthermore, the target of the chimeric antigen receptor may be GPC3.

[0031] Furthermore, the amino acid sequence of the chimeric antigen receptor may be SEQ ID No.4.

[0032] The amino acid sequence of the IFNα may be SEQ ID No.5.

[0033] Furthermore, the gene encoding the chimeric antigen receptor targeting GPC3 may be any of the following:

[0034] G1) a DNA molecule encoding a protein having an amino acid sequence of SEQ ID No. 4;

[0035] G2) the coding sequence is the DNA molecule of SEQ ID No. 3, positions 1-1473;

[0036] G3) The nucleotide sequence is the DNA molecule of position 1-1473 of SEQ ID No.3.

[0037] Furthermore, the nucleic acid molecule may be any of the following:

[0038] C1) a DNA molecule having a nucleotide sequence of SEQ ID No. 3;

[0039] C2) A DNA molecule having more than 70% identity with the DNA molecule shown in C1) and having the same function as obtained by modifying the nucleotide sequence shown in SEQ ID No.3 and / or replacing and / or deleting and / or adding one or more nucleotides.

[0040] 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.

[0041] The GPC3 CAR-IC cells described in this article can be effector T cells in adoptive cellular immunotherapy.

[0042] The GPC3 CAR-IC cells described herein contain the DNA molecule shown in SEQ ID No. 3 and express the GPC3 CAR-IC gene (SEQ ID No. 3).

[0043] The GPC3 CAR-IC cell described herein may be a recombinant cell obtained by introducing the DNA molecule shown in SEQ ID No. 3 into a T cell. Specifically, in one embodiment of the present invention, the GPC3 CAR-IC cell is a recombinant cell obtained by packaging the recombinant retroviral vector MP71-GPC3 CAR-IC with a packaging cell to obtain a recombinant retrovirus, and then introducing the recombinant retrovirus into a T cell, and the packaging cell may be a Phoenix Ecotropic (ECO) cell and a PG13 cell.

[0044] 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 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.

[0045] Furthermore, the ionizing radiation described herein may be X-rays, the duration of the irradiation treatment may be 20-3000 seconds, and the dose of the irradiation treatment may be 1-50 Gy.

[0046] 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 seconds.

[0047] The 1-50 Gy mentioned herein may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 Gy.

[0048] Furthermore, the irradiation treatment conditions may be: an irradiation dose of 10 Gy and a dose rate of 1.199 Gy / min.

[0049] Further, the irradiation treatment conditions may be: voltage of 160KV, current of 25mA, and 0.3mm copper filtration. The 0.3mm copper filtration means that the X-ray tube has an additional copper filter plate to produce 0.3mm Cu half value layer (HVL) irradiation quality.

[0050] The present invention also provides any of the following uses of any of the irradiated CAR-T cells described herein:

[0051] D1) Use in the preparation of drugs for preventing or treating tumors;

[0052] D2) Use in the preparation of a medicament for preventing or treating a tumor expressing the GPC3 antigen;

[0053] D3) Use in the preparation of a medicament for preventing or treating 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.

[0054] 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.

[0055] Furthermore, the liver cancer may be hepatocellular carcinoma (HCC).

[0056] Furthermore, the squamous cell carcinoma may be lung squamous cell carcinoma.

[0057] Furthermore, the melanoma may be malignant melanoma.

[0058] Furthermore, the glioma may be a glioblastoma.

[0059] Furthermore, the tumor expressing the GPC3 antigen may be a tumor with high expression of GPC3.

[0060] Furthermore, the D1) may be used in the preparation of drugs for inhibiting and / or killing tumors.

[0061] Furthermore, the D2) may be used in the preparation of a drug for inhibiting and / or killing a tumor expressing the GPC3 antigen.

[0062] The inhibition may be inhibition of tumor proliferation and / or growth.

[0063] The tumor described herein may be a solid tumor (eg, pancreatic cancer, lung cancer, kidney cancer, liver cancer, or brain glioma).

[0064] The present invention also provides a method for preparing any of the irradiated CAR-T cells described herein, which may include any of the following:

[0065] E1) irradiating any of the GPC3 CAR-IC cells described herein with ionizing radiation to obtain irradiated CAR-T cells, wherein the ionizing radiation is selected from one or more of α-rays, β-rays, γ-rays, X-rays, electron beams, proton beams, heavy ion beams, and neutron beams;

[0066] E2) irradiating any of the GPC3 CAR-IC cells described herein with ionizing radiation to obtain irradiated CAR-T cells, wherein the ionizing radiation is X-rays, the irradiation time is 20-3000 seconds, and the irradiation dose is 1-50 Gy;

[0067] E3) irradiating any of the GPC3 CAR-IC cells described herein with ionizing radiation to obtain irradiated CAR-T cells, wherein the ionizing radiation is X-rays, and the irradiation treatment conditions are: an irradiation dose of 10 Gy and a dose rate of 1.199 Gy / min.

[0068] The present invention also provides a method for inhibiting proliferation of any of the GPC3 CAR-IC cells described herein, which may include any of the following:

[0069] F1) irradiating the GPC3 CAR-IC cells with ionizing radiation to inhibit the proliferation of the GPC3 CAR-IC cells, wherein the ionizing radiation is selected from one or more of α-rays, β-rays, γ-rays, X-rays, electron beams, proton beams, heavy ion beams, and neutron beams;

[0070] F2) irradiating the GPC3 CAR-IC cells with ionizing radiation to inhibit the proliferation of the GPC3 CAR-IC cells, wherein the ionizing radiation is X-rays, the irradiation time is 20-3000 seconds, and the irradiation dose is 1-50 Gy;

[0071] F3) irradiating the GPC3 CAR-IC cells with ionizing radiation to inhibit the proliferation of the GPC3 CAR-IC cells, wherein the ionizing radiation is X-rays, and the irradiation treatment conditions are: an irradiation dose of 10 Gy and a dose rate of 1.199 Gy / min.

[0072] Furthermore, the inhibiting of GPC3 CAR-IC cell proliferation may be inhibiting excessive proliferation of GPC3 CAR-IC cells.

[0073] Furthermore, the inhibiting of excessive proliferation of GPC3 CAR-IC cells may be inhibiting excessive proliferation of GPC3 CAR-IC cells in vivo after reinfusion.

[0074] The nucleic acid molecules 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.

[0075] 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:

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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, 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 cells. The GPC3CAR-IC 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 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.

[0089] The present invention constructs CAR-T cells by designing a fusion gene containing a chimeric antigen receptor and a chimeric switch receptor encoding gene, converts the inhibitory anti-tumor T cell response activated by PD-L1 and TGFβ into a CD27-mediated activation signal, endows CAR-T cells with the ability to resist the immunosuppressive tumor microenvironment, and makes them have stronger in vivo persistence, promotes the expansion of CAR-T cells, prolongs the in vivo survival time, and promotes cytokine secretion, and achieves the regulation of the microenvironment near the tumor by promoting the secretion of cytokines IFN-γ and IFN-α2, relieves its immunosuppression, and improves the anti-tumor effect of CAR-T cells by mobilizing the body's own immunity to participate in the killing of tumor cells.

[0090] The GPC3 CAR-IC cells designed and constructed by the present invention can be better used for adoptive cell immunotherapy through modification at the genetic level. Another important difficulty of adoptive cell immunotherapy is the subsequent adverse reaction treatment and quality control. In order to realize the industrialization of GPC3 CAR-IC cells and develop a complete immunotherapy technology, the inventors of the present invention further developed an irradiated GPC3 CAR-IC cell.

[0091] The proliferation activity of the irradiated GPC3 CAR-IC cells of the present invention is inhibited to a certain extent (proliferation activity is reduced), thereby inhibiting the excessive proliferation of the cells in the body after reinfusion to reduce the GVHD reaction, while still maintaining good specific secretion of T cell effector molecule IFN-γ, cytotoxicity, and specific killing of GPC3 +The effects of irradiated GPC3 CAR-IC cells on tumor cells, the effects of specifically secreting the T cell effector molecule IFNα2, the effects of inducing NK cells to secrete the functional effector molecule IFN-γ, and / or the in vivo tumor inhibition effects did not affect the killing activity of irradiated GPC3 CAR-IC cells on tumor cells.

[0092] The irradiated GPC3 CAR-IC cells of the present invention can survive in vivo for 5-10 days.

[0093] In summary, the irradiated GPC3 CAR-IC cells of the present invention have the following advantages:

[0094] (1) By modifying the CAR gene at the genetic level, the immunosuppressive effect of the tumor microenvironment was reduced or inhibited, which promoted the proliferation of GPC3 CAR-IC cells, prolonged their survival time in vivo, and promoted cytokine secretion, thereby improving the anti-tumor effect of GPC3 CAR-IC cells.

[0095] (2) The prior art uses CRISPR gene editing technology to knock out related HLA I and TCR to reduce the risk of GVHD, but GVHD may still occur and there are related gene editing risks. The irradiated GPC3 CAR-IC cells of the present invention have a relatively short survival time in the body after irradiation, which is not enough to produce a GVHD reaction. Therefore, there is no need for gene editing, which avoids the risks associated with gene editing and is safer.

[0096] (3) The viral vectors used in the preparation of CAR-T cells will further lose their ability to amplify after irradiation, and the viruses will be killed, which can better avoid the risk of CAR-T products being contaminated with replication-competent retrovirus (RCR).

[0097] (4) By controlling the therapeutic dose of irradiated GPC3 CAR-IC cells, the toxic and side effects that may occur during the treatment of tumor patients, such as cytokine release syndrome (CRS), CAR-T cell-related encephalopathy syndrome (CRES), and graft versus host disease (GVHD), can be more effectively controlled.

[0098] (5) Efficiency: Irradiation is easy to scale up and can achieve large-scale industrial production.

[0099] (6) Universal and low-cost: Ready-to-use "off-the-shelf finished products" can be mass-produced in advance and can be used for treatment when suitable patients need them. This can effectively avoid missing the best treatment opportunity for patients with rapidly progressive cancer due to the long production cycle of conventional CAR-T cells. It can be widely and conveniently supplied to suitable patients and greatly reduce the production cost of cell products and the treatment cost of patients.

[0100] The irradiated GPC3 CAR-IC cells and the preparation method thereof developed by the present invention have important significance and wide clinical application value for CAR-T treatment of tumors (such as hepatocellular carcinoma). BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 Schematic diagram of the GPC3 CAR-IC gene structure.

[0102] Figure 2 These are the expression detection results of various parts of GPC3 CAR-IC cells in Example 2.

[0103] Figure 3 The effect of irradiation on the survival and proliferation of GPC3 CAR-IC cells. Figure 3 A and B show the effects of irradiation on the survival of GPC3CAR-IC cells; Figure 3 Center C shows the effect of irradiation on the proliferation of GPC3 CAR-IC cells.

[0104] Figure 4 The effect of irradiation on the secretion of functional effector molecule IFN-γ by GPC3 CAR-IC cells.

[0105] Figure 5 The results of cytotoxic (expression of degranulated CD107a) function test of irradiated GPC3 CAR-IC cells.

[0106] Figure 6 The results of the cytotoxicity test of irradiated GPC3 CAR-IC cells in killing tumor cells.

[0107] Figure 7 These are the results of cell function testing of irradiated GPC3 CAR-IC cells secreting IFN-γ and IFN-α2.

[0108] Figure 8 To detect the induction effect of IFN-α2 secreted by irradiated GPC3 CAR-IC cells co-cultured with target cells on NK cells in vitro.

[0109] Fig. 9 The in vivo tumoricidal effect and animal survival of irradiated GPC3 CAR-IC cells were evaluated in the HepG2-NSG xenograft model. DETAILED DESCRIPTION

[0110] 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.

[0111] 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.

[0112] The pUC57 vector in the following examples is a product of Beijing Qingke Biotechnology Co., Ltd.

[0113] 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.

[0114] The human peripheral blood mononuclear cells (PBMC) in the following examples were derived from the venous blood of healthy volunteers.

[0115] 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.

[0116] The NSG mice (6-7 weeks old, weighing 18-25 g) in the following examples were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.

[0117] The RPMI-1640 culture medium in the following examples is a product of Sigma, with the product number R8758.

[0118] In the following examples, the RPMI-1640 medium containing 10% fetal bovine serum (FBS) (10% FBS RPMI-1640 medium) is prepared using RPMI-1640 medium (R8758) as a solvent and fetal bovine serum as a solute, and is also referred to as a complete medium.

[0119] The sequences involved in the following examples are shown in Table 1.

[0120] Table 1. Related sequences of GPC3 CAR-IC genes

[0121]

[0122]

[0123]

[0124] Example 1. Construction of GPC3 CAR-IC cells

[0125] The GPC3 CAR-IC cells constructed in this embodiment use GPC3 as a target 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:

[0126] 1. GPC3 CAR-IC gene design

[0127] 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:

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 2. GPC3 CAR-IC gene synthesis

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 3. Construction of GPC3 CAR-IC cells

[0144] 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 cells. The specific steps are as follows:

[0145] 3-1. Construction of recombinant retroviral vector

[0146] (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.

[0147] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel excision.

[0148] (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.

[0149] (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.

[0150] 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.

[0151] 3-2. Retroviral packaging

[0152] The MP71-GPC3 CAR-IC plasmid prepared in step 3-1 is introduced into packaging cells for packaging to complete virus assembly and obtain retrovirus. The specific steps of virus packaging are as follows:

[0153] 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;

[0154] b) Day 2: Transfection is performed when the confluency of ECO cells reaches about 90% (usually about 14-18 hours after plating); prepare 12.5 μg of plasmid, 1.25 M CaCl 2 250 μL, HO 2 O 1mL, total volume 1.25mL; add 2×HBS with the same volume as the plasmid complex in another tube, vortex and shake for 20s while adding the plasmid complex. Gently add the mixture along the edge to the ECO dish, culture at 37℃ for 4h, remove the culture medium, wash once with PBS, and add preheated fresh culture medium again;

[0155] 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;

[0156] 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;

[0157] 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, 2000×g for 2 h, and discard the unbound virus supernatant;

[0158] 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;

[0159] g) Add 5 ml of complete medium containing 10% FBS to terminate digestion, and centrifuge at 1500 rpm for 5 min;

[0160] 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;

[0161] i) Centrifuge at 1000 rpm for 1 min at 37°C and 5% CO 2 Culture for 48 hours;

[0162] 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;

[0163] 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.

[0164] 3-3. Retrovirus infection of human T cells

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] g) Thus, recombinant cells infected with the retrovirus prepared in step 3-2 are obtained, which are named GPC3 CAR-IC cells (ie, T cells expressing the GPC3 CAR-IC gene with the nucleotide sequence of SEQ ID No. 3).

[0172] Example 2. Detection of CAR expression in GPC3 CAR-IC cells

[0173] The constructed GPC3 CAR-IC cells and 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. CAR expression was detected until D8 day.

[0174] 1. Expression detection of GPC3 CAR, μPD-1 and TRⅡ:

[0175] (1) After centrifugation (1500 rpm × 5 min), the supernatant was discarded and 200 μL FACS buffer (1× PBS containing 0.1% NaN) was added to each well of a 96-well round-bottom plate. 3 and 2% FBS) and centrifuged at 1500 rpm for 5 min;

[0176] (2) Add 60 μL of prepared fluorescent antibody (anti-human FITC-GPC3(rp) / PE-PD-L1 / APC-TRII) to each well, resuspend and mix, and incubate at 4°C for 30 min;

[0177] (3) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0178] (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 percentages of GPC3 CAR, μPD-1, and TRⅡ in T cells.

[0179] 2. Interferon α2 (IFNα2) expression detection:

[0180] (1) After centrifugation (1500 rpm × 5 min), the supernatant was discarded and 200 μL FACS buffer (1× PBS containing 0.1% NaN) was added to each well of a 96-well round-bottom plate. 3 and 2% FBS) and centrifuged at 1500 rpm for 5 min;

[0181] (2) Add 60 μL of prepared fluorescent antibody (anti-human FITC-GPC3(rp)) to each well, resuspend and mix, and incubate at 4°C for 30 min;

[0182] (3) Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min;

[0183] (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.

[0184] (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;

[0185] (6) Add 20 μL of prepared anti-human Biotin-IFNα2 / SA-APC to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0186] (7) Add 200 μL of 0.5% BSA to each well, centrifuge at 1500 rpm for 5 min, and wash twice;

[0187] (8) Add 20 μL of prepared SA-APC to each well, resuspend and mix, and incubate at 4°C for 10 min;

[0188] (9) Add 200 μL of 0.5% BSA to each well and centrifuge at 1500 rpm for 5 min. Discard 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.

[0189] Test results such as Figure 2 As shown, the prepared GPC3 CAR-IC cells simultaneously express CAR (ie, GPC3 CAR), IFNα2, μPD-1, and TRⅡ, indicating that the expression of each part in the GPC3 CAR-IC cells meets the expected design.

[0190] Example 3: Effect of irradiation on GPC3 CAR-IC cell survival

[0191] The constructed GPC3 CAR-IC cells were resuspended in RPMI-1640 medium containing 10% FBS and the cell density was adjusted to 1×10 7 / mL, and different irradiation doses were adopted, the radiation doses were 0Gy and 10Gy respectively, the irradiation conditions were: 160KV, 25mA, 0.3mm copper filtration, the default dose rate was 1.199Gy / min, and the irradiation time = irradiation dose / (1.199Gy / min).

[0192] 1×10 of 0Gy irradiated GPC3 CAR-IC were added to each well. 6 10 Gy irradiated GPC3 CAR-IC cells were plated at 2×10 6 The cells were cultured on 24-well TC plates in a 10% FBS RPMI-1640 medium containing IL-2 (500 U / mL) and placed in a 37° C. incubator, and the day was recorded as D0.

[0193] The irradiated cells were counted for six consecutive days (D0, D1, D2, D3, D4, and D5) using APC Annexin V (Biolegend, Catalog No. 640920) and 7-AAD Viability Staining Solution (Biolegend, Catalog No. 420404), and the cell viability was calculated.

[0194] The results are as follows Figure 3 As shown in A and B, after irradiation, the cell viability and the number of live cells gradually decreased. On D5, the cell viability was about 35% of the original value, and the number of live cells was about 38% of the original value.

[0195] Example 4: Effect of irradiation on the proliferation of GPC3 CAR-IC cells

[0196] 1. CFSE dye labeling of GPC3 CAR-IC cells:

[0197] (1) GPC3 CAR-IC cells were collected, the culture supernatant was discarded by centrifugation, and the cells were washed three times with 1× PBS buffer to remove the effect of FBS on CFSE labeling;

[0198] (2) Resuspend the cells to 1×10 7 pcs / mL, add CFSE dye (Invitrogen, catalog number 65-0850-84) to 5 μM, mix quickly, and incubate in a 37°C incubator for 10 min;

[0199] (3) Add at least 2 volumes of cold RPMI-1640 medium containing 10% FBS to terminate CFSE labeling, and then centrifuge to remove the supernatant;

[0200] (4) Resuspend in RPMI-1640 medium containing 10% FBS and wash twice by centrifugation;

[0201] (5) Finally, the labeled GPC3 CAR-IC cells were resuspended in RPMI-1640 medium containing 10% FBS according to the required density.

[0202] 2. Irradiate the labeled GPC3 CAR-IC cells according to the dose required by the experiment.

[0203] Specifically, the labeled GPC3 CAR-IC cells were resuspended in RPMI-1640 medium containing 10% FBS and the cell density was adjusted to 1×10 7 / mL, and different irradiation doses were adopted, the radiation doses were 0Gy, 5Gy, 7.5Gy and 10Gy respectively, the irradiation conditions were: 160KV, 25mA, 0.3mm copper filtration, the default dose rate was 1.199Gy / min, and the irradiation time = irradiation dose / (1.199Gy / min).

[0204] 3. GPC3 CAR-IC cells irradiated with different doses were plated at 2×10 per well. 6 The cells were cultured on a 24-well TC plate in a cell culture medium of 10% FBS RPMI-1640 medium containing IL-2 (500 U / mL). The plates were cultured in a 37° C. incubator and recorded as day D0.

[0205] 4. Collect cells on day 4 and detect the proliferation of GPC3 CAR-IC cells by flow cytometry.

[0206] The results are as follows Figure 3 As shown in C, irradiation inhibited the proliferation of GPC3 CAR-IC cells, with a dose effect. On D4, GPC3 CAR-IC cells irradiated with a dose of 10 Gy did not proliferate, while CAR-IC cells irradiated with doses of 5 Gy and 7.5 Gy had a small amount of cell division.

[0207] Example 5. Effect of irradiation on GPC3 CAR-IC cell function

[0208] GPC3 CAR-IC cells were resuspended in RPMI-1640 medium containing 10% FBS and the cell density was adjusted to 1×10 7 / mL, and different irradiation doses were adopted, the radiation doses were 0Gy and 10Gy respectively, the irradiation conditions were: 160KV, 25mA, 0.3mm copper filtration, the default dose rate was 1.199Gy / min, and the irradiation time = irradiation dose / (1.199Gy / min).

[0209] GPC3 CAR-IC cells irradiated with different doses were plated at 2×10 per well. 6 The cells were cultured on a 24-well TC plate in a cell culture medium of 10% FBS RPMI-1640 medium containing IL-2 (500 U / mL). The plates were cultured in a 37° C. incubator and recorded as day D0.

[0210] After culturing, various functional tests were performed as described below.

[0211] 5.1 Effect of irradiation on the secretion of the functional effector molecule IFN-γ by GPC3 CAR-IC cells

[0212] For four consecutive days (D0, D1, D2, and D3), the irradiated GPC3 CAR-IC cells were taken to detect the secreted IFN-γ. The specific steps are as follows:

[0213] 1. Take the GPC3 CAR-IC cells cultured above at different irradiation doses on D0, D1, D2 and D3, and adjust the cell density to 2×10 6 / mL, take 100 μL and add it to a 96-well U-bottom plate, add HepG2 cells at a ratio of test cells: target cells = 1:1, then 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 6 hours.

[0214] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0215] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN 3 and 2% FBS), resuspended, centrifuged at 1500 rpm for 5 min, and repeated this step twice;

[0216] (2) Add 60 μL of prepared fluorescent antibodies (fluorescently labeled anti-human CD3 / CD4 / CD8 antibodies and GPC3-hFc recombinant protein) to each well, resuspend and mix, and incubate at room temperature for 10 min in the dark;

[0217] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0218] (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.

[0219] (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;

[0220] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Catalog No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark;

[0221] (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 the functional effector molecule IFN-γ in CAR-T cells.

[0222] The results are as follows Figure 4 As shown, GPC3 CAR-IC cells on D0 / D1 / D2 / D3 days can specifically secrete the T cell effector molecule IFN-γ, and GPC3 CAR-IC cells 3 days after irradiation still retain CAR-specific effector function (IFN-γ secretion).

[0223] 5.2. Cytotoxicity (expression of degranulated CD107a) function detection of irradiated GPC3 CAR-IC cells

[0224] The irradiated GPC3 CAR-IC cells were collected for four consecutive days (D0, D1, D2, and D3) to detect the degranulation ability, namely, the expression of CD107a.

[0225] 1) The above cells were cultured until D15 and the cell density was adjusted to 2×10 6 / mL, take 100μL and add it into a 96-well U-bottom plate;

[0226] 2) HepG2 cells were added to each well at a ratio of test cells: target cells = 1:1 as CAR antigen-specific stimulation;

[0227] 3) Add 1 μL of APC-labeled anti-human CD107a antibody (Biolegend, Cat. No. 328620) to each well and incubate at 37°C for 4 h;

[0228] 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;

[0229] 5) After incubation, flow cytometry staining is performed. The steps are as follows:

[0230] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN 3 and 2% FBS) and resuspended and mixed, centrifuged at 1500 rpm for 5 min, and repeated this step twice;

[0231] (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;

[0232] (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.

[0233] Test results such as Figure 5 As shown, GPC3 CAR-IC cells can specifically upregulate CD107a after irradiation on D0 / D1 / D2 / D3 days, and GPC3 CAR-IC cells 3 days after irradiation still retain CAR-specific killing function.

[0234] 5.3. Cytotoxicity test of irradiated GPC3 CAR-IC

[0235] The irradiated GPC3 CAR-IC cells were collected for four consecutive days (D0, D1, D2, and D3) to detect the cytotoxic effect of killing tumor cells.

[0236] 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;

[0237] 2) Add target cells HepG2 at different effector-target ratios (1:1, 1:3, 1:9, 1:27) and culture at 37°C for 16 h.

[0238] 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 GPC3 CAR-IC cells:

[0239] Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0240] Eexp: RLU value when effector cells and target cells are co-cultured;

[0241] Emin: RLU value of spontaneous death of effector cells in the absence of cells;

[0242] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0243] Tmin: RLU value under the condition of maximum killing rate.

[0244] Test results such as Figure 6 As shown in Figure 2, both non-irradiated and irradiated GPC3 CAR-IC cells can specifically kill GPC3 + target cells, and the killing effects of both on D0, D1, D2, and D3 were similar; the control CTR T had a negative effect on GPC3 + There was no killing effect on target cells.

[0245] 5.4. Cellular Function Detection of IFN-γ and IFN-α2 Secretion by Irradiated GPC3 CAR-IC Cells

[0246] The irradiated GPC3 CAR-IC cells were cultured until D15, and 1×10 cells were cultured in 1 mL per well. 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.

[0247] The co-cultured cells were collected for five consecutive days (D1, D2, D3, D4, and D5) to detect the secreted IFN-γ and IFN-α2.

[0248] The test groups and controls are as follows:

[0249] CTR 0Gy-HepG2: 1×10 per well 6 Virus-free, non-irradiated T cells (CTR T cells) were cultured without HepG2 cells;

[0250] CTR 0Gy+HepG2: 1×10 per well 6 1×106 HepG2 cells were co-cultured;

[0251] GPC3 CAR-IC 0Gy-HepG2: 1×10 per well 6 Unirradiated GPC3 CAR-IC cells, no HepG2 cells co-cultured;

[0252] GPC3 CAR-IC 0Gy+HepG2: 1×10 per well 6 1×10 non-irradiated GPC3 CAR-IC cells were added 6 HepG2 cells;

[0253] GPC3 CAR-IC 10Gy-HepG2: 1×10 per well 6 10Gy irradiated GPC3 CAR-IC cells, without HepG2 cells co-culture;

[0254] GPC3 CAR-IC 10Gy+HepG2: 1×10 per well 6 1×10 GPC3 CAR-IC cells irradiated with 10 Gy were added 6 HepG2 cells.

[0255] The specific detection steps are as follows:

[0256] 1. The co-cultured cells on days D1, D2, D3, D4, and D5 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.

[0257] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0258] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN 3 and 2% FBS), resuspended, centrifuged at 1500 rpm for 5 min, and repeated this step twice;

[0259] (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;

[0260] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0261] (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.

[0262] (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;

[0263] (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;

[0264] (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 cells and control cells.

[0265] Test results such as Figure 7 As shown in the figure, after co-culture with target cell antigens, both non-irradiated and irradiated GPC3 CAR-IC cells can specifically secrete the T cell effector molecule IFN-γ; GPC3 CAR-IC cells can secrete low levels of IFNα2 under conditions without antigen stimulation, and can secrete high levels of IFNα2 under conditions with antigen stimulation.

[0266] 5.5. In vitro detection of the induction effect of IFN-α2 secreted by irradiated GPC3 CAR-IC cells co-cultured with target cells on NK cells

[0267] The test cells were irradiated GPC3 CAR-IC cells and CTR T cells (i.e., T cells without virus transfection).

[0268] 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 1×10 cells to each well at a ratio of 1:1 between test cells and target cells. 6 HepG2 cells were cultured for 3 days;

[0271] 3) Collect the supernatant by centrifugation and incubate with PBMC cells, where PBMC: 1×10 6 / 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 and add 200 μL FACS buffer (1× PBS containing 0.1% NaN 3 and 2% FBS) and centrifuged 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) 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 (BD Canto-II) to read the cells and analyze the percentage of effector molecule IFN-γ in NK cells.

[0281] Test results such as Figure 8 As shown, IFN-α2 secreted by irradiated GPC3 CAR-IC cells can induce NK cells to secrete the functional effector molecule IFN-γ.

[0282] 5.6. Evaluation of the in vivo tumoricidal effect and animal survival of irradiated GPC3 CAR-IC cells in the HepG2-NSG transplant model

[0283] The HepG2 subcutaneous tumor-bearing NSG mouse model (HepG2-NSG model) was used to evaluate the in vivo tumoricidal effect of irradiated GPC3 CAR-IC 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 After the injection of irradiated GPC3 CAR-IC cells, the day was recorded as D0. During this period, the tumor-bearing mice were randomly divided into 3 groups, 5 mice / group, and the groups were divided as follows:

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

[0287] GPC3 CAR-IC 0Gy group: injected with non-irradiated GPC3 CAR-IC cells, 3×10 6 Cells / piece;

[0288] GPC3 CAR-IC 10Gy group: 10Gy irradiated GPC3 CAR-IC cells, 3×10 6 Cells / piece.

[0289] 3) On D5, the GPC3 CAR-IC 10Gy group received the second injection.

[0290] 4) The tumor size was measured with a vernier caliper on D7, D14, D21, D28, and D35 after administration.

[0291] 5) Blood was collected from the submandibular area of ​​the mice on D1, D3, D5, D6, D8, D10, and D14 days after administration, and the conventional qPCR method was used to detect the retention of GPC3 CAR-IC cells in vivo after irradiation.

[0292] Test results such as Fig. 9As shown in the figure, compared with the control group, GPC3 CAR-IC cells injected with high doses of radiation twice on D0 and D5 had a significant tumor inhibition effect, although the effect was slightly worse than that of non-irradiated GPC3 CAR-IC cells. After cell injection, irradiated GPC3 CAR-IC cells were detected in the blood on D1 day, reaching a peak on D3 day and then gradually decreasing, and still detectable on D5 day. This proves that irradiated GPC3 CAR-IC cells can survive in vivo for at least 5 days.

[0293] 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. Irradiated CAR-T cells, the cells are prepared according to a method comprising the following steps: irradiating in vitro CAR-T cells with X-rays to obtain irradiated CAR-T cells, the in vitro CAR-T cells are GPC3 CAR-IC cells, the GPC3CAR-IC cells contain nucleic acid molecules, and the nucleic acid molecules are, from the 5' end to the 3' end, a chimeric antigen receptor coding gene, a P2A gene, an IFNα gene, a promoter element gene, and a chimeric switch receptor coding gene; the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.4, the amino acid sequence of the chimeric switch receptor is shown in SEQ ID No.1; the amino acid sequence of IFNα is shown in SEQ ID No.

5.

2. The irradiated CAR-T cell according to claim 1, characterized in that The irradiation treatment conditions are: irradiation dose of 10 Gy, dose rate of 1.199 Gy / min.

3. The irradiated CAR-T cell according to claim 2, characterized in that: The irradiated CAR-T cells have reduced proliferation activity compared to the GPC3CAR-IC cells, while maintaining the effects of specific secretion of T cell effector molecule IFN-γ, cytotoxicity, and specific killing of GPC3 + The effects on tumor cells, the effects on specific secretion of T cell effector molecule IFNα2, the effects on inducing NK cells to secrete functional effector molecule IFN-γ and / or the in vivo tumor inhibition effects.

4. The irradiated CAR-T cell according to any one of claims 1 to 3, characterized in that: The encoding gene of the chimeric switch receptor is a DNA molecule encoding a fusion protein, and the fusion protein is any one of the following: B1) a protein whose amino acid sequence is SEQ ID No. 1; B2) A fusion protein with the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

5. The irradiated CAR-T cell according to claim 4, characterized in that The DNA molecule encoding the fusion protein is a DNA molecule whose coding sequence is SEQ ID No.

2.

6. The irradiated CAR-T cell according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

3.

7. Use of the irradiated CAR-T cells according to any one of claims 1 to 6 in the preparation of a drug for treating liver cancer.

8. A method for preparing the irradiated CAR-T cells according to any one of claims 1 to 6, characterized in that: The method comprises irradiating the GPC3 CAR-IC cells of any one of claims 1 to 6 with X-rays to obtain irradiated CAR-T cells, wherein the irradiation treatment conditions are: an irradiation dose of 10 Gy and a dose rate of 1.199 Gy / min.

9. A method for inhibiting the proliferation activity of the GPC3 CAR-IC cells according to any one of claims 1 to 6 in vitro, characterized in that: The method comprises irradiating the GPC3 CAR-IC cells with X-rays to inhibit the proliferation activity of the GPC3 CAR-IC cells, and the irradiation treatment conditions are: an irradiation dose of 10 Gy and a dose rate of 1.199 Gy / min.

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