WO2023104168A1 - Chimeric antigen receptor and chimeric antigen receptor t cell targeting both gpc3 and cd276, preparation method therefor and use thereof - Google Patents
Chimeric antigen receptor and chimeric antigen receptor t cell targeting both gpc3 and cd276, preparation method therefor and use thereof Download PDFInfo
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- WO2023104168A1 WO2023104168A1 PCT/CN2022/137669 CN2022137669W WO2023104168A1 WO 2023104168 A1 WO2023104168 A1 WO 2023104168A1 CN 2022137669 W CN2022137669 W CN 2022137669W WO 2023104168 A1 WO2023104168 A1 WO 2023104168A1
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- chimeric antigen
- antigen receptor
- gpc3
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Classifications
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Definitions
- the present invention relates to biological products, in particular to a chimeric antigen receptor targeting GPC3 and CD276, a chimeric antigen receptor T cell, and a preparation method and application thereof.
- Hepatocellular carcinoma is a common malignancy, accounting for 90% of all primary liver cancers, and is the second most deadly cancer worldwide.
- Most patients with HCC receive local or systemic therapies such as chemotherapy, radiotherapy, hepatectomy, dermal alcohol injections, radiofrequency ablation, various embolization therapies, antibody therapies such as immune checkpoint inhibitors, vascular endothelial growth factor receptor Inhibitors and targeted therapies such as sorafenib, however the efficacy of these therapies is insufficient (Lee, Y.H., et al., Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, Immune Checkpoint Blockade and Beyond. Frontiers in Immunology, 2020. 11), causing irreparable harm to patients and their families. Therefore, new drugs for the treatment of HCC are urgently needed.
- CAR-T Chimeric Antigen Receptor T Cell
- CAR-T Chimeric Antigen Receptor T Cell
- Genetic engineering such as transduction based on recombinant virus vectors
- chimeric antigen receptors on the surface of T cells.
- they can activate the autoimmune system and effectively kill tumor cells (Rafiq, S., C.S. hackett, and R.J. Brentjens, Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nature Reviews Clinical Oncology, 2020. 17(3): p. 147-167.
- Chimeric antigen receptor T cell technology is considered to be the most likely therapy to overcome cancer, and CAR-T cells targeting CD19 have produced good results in blood tumors.
- CAR-T drugs have been approved for marketing in the world, showing the great application prospects of CAR-T cells in the treatment of malignant tumors.
- CAR-T cell therapy faces the problems of heterogeneous expression of tumor antigens and loss of antigens.
- CD19-negative cell populations can escape the recognition of CD19-targeted chimeric antigen receptor T cells, leading to overgrowth of cell populations and relapse of the disease.
- chimeric antigen receptor T cells targeting single antigens failed to eradicate tumors because chimeric antigen receptor T cells could not recognize these tumor cells
- Low-density expression of target antigens (Anurathapan, U.; Chan, RC; Hindi, HF; Mucharla, R.; Bajgain, P.; Hayes, BC; Fisher, WE; Heslop, HE; Rooney, CM; Brenner, MK ; et al. Kinetics of tumor destruction by chimeric antigen receptor-modified t cells. Mol. Ther. 2014, 22 , 623–633.
- CD276, also known as B7-H3 is a type of transmembrane protein belonging to the B7 family.
- CD276 is highly expressed in a variety of cancers, including lung adenocarcinoma, glioma, neuroblastoma, pancreatic cancer, and ovarian cancer, and is hardly expressed in normal tissues.
- Monoclonal antibodies that specifically recognize CD276 mediate efficient tumor clearance in a variety of tumor models.
- CD276 is highly expressed in hepatocellular carcinoma and is associated with the progression of liver cancer and the poor prognosis of tumor patients.
- CAR-T cells targeting CD276 can effectively kill hepatocellular carcinoma cell lines in vitro.
- the single-target CAR-T cell therapy for solid tumors (such as hepatocellular carcinoma) faces the obstacle of heterogeneous expression of antigens.
- the present invention provides a chimeric antigen receptor targeting GPC3 and CD276 at the same time, chimeric antigen receptor T cells, preparation methods and applications thereof.
- the present invention adopts the following technical solutions:
- the present invention provides a chimeric antigen receptor targeting both GPC3 and CD276, and the chimeric antigen receptor is named GPC3-CD276 TanCAR.
- the chimeric antigen receptor includes a single-chain antibody targeting GPC3, a linker, a single-chain antibody targeting CD276, an extracellular hinge region, and a transmembrane region arranged in sequence from the amino terminal to the carboxyl terminal , co-stimulatory signal region and CD3 ⁇ intracellular region.
- amino acid sequence of the GPC3-targeting single-chain antibody is shown in SEQ.ID.NO.3.
- nucleotide sequence of the gene encoding the GPC3-targeting single-chain antibody is shown in SEQ.ID.NO.4.
- the linker includes repeating tandem G4S, for example (G4S)3 whose amino acid sequence is shown in SEQ.ID.NO.5.
- the linker for example, the nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6.
- amino acid sequence of the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.7.
- nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8.
- the extracellular hinge region is selected from CD8 ⁇ hinge region, CD28 hinge region, IgG1 hinge region or IgG4 hinge region.
- amino acid sequence of the CD8 ⁇ hinge region is shown in SEQ.ID.NO.9.
- nucleotide sequence of the gene encoding the hinge region of CD8 ⁇ is shown in SEQ.ID.NO.10.
- the transmembrane region is selected from CD8 ⁇ transmembrane region, CD4 transmembrane region, CD28 transmembrane region, CD3 ⁇ transmembrane region or ICOS transmembrane region.
- amino acid sequence of the CD8 ⁇ transmembrane region is shown in SEQ.ID.NO.11.
- nucleotide sequence of the gene encoding the CD8 ⁇ transmembrane region is shown in SEQ.ID.NO.12.
- the co-stimulatory signal region is selected from 4-1BB intracellular region, CD28 intracellular region, OX40 intracellular region, ICOS intracellular region or CD27 intracellular region.
- amino acid sequence of the 4-1BB intracellular region is shown in SEQ.ID.NO.13.
- nucleotide sequence of the gene encoding the 4-1BB intracellular region is shown in SEQ.ID.NO.14.
- amino acid sequence of the intracellular region of CD3 ⁇ is shown in SEQ.ID.NO.15.
- nucleotide sequence of the gene encoding the intracellular region of CD3 ⁇ is shown in SEQ.ID.NO.16.
- the chimeric antigen receptor includes the amino acid sequence shown in SEQ.ID.NO.1.
- the gene sequence of the chimeric antigen receptor includes the nucleotide sequence shown in SEQ.ID.NO.2.
- the present invention provides a chimeric antigen receptor T cell targeting GPC3 and CD276 at the same time, the T cell includes the above-mentioned chimeric antigen receptor targeting GPC3 and CD276 at the same time (that is, the surface of the T cell has a simultaneous target chimeric antigen receptors to GPC3 and CD276).
- the present invention provides a recombinant virus vector and a recombinant virus.
- the recombinant virus vector includes the gene encoding the above-mentioned chimeric antigen receptor, and the recombinant virus is packaged by lentivirus using the recombinant virus vector.
- the coding gene of the chimeric antigen receptor includes a signal peptide coding gene, a GPC3-targeting single-chain antibody coding gene, a linker (such as (G4S)3 ), coding genes of single-chain antibodies targeting CD276, coding genes of extracellular hinge region (such as CD8 ⁇ extracellular hinge region), coding genes of transmembrane region (such as CD8 ⁇ transmembrane region), co-stimulatory signal region (eg, 4-1BB intracellular region) and the gene encoding the CD3 ⁇ intracellular region.
- a linker such as (G4S)3
- the signal peptide is a CD8 ⁇ signal peptide
- the nucleotide sequence of the gene encoding the CD8 ⁇ signal peptide is shown in SEQ.ID.NO.19.
- the amino acid sequence of the CD8 ⁇ signal peptide is shown in SEQ.ID.NO.20.
- the gene sequence of the chimeric antigen receptor targeting GPC3 and CD276 in the recombinant virus vector and the recombinant virus is shown in SEQ.ID.NO.17, and the gene sequence includes the chimeric antigen receptor The nucleotide sequence of the coding gene.
- the nucleotide sequence shown in SEQ.ID.NO.17 has a gene encoding CD8 ⁇ signal peptide added. The gene encoding the signal peptide can better guide the expression of the chimeric antigen receptor to the cell surface.
- amino acid sequence of the chimeric antigen receptor is shown in SEQ.ID.NO.18, specifically as follows:
- the recombinant viral vector is pWPXLd lentiviral vector.
- the present invention provides a method for preparing chimeric antigen receptor T cells simultaneously targeting GPC3 and CD276, comprising the following steps:
- TanCAR Construct or artificially synthesize the gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR targeting both GPC3 and CD276 by gene cloning, the GPC3-CD276
- the gene sequence of TanCAR includes the nucleotide sequence shown in SEQ.ID.NO.2 (for example, the gene sequence of GPC3-CD276 TanCAR is shown in SEQ.ID.NO.17);
- TanCAR co-transfects host cells with envelope plasmids and packaging plasmids to obtain recombinant lentiviruses
- the recombinant lentivirus was transfected into CD3-positive T lymphocytes (CD3-positive T cells for short), and chimeric antigen receptor T cells targeting both GPC3 and CD276 were obtained after isolation.
- the envelope plasmid is PMD2G
- the packaging plasmid is psPAX2
- the host cell is HEK293T cells.
- the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells.
- the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood or placental blood.
- the present invention provides a drug for treating hepatocellular carcinoma, which includes the above-mentioned chimeric antigen receptor T cells targeting both GPC3 and CD276.
- the chimeric antigen receptor T cells that simultaneously target GPC3 and CD276 proposed by the present invention not only have a killing effect on tumor cells expressing GPC3 and CD276, but also have killing activity on GPC3-negative CD276-positive tumor cells, which solves the problem of antigen heterogeneity Chimeric antigen receptor T cells caused by sexual expression or antigen loss are not effective in treating solid tumors such as hepatocellular carcinoma.
- Figure 1 is a schematic diagram of the structure of the gene encoding GPC3-CD276 TanCAR.
- FIG. 2 shows the results of flow cytometric analysis of the expression of GPC3-CD276 TanCAR on T cells; among them: UTD group is CD3 positive T cells without transduction virus, GPC3-CD276 The TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276.
- Figure 3 shows the results of the analysis of the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on Huh7 cell lines; among them: UTD group is CD3 positive T cells without transduction virus, GPC3-CD276 The TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276.
- Figure 4 shows the results of the analysis of the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on SK-HEP-1-CD276 cell line; among them: UTD group is CD3 positive T cells without transduction virus, GPC3 The CAR-T group is chimeric antigen receptor T cells targeting GPC3 with a single target, GPC3-CD276 TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276; * indicates significant difference.
- chimeric antigen receptor GPC3-CD276 targeting both GPC3 and CD276 In the gene sequence of TanCAR, from the 5' end to the 3' end, the encoding genes of the following elements are included in sequence: the encoding gene of CD8 ⁇ signal peptide (CD8 ⁇ SP), the encoding gene of single-chain antibody targeting GPC3 (GPC3-scFv), the linker The coding gene of "(G4S)3", the coding gene of CD276-targeting single-chain antibody (CD276-scFv), the coding gene of CD8 ⁇ hinge region (CD8 ⁇ hinge), the coding gene of CD8 ⁇ transmembrane region (CD8 ⁇ TM), The gene encoding the intracellular region of 4-1BB (4-1BB) and the gene encoding the intracellular region of CD3 ⁇ (CD3-zeta).
- the nucleotide sequence of the gene encoding the CD8 ⁇ signal peptide is shown in SEQ.ID.NO.19, the nucleotide sequence of the gene encoding the single-chain antibody targeting GPC3 is shown in SEQ.ID.NO.4, The nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6, the nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8, CD8 ⁇
- the nucleotide sequence of the gene encoding the hinge region is shown in SEQ.ID.NO.10, the nucleotide sequence of the gene encoding the CD8 ⁇ transmembrane region is shown in SEQ.ID.NO.12, and the 4-1BB intracellular region
- the nucleotide sequence of the coding gene of CD3 ⁇ is shown in SEQ.ID.NO.14, and the nucleotide sequence of the coding gene of CD3 ⁇ intracellular
- the chimeric antigen receptor GPC3-CD276 The gene sequence of TanCAR was synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd.
- Synthetic GPC3-CD276 The gene sequence of TanCAR was inserted between the BamH1 and EcoR1 restriction sites of the pWPXLd vector, and then transformed into Escherichia coli competent cells DH5 ⁇ for positive clone PCR identification and sequencing identification. The verified correct plasmid is labeled as recombinant plasmid pWPXLd-GPC3-CD276 TanCAR was used in follow-up experiments.
- the recombinant plasmid pWPXLd-GPC3-CD276 TanCAR, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine3000 transfection reagent.
- the virus-containing supernatant was collected at 48 hours: first, the culture system was centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was taken, and then filtered through a 0.45 ⁇ m filter membrane, and the obtained recombinant lentivirus supernatant was used for T cell infection.
- PBMC Peripheral Blood Mononuclear Cells
- Source of PBMC autologous venous blood of healthy volunteers.
- the operation process for separating PBMCs extract the blood of the healthy volunteers, use Ficoll to collect peripheral blood mononuclear cells, and take the middle layer cells after centrifugation; wash with PBS and count to obtain PBMCs.
- the CD3-positive T lymphocytes separated by the immunomagnetic bead method were taken, and the recombinant lentivirus corresponding to the number of CD3-positive T lymphocytes was added for culture.
- RTCA system was used to analyze the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on liver cancer cell lines
- the target cell Huh7 a liver cancer cell line expressing GPC3 and CD276 at the same time
- Huh7 a liver cancer cell line expressing GPC3 and CD276 at the same time
- UTD group CD3 positive T lymphocytes added with untransduced virus
- GPC3-CD276 TanCAR-T group chimeric antigen receptor T cells targeting both GPC3 and CD276
- control group control, medium group
- the different effector cells added to the target cell SK-HEP-1-CD276 (a liver cancer cell line that overexpresses CD276 but does not express GPC3), it is divided into: UTD group (CD3 positive T lymphocytes added with untransduced virus), GPC3 CAR-T group (the gene sequence corresponding to chimeric antigen receptor GPC3 CAR lacks linker-CD276-scFv compared with GPC3-CD276Tan CAR; the production method of CAR-T cells is the same, and the positive rate of GPC3 CAR expression is about 30%), GPC3 -CD276 TanCAR-T group (chimeric antigen receptor T cells targeting both GPC3 and CD276), and a control group (control, medium group).
- UTD group CD3 positive T lymphocytes added with untransduced virus
- GPC3 CAR-T group the gene sequence corresponding to chimeric antigen receptor GPC3 CAR lacks linker-CD276-scFv compared with
- the ratio of different effector cells such as collected GPC3-CD276 TanCAR-T cells
- target cells E:T ) 20:1, 10:1, 5:1
- 100 ⁇ L of effector cell suspension corresponding to the number of cells into the target cells set at least 2 duplicate wells for each group, and then put them into the RTCA resistance system, after a certain period of time Assays for killing activity were performed.
- Effector cell killing rate analysis was performed using the cell index at the indicated time points.
- UTD kill rate (control cell index -UTD cell index )/control cell index ⁇ 100
- CAR-T killing rate (control cell index - CAR-T cell index )/control cell index ⁇ 100
- the chimeric antigen receptor T cells targeting both GPC3 and CD276 constructed by the present invention have stronger killing activity against GPC3 and CD276 positive tumor cell lines, and compared with GPC3 CAR-T, they are more effective against GPC3 negative CD276 positive tumors.
- the cell line has a stronger killing effect and overcomes the problem of tumor antigen escape caused by the heterogeneous expression of GPC3. Therefore, the chimeric antigen receptor T cells targeting GPC3 and CD276 at the same time have great application prospects in the treatment of solid tumors such as hepatocellular carcinoma.
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Abstract
Disclosed are a chimeric antigen receptor and a chimeric antigen receptor T cell targeting both GPC3 and CD276, a preparation method therefor and use thereof. Experiments show that a chimeric antigen receptor T cell targeting both GPC3 and CD276 has strong killing activity against a tumor cell line expressing both GPC3 and CD276, and against a GPC3-negative CD276-positive tumor cell line. Therefore, the chimeric antigen receptor T cell has great prospects for use in treating hepatocellular carcinoma and the like.
Description
本发明涉及生物制品，具体涉及同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。The present invention relates to biological products, in particular to a chimeric antigen receptor targeting GPC3 and CD276, a chimeric antigen receptor T cell, and a preparation method and application thereof.
肝细胞癌是常见的恶性肿瘤，占所有原发性肝癌的90%，是全球第二大致死性癌症。大多数肝细胞癌病人接受局部或系统性疗法，比如化疗、放疗、肝切除术、皮肤乙醇注射、射频消融术、各种栓塞疗法、抗体疗法如免疫检查点抑制剂、血管内皮生长因子受体抑制剂和靶向疗法如索拉菲尼，然而这些疗法的疗效都是不够的（Lee, Y.H.,
et al., Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy,
Immune Checkpoint Blockade and Beyond. Frontiers in Immunology, 2020. 11），对病人和病人的家庭造成了难以挽回的伤害。因此迫切需要新的治疗肝细胞癌的药物。Hepatocellular carcinoma is a common malignancy, accounting for 90% of all primary liver cancers, and is the second most deadly cancer worldwide. Most patients with HCC receive local or systemic therapies such as chemotherapy, radiotherapy, hepatectomy, dermal alcohol injections, radiofrequency ablation, various embolization therapies, antibody therapies such as immune checkpoint inhibitors, vascular endothelial growth factor receptor Inhibitors and targeted therapies such as sorafenib, however the efficacy of these therapies is insufficient (Lee, Y.H.,
et al., Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy,
Immune Checkpoint Blockade and Beyond. Frontiers in Immunology, 2020. 11), causing irreparable harm to patients and their families. Therefore, new drugs for the treatment of HCC are urgently needed.
CAR-T(嵌合抗原受体T细胞)技术是近年来发展火热的细胞免疫疗法，它通过基因工程手段（例如基于重组病毒载体的转导）使嵌合抗原受体表达在T细胞表面，通过在体外制备和扩增嵌合抗原受体T细胞，然后回输至人体，激活自身免疫系统，发挥对肿瘤细胞的高效杀伤（Rafiq, S., C.S.
Hackett, and R.J. Brentjens, Engineering strategies to overcome the current
roadblocks in CAR T cell therapy. Nature Reviews Clinical Oncology, 2020.
17(3): p. 147-167. Benmebarek, M.R., et al., Killing Mechanisms of Chimeric
Antigen Receptor (CAR) T Cells. International Journal of Molecular Sciences,
2019. 20(6).）。嵌合抗原受体T细胞技术被认为是最有可能攻克癌症的疗法，并且靶向CD19的CAR-T细胞在血液肿瘤中产生了良好的效果。目前全球已有6款CAR-T药物获批上市，展现了CAR-T细胞在治疗恶性肿瘤中的巨大应用前景。CAR-T (Chimeric Antigen Receptor T Cell) technology is a hot cellular immunotherapy developed in recent years. It uses genetic engineering (such as transduction based on recombinant virus vectors) to express chimeric antigen receptors on the surface of T cells. By preparing and expanding chimeric antigen receptor T cells in vitro, and then infusing them back into the human body, they can activate the autoimmune system and effectively kill tumor cells (Rafiq, S., C.S.
Hackett, and R.J. Brentjens, Engineering strategies to overcome the current
roadblocks in CAR T cell therapy. Nature Reviews Clinical Oncology, 2020.
17(3): p. 147-167. Benmebarek, M.R., et al., Killing Mechanisms of Chimeric
Antigen Receptor (CAR) T Cells. International Journal of Molecular Sciences,
2019. 20(6).). Chimeric antigen receptor T cell technology is considered to be the most likely therapy to overcome cancer, and CAR-T cells targeting CD19 have produced good results in blood tumors. At present, 6 CAR-T drugs have been approved for marketing in the world, showing the great application prospects of CAR-T cells in the treatment of malignant tumors.
然而CAR-T细胞治疗面临肿瘤抗原异质性表达、抗原丢失的难题。首先在急性淋巴细胞白血病中，CD19阴性细胞群可以逃避靶向CD19嵌合抗原受体T细胞的识别，导致细胞群的过度生长，产生疾病的复发。另外在胰腺癌、前列腺癌和神经母细胞瘤的临床前研究中，单抗原靶向的嵌合抗原受体T细胞并不能根除肿瘤，这是因为嵌合抗原受体T细胞不能识别这些肿瘤细胞表达的低密度的靶抗原（Anurathapan, U.;
Chan, R.C.; Hindi, H.F.; Mucharla, R.; Bajgain, P.; Hayes, B.C.; Fisher, W.E.;
Heslop, H.E.;Rooney, C.M.; Brenner, M.K.; et al. Kinetics of tumor destruction
by chimeric antigen receptor-modified t cells.
Mol. Ther. 2014,
22,
623–633. O’Rourke, D.M.; Nasrallah, M.P.; Desai, A.; Melenhorst, J.J.;
Mansfield, K.; Morrissette, J.J.D.; Martinez-Lage, M.; Brem, S.; Maloney, E.;
Shen, A.; et al. A single dose of peripherally infused egfrviii-directed car t
cells mediates antigen loss and induces adaptive resistance in patients with
recurrent glioblastoma.
Sci. Transl. Med. 2017,
9, eaaa0984）。在肝细胞癌中，血清中GPC3含量升高，抑制了靶向GPC3的嵌合抗原受体T细胞与细胞表面GPC3的结合，阻碍了嵌合抗原受体T细胞的疗效（Sun, L.,
et al., Shed antigen-induced blocking effect on CAR-T cells targeting
Glypican-3 in Hepatocellular Carcinoma. Journal for Immunotherapy of Cancer,
2021. 9(4)）。
However, CAR-T cell therapy faces the problems of heterogeneous expression of tumor antigens and loss of antigens. First, in acute lymphoblastic leukemia, CD19-negative cell populations can escape the recognition of CD19-targeted chimeric antigen receptor T cells, leading to overgrowth of cell populations and relapse of the disease. In addition, in preclinical studies of pancreatic cancer, prostate cancer, and neuroblastoma, chimeric antigen receptor T cells targeting single antigens failed to eradicate tumors because chimeric antigen receptor T cells could not recognize these tumor cells Low-density expression of target antigens (Anurathapan, U.; Chan, RC; Hindi, HF; Mucharla, R.; Bajgain, P.; Hayes, BC; Fisher, WE; Heslop, HE; Rooney, CM; Brenner, MK ; et al. Kinetics of tumor destruction by chimeric antigen receptor-modified t cells. Mol. Ther. 2014, 22 , 623–633. O'Rourke, DM; Nasrallah, MP; Desai, A.; Melenhorst, JJ; K.; Morrissette, JJD; Martinez-Lage, M.; Brem, S.; Maloney, E.; Shen, A.; et al. A single dose of peripherally infused egfrviii-directed car t cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci. Transl. Med. 2017, 9 , eaaa0984). In hepatocellular carcinoma, elevated levels of GPC3 in serum inhibit the binding of GPC3-targeting CAR T cells to cell surface GPC3, hindering the efficacy of CAR T cells (Sun, L., et al., Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for Immunotherapy of Cancer, 2021. 9(4)).
CD276又称为B7-H3，是属于B7家族的一类跨膜蛋白。CD276在多种癌症中高表达，包括肺腺癌、胶质瘤、神经母细胞瘤、胰腺癌、卵巢癌，在正常组织中几乎不表达。特异性识别CD276的单克隆抗体在多种肿瘤模型中介导肿瘤的有效清除。CD276在肝细胞癌高表达并且与肝癌的进展和肿瘤病人的不良预后相关，靶向CD276的CAR-T细胞在体外能有效的杀伤肝细胞癌细胞系。但单靶点的CAR-T细胞治疗实体瘤（例如肝细胞癌）面临抗原异质性表达的障碍。CD276, also known as B7-H3, is a type of transmembrane protein belonging to the B7 family. CD276 is highly expressed in a variety of cancers, including lung adenocarcinoma, glioma, neuroblastoma, pancreatic cancer, and ovarian cancer, and is hardly expressed in normal tissues. Monoclonal antibodies that specifically recognize CD276 mediate efficient tumor clearance in a variety of tumor models. CD276 is highly expressed in hepatocellular carcinoma and is associated with the progression of liver cancer and the poor prognosis of tumor patients. CAR-T cells targeting CD276 can effectively kill hepatocellular carcinoma cell lines in vitro. However, the single-target CAR-T cell therapy for solid tumors (such as hepatocellular carcinoma) faces the obstacle of heterogeneous expression of antigens.
为了解决以上的问题，本发明提供一种同时靶向GPC3和CD276的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。In order to solve the above problems, the present invention provides a chimeric antigen receptor targeting GPC3 and CD276 at the same time, chimeric antigen receptor T cells, preparation methods and applications thereof.
为达到上述目的，本发明采用了以下技术方案：To achieve the above object, the present invention adopts the following technical solutions:
第一方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体，该嵌合抗原受体命名为GPC3-CD276 TanCAR。In a first aspect, the present invention provides a chimeric antigen receptor targeting both GPC3 and CD276, and the chimeric antigen receptor is named GPC3-CD276 TanCAR.
优选的，所述嵌合抗原受体包括从氨基端到羧基端依次排列的靶向GPC3的单链抗体、连接子（linker）、靶向CD276的单链抗体、胞外铰链区、跨膜区、共刺激信号区以及CD3ζ胞内区。Preferably, the chimeric antigen receptor includes a single-chain antibody targeting GPC3, a linker, a single-chain antibody targeting CD276, an extracellular hinge region, and a transmembrane region arranged in sequence from the amino terminal to the carboxyl terminal , co-stimulatory signal region and CD3ζ intracellular region.
优选的，所述靶向GPC3的单链抗体的氨基酸序列如SEQ.ID.NO.3所示。Preferably, the amino acid sequence of the GPC3-targeting single-chain antibody is shown in SEQ.ID.NO.3.
优选的，所述靶向GPC3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.4所示。Preferably, the nucleotide sequence of the gene encoding the GPC3-targeting single-chain antibody is shown in SEQ.ID.NO.4.
优选的，所述连接子包括重复串联的G4S，例如氨基酸序列如SEQ.ID.NO.5所示的（G4S）3。Preferably, the linker includes repeating tandem G4S, for example (G4S)3 whose amino acid sequence is shown in SEQ.ID.NO.5.
优选的，所述连接子，例如（G4S）3的编码基因的核苷酸序列如SEQ.ID.NO.6所示。Preferably, the linker, for example, the nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6.
优选的，所述靶向CD276的单链抗体的氨基酸序列如SEQ.ID.NO.7所示。Preferably, the amino acid sequence of the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.7.
优选的，所述靶向CD276的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.8所示。Preferably, the nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8.
优选的，所述胞外铰链区选自CD8α铰链区、CD28铰链区、IgG1铰链区或IgG4铰链区。Preferably, the extracellular hinge region is selected from CD8α hinge region, CD28 hinge region, IgG1 hinge region or IgG4 hinge region.
优选的，所述CD8α铰链区的氨基酸序列如SEQ.ID.NO.9所示。Preferably, the amino acid sequence of the CD8α hinge region is shown in SEQ.ID.NO.9.
优选的，所述CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.10所示。Preferably, the nucleotide sequence of the gene encoding the hinge region of CD8α is shown in SEQ.ID.NO.10.
优选的，所述跨膜区选自CD8α跨膜区、CD4跨膜区、CD28跨膜区、CD3ζ跨膜区或ICOS跨膜区。Preferably, the transmembrane region is selected from CD8α transmembrane region, CD4 transmembrane region, CD28 transmembrane region, CD3ζ transmembrane region or ICOS transmembrane region.
优选的，所述CD8α跨膜区的氨基酸序列如SEQ.ID.NO.11所示。Preferably, the amino acid sequence of the CD8α transmembrane region is shown in SEQ.ID.NO.11.
优选的，所述CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.12所示。Preferably, the nucleotide sequence of the gene encoding the CD8α transmembrane region is shown in SEQ.ID.NO.12.
优选的，所述共刺激信号区选自4-1BB胞内区、CD28胞内区、OX40胞内区、ICOS胞内区或CD27胞内区。Preferably, the co-stimulatory signal region is selected from 4-1BB intracellular region, CD28 intracellular region, OX40 intracellular region, ICOS intracellular region or CD27 intracellular region.
优选的，所述4-1BB胞内区的氨基酸序列如SEQ.ID.NO.13所示。Preferably, the amino acid sequence of the 4-1BB intracellular region is shown in SEQ.ID.NO.13.
优选的，所述4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.14所示。Preferably, the nucleotide sequence of the gene encoding the 4-1BB intracellular region is shown in SEQ.ID.NO.14.
优选的，所述CD3ζ胞内区的氨基酸序列如SEQ.ID.NO.15所示。Preferably, the amino acid sequence of the intracellular region of CD3ζ is shown in SEQ.ID.NO.15.
优选的，所述CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.16所示。Preferably, the nucleotide sequence of the gene encoding the intracellular region of CD3ζ is shown in SEQ.ID.NO.16.
优选的，所述嵌合抗原受体包括如SEQ.ID.NO.1所示的氨基酸序列。Preferably, the chimeric antigen receptor includes the amino acid sequence shown in SEQ.ID.NO.1.
优选的，所述嵌合抗原受体的基因序列包括如SEQ.ID.NO.2所示的核苷酸序列。Preferably, the gene sequence of the chimeric antigen receptor includes the nucleotide sequence shown in SEQ.ID.NO.2.
第二方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体T细胞，该T细胞包括上述同时靶向GPC3和CD276的嵌合抗原受体（即T细胞表面具有同时靶向GPC3和CD276的嵌合抗原受体）。In a second aspect, the present invention provides a chimeric antigen receptor T cell targeting GPC3 and CD276 at the same time, the T cell includes the above-mentioned chimeric antigen receptor targeting GPC3 and CD276 at the same time (that is, the surface of the T cell has a simultaneous target chimeric antigen receptors to GPC3 and CD276).
第三方面，本发明提供了一种重组病毒载体及重组病毒，该重组病毒载体包括上述嵌合抗原受体的编码基因，该重组病毒采用所述重组病毒载体进行慢病毒包装而成。In a third aspect, the present invention provides a recombinant virus vector and a recombinant virus. The recombinant virus vector includes the gene encoding the above-mentioned chimeric antigen receptor, and the recombinant virus is packaged by lentivirus using the recombinant virus vector.
优选的，所述嵌合抗原受体的编码基因包括从5’端到3’端依次连接的信号肽的编码基因、靶向GPC3的单链抗体的编码基因、连接子（例如（G4S）3）的编码基因、靶向CD276的单链抗体的编码基因、胞外铰链区（例如CD8α胞外铰链区）的编码基因、跨膜区（例如CD8α跨膜区）的编码基因、共刺激信号区（例如4-1BB胞内区）的编码基因以及CD3ζ胞内区的编码基因。Preferably, the coding gene of the chimeric antigen receptor includes a signal peptide coding gene, a GPC3-targeting single-chain antibody coding gene, a linker (such as (G4S)3 ), coding genes of single-chain antibodies targeting CD276, coding genes of extracellular hinge region (such as CD8α extracellular hinge region), coding genes of transmembrane region (such as CD8α transmembrane region), co-stimulatory signal region (eg, 4-1BB intracellular region) and the gene encoding the CD3ζ intracellular region.
优选的，所述信号肽为CD8α信号肽，CD8α信号肽的编码基因的核苷酸序列如SEQ.ID.NO.19所示。Preferably, the signal peptide is a CD8α signal peptide, and the nucleotide sequence of the gene encoding the CD8α signal peptide is shown in SEQ.ID.NO.19.
优选的，所述CD8α信号肽的氨基酸序列如SEQ.ID.NO.20所示。Preferably, the amino acid sequence of the CD8α signal peptide is shown in SEQ.ID.NO.20.
优选的，所述重组病毒载体及重组病毒中，同时靶向GPC3和CD276的嵌合抗原受体的基因序列如SEQ.ID.NO.17所示，该基因序列包括所述嵌合抗原受体的编码基因的核苷酸序列。如SEQ.ID.NO.17所示的核苷酸序列与第一方面中如SEQ.ID.NO.2所示的核苷酸序列相比，增加了CD8α信号肽的编码基因。所述信号肽的编码基因可以较好地指导所述嵌合抗原受体表达到细胞表面。Preferably, the gene sequence of the chimeric antigen receptor targeting GPC3 and CD276 in the recombinant virus vector and the recombinant virus is shown in SEQ.ID.NO.17, and the gene sequence includes the chimeric antigen receptor The nucleotide sequence of the coding gene. Compared with the nucleotide sequence shown in SEQ.ID.NO.2 in the first aspect, the nucleotide sequence shown in SEQ.ID.NO.17 has a gene encoding CD8α signal peptide added. The gene encoding the signal peptide can better guide the expression of the chimeric antigen receptor to the cell surface.
优选的，所述嵌合抗原受体的氨基酸序列如SEQ.ID.NO.18所示，具体如下：Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ.ID.NO.18, specifically as follows:
MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNANTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTF TDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSL YLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSFLASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
优选的，所述重组病毒载体为pWPXLd慢病毒载体。Preferably, the recombinant viral vector is pWPXLd lentiviral vector.
第四方面，本发明提供了一种同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备方法，包括以下步骤：In a fourth aspect, the present invention provides a method for preparing chimeric antigen receptor T cells simultaneously targeting GPC3 and CD276, comprising the following steps:
1）通过基因克隆构建或人工合成同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276 TanCAR的基因序列，所述GPC3-CD276
TanCAR的基因序列包括如SEQ.ID.NO.2所示的核苷酸序列（例如GPC3-CD276 TanCAR的基因序列如SEQ.ID.NO.17所示）；1) Construct or artificially synthesize the gene sequence of the chimeric antigen receptor GPC3-CD276 TanCAR targeting both GPC3 and CD276 by gene cloning, the GPC3-CD276
The gene sequence of TanCAR includes the nucleotide sequence shown in SEQ.ID.NO.2 (for example, the gene sequence of GPC3-CD276 TanCAR is shown in SEQ.ID.NO.17);
2）将所述GPC3-CD276
TanCAR的基因序列插入到pWPXLd载体中，得到重组质粒pWPXLd-GPC3-CD276
TanCAR；2) The GPC3-CD276
The gene sequence of TanCAR was inserted into the pWPXLd vector to obtain the recombinant plasmid pWPXLd-GPC3-CD276
TanCAR;
3）将所述重组质粒pWPXLd-GPC3-CD276
TanCAR与包膜质粒、包装质粒共转染宿主细胞，得到重组慢病毒；3) The recombinant plasmid pWPXLd-GPC3-CD276
TanCAR co-transfects host cells with envelope plasmids and packaging plasmids to obtain recombinant lentiviruses;
4）将所述重组慢病毒转染CD3阳性T淋巴细胞（简称CD3阳性T细胞），经分离获得同时靶向GPC3和CD276的嵌合抗原受体T细胞。4) The recombinant lentivirus was transfected into CD3-positive T lymphocytes (CD3-positive T cells for short), and chimeric antigen receptor T cells targeting both GPC3 and CD276 were obtained after isolation.
优选的，所述包膜质粒为PMD2G，包装质粒为psPAX2，宿主细胞为HEK293T细胞。Preferably, the envelope plasmid is PMD2G, the packaging plasmid is psPAX2, and the host cell is HEK293T cells.
优选的，所述步骤4中，CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。Preferably, in step 4, the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells.
优选的，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血或胎盘血等。Preferably, the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood or placental blood.
第五方面，本发明提供了一种治疗肝细胞癌的药物，该药物包括上述同时靶向GPC3和CD276的嵌合抗原受体T细胞。In the fifth aspect, the present invention provides a drug for treating hepatocellular carcinoma, which includes the above-mentioned chimeric antigen receptor T cells targeting both GPC3 and CD276.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明所提出的同时靶向GPC3和CD276的嵌合抗原受体T细胞，不仅对表达GPC3和CD276的肿瘤细胞有杀伤作用，同时对GPC3阴性CD276阳性肿瘤细胞有杀伤活性，解决了抗原异质性表达或抗原丢失造成的嵌合抗原受体T细胞治疗肝细胞癌等实体瘤效果不佳的难题。The chimeric antigen receptor T cells that simultaneously target GPC3 and CD276 proposed by the present invention not only have a killing effect on tumor cells expressing GPC3 and CD276, but also have killing activity on GPC3-negative CD276-positive tumor cells, which solves the problem of antigen heterogeneity Chimeric antigen receptor T cells caused by sexual expression or antigen loss are not effective in treating solid tumors such as hepatocellular carcinoma.
图1为GPC3-CD276 TanCAR的编码基因的结构示意图。Figure 1 is a schematic diagram of the structure of the gene encoding GPC3-CD276 TanCAR.
图2为GPC3-CD276 TanCAR在T细胞上表达的流式分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3-CD276
TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞。Figure 2 shows the results of flow cytometric analysis of the expression of GPC3-CD276 TanCAR on T cells; among them: UTD group is CD3 positive T cells without transduction virus, GPC3-CD276
The TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276.
图3为同时靶向GPC3和CD276的嵌合抗原受体T细胞对Huh7细胞系的杀伤活性分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3-CD276
TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞。Figure 3 shows the results of the analysis of the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on Huh7 cell lines; among them: UTD group is CD3 positive T cells without transduction virus, GPC3-CD276
The TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276.
图4为同时靶向GPC3和CD276的嵌合抗原受体T细胞对SK-HEP-1-CD276细胞系杀伤活性分析结果；其中：UTD组为未转导病毒的CD3阳性T细胞，GPC3
CAR-T组为单靶点靶向GPC3的嵌合抗原受体T细胞，GPC3-CD276
TanCAR-T组为同时靶向GPC3和CD276的嵌合抗原受体T细胞；*表示差异显著。Figure 4 shows the results of the analysis of the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on SK-HEP-1-CD276 cell line; among them: UTD group is CD3 positive T cells without transduction virus, GPC3
The CAR-T group is chimeric antigen receptor T cells targeting GPC3 with a single target, GPC3-CD276
TanCAR-T group is chimeric antigen receptor T cells targeting both GPC3 and CD276; * indicates significant difference.
以下结合附图和实施例对本发明做进一步详细说明。所述实施例仅用于解释本发明，而非对本发明保护范围的限制。The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The examples are only used to explain the present invention, not to limit the protection scope of the present invention.
（一）同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备(1) Preparation of chimeric antigen receptor T cells targeting GPC3 and CD276 simultaneously
（1）制备同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276
TanCAR的基因序列(1) Preparation of chimeric antigen receptor GPC3-CD276 targeting both GPC3 and CD276
Gene sequence of TanCAR
参见图1，同时靶向GPC3和CD276的嵌合抗原受体GPC3-CD276
TanCAR的基因序列中，从5’端到3’端依次包括以下元件的编码基因：CD8α信号肽（CD8α SP）的编码基因、靶向GPC3的单链抗体（GPC3-scFv）的编码基因、连接子“（G4S）3”的编码基因、靶向CD276的单链抗体（CD276-scFv）的编码基因、CD8α铰链区（CD8α hinge）的编码基因、CD8α跨膜区（CD8α TM）的编码基因、4-1BB胞内区（4-1BB）的编码基因以及CD3ζ胞内区（CD3-zeta）的编码基因。See Figure 1, chimeric antigen receptor GPC3-CD276 targeting both GPC3 and CD276
In the gene sequence of TanCAR, from the 5' end to the 3' end, the encoding genes of the following elements are included in sequence: the encoding gene of CD8α signal peptide (CD8α SP), the encoding gene of single-chain antibody targeting GPC3 (GPC3-scFv), the linker The coding gene of "(G4S)3", the coding gene of CD276-targeting single-chain antibody (CD276-scFv), the coding gene of CD8α hinge region (CD8α hinge), the coding gene of CD8α transmembrane region (CD8α TM), The gene encoding the intracellular region of 4-1BB (4-1BB) and the gene encoding the intracellular region of CD3ζ (CD3-zeta).
所述CD8α信号肽的编码基因的核苷酸序列如SEQ.ID.NO.19所示、靶向GPC3的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.4所示、（G4S）3的编码基因的核苷酸序列如SEQ.ID.NO.6所示、靶向CD276的单链抗体的编码基因的核苷酸序列如SEQ.ID.NO.8所示、CD8α铰链区的编码基因的核苷酸序列如SEQ.ID.NO.10所示、CD8α跨膜区的编码基因的核苷酸序列如SEQ.ID.NO.12所示、4-1BB胞内区的编码基因的核苷酸序列如SEQ.ID.NO.14所示、CD3ζ胞内区的编码基因的核苷酸序列如SEQ.ID.NO.16所示。通过在CD3ζ胞内区的编码基因的3’端连接终止密码子，即得到所述嵌合抗原受体GPC3-CD276
TanCAR的基因序列，具体如SEQ.ID.NO.17所示。The nucleotide sequence of the gene encoding the CD8α signal peptide is shown in SEQ.ID.NO.19, the nucleotide sequence of the gene encoding the single-chain antibody targeting GPC3 is shown in SEQ.ID.NO.4, The nucleotide sequence of the gene encoding (G4S)3 is shown in SEQ.ID.NO.6, the nucleotide sequence of the gene encoding the single-chain antibody targeting CD276 is shown in SEQ.ID.NO.8, CD8α The nucleotide sequence of the gene encoding the hinge region is shown in SEQ.ID.NO.10, the nucleotide sequence of the gene encoding the CD8α transmembrane region is shown in SEQ.ID.NO.12, and the 4-1BB intracellular region The nucleotide sequence of the coding gene of CD3ζ is shown in SEQ.ID.NO.14, and the nucleotide sequence of the coding gene of CD3ζ intracellular region is shown in SEQ.ID.NO.16. The chimeric antigen receptor GPC3-CD276 is obtained by connecting a stop codon at the 3' end of the gene encoding the CD3ζ intracellular region
The gene sequence of TanCAR is specifically shown in SEQ.ID.NO.17.
所述嵌合抗原受体GPC3-CD276
TanCAR的基因序列由江苏金唯智生物技术有限公司进行基因合成。The chimeric antigen receptor GPC3-CD276
The gene sequence of TanCAR was synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd.
（2）构建重组质粒pWPXLd-GPC3-CD276
TanCAR (2) Construction of recombinant plasmid pWPXLd-GPC3-CD276
TanCAR
将合成的GPC3-CD276
TanCAR的基因序列插入到pWPXLd载体的BamH1和EcoR1酶切位点之间，然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经验证正确的质粒标记为重组质粒pWPXLd-GPC3-CD276
TanCAR用于后续实验。Synthetic GPC3-CD276
The gene sequence of TanCAR was inserted between the BamH1 and EcoR1 restriction sites of the pWPXLd vector, and then transformed into Escherichia coli competent cells DH5α for positive clone PCR identification and sequencing identification. The verified correct plasmid is labeled as recombinant plasmid pWPXLd-GPC3-CD276
TanCAR was used in follow-up experiments.
（3）重组慢病毒构建(3) Construction of recombinant lentivirus
将重组质粒pWPXLd-GPC3-CD276
TanCAR、包装质粒psPAX2及包膜质粒pMD2G使用lipofectamine3000转染试剂共转染入培养好的HEK293T细胞。第48h收集含病毒的上清：首先2000rpm室温离心培养体系5分钟，取上层清液，然后经0.45μm滤膜过滤，得到的重组慢病毒上清用于T细胞感染。The recombinant plasmid pWPXLd-GPC3-CD276
TanCAR, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine3000 transfection reagent. The virus-containing supernatant was collected at 48 hours: first, the culture system was centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was taken, and then filtered through a 0.45 μm filter membrane, and the obtained recombinant lentivirus supernatant was used for T cell infection.
（4）嵌合抗原受体T细胞的制备(4) Preparation of chimeric antigen receptor T cells
a)
PBMC(外周血单个核细胞)的分离a)
Isolation of PBMC (Peripheral Blood Mononuclear Cells)
PBMC的来源：健康志愿者的自体静脉血。Source of PBMC: autologous venous blood of healthy volunteers.
分离PBMC的操作流程：抽取所述健康志愿者血液，使用Ficoll收集外周血单个核细胞，离心分离后取中间层细胞；经PBS洗涤、计数后得到PBMC。The operation process for separating PBMCs: extract the blood of the healthy volunteers, use Ficoll to collect peripheral blood mononuclear cells, and take the middle layer cells after centrifugation; wash with PBS and count to obtain PBMCs.
b)
免疫磁珠法分离抗原特异性T淋巴细胞b)
Separation of Antigen-specific T Lymphocytes by Immunomagnetic Beads
取上述PBMC，加入含10%血清和适量IL-2的KBM581培养基，配成细胞悬液；按磁珠与细胞的数目比例为1:1，加入CD3/CD28免疫磁珠，室温下于摇床以20rpm的转速孵育45分钟；采用磁铁对孵育磁珠的细胞进行筛选，去除未吸附的细胞悬液后，加入上述KBM581培养基重悬磁珠-细胞混合物，得到CD3阳性T淋巴细胞，继续培养24小时后用于慢病毒感染。Take the above PBMC, add KBM581 medium containing 10% serum and appropriate amount of IL-2 to make a cell suspension; according to the ratio of the number of magnetic beads to cells is 1:1, add CD3/CD28 immunomagnetic beads, shake at room temperature The bed was incubated at a speed of 20 rpm for 45 minutes; the cells incubated with magnetic beads were screened with a magnet, and after removing the unadsorbed cell suspension, the above-mentioned KBM581 medium was added to resuspend the magnetic bead-cell mixture to obtain CD3 positive T lymphocytes, continue After 24 hours of culture, it was used for lentivirus infection.
c)
病毒转染法制备抗原特异性T淋巴细胞c)
Preparation of antigen-specific T lymphocytes by virus transfection
取上述经过免疫磁珠法分离得到的CD3阳性T淋巴细胞，加入与CD3阳性T淋巴细胞数相应的所述重组慢病毒进行培养。The CD3-positive T lymphocytes separated by the immunomagnetic bead method were taken, and the recombinant lentivirus corresponding to the number of CD3-positive T lymphocytes was added for culture.
培养的第3天，收集一定数量的感染重组慢病毒的CD3阳性T淋巴细胞，流式细胞术分析该细胞表面CAR的表达，结果如图2所示，与未转导病毒的CD3阳性T淋巴细胞相比，感染重组慢病毒的T细胞中GPC3-CD276 TanCAR有约14%的表面表达，表明同时靶向GPC3和CD276的嵌合抗原受体T细胞制备成功。继续培养48小时，收集同时靶向GPC3和CD276的嵌合抗原受体T细胞用于杀伤实验分析，或保存在细胞冻存液中，并放置于程序降温盒中-80℃保存24小时后转移至液氮罐长期保存。On the third day of culture, a certain number of CD3-positive T lymphocytes infected with the recombinant lentivirus were collected, and the expression of CAR on the surface of the cells was analyzed by flow cytometry. Compared with cells, about 14% of GPC3-CD276 TanCAR was expressed on the surface of T cells infected with recombinant lentivirus, indicating that chimeric antigen receptor T cells targeting both GPC3 and CD276 were successfully prepared. Continue to culture for 48 hours, collect chimeric antigen receptor T cells targeting both GPC3 and CD276 for killing assay analysis, or store in cell cryopreservation solution, and place in a programmed cooling box at -80°C for 24 hours before transfer Long-term storage in liquid nitrogen tanks.
（二）使用RTCA系统分析同时靶向GPC3和CD276的嵌合抗原受体T细胞对肝癌细胞系的杀伤活性(2) RTCA system was used to analyze the killing activity of chimeric antigen receptor T cells targeting GPC3 and CD276 on liver cancer cell lines
2.1
实验1对照及效应细胞分组2.1
Experiment 1 Control and Effector Cell Grouping
按照针对靶细胞Huh7（同时表达GPC3和CD276的肝癌细胞系）所加入的效应细胞不同分为：UTD组（加入未转导病毒的CD3阳性T淋巴细胞）和GPC3-CD276
TanCAR-T组（同时靶向GPC3和CD276的嵌合抗原受体T细胞），同时设置对照组（control，培养基组）。According to the different effector cells added to the target cell Huh7 (a liver cancer cell line expressing GPC3 and CD276 at the same time), it is divided into: UTD group (CD3 positive T lymphocytes added with untransduced virus) and GPC3-CD276
TanCAR-T group (chimeric antigen receptor T cells targeting both GPC3 and CD276), and a control group (control, medium group).
2.2
实验2对照及效应细胞分组2.2
Experiment 2 Control and Effector Cell Grouping
按照针对靶细胞SK-HEP-1-CD276(过表达CD276而GPC3不表达的肝癌细胞系)所加入的效应细胞不同分为：UTD组（加入未转导病毒的CD3阳性T淋巴细胞）、GPC3 CAR-T组（对应嵌合抗原受体GPC3 CAR的基因序列与GPC3-CD276Tan CAR相比缺少linker-CD276-scFv；CAR-T细胞生产方式一致，GPC3 CAR表达阳性率约为30%）、GPC3-CD276
TanCAR-T组（同时靶向GPC3和CD276的嵌合抗原受体T细胞），同时设置对照组（control，培养基组）。According to the different effector cells added to the target cell SK-HEP-1-CD276 (a liver cancer cell line that overexpresses CD276 but does not express GPC3), it is divided into: UTD group (CD3 positive T lymphocytes added with untransduced virus), GPC3 CAR-T group (the gene sequence corresponding to chimeric antigen receptor GPC3 CAR lacks linker-CD276-scFv compared with GPC3-CD276Tan CAR; the production method of CAR-T cells is the same, and the positive rate of GPC3 CAR expression is about 30%), GPC3 -CD276
TanCAR-T group (chimeric antigen receptor T cells targeting both GPC3 and CD276), and a control group (control, medium group).
2.3
实验操作2.3
Experimental operation
首先用50 μL DMEM或RPMI1640培养基进行RTCA单板的平衡，然后收集培养好的靶细胞Huh7或SK-HEP-1-CD276，细胞计数后在每个孔中加入含有5000个靶细胞的50 μL细胞悬液，37℃培养箱中放置15分钟，然后放置在RTCA电阻系统中，24小时后按不同的效应细胞（例如收集的GPC3-CD276 TanCAR-T细胞）与靶细胞的比例（E:T）20:1、10:1、5:1，把相应细胞数目的效应细胞悬液100μL加入到靶细胞中，每组至少设置2个复孔，然后放入RTCA电阻系统中，在一定时间后进行杀伤活性分析。First use 50 μL of DMEM or RPMI1640 medium to balance the RTCA single plate, then collect the cultured target cells Huh7 or SK-HEP-1-CD276, and add 50 μL of 5000 target cells to each well after cell counting The cell suspension was placed in a 37°C incubator for 15 minutes, and then placed in the RTCA resistance system. After 24 hours, the ratio of different effector cells (such as collected GPC3-CD276 TanCAR-T cells) to target cells (E:T ) 20:1, 10:1, 5:1, add 100 μL of effector cell suspension corresponding to the number of cells into the target cells, set at least 2 duplicate wells for each group, and then put them into the RTCA resistance system, after a certain period of time Assays for killing activity were performed.
2.4
结果分析2.4
Result analysis
使用指定时间点的细胞指数（cell index）进行效应细胞杀伤率分析。Effector cell killing rate analysis was performed using the cell index at the indicated time points.
所述UTD组杀伤率计算公式：The formula for calculating the killing rate of the UTD group:
UTD杀伤率=（control
cell
index-UTD
cell index）/control
cell index×100
UTD kill rate = (control cell index -UTD cell index )/control cell index × 100
所述GPC3-CD276 TanCAR-T组、GPC3 CAR-T组杀伤率计算公式：The formula for calculating the killing rate of the GPC3-CD276 TanCAR-T group and the GPC3 CAR-T group:
CAR-T杀伤率=（control
cell
index -CAR-T
cell index）/control
cell index×100
CAR-T killing rate = (control cell index - CAR-T cell index )/control cell index × 100
实验结果表明（图3），GPC3-CD276
TanCAR-T与UTD相比（杀伤24小时），同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3阳性CD276阳性肝癌细胞系Huh7有更强的杀伤活性，杀伤效应在效靶比（E:T）为10:1、20:1时较优。The experimental results showed (Figure 3), GPC3-CD276
Compared with UTD (killed for 24 hours), TanCAR-T and chimeric antigen receptor T cells targeting both GPC3 and CD276 had stronger killing activity against the GPC3-positive CD276-positive liver cancer cell line Huh7, and the killing effect was at the target ratio ( E:T) is better when it is 10:1 or 20:1.
另外实验结果还表明（图4），在效靶比20:1时，GPC3-CD276 TanCAR-T与GPC3 CAR-T相比（杀伤20小时），同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3阴性CD276阳性肝癌细胞系SK-HEP-1-CD276有更强的杀伤效果。In addition, the experimental results also showed (Figure 4) that when the effect-to-target ratio was 20:1, GPC3-CD276 TanCAR-T, compared with GPC3 CAR-T (killed for 20 hours), simultaneously targeted the chimeric antigen receptors of GPC3 and CD276 T cells have a stronger killing effect on the GPC3-negative CD276-positive liver cancer cell line SK-HEP-1-CD276.
总之，本发明构建的同时靶向GPC3和CD276的嵌合抗原受体T细胞对GPC3和CD276阳性肿瘤细胞系有较强的杀伤活性，并且与GPC3 CAR-T相比，对GPC3阴性CD276阳性肿瘤细胞系有更强的杀伤效果，克服了GPC3异质性表达导致的肿瘤抗原逃逸的问题。因此所述同时靶向GPC3和CD276的嵌合抗原受体T细胞在肝细胞癌等实体瘤的治疗中有较大的应用前景。In conclusion, the chimeric antigen receptor T cells targeting both GPC3 and CD276 constructed by the present invention have stronger killing activity against GPC3 and CD276 positive tumor cell lines, and compared with GPC3 CAR-T, they are more effective against GPC3 negative CD276 positive tumors. The cell line has a stronger killing effect and overcomes the problem of tumor antigen escape caused by the heterogeneous expression of GPC3. Therefore, the chimeric antigen receptor T cells targeting GPC3 and CD276 at the same time have great application prospects in the treatment of solid tumors such as hepatocellular carcinoma.
Claims (10)
- 一种同时靶向GPC3和CD276的嵌合抗原受体，其特征在于：该嵌合抗原受体包括靶向GPC3的单链抗体、连接子、靶向CD276的单链抗体、胞外铰链区、跨膜区、共刺激信号区以及CD3ζ胞内区。 A chimeric antigen receptor targeting GPC3 and CD276 simultaneously, characterized in that: the chimeric antigen receptor includes a single-chain antibody targeting GPC3, a linker, a single-chain antibody targeting CD276, an extracellular hinge region, Transmembrane region, co-stimulatory signal region and CD3ζ intracellular region.
- 根据权利要求1所述一种同时靶向GPC3和CD276的嵌合抗原受体，其特征在于：所述胞外铰链区选自CD8α铰链区、CD28铰链区、IgG1铰链区或IgG4铰链区。 A chimeric antigen receptor targeting GPC3 and CD276 simultaneously according to claim 1, characterized in that: the extracellular hinge region is selected from a CD8α hinge region, a CD28 hinge region, an IgG1 hinge region or an IgG4 hinge region.
- 根据权利要求1所述一种同时靶向GPC3和CD276的嵌合抗原受体，其特征在于：所述跨膜区选自CD8α跨膜区、CD4跨膜区、CD28跨膜区、CD3ζ跨膜区或ICOS跨膜区。 A chimeric antigen receptor targeting GPC3 and CD276 simultaneously according to claim 1, wherein the transmembrane region is selected from the group consisting of CD8α transmembrane region, CD4 transmembrane region, CD28 transmembrane region, and CD3ζ transmembrane region region or ICOS transmembrane region.
- 根据权利要求1所述一种同时靶向GPC3和CD276的嵌合抗原受体，其特征在于：所述共刺激信号区选自4-1BB胞内区、CD28胞内区、OX40胞内区、ICOS胞内区或CD27胞内区。 A chimeric antigen receptor targeting GPC3 and CD276 simultaneously according to claim 1, wherein the co-stimulatory signal region is selected from the group consisting of 4-1BB intracellular region, CD28 intracellular region, OX40 intracellular region, ICOS intracellular region or CD27 intracellular region.
- 一种同时靶向GPC3和CD276的嵌合抗原受体T细胞，其特征在于：该T细胞包括如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体。 A chimeric antigen receptor T cell targeting GPC3 and CD276 simultaneously, characterized in that: the T cell comprises the chimeric antigen receptor targeting GPC3 and CD276 simultaneously according to claim 1.
- 一种重组病毒载体，其特征在于：该重组病毒载体包括如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体的对应编码基因。 A recombinant virus vector, characterized in that: the recombinant virus vector comprises the corresponding coding gene of the chimeric antigen receptor targeting GPC3 and CD276 as claimed in claim 1.
- 根据权利要求6所述一种重组病毒载体，其特征在于：所述重组病毒载体中，同时靶向GPC3和CD276的嵌合抗原受体的基因序列如SEQ.ID.NO.17所示。 The recombinant viral vector according to claim 6, characterized in that: in the recombinant viral vector, the gene sequence of the chimeric antigen receptor targeting GPC3 and CD276 is shown in SEQ.ID.NO.17.
- 一种同时靶向GPC3和CD276的嵌合抗原受体T细胞的制备方法，其特征在于：包括以下步骤： A method for preparing chimeric antigen receptor T cells targeting GPC3 and CD276 simultaneously, characterized in that it comprises the following steps:1）将如权利要求1所述的同时靶向GPC3和CD276的嵌合抗原受体的对应编码基因插入到pWPXLd载体中，得到重组质粒；1) Insert the corresponding coding gene of the chimeric antigen receptor targeting GPC3 and CD276 as claimed in claim 1 into the pWPXLd vector to obtain a recombinant plasmid;2）将所述重组质粒与包膜质粒、包装质粒共转染，得到重组慢病毒；2) co-transfecting the recombinant plasmid with the envelope plasmid and the packaging plasmid to obtain a recombinant lentivirus;3）将所述重组慢病毒转染CD3阳性T淋巴细胞，经分离获得同时靶向GPC3和CD276的嵌合抗原受体T细胞。3) The recombinant lentivirus was transfected into CD3-positive T lymphocytes, and chimeric antigen receptor T cells targeting GPC3 and CD276 were obtained after isolation.
- 一种治疗肝细胞癌的药物，其特征在于：该药物包括如权利要求5所述的同时靶向GPC3和CD276的嵌合抗原受体T细胞。 A medicine for treating hepatocellular carcinoma, characterized in that the medicine comprises chimeric antigen receptor T cells targeting GPC3 and CD276 as claimed in claim 5.
- 一种如权利要求5所述的同时靶向GPC3和CD276的嵌合抗原受体T细胞在制备抗肿瘤药物中的应用。 An application of a chimeric antigen receptor T cell targeting GPC3 and CD276 as claimed in claim 5 in the preparation of antitumor drugs.
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