WO2021093251A1 - 一种靶向fgfr4和dr5的嵌合抗原受体t细胞及其制备方法和应用 - Google Patents

一种靶向fgfr4和dr5的嵌合抗原受体t细胞及其制备方法和应用 Download PDF

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WO2021093251A1
WO2021093251A1 PCT/CN2020/081368 CN2020081368W WO2021093251A1 WO 2021093251 A1 WO2021093251 A1 WO 2021093251A1 CN 2020081368 W CN2020081368 W CN 2020081368W WO 2021093251 A1 WO2021093251 A1 WO 2021093251A1
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fgfr4
amino acid
acid sequence
car
chain antibody
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French (fr)
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许晨光
张宏玲
温丽联
黎琴子
刘婉
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深圳宾德生物技术有限公司
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Definitions

  • the present invention claims the priority of the prior application of the application number 201911120424.0 filed on November 15, 2019 under the title "A chimeric antigen receptor T cell targeting FGFR4 and DR5 and its preparation method and application".
  • the content of the first application is incorporated into this text by way of introduction.
  • the invention relates to the field of medical biology, in particular to a chimeric antigen receptor T cell targeting FGFR4 and DR5, and a preparation method and application thereof.
  • liver cancer is one of the most common malignant tumors and the second leading cause of death from cancer in the world. It has seriously threatened people's health and lives.
  • the common treatment methods for liver cancer in clinic mainly include surgical resection, liver transplantation, chemotherapy and radiotherapy.
  • liver transplantation mainly include surgical resection, liver transplantation, chemotherapy and radiotherapy.
  • the existing commonly used anti-tumor drugs can effectively prolong the survival period of patients, they generally have problems such as low selectivity and large toxic side effects in clinical applications.
  • targeted drugs have the problem of easily inducing tumor drug resistance after long-term use. For example, sorafenib, a multi-kinase inhibitor approved in 2007, only improved overall survival by 3 months. It can be seen that finding a safe and effective treatment for liver cancer clinically is still a difficult problem.
  • Death receptor 5 also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2)
  • TRAIL-R2 tumor necrosis factor-related apoptosis-inducing ligand receptor 2
  • FGFR1-4 fibroblast growth factor receptor family
  • FGFR4 is different from other members at amino acid 552.
  • FGFR4 is cysteine, while other members are tyrosine at this position.
  • FGFR4 is overexpressed in a variety of tumors, such as liver cancer, breast cancer, and gastric cancer.
  • FGFR4 inhibitor H3B-6527 has obtained orphan drug designation granted by the US FDA.
  • FGFR4 selective inhibitors are basically covalent reversible or irreversible inhibitors based on Cys552, once this site mutation is likely to lead to the emergence of drug resistance.
  • CAR-T Chimeric antigen receptor T cell technology
  • the present invention provides a chimeric antigen receptor T cell targeting FGFR4 and DR5, including the chimeric antigen receptor CAR-FGFR4 targeting FGFR4 and the chimeric antigen receptor CAR-DR5 targeting DR5 , CAR-FGFR4 specifically targets FGFR4, CAR-DR5 specifically targets DR5, thereby promoting the expansion of T cells in patients, killing tumor cells efficiently and specifically, and effectively inhibiting the occurrence of tumor cell escape , Better maintain the vitality and lethality of chimeric antigen receptor T cells, and will not cause damage to normal cells.
  • the invention also provides a preparation method and application of chimeric antigen receptor T cells targeting FGFR4 and DR5.
  • the present invention provides a chimeric antigen receptor T cell targeting FGFR4 and DR5, including a chimeric antigen receptor CAR-FGFR4 targeting FGFR4 and a chimeric antigen receptor CAR-DR5 targeting DR5
  • the amino acid sequence of the CAR-FGFR4 includes the amino acid sequence of the single-chain antibody targeting FGFR4, the extracellular hinge region, the transmembrane region, and the intracellular signal region, which are sequentially linked from the amino terminal to the carboxyl terminal, and the CAR-DR5
  • the amino acid sequence of includes the amino acid sequence of the DR5-targeting single-chain antibody, the extracellular hinge region, the transmembrane region and the intracellular signal region that are sequentially connected from the amino terminal to the carboxy terminal;
  • amino acid sequence of the single-chain antibody targeting FGFR4 includes at least one of the following:
  • the amino acid sequence of the single-chain antibody targeting FGFR4 includes the amino acid sequence of the single-chain antibody heavy chain VH shown in SEQ ID NO: 1 and the single-chain antibody light chain antibody shown in SEQ ID NO: 2
  • the amino acid sequence of the chain VL, or the amino acid sequence of the single-chain antibody targeting FGFR4 includes the amino acid sequence of the heavy chain VH of the single-chain antibody as shown in SEQ ID NO: 3 and the single-chain antibody as shown in SEQ ID NO: 4
  • the amino acid sequence of the antibody light chain VL, or the amino acid sequence of the single-chain antibody targeting FGFR4 includes the amino acid sequence of the single-chain antibody heavy chain VH shown in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH shown in SEQ ID NO: 2
  • the single chain antibody heavy chain VH shown in SEQ ID NO: 1 and the single chain antibody light chain VL shown in SEQ ID NO: 2 are sequentially connected to form a single chain antibody targeting FGFR4, such as
  • the single chain antibody heavy chain VH shown in SEQ ID NO: 3 and the single chain antibody light chain VL shown in SEQ ID NO: 4 are sequentially connected to form a single chain antibody targeting FGFR4.
  • amino acid sequence of the heavy chain VH of the single-chain antibody as shown in SEQ ID NO: 1 and the amino acid sequence of the light chain VL of the single-chain antibody as shown in SEQ ID NO: 2 pass through the first connecting peptide connection.
  • amino acid sequence of the heavy chain VH of the single-chain antibody as shown in SEQ ID NO: 3 and the amino acid sequence of the light chain VL of the single-chain antibody as shown in SEQ ID NO: 4 pass through a second connecting peptide connection.
  • the first connecting peptide and the second connecting peptide are used to connect the amino acid sequence of the heavy chain VH of the single-chain antibody and the amino acid sequence of the light chain VL of the single-chain antibody to make the linked single-chain antibody heavy chain
  • the light chain and the light chain maintain their respective spatial conformations to maintain the function and activity of the overall single-chain antibody.
  • the first connecting peptide and the second connecting peptide can be, but are not limited to, a polypeptide sequence mainly composed of glycine and serine.
  • glycine has the smallest molecular weight and is the amino acid with the shortest side chain, which can increase The flexibility of the side chain; Serine is the most hydrophilic amino acid, which can increase the hydrophilicity of the peptide chain.
  • the amino acid sequence of the first connecting peptide is GGGGSGGGGSGGGGS
  • the amino acid sequence of the second connecting peptide is GGGGSGGGGSGGGGS.
  • connection sequence of the amino acid sequence of the single-chain antibody heavy chain VH, the single-chain antibody light chain VL and the connecting peptide linker from the amino acid end to the carboxyl end is VH-linker-VL and/or VL-linker-VH.
  • amino acid sequence of the single-chain antibody targeting FGFR4 includes at least one of the following situations:
  • the sequential position relationship of the single-chain antibody light chain VL and the single-chain antibody heavy chain VH does not affect the targeting and activity of the single-chain antibody.
  • amino acid sequence of the single-chain antibody targeting FGFR4 includes any amino acid sequence shown in SEQ ID NO: 6-7.
  • the encoding gene of the single-chain antibody targeting FGFR4 includes a nucleotide sequence corresponding to the amino acid sequence of the single-chain antibody targeting FGFR4.
  • amino acid sequence of the single-chain antibody targeting DR5 includes the amino acid sequence shown in SEQ ID NO: 5.
  • the "sequential connection from the amino terminus to the carboxy terminus” specifically means: the carboxy terminus of the amino acid sequence of the single chain antibody is connected to the amino terminus of the amino acid sequence of the extracellular hinge region, and the The carboxy terminus of the amino acid sequence of the region is connected to the amino terminus of the amino acid sequence of the transmembrane region, and the carboxy terminus of the amino acid sequence of the transmembrane region is connected to the amino terminus of the amino acid sequence of the intracellular signal region.
  • the extracellular hinge region is used to promote the binding of the single-chain antibody targeting FGFR4 to FGFR4 on the tumor.
  • the extracellular hinge region includes one or a combination of CD8 ⁇ hinge region, CD28 hinge region, CD4 hinge region, CD5 hinge region, CD134 hinge region, CD137 hinge region, and ICOS hinge region. Further optionally, the extracellular hinge region includes a CD8 ⁇ hinge region.
  • the transmembrane region is used to immobilize the FGFR4-targeting chimeric antigen receptor CAR-FGFR4.
  • the transmembrane region includes one or a combination of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region, and a CD28 transmembrane region. Further optionally, the transmembrane region includes a CD8 transmembrane region.
  • the intracellular signal region is used to provide signals for T cell activation, maintain the survival time of T cells and activate T cell proliferation signal pathways.
  • the intracellular signal area includes any of the 4-1BB signal area, the CD3 ⁇ signal area, the ICOS signal area, the CD27 signal area, the OX40 signal area, the CD28 signal area, the IL1R1 signal area, the CD70 signal area, and the TNFRSF19L signal area.
  • the intracellular signal area includes a 4-1BB signal area and a CD3 ⁇ signal area.
  • the hinge region, transmembrane region, and signal region in the CAR-FGFR4 and the CAR-DR5 can be the same or different, and the selection can be made according to actual needs.
  • the amino acid sequence of the CAR-FGFR4 includes FGFR4-targeting single-chain antibody, CD8 ⁇ hinge region, CD8 transmembrane region, 4-1BB signal region and CD3 ⁇ signal region which are sequentially connected from the amino terminal to the carboxy terminal. Amino acid sequence.
  • amino acid sequence of CAR-FGFR4 includes any amino acid sequence shown in SEQ ID NO: 8-9.
  • connection sequence of the single-chain antibody heavy chain VH and the single-chain antibody light chain VL in the single-chain antibody targeting FGFR4 does not affect the targeting and activity of CAR-FGFR4, and the prepared CAR-FGFR4 has targeting The role of FGFR4.
  • the encoding gene of the FGFR4 targeting chimeric antigen receptor CAR-FGFR4 includes the nucleotide sequence corresponding to the amino acid sequence of the FGFR4 targeting chimeric antigen receptor CAR-FGFR4.
  • the amino acid sequence of the CAR-DR5 includes a single chain antibody targeting DR5, a CD8 ⁇ hinge region, a CD8 transmembrane region, a 4-1BB signal region, and a CD3 ⁇ signal region connected sequentially from the amino terminal to the carboxy terminal. Amino acid sequence.
  • amino acid sequence of the CAR-DR5 includes the amino acid sequence shown in SEQ ID NO: 10.
  • the CAR-DR5 encoding gene includes a nucleotide sequence corresponding to the amino acid sequence of the chimeric antigen receptor CAR-DR5 targeting DR5.
  • the chimeric antigen receptor T cell targeting FGFR4 and DR5 may be a dual-target chimeric antigen receptor T cell with CAR-FGFR4 and CAR-DR5 (ie, FGFR4 and DR5 targeting FGFR4 and DR5).
  • Dual target chimeric antigen receptor T cells or a mixture of CAR-FGFR4 chimeric antigen receptor T cells and CAR-DR5 chimeric antigen receptor T cells, or CAR-FGFR4 and CAR -DR5 dual-target chimeric antigen receptor T cells mixed with at least one of CAR-FGFR4 chimeric antigen receptor T cells and CAR-DR5 chimeric antigen receptor T cells.
  • chimeric antigen receptor T cells can recognize tumor cells that express FGFR4 antigen protein on their surface, and they can also recognize tumor cells that express DR5 antigen protein on their surface.
  • FGFR4 antigen protein can also recognize tumor cells that express DR5 antigen protein on their surface.
  • DR5 antigen protein can also recognize tumor cells that express DR5 antigen protein on their surface.
  • the recognition is also good, which can effectively avoid the immune escape phenomenon of tumor cells.
  • the chimeric antigen receptor T cell targeting FGFR4 and DR5 is a dual-target chimeric antigen receptor T cell with CAR-FGFR4 and CAR-DR5
  • the position distribution of CAR-FGFR4 and CAR-DR5 Not limited.
  • the CAR-FGFR4 and the CAR-DR5 are alternately distributed. There is no chemical bond between the CAR-FGFR4 single-chain antibody and the CAR-DR5 single-chain antibody to ensure better recognition ability.
  • the chimeric antigen receptor T cells targeting FGFR4 provided in the first aspect of the present invention can specifically target tumor cells expressing FGFR4. After CAR-FGFR4 is combined with FGFR4, the intracellular signal region of T cells is activated. Promote the expansion of T cells in the patient's body, and efficiently and specifically kill tumor cells, while causing almost no damage to normal cells.
  • the present invention provides a recombinant vector comprising the CAR-FGFR4 and CAR-DR5 codes in the chimeric antigen receptor T cells targeting FGFR4 and DR5 as described in the first aspect gene.
  • the CAR-FGFR4 and CAR-DR5 encoding genes may be contained in the same recombinant vector, or the CAR-FGFR4 and CAR-DR5 encoding genes may be contained in different vectors.
  • the vector can be, but is not limited to, a gene delivery vector.
  • the vector is at least one of a viral vector and a non-viral vector.
  • the non-viral vectors include plasmid vectors and phage vectors.
  • the plasmid vector can be, but is not limited to, eukaryotic plasmid vector, prokaryotic plasmid vector, minicircle DNA, transposon and the like.
  • the vector is minicircle DNA
  • the recombinant minicircle DNA inserted into the coding gene of the chimeric antigen receptor CAR-FGFR4 targeting FGFR4 can be directly transfected into CD3-positive T lymphocytes to prepare a chimera targeting FGFR4 Antigen receptor T cells.
  • the viral vector includes a lentiviral vector, an adenoviral vector or a retroviral vector. Furthermore, the viral vector is a lentiviral vector.
  • the recombinant vector provided by the second aspect of the present invention is safe and efficient, can stably realize the introduction or replication of CAR-FGFR4 and CAR-DR5 encoding genes into host cells, and can be used for the preparation of chimeric antigen receptor T cells.
  • the present invention provides a host cell, which includes the recombinant vector as described in the second aspect.
  • the host cell can be used to assemble the recombinant viral vector to make it infectious.
  • the host cells may include HEK293T cells, 293 cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells or COS7 cells, etc., but are not limited thereto.
  • the host cell is HEK293T cell.
  • the host cell is a corresponding eukaryotic host cell or a prokaryotic host cell.
  • the host cell provided in the third aspect of the present invention can stably store the CAR-FGFR4 and CAR-DR5 encoding genes that target FGFR4 and DR5, and is beneficial to the preparation of chimeric antigen receptor T cells that target FGFR4 and DR5.
  • the present invention provides a method for preparing chimeric antigen receptor T cells targeting FGFR4 and DR5, including:
  • the first recombinant lentivirus and the second recombinant lentivirus are jointly transfected into CD3-positive T lymphocytes sequentially or simultaneously, and chimeric antigen receptor T cells targeting FGFR4 and DR5 are obtained after isolation .
  • the above-mentioned "sequential connection from the 5'end to the 3'end” specifically means: the 3'end of the signal peptide encoding gene sequence is connected to the 5'end of the single-chain antibody encoding gene, and the single-chain antibody encoding gene
  • the 3'end of the gene encoding the extracellular hinge region is connected to the 5'end of the gene encoding the extracellular hinge region
  • the 3'end of the gene encoding the extracellular hinge region is connected to the 5'end of the gene encoding the transmembrane region.
  • the 3'end of the coding gene of the transmembrane region is connected to the 5'end of the coding gene of the intracellular signal region.
  • the signal peptide is used to guide the expression of the chimeric antigen receptor CAR-FGFR4 on the cell surface, and the signal peptide is cleaved by a signal peptidase during protein translation and maturation.
  • amino acid sequence of the signal peptide includes the amino acid sequence shown in SEQ ID NO: 11.
  • the CAR-FGFR4 encoding gene includes a signal peptide encoding gene that is sequentially connected from 5'end to 3'end, a single-chain antibody encoding gene targeting FGFR4, a CD8 ⁇ hinge region encoding gene, and CD8 The coding gene of the transmembrane region, the coding gene of the 4-1BB signal region and the coding gene of the CD3 ⁇ signal region.
  • the CAR-FGFR4 encoding gene includes a nucleotide sequence corresponding to any amino acid sequence shown in SEQ ID NO: 8-9.
  • the CAR-DR5 encoding gene includes a signal peptide encoding gene that is sequentially connected from 5'end to 3'end, a single chain antibody encoding gene targeting DR5, a CD8 ⁇ hinge region encoding gene, and CD8
  • the CAR-DR5 encoding gene includes a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 10.
  • the first gene delivery vector and the second gene delivery vector can be the same or different, and can be selected according to actual needs.
  • the encoding gene of CAR-FGFR4 and the encoding gene of CAR-DR5 can be inserted into different genes.
  • the same gene delivery vector can also be inserted.
  • the connection sequence of the CAR-FGFR4 encoding gene and the CAR-DR5 encoding gene is not limited.
  • the CAR-FGFR4 encoding gene is inserted between the BamH I and EcoR I restriction sites in the pCDH-EF1-MCS vector, and is located after the EF1 ⁇ of the pCDH-EF1-MCS vector, and starts with EF1 ⁇ child.
  • the 5'end of the CAR-FGFR4 coding gene can be added with a start codon (such as ATG) and BamH I in the pCDH-EF1-MCS vector
  • a stop codon such as TAA
  • the CAR-DR5 encoding gene is inserted between the BamH I and EcoR I restriction sites in the pCDH-EF1-MCS vector, and is located after the EF1 ⁇ of the pCDH-EF1-MCS vector, starting with EF1 ⁇ child.
  • the 5'end of the CAR-DR5 encoding gene can be added with a start codon (such as ATG) and BamH I in the pCDH-EF1-MCS vector
  • a stop codon such as TAA
  • the first recombinant gene delivery vector and the second recombinant gene delivery vector when packaged, they can be packaged separately or at the same time.
  • the first recombinant gene delivery vector and the second recombinant gene delivery vector are packaged to obtain the first recombinant lentivirus with the CAR-FGFR4 encoding gene and the second recombinant lentivirus with the CAR-DR5 encoding gene.
  • Viruses including:
  • the first recombinant gene delivery vector, the envelope plasmid and the packaging plasmid are co-transfected into host cells to obtain the first recombinant lentivirus; and the second recombinant gene delivery vector is co-transfected with the envelope plasmid and the packaging plasmid Infect the host cell to obtain the second recombinant lentivirus.
  • the envelope plasmid is PMD2G
  • the packaging plasmid is psPAX2
  • the host cell is a HEK293T cell.
  • the envelope plasmid PMD2G encodes the vesicular stomatitis virus glycoprotein capsid, which assists the recombinant lentivirus to adhere to the cell membrane and maintains the infectivity of the recombinant lentivirus.
  • the recombinant lentivirus of the present invention may further contain envelope proteins from other viruses.
  • the protein is preferably a viral envelope protein that infects human cells.
  • this protein such as retrovirus facultative virus hand skin membrane protein, etc.
  • an envelope protein derived from mouse leukemia virus (MuMLV) 4070A strain can be used.
  • the envelope protein from MuMLV 10Al can also be used.
  • proteins of the herpesvirus family such as the gB, gD, gH, and gp85 proteins of the herpes simplex virus, and the gp350 and gp220 proteins of the Epstein-Barr virus.
  • a protein of the Hepatoviridae family such as the S protein of hepatitis B virus.
  • the envelope protein can also be formed after the measles virus glycoprotein is fused with other single-chain antibodies.
  • the packaging of recombinant lentivirus usually adopts transient transfection or cell line packaging.
  • Human cell lines that can be used as packaging cells during transient transfection such as 293 cells, 293T cells, 293FT cells, 293LTV cells, 293EBNA cells and other clones isolated from 293 cells; SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, TE671 cells, etc.
  • Cell lines derived from monkeys for example, COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, etc. can also be used.
  • the commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also frequently used.
  • Recombinant lentivirus packaging also uses some lentiviral packaging cell lines, such as stable cell lines produced using the most common Env glycoprotein, VSVG protein or HIV-1 gag-pol protein.
  • large-scale lentiviral vector systems all adopt the method of segmenting the genome, which means that genes with different auxiliary functions are located on different plasmids.
  • genes with different auxiliary functions are located on different plasmids.
  • there are four-plasmid system encoding gag-pol gene, Rev gene, VSVG gene, and SIN transfer gene are located in four different plasmids
  • three-plasmid system the plasmid encoding Rev gene is removed, and the gag-pol plasmid is gag-pol.
  • the pol gene uses codons that are preferred in human cells) and a two-plasmid system (the auxiliary genes necessary for lentiviral vector packaging are located on the same plasmid, these auxiliary genes are a single gene sequence; the other is a transgenic plasmid) .
  • lentivirus packaging systems with more than four plasmid systems in use.
  • the first recombinant lentivirus and the second recombinant lentivirus are jointly transfected into CD3-positive T lymphocytes sequentially or simultaneously, the first recombinant lentivirus and the second recombinant lentivirus are The titer ratio is 1: (0.5-2).
  • the CD3-positive T lymphocytes are obtained from human peripheral blood mononuclear cells.
  • the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like. Further, it is derived from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • CD3 positive T lymphocytes are obtained.
  • CD3/CD28 immunomagnetic beads are added to the peripheral blood mononuclear cells in a certain proportion, after a period of incubation, they are placed in a magnet for screening, and the immunomagnetic bead coating is obtained After removing the magnetic beads, CD3 positive T lymphocytes can be obtained.
  • the combined transfection of the first recombinant lentivirus and the second recombinant lentivirus into CD3-positive T lymphocytes sequentially or simultaneously includes:
  • the second recombinant lentivirus is transfected; or after the second recombinant lentivirus is first transfected into CD3 positive T lymphocytes, Then the first recombinant lentivirus is transfected; or the first recombinant lentivirus and the second recombinant lentivirus are simultaneously transfected into CD3 positive T lymphocytes.
  • the chimeric antigen receptor T cell targeting FGFR4 and DR5 includes a dual target chimeric antigen receptor T cell carrying the CAR-FGFR4 and the CAR-DR5, or includes the CAR-FGFR4 and the CAR-DR5.
  • -A mixture of FGFR4 chimeric antigen receptor T cells and chimeric antigen receptor T cells carrying the CAR-DR5, or including a dual target chimeric antigen receptor carrying the CAR-FGFR4 and the CAR-DR5 A mixture of somatic T cells and at least one of chimeric antigen receptor T cells carrying the CAR-FGFR4 and chimeric antigen receptor T cells carrying the CAR-DR5.
  • the targeting T lymphocyte There are two independent, unbonded chimeric antigen receptors on the surface (that is, two independent single-chain antibodies), which does not affect their recognition and binding to their respective targets, and can simultaneously and efficiently recognize tumor cells.
  • the above FGFR4 and DR5 can identify and kill tumor cells expressing one or both of FGFR4 and DR5, avoid the occurrence of tumor cell escape, improve the breadth and breadth of targeted recognition, and the broad spectrum of killing , It also has strong tumor killing ability in complex tumor microenvironment.
  • the present invention provides a pharmaceutical composition, comprising the chimeric antigen receptor T cells targeting FGFR4 and DR5 prepared as described in the first aspect or prepared by the preparation method as described in the fourth aspect, such as The recombinant vector as described in the second aspect or the host cell as described in the third aspect.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or adjuvant.
  • a pharmaceutically acceptable carrier is to transport the pharmaceutical composition to make it play its due role.
  • the carrier and/or adjuvant must be compatible with the components of the pharmaceutical composition, not affect the biological activity of the pharmaceutical composition, and it is relatively non-toxic, and does not cause toxic and side effects with the pharmaceutical composition it carries.
  • the carrier includes at least one of a solvent, a polymer, and a liposome.
  • the auxiliary material includes at least one of a diluent, an excipient and a stabilizer.
  • the pharmaceutical composition can be, but not limited to, the preparation of drugs for preventing, diagnosing, and treating malignant tumors.
  • the malignant tumors include tumors expressing FGFR4 and/or DR5, and further include high-expressing FGFR4 and/or tumors.
  • DR5 tumor is optionally, the malignant tumor includes at least one of liver cancer, glioma, lung cancer, gastric cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, and cervical cancer.
  • the present invention provides chimeric antigen receptor T cells targeting FGFR4 and DR5 prepared as described in the first aspect or prepared by the preparation method described in the fourth aspect, and the recombinant T cells as described in the second aspect
  • the mode of administration can be, but is not limited to, intravenous injection, tumor in situ injection, subcutaneous injection, etc.
  • the dosage and frequency selected in the specific application are selected according to actual needs, and there is no limitation on this.
  • kits includes the chimeric antigen receptor T cell targeting FGFR4 and DR5 in the first aspect, the recombinant vector in the second aspect, and the third aspect One or more of the host cell or the pharmaceutical composition of the fifth aspect.
  • the malignant tumors include tumors that express FGFR4 and/or DR5, and further include tumors that highly express FGFR4 and/or DR5.
  • the malignant tumor includes at least one of liver cancer, glioma, lung cancer, gastric cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, and cervical cancer.
  • the chimeric antigen receptor T cells targeting FGFR4 and DR5 of the present invention can efficiently recognize and kill cancer cells expressing FGFR4 and/or DR5, and are especially suitable for liver cancer cells.
  • the present invention provides chimeric antigen receptor T cells targeting FGFR4 and DR5, specifically targeting FGFR4 and DR5, activating the signal area in T cells, and promoting the expansion of T cells in patients, with high efficiency and specificity Killing tumor cells, it can recognize and kill tumor cells that express one or both of FGFR4 and DR5, effectively inhibit the occurrence of tumor cell escape, improve the breadth and breadth of targeted recognition, and broad-spectrum killing It also has strong tumor killing ability in the complex tumor microenvironment, can self-replicate and reproduce, has a long half-life, and forms memory cells, which play a continuous targeting effect and will not cause damage to normal cells.
  • Figure 1 is a plasmid map of the pCDH-EF1-FGFR4-CAR recombinant plasmid provided in the embodiment of the present invention.
  • Figure 2 is a plasmid map of the pCDH-EF1-DR5-CAR recombinant plasmid provided in the embodiment of the present invention.
  • nucleotide sequence corresponding to any amino acid sequence shown in SEQ ID NO: 8-9 (gene sequence of CAR-FGFR4), and add a core corresponding to the amino acid sequence shown in SEQ ID NO: 11 at its 5'end Nucleotide sequence (gene sequence of signal peptide).
  • step (1) Insert the nucleotide sequence of step (1) between the BamH I and EcoR I restriction sites of the pCDH-EF1-MCS vector, and use EF1 ⁇ as the promoter after the pCDH-EF1-MCS vector EF1 ⁇ .
  • a start codon such as ATG
  • a stop codon such as TAA
  • the pCDH-EF1-FGFR4-CAR recombinant plasmid was successfully constructed, as shown in Figure 1 for the pCDH-EF1-FGFR4-CAR recombinant plasmid.
  • step (2) Insert the nucleotide sequence of step (2) between the BamH I and EcoR I restriction sites of the pCDH-EF1-MCS vector, and use EF1 ⁇ as the promoter after the pCDH-EF1-MCS vector EF1 ⁇ .
  • a start codon such as ATG
  • a stop codon such as TAA
  • the pCDH-EF1-DR5-CAR recombinant plasmid was successfully constructed, as shown in Figure 2 for the pCDH-EF1-DR5-CAR recombinant plasmid.
  • the pCDH-EF1-FGFR4-CAR recombinant plasmid, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells.
  • the virus-containing supernatant was harvested at 48h, filtered through a 0.45 ⁇ m filter, and stored in an ultra-low temperature refrigerator at -80°C; the virus-containing supernatant was harvested for the second time at 72h, filtered with a 0.45 ⁇ m filter, and combined with the virus supernatant harvested at 48h Put them into the ultracentrifuge tube together, put them into the Beckman ultracentrifuge one by one, set the centrifugation parameters to 25000rpm, the centrifugation time to 2h, and the centrifugation temperature to be controlled at 4°C; after centrifugation, discard the supernatant and try to remove the residue on the tube wall Add the virus preservation solution, gently pipetting to resuspend; after fully dissolving, centrifuge at high speed at 10,000 rpm, centrifuge for 5 min, take the supernatant and determine the titer by fluorescence method.
  • the virus is 100 ⁇ l, 2 ⁇ 10 8 cells/mL. Installed and stored in
  • the pCDH-EF1-DR5-CAR recombinant plasmid, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells.
  • the virus-containing supernatant was harvested at 48h, filtered through a 0.45 ⁇ m filter, and stored in an ultra-low temperature refrigerator at -80°C; the virus-containing supernatant was harvested for the second time at 72h, filtered with a 0.45 ⁇ m filter, and combined with the virus supernatant harvested at 48h Put them into the ultracentrifuge tube together, put them into the Beckman ultracentrifuge one by one, set the centrifugation parameters to 25000rpm, the centrifugation time to 2h, and the centrifugation temperature to be controlled at 4°C; after centrifugation, discard the supernatant and try to remove the residue on the tube wall Add the virus preservation solution, gently pipetting to resuspend; after fully dis
  • PBMC peripheral blood mononuclear cells
  • PBMC comes from autologous venous blood, autologous bone marrow, umbilical cord blood and placental blood. It is best derived from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy. The patient's blood is drawn and sent to the blood separation chamber; the peripheral blood mononuclear cells are collected, Ficoll centrifugal separation, and the middle layer cells are collected; after washing with PBS, PBMCs are obtained.
  • PBMC blood pressure
  • serum-free basal medium to prepare a cell suspension
  • CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells of 3:1, and incubate for 1-2h at room temperature; incubate with a magnet pair.
  • the cells of the magnetic beads are screened; after washing with PBS and removing the immunomagnetic beads, CD3 positive T lymphocytes are obtained.
  • the titer ratio of the first recombinant lentivirus with CAR-FGFR4 and the second recombinant lentivirus with CAR-DR5 is 1:1.
  • the diluted cell concentration is 1 ⁇ 10 6 cells/mL, check cell viability, and continue to culture. After expansion and culture to day 9-11, cells were collected to prepare chimeric antigen receptor T cells targeting FGFR4 and DR5, which were stored in a special cell cryopreservation solution for reinfusion.
  • the chimeric antigen receptor T cells targeting FGFR4 and DR5 (experimental group) prepared by the method of the present invention were subjected to FGFR4 chimeric antigen receptor T cells (control group 1) and chimeric antigen targeting DR5.
  • control group 1 FGFR4 chimeric antigen receptor T cells
  • chimeric antigen targeting DR5 chimeric antigen targeting DR5.
  • control group 2 somatic T cells
  • T lymphocytes negative control group
  • Huh7 was used as the blank control group.
  • the effector cells targeted FGFR4 chimeric
  • the ratio of antigen receptor T cells/targeting FGFR4 chimeric antigen receptor T cells/targeting DR5 chimeric antigen receptor T cells/T lymphocytes) to target cells solid tumor cell line Huh7
  • the ratios of 5:1, 2.5:1, 1.25:1 and 0.625:1 were co-cultured in cell plates at 37°C and 5% CO 2 to observe the adherence of the cells and evaluate their killing ability. The results showed that it was compared with the control group 1.
  • the chimeric antigen receptor T cells targeting FGFR4 and DR5 prepared by the method of the present invention have excellent tumor killing ability, and the killing ability is much higher than that of the control group 1
  • the control group 2 and the negative control group therefore, the chimeric antigen receptor T cells targeting FGFR4 and DR5 prepared by the method of the present invention have very considerable application prospects in the preparation of drugs for preventing, diagnosing and treating malignant tumors.

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Abstract

提供了一种靶向FGFR4和DR5的嵌合抗原受体T细胞，该细胞包括靶向FGFR4的嵌合抗原受体CAR-FGFR4和靶向DR5的嵌合抗原受体CAR-DR5，该CAR-FGFR4包括氨基端到羧基端顺次连接的靶向FGFR4单链抗体、胞外铰链区、跨膜区和胞内信号区，该CAR-DR5包括氨基端到羧基端顺次连接的靶向DR5单链抗体、胞外铰链区、跨膜区和胞内信号区；该靶向FGFR4的单链抗体包括以下中的至少一种：(a)如SEQ ID NO：1所示的单链抗体重链VH以及如SEQ ID NO：2所示的单链抗体轻链VL；(b)如SEQ ID NO：3所示的单链抗体重链VH以及如SEQ ID NO：4所示的单链抗体轻链VL。

Description

一种靶向FGFR4和DR5的嵌合抗原受体T细胞及其制备方法和应用
本发明要求2019年11月15日递交的发明名称为“一种靶向FGFR4和DR5的嵌合抗原受体T细胞及其制备方法和应用”的申请号201911120424.0的在先申请优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及医学生物领域，特别涉及一种靶向FGFR4和DR5的嵌合抗原受体T细胞及其制备方法和应用。
背景技术
原发性肝癌是最常见的恶性肿瘤之一，是全世界范围内第二大癌症死亡原因，己经严重威胁到了人们的健康和生活。临床中常见的肝癌治疗方法主要有手术切除、肝移植、化学药物治疗和放射治疗等。然而，由于肝癌早期病症不明显因而不易被检测，大部分病人确诊时己处于中晚期，失去手术治疗和肝移植的机会。实际治疗中约有90％的肝癌患者需要接受药物治疗。现有常用的抗肿瘤药物虽然可以有效延长患者生存期，但在临床应用中普遍存在选择性低、毒副作用大等问题。即便是靶向药物也存在长期使用后容易诱发肿瘤耐药性的问题。例如2007年批准的多激酶抑制剂索拉菲尼，但仅仅将总生存期提高3个月。由此可见，临床上为肝癌寻找安全有效的疗法依然是个难题。
死亡受体5(DR5)又名肿瘤坏死因子相关凋亡诱导配体受体2(TRAIL-R2)，在多种肿瘤组织高表达，尤其是肺癌、乳腺癌、卵巢癌、直肠癌、宫颈癌等，而在大多数正常细胞几乎不表达，成为理想的靶点。成纤维细胞生长因子受体家族有四个成员，FGFR1-4，其激酶域同源性高达74-92％。通过分析FGFR家族的结构发现在552号氨基酸上FGFR4与其他成员不同，FGFR4上是半胱氨酸，而其他成员在该位点是酪氨酸。可以尝试利用这半胱氨酸的特性来设计抑制剂，达到高选择性的靶向FGFR4。FGFR4在多种肿瘤中存在过表达，例如肝癌、乳腺癌、胃癌等。目前在制药产业领域，FGFR4的抑制剂H3B-6527已获得美国FDA授予的孤儿药资格。FGFR4选择性抑制剂基本都是基于Cys552的共价可逆或不可逆抑制剂，一旦这个位点突变很可能导致耐药情况的产生。
免疫细胞治疗是现有技术中唯一有可能彻底清除癌细胞的方法，其治疗肿瘤具有特异性强、几乎无毒副作用的巨大优势弥补了传统疗法的弊端，在国内外已经用于临床治疗恶性肿瘤，嵌合抗原受体T细胞技术(CAR-T)为当前过继性细胞回输治疗技术最新的免疫细胞技术之一，因其能够在体内能激活自身免疫系统，持续性地靶向肿瘤细胞进行杀伤，最终达到清除恶性肿瘤细胞的目的而受到广泛的关注和研究。目前还未有同时靶向FGFR4和DR5的嵌合抗原受体T细胞的制备和研究。
发明内容
有鉴于此，本发明提供了一种靶向FGFR4和DR5的嵌合抗原受体T细胞，包括靶向FGFR4的嵌合抗原受体CAR-FGFR4和靶向DR5的嵌合抗原受体CAR-DR5，CAR-FGFR4专一性的靶向FGFR4，CAR-DR5专一性的靶向DR5，从而促进T细胞在患者体内的扩增，高效且特异性的杀伤肿瘤细胞，有效抑制肿瘤细胞逃逸的发生，更好的维持嵌合抗原受体T细胞的活力和杀伤力，并且对正常细胞不 会造成损伤。本发明还提供了靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法和应用。
第一方面，本发明提供了一种靶向FGFR4和DR5的嵌合抗原受体T细胞，包括靶向FGFR4的嵌合抗原受体CAR-FGFR4和靶向DR5的嵌合抗原受体CAR-DR5，所述CAR-FGFR4的氨基酸序列包括从氨基端到羧基端顺次连接的靶向FGFR4的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，所述CAR-DR5的氨基酸序列包括从氨基端到羧基端顺次连接的靶向DR5的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列；
其中，所述靶向FGFR4的单链抗体的氨基酸序列包括以下中的至少一种：
(a)如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；
(b)如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列。
在本发明中，所述靶向FGFR4的单链抗体的氨基酸序列包括如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列，或所述靶向FGFR4的单链抗体的氨基酸序列包括如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列，或所述靶向FGFR4的单链抗体的氨基酸序列包括如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；和如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列。
在本发明中，如SEQ ID NO：1所示的单链抗体重链VH以及如SEQ ID NO：2所示的单链抗体轻链VL顺次连接形成具有靶向FGFR4的单链抗体，如SEQ ID NO：3所示的单链抗体重链VH以及如SEQ ID NO：4所示的单链抗体轻链VL顺次连接形成具有靶向FGFR4的单链抗体。
可选的，所述如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列和所述如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列通过第一连接肽连接。
可选的，所述如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列和所述如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列通过第二连接肽连接。
在本发明中，所述第一连接肽和所述第二连接肽用于连接单链抗体重链VH的氨基酸序列和单链抗体轻链VL的氨基酸序列，使所连接的单链抗体重链和轻链保持各自的空间构象，以维持整体单链抗体的功能和活性。在本发明中，所述第一连接肽和所述第二连接肽可以但不限于为以甘氨酸和丝氨酸为主构成的多肽序列，其中，甘氨酸的分子量最小，是侧链最短的氨基酸，可以增加侧链的柔韧性；丝氨酸是亲水性最强的氨基酸，可以增加肽链的亲水性。可选的，所述第一连接肽的氨基酸序列为GGGGSGGGGSGGGGS，所述第二连接肽的氨基酸序列为GGGGSGGGGSGGGGS。
在本发明中，单链抗体重链VH、单链抗体轻链VL和连接肽linker的氨基酸序列从氨基酸端到羧基端的连接顺序为VH-linker-VL和/或VL-linker-VH。
也就是说，所述靶向FGFR4的单链抗体的氨基酸序列包括以下情况中的至少一种：
(i)从氨基酸端到羧基端依次包括：所述如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列、所述第一连接肽的氨基酸序列和所述如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；
(ii)从氨基酸端到羧基端依次包括：所述如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列、所述第一连接肽的氨基酸序列和所述如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列；
(iii)从氨基酸端到羧基端依次包括：所述如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列、所述第二连接肽的氨基酸序列和所述如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列；
(iiii)从氨基酸端到羧基端依次包括：所述如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列、所述第二连接肽的氨基酸序列和所述如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列。
在本发明中，单链抗体轻链VL和单链抗体重链VH的先后位置关系不影响单链抗体的靶向性和活性。
可选的，所述靶向FGFR4的单链抗体的氨基酸序列包括如SEQ ID NO：6-7所示的任一氨基酸序列。
在本发明中，所述靶向FGFR4的单链抗体的编码基因包括所述靶向FGFR4的单链抗体的氨基酸序列对应的核苷酸序列。
可选的，所述靶向DR5的单链抗体的氨基酸序列包括如SEQ ID NO：5所示的氨基酸序列。
在本发明中，所述“从氨基端到羧基端顺次连接”具体为：单链抗体的氨基酸序列的羧基端与所述胞外铰链区的氨基酸序列的氨基端相连，所述胞外铰链区的氨基酸序列的羧基端与所述跨膜区的氨基酸序列的氨基端相连，所述跨膜区的氨基酸序列的羧基端与所述胞内信号区的氨基酸序列的氨基端相连。
在本发明中，所述胞外铰链区用于促进所述靶向FGFR4的单链抗体与肿瘤上的FGFR4结合。
可选的，所述胞外铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。进一步可选的，所述胞外铰链区包括CD8α铰链区。
在本发明中，所述跨膜区用于固定所述靶向FGFR4的嵌合抗原受体CAR-FGFR4。
可选的，所述跨膜区包括CD3跨膜区、CD4跨膜区、CD8跨膜区、CD28跨膜区中的一种或多种的组合。进一步可选的，所述跨膜区包括CD8跨膜区。
在本发明中，所述胞内信号区用于提供T细胞活化的信号，维持T细胞的生存时间和激活T细胞增殖信号通路。
可选的，所述胞内信号区包括4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区、TNFRSF19L信号区中的一种或多种的组合。可选的，所述胞内信号区包括4-1BB信号区和CD3ζ信号区。
在本发明中，所述CAR-FGFR4和所述CAR-DR5中的铰链区、跨膜区和信号区可以相同，也可以不同，根据实际需要进行选择。
可选的，所述CAR-FGFR4的氨基酸序列包括从氨基端到羧基端顺次连接的靶向FGFR4的单链抗体、CD8α铰链区、CD8跨膜区、4-1BB信号区和CD3ζ信号区的氨基酸序列。
可选的，所述CAR-FGFR4的氨基酸序列包括如SEQ ID NO：8-9所示的任一氨基酸序列。
在本发明中，靶向FGFR4的单链抗体中单链抗体重链VH和单链抗体轻链VL的连接顺序不影响CAR-FGFR4靶向性和活性，制得的CAR-FGFR4均具有靶向FGFR4的作用。
在本发明中，所述靶向FGFR4的嵌合抗原受体CAR-FGFR4的编码基因包括所述靶向FGFR4的嵌合抗原受体CAR-FGFR4的氨基酸序列对应的核苷酸序列。
可选的，所述CAR-DR5的氨基酸序列包括从氨基端到羧基端顺次连接的靶向DR5的单链抗体、CD8α铰链区、CD8跨膜区、4-1BB信号区和CD3ζ信号区的氨基酸序列。
可选的，所述CAR-DR5的氨基酸序列包括如SEQ ID NO：10所示的氨基酸序列。
在本发明中，所述CAR-DR5的编码基因包括所述靶向DR5的嵌合抗原受体CAR-DR5的氨基酸序列对应的核苷酸序列。
在本发明中，所述靶向FGFR4和DR5的嵌合抗原受体T细胞可以为带CAR-FGFR4和CAR-DR5的双靶点嵌合抗原受体T细胞(即，靶向FGFR4和DR5的双靶点嵌合抗原受体T细胞)，也可以为带CAR-FGFR4的嵌合抗原受体T细胞和带CAR-DR5的嵌合抗原受体T细胞的混合或为带CAR-FGFR4和CAR-DR5的双靶点嵌合抗原受体T细胞与带CAR-FGFR4的嵌合抗原受体T细胞和带CAR-DR5的嵌合抗原受体T细胞中的至少一种的混合。此时，嵌合抗原受体T细胞既可以识别表面表达有FGFR4抗原蛋白的肿瘤细胞，也可以识别表面表达有DR5抗原蛋白的肿瘤细胞，当然对同时有FGFR4抗原蛋白和DR5抗原蛋白的肿瘤细胞的识别性也较好，能够有效避免肿瘤细胞出现免疫逃逸现象。
其中，当所述靶向FGFR4和DR5的嵌合抗原受体T细胞为带CAR-FGFR4和CAR-DR5的双靶点嵌合抗原受体T细胞时，CAR-FGFR4和CAR-DR5的位置分布不作限定。可选的，所述CAR-FGFR4和所述CAR-DR5交替分布。所述CAR-FGFR4的单链抗体和所述CAR-DR5的单链抗体之间没有化学键相连，以确保更好的识别能力。
本发明第一方面提供的靶向FGFR4的嵌合抗原受体T细胞可以专一性地靶向表达FGFR4的肿瘤细胞，在CAR-FGFR4与FGFR4结合后，T细胞的胞内信号区被激活，促进T细胞在患者体内的扩增，并高效且特异性的杀伤肿瘤细胞，而对正常细胞几乎不会造成损伤。
第二方面，本发明提供了一种重组载体，所述重组载体包括如第一方面所述的靶向FGFR4和DR5的嵌合抗原受体T细胞中所述CAR-FGFR4和CAR-DR5的编码基因。
在本发明中，可以在同一重组载体含有所述CAR-FGFR4和CAR-DR5的编码基因，也可以在不同载体中分别含所述CAR-FGFR4和CAR-DR5的编码基因。
在本发明中，所述载体可以但不限于为基因传递载体。
可选的，所述载体为病毒载体和非病毒载体中的至少一种。进一步的，所述非病毒载体包括质粒载体和噬菌体载体。具体的，所述质粒载体可以但不限于为真核质粒载体、原核质粒载体、微环DNA、转座子等。当所述载体为微环DNA时，可以将插入靶向FGFR4的嵌合抗原受体CAR-FGFR4的编码基因的重组微环DNA直接转染CD3阳性T淋巴细胞，制得靶向FGFR4的嵌合抗原受体T细胞。
进一步的，所述病毒载体包括慢病毒载体、腺病毒载体或逆转录病毒载体。更进一步的，所述病毒载体为慢病毒载体。
本发明第二方面提供的重组载体安全高效，可以稳定地实现将CAR-FGFR4和CAR-DR5的编码基因导入宿主细胞中或复制，并可以用于嵌合抗原受体T细胞的制备。
第三方面，本发明提供了一种宿主细胞，所述宿主细胞包括如第二方面所述的重组载体。
可选的，当所述重组载体为重组病毒载体时，所述宿主细胞可以用于组装所述重组病毒载体，使其具有感染性。进一步的，所述宿主细胞可以包括HEK293T细胞、293细胞、293T细胞、293FT细胞、SW480细胞、u87MG细胞、HOS细胞或COS7细胞等，但不限于此。更进一步的，所述宿主细胞为HEK293T细胞。
可选的，当所述重组质粒为重组真核质粒载体、重组原核质粒载体和重组微环DNA时，所述宿主细胞为相应的真核宿主细胞或原核宿主细胞。
本发明第三方面提供的宿主细胞可以稳定保存具有靶向FGFR4和DR5的CAR-FGFR4和CAR-DR5的编码基因，有利于靶向FGFR4和DR5的嵌合抗原受体T细胞的制备。
第四方面，本发明提供了一种靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法，包括：
(1)提供靶向FGFR4的嵌合抗原受体CAR-FGFR4的编码基因，包括从5’端到3’端顺次连接的第一信号肽的编码基因、靶向FGFR4的单链抗体的编码基因、第一胞外铰链区的编码基因、第一跨膜区的编码基因、第一胞内信号区的编码基因，其中，所述靶向FGFR4的单链抗体的氨基酸序列包括以下中的至少一种：
(a)如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；
(b)如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列；
(2)提供靶向DR5的嵌合抗原受体CAR-DR5的编码基因，包括从5’端到3’端顺次连接的第二信号肽的编码基因、靶向DR5的单链抗体的编码基因、第二胞外铰链区的编码基因、第二跨膜区的编码基因、第二胞内信号区的编码基因；
(3)将所述CAR-FGFR4的编码基因到插入到第一基因传递载体中，得到第一重组基因传递载体，将所述CAR-DR5的编码基因到插入到第二基因传递载体中，得到第二重组基因传递载体；
(4)对所述第一重组基因传递载体和所述第二重组基因传递载体进行包装，得到带CAR-FGFR4编码基因的第一重组慢病毒以及带CAR-DR5编码基因的第二重组慢病毒；
(5)将所述第一重组慢病毒和所述第二重组慢病毒按顺序地或同时地联合转染CD3阳性T淋巴细胞，经分离获得靶向FGFR4和DR5的嵌合抗原受体T细胞。
上述“从5’端到3’端顺次连接”具体为：所述信号肽的编码基因序列的3’端与单链抗体的编码基因的5’端相连，所述单链抗体的编码基因的3’端与所述胞外铰链区的编码基因的5’端相连，所述胞外铰链区的编码基因的3’端与所述跨膜区的编码基因的5’端相连，所述跨膜区的编码基因的3’端与所述胞内信号区的编码基因的5’端相连。
在本发明中，所述信号肽用于指导所述嵌合抗原受体CAR-FGFR4表达到细胞表面，所述信号肽在蛋白翻译成熟过程中被信号肽酶切割。
可选的，所述信号肽的氨基酸序列包括如SEQ ID NO：11所示的氨基酸序列。
所述胞外铰链区、跨膜区、胞内信号区的具体选择及相应的编码基因序列如本发明第二方面部分所述，这里不再赘述。
可选的，所述CAR-FGFR4的编码基因包括从5’端到3’端顺次连接的信号肽的编码基因、靶向 FGFR4的单链抗体的编码基因、CD8α铰链区的编码基因、CD8跨膜区的编码基因、4-1BB信号区的编码基因和CD3ζ信号区的编码基因。
可选的，所述CAR-FGFR4的编码基因包括如SEQ ID NO：8-9所示的任一氨基酸序列对应的核苷酸序列。
可选的，所述CAR-DR5的编码基因包括从5’端到3’端顺次连接的信号肽的编码基因、靶向DR5的单链抗体的编码基因、CD8α铰链区的编码基因、CD8跨膜区的编码基因、4-1BB信号区的编码基因和CD3ζ信号区的编码基因。
可选的，所述CAR-DR5的编码基因包括如SEQ ID NO：10所示的氨基酸序列对应的核苷酸序列。
在本发明中，第一基因传递载体和第二基因传递载体可以相同，也可以不同，根据实际需要进行选择，所述CAR-FGFR4的编码基因和所述CAR-DR5的编码基因可以插入不同基因传递载体中，也可以插入相同基因传递载体，当插入相同基因传递载体时，对所述CAR-FGFR4的编码基因和所述CAR-DR5的编码基因的连接顺序不做限定。
在本发明中，所述CAR-FGFR4的编码基因插入到pCDH-EF1-MCS载体中BamH Ⅰ和EcoR Ⅰ酶切位点之间，且位于pCDH-EF1-MCS载体的EF1α之后，以EF1α为启动子。所述CAR-FGFR4的编码基因插入到pCDH-EF1-MCS载体时，所述CAR-FGFR4的编码基因的5’端可加入起始密码子(如ATG)与pCDH-EF1-MCS载体中BamH Ⅰ酶切位点相连，3’端可加入终止密码子(如TAA)与pCDH-EF1-MCS载体中EcoR Ⅰ酶切位点相连。
在本发明中，所述CAR-DR5的编码基因插入到pCDH-EF1-MCS载体中BamH Ⅰ和EcoR Ⅰ酶切位点之间，且位于pCDH-EF1-MCS载体的EF1α之后，以EF1α为启动子。所述CAR-DR5的编码基因插入到pCDH-EF1-MCS载体时，所述CAR-DR5的编码基因的5’端可加入起始密码子(如ATG)与pCDH-EF1-MCS载体中BamH Ⅰ酶切位点相连，3’端可加入终止密码子(如TAA)与pCDH-EF1-MCS载体中EcoR Ⅰ酶切位点相连。
在本发明中，对所述第一重组基因传递载体和所述第二重组基因传递载体进行包装时，可以分别进行包装，也可以同时进行包装。
可选的，对所述第一重组基因传递载体和所述第二重组基因传递载体进行包装，得到带CAR-FGFR4编码基因的第一重组慢病毒以及带CAR-DR5编码基因的第二重组慢病毒，包括：
将所述第一重组基因传递载体与包膜质粒和包装质粒共同转染宿主细胞，得到所述第一重组慢病毒；以及将所述第二重组基因传递载体与包膜质粒和包装质粒共同转染宿主细胞，得到所述第二重组慢病毒。
可选的，所述包膜质粒为PMD2G，所述包装质粒为psPAX2，所述宿主细胞为HEK293T细胞。所述包膜质粒PMD2G编码水疱性口炎病毒糖蛋白衣壳，所述水疱性口炎病毒糖蛋白衣壳协助重组慢病毒向细胞膜粘附，并保持重组慢病毒的感染性。
本发明所述重组慢病毒可以进一步含有来自其它病毒的被膜蛋白。例如，作为这种蛋白质，最好是来自感染人类细胞的病毒被膜蛋白。对这种蛋白质没有特别的限定，如逆转录病毒的兼嗜性病毒手皮膜蛋白等，例如可以使用来自小鼠白血病病毒(MuMLV)4070A株的被膜蛋白。另外，也可以使用来自MuMLV 10Al的被膜蛋白。另外，作为疱疹病毒科的蛋白，如单纯性疱疹病毒的gB、gD、gH、 gp85蛋白，EB病毒的gp350、gp220蛋白等。作为嗜肝病毒科的蛋白，如B型肝炎病毒的S蛋白等。所述被膜蛋白还可为麻疹病毒糖蛋白与其他单链抗体融合后形成。
重组慢病毒的包装通常采用瞬时转染或采用细胞系包装。瞬时转染时可以用作包装细胞使用的人类细胞株，例如包括293细胞、293T细胞、293FT细胞、293LTV细胞、293EBNA细胞及其他的从293细胞分离的克隆；SW480细胞、u87MG细胞、HOS细胞、C8166细胞、MT-4细胞、Molt-4细胞、HeLa细胞、HT1080细胞、TE671细胞等。也可以采用来源于猴子的细胞株，例如，COS1细胞、COS7细胞、CV-1细胞、BMT10细胞等。而且，通常采用的磷酸钙和PEI转染试剂，还有一些转染试剂如Lipofectamine2000、FuGENE和S93fectin也被经常使用。
重组慢病毒的包装也采用一些慢病毒包装细胞系，如使用最普遍的Env糖蛋白、VSVG蛋白或HIV-1gag-pol蛋白所产生的稳定细胞系。
为了安全起见，大规模使用的慢病毒载体系统都是采用分割基因组的方法，即将起不同辅助功能的基因定位于不同的质粒。目前有四质粒系统(编码gag-pol基因、Rev基因、VSVG基因、SIN转移基因分别位于四个不同的质粒)、三质粒系统(去掉了编码Rev基因的质粒，在gag-pol质粒中gag-pol基因采用了在人细胞中偏爱性的密码子)和二质粒系统(慢病毒载体包装所必需的辅助基因位于同一个质粒上，这些辅助基因是单一的基因序列；另一个则是转基因质粒)。也有超过四质粒系统的慢病毒包装系统在使用。
可选的，所述第一重组慢病毒和所述第二重组慢病毒按顺序地或同时地联合转染CD3阳性T淋巴细胞时，所述第一重组慢病毒和所述第二重组慢病毒的滴度比为1：(0.5-2)。
可选的，所述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。进一步的，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。进一步的，来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
具体的，所述CD3阳性T淋巴细胞的获得过程如下：向外周血单个核细胞中按一定比例加入CD3/CD28免疫磁珠，孵育一段时间后，放入磁铁进行筛选，得到免疫磁珠包被的CD3阳性T淋巴细胞，去除磁珠后，获得CD3阳性T淋巴细胞。
可选的，将所述第一重组慢病毒和所述第二重组慢病毒按顺序地或同时地联合转染CD3阳性T淋巴细胞，包括：
先将所述第一重组慢病毒转染CD3阳性T淋巴细胞后，再将所述第二重组慢病毒进行转染；或先将所述第二重组慢病毒转染CD3阳性T淋巴细胞后，再将所述第一重组慢病毒进行转染；或将所述第一重组慢病毒和所述第二重组慢病毒同时地联合转染CD3阳性T淋巴细胞。
可选的，所述靶向FGFR4和DR5的嵌合抗原受体T细胞包括带所述CAR-FGFR4和所述CAR-DR5的双靶点嵌合抗原受体T细胞，或者包括带所述CAR-FGFR4的嵌合抗原受体T细胞和带所述CAR-DR5的嵌合抗原受体T细胞的混合，或者包括带所述CAR-FGFR4和所述CAR-DR5的双靶点嵌合抗原受体T细胞与带所述CAR-FGFR4的嵌合抗原受体T细胞和带所述CAR-DR5的嵌合抗原受体T细胞中的至少一种的混合。
当所述靶向FGFR4和DR5的嵌合抗原受体T细胞为带所述CAR-FGFR4和所述CAR-DR5的双靶点嵌合抗原受体T细胞时，此时靶向性T淋巴细胞的表面具有两个独立的、未键合的嵌合抗原受体 (也即是有两个独立的单链抗体)，不影响它们对各自靶标的识别、结合，能同时、高效地识别肿瘤细胞上的FGFR4和DR5，对表达FGFR4和DR5中一种或两种的肿瘤细胞均可以进行识别和杀伤，避免出现肿瘤细胞逃逸的发生，提高了靶向识别的广度和宽度，以及杀伤广谱性，在复杂的肿瘤微环境下也具有较强的肿瘤杀伤能力。
第五方面，本发明提供了一种药物组合物，包括如第一方面所述的或如第四方面所述的制备方法制得的靶向FGFR4和DR5的嵌合抗原受体T细胞、如第二方面所述的重组载体或如第三方面所述的宿主细胞。
可选的，所述药物组合物还包括药学上可接受的载体和/或辅料。在本申请中，“药学上可接受的载体”的作用是运输所述药物组合物，使其发挥应有的作用。载体和/或辅料必须和药物组合物成分相兼容，不影响药物组合物的生物学活性，而且本身是相对无毒的，且不与其携带的药物组合物发生引起毒副作用的反应。进一步的，所述载体包括溶剂、聚合物和脂质体中的至少一种。进一步的，所述辅料包括稀释剂、赋形剂和稳定剂中的至少一种。
在本申请中，所述药物组合物可以但不限于用于制备预防、诊断和治疗恶性肿瘤的药物，所述恶性肿瘤包括表达FGFR4和/或DR5的肿瘤，进一步的包括高表达FGFR4和/或DR5的肿瘤。可选的，所述恶性肿瘤包括肝癌、脑胶质瘤、肺癌、胃癌、结肠癌、胰腺癌、乳腺癌、卵巢癌和宫颈癌中的至少一种。
第六方面，本发明提供了如第一方面所述的或如第四方面所述的制备方法制得的靶向FGFR4和DR5的嵌合抗原受体T细胞、如第二方面所述的重组载体、如第三方面所述的宿主细胞或第五方面所述的药物组合物在制备预防、诊断和治疗恶性肿瘤的药物中的应用。
在应用中，给药方式可以但不限于为静脉注射、肿瘤原位注射、皮下注射等。具体应用时选择的剂量、次数等根据实际需要进行选择，对此不做限定。
所述应用具体为：提供了一种试剂盒，所述试剂盒包括第一方面所述的靶向FGFR4和DR5的嵌合抗原受体T细胞、第二方面所述的重组载体、第三方面所述的宿主细胞或第五方面所述的药物组合物中的一种或多种。
在本发明中，所述恶性肿瘤包括表达FGFR4和/或DR5的肿瘤，进一步的包括高表达FGFR4和/或DR5的肿瘤。可选的，所述恶性肿瘤包括肝癌、脑胶质瘤、肺癌、胃癌、结肠癌、胰腺癌、乳腺癌、卵巢癌和宫颈癌中的至少一种。本发明的靶向FGFR4和DR5的嵌合抗原受体T细胞可以高效识别并杀伤表达有FGFR4和/或DR5的癌细胞，尤其适用于肝癌细胞。
本发明的有益效果：
本发明提供了靶向FGFR4和DR5的嵌合抗原受体T细胞，专一性的靶向FGFR4和DR5，激活T细胞内的信号区，促进T细胞在患者体内的扩增，高效且特异性的杀伤肿瘤细胞，对表达FGFR4和DR5中一种或两种的肿瘤细胞均可以进行识别和杀伤，有效的抑制肿瘤细胞逃逸的发生，提高了靶向识别的广度和宽度，以及杀伤广谱性，在复杂的肿瘤微环境下也具有较强的肿瘤杀伤能力，能够自我复制繁殖，半衰期长，并形成记忆细胞，起到持续的靶向作用并且对正常细胞不会造成损伤。
附图说明
图1为本发明实施例提供的pCDH-EF1-FGFR4-CAR重组质粒的质粒图谱。
图2为本发明实施例提供的pCDH-EF1-DR5-CAR重组质粒的质粒图谱。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
实施例一
一种靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法，包括：
(1)制备靶向FGFR4的嵌合抗原受体CAR-FGFR4的基因序列
提供SEQ ID NO：8-9所示的任一氨基酸序列对应的核苷酸序列(CAR-FGFR4的基因序列)，在其5’端添加如SEQ ID NO：11所示的氨基酸序列对应的核苷酸序列(信号肽的基因序列)。
(2)制备靶向DR5的嵌合抗原受体CAR-DR5的基因序列
提供SEQ ID NO：10所示的氨基酸序列对应的核苷酸序列(CAR-FGFR4的基因序列)，在其5’端添加如SEQ ID NO：11所示的氨基酸序列对应的核苷酸序列(信号肽的基因序列)。
(3)构建pCDH-EF1-FGFR4-CAR重组质粒和pCDH-EF1-DR5-CAR重组质粒
将步骤(1)的核苷酸序列插入到pCDH-EF1-MCS载体的BamH Ⅰ和EcoR Ⅰ酶切位点之间，并在pCDH-EF1-MCS载体EF1α之后，以EF1α为启动子。上述核苷酸序列插入到pCDH-EF1-MCS载体时，在上述核苷酸序列的5’端可加入起始密码子(如ATG)与pCDH-EF1-MCS载体中BamH Ⅰ酶切位点相连，3’端可加入终止密码子(如TAA)与pCDH-EF1-MCS载体中EcoR Ⅰ酶切位点相连。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。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建pCDH-EF1-FGFR4-CAR重组质粒，如图1所示为pCDH-EF1-FGFR4-CAR重组质粒。
将步骤(2)的核苷酸序列插入到pCDH-EF1-MCS载体的BamH Ⅰ和EcoR Ⅰ酶切位点之间，并在pCDH-EF1-MCS载体EF1α之后，以EF1α为启动子。上述核苷酸序列插入到pCDH-EF1-MCS载体时，在上述核苷酸序列的5’端可加入起始密码子(如ATG)与pCDH-EF1-MCS载体中BamH Ⅰ酶切位点相连，3’端可加入终止密码子(如TAA)与pCDH-EF1-MCS载体中EcoR Ⅰ酶切位点相连。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。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建pCDH-EF1-DR5-CAR重组质粒，如图2所示为pCDH-EF1-DR5-CAR重组质粒。
(4)重组慢病毒构建
将pCDH-EF1-FGFR4-CAR重组质粒、包装质粒psPAX2、包膜质粒pMD2G三者共转染入培养好的HEK293T细胞。第48h收获含病毒的上清，经0.45μm滤膜过滤，-80℃超低温冰箱中保存；第72h二次收获含病毒的上清，0.45μm滤膜过滤，与第48h收获的病毒上清合并一起加入超速离心管中，逐一放入至Beckman超速离心机内，设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000rpm，离心5min后，取上清荧光法测定滴度，病毒按照100μl，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到带CAR-FGFR4的第一重组慢病毒。
将pCDH-EF1-DR5-CAR重组质粒、包装质粒psPAX2、包膜质粒pMD2G三者共转染入培养好的HEK293T细胞。第48h收获含病毒的上清，经0.45μm滤膜过滤，-80℃超低温冰箱中保存；第72h二次收获含病毒的上清，0.45μm滤膜过滤，与第48h收获的病毒上清合并一起加入超速离心管中，逐一放入至Beckman超速离心机内，设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000rpm，离心5min后，取上清荧光法测定滴度，病毒按照100μl，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到带CAR-DR5的第二重组慢病毒。
(5)靶向FGFR4和DR5的嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间层细胞；经PBS洗涤后，得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为3:1，加入CD3/CD28免疫磁珠，室温孵1-2h；采用磁铁对孵育好磁珠的细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c)病毒转染法制备抗原特异性T淋巴细胞
取上述经过免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的病毒滴度的带CAR-FGFR4的第一重组慢病毒和带CAR-DR5的第二重组慢病毒进行共同培养，带CAR-FGFR4的第一重组慢病毒和带CAR-DR5的第二重组慢病毒的滴度比为1:1。培养的第3天，进行细胞计数和换液，调整细胞浓度为1×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为1×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，制得靶向FGFR4和DR5的嵌合抗原受体T细胞，将其保存在回输专用的细胞冻存液中。
效果实施例
为了评估本发明所描述的上述方法制备的靶向FGFR4和DR5的嵌合抗原受体T细胞，进行如下效果实施例。
将经过本发明方法制得的靶向FGFR4和DR5的嵌合抗原受体T细胞(实验组)与靶向FGFR4嵌合抗原受体T细胞(对照组1)、靶向DR5的嵌合抗原受体T细胞(对照组2)、T淋巴细胞(阴性对照组)的体外肿瘤杀伤效果进行比较，实体瘤细胞系Huh7作为空白对照组，具体的：在体外将效应细胞(靶向FGFR4的嵌合抗原受体T细胞/靶向FGFR4嵌合抗原受体T细胞/靶向DR5的嵌合抗原受体T细胞/T淋巴细胞)与靶细胞(实体瘤细胞系Huh7)数量比为10:1、5:1、2.5:1、1.25:1和0.625:1比例，在37℃，5％CO 2的细胞板中共培养，观察细胞贴壁情况，并评估其杀伤能力，结果表明，与对照组1、对照组2、阴性对照组相比，经过本发明所述的方法制备的靶向FGFR4和DR5的嵌合抗原受体T细胞具有优异的肿瘤杀伤力，且杀伤能力远远高于对照组1、对照组2以及阴性对照组，因此经本发明方法制备的靶向FGFR4和DR5的嵌合抗原受体T细胞在制备预防、诊断和治疗恶性肿瘤的药物中具有非常可观的应用前景。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (16)

  1. 一种靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，包括靶向FGFR4的嵌合抗原受体CAR-FGFR4和靶向DR5的嵌合抗原受体CAR-DR5，所述CAR-FGFR4的氨基酸序列包括从氨基端到羧基端顺次连接的靶向FGFR4的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，所述CAR-DR5的氨基酸序列包括从氨基端到羧基端顺次连接的靶向DR5的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列；
    其中，所述靶向FGFR4的单链抗体的氨基酸序列包括以下中的至少一种：
    (a)如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；
    (b)如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列。
  2. 如权利要求1所述的靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，所述如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列和所述如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列通过第一连接肽连接，所述如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列和所述如SEQ ID NO：4所示的单链抗体轻链VL的氨基酸序列通过第二连接肽连接。
  3. 如权利要求2所述的靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，所述第一连接肽的氨基酸序列为GGGGSGGGGSGGGGS，所述第二连接肽的氨基酸序列为GGGGSGGGGSGGGGS。
  4. 如权利要求1所述的靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，所述靶向DR5的单链抗体的氨基酸序列包括如SEQ ID NO：5所示的氨基酸序列。
  5. 如权利要求1所述的靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，所述靶向FGFR4的单链抗体的氨基酸序列包括如SEQ ID NO：6-7所示的任一氨基酸序列。
  6. 如权利要求1所述的靶向FGFR4和DR5的嵌合抗原受体T细胞，其特征在于，所述CAR-FGFR4的氨基酸序列包括如SEQ ID NO：8-9所示的任一氨基酸序列，所述CAR-DR5的氨基酸序列包括如SEQ ID NO：10所示的氨基酸序列。
  7. 一种重组载体，其特征在于，包括如权利要求1-6任一项所述的靶向FGFR4和DR5的嵌合抗原受体T细胞中所述CAR-FGFR4和所述CAR-DR5的编码基因。
  8. 一种宿主细胞，其特征在于，包括如权利要求7所述的重组载体。
  9. 一种靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法，其特征在于，包括：
    (1)提供靶向FGFR4的嵌合抗原受体CAR-FGFR4的编码基因，包括从5’端到3’端顺次连接的第一信号肽的编码基因、靶向FGFR4的单链抗体的编码基因、第一胞外铰链区的编码基因、第一跨膜区的编码基因、第一胞内信号区的编码基因，其中，所述靶向FGFR4的单链抗体的氨基酸序列包括以下中的至少一种：
    (a)如SEQ ID NO：1所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：2所示的单链抗体轻链VL的氨基酸序列；
    (b)如SEQ ID NO：3所示的单链抗体重链VH的氨基酸序列以及如SEQ ID NO：4所示的单链 抗体轻链VL的氨基酸序列；
    (2)提供靶向DR5的嵌合抗原受体CAR-DR5的编码基因，包括从5’端到3’端顺次连接的第二信号肽的编码基因、靶向DR5的单链抗体的编码基因、第二胞外铰链区的编码基因、第二跨膜区的编码基因、第二胞内信号区的编码基因；
    (3)将所述CAR-FGFR4的编码基因到插入到第一基因传递载体中，得到第一重组基因传递载体，将所述CAR-DR5的编码基因到插入到第二基因传递载体中，得到第二重组基因传递载体；
    (4)对所述第一重组基因传递载体和所述第二重组基因传递载体进行包装，得到带CAR-FGFR4编码基因的第一重组慢病毒以及带CAR-DR5编码基因的第二重组慢病毒；
    (5)将所述第一重组慢病毒和所述第二重组慢病毒按顺序地或同时地联合转染CD3阳性T淋巴细胞，经分离获得靶向FGFR4和DR5的嵌合抗原受体T细胞。
  10. 如权利要求9所述的靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法，其特征在于，所述CAR-FGFR4的编码基因包括如SEQ ID NO：8-9所示的任一氨基酸序列对应的核苷酸序列，所述CAR-DR5的编码基因包括如SEQ ID NO：10所示的氨基酸序列对应的核苷酸序列。
  11. 如权利要求9所述的靶向FGFR4和DR5的嵌合抗原受体T细胞的制备方法，其特征在于，所述第一重组慢病毒和所述第二重组慢病毒的滴度比为1：(0.5-2)。
  12. 一种药物组合物，其特征在于，包括如权利要求1-6任一项所述或如权利要求9-11任一项所述的制备方法制得的靶向FGFR4和DR5的嵌合抗原受体T细胞、如权利要求7所述的重组载体或如权利要求8所述的宿主细胞。
  13. 如权利要求12所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体和/或辅料。
  14. 如权利要求13所述的药物组合物，其特征在于，所述载体包括溶剂、聚合物和脂质体中的至少一种，所述辅料包括稀释剂、赋形剂和稳定剂中的至少一种。
  15. 如权利要求1-6任一项所述或如权利要求9-11任一项所述的制备方法制得的靶向FGFR4和DR5的嵌合抗原受体T细胞、如权利要求7所述的重组载体、如权利要求8所述的宿主细胞或如权利要求12-14任一项所述的药物组合物在制备预防、诊断和治疗恶性肿瘤的药物中的应用。
  16. 如权利要求15所述的应用，其特征在于，所述恶性肿瘤包括肝癌、脑胶质瘤、肺癌、胃癌、结肠癌、胰腺癌、乳腺癌、卵巢癌和宫颈癌中的至少一种。
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