CN112813030A

CN112813030A - Chimeric antigen receptor T cell targeting FGFR4 and DR5 and preparation method and application thereof

Info

Publication number: CN112813030A
Application number: CN201911120424.0A
Authority: CN
Inventors: 许晨光; 张宏玲; 温丽联; 黎琴子; 刘婉
Original assignee: Shenzhen Bindebio Technology Co ltd
Current assignee: Shenzhen Bindebio Technology Co ltd
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2021-05-18
Also published as: WO2021093251A1

Abstract

The invention provides a chimeric antigen receptor T cell targeting FGFR4 and DR5, which comprises a chimeric antigen receptor CAR-FGFR4 targeting FGFR4 and a chimeric antigen receptor CAR-DR5 targeting DR5, wherein CAR-FGFR4 comprises a targeted FGFR4 single-chain antibody, an extracellular hinge region, a transmembrane region and an intracellular signal region which are sequentially connected from an amino terminal to a carboxyl terminal, and CAR-DR5 comprises a targeted DR5 single-chain antibody, an extracellular hinge region, a transmembrane region and an intracellular signal region which are sequentially connected from an amino terminal to a carboxyl terminal; single chain antibodies targeting FGFR4 include at least one of: (a) as shown in SEQ ID NO: 1 and a heavy chain VH as shown in SEQ ID NO: 2, a single chain antibody light chain VL; (b) as shown in SEQ ID NO: 3 and a single chain antibody heavy chain VH as shown in SEQ ID NO: 4, and a single chain antibody light chain VL.

Description

Chimeric antigen receptor T cell targeting FGFR4 and DR5 and preparation method and application thereof

Technical Field

The invention relates to the field of medical biology, in particular to a chimeric antigen receptor T cell targeting FGFR4 and DR5, a preparation method and application thereof.

Background

Primary liver cancer is one of the most common malignant tumors, is the second leading cause of cancer death worldwide, and seriously threatens the health and life of people. The common clinical liver cancer treatment methods mainly comprise surgical resection, liver transplantation, chemical drug therapy, radiotherapy and the like. However, since early liver cancer is not obvious and thus not easy to detect, most patients have been diagnosed in the middle and late stages, and the chance of surgical treatment and liver transplantation is lost. About 90% of liver cancer patients in actual treatment need to receive drug treatment. The existing common antitumor drugs can effectively prolong the life of patients, but have the problems of low selectivity, large toxic and side effects and the like in clinical application. Even targeted drugs have a problem that they are likely to induce tumor resistance after long-term use. E.g. the multi-kinase inhibitor sorafenib approved in 2007, but only improved overall survival by 3 months. Therefore, the clinical search for a safe and effective therapy for liver cancer is still a difficult problem.

Death receptor 5(DR5), also known as TRAIL-R2, is highly expressed in a variety of tumor tissues, especially lung cancer, breast cancer, ovarian cancer, rectal cancer, cervical cancer and the like, but is hardly expressed in most normal cells, and becomes an ideal target. The fibroblast growth factor receptor family has four members, FGFR1-4, and the homology of the kinase domain is as high as 74-92%. By analyzing the structure of the FGFR family, it was found that FGFR4 differs from other members at amino acid 552, cysteine being present in FGFR4, and tyrosine being present in other members at this site. The characteristics of the cysteine can be used for designing an inhibitor to achieve high selectivity targeting FGFR 4. FGFR4 is overexpressed in various tumors, such as liver cancer, breast cancer, gastric cancer, and the like. Currently in the pharmaceutical industry, inhibitors of FGFR4, H3B-6527, have acquired U.S. FDA-granted orphan drug qualification. FGFR4 selective inhibitors are essentially all covalent reversible or irreversible inhibitors based on Cys552, once this site mutation is likely to lead to the development of a drug-resistant situation.

Immune cell therapy is the only method which has the possibility of completely eliminating cancer cells in the prior art, has the great advantages of strong specificity and almost no toxic or side effect in treating tumors, overcomes the defects of the traditional therapy, is already used for clinically treating malignant tumors at home and abroad, and a chimeric antigen receptor T cell technology (CAR-T) is one of the latest immune cell technologies of the current adoptive cell reinfusion therapy technology, and is widely concerned and researched because the CAR-T can activate an autoimmune system in vivo and continuously target tumor cells to kill the tumor cells, and finally the aim of eliminating the malignant tumor cells is fulfilled. Currently, there is no preparation and study of chimeric antigen receptor T cells targeting both FGFR4 and DR 5.

Disclosure of Invention

In view of this, the 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, a CAR-FGFR 4-specific targeting FGFR4, and a CAR-DR 5-specific targeting DR5, thereby promoting T cell expansion in a patient, killing tumor cells with high efficiency and specificity, effectively inhibiting tumor cell escape, better maintaining the viability and lethality of the chimeric antigen receptor T cell, and not causing damage to normal cells. The invention also provides a preparation method and application of the chimeric antigen receptor T cell targeting FGFR4 and DR 5.

In a first aspect, the invention provides a chimeric antigen receptor T cell targeting FGFR4 and DR5, comprising a chimeric antigen receptor CAR-FGFR4 targeting FGFR4 and a chimeric antigen receptor CAR-DR5 targeting DR5, the amino acid sequence of CAR-FGFR4 comprises, sequentially connected from amino terminus to carboxyl terminus, an amino acid sequence of a single-chain antibody targeting FGFR4, an extracellular hinge region, a transmembrane region and an intracellular signal region, and the amino acid sequence of CAR-DR5 comprises, sequentially connected from amino terminus to carboxyl terminus, an amino acid sequence of a single-chain antibody targeting DR5, an extracellular hinge region, a transmembrane region and an intracellular signal region;

wherein the amino acid sequence of the single chain antibody targeting FGFR4 comprises at least one of:

(a) as shown in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2, the amino acid sequence of a light chain VL of the single-chain antibody;

(b) as shown in SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4, and the amino acid sequence of a single-chain antibody light chain VL.

In the present invention, the amino acid sequence of the single chain antibody targeting FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2, or the amino acid sequence of the single-chain antibody targeting FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4, or the amino acid sequence of the single-chain antibody targeting FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2, the amino acid sequence of a light chain VL of the single-chain antibody; and as shown in SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4, and the amino acid sequence of a single-chain antibody light chain VL.

In the present invention, the amino acid sequence shown in SEQ ID NO: 1 and a heavy chain VH as shown in SEQ ID NO: 2 to form a single-chain antibody with a targeting FGFR4, as shown in SEQ ID NO: 3 and a single chain antibody heavy chain VH as shown in SEQ ID NO: 4 to form a single-chain antibody targeting FGFR 4.

Optionally, the amino acid sequence as set forth in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2 is linked by a first linking peptide.

Optionally, the amino acid sequence as set forth in SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4 is connected through a second connecting peptide.

In the present invention, 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, so that the connected heavy chain and light chain of the single-chain antibody maintain their respective spatial conformations to maintain the function and activity of the whole single-chain antibody. In the present invention, the first linker peptide and the second linker peptide may be, but not limited to, polypeptide sequences mainly composed of glycine and serine, wherein the 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 and increases the hydrophilicity of the peptide chain. Optionally, the amino acid sequence of the first connecting peptide is GGGGSGGGGSGGGGS, and the amino acid sequence of the second connecting peptide is ggggsggggsggs.

In the present invention, the amino acid sequences of the single-chain antibody heavy chain VH, the single-chain antibody light chain VL and the linker peptide linker are linked in the order of VH-linker-VL and/or VL-linker-VH from the amino acid terminus to the carboxyl terminus.

That is, the amino acid sequence of the single chain antibody targeting FGFR4 comprises at least one of:

(i) comprises the following components from the amino acid end to the carboxyl end in sequence: the nucleotide sequence shown as SEQ ID NO: 1, the amino acid sequence of the first connecting peptide and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2, the amino acid sequence of a light chain VL of the single-chain antibody;

(ii) comprises the following components from the amino acid end to the carboxyl end in sequence: the nucleotide sequence shown as SEQ ID NO: 2, the amino acid sequence of the first linking peptide, and the amino acid sequence of the single-chain antibody light chain VL shown in SEQ ID NO: 1 of a single-chain antibody heavy chain VH;

(iii) comprises the following components from the amino acid end to the carboxyl end in sequence: the nucleotide sequence shown as SEQ ID NO: 3, the amino acid sequence of the heavy chain VH of the single-chain antibody, the amino acid sequence of the second connecting peptide, and the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of a light chain VL of the single-chain antibody;

(iiii) comprises, in order from amino terminus to carboxy terminus: the nucleotide sequence shown as SEQ ID NO: 4, the amino acid sequence of the second connecting peptide and the amino acid sequence of the single-chain antibody light chain VL shown in SEQ ID NO: 3, and the amino acid sequence of the heavy chain VH of the single-chain antibody.

In the invention, the sequential position relationship of the single-chain antibody light chain VL and the single-chain antibody heavy chain VH does not influence the targeting property and the activity of the single-chain antibody.

Alternatively, the amino acid sequence of the single chain antibody targeting FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 6-7.

In the invention, the encoding gene of the single-chain antibody targeting FGFR4 comprises a nucleotide sequence corresponding to the amino acid sequence of the single-chain antibody targeting FGFR 4.

Alternatively, the amino acid sequence of the DR 5-targeting single chain antibody comprises the amino acid sequence set forth in SEQ ID NO: 5.

In the present invention, the "connecting in sequence from amino terminus to carboxyl terminus" specifically includes: the carboxyl terminal of the amino acid sequence of the single chain antibody is connected with the amino terminal of the amino acid sequence of the extracellular hinge region, the carboxyl terminal of the amino acid sequence of the extracellular hinge region is connected with the amino terminal of the amino acid sequence of the transmembrane region, and the carboxyl terminal of the amino acid sequence of the transmembrane region is connected with the amino terminal of the amino acid sequence of the intracellular signal region.

In the present invention, the extracellular hinge region is used to facilitate binding of the single chain antibody targeting FGFR4 to FGFR4 on tumors.

Optionally, the extracellular hinge region comprises a combination of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, an ICOS hinge region. Further optionally, the extracellular hinge region comprises a CD8 a hinge region.

In the present invention, the transmembrane region is used to immobilize the chimeric antigen receptor CAR-FGFR4 targeting FGFR 4.

Optionally, the transmembrane region comprises one or more of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region, and a CD28 transmembrane region. Further optionally, the transmembrane region comprises a CD8 transmembrane region.

In the present invention, the intracellular signaling region is used to provide a signal for T cell activation, maintain the survival time of T cells, and activate T cell proliferation signaling pathways.

Optionally, the intracellular signaling region comprises a combination of one or more of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region. Optionally, the intracellular signaling region comprises a 4-1BB signaling region and a CD3 zeta signaling region.

In the present invention, the hinge region, transmembrane region and signal region of the CAR-FGFR4 and CAR-DR5 may be the same or different, and are selected according to actual needs.

Optionally, the amino acid sequence of CAR-FGFR4 comprises, sequentially linked from amino terminus to carboxy terminus, an amino acid sequence targeting a single chain antibody of FGFR4, a CD8 a hinge region, a CD8 transmembrane region, a 4-1BB signal region, and a CD3 zeta signal region.

Alternatively, the amino acid sequence of CAR-FGFR4 comprises the amino acid sequence set forth as SEQ ID NO: 8-9.

In the invention, the 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 influence the targeting property and the activity of CAR-FGFR4, and the prepared CAR-FGFR4 has the effect of targeting FGFR 4.

In the invention, the encoding gene of the chimeric antigen receptor CAR-FGFR4 targeting FGFR4 comprises a nucleotide sequence corresponding to the amino acid sequence of the chimeric antigen receptor CAR-FGFR4 targeting FGFR 4.

Optionally, the amino acid sequence of CAR-DR5 includes, connected in sequence from amino-terminus to carboxy-terminus, an amino acid sequence of a single chain antibody targeting DR5, a CD8 a hinge region, a CD8 transmembrane region, a 4-1BB signal region, and a CD3 zeta signal region.

Alternatively, the amino acid sequence of CAR-DR5 includes the amino acid sequence set forth as SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

In the invention, the encoding gene of the CAR-DR5 comprises a nucleotide sequence corresponding to the amino acid sequence of the DR5 targeted chimeric antigen receptor CAR-DR 5.

In the present invention, the chimeric antigen receptor T cells targeting FGFR4 and DR5 may be dual-target chimeric antigen receptor T cells with CAR-FGFR4 and CAR-DR5 (i.e., dual-target chimeric antigen receptor T cells targeting FGFR4 and DR5), may also be a mixture of chimeric antigen receptor T cells with CAR-FGFR4 and chimeric antigen receptor T cells with CAR-DR5, or a mixture of dual-target chimeric antigen receptor T cells with CAR-4 and CAR-DR5 and at least one of chimeric antigen receptor T cells with CAR-FGFR4 and chimeric antigen receptor T cells with CAR-DR 5. In this case, the chimeric antigen receptor T cell can recognize both a tumor cell expressing the FGFR4 antigen protein on the surface and a tumor cell expressing the DR5 antigen protein on the surface, and certainly, has a good ability to recognize a tumor cell expressing both the FGFR4 antigen protein and the DR5 antigen protein, and can effectively prevent the tumor cell from immune escape.

Wherein, when 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 location distribution of CAR-FGFR4 and CAR-DR5 is not limited. Optionally, the CAR-FGFR4 and the CAR-DR5 are distributed alternately. There is no chemical bond linkage between the single chain antibody of CAR-FGFR4 and the single chain antibody of CAR-DR5 to ensure better recognition ability.

The chimeric antigen receptor T cell targeting FGFR4 provided by the first aspect of the invention can specifically target tumor cells expressing FGFR4, and after the CAR-FGFR4 is combined with the FGFR4, the intracellular signal region of the T cell is activated, so that the expansion of the T cell in a patient body is promoted, the tumor cells are killed efficiently and specifically, and the normal cells are hardly damaged.

In a second aspect, the invention provides a recombinant vector comprising the CAR-FGFR4 and CAR-DR5 encoding genes in a chimeric antigen receptor T cell targeting FGFR4 and DR5 as described in the first aspect.

In the invention, the genes encoding the CAR-FGFR4 and the CAR-DR5 can be contained in the same recombinant vector, or the genes encoding the CAR-FGFR4 and the CAR-DR5 can be contained in different vectors.

In the present invention, the vector may be, but is not limited to, a gene delivery vector.

Optionally, the vector is at least one of a viral vector and a non-viral vector.

Further, the non-viral vector includes a plasmid vector and a phage vector. Specifically, the plasmid vector may be, but not limited to, a eukaryotic plasmid vector, a prokaryotic plasmid vector, a micro-circle DNA, a transposon, etc. When the vector is micro-ring DNA, the CD3 positive T lymphocyte can be directly transfected by the recombinant micro-ring DNA inserted with the coding gene of the chimeric antigen receptor CAR-FGFR4 targeting FGFR4, so that the chimeric antigen receptor T cell targeting FGFR4 is prepared.

Further, the viral vector includes a lentiviral vector, an adenoviral vector or a retroviral vector. Further, the viral vector is a lentiviral vector.

The recombinant vector provided by the second aspect of the invention is safe and efficient, can stably realize the introduction or replication of the coding genes of CAR-FGFR4 and CAR-DR5 into host cells, and can be used for preparing chimeric antigen receptor T cells.

In a third aspect, the present invention provides a host cell comprising a recombinant vector as described in the second aspect.

Alternatively, when the recombinant vector is a recombinant viral vector, the host cell can be used to assemble the recombinant viral vector so that it is infectious. Further, the host cell may include, but is not limited to, HEK293T cell, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, or COS7 cell, and the like. Further, the host cell is a HEK293T cell.

Optionally, when the recombinant plasmid is a recombinant eukaryotic plasmid vector, a recombinant prokaryotic plasmid vector, or a recombinant minicircle DNA, the host cell is a corresponding eukaryotic host cell or prokaryotic host cell.

The host cell provided by the third aspect of the invention can stably store the coding genes of CAR-FGFR4 and CAR-DR5 which target FGFR4 and DR5, and is beneficial to the preparation of chimeric antigen receptor T cells which target FGFR4 and DR 5.

In a fourth aspect, the present invention provides a method for preparing a chimeric antigen receptor T cell targeting FGFR4 and DR5, comprising:

(1) providing a gene encoding a chimeric antigen receptor CAR-FGFR4 targeting FGFR4, comprising a gene encoding a first signal peptide, a gene encoding a single chain antibody targeting FGFR4, a gene encoding a first extracellular hinge region, a gene encoding a first transmembrane region, a gene encoding a first intracellular signal region, connected in sequence from the 5 'end to the 3' end, wherein the amino acid sequence of the single chain antibody targeting FGFR4 comprises at least one of:

(b) as shown in SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4, the amino acid sequence of a light chain VL of the single-chain antibody;

(2) providing genes encoding a chimeric antigen receptor CAR-DR5 targeting DR5, comprising a gene encoding a second signal peptide, a gene encoding a single chain antibody targeting DR5, a gene encoding a second extracellular hinge region, a gene encoding a second transmembrane region, a gene encoding a second intracellular signal region, linked in sequence from the 5 'end to the 3' end;

(3) inserting the encoding gene of the CAR-FGFR4 into a first gene delivery vector to obtain a first recombinant gene delivery vector, and inserting the encoding gene of the CAR-DR5 into a second gene delivery vector to obtain a second recombinant gene delivery vector;

(4) packaging the first recombinant gene delivery vector and the second recombinant gene delivery vector to obtain a first recombinant lentivirus with a CAR-FGFR4 encoding gene and a second recombinant lentivirus with a CAR-DR5 encoding gene;

(5) and jointly transfecting the first recombinant lentivirus and the second recombinant lentivirus with CD3 positive T lymphocytes sequentially or simultaneously, and separating to obtain the chimeric antigen receptor T cells targeting FGFR4 and DR 5.

The "connecting in sequence from 5 'end to 3' end" is specifically: the 3 'end of the coding gene sequence of the signal peptide is connected with the 5' end of the coding gene of the single-chain antibody, the 3 'end of the coding gene of the single-chain antibody is connected with the 5' end of the coding gene of the extracellular hinge region, the 3 'end of the coding gene of the extracellular hinge region is connected with the 5' end of the coding gene of the transmembrane region, and the 3 'end of the coding gene of the transmembrane region is connected with the 5' end of the coding gene of the intracellular signal region.

In the present invention, the signal peptide is used to direct the expression of the chimeric antigen receptor CAR-FGFR4 to the cell surface, which signal peptide is cleaved by a signal peptidase during the translational maturation of the protein.

Alternatively, the amino acid sequence of the signal peptide comprises the amino acid sequence set forth as SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

The specific selection of the extracellular hinge region, transmembrane region and intracellular signal region and the corresponding coding gene sequence are as described in the section of the second aspect of the present invention and will not be described herein again.

Optionally, the CAR-FGFR4 encoding genes include a signal peptide encoding gene, a single-chain antibody targeting FGFR4 encoding gene, a CD8 α hinge region encoding gene, a CD8 transmembrane region encoding gene, a 4-1BB signal region encoding gene, and a CD3 zeta signal region encoding gene, which are linked in sequence from the 5 'end to the 3' end.

Alternatively, the gene encoding CAR-FGFR4 comprises the amino acid sequence set forth as SEQ ID NO: 8-9, or a nucleotide sequence corresponding to any one of the amino acid sequences shown in the specification.

Optionally, the CAR-DR5 encoding genes include a signal peptide encoding gene, a DR5 targeting single chain antibody encoding gene, a CD8 a hinge region encoding gene, a CD8 transmembrane region encoding gene, a 4-1BB signal region encoding gene, and a CD3 zeta signal region encoding gene, which are linked in sequence from the 5 'end to the 3' end.

Alternatively, the gene encoding CAR-DR5 comprises the amino acid sequence set forth in SEQ ID NO: 10, or a nucleotide sequence corresponding to the amino acid sequence shown in the figure.

In the present invention, the first gene delivery vector and the second gene delivery vector may be the same or different, and may be selected according to actual needs, the coding gene of CAR-FGFR4 and the coding gene of CAR-DR5 may be inserted into different gene delivery vectors, or may be inserted into the same gene delivery vector, and when the same gene delivery vector is inserted, the order of linkage between the coding gene of CAR-FGFR4 and the coding gene of CAR-DR5 is not limited.

In the invention, the coding gene of CAR-FGFR4 is inserted between BamHI and EcoRI enzyme cutting sites in pCDH-EF1-MCS vector, and is positioned behind EF1 alpha of pCDH-EF1-MCS vector, and EF1 alpha is used as promoter. When the coding gene of the CAR-FGFR4 is inserted into a pCDH-EF1-MCS vector, the 5 'end of the coding gene of the CAR-FGFR4 can be added with an initiation codon (such as ATG) to be connected with a BamHI enzyme cutting site in the pCDH-EF1-MCS vector, and the 3' end can be added with a termination codon (such as TAA) to be connected with an EcoRI enzyme cutting site in the pCDH-EF1-MCS vector.

In the invention, the coding gene of CAR-DR5 is inserted between BamHI and EcoRI enzyme cutting sites in pCDH-EF1-MCS vector, and is positioned behind EF1 alpha of pCDH-EF1-MCS vector, and EF1 alpha is used as a promoter. When the coding gene of the CAR-DR5 is inserted into a pCDH-EF1-MCS vector, the 5 'end of the coding gene of the CAR-DR5 can be added with an initiation codon (such as ATG) to be connected with a BamHI enzyme cutting site in the pCDH-EF1-MCS vector, and the 3' end can be added with a termination codon (such as TAA) to be connected with an EcoRI enzyme cutting site in the pCDH-EF1-MCS vector.

In the present invention, the first recombinant gene delivery vector and the second recombinant gene delivery vector may be packaged separately or simultaneously.

Optionally, packaging the first recombinant gene delivery vector and the second recombinant gene delivery vector to obtain a first recombinant lentivirus having a gene encoding CAR-FGFR4 and a second recombinant lentivirus having a gene encoding CAR-DR5, comprising:

co-transfecting the first recombinant gene delivery vector with an envelope plasmid and a packaging plasmid to obtain the first recombinant lentivirus; and co-transfecting the second recombinant gene delivery vector with an envelope plasmid and a packaging plasmid to obtain the second recombinant lentivirus.

Optionally, the envelope plasmid is PMD2G, the packaging plasmid is psPAX2, and the host cell is HEK293T cell. The enveloped plasmid PMD2G encodes a vesicular stomatitis virus glycoprotein capsid that aids in the adhesion of the recombinant lentivirus to the cell membrane and maintains infectivity of the recombinant lentivirus.

The recombinant lentivirus of the present invention may further contain envelope proteins from other viruses. For example, a viral envelope protein derived from a human cell infected with the protein is preferable. Such a protein is not particularly limited, and examples thereof include retroviral amphotropic virus hand membrane proteins, and envelope proteins derived from, for example, murine leukemia virus (MuMLV)4070A strain can be used. In addition, envelope proteins from MuMLV10Al may also be used. The proteins of the herpesviridae family include proteins gB, gD, gH and gp85 of herpes simplex virus, and proteins gp350 and gp220 of EB virus. As the proteins of the hepadnaviridae family, S proteins of hepatitis B virus and the like are exemplified. The envelope protein may also be formed by fusion of measles virus glycoprotein with other single chain antibodies.

Packaging of recombinant lentiviruses is usually by transient transfection or by cell line packaging. Human cell lines that can be used as packaging cells upon transient transfection include, for example, 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, and the like. Monkey-derived cell lines, for example, COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, and the like can also be used. Furthermore, commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin, are also commonly used.

Packaging of recombinant lentiviruses also employs some lentivirus packaging cell lines, such as stable cell lines produced using the most common Env glycoprotein, VSVG protein, or HIV-1gag-pol protein.

For safety reasons, the lentivirus vector systems used on a large scale all use a method of splitting the genome, i.e. locating genes with different helper functions on different plasmids. Currently, there are four-plasmid systems (where the coding gag-pol gene, Rev gene, VSVG gene, SIN transgene are located on four different plasmids), three-plasmid systems (where the plasmid coding for Rev gene is removed and the gag-pol gene in the gag-pol plasmid employs codons preferred in human cells), and two-plasmid systems (where the helper genes necessary for lentiviral vector packaging are located on the same plasmid, these helper genes being single gene sequences, and the other being a transgenic plasmid). There are also lentiviral packaging systems in use that exceed the four plasmid system.

Optionally, when the first recombinant lentivirus and the second recombinant lentivirus are co-transfected with CD3 positive T lymphocytes sequentially or simultaneously, the titer ratio of the first recombinant lentivirus to the second recombinant lentivirus is 1: (0.5-2).

Alternatively, the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells. Furthermore, the human-derived peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like. Further, the blood is derived from fresh peripheral blood or bone marrow collected after one month of operation and one month of chemotherapy for cancer patients.

Specifically, the process for obtaining the CD3 positive T lymphocyte is as follows: adding CD3/CD28 immunomagnetic beads into peripheral blood mononuclear cells according to a certain proportion, incubating for a period of time, putting a magnet for screening to obtain CD3 positive T lymphocytes coated by the immunomagnetic beads, and removing the magnetic beads to obtain CD3 positive T lymphocytes.

Optionally, co-transfecting the first recombinant lentivirus and the second recombinant lentivirus sequentially or simultaneously with a CD3 positive T lymphocyte comprising:

transfecting the first recombinant lentivirus with CD3 positive T lymphocytes, and then transfecting the second recombinant lentivirus; or after the second recombinant lentivirus is transfected with CD3 positive T lymphocytes, the first recombinant lentivirus is transfected; or co-transfecting the first recombinant lentivirus and the second recombinant lentivirus with a CD3 positive T lymphocyte simultaneously.

Optionally, the chimeric antigen receptor T cells targeting FGFR4 and DR5 comprise dual-targeted chimeric antigen receptor T cells with the CAR-FGFR4 and the CAR-DR5, or comprise a mixture of chimeric antigen receptor T cells with the CAR-FGFR4 and chimeric antigen receptor T cells with the CAR-DR5, or comprise a mixture of dual-targeted chimeric antigen receptor T cells with the CAR-FGFR4 and the CAR-DR5 and at least one of chimeric antigen receptor T cells with the CAR-FGFR4 and chimeric antigen receptor T cells with the CAR-DR 5.

When the chimeric antigen receptor T cell targeting FGFR4 and DR5 is a double-target chimeric antigen receptor T cell with the CAR-FGFR4 and the CAR-DR5, the surface of the targeting T lymphocyte has two independent and unbonded chimeric antigen receptors (namely two independent single-chain antibodies) without influencing the recognition and combination of the two targets, the targeting T lymphocyte can simultaneously and efficiently recognize the FGFR4 and DR5 on tumor cells, can recognize and kill the tumor cells expressing one or two of the FGFR4 and DR5, avoids the escape of the tumor cells, improves the breadth and width of targeted recognition, has broad spectrum of killing microenvironment, and has stronger tumor killing capability under complex tumor.

In a fifth aspect, the present invention provides a chimeric antigen receptor T cell targeting FGFR4 and DR5, the recombinant vector according to the second aspect, or the host cell according to the third aspect, prepared by the preparation method according to the first aspect or the fourth aspect, for use in the preparation of a medicament for the prevention, diagnosis, and treatment of malignant tumors.

In application, the administration mode can be, but is not limited to, intravenous injection, tumor in situ injection, subcutaneous injection and the like. The dosage, frequency and the like selected in the specific application are selected according to actual needs, and are not limited.

The application specifically comprises the following steps: there is provided a kit comprising one or more of a chimeric antigen receptor T cell targeting FGFR4 and DR5 of the first aspect, a recombinant vector of the second aspect, or a host cell of the third aspect.

In the present invention, the malignant tumor includes a tumor expressing FGFR4 and/or DR5, and further includes a tumor highly expressing FGFR4 and/or DR 5. Optionally, the malignant tumor comprises at least one of liver cancer, brain glioma, lung cancer, gastric cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer and cervical cancer. The chimeric antigen receptor T cell targeting FGFR4 and DR5 can efficiently recognize and kill cancer cells expressing FGFR4 and/or DR5, and is particularly suitable for liver cancer cells.

The invention has the beneficial effects that:

the invention provides chimeric antigen receptor T cells targeting FGFR4 and DR5, specifically targeting FGFR4 and DR5, activating a signal region in the T cells, promoting the expansion of the T cells in a patient body, efficiently and specifically killing tumor cells, identifying and killing the tumor cells expressing one or two of FGFR4 and DR5, effectively inhibiting the escape of the tumor cells, improving the breadth and width of targeted identification and killing broad spectrum, having stronger tumor killing capacity under a complex tumor microenvironment, being capable of self-replicating and propagating, having long half-life, forming memory cells, playing a role of continuous targeting and not damaging normal cells.

Drawings

FIG. 1 is a plasmid map of pCDH-EF1-FGFR4-CAR recombinant plasmid provided by the embodiment of the invention.

FIG. 2 is a plasmid map of a pCDH-EF1-DR5-CAR recombinant plasmid provided by an embodiment of the present invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Example one

A method of making a chimeric antigen receptor T cell targeted to FGFR4 and DR5, comprising:

(1) preparation of chimeric antigen receptor CAR-FGFR4 Gene sequence targeting FGFR4

Providing SEQ ID NO: 8-9 (CAR-FGFR4 gene sequence), and at the 5' end, a nucleotide sequence corresponding to any one of the amino acid sequences shown in SEQ ID NO: 11 (SEQ ID NO: 11) in the sequence of a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 11.

(2) Preparation of chimeric antigen receptor CAR-DR5 Gene sequence targeting DR5

Providing SEQ ID NO: 10 (CAR-FGFR 4), and a nucleotide sequence (CAR-FGFR4 gene sequence) corresponding to the amino acid sequence shown in SEQ ID NO: 11 (SEQ ID NO: 11) in the sequence of a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 11.

(3) Construction of pCDH-EF1-FGFR4-CAR recombinant plasmid and pCDH-EF1-DR5-CAR recombinant plasmid

The nucleotide sequence of the step (1) is inserted between BamHI and EcoRI enzyme cutting sites of pCDH-EF1-MCS vector, and EF1 alpha is used as a promoter after pCDH-EF1-MCS vector EF1 alpha. When the nucleotide sequence is inserted into a pCDH-EF1-MCS vector, an initiation codon (such as ATG) can be added at the 5 'end of the nucleotide sequence to be connected with a BamHI enzyme cutting site in the pCDH-EF1-MCS vector, and a termination codon (such as TAA) can be added at the 3' end to be connected with an EcoRI enzyme cutting site in the pCDH-EF1-MCS vector. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. The size and the sequence of the fragment which meets the target are identified through PCR product gel electrophoresis detection and sequencing, and the pCDH-EF1-FGFR4-CAR recombinant plasmid is successfully constructed, and is shown as a pCDH-EF1-FGFR4-CAR recombinant plasmid in figure 1.

The nucleotide sequence of the step (2) is inserted between BamHI and EcoRI enzyme cutting sites of pCDH-EF1-MCS vector, and EF1 alpha is used as a promoter after pCDH-EF1-MCS vector EF1 alpha. When the nucleotide sequence is inserted into a pCDH-EF1-MCS vector, an initiation codon (such as ATG) can be added at the 5 'end of the nucleotide sequence to be connected with a BamHI enzyme cutting site in the pCDH-EF1-MCS vector, and a termination codon (such as TAA) can be added at the 3' end to be connected with an EcoRI enzyme cutting site in the pCDH-EF1-MCS vector. Then transferred into escherichia coli competent cell DH5 alpha, and positive clone PCR identification and sequencing identification are carried out. The size and the sequence of the fragment which meets the target are identified through PCR product gel electrophoresis detection and sequencing, and the pCDH-EF1-DR5-CAR recombinant plasmid is successfully constructed, as shown in figure 2, the pCDH-EF1-DR5-CAR recombinant plasmid is shown.

(4) Recombinant lentivirus construction

The pCDH-EF1-FGFR4-CAR recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into the cultured HEK293T cells. Collecting virus-containing supernatant in 48h, filtering with 0.45 μm filter membrane, and storing in an ultra-low temperature refrigerator at-80 deg.C; harvesting virus-containing supernatants for the second 72h, filtering with 0.45 μm filter membrane, mixing with the virus supernatants harvested for the 48h, adding into an ultracentrifuge tube, placing into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after fully dissolving, centrifuging at high speed 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, and measuring virus according to 100 mul, 2 multiplied by 10⁸Subpackaging each/mL, and storing in an ultra-low temperature refrigerator at-80 ℃ to obtain the first recombinant lentivirus with CAR-FGFR 4.

The pCDH-EF1-DR5-CAR recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into the cultured HEK293T cells. Collecting virus-containing supernatant in 48h, filtering with 0.45 μm filter membrane, and storing in an ultra-low temperature refrigerator at-80 deg.C; harvesting virus-containing supernatants for the second 72h, filtering with 0.45 μm filter membrane, mixing with the virus supernatants harvested for the 48h, adding into an ultracentrifuge tube, placing into a Beckman ultracentrifuge one by one, setting the centrifugation parameters to be 25000rpm, the centrifugation time to be 2h, and controlling the centrifugation temperature to be 4 ℃; after the centrifugation is finished, removing the supernatant, removing the liquid remained on the tube wall as much as possible, adding a virus preservation solution, and lightly and repeatedly blowing and resuspending; after fully dissolving, centrifuging at high speed 10000rpm for 5min, taking supernatant to measure titer by a fluorescence method, and measuring virus according to 100 mul, 2 multiplied by 10⁸Subpackaging each strain/mL, and storing in an ultra-low temperature refrigerator at-80 ℃ to obtain the second recombinant lentivirus with CAR-DR 5.

(5) Preparation of chimeric antigen receptor T cells targeting FGFR4 and DR5

a) Isolation of PBMC (peripheral blood mononuclear cells)

PBMC is derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. Preferably fresh peripheral blood or bone marrow taken from cancer patients after one month of surgery and one month of chemotherapy.

Drawing blood from a patient and sending the blood to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking intermediate layer cells after Ficoll centrifugal separation; PBMC were obtained after PBS wash.

b) Separation of antigen specific T lymphocyte by immunomagnetic bead method

Taking the PBMC, adding a serum-free basal culture medium to prepare a cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 3:1, and incubating for 1-2h at room temperature; screening the cells incubated with the magnetic beads by using a magnet; after washing with PBS and removal of immunomagnetic beads, CD 3-positive T lymphocytes were obtained.

c) Preparation of antigen-specific T lymphocytes by virus transfection method

And (3) adding the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method into the first recombinant lentivirus with CAR-FGFR4 and the second recombinant lentivirus with CAR-DR5 with virus titer corresponding to the number of the CD3 positive cells for co-culture, wherein the titer ratio of the first recombinant lentivirus with CAR-FGFR4 to the second recombinant lentivirus with CAR-DR5 is 1: 1.

On the 3 rd day of the culture, cell counting and medium exchange were performed to adjust the cell concentration to 1X 10⁶Inoculating and culturing the seeds per mL; on the 5 th day of culture, the state of cells was observed, and if the cell density increased, the cell concentration was diluted to 1X 10⁶And (4) detecting the activity of the cells per mL, and continuing to culture. Expanding and culturing to 9-11 days, collecting cells, preparing chimeric antigen receptor T cells targeting FGFR4 and DR5, and storing the chimeric antigen receptor T cells in a cell freezing medium special for reinfusion.

Effects of the embodiment

To evaluate the chimeric antigen receptor T cells targeting FGFR4 and DR5 prepared by the above-described method described in the present invention, the following effect examples were performed.

Comparing the in vitro tumor killing effects of the chimeric antigen receptor T cells (experimental group) targeting FGFR4 and DR5 prepared by the method with the chimeric antigen receptor T cells (control group 1) targeting FGFR4, the chimeric antigen receptor T cells (control group 2) targeting DR5 and the T lymphocytes (negative control group), wherein a solid tumor cell line Huh7 is used as a blank control group, and specifically: the number ratio of effector cells (chimeric antigen receptor T cells targeting FGFR 4/chimeric antigen receptor T cells targeting FGFR 4/chimeric antigen receptor T cells targeting DR 5/T lymphocytes) to target cells (solid tumor cell line Huh7) was 10:1, 5:1, 2.5:1, 1.25:1 and 0.625:1 in vitro at 37 ℃, 5% CO₂The cell plates are co-cultured, the cell adherence condition is observed, and the killing capability of the cell plates is evaluated, the result shows that compared with a control group 1, a control group 2 and a negative control group, the chimeric antigen receptor T cell targeting FGFR4 and DR5 prepared by the method has excellent tumor killing capability, and the killing capability is far higher than that of the control group 1, the control group 2 and the negative control group, so that the chimeric antigen receptor T cell targeting FGFR4 and DR5 prepared by the method has very considerable application prospect in preparing the medicines for preventing, diagnosing and treating malignant tumors.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shenzhen Binje Biotechnology Limited

<120> chimeric antigen receptor T cell targeting FGFR4 and DR5, and preparation method and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 1

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Asn Gly Gly Arg Tyr Ile Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Glu Lys Thr Ser Gly Tyr Val Trp Phe Ala Tyr Trp

100 105 110

Gly Glu Gly Thr Leu Val Thr Val

115 120

<210> 2

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 2

Asp Ile Gln Met Thr Gln Thr Pro Ala Ser Leu Ser Ala Ser Val Glu

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Arg Ser Pro Gln Leu Leu Leu

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His Phe Ser Gly Thr Pro Tyr

85 90 95

Thr Phe Gly Glu Gly Thr Lys Leu Glu Ile

100 105

<210> 3

<211> 116

<212> PRT

<213> Artificial Sequence

<400> 3

Glu Val Met Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Leu Cys

85 90 95

Val Arg Ser Ser Ser Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ile Val Thr Val

115

<210> 4

<211> 111

<212> PRT

<213> Artificial Sequence

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Leu Thr Phe Gly Glu Gly Thr Lys Leu Glu Ile

100 105 110

<210> 5

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 5

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

130 135 140

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Thr Cys

145 150 155 160

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Glu Ile Asn Pro Asp Ser Ser Arg Ile Asn Tyr Met Pro Ser Leu

180 185 190

Lys Glu Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

210 215 220

Ala Arg Gly Gly Thr Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 6

<211> 241

<212> PRT

<213> Artificial Sequence

<400> 6

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Asn Gly Gly Arg Tyr Ile Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Glu Lys Thr Ser Gly Tyr Val Trp Phe Ala Tyr Trp

100 105 110

Gly Glu Gly Thr Leu Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Pro Ala

130 135 140

Ser Leu Ser Ala Ser Val Glu Glu Thr Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly

165 170 175

Arg Ser Pro Gln Leu Leu Leu Tyr Asn Ala Lys Thr Leu Ala Glu Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu

195 200 205

Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Ile Tyr Tyr Cys Gln

210 215 220

His Phe Ser Gly Thr Pro Tyr Thr Phe Gly Glu Gly Thr Lys Leu Glu

225 230 235 240

Ile

<210> 7

<211> 242

<212> PRT

<213> Artificial Sequence

<400> 7

Glu Val Met Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Leu Cys

85 90 95

Val Arg Ser Ser Ser Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ile Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val

130 135 140

Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

145 150 155 160

Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

210 215 220

Cys Lys Gln Ser Tyr Asn Leu Leu Thr Phe Gly Glu Gly Thr Lys Leu

225 230 235 240

Glu Ile

<210> 8

<211> 464

<212> PRT

<213> Artificial Sequence

<400> 8

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Glu Ile Ser Asn Gly Gly Arg Tyr Ile Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg Gly Gly Glu Lys Thr Ser Gly Tyr Val Trp Phe Ala Tyr Trp

100 105 110

Gly Glu Gly Thr Leu Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Pro Ala

130 135 140

Ser Leu Ser Ala Ser Val Glu Glu Thr Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly

165 170 175

Arg Ser Pro Gln Leu Leu Leu Tyr Asn Ala Lys Thr Leu Ala Glu Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu

195 200 205

Lys Ile Asn Ser Leu Gln Pro Glu Asp Phe Gly Ile Tyr Tyr Cys Gln

210 215 220

His Phe Ser Gly Thr Pro Tyr Thr Phe Gly Glu Gly Thr Lys Leu Glu

225 230 235 240

Ile Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

245 250 255

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

260 265 270

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

275 280 285

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

290 295 300

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

305 310 315 320

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

325 330 335

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

340 345 350

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

355 360 365

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

370 375 380

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

385 390 395 400

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

405 410 415

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

420 425 430

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

435 440 445

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

450 455 460

<210> 9

<211> 465

<212> PRT

<213> Artificial Sequence

<400> 9

Glu Val Met Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Leu Cys

85 90 95

Val Arg Ser Ser Ser Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ile Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val

130 135 140

Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

145 150 155 160

Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

210 215 220

Cys Lys Gln Ser Tyr Asn Leu Leu Thr Phe Gly Glu Gly Thr Lys Leu

225 230 235 240

Glu Ile Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

245 250 255

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

260 265 270

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

275 280 285

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

290 295 300

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr

305 310 315 320

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

325 330 335

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

340 345 350

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 10

<211> 469

<212> PRT

<213> Artificial Sequence

<400> 10

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

130 135 140

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Thr Cys

145 150 155 160

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

165 170 175

Gly Glu Ile Asn Pro Asp Ser Ser Arg Ile Asn Tyr Met Pro Ser Leu

180 185 190

Lys Glu Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

195 200 205

Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

210 215 220

Ala Arg Gly Gly Thr Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

305 310 315 320

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

325 330 335

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

340 345 350

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

355 360 365

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

370 375 380

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

385 390 395 400

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

405 410 415

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

420 425 430

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

435 440 445

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

450 455 460

Ala Leu Pro Pro Arg

465

<210> 11

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

Claims

1. A chimeric antigen receptor T cell targeting FGFR4 and DR5, comprising a chimeric antigen receptor CAR-FGFR4 targeting FGFR4 and a chimeric antigen receptor CAR-DR5 targeting DR5, the amino acid sequence of CAR-FGFR4 comprising, sequentially linked from amino terminus to carboxy terminus, the amino acid sequence of a single-chain antibody targeting FGFR4, an extracellular hinge region, a transmembrane region and an intracellular signal region, the amino acid sequence of CAR-DR5 comprising, sequentially linked from amino terminus to carboxy terminus, the amino acid sequence of a single-chain antibody targeting DR5, an extracellular hinge region, a transmembrane region and an intracellular signal region;

2. The chimeric antigen receptor T cell targeting FGFR4 and DR5 of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 2 is connected through a first connecting peptide, and the amino acid sequence of the light chain VL of the single-chain antibody is shown as SEQ ID NO: 3 and the amino acid sequence of the heavy chain VH of the single-chain antibody shown as SEQ ID NO: 4 is connected through a second connecting peptide.

3. The chimeric antigen receptor T cell targeting FGFR4 and DR5 of claim 1, wherein the amino acid sequence of the single chain antibody targeting DR5 comprises the amino acid sequence set forth in SEQ ID NO: 5.

4. The chimeric antigen receptor T cell targeting FGFR4 and DR5 of claim 1, wherein the amino acid sequence of the single chain antibody targeting FGFR4 comprises an amino acid sequence as set forth in SEQ ID NO: 6-7.

5. The chimeric antigen receptor T cell targeting FGFR4 and DR5 of claim 1, wherein the amino acid sequence of CAR-FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 8-9, the amino acid sequence of CAR-DR5 comprises the amino acid sequence as set forth in SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

6. A recombinant vector comprising the CAR-FGFR4 and the CAR-DR5 encoding genes in the chimeric antigen receptor T cell targeting FGFR4 and DR5 of any of claims 1-4.

7. A host cell comprising the recombinant vector of claim 6.

8. A method of making a chimeric antigen receptor T cell targeted to FGFR4 and DR5, comprising:

9. The method of making a chimeric antigen receptor T cell targeting FGFR4 and DR5 of claim 8, wherein the gene encoding CAR-FGFR4 comprises the amino acid sequence set forth in SEQ ID NO: 8-9, and the gene encoding the CAR-DR5 comprises a nucleotide sequence corresponding to any one of the amino acid sequences shown in SEQ ID NO: 10, or a nucleotide sequence corresponding to the amino acid sequence shown in the figure.

10. Use of a chimeric antigen receptor T cell targeting FGFR4 and DR5, produced by the process according to any one of claims 1 to 5 or by the process according to any one of claims 8 to 9, a recombinant vector according to claim 6 or a host cell according to claim 7 for the preparation of a medicament for the prophylaxis, diagnosis and treatment of malignancies.