CN110157675B

CN110157675B - Targeting T lymphocyte and preparation method and application thereof

Info

Publication number: CN110157675B
Application number: CN201810148269.2A
Authority: CN
Inventors: 张宏玲; 龙丽梅
Original assignee: Shenzhen Bindebiotech Co ltd
Current assignee: Shenzhen Bindebiotech Co ltd
Priority date: 2018-02-12
Filing date: 2018-02-12
Publication date: 2022-05-27
Anticipated expiration: 2038-02-12
Also published as: CN110157675A

Abstract

The invention provides a targeting T lymphocyte, which comprises a chimeric antigen receptor CAR-CD19 targeting CD19 and/or a chimeric antigen receptor CAR-BCMA targeting BCMA, has wide and strong targeting identification on tumor cells, can prevent the tumor cells from target escape, kills the tumor cells efficiently and specifically, and expands the broad-spectrum killing property of the tumor cells. The invention also provides a preparation method and application of the targeting T lymphocyte.

Description

Targeting T lymphocyte and preparation method and application thereof

Technical Field

The invention relates to the field of medical biology, in particular to a targeting T lymphocyte and a preparation method and application thereof.

Background

Leukemia is a malignant disease of the hematopoietic system and has a high mortality rate among malignant tumors of various age groups in China. CAR-T (chimeric antigen receptor T cell) technology is a novel immune cell therapy, and is characterized in that T cells modified by CAR are infused back to a human body to activate an autoimmune system and kill tumor cells so as to achieve the purpose of eliminating malignant tumor cells.

Although the CAR-T technology achieves significant efficacy in the treatment of hematological tumors and the like, in a complex tumor microenvironment (for example, multiple tumor antigens appear simultaneously), the recognition and killing capabilities of the CAR-T technology on tumor cells are greatly reduced, and the clinical application of the CAR-T technology is limited. For example, in lymphocytic leukemia, most patient tumor cells express CD19, but there are also cases where CD19 is not expressed (i.e., CD19 negative escape). Therefore, there is a need to provide T lymphocytes with a strong target for malignant tumors.

Disclosure of Invention

In view of the above, the invention provides a targeting T lymphocyte and a preparation method and application thereof. The T lymphocyte can target two tumor targets of B cell malignant tumor (such as lymphocyte leukemia), has high targeting property and wide and strong identification property on tumor cells, can prevent the tumor cells from escaping from the targets, and expands the broad-spectrum killing property of the T cells.

In a first aspect, the present invention provides a targeted T lymphocyte, comprising a CD 19-targeting chimeric antigen receptor CAR-CD19 and/or a BCMA-targeting chimeric antigen receptor CAR-BCMA, wherein the CAR-CD19 comprises, connected in sequence from amino terminus to carboxy terminus, the amino acid sequence of a CD 19-targeting single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region, and the CAR-BCMA comprises, connected in sequence from amino terminus to carboxy terminus, the amino acid sequence of a BCMA-targeting single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region; wherein the amino acid sequence of the CD 19-targeting single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the BCMA-targeted single-chain antibody comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

Preferably, the targeted T lymphocyte can be a dual-target chimeric antigen receptor T cell with CAR-CD19 and CAR-BCMA (i.e., a dual-target chimeric antigen receptor T cell targeted to CD19 and BCMA), can also be a mixture of a chimeric antigen receptor T cell with CAR-CD19 and a chimeric antigen receptor T cell with CAR-BCMA, can also be a dual-target chimeric antigen receptor T cell with CAR-CD19 and CAR-BCMA, a chimeric antigen receptor T cell with CAR-CD19, and a mixture of a chimeric antigen receptor T cell with CAR-BCMA.

At this time, the targeting T lymphocyte can recognize tumor cells with CD19 antigen protein expressed on the surface, and can also recognize tumor cells with BCMA antigen protein expressed on the surface, certainly, the targeting T lymphocyte has better recognition performance on tumor cells with both CD19 antigen protein and BCMA antigen protein, and can effectively avoid the occurrence of CD19 immune escape of tumor cells.

Wherein, when the targeted T lymphocyte is a double-target CAR-T with CAR-CD19 and CAR-BCMA, the distribution positions of the chimeric antigen receptors CAR-CD19 and CAR-BCMA are not limited, and can be alternatively distributed (such as ABAB … and AABABB …) or sequentially distributed (such as AAAA … BBMA B …), but the two are not covalently connected, and the corresponding CD19 single-chain antibody and the BCMA single-chain antibody are not covalently connected, so that the two can keep better recognition capability.

The "connecting in sequence from amino terminus to carboxyl terminus" is specifically: the carboxyl end of the amino acid sequence of the single-chain antibody targeting CD19 or the single-chain antibody targeting BCMA is linked to the amino end of the amino acid sequence of the extracellular hinge region, the carboxyl end of the amino acid sequence of the extracellular hinge region is linked to the amino end of the amino acid sequence of the transmembrane region, and the carboxyl end of the amino acid sequence of the transmembrane region is linked to the amino end of the amino acid sequence of the intracellular signal region.

The above-mentioned CD19 is a B cell line marker in the blood system, and is involved in processes such as B cell development, intracellular signaling, etc. by binding to a B cell receptor on a B cell. CD19 is a more common target for B-lineage malignancies (e.g., lymphocytic leukemia), but there are also a number of malignant B cells that do not express CD 19. BCMA is a member of the TNF superfamily of tumor necrosis factor receptors, is a major B cell biomarker, is not expressed in most normal cells and organs, and is widely present on Multiple Myeloma (MM) cells and the surface of B-line malignant tumors in the blood system. Therefore, BCMA is selected as a target of B-line malignant tumor cells with negative escape of CD19 or loss of CD19 antigen, and the CD19 escape of the tumor cells can be avoided.

Alternatively, the gene encoding the CD 19-targeting single chain antibody comprises the amino acid sequence as set forth in SEQ ID NO:3, and the coding gene of the BCMA-targeted single-chain antibody comprises the nucleotide sequence shown in SEQ ID NO: 4. The CD 19-targeted single-chain antibody disclosed by the invention has the affinity activity for a CD19 antigen, and can efficiently recognize tumor cells with CD19 antigens expressed on the surface. Similarly, the single-chain antibody targeting BCMA also retains the affinity activity to the BCMA antigen, and can efficiently recognize tumor cells with the BCMA antigen expressed on the surface.

Alternatively, the coding gene of the amino acid sequence of the single-chain antibody targeting CD19 should take into consideration degenerate bases, that is, the coding gene of the amino acid sequence shown as SEQ ID NO. 1 includes the nucleotide sequence shown as SEQ ID NO. 3, and the protection scope should also protect the nucleotide sequence having base degeneracy with SEQ ID NO. 3, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 1. The genes encoding the amino acid sequences of said BCMA-targeting single chain antibodies should likewise take into account degenerate bases.

In the present invention, the extracellular hinge region in the CAR-CD19 is used to facilitate the binding of the CD19 targeted single chain antibody to CD19 on tumor cells; similarly, the extracellular hinge region in the CAR-BCMA is used to facilitate binding of the BCMA-targeted single chain antibody to BCMA on tumor cells.

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 is a CD8 a hinge region.

Alternatively, the amino acid sequence of the CD8 a hinge region comprises the amino acid sequence as set forth in SEQ ID NO:9, or a pharmaceutically acceptable salt thereof.

Alternatively, the gene encoding the CD8 a hinge region comprises the amino acid sequence as set forth in SEQ ID NO:10, or a nucleotide sequence shown in the figure.

Alternatively, the gene encoding the CD8 alpha hinge region should take into account degenerate bases, i.e., the gene encoding the amino acid sequence shown in SEQ ID NO. 9 includes the nucleotide sequence shown in SEQ ID NO. 10, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 10, and the amino acid sequences corresponding to these nucleotide sequences are still SEQ ID NO. 9.

In the present invention, the transmembrane region in CAR-CD19 is used to immobilize the CD19 targeted chimeric antigen receptor CAR-CD 19; similarly, the transmembrane region in the CAR-BCMA is used to immobilize the BCMA-targeted chimeric antigen receptor CAR-BCMA.

Optionally, the transmembrane region comprises a combination of 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 is the CD8 transmembrane region.

Alternatively, the amino acid sequence of the CD8 transmembrane region comprises the amino acid sequence set forth as SEQ ID NO:11, or a pharmaceutically acceptable salt thereof.

Alternatively, the gene encoding the CD8 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 12.

Alternatively, the gene encoding the transmembrane region of CD8 should take into account degenerate bases, i.e., the gene encoding the amino acid sequence shown in SEQ ID NO. 11 includes the nucleotide sequence shown in SEQ ID NO. 12, and the protection scope should also protect the nucleotide sequence having the base degeneracy property with SEQ ID NO. 12, and the corresponding amino acid sequence of these nucleotide sequences is still SEQ ID NO. 11.

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 a T cell proliferation signaling pathway.

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, a TNFRSF19L signaling region.

In one embodiment of the present invention, the intracellular signaling region is a 4-1BB signaling region and a CD3 zeta signaling region sequentially linked from the amino terminus to the carboxy terminus. Accordingly, the genes encoding the intracellular signaling region include a gene encoding 4-1BB signaling region and a gene encoding CD3 zeta signaling region, which are sequentially linked from the 5 'end to the 3' end.

In another embodiment of the present invention, the intracellular signaling region may further comprise a CD27 signaling region and a CD3 zeta signaling region sequentially linked from an amino terminus to a carboxy terminus.

Alternatively, the amino acid sequence of the 4-1BB signal region comprises the amino acid sequence set forth as SEQ ID NO:13, or a pharmaceutically acceptable salt thereof.

Optionally, the gene encoding the 4-1BB signal region comprises a nucleotide sequence as set forth in SEQ ID NO:14, or a nucleotide sequence as set forth in fig. 14.

Alternatively, the gene encoding the 4-1BB signal region should take into account degenerate bases, i.e., the gene encoding the amino acid sequence shown in SEQ ID NO. 13 includes the nucleotide sequence shown in SEQ ID NO. 14, and the protection scope should also protect the nucleotide sequence having the base degeneracy property with SEQ ID NO. 14, and the amino acid sequence corresponding to these nucleotide sequences is still SEQ ID NO. 13.

Alternatively, the amino acid sequence of the CD3 zeta signal region comprises the amino acid sequence set forth in SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.

Alternatively, the gene encoding the CD3 zeta signaling region comprises the amino acid sequence set forth in SEQ ID NO: 16.

Alternatively, the gene encoding the CD3 zeta signal region should take into account degenerate bases, i.e. the gene encoding the amino acid sequence shown in SEQ ID NO. 15 comprises the nucleotide sequence shown in SEQ ID NO. 16, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 16, and the amino acid sequences corresponding to these nucleotide sequences are still SEQ ID NO. 15.

In the present invention, the amino acid sequences of the extracellular hinge region, transmembrane region and intracellular signal region in CAR-CD19 may be the same as or different from the amino acid sequences of the corresponding extracellular hinge region, transmembrane region and intracellular signal region in CAR-BCMA.

In one embodiment of the invention, the amino acid sequence of CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 5.

Optionally, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 17.

Alternatively, the CAR-CD19 encoding gene should take into account degenerate bases, i.e. the encoding gene of the amino acid sequence shown in SEQ ID No. 5 comprises the nucleotide sequence shown in SEQ ID No. 17, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID No. 17, the corresponding amino acid sequences of these nucleotide sequences still being SEQ ID No. 5.

In one embodiment of the invention, the amino acid sequence of CAR-BCMA comprises the amino acid sequence as set forth in SEQ ID NO: 6.

Optionally, the gene encoding CAR-BCMA comprises the sequence as set forth in SEQ ID NO:18, or a nucleotide sequence shown in the specification.

Alternatively, the gene encoding the CAR-BCMA should take into account degenerate bases, i.e., the gene encoding the amino acid sequence shown in SEQ ID NO. 6 comprises the nucleotide sequence shown in SEQ ID NO. 18, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 18, and the amino acid sequences corresponding to these nucleotide sequences are still SEQ ID NO. 6.

The targeting T lymphocyte provided by the first aspect of the invention comprises a chimeric antigen receptor CAR-CD19 targeting CD19 and/or a chimeric antigen receptor CAR-BCMA targeting BCMA, and can recognize two tumor targets, enhance the recognition of tumor cells and avoid the escape of the tumor cells. When CAR-CD19 and/or CAR-BCMA bind to the corresponding antigenic protein on the tumor cell, the intracellular signaling region of the targeted T lymphocyte is activated, promoting expansion of the T cell in the patient, and killing the tumor cell, particularly tumor cell expressing CD19 and/or BCMA, with high efficiency and specificity, expanding the broad spectrum killing of the T cell. In addition, the single-chain antibody of the CD19 and the BCMA is a humanized single-chain antibody, so that the targeting T cell avoids causing immune response of human bodies and has lasting in-vivo maintenance capacity such as activity and lethality.

The targeting T lymphocyte can effectively identify and kill tumor cells expressing CD19 and/or BCMA, especially malignant B lymphocytes, such as lymphocytic leukemia cells.

In a second aspect, the invention provides a recombinant viral vector comprising a gene encoding CAR-CD19 and/or CAR-BCMA in a targeted T lymphocyte as described in the first aspect.

Optionally, when the recombinant viral vector contains a gene encoding CAR-CD19 and a gene encoding CAR-BCMA, a specific sequence is included between the gene encoding CAR-CD19 and the gene encoding CAR-BCMA on the recombinant viral vector, such that the gene encoding CAR-CD19 and the gene encoding CAR-BCMA can obtain two separate proteins CAR-CD19 and CAR-BCMA after transcription and translation. After transfection of CD3 positive T lymphocytes with such recombinant viral vectors, the resulting targeted T lymphocytes are dual-target chimeric antigen receptor T cells with CAR-CD19 and CAR-BCMA. Further alternatively, the specific sequence may be an RBS sequence, an IRES sequence, a T2A sequence, or other protease sequence, etc.

Optionally, the recombinant viral vector carries the gene encoding the CAR-CD19, or carries the gene encoding the CAR-BCMA. Preferably, the targeting T lymphocyte according to the first aspect of the present invention can be obtained by co-infecting the T lymphocyte with a recombinant viral vector comprising a gene encoding CAR-CD19 and a recombinant viral vector comprising a gene encoding CAR-BCMA separately or simultaneously, and the targeting T lymphocyte can sufficiently express CAR-CD19 and/or CAR-BCMA.

Preferably, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 19. As shown in SEQ ID NO: 19 and the nucleotide sequence shown as SEQ ID NO:17, the gene encoding the linker peptide described below is increased as compared with the nucleotide sequence shown in FIG. 17. The encoding gene of the signal peptide can better guide the chimeric antigen receptor CAR-CD19 to reach the cell surface.

Preferably, the gene encoding CAR-BCMA comprises the sequence as set forth in SEQ ID NO: 20, or a nucleotide sequence shown in the specification. As shown in SEQ ID NO: 20 and the nucleotide sequence shown as SEQ ID NO:18, and a gene encoding a linker peptide described below is added to the nucleotide sequence shown in FIG. 18. The encoding gene of the signal peptide can better guide the chimeric antigen receptor CAR-BCMA to reach the cell surface.

Optionally, the viral vector in the recombinant viral vector comprises a lentiviral vector, an adenoviral vector or a retroviral vector. Further optionally, the viral vector is a lentiviral vector. Steps (1) to (3) of the production method provided in the fourth aspect of the present invention show the production process of the recombinant viral vector.

The recombinant virus vector provided by the second aspect of the invention has higher infection efficiency and transcription efficiency, wherein the coding gene segment of CAR-CD19 and/or CAR-BCMA can be inserted into a host genome through gene recombination to obtain the targeted T lymphocyte, so that the targeted T lymphocyte can continuously and stably exert targeting and killing effects.

In a third aspect, the present invention provides a host cell comprising a recombinant lentiviral vector according to the second aspect.

The host cell provided by the third aspect of the invention is used to assemble the recombinant viral vector of the second aspect so that it is infectious.

Alternatively, the host cells include, but are not limited to, HEK293T cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells, COS1 cells, and COS7 cells.

In a fourth aspect, the present invention provides a method for preparing a targeting T lymphocyte, comprising:

(1) providing a gene encoding a chimeric antigen receptor CAR-CD19 targeting CD19 and a gene encoding a chimeric antigen receptor CAR-BCMA targeting BCMA, respectively;

the coding gene of the CAR-CD19 comprises a coding gene which is sequentially connected with a signal peptide from a 5 'end to a 3' end, a coding gene of a single-chain antibody targeting CD19, a coding gene of an extracellular hinge region, a coding gene of a transmembrane region and a coding gene of an intracellular signal region; the coding gene of the CAR-BCMA comprises a coding gene which is sequentially connected with a signal peptide from a 5 'end to a 3' end, a coding gene of a single-chain antibody targeting the BCMA, a coding gene of an extracellular hinge region, a coding gene of a transmembrane region and a coding gene of an intracellular signal region;

wherein, the coding gene of the CD 19-targeted single-chain antibody comprises the sequence shown in SEQ ID NO:1, and the coding gene of the single-chain antibody targeting BCMA comprises a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO:2, and the nucleotide sequence corresponds to the amino acid sequence shown in the figure;

(2) inserting the coding gene of the CAR-CD19 and the coding gene of the CAR-BCMA into a pWPXLD vector respectively to obtain a pWPXLD-CAR-CD19 recombinant plasmid and a pWPXLD-CAR-BCMA recombinant plasmid;

(3) packaging the pWPXLD-CAR-CD19 recombinant plasmid and the pWPXLD-CAR-BCMA recombinant plasmid respectively to obtain a first recombinant lentivirus with a CAR-CD19 encoding gene and a second recombinant lentivirus with a CAR-BCMA encoding gene;

(4) and (3) transfecting the first recombinant lentivirus and the second recombinant lentivirus separately or simultaneously with the first recombinant lentivirus and the second recombinant lentivirus in a combined manner to obtain CD3 positive T lymphocytes, and separating to obtain the targeted T lymphocytes.

Taking the coding gene of CAR-CD19 as an example, the "sequence connection 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 targeting CD19, the 3 'end of the coding gene of the single-chain antibody targeting CD19 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 chimeric antigen receptor CAR-CD19 or CAR-BCMA expression to the cell surface, which is cleaved by a signal peptidase during the protein translation maturation. The signal peptide in the gene encoding CAR-CD19 may be identical to or different from the signal peptide in the gene encoding CAR-BCMA in amino acid sequence, or the signal peptides may be identical to or different from each other in amino acid sequence.

Alternatively, the amino acid sequence of the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:21, or a pharmaceutically acceptable salt thereof.

Optionally, the gene encoding the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO: 22.

Optionally, the gene encoding the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:23, or a pharmaceutically acceptable salt thereof.

Alternatively, the coding gene for the signal peptide should take into account degenerate bases, i.e., the coding gene for the amino acid sequence shown in SEQ ID NO. 21 comprises the nucleotide sequence shown in SEQ ID NO. 22 or SEQ ID NO. 23, and the scope of protection should also protect nucleotide sequences having base degeneracy with SEQ ID NO. 22 or SEQ ID NO. 23, and the corresponding amino acid sequences of these nucleotide sequences are still SEQ ID NO. 21.

For the specific selection of the extracellular hinge region, transmembrane region and intracellular signal region and the corresponding coding gene sequence, reference is made to the first aspect of the present invention, which is not described herein in detail.

Optionally, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 7, or a nucleotide sequence corresponding to the amino acid sequence shown in the specification.

Optionally, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 19. Of course, the gene encoding CAR-CD19 may also include a sequence identical to SEQ ID NO: 19 has base degeneracy.

Optionally, the gene encoding CAR-BCMA comprises the sequence as set forth in SEQ ID NO: 8, and the nucleotide sequence corresponding to the amino acid sequence shown in the figure.

Optionally, the gene encoding CAR-BCMA comprises the sequence as set forth in SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof. Of course, the gene encoding the CAR-BCMA may also include a sequence identical to SEQ ID NO: 20 has the base degeneracy.

Using CAR-CD19 as an example, SEQ ID NO: 19 and SEQ ID NO:17, but when the chimeric antigen receptor CAR-CD19 reaches the surface of a T cell, the signal peptide is cut by a signal peptidase in the process of protein translation maturation. Thus, the translated amino acid sequence of the chimeric antigen receptor CAR-CD19 (SEQ ID NO: 5) does not have a sequence as shown in SEQ ID NO:21, or a pharmaceutically acceptable salt thereof. The case of CAR-BCMA is similar.

Taking CAR-CD19 as an example, the coding gene sequence of CAR-CD19 is inserted between BamHI and EcoRI enzyme cutting sites in pWPXLD vector, and is positioned behind elongation factor 1 alpha (EF1 alpha) of pWPXLD vector, and EF1 alpha is taken as a promoter. When the coding gene sequence of the CAR-CD19 is inserted into a pWPXLD vector, the 5 'end of the gene sequence of the CAR-CD19 can be added with an initiation codon (such as ATG) to be connected with the enzyme cutting site BamH1 in the pWPXLD vector, and the 3' end can be added with a termination codon to be connected with the enzyme cutting site EcoR1 in the pWPXLD vector. The same is true for CAR-BCMA.

Optionally, in step (3), the "packaging the pwxld-CAR-CD 19 recombinant plasmid and the pwxld-CAR-BCMA recombinant plasmid, respectively, to obtain a first recombinant lentivirus with a CAR-CD19 encoding gene and a second recombinant lentivirus with a CAR-BCMA encoding gene" comprises:

co-transfecting the pWPXLD-CAR-CD19 recombinant plasmid with an envelope plasmid and a packaging plasmid to obtain a host cell to obtain the first recombinant lentivirus; co-transfecting the pWPXLD-CAR-BCMA recombinant plasmid, an envelope plasmid and a packaging plasmid into a host cell to obtain the second recombinant lentivirus.

The recombinant plasmid and the packaged virus are prepared by adopting the method, wherein on the pWPXLD-CAR-CD19 recombinant plasmid and the pWPXLD-CAR-CD22 recombinant plasmid, the encoding gene of CAR-CD19 and the encoding gene of CAR-BCMA are subjected to codon optimization, the molecular weight is proper, the packaging efficiency of the recombinant lentivirus is high, and the concentration of the virus prepared by host cells is high. Correspondingly, when the first recombinant lentivirus and the second recombinant lentivirus are used for combined transfection of the CD3 positive T lymphocytes, the two recombinant lentiviruses are used in a lower amount, so that the experiment cost can be reduced.

In one embodiment of the invention, the envelope plasmid is PMD2G, the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

The envelope plasmid PMD2G encodes a vesicular stomatitis virus glycoprotein capsid that can assist in the adhesion of recombinant lentiviruses to the cell membrane and maintain the infectivity of the recombinant lentiviruses.

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 amphotropic virus hand membrane proteins of retroviruses, and envelope proteins derived from mouse leukemia virus (MuMLV)4070A strain can be used. In addition, envelope proteins derived from MuMLV 10Al may also be used. Examples of 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. Examples of the hepadnaviridae protein include hepatitis B virus S protein. 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 lentiviral vector systems used on a large scale all use methods for partitioning the genome, i.e., targeting 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, in step (4), the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells. 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 optionally, the source is fresh peripheral blood or bone marrow collected after one month of surgery or one month of chemotherapy for the cancer patient.

Specifically, the process for obtaining the CD3 positive T lymphocyte is as follows: adding peripheral blood mononuclear cells into CD3/CD28 immunomagnetic beads 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.

Wherein, in step (4), the first recombinant lentivirus and the second recombinant lentivirus are co-transfected with CD3 positive T lymphocytes separately or simultaneously, comprising:

transfecting CD3 positive T lymphocytes by using the first recombinant lentivirus, and then transfecting by using the second recombinant lentivirus; or after the second recombinant lentivirus is used for transfecting CD3 positive T lymphocytes, the first recombinant lentivirus is used for transfecting; or simultaneously transfecting a CD3 positive T lymphocyte with the first recombinant lentivirus and the second recombinant lentivirus. Here, "co-transfection" refers to the same group of cells.

Further, in the step (4), the ratio of the virus titer of the first recombinant lentivirus to that of the first recombinant lentivirus is 1: (0.5-2).

In the present invention, the prepared targeting T lymphocyte comprises at least one of a dual-target chimeric antigen receptor T cell carrying the CAR-CD19 and the CAR-BCMA, a chimeric antigen receptor T cell carrying the CAR-CD19, and a chimeric antigen receptor T cell carrying the CAR-BCMA. Optionally, a dual-target chimeric antigen receptor T cell bearing said CAR-CD19 and said CAR-BCMA, or a mixture of a chimeric antigen receptor T cell bearing said CAR-CD19 and a chimeric antigen receptor T cell bearing said CAR-BCMA, or a mixture of one or both of a chimeric antigen receptor T cell bearing said CAR-CD19 and a chimeric antigen receptor T cell bearing said CAR-BCMA with said dual-target chimeric antigen receptor T cell.

Preferably, said targeting T lymphocyte is a dual-targeted chimeric antigen receptor T cell bearing said CAR-CD19 and said CAR-BCMA, or is a mixture of a chimeric antigen receptor T cell bearing said CAR-CD19 and a chimeric antigen receptor T cell bearing said CAR-BCMA, or is a mixture of three of a dual-targeted chimeric antigen receptor T cell bearing said CAR-CD19 and said CAR-BCMA, a chimeric antigen receptor T cell bearing said CAR-CD19, and a chimeric antigen receptor T cell bearing said CAR-BCMA.

In this case, the surface of the targeting T lymphocyte has two independent and non-covalently bound chimeric antigen receptors (i.e., two independent single-chain antibodies), which can simultaneously and efficiently recognize CD19 and BCMA targets on tumor cells without affecting their recognition and binding to their respective targets. The targeting T lymphocyte can identify and kill tumor cells expressing one or two of CD19 and BCMA, can avoid target escape of the tumor cells, improves the target identification breadth and strength, kills the broad spectrum, and has strong tumor killing capability in a complex tumor microenvironment.

In another embodiment of the invention, when the desired targeting T lymphocyte is a mixture of a chimeric antigen receptor T cell bearing said CAR-CD19 and a chimeric antigen receptor T cell bearing said CAR-BCMA, it can also be prepared in the following manner: transfecting CD3 positive T lymphocytes by using the first recombinant lentivirus to obtain chimeric antigen receptor T cells with CAR-CD 19; transfecting CD3 positive T lymphocytes by using the second recombinant lentivirus to obtain chimeric antigen receptor T cells with the CAR-BCMA; the two chimeric antigen receptor T cells are then mixed. This also applies to the diagnosis and/or treatment of B-line malignancies.

In the preparation method of the targeting T lymphocyte provided by the fourth aspect of the invention, the CD3 positive T lymphocyte is transfected by the first recombinant lentivirus with the CAR-CD19 encoding gene and the second recombinant lentivirus with the CAR-BCMA encoding gene respectively or simultaneously in a combined manner, so that the expression efficiency of the chimeric antigen receptor CAR-CD19 and/or CAR-BCMA on the prepared targeting T lymphocyte is higher, and the prepared targeting T lymphocyte has better tumor recognition and killing capabilities.

In a fifth aspect, the present invention provides a use of the targeting T lymphocyte according to the first aspect of the present invention, the recombinant viral vector according to the second aspect of the present invention, the host cell according to the third aspect of the present invention, or the targeting T lymphocyte according to the preparation method of the fourth aspect of the present invention in the preparation of a medicament for diagnosing and/or treating malignant tumor.

In particular, it is applicable to the diagnosis and treatment of malignancies expressing CD19 and/or BCMA, especially malignant B lymphocyte related tumors, such as B lymphocyte leukemias, B cell lymphomas and the like.

The application may specifically be: there is provided a kit comprising one or more of the targeted T lymphocyte according to the first aspect, or the targeted T lymphocyte transfected by the recombinant viral vector according to the second aspect, or the targeted T lymphocyte prepared by the preparation method according to the fourth aspect, the recombinant viral vector according to the second aspect, or the host cell according to the fourth aspect.

Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

FIG. 1 is a plasmid map of pWPXld-CAR-CD19 recombinant plasmid provided by the embodiment of the present invention.

FIG. 2 is a plasmid map of pWPXld-CAR-BCMA recombinant plasmid provided by the embodiment of the present invention.

FIG. 3 is a graph showing the in vitro tumor cell killing effect of the targeting T lymphocytes provided by the embodiment of the present invention.

FIG. 4 is a graph showing the effect of the targeting T lymphocytes provided by the embodiment of the invention on treating tumor mice.

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.

Embodiments are a method for preparing a targeted T lymphocyte, comprising the steps of:

(1) preparation of chimeric antigen receptor CAR-CD19 Gene sequence targeting CD19

Preparing genes encoding a signal peptide, a single-chain antibody targeting CD19, a CD8 alpha hinge region, a CD8 transmembrane region, a 4-1BB signal region and a CD3 zeta signal region respectively, wherein in the CAR-CD19, the gene encoding the signal peptide is shown as SEQ ID NO:22, the coding gene of the single-chain antibody targeting the CD19 is shown as SEQ ID NO:3, the encoding gene of the CD8 alpha hinge region is shown as SEQ ID NO:10, the coding gene of the CD8 transmembrane region is shown as SEQ ID NO:12, and the coding gene of the 4-1BB signal region is shown as SEQ ID NO:14, the coding gene of the CD3 zeta signal region is shown as SEQ ID NO: shown at 16.

Connecting the signal peptide, the single-chain antibody targeting the CD19, the CD8 alpha hinge region, the CD8 transmembrane region, the 4-1BB signal region and the coding gene of the CD3 zeta signal region together from the 5 'end to the 3' end sequentially by a PCR method to obtain the coding gene of CAR-CD19, wherein the coding gene of CAR-CD19 is shown as SEQ ID NO: as shown at 19.

(2) Genetic sequence for preparing BCMA-targeted chimeric antigen receptor CAR-BCMA

Separately preparing genes encoding a signal peptide, a single chain antibody targeting BCMA, a CD8 α hinge region, a CD8 transmembrane region, a 4-1BB signal region, and a CD3 zeta signal region, wherein the gene encoding the signal peptide for CAR-BCMA is as set forth in SEQ ID NO:23, the coding gene of the single-chain antibody targeting BCMA is shown as SEQ ID NO: 4, the encoding gene of the CD8 alpha hinge region is shown as SEQ ID NO:10, the coding gene of the CD8 transmembrane region is shown as SEQ ID NO:12, and the coding gene of the 4-1BB signal region is shown as SEQ ID NO:14, the coding gene of the CD3 zeta signal region is shown as SEQ ID NO: shown at 16.

Connecting the signal peptide, the single-chain antibody targeting the BCMA, the CD8 alpha hinge region, the CD8 transmembrane region, the 4-1BB signal region and the coding gene of the CD3 zeta signal region together from the 5 'end to the 3' end in sequence by a PCR method to obtain the coding gene of the CAR-BCMA, wherein the coding gene of the CAR-BCMA is shown as SEQ ID NO: shown at 20.

(3) Construction of pWPXld-CAR-CD19 recombinant plasmid and pWPXld-CAR-BCMA recombinant plasmid

The gene coding for CAR-CD19 was inserted between the BamHI and EcoRI sites of pWPXLD vector, and after pWPXLD vector EF1 α, EF1 α was used as promoter. When the coding gene of the CAR-CD19 is inserted into a pWPXLD vector, the 5 'end of the coding gene of the CAR-CD19 can be added with an initiation codon (such as ATG) to be connected with a BamHI enzyme cutting site in the pWPXLD 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 pWPXLD 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 pWPXld-CAR-CD19 recombinant plasmid shown in figure 1 is successfully constructed.

The coding gene of CAR-BCMA is inserted between BamHI and EcoRI enzyme cutting sites of pWPXLD vector, and EF1 alpha is used as a promoter after pWPXLD vector EF1 alpha. When the coding gene of the CAR-BCMA is inserted into a pWPXLD vector, the 5 'end of the coding gene of the CAR-BCMA can be added with an initiation codon (such as ATG) to be connected with a BamHI enzyme cutting site in the pWPXLD 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 pWPXLD 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 pWPXld-CAR-BCMA recombinant plasmid shown in figure 2 is successfully constructed.

(4) Recombinant lentivirus construction

The pWPXld-CAR-BCMA recombinant plasmid, the packaging plasmid psPAX2 and the envelope are addedThe three plasmids pMD2G were 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 subjecting the virus to a virus detection method of 100 mu L and 2X 10⁸And (5) subpackaging TU/mL, and storing at an ultralow temperature of-80 ℃ to obtain the first recombinant lentivirus with CAR-CD 19.

The pWPXLD-CAR-BCMA recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into 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 subjecting the virus to a virus detection method of 100 mu L and 2X 10⁸And (5) subpackaging TU/mL, and storing in an ultra-low temperature refrigerator at-80 ℃ to obtain the second recombinant lentivirus with CAR-BCMA.

(5) Preparation of targeting T lymphocyte

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) taking the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method, and adding the first recombinant lentivirus with CAR-CD19 and the second recombinant lentivirus with CAR-BCMA with the virus titer corresponding to the number of the CD3 positive cells for co-culture, wherein the ratio of the dosage (titer) of the first recombinant lentivirus to the dosage (titer) of the second recombinant lentivirus 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, obtaining targeting T lymphocytes, namely CAR-CD19 single positive T lymphocytes and CAR-BCMA single positive T lymphocytes, and CAR-19/CAR-BCMA double positive T lymphocytes, and storing in a cell freezing stock solution special for back transfusion.

Effects of the embodiment

First embodiment of effects: evaluation of in vitro tumor cell killing of Targeted T lymphocytes of the invention

Comparing the targeting T lymphocytes (experimental group) prepared by the first inventive example with the T lymphocytes (negative control group) that were not prepared, the T cells with CAR-CD19 alone (CD19 CAR-T alone group), and the T cells with CAR-BCMA alone (BCMA CAR-T alone group), the four groups of effector cells were compared with the target cells (Raji cells or Nalm-6 cells) in vitro in a number ratio of 1:10, 1:3. 1:1, 3:1 and 10:1, 5% CO at 37 ℃₂Then, co-culture was performed, and at 15 to 18 hours after the culture, cells were collected, flow-stained, and cell killing was examined, and the results are shown in FIG. 3. As can be seen from FIG. 3, the tumor killing power of the targeting T lymphocyte prepared by the method of the present invention is over 60%, even up to 99%, which is much higher than that of other control groups, which indicates that the targeting T lymphocyte prepared by the method of the present invention has super strong tumor killing capability.

Second Effect example, evaluation of the killing of tumor cells in mice by the targeting T lymphocytes of the present invention

The targeting T lymphocytes prepared according to the first embodiment of the invention (experimental group) were intravenously injected with 1 × 10 cells per mouse tail in a mouse lymphocytic leukemia model, together with unprepared T lymphocytes (negative control group), T cells with CAR-CD19 alone (CD19 CAR-T alone group), and T cells with CAR-BCMA alone (BCMACAR-T alone group)⁶The survival curve of each cell (n-9) was obtained and the results are shown in fig. 4. As can be seen from FIG. 4, the targeting T lymphocyte prepared by the invention can stabilize the survival rate of the mouse at about 80% after being injected into the mouse for 30 days, which far exceeds the negative control group and the two independent groups. The results in fig. 4 show that the provided targeting T lymphocytes are able to better protect mice from death due to lymphocytic leukemia.

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> targeting T lymphocyte and preparation method and application thereof

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

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

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 2

<211> 248

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

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

20 25 30

Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

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

130 135 140

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

145 150 155 160

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

165 170 175

Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala

180 185 190

Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

195 200 205

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

210 215 220

Tyr Phe Cys Ala Leu Ala Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln

225 230 235 240

Gly Thr Ser Val Thr Val Ser Ser

245

<210> 3

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gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacatcgtgc tgacccagag ccctcctagc ctggccatga gcctgggcaa gagggccacc 60

atcagctgca gggccagcga aagcgtgacc atcctgggca gccacctgat ccactggtac 120

cagcagaagc ctggccagcc ccctaccctg ctgatccagc tggccagcaa cgtgcagaca 180

ggcgtgcctg ccaggtttag cggcagcggc agcaggaccg acttcaccct gaccatcgac 240

cctgtggagg aggacgacgt ggccgtgtac tactgcctgc agagcaggac catccctagg 300

accttcggcg gcggcaccaa gctggagatt aagggaggcg gaggatctgg cggcggagga 360

agtggcggag ggggatctgg gggaggcgga agccagatcc agctggtgca gagcggccct 420

gagctgaaga agcccggcga gaccgtgaag atcagctgca aggccagcgg ctacaccttc 480

accgactaca gcatcaactg ggtgaagagg gcccctggca agggcctgaa gtggatgggc 540

tggatcaaca ccgagaccag ggagcccgcc tacgcctacg acttcagggg caggttcgcc 600

ttcagcctgg agacaagcgc cagcaccgcc accctgcaga tcaacaacct gaagtacgag 660

gacaccgcca catacttctg cgccctggcc tacagctacg ccatggacta ctggggccag 720

ggcacatccg tgaccgtgag cagc 744

<210> 5

<211> 465

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

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

100 105 110

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

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

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

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser 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> 6

<211> 471

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

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

20 25 30

Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

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

130 135 140

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

145 150 155 160

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

165 170 175

Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala

180 185 190

Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

195 200 205

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

210 215 220

Tyr Phe Cys Ala Leu Ala Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln

225 230 235 240

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

245 250 255

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

260 265 270

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

405 410 415

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

420 425 430

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

435 440 445

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

450 455 460

Met Gln Ala Leu Pro Pro Arg

465 470

<210> 7

<211> 485

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val

50 55 60

Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

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

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn

100 105 110

Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

130 135 140

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

145 150 155 160

Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val

165 170 175

Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val

180 185 190

Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg

195 200 205

Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met

210 215 220

Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His

225 230 235 240

Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu Pro Pro Arg

485

<210> 8

<211> 491

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala

20 25 30

Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

35 40 45

Val Thr Ile Leu Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr

65 70 75 80

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

85 90 95

Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys

100 105 110

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

115 120 125

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln Leu Val Gln Ser Gly Pro

145 150 155 160

Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro

180 185 190

Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu

195 200 205

Pro Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu

210 215 220

Thr Ser Ala Ser Thr Ala Thr Leu Gln Ile Asn Asn Leu Lys Tyr Glu

225 230 235 240

Asp Thr Ala Thr Tyr Phe Cys Ala Leu Ala Tyr Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

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

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

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

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

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

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

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

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 9

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 10

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 11

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 12

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 13

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 16

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 17

<211> 1395

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 780

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 840

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 960

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1020

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1080

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1140

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1260

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1380

gccctgcccc ctcgc 1395

<210> 18

<211> 1413

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gacatcgtgc tgacccagag ccctcctagc ctggccatga gcctgggcaa gagggccacc 60

atcagctgca gggccagcga aagcgtgacc atcctgggca gccacctgat ccactggtac 120

cagcagaagc ctggccagcc ccctaccctg ctgatccagc tggccagcaa cgtgcagaca 180

ggcgtgcctg ccaggtttag cggcagcggc agcaggaccg acttcaccct gaccatcgac 240

cctgtggagg aggacgacgt ggccgtgtac tactgcctgc agagcaggac catccctagg 300

accttcggcg gcggcaccaa gctggagatt aagggaggcg gaggatctgg cggcggagga 360

agtggcggag ggggatctgg gggaggcgga agccagatcc agctggtgca gagcggccct 420

gagctgaaga agcccggcga gaccgtgaag atcagctgca aggccagcgg ctacaccttc 480

accgactaca gcatcaactg ggtgaagagg gcccctggca agggcctgaa gtggatgggc 540

tggatcaaca ccgagaccag ggagcccgcc tacgcctacg acttcagggg caggttcgcc 600

ttcagcctgg agacaagcgc cagcaccgcc accctgcaga tcaacaacct gaagtacgag 660

gacaccgcca catacttctg cgccctggcc tacagctacg ccatggacta ctggggccag 720

ggcacatccg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 780

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 840

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 900

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 960

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1020

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1080

gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1140

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1200

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1260

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1320

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1380

gacgcccttc acatgcaggc cctgccccct cgc 1413

<210> 19

<211> 1455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 120

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 180

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 240

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 300

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 360

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 420

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 480

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 540

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 600

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 660

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 720

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 780

tcctcaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgc 1455

<210> 20

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gacatcgtgc tgacccagag ccctcctagc ctggccatga gcctgggcaa gagggccacc 120

atcagctgca gggccagcga aagcgtgacc atcctgggca gccacctgat ccactggtac 180

cagcagaagc ctggccagcc ccctaccctg ctgatccagc tggccagcaa cgtgcagaca 240

ggcgtgcctg ccaggtttag cggcagcggc agcaggaccg acttcaccct gaccatcgac 300

cctgtggagg aggacgacgt ggccgtgtac tactgcctgc agagcaggac catccctagg 360

accttcggcg gcggcaccaa gctggagatt aagggaggcg gaggatctgg cggcggagga 420

agtggcggag ggggatctgg gggaggcgga agccagatcc agctggtgca gagcggccct 480

gagctgaaga agcccggcga gaccgtgaag atcagctgca aggccagcgg ctacaccttc 540

accgactaca gcatcaactg ggtgaagagg gcccctggca agggcctgaa gtggatgggc 600

tggatcaaca ccgagaccag ggagcccgcc tacgcctacg acttcagggg caggttcgcc 660

ttcagcctgg agacaagcgc cagcaccgcc accctgcaga tcaacaacct gaagtacgag 720

gacaccgcca catacttctg cgccctggcc tacagctacg ccatggacta ctggggccag 780

ggcacatccg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 840

cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 900

ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccttg 960

gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caaacggggc 1020

agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080

gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140

gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1200

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1320

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1380

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440

gacgcccttc acatgcaggc cctgccccct cgc 1473

<210> 21

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro

20

<210> 22

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

<210> 23

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims

1. A targeted T lymphocyte comprising a CD 19-targeting chimeric antigen receptor CAR-CD19 and a BCMA-targeting chimeric antigen receptor CAR-BCMA, wherein the CAR-CD19 comprises, sequentially joined from amino terminus to carboxy terminus, an amino acid sequence of a CD 19-targeting single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region, and the CAR-BCMA comprises, sequentially joined from amino terminus to carboxy terminus, an amino acid sequence of a BCMA-targeting single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region;

wherein the amino acid sequence of the single-chain antibody targeting CD19 comprises the amino acid sequence shown as SEQ ID NO:1, and the amino acid sequence of the BCMA-targeted single-chain antibody comprises the amino acid sequence shown as SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

2. The targeted T lymphocyte of claim 1, wherein the amino acid sequence of CAR-CD19 comprises the amino acid sequence set forth in SEQ ID NO:5, the amino acid sequence of CAR-BCMA comprises the amino acid sequence as set forth in SEQ ID NO: 6.

3. A recombinant viral vector comprising the genes encoding CAR-CD19 and CAR-BCMA in the targeted T lymphocyte of any of claims 1-2.

4. A host cell comprising the recombinant viral vector of claim 3.

5. A method for preparing a targeting T lymphocyte is characterized by comprising the following steps:

(4) and (3) transfecting CD3 positive T lymphocytes by combining the first recombinant lentivirus and the second recombinant lentivirus separately or simultaneously, and separating to obtain the targeted T lymphocytes.

6. The method of claim 5, wherein the gene encoding CAR-CD19 comprises the amino acid sequence set forth in SEQ ID NO: 7, and the coding gene of the CAR-BCMA comprises a nucleotide sequence corresponding to an amino acid sequence shown as SEQ ID NO: 8, and the nucleotide sequence corresponding to the amino acid sequence shown in the figure.

7. The method of claim 5, wherein in step (4), the ratio of viral titers of the first recombinant lentivirus to the second recombinant lentivirus is 1: (0.5-2).

8. The method of claim 7, wherein the targeted T lymphocyte is a dual-target chimeric antigen receptor T cell comprising the CAR-CD19 and the CAR-BCMA, or a mixture of a chimeric antigen receptor T cell comprising the CAR-CD19 and a chimeric antigen receptor T cell comprising the CAR-BCMA, or a mixture of a dual-target chimeric antigen receptor T cell comprising the CAR-CD19 and the CAR-BCMA, a chimeric antigen receptor T cell comprising the CAR-CD19, and a chimeric antigen receptor T cell comprising the CAR-BCMA.

9. Use of the recombinant viral vector according to claim 3, the host cell according to claim 4, the targeted T lymphocyte according to any one of claims 1-2 or the targeted T lymphocyte produced by the process according to any one of claims 5-8 for the preparation of a medicament for the diagnosis and/or treatment of malignancies expressing CD19 and/or BCMA antigen protein.