CN110623979A

CN110623979A - Application of chimeric antigen receptor T cell targeting CD19 in autoimmune diseases

Info

Publication number: CN110623979A
Application number: CN201810666586.3A
Authority: CN
Inventors: 张宏玲; 曾滢; 钟春颖; 李宇桃
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2018-06-25
Filing date: 2018-06-25
Publication date: 2019-12-31

Abstract

The invention provides an application of a chimeric antigen receptor T cell targeting CD19 in preparation of a medicament for preventing and/or treating autoimmune diseases, and also provides a kit and a method for preventing and/or treating autoimmune diseases.

Description

Application of chimeric antigen receptor T cell targeting CD19 in autoimmune diseases

Technical Field

The invention relates to the field of medical biology, in particular to application of chimeric antigen receptor T cells targeting CD19 in preparation of a medicine for preventing and/or treating autoimmune diseases, a kit for preventing and/or treating autoimmune diseases and a method.

Background

Autoimmune diseases refer to diseases caused by the damage of self tissues caused by the immune reaction of the body to self antigens, and are generally caused by various genetic and environmental factors and the like. For example, Systemic Lupus Erythematosus (SLE), an autoimmune inflammatory connective tissue disease that is frequently encountered in many organs of young women, has an unknown etiology, and causes T lymphocyte depletion, T suppressor cell function reduction, B cell hyperproliferation, production of a large amount of autoantibodies, and binding with corresponding autoantigens in the body to form corresponding immune complexes that are deposited on the skin, joints, small blood vessels, glomeruli, etc., and trigger inflammation, tissue destruction, or premature death.

The therapeutic efficacy of treatments for autoimmune diseases is quite limited. Traditional therapeutic approaches rely on the action of steroids and various cytotoxic and cytostatic immunosuppressive agents, which rapidly eliminate the amplifying autoreactive immune cells and thereby delay the development of the autoimmune process. The most commonly used autoimmune disease treatments (e.g. cortisone, methotrexate, mycophenolate mofetil, etc.) have limited efficacy and are associated with a number of side effects. Therefore, there is a need for a drug and method for treating autoimmune diseases with less side effects.

Immune cell therapy has become a treatment method with great prospect in tumor treatment after surgery, radiotherapy and chemotherapy, has the great advantages of strong specificity and almost no toxic or side effect, makes up the defects of the traditional treatment method, and has the common chimeric antigen receptor T cell (CAR-T) technology and T Cell Receptor (TCR) gene modified T cell (TCR-T) technology. However, no application of immunocytotherapy to autoimmune diseases is known at present.

Disclosure of Invention

In view of the above, the present invention provides an application of a chimeric antigen receptor T cell targeting CD19 in autoimmune diseases, so as to provide a new prevention and treatment means for autoimmune diseases, and the toxic and side effects are small.

The invention provides an application of a chimeric antigen receptor T cell targeting CD19 in preparation of a medicament for preventing and/or treating autoimmune diseases.

Wherein the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis.

Optionally, the autoimmune disease is systemic lupus erythematosus.

The chimeric antigen receptor T cell targeting CD19 comprises: a chimeric antigen receptor CAR-CD19 targeting CD19, the CAR-CD19 comprising, sequentially joined from amino terminus to carboxy terminus, an amino acid sequence of a single chain antibody targeting CD19, an extracellular hinge region, a transmembrane region, and an intracellular signal region, wherein the single chain antibody targeting CD19 comprises the amino acid sequence as set forth in SEQ ID NO:1 and the single-chain antibody targeting the CD19 is a humanized single-chain antibody.

The single chain antibody targeting CD19 specifically binds to CD19 protein on B cells in a subject at risk for developing or already having an autoimmune disease. In addition, the single-chain antibody targeting the CD19 is a humanized single-chain antibody, and can avoid causing immune response of human bodies.

Alternatively, the gene encoding the CD 19-targeting single chain antibody comprises the amino acid sequence as set forth in SEQ ID NO: 2.

Optionally, the amino acid sequence of CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 3.

Optionally, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 4.

Further, the encoding genes of the CAR-CD19 comprise a gene encoding a signal peptide, a gene encoding a single-chain antibody targeting CD19, a gene encoding an extracellular hinge region, a gene encoding a transmembrane region, and a gene encoding an intracellular signal region, which are sequentially connected from the 5 'end to the 3' end; the CD 19-targeting single-chain antibody is a humanized single-chain antibody, and the encoding gene of the CD 19-targeting single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:1, or a nucleotide sequence corresponding to the amino acid sequence shown in the specification.

Preferably, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 5.

CD19 is an important marker of B cells in the blood system, is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and is involved in processes such as B cell development and intracellular signaling through binding to B cell receptors on B cells. When the chimeric antigen receptor T cell targeting CD19 is returned to a subject, the chimeric antigen receptor T cell is specifically combined with B cells expressing CD19 in the subject at risk or suffering from autoimmune diseases, and is effectively expanded in the subject, so that the B cells (especially mature B lymphocytes) are efficiently and specifically killed, and the aim of preventing and/or treating the autoimmune diseases is fulfilled.

The invention also provides a kit for preventing and/or treating autoimmune diseases, wherein the kit comprises the chimeric antigen receptor T cell targeting CD 19.

Wherein the kit further comprises a pharmaceutically acceptable carrier. Further, the pharmaceutically acceptable carrier includes water or physiological saline, but is not limited thereto.

Wherein the kit further comprises instructions for administering to the subject the CD 19-targeted chimeric antigen receptor T cell and the pharmaceutically acceptable carrier in combination to prevent and/or treat the subject from developing one or more symptoms of an autoimmune disease.

The present invention also provides a method for preventing and/or treating an autoimmune disease, comprising: the subject is administered chimeric antigen receptor T cells targeted to CD 19. This may prevent the subject from developing one or more symptoms of the autoimmune disease, or may ameliorate an established condition.

Optionally, the subject is asymptomatic but at risk for experiencing one or more symptoms of an autoimmune disease.

Further, the subject is at risk for experiencing one or more symptoms of Systemic Lupus Erythematosus (SLE).

Optionally, the subject is a subject exhibiting one or more symptoms of an autoimmune disease.

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 graph showing the effect of CD 19-targeted chimeric antigen receptor T cells of the present invention on killing CD 19-expressing cells in vitro.

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.

The embodiment of the invention provides application of a chimeric antigen receptor T cell targeting CD19 in preparation of a medicament for preventing and/or treating autoimmune diseases.

The autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis, but are not limited to these autoimmune diseases associated with B cell hyperproliferation and the like.

Optionally, the autoimmune disease is systemic lupus erythematosus.

The autoimmune disease has a certain relationship with B cell hyperproliferation, abnormal antibody secretion and the like, and the autoimmune disease can be prevented and/or treated by clearing B cells or inhibiting the function of the B cells. CD19 is an important marker of B cells in the blood system, is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and is involved in processes such as B cell development and intracellular signaling through binding to B cell receptors on B cells. When the chimeric antigen receptor T cell targeting CD19 is returned to a subject, the chimeric antigen receptor T cell can be specifically combined with B cells expressing CD19 in the subject at risk or suffering from autoimmune diseases, can be effectively expanded in the subject, and can efficiently and specifically kill the B cells (especially mature B lymphocytes) so as to achieve the aim of preventing and/or treating the autoimmune diseases.

The embodiment of the invention also provides a kit for preventing and/or treating autoimmune diseases, wherein the kit comprises the chimeric antigen receptor T cell targeting CD 19.

The embodiment of the invention also provides a method for preventing and/or treating autoimmune diseases, which comprises the following steps: the subject is administered chimeric antigen receptor T cells targeted to CD 19. This may prevent the subject from developing one or more symptoms of the autoimmune disease, or may ameliorate an established condition.

Wherein the amount of the chimeric antigen receptor T cells targeting CD19 administered may be administered according to their specific purpose.

The chimeric antigen receptor T cell targeting CD19 can be specifically combined with B cells in a subject at risk or already suffering from autoimmune diseases through the single-chain antibody targeting CD19 in CAR-CD19, the intracellular signal region of the T cell is activated, the T cell can be effectively expanded in the body of the subject, B cells (especially mature B lymphocytes) are efficiently and specifically killed, and the purpose of preventing and/or treating autoimmune diseases is achieved. In addition, the single-chain antibody targeting the CD19 is a humanized single-chain antibody, and can avoid causing immune response of human bodies.

Alternatively, the gene encoding the CD 19-targeted single-chain antibody should take into account degenerate bases, i.e., the gene encoding the amino acid sequence shown in SEQ ID NO. 1 includes the nucleotide sequence shown in SEQ ID NO. 2, and the protection scope should also protect the nucleotide sequences having base degeneracy with SEQ ID NO. 2, and the corresponding amino acid sequences of these nucleotide sequences are still SEQ ID NO. 1.

The sequence connection from the amino terminal to the carboxyl terminal is specifically as follows: 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.

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 CD19 expressing B lymphocytes.

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 C D5 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: 6.

Alternatively, the gene encoding the CD8 a hinge region comprises the amino acid sequence as set forth in SEQ ID NO: 7. The coding gene of the CD8 alpha hinge region should consider degenerate bases, namely the coding gene of the amino acid sequence shown as SEQ ID NO. 6 comprises the nucleotide sequence shown as SEQ ID NO. 7, the protection scope should also protect the nucleotide sequence with base degeneracy property with the SEQ ID NO. 7, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 6.

In the invention, the transmembrane region is used for fixing the chimeric antigen receptor CAR-CD19 targeting CD 19.

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: 8.

Alternatively, the gene encoding the CD8 transmembrane region comprises the amino acid sequence set forth in SEQ ID NO:9, or a nucleotide sequence shown in the specification. The coding gene of the CD8 transmembrane region should take degenerate bases into consideration, namely the coding gene of the amino acid sequence shown as SEQ ID NO. 8 comprises the nucleotide sequence shown as SEQ ID NO. 9, the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 9, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 8.

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 C D27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, a TNFRS F19L 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.

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:10, 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: 11. The coding gene of the 4-1BB signal region should take degenerate bases into consideration, that is, the coding gene of the amino acid sequence shown in SEQ ID NO. 10 includes the nucleotide sequence shown in SEQ ID NO. 11, the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 11, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 10.

Alternatively, the amino acid sequence of the CD3 zeta signal region comprises the amino acid sequence set forth in SEQ ID NO: 12.

Alternatively, the gene encoding the CD3 zeta signaling region comprises the amino acid sequence set forth in SEQ ID NO:13, or a nucleotide sequence as set forth in seq id no. The coding gene of the CD3 zeta signal region should take degenerate bases into consideration, that is, the coding gene of the amino acid sequence shown in SEQ ID NO. 12 comprises the nucleotide sequence shown in SEQ ID NO. 13, and the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 13, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 12.

Optionally, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 4. The coding gene of the CAR-CD19 should consider degenerate bases, that is, the coding gene of the amino acid sequence shown in SEQ ID NO. 3 includes the nucleotide sequence shown in SEQ ID NO. 4, and the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 4, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 3.

Wherein the gene encoding the signal peptide directs the CAR-CD19 to the cell surface, but when the chimeric antigen receptor CAR-CD19 reaches the surface of a T cell, the signal peptide is cleaved by the signal peptidase during the maturation of protein translation. Thus, the translated amino acid sequence of CAR-CD19 (SEQ ID NO: 3) does not have the amino acid sequence of the signal peptide.

Alternatively, the amino acid sequence of the signal peptide comprises the amino acid sequence as set forth in SEQ ID NO:14, 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:15, or a nucleotide sequence as set forth in seq id no. The coding gene sequence of the signal peptide should take degenerate bases into consideration, namely, the coding gene of the amino acid sequence shown as SEQ ID NO. 14 comprises the nucleotide sequence shown as SEQ ID NO. 15, the protection scope should also protect the nucleotide sequence with base degeneracy with the SEQ ID NO. 15, and the corresponding amino acid sequence of the nucleotide sequence is still SEQ ID NO. 14.

Preferably, the gene encoding CAR-CD19 comprises the amino acid sequence set forth as SEQ ID NO: 5. And SEQ ID NO:4, compared with the nucleotide sequence shown in SEQ ID NO: 5, the coding gene of the signal peptide is added in the nucleotide sequence shown in the figure.

In one embodiment of the present invention, the chimeric antigen receptor T cell targeting CD19 can be prepared by the following method:

(1) providing a chimeric antigen receptor CAR-CD19 encoding gene targeting CD19, the CAR-CD19 encoding gene comprising a gene encoding a signal peptide, a gene encoding a single chain antibody targeting CD19, a gene encoding an extracellular hinge region, a gene encoding a transmembrane region, and a gene encoding an intracellular signal region, sequentially linked from 5 'to 3'; the CD 19-targeting single-chain antibody is a humanized single-chain antibody, and the encoding gene of the CD 19-targeting single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 2;

(2) inserting the encoding gene of the CAR-CD19 into a pWPXLD vector to obtain a pWPXLD-CAR-CD19 recombinant plasmid;

(3) co-transfecting the pWPXLD-CAR-CD19 recombinant plasmid, an envelope plasmid and a packaging plasmid to a host cell, and packaging to obtain a recombinant lentivirus;

(4) transfecting the recombinant lentivirus with CD3 positive T lymphocytes to obtain chimeric antigen receptor T cells targeting CD 19.

The recombinant lentivirus can be packaged by a three-plasmid system or a four-plasmid system, and enveloped plasmids and packaging plasmids are substances commonly used in the field.

The preparation of chimeric antigen receptor T cells targeting CD19 is described below by way of a specific example, which specifically includes the steps of:

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

Respectively preparing coding genes of 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, wherein the coding gene sequence of the signal peptide is shown as SEQ ID NO:15, the coding gene sequence of the single-chain antibody targeting the CD19 is shown as SEQ ID NO:2, the coding gene sequence of the CD8 alpha hinge region is shown as SEQ ID NO:7, the coding gene sequence of the CD8 transmembrane region is shown as SEQ ID NO:9, and the coding gene sequence of the 4-1BB signal region is shown as SEQ ID NO:11, the coding gene sequence of the CD3 zeta signal region is shown as SEQ ID NO: shown at 13.

Connecting the coding genes of 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 CD3 zeta signal region together from 5 'end to 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: 5, the single chain antibody targeting CD19 is a humanized single chain antibody.

(2) Construction of pWPXld-CAR-CD19 recombinant plasmid

The gene coding for CAR-CD19 was inserted between the BamHI and EcoRI sites of pWPXLD vector, after elongation factor 1 α (EF1 α) of pWPXLD vector, using EF1 α 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.

(3) Recombinant lentivirus construction

Co-transfecting the pWPXld-CAR-CD19 recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G into a culture mediumHEK293T cell. 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 a 0.45-micrometer 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 recombinant lentivirus with the CAR-CD19 encoding gene.

(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) adding the recombinant lentivirus with the virus titer corresponding to the number of the CD3 positive cells into the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method for culture.

On day 3 of culture, cell count and fluid change were performed to adjust cellsThe concentration is 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 to obtain chimeric antigen receptor T cells targeting CD19, and storing in cell freezing stock special for reinfusion for patients.

Effects of the embodiment

To evaluate the effect of the chimeric antigen receptor T cells targeting CD19 prepared by the above method described in the present invention on autoimmune diseases, the following effect examples were performed.

Assessing the ability of chimeric antigen receptor T cells targeting CD19 to kill CD19 expressing cells in vitro

The effect of killing CD 19-expressing cells in vitro by using the chimeric antigen receptor T cells targeting CD19 prepared in the examples of the present invention (abbreviated as experimental group) and T lymphocytes not prepared (negative control group) was compared with that of the non-prepared T lymphocytes by using human peripheral blood, specifically: the two groups of effector cells (i.e., chimeric antigen receptor T cells targeting CD19, unproductive T lymphocytes) were mixed in vitro with the target cells (Raji cells) at a ratio of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1 and 0.31:1 by number at 37 deg.C and 5% CO₂Then, co-culture was performed, and at 4 hours after the culture, cells were collected, flow-stained, and the killing of Raji cells was examined, and the results are shown in fig. 2. As can be seen from FIG. 2, the chimeric antigen receptor T cells targeting CD19 in the experimental group have very strong ability to kill cells expressing CD19, which indicates that the chimeric antigen receptor T cells are expected to kill B cells with CD19 targets over-expressed in patients with autoimmune diseases, thereby achieving the effect of treating the autoimmune diseases.

The above examples only show some 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.

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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> 5

<211> 1455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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> 6

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

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> 7

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 8

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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> 9

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 10

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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> 11

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 12

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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> 13

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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> 14

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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> 15

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 60

Claims

1. Application of chimeric antigen receptor T cells targeting CD19 in preparation of drugs for preventing and/or treating autoimmune diseases.

2. The use of claim 1, wherein the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis.

3. The use of claim 1, wherein the CD 19-targeted chimeric antigen receptor T cell comprises a CD 19-targeted chimeric antigen receptor CAR-CD19, wherein the CAR-CD19 comprises, connected sequentially from amino terminus to carboxy terminus, an amino acid sequence of a CD 19-targeted single-chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular signal region; the amino acid sequence of the CD 19-targeting single-chain antibody comprises the amino acid sequence shown as SEQ ID NO:1 and the single-chain antibody targeting the CD19 is a humanized single-chain antibody.

4. The use of claim 3, wherein the gene encoding the CD 19-targeting single chain antibody comprises the amino acid sequence set forth in SEQ ID NO: 2.

5. The use of claim 3, wherein the amino acid sequence of CAR-CD19 comprises the amino acid sequence set forth in SEQ ID NO: 3.

6. The use of claim 3, wherein the gene sequence encoding CAR-CD19 comprises the sequence set forth in SEQ ID NO: 4.

7. A kit for the prevention and/or treatment of an autoimmune disease, the kit comprising chimeric antigen receptor T cells targeted to CD 19.

8. The kit of claim 7, further comprising a pharmaceutically acceptable carrier.

9. The kit of claim 7, further comprising instructions for administering to the subject the CD 19-targeted chimeric antigen receptor T cell and the pharmaceutically acceptable carrier in combination to prevent and/or treat one or more symptoms of an autoimmune disease in the subject.

10. A method for preventing and/or treating an autoimmune disease, comprising: the subject is administered chimeric antigen receptor T cells targeted to CD 19.