CN107312097B

CN107312097B - Chimeric antigen receptor based on CD30 and application thereof

Info

Publication number: CN107312097B
Application number: CN201710586040.2A
Authority: CN
Inventors: 李俊芳; 章睿
Original assignee: Shenzhen Geno-Immune Medical Institute
Current assignee: Shenzhen Geno-Immune Medical Institute
Priority date: 2017-07-18
Filing date: 2017-07-18
Publication date: 2020-06-16
Anticipated expiration: 2037-07-18
Also published as: CN107312097A

Abstract

The invention relates to a chimeric antigen receptor based on CD30 and application thereof, in particular to a construction method of a chimeric antigen receptor T (CAR-T) cell technology based on a tumor specific target CD30 and application thereof in anti-tumor treatment, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signal transduction region and a CD3 zeta signal transduction domain which are connected in series; wherein the antigen binding domain binds to a tumor surface antigen that is CD 30. The chimeric antigen receptor of the invention carries out specific gene modification on the single-chain antibody aiming at the tumor surface antigen CD30, the modified antibody can enable the antigen-antibody to have stronger binding force and difficult mutation, and has better effect compared with other chimeric antigen receptors and other tumor antigens, the expression amount of the target spot is high, so that the immune effect of CAR-T cells is enhanced, and the treatment effect of the CAR-T cells is enhanced.

Description

Chimeric antigen receptor based on CD30 and application thereof

Technical Field

The invention relates to the field of tumor cell immunotherapy, in particular to a chimeric antigen receptor based on CD30 and application thereof, and specifically relates to a construction method of a chimeric antigen receptor T (CAR-T) cell technology based on a tumor specific target CD30 and application thereof in anti-tumor therapy.

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) is one of the most promising tumor immunotherapy. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Typically, the CAR comprises a Single chain fragment variable (scFv) region of an antibody or a binding domain specific for a Tumor Associated Antigen (TAA), which is coupled to the cytoplasmic domain of a T cell signaling molecule by a hinge and a transmembrane region. The most common lymphocyte activation moiety comprises a T cell costimulatory domain in tandem with a T cell effector function triggering (e.g., CD3 ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-transplanted T cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.

Most patients with lymphocytic malignancies, including Hodgkin's Lymphoma, B-cell Lymphoma and acute lymphocytic leukemia (B-cell Lymphoma and B-cell access lymphocytic leukemia, leukemia, B-ALL) and Chronic Lymphocytic Leukemia (CLL) will die as a result of their disease. One approach to treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through expression of a CAR. CARs are antigen receptors designed to recognize cell surface antigens in a Human Leukocyte Antigen (HLA) independent manner. Attempts to treat these types of patients using genetically modified cells expressing CARs have met with promising success.

The CD19 molecule is a potential target for treatment of B lymphocyte lineage tumors and is also a hotspot in CAR research, and expression of CD19 is restricted to normal and malignant B cells and is a widely accepted CAR target for safety testing. Chimeric antigen receptor gene-modified T cells targeting the CD19 molecule (CD19CAR-T) have had great success in treating multiple, refractory acute B-lymphocyte leukemia, while being significantly less effective in the treatment of refractory, relapsed chronic B-lymphocyte leukemia and B-lymphocyte lineage lymphoma.

CN 104788573A discloses a chimeric antigen receptor hCD19scFv-CD8 α -CD28-CD3 zeta and the application thereof, the chimeric antigen receptor is composed of anti-human CD19 monoclonal antibody HI19a light chain and heavy chain variable region (hCD19scFv), human CD8 α hinge region, human CD28 transmembrane region and intracellular region, and human CD3 zeta intracellular region structure in series, after the CD19 in the patent is subjected to CAR-T cell transfusion once, the expression level of CD19 is reduced, and immune mechanism is easy to escape.

Therefore, it is important to prepare a chimeric antigen receptor that can solve the problems of easy mutation and reduced expression of CD 19.

Disclosure of Invention

Aiming at the situations that the targeting in the current CAR-T technology for treating the tumor is not ideal and the tumor microenvironment influences the treatment effect of the CAR-T technology, the invention provides the chimeric antigen receptor based on the CD30 and the application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a CD 30-based chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, and an inducible suicide fusion domain in tandem;

wherein the antigen binding domain binds to a tumor surface antigen that is CD 30.

According to the invention, the antigen binding domain is combined with the tumor surface antigen CD30, and then the antigen binding domain, namely the single-chain antibody aiming at the tumor surface antigen CD30, is subjected to specific human genetic code optimization modification, so that the tumor surface antigen CD30 can be specifically combined on the chimeric antigen receptor of the application, and compared with other chimeric antigen receptors and other tumor antigens, the chimeric antigen receptor has a better effect, the expression amount of a target spot is high, and the immune effect of CAR-T cells is enhanced.

According to the invention, the antigen binding domain is a single chain antibody (scFv) against the tumor surface antigen CD30, the amino acid sequence of the single chain antibody against the tumor surface antigen CD30 is shown as SEQ ID NO.1, and the amino acid sequence (SEQ ID NO.1) of the single chain antibody against the tumor surface antigen CD30 is as follows:

QIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIK.

in the invention, the single-chain antibody aiming at the tumor surface antigen CD30 is subjected to specificity transformation, so that the antigen-antibody binding force of the antibody expressed by the transformed sequence is stronger.

According to the invention, the antigen binding domain also comprises a single-chain antibody of the mutant aiming at the tumor surface antigen CD30, and the amino acid sequence of the single-chain antibody of the mutant aiming at the tumor surface antigen CD30 has more than 90 percent of similarity with the amino acid sequence shown in SEQ ID NO. 1.

According to the present invention, the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain, which may be selected or modified by amino acid substitutions in some embodiments.

According to the present invention, the co-stimulatory signaling region is any one or a combination of at least two of CD28 signaling domain, CD27 signaling domain and CD137 signaling domain, preferably a combination of CD28 signaling domain, CD137 signaling domain and CD27 signaling domain, the arrangement of the CD28 signaling domain, CD137 signaling domain and CD27 signaling domain can be adjusted as required by those skilled in the art, the different arrangement of CD28 signaling domain, CD27 signaling domain and CD137 signaling domain does not affect the chimeric antigen receptor, and the sequential combination of CD28-CD27-CD137 is preferably adopted in the present application.

According to the invention, the inducible suicide fusion domain comprises a caspase 9 domain, the amino acid sequence of the caspase 9 domain is shown as SEQ ID NO.4, and the amino acid sequence of the caspase 9 domain (SEQ ID NO.4) is as follows:

GSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

according to the invention, the inducible suicide fusion domain is concatenated with the CD3 zeta signaling domain via a 2A sequence, the 2A sequence cleaving the protein expressed by the inducible suicide fusion domain from the chimeric antigen receptor protein, thereby enabling the chimeric antigen receptor to function, and by injecting an activator, thereby enabling activation of the inducible suicide fusion domain, thereby disabling the chimeric antigen receptor.

According to the present invention, the chimeric antigen receptor further comprises a signal peptide, wherein the signal peptide is a signal peptide capable of directing the transmembrane transfer of the chimeric antigen receptor, one skilled in the art can select a signal peptide which is conventional in the art according to needs, the signal peptide can be a signal peptide of any Secretory protein gene, the signal peptide of the present invention is a secretor signal peptide, and the amino acid sequence of the secretor signal peptide is shown in SEQ ID No. 5-6.

Preferably, the secretor signal peptide is a signal peptide of the CD8a gene, the amino acid sequence of the secretor signal peptide is shown as SEQ ID No.5, and the amino acid sequence of SEQ ID No.5 is as follows: MALPVTALLLPLALLLHAARP are provided.

Preferably, the secretor signal peptide is a signal peptide of the GMCSFR gene, the amino acid sequence of the secretor signal peptide is shown as SEQ ID NO.6, and the amino acid sequence of the SEQ ID NO.6 is as follows: MLLLVTSLLLCELPHPAFLLIP

The chimeric antigen receptor of the present invention may further include a hinge region, which may be selected by those skilled in the art according to practical circumstances, and is not particularly limited herein, and the presence of the hinge region does not affect the performance of the chimeric antigen receptor of the present invention.

According to the present invention, the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, a 2A sequence, and an inducible suicide fusion domain in tandem.

Preferably, the chimeric antigen receptor is a secretor signal peptide, a CD30 antigen binding domain, a CD8 α and/or CD28 transmembrane domain, a CD28 signaling domain, a CD27 signaling domain and a CD137 signaling domain, and a CD3 zeta signaling domain, a 2A sequence and a caspase 9 domain are connected in series, and the specific arrangement is as follows:

Secretory-CD30-CD28-CD27-CD137-CD3ζ-2A-FBKP.Casp9。

according to the invention, the amino acid sequence of the chimeric antigen receptor Secretory-CD30-CD28-CD27-CD137-CD3 zeta-2A-FBKP.Casp9 is shown in SEQ ID NO.2, and the amino acid sequence (SEQ ID NO.2) of the chimeric antigen receptor is as follows:

MLLLVTSLLLCELPHPAFLLIPQIQLVQSGAEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPGGGGSGGGGSTSGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

according to the invention, the nucleotide sequence of the chimeric antigen receptor Secretory-CD30-CD28-CD27-CD137-CD3 zeta-2A-FBKP.Casp9 is shown in SEQ ID NO.3, and the nucleotide sequence (SEQ ID NO.3) of the chimeric antigen receptor is as follows:

ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATCCCCCAGATCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATCACCTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTGGATCTACCCCGGCAGCGGCAACACCAAGTACAACGAGAAGTTCAAGGGCAGAGTGACCATGACCAGAGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCAGACTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCAACTACGGCAACTACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCAGCACCAGCGGCAGCGGCAAGCCCGGCAGCAGCGAGGGCAGCACCAAGGGCGACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGAGAGCCACCATCAACTGCAAGGCCAGCCAGAGCGTGGACTTCGACGGCGACAGCTACATGAACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGCCGCCAGCAACCTGGAGAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCAACGAGGACCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGCCGCCGCCATCGAGGTGATGTACCCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGAGAAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCAGAAGACCCGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCCCCCAGAGACTTCGCCGCCTACAGAAGCGCCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGAGAAGAAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAGCCCGCCGAGCCCTGCCACTACAGCTGCCCCAGAGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGACTACAGAAAGCCCGAGCCCGCCTGCAGCCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCACCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGAACCAGCGGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGGCGTGCAGGTGGAGACCATCAGCCCCGGCGACGGCAGAACCTTCCCCAAGAGAGGCCAGACCTGCGTGGTGCACTACACCGGCATGCTGGAGGACGGCAAGAAGGTGGACAGCAGCAGAGACAGAAACAAGCCCTTCAAGTTCATGCTGGGCAAGCAGGAGGTGATCAGAGGCTGGGAGGAGGGCGTGGCCCAGATGAGCGTGGGCCAGAGAGCCAAGCTGACCATCAGCCCCGACTACGCCTACGGCGCCACCGGCCACCCCGGCATCATCCCCCCCCACGCCACCCTGGTGTTCGACGTGGAGCTGCTGAAGCTGGAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGCCATGGTGGGCGCCCTGGAGAGCCTGAGAGGCAACGCCGACCTGGCCTACATCCTGAGCATGGAGCCCTGCGGCCACTGCCTGATCATCAACAACGTGAACTTCTGCAGAGAGAGCGGCCTGAGAACCAGAACCGGCAGCAACATCGACTGCGAGAAGCTGAGAAGAAGATTCAGCAGCCTGCACTTCATGGTGGAGGTGAAGGGCGACCTGACCGCCAAGAAGATGGTGCTGGCCCTGCTGGAGCTGGCCAGACAGGACCACGGCGCCCTGGACTGCTGCGTGGTGGTGATCCTGAGCCACGGCTGCCAGGCCAGCCACCTGCAGTTCCCCGGCGCCGTGTACGGCACCGACGGCTGCCCCGTGAGCGTGGAGAAGATCGTGAACATCTTCAACGGCACCAGCTGCCCCAGCCTGGGCGGCAAGCCCAAGCTGTTCTTCATCCAGGCCTGCGGCGGCGAGCAGAAGGACCACGGCTTCGAGGTGGCCAGCACCAGCCCCGAGGACGAGAGCCCCGGCAGCAACCCCGAGCCCGACGCCACCCCCTTCCAGGAGGGCCTGAGAACCTTCGACCAGCTGGACGCCATCAGCAGCCTGCCCACCCCCAGCGACATCTTCGTGAGCTACAGCACCTTCCCCGGCTTCGTGAGCTGGAGAGACCCCAAGAGCGGCAGCTGGTACGTGGAGACCCTGGACGACATCTTCGAGCAGTGGGCCCACAGCGAGGACCTGCAGAGCCTGCTGCTGAGAGTGGCCAACGCCGTGAGCGTGAAGGGCATCTACAAGCAGATGCCCGGCTGCTTCAACTTCCTGAGAAAGAAGCTGTTCTTCAAGACCAGCGCCAGCTGA.

in the invention, the chimeric antigen receptor also comprises a promoter, wherein the promoter is any one or the combination of at least two of EF1a, CMV-TAR or CMV.

According to the invention, the chimeric antigen receptor is expressed by transfection of its encoded nucleic acid sequence into a T cell.

According to the invention, the transfection is by transfection into T cells by any one of or a combination of at least two of viral vectors, eukaryotic expression plasmids or mRNA sequences, preferably by viral vectors.

Preferably, the viral vector is a lentiviral vector and/or a retroviral vector, preferably a lentiviral vector.

In a second aspect, the present invention provides a recombinant lentivirus obtained by co-transfecting a mammalian cell with a viral vector comprising the chimeric antigen receptor of the first aspect and the packaging helper plasmids pNHP and pHEF-VSVG.

According to the present invention, the mammalian cell is any one of 293 cell, 293T cell or TE671 cell or a combination of at least two thereof.

In a third aspect, the present invention provides a composition comprising a chimeric antigen receptor according to the first aspect and/or a recombinant lentivirus according to the second aspect.

In a fourth aspect, the present invention provides a chimeric antigen receptor according to the first aspect, a recombinant lentivirus according to the second aspect or a composition according to the third aspect, for preparing chimeric antigen receptor T cells and the use thereof in drugs for treating tumors;

preferably, the tumor is a blood-related tumor disease selected from, but not limited to, leukemia and/or a solid tumor.

Compared with the prior art, the invention has the following beneficial effects:

(1) the chimeric antigen receptor of the invention carries out specific gene modification on CD30 tumor surface antigen, and the modified antibody can make the antigen-antibody binding force stronger;

(2) the chimeric antigen receptor can specifically recognize the tumor surface antigen CD30, the expression level of CD30 in leukemia and lymphoma is high, and the single-chain antibody aiming at the tumor surface antigen CD30 on the chimeric antigen receptor is not easy to mutate, so that the chimeric antigen receptor has better effect compared with other chimeric antigen receptors and other tumor antigens, the immune effect of CAR-T cells is enhanced, and the treatment effect of the CAR-T cells is enhanced;

(3) after CAR-T cell reinfusion is carried out on the chimeric antigen receptor, the expression level of CD30 on the surface of a tumor cannot be reduced, an immune mechanism cannot easily escape, and better treatment can be carried out.

Drawings

FIG. 1 is a synthetic gene sequence map of a chimeric antigen receptor of the present invention;

FIG. 2 is a graph of the results of flow cytometric analysis of CD30 antibody in primary lymphoma cells, wherein the gray areas are isotype negative controls;

FIG. 3(a) is a diagram showing the results of flow cytometry analysis of an immunohistochemical negative control; FIG. 3(b) is a graph showing the results of flow cytometry analysis of CD30 antibody staining of primary lymphoma cells;

FIG. 4(a) is a graph showing the results of flow cytometry analysis of ALCL tumor (anaplastic large cell lymphoma) target cells, and FIG. 4(b) is a graph showing the results of apoptosis of ALCL tumor target cells;

FIG. 5(a) is a graph showing the results of flow cytometry analysis of co-culture of normal T cells and ALCL tumor target cells, and FIG. 5(b) is a graph showing the results of apoptosis of co-culture of normal T cells and ALCL tumor target cells;

FIG. 6(a) is a graph of the results of flow cytometry analysis of GD2CAR-T cells co-cultured with ALCL tumor target cells, and FIG. 6(b) is a graph of the results of apoptosis of GD2CAR-T cells co-cultured with ALCL tumor target cells;

FIG. 7(a) is a graph showing the results of flow cytometry analysis of the co-culture of CD30 chimeric antigen receptor and ALCL tumor target cells, and FIG. 7(b) is a graph showing the results of apoptosis of CD30 chimeric antigen receptor T cells co-cultured with ALCL tumor target cells;

fig. 8 is a graph showing the results of GFP staining after 12 days of co-culture, wherein the green fluorescence of the right panel in each result graph indicates the survival cells of the primary lymphoma cells, the first column is a graph showing the GFP staining of the primary lymphoma cells and the GFP staining of the primary lymphoma cells after GD2CAR-T cell poisoning, and the second column is a graph showing the GFP staining of the primary lymphoma cells after general T cell poisoning and the GFP staining of the primary lymphoma cells after CD30 CAR T cell poisoning prepared.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1: construction of chimeric antigen receptors

(1) Synthesizing a secretor signal peptide, a CD30 antigen binding domain, a CD8 α and/or CD28 transmembrane domain, a CD28 signaling domain, a CD27 signaling domain and a CD137 signaling domain, a CD3 zeta signaling domain, a 2A sequence and a caspase 9 domain through a whole gene as shown in fig. 1, i.e., secretor-CD 30-CD28-CD27-CD137-CD3 zeta-2A-fbkp.casp 9;

the nucleotide sequence SEQ ID NO.3 of the chimeric antigen receptor is as follows:

example 2: lentiviral packaging

(1) 293T cells, 1X 10, were cultured separately in six-well plates⁶Culturing each cell/well for 17-18 hours;

(2) add 600. mu.L/well fresh DMEM containing 10% FBS;

(3) the following reagents were added to sterile centrifuge tubes: each well was taken 75. mu.L of supernatant of DMEM, 2.7. mu.g of helperDNA mix (1.8. mu.g of pNHP, 0.5. mu.g of pHEF-VSV-G, 0.2. mu.g of pHEF-GFP) and 0.8. mu.g of pTYF DNA vector, vortexed;

(4) sucking 7 mu L of Superfect from the center of each pore plate, adding the Superfect into a centrifugal tube, blowing and beating for 5 times, and standing for 7-10 minutes at room temperature;

(5) dropwise adding the DNA-Superfect mixed solution in the centrifugal tube into each culture hole, and uniformly swirling;

(6)37℃3％CO₂culturing in an incubator for 4-5 hours;

(7) sucking the culture solution of the culture medium, flushing the culture medium with 1.5mL of AIM-V, and adding 1.5mL of AIM-V for continuous culture;

(8) the medium was returned to 3% CO₂The cells were incubated overnight in an incubator and the next morning the transfection efficiency was observed using a fluorescence microscope.

Example 3: lentivirus purification and concentration

1) Virus purification

Removing cell debris by centrifugation (1000g, 5 min) to obtain viral supernatant, filtering the viral supernatant with a 0.45 micron low protein binding filter, dividing the virus into small portions, and storing at-80 deg.C;

generally, transfected cells can produce 10 cells per ml of medium⁶To 10⁷Transduction unit titrated lentiviral vectors.

2) Concentration of lentiviral vectors with Centricon Filter

(1) In a biological safety cabinet, taking a Centricon tube, disinfecting the tube for 1 time by using 70% alcohol, and then cleaning the tube for 3 times by using sterile PBS (phosphate buffer solution);

(2) add 18ml of virus supernatant to each Centricon P-20 filter tube, then centrifuge at 2500g for 30 minutes or until the virus volume is reduced to 0.5 ml;

(3) the filter tube was shaken and then centrifuged at 400g for 2 minutes to collect the concentrated virus in a collection cup. Finally, the viruses in all tubes are collected in a centrifuge tube.

Example 4: transfection of CAR-T cells

Activating the T cells at 5X 10⁶Inoculated into 24-well plates and 50. mu.l of concentrate was addedCentrifuging the target gene-contracted lentivirus at 100g centrifugal speed at room temperature for 100 min, culturing at 37 deg.C for 24 hr, adding 1ml AIM-V medium containing 2% human serum and cell culture factor, culturing for 2-3 days, harvesting and counting cells, and culturing at 1 × 10⁷Inoculated into 12-well plates, cultured for 2-3 days, target cells were infected with lentiviral vector carrying GFP and the cytotoxic effect was observed by annexin V/PI staining, the results are shown in FIGS. 2-3.

As can be seen from FIGS. 2, 3(a) -3 (b), the expression level of CD30 on the cell surface of primary lymphoma is high, and the CD30 chimeric antigen receptor selected by the invention can be used for treating primary lymphoma.

Example 5 in vitro tumor killing of CAR-T cells

(1) Non-specific 4GS-meso CART cells, GD2CAR-T cells, and specific 4GS-CD30 CART cells prepared herein were CO-cultured with primary lymphoma cells, placed at 37 degrees 5% CO₂Co-culturing for 18h in an incubator;

(2) evaluating the recognition and killing functions of the CAR-T cells on target cells in vitro, wherein the target cells are calcein markers or infected with LV-GFP;

as shown in FIGS. 4 to 8, the fluorescence intensity was 10 as seen by comparing FIGS. 4(a), 5(a), 6(a) and 7(a)³The above cells were selected as ALCL tumor target cells, and the fluorescence intensity was 10 in FIGS. 4(a), 5(a), 6(a) and 7(a)³Upon further analysis of the above cells, 8.1% of the target cells were imminent to apoptosis, 3.3% of the target cells were apoptotic, as seen in FIG. 4(b), 15.4% of the target cells were imminent to apoptosis, 8.1% of the target cells were apoptotic, as seen in FIG. 6(b), 15.8% of the target cells were imminent to apoptosis, 10.0% of the target cells were apoptotic, as seen in FIG. 7(b), 44.5% of the target cells were imminent to apoptosis, and 29.1% of the target cells were apoptotic; therefore, the target cells of the CD30 chimeric antigen receptor T cells are threatened to be apoptotic, and the apoptosis number of the target cells is obviously higher than that of other control groups, so that the CD30 chimeric antigen receptor has better poisoning effect on ALCL tumor target cells.

As can be seen from fig. 8, the fluorescence intensity of the primary lymphoma cells cultured alone, the primary lymphoma cells cultured with the general T cells, and the primary lymphoma cells cultured with GD2-CAR T cells after 12 days did not change significantly, and after the primary lymphoma cells cultured with the CD30 CAR T cells of the present application for 12 days, the green fluorescence was substantially disappeared, i.e., there were no surviving primary lymphoma cells, indicating that the CD30 chimeric antigen receptor has a very good effect on the poisoning effect of the primary lymphoma cells.

In conclusion, the single-chain antibody of the chimeric antigen receptor, which is directed to the CD30 tumor surface antigen, is not easy to mutate, and has better effect compared with other chimeric antigen receptors and other tumor antigens, so that the immune effect of the CAR-T cell is enhanced, and the therapeutic effect of the CAR-T cell is enhanced.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Shenzhen City institute of immune gene therapy

<120> CD 30-based chimeric antigen receptor and application thereof

<130>2017

<160>6

<170>PatentIn version 3.3

<210>1

<211>246

<212>PRT

<213> artificially synthesized sequence

<400>1

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser

115 120 125

Ser Glu Gly Ser Thr Lys Gly AspIle Val Met Thr Gln Ser Pro Asp

130 135 140

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

145 150 155 160

Ser Gln Ser Val Asp Phe Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn

180 185 190

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

195 200 205

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

210 215 220

Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Trp Thr Phe Gly Gln Gly

225 230 235 240

Thr Lys Val Glu Ile Lys

245

<210>2

<211>1056

<212>PRT

<213> artificially synthesized sequence

<400>2

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu LeuIle Pro Gln Ile Gln Leu Val Gln Ser Gly Ala Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr Asp Tyr Tyr Ile Thr Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr

65 70 75 80

Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr Ser Gly

130 135 140

Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala

165 170 175

Thr Ile Asn Cys Lys Ala SerGln Ser Val Asp Phe Asp Gly Asp Ser

180 185 190

Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

195 200 205

Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Asp Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro

245 250 255

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ala Ala Ile

260 265 270

Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly

275 280 285

Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe

290 295 300

Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val

305 310 315 320

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

325 330 335

Val Arg Ser Lys Arg Ser Arg Leu LeuHis Ser Asp Tyr Met Asn Met

340 345 350

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

355 360 365

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

370 375 380

Ser Gly Gly Gly Gly Ser Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly

385 390 395 400

Glu Ser Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg

405 410 415

Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro

420 425 430

Glu Pro Ala Cys Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

435 440 445

Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Val Lys Arg

450 455 460

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

465 470 475 480

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

485 490 495

Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly GlyGly Ser Gly Gly Gly

500 505 510

Gly Ser Gly Gly Gly Gly Ser Arg Val Lys Phe Ser Arg Ser Ala Asp

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Met Gln Ala Leu Pro Pro Arg Thr Ser Gly Ser Gly Ala Thr Asn Phe

625 630 635 640

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met

645 650 655

Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly ArgThr Phe Pro

660 665 670

Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp

675 680 685

Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe

690 695 700

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

705 710 715 720

Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr

725 730 735

Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr

740 745 750

Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly Gly Ser

755 760 765

Gly Gly Gly Gly Ser Gly Ala Met Val Gly Ala Leu Glu Ser Leu Arg

770 775 780

Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His

785 790 795 800

Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg

805 810 815

Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg ArgPhe

820 825 830

Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys

835 840 845

Lys Met Val Leu Ala Leu Leu Glu Leu Ala Arg Gln Asp His Gly Ala

850 855 860

Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln Ala Ser

865 870 875 880

His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val

885 890 895

Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser

900 905 910

Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu

915 920 925

Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu

930 935 940

Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly

945 950 955 960

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

965 970 975

Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp

980 985 990

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

995 1000 1005

Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu

1010 1015 1020

Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met

1025 1030 1035

Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr

1040 1045 1050

Ser Ala Ser

1055

<210>3

<211>3171

<212>DNA

<213> artificially synthesized sequence

<400>3

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60

atcccccaga tccagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg 120

aaggtgagct gcaaggccag cggctacacc ttcaccgact actacatcac ctgggtgaga 180

caggcccccg gccagggcct ggagtggatg ggctggatct accccggcag cggcaacacc 240

aagtacaacg agaagttcaa gggcagagtg accatgacca gagacaccag catcagcacc 300

gcctacatgg agctgagcag actgagaagc gacgacaccg ccgtgtacta ctgcgccaac 360

tacggcaact actggttcgc ctactggggc cagggcaccc tggtgaccgtgagcagcggc 420

agcaccagcg gcagcggcaa gcccggcagc agcgagggca gcaccaaggg cgacatcgtg 480

atgacccaga gccccgacag cctggccgtg agcctgggcg agagagccac catcaactgc 540

aaggccagcc agagcgtgga cttcgacggc gacagctaca tgaactggta ccagcagaag 600

cccggccagc cccccaagct gctgatctac gccgccagca acctggagag cggcgtgccc 660

gacagattca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctgcag 720

gccgaggacg tggccgtgta ctactgccag cagagcaacg aggacccctg gaccttcggc 780

cagggcacca aggtggagat caaggccgcc gccatcgagg tgatgtaccc ccccccctac 840

ctggacaacg agaagagcaa cggcaccatc atccacgtga agggcaagca cctgtgcccc 900

agccccctgt tccccggccc cagcaagccc ttctgggtgc tggtggtggt gggcggcgtg 960

ctggcctgct acagcctgct ggtgaccgtg gccttcatca tcttctgggt gagaagcaag 1020

agaagcagac tgctgcacag cgactacatg aacatgaccc ccagaagacc cggccccacc 1080

agaaagcact accagcccta cgcccccccc agagacttcg ccgcctacag aagcgccagc 1140

ggcggcggcg gcagcggcgg cggcggcagc cagagaagaa agtacagaag caacaagggc 1200

gagagccccg tggagcccgc cgagccctgc cactacagct gccccagaga ggaggagggc 1260

agcaccatcc ccatccagga ggactacaga aagcccgagc ccgcctgcag ccccggcggc 1320

ggcggcagcg gcggcggcgg cagcaccagc ggcggcggcg gcagcggcgg cggcggcagc 1380

gtggtgaaga gaggcagaaa gaagctgctg tacatcttca agcagccctt catgagaccc 1440

gtgcagacca cccaggagga ggacggctgc agctgcagat tccccgagga ggaggagggc 1500

ggctgcgagc tgggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcaga 1560

gtgaagttca gcagaagcgc cgacgccccc gcctaccagc agggccagaa ccagctgtac 1620

aacgagctga acctgggcag aagagaggag tacgacgtgc tggacaagag aagaggcaga 1680

gaccccgaga tgggcggcaa gcccagaaga aagaaccccc aggagggcct gtacaacgag 1740

ctgcagaagg acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagagaaga 1800

agaggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggacacctac 1860

gacgccctgc acatgcaggc cctgcccccc agaaccagcg gcagcggcgc caccaacttc 1920

agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccatggg cgtgcaggtg 1980

gagaccatca gccccggcga cggcagaacc ttccccaaga gaggccagac ctgcgtggtg 2040

cactacaccg gcatgctgga ggacggcaag aaggtggaca gcagcagaga cagaaacaag 2100

cccttcaagt tcatgctggg caagcaggag gtgatcagag gctgggagga gggcgtggcc 2160

cagatgagcg tgggccagag agccaagctg accatcagcc ccgactacgc ctacggcgcc 2220

accggccacc ccggcatcat ccccccccac gccaccctgg tgttcgacgt ggagctgctg 2280

aagctggagg gcggcggcgg cagcggcggc ggcggcagcg gcgccatggt gggcgccctg 2340

gagagcctga gaggcaacgc cgacctggcc tacatcctga gcatggagcc ctgcggccac 2400

tgcctgatca tcaacaacgt gaacttctgc agagagagcg gcctgagaac cagaaccggc 2460

agcaacatcg actgcgagaa gctgagaaga agattcagca gcctgcactt catggtggag 2520

gtgaagggcg acctgaccgc caagaagatg gtgctggccc tgctggagct ggccagacag 2580

gaccacggcg ccctggactg ctgcgtggtg gtgatcctga gccacggctg ccaggccagc 2640

cacctgcagt tccccggcgc cgtgtacggc accgacggct gccccgtgag cgtggagaag 2700

atcgtgaaca tcttcaacgg caccagctgc cccagcctgg gcggcaagcc caagctgttc 2760

ttcatccagg cctgcggcgg cgagcagaag gaccacggct tcgaggtggc cagcaccagc 2820

cccgaggacg agagccccgg cagcaacccc gagcccgacg ccaccccctt ccaggagggc 2880

ctgagaacct tcgaccagct ggacgccatc agcagcctgc ccacccccag cgacatcttc 2940

gtgagctaca gcaccttccc cggcttcgtg agctggagag accccaagag cggcagctgg 3000

tacgtggaga ccctggacga catcttcgag cagtgggccc acagcgagga cctgcagagc 3060

ctgctgctga gagtggccaa cgccgtgagc gtgaagggca tctacaagca gatgcccggc 3120

tgcttcaact tcctgagaaa gaagctgttc ttcaagacca gcgccagctg a 3171

<210>4

<211>423

<212>PRT

<213> artificially synthesized sequence

<400>4

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr Ile Ser Pro

20 25 30

Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His

35 40 45

Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp

50 55 60

Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg

65 70 75 80

Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys

85 90 95

Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly

100 105 110

Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys

115 120 125

Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Met Val

130 135 140

Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu

145 150 155 160

Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe

165 170 175

Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys

180 185 190

Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val

195 200 205

Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu

210 215 220

Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu

225 230 235 240

Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr

245 250 255

Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe

260 265 270

Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe

275 280 285

Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala

290 295 300

Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp

305 310 315 320

Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala

325 330 335

Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr

340 345 350

Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr

355 360 365

Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp

370 375 380

Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly

385 390 395 400

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

405 410 415

Phe Phe Lys Thr Ser Ala Ser

420

<210>5

<211>21

<212>PRT

<213> artificially synthesized sequence

<400>5

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

1 5 10 15

His Ala Ala Arg Pro

20

<210>6

<211>22

<212>PRT

<213> artificially synthesized sequence

<400>6

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

Claims

1. A CD 30-based chimeric antigen receptor, wherein the chimeric antigen receptor is secretor-CD 30-CD28-CD27-CD137-CD3 ζ -2A-fbkp.casp 9;

the amino acid sequence of the chimeric antigen receptor Secretory-CD30-CD28-CD27-CD137-CD3 zeta-2A-FBKP.Casp9 is shown in SEQ ID NO. 2.

2. A recombinant lentivirus obtained by co-transfecting a mammalian cell with a viral vector comprising the chimeric antigen receptor of claim 1 and the packaging helper plasmids pNHP and pHEF-VSVG.

3. A composition comprising the chimeric antigen receptor of claim 1 and/or the recombinant lentivirus of claim 2.

4. Use of the chimeric antigen receptor of claim 1, the recombinant lentivirus of claim 2 or the composition of claim 3 for the preparation of chimeric antigen receptor T-cells and for the preparation of a medicament for the treatment of leukemia and/or lymphoma.