CN110903401A

CN110903401A - A second-generation chimeric antigen receptor targeting CD19 and its expression vector and application

Info

Publication number: CN110903401A
Application number: CN201911141911.5A
Authority: CN
Inventors: 孙洁; 荆瑞瑞; 周春
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-03-24

Abstract

The invention discloses a second-generation chimeric antigen receptor targeting CD19, an expression vector and application thereof, and relates to the field of preparation of antitumor drugs. The second generation chimeric antigen receptors include: a tumor antigen binding domain, an extracellular hinge and transmembrane domain, an intracellular costimulatory signal domain, a signal transduction domain; wherein, the signal transduction structural domain in the cell has three immunoreceptor tyrosine activation motifs, three ITAMs respectively have two tyrosines, and two tyrosines are mutated into proline respectively. The invention also provides an expression vector of the second-generation chimeric antigen receptor and application of the expression vector in antitumor drugs. The signal transduction structural domain of the chimeric antigen receptor provided by the invention has basically consistent killing capacity in vitro with that of the non-mutated chimeric antigen receptor, but the in vivo mouse model is obviously superior to that of the non-mutated chimeric antigen receptor. Provides a new tumor cell medicament for clinically treating leukemia and also provides a new design idea for clinically treating solid tumor by using the chimeric antigen therapy.

Description

Second-generation chimeric antigen receptor targeting CD19, and expression vector and application thereof

Technical Field

The application relates to the field of preparation of antitumor drugs, in particular to a second-generation chimeric antigen receptor targeting CD19, an expression vector and application thereof.

Background

Malignant tumors are one of diseases seriously threatening human health, and particularly, the incidence rate of blood tumors such as leukemia, lymphoma and the like is high. Among the drugs that have been used clinically for the treatment of leukemia and lymphoma are kymeriah and yescata. They achieve certain treatment effect clinically, but the leukemia and the lymphoma treated by the drugs still relapse; and has side effects such as cytokine storm and neurotoxicity. And the remission rate of the patient is not high enough, and other treatment means such as bridging bone marrow transplantation and the like are needed.

Currently, chimeric antigen receptors have been developed that mainly comprise four parts, a tumor antigen binding domain, an extracellular hinge and transmembrane domain, an intracellular costimulatory signaling domain, a signaling domain. Kymriah which has been applied to clinical application at present^TMAnd the costimulatory signaling domains of Yescata are 4-1BB and CD28, and the intracellular signaling region CD3 ζ are both signals mediating T cell activation. One reason why the current post-treatment leukemias and lymphomas still have a high recurrence rate may be that dual signaling stimulates over-activation of T cells, exacerbating T cell depletion. This hypothesis has been experimentally confirmed in a recent paper by mutating tyrosine in each ITAM to prevent its phosphorylation and downstream signal transduction, generating 1XX and XX3 these 1928 ζ mutants containing a single ITAM to analyze CAR-T cell function, differentiation and efficacy.

In the acute lymphoblastic leukemia mouse model, ITAM mutated CAR produces CAR-T cells with significant differences in therapeutic efficacy from 1928 ζ. The efficacy of different CAR-T cells was compared by injecting low doses of CAR-T cells. XX3 significantly reduced the antitumor efficacy compared to 1928 ζ; whereas 1XX exceeded 1928 ζ, long-term remission was induced in mice. The efficacy of tumor eradication gradually decreased as the location of ITAMs moved distally (1XX > XX 3). The 1XX CAR is capable of rapid tumor eradication and CAR design that achieves sustained and complete remission at the lowest T cell dose, and whether this phenomenon is a result of co-development at different tumor antigen binding domains is currently unknown.

Disclosure of Invention

Aiming at the problems in the prior art, the application provides a second-generation chimeric antigen receptor targeting CD19, an expression vector and application thereof, which can prolong the survival time of T cells in vivo, improve the tumor treatment effect and reduce the recurrence rate of tumors.

The application is realized by the following technical scheme: a second-generation chimeric antigen receptor targeting CD19, the second-generation chimeric antigen receptor comprising: a tumor antigen binding domain, an extracellular hinge and transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain. Wherein the intracellular signal transduction domain comprises a first immunoreceptor tyrosine activation motif, a second immunoreceptor tyrosine activation motif, and a third immunoreceptor tyrosine activation motif, wherein each of the three immunoreceptor tyrosine activation motifs has a tyrosine, and wherein the tyrosine in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif is mutated to proline, or wherein the tyrosine in the first immunoreceptor tyrosine activation motif and the second immunoreceptor tyrosine activation motif is mutated to proline.

Further, the tumor antigen binding domain comprises a single chain antibody targeting CD19, and the sequence of the single chain antibody is SEQ ID NO. 1.

Further, the intracellular costimulatory signal domain comprises CD28, and the sequence of the intracellular costimulatory domain is SEQ ID No. 2.

Further, tyrosine in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif is mutated into proline, and the amino acid sequence of the second-generation chimeric antigen receptor is SEQ ID No. 3.

Further, tyrosine in the first immunoreceptor tyrosine activation motif and the second immunoreceptor tyrosine activation motif is mutated into proline, and the amino acid sequence of the second-generation chimeric antigen receptor is SEQ ID No. 4.

Further, the intracellular signaling co-stimulatory domain may also be 4-1BB, and the tyrosine in the second and third immunoreceptor tyrosine-activation motifs in the intracellular signaling domain is mutated to proline. The amino acid sequence of the second generation chimeric antigen receptor is SEQ ID NO. 6.

Further, the intracellular signaling co-stimulation domain may be 4-1BB, the tyrosine in the first and second immunoreceptor tyrosine activation motifs in the intracellular signaling domain is mutated to proline, and the amino acid sequence of the second-generation chimeric antigen receptor is SEQ ID No. 7.

An expression vector for a secondary chimeric antigen receptor comprising nucleotides encoding said secondary chimeric antigen receptor.

An application of the expression vector of the second generation chimeric antigen receptor in preparing antineoplastic medicines.

Furthermore, the anti-tumor drug is obtained by expressing a carrier on T cells through a second-generation chimeric antigen receptor.

Compared with the prior art, the invention has the following beneficial effects: the intracellular signal transduction structural domain of the chimeric antigen receptor provided by the application has almost no difference from the proliferation and killing capability of the existing second-generation chimeric antigen receptor in vitro, but has obviously enhanced anti-tumor capability in vivo, thereby providing a new tumor cell medicament for clinically treating leukemia and providing a new design idea for clinically treating solid tumor by using the chimeric antigen therapy. The application modifies the co-stimulation signal structure domain of the chimeric antigen receptor, can stimulate the T cells to quickly proliferate, and prolongs the in vivo survival time of the T cells, thereby improving the tumor treatment effect and reducing the recurrence rate of tumors.

Drawings

FIG. 1 is a schematic diagram of the structure of different chimeric antigen receptor mutated ITAMs provided in example 1;

FIG. 2 is a schematic diagram of the construction of the nucleotides of the chimeric antigen receptor provided in example 2 into a lentiviral vector;

FIG. 3 is a schematic diagram of the reprogramming of the chimeric antigen receptor to the TCR α gene provided in example 3;

fig. 4 is a graph of flow results of CRISPR system and adeno-associated virus in example 4 detected three days after the site-specific insertion of three different CARs, FMC28Z, FMC1XX, FMCXX3, into the TRAC site.

FIG. 5 is a graph of the lysis of T cell effector cells expressing the chimeric antigen receptor provided in example 5 with the target leukemia tumor cell line NALM6-GFP-Luciferase at different E: T ratios.

FIG. 6 is a tumor cure in a mouse model of the target leukemia tumor cell line NALM6-GFP-Luciferase following injection of the chimeric antigen receptor T cells provided in example 5.

Detailed Description

The technical solution of the present application is further described in detail by the accompanying drawings and examples.

Chimeric antigen receptor T cell (CAR-T) technology is a novel cancer treatment approach. CAR-T cells are constructed by introducing a nucleic acid fused with a CAR gene into the genome of autologous or allogeneic T lymphocytes. The development of Chimeric Antigen Receptors (CARs) goes through different stages. The first generation of CARs did not have a costimulatory signaling domain, and thus CAR-T cells had less proliferative effects and poor antitumor effects. Second generation CARs contain CARs with a single costimulatory signaling domain.

The embodiment of the application provides a CAR with a mutated intracellular signal transduction structural domain, which can stimulate the rapid proliferation of T cells and prolong the survival time of the T cells in vivo, so that the tumor treatment effect can be improved, and the recurrence rate of tumors is reduced.

In the present embodiment, "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-or double-stranded form, or a combination of DNA or RNA thereof, which is synthetic or recombinant, and polymers thereof.

An "expression vector" refers to a vector comprising recombinant nucleotides including an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes cis-acting elements sufficient for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), among others, that incorporate recombinant polynucleotides.

"lentivirus" refers to a genus of the family Retroviridae, lentiviruses that are unique among retroviruses, are capable of infecting non-dividing cells, and can transfer large amounts of genetic information into the DNA of host cells, and thus they are among the most effective means in gene delivery vectors, such as HIV, SIV, and FIV, among others.

Next, the CAR provided in the embodiments of the present application will be specifically described in the embodiments.

Before the present embodiments are further described, it is to be understood that the scope of the present application is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application; in the specification and claims of this application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected from the group consisting of the endpoints unless otherwise indicated herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the present application, in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and the description of the present application.

Example 1

This example provides a second generation chimeric antigen receptor targeting CD19, comprising: a tumor antigen binding domain, an extracellular hinge and transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain, wherein the costimulatory signaling domain is a precisely combined costimulatory signaling domain. The intracellular signal transduction domain comprises a first immune receptor tyrosine activation motif, a second immune receptor tyrosine activation motif and a third immune receptor tyrosine activation motif, two tyrosines are respectively arranged on the three immune receptor tyrosine activation motifs, tyrosine in the second immune receptor tyrosine activation motif and the third immune receptor tyrosine activation motif is mutated into proline, or tyrosine in the first immune receptor tyrosine activation motif and the second immune receptor tyrosine activation motif is mutated into proline, the amino acid sequences can be obtained by means of gene synthesis, and the correctness of the mutation sites is verified by gene sequencing.

In this example, the tumor antigen binding domain is a single chain antibody targeting CD19, and the sequence of the CD 19-targeting single chain antibody is SEQ ID No. 1:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS。

in this embodiment, the intracellular costimulatory signaling domain comprises CD28, and the sequence of the intracellular costimulatory signaling domain is SEQ ID No.2, specifically:

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。

in the present embodiment, the ITAM sequence containing different CD3 ζ is mutated in the intracellular signaling domain to include a first immunoreceptor tyrosine activation motif, a second immunoreceptor tyrosine activation motif, a third immunoreceptor tyrosine activation motif, two tyrosines in each of the three immunoreceptor tyrosine activation motifs, the tyrosines in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif are mutated to proline, and the amino acid sequence of the second-generation chimeric antigen receptor is SEQ ID No. 3; or the tyrosine in the first immune receptor tyrosine activation motif and the tyrosine in the second immune receptor tyrosine activation motif are mutated into proline, and the amino acid sequence of the second generation chimeric antigen receptor is SEQ ID NO. 4.

Wherein the structural schematic diagram of the mutation is shown in figure 1, and the detection by the gene sequencing method shows that the chimeric antigen receptor of which the tyrosine in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif is changed into proline and the tyrosine in the first immunoreceptor tyrosine activation motif and the second immunoreceptor tyrosine activation motif is changed into proline does occur.

Example 2

In this embodiment, the intracellular costimulatory signal domain comprises 4-1BB, and the sequence of the intracellular costimulatory domain is SEQ ID No.5, specifically: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL are provided.

In the present embodiment, the ITAM sequence containing different CD3 ζ is mutated in the intracellular signaling domain to include a first immunoreceptor tyrosine activation motif, a second immunoreceptor tyrosine activation motif, a third immunoreceptor tyrosine activation motif, one tyrosine in each of the three immunoreceptor tyrosine activation motifs, wherein the tyrosine in the second immunoreceptor tyrosine activation motif and the tyrosine in the third immunoreceptor tyrosine activation motif is mutated to proline, and the amino acid sequence of the second-generation chimeric antigen receptor is SEQ ID No. 6; or the tyrosine in the first immune receptor tyrosine activation motif and the tyrosine in the second immune receptor tyrosine activation motif are mutated into proline, and the amino acid sequence of the second generation chimeric antigen receptor is SEQ ID NO. 7.

The gene sequencing method shows that the chimeric antigen receptor of which tyrosine in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif is mutated into proline and the tyrosine in the first immunoreceptor tyrosine activation motif and the second immunoreceptor tyrosine activation motif is mutated into proline does occur.

Example 3

Performing PCR amplification by using each chimeric antigen receptor template synthesized in the embodiment 1 or the embodiment 2, wherein the CAR forward primer is shown as SEQ ID NO.8, specifically CATTTCAGGTGTCGTGATTCGAATTCGCCGCCACCATG; the CAR negative primer is shown as SEQ ID NO.9, specifically TGCTCACCATGGGTCCGGGATTCTCCTC.

Meanwhile, an EGFP (enhanced Green Fluorescent Protein) gene is amplified by PCR, wherein an EGFP forward primer is shown as SEQ ID No.10, specifically GGCCCCTCGCGGATCTGGAGCAACAAAC, EGFP forward primer is shown as SEQ ID No.11, and specifically GACGCGGTCTAGAGTCGCGGGATCCTTACTTGTACAGCTCGTCCATG. Wherein EGFP is used to detect the expression of the CAR gene.

The CAR gene segment can be connected with the EGFP gene segment through a P2A linker to obtain a CAR-P2A-EGFP DNA segment, and the CAR-P2A-EGFP DNA segment is constructed into a lentiviral vector by using 2-hieff mix (lenti) and verified to be successfully constructed into the lentiviral vector through a gene sequencing method. The constructed map is shown in FIG. 2.

Example 4

The chimeric antigen receptor templates synthesized in example 1 or example 2 were synthesized by gene synthesis to form left and right homology arms (left homology arms) on adeno-associated virus vectors, three CARs containing different ITAMs were constructed on both sides of the homology arms of adeno-associated virus vectors by molecular cloning, the adeno-associated virus vectors were digested with AvrII and NcoI-HF, forward primers for cloning the three CARs were shown in SEQ id No.12, GTGGAGGAGAATCCCGGCCCCATGGCCTTACCAGTGACCGCCTTG, reverse primers were shown in SEQ id No.13, CAACTAGAAGGCACAGTCGCCTAGGGATTTAGCGAGGGGGCAGGGCCTG, and the successful construction of the three CARs on adeno-associated virus vectors was verified by gene sequencing. The three CAR genes were then site-directed integrated into the TRAC site by CRISPR-cas9 technique, and the schematic structural diagram of the integration is shown in fig. 3.

Example 5

Taking several CARs in example 2 as an example, several CARs were loaded into human primary T cells.

1) Three generations of CAR transfection plasmids prepared and two virus packaging plasmids of psPAX2 and PMD.2G were extracted by a Plasmid Maxi Plasmid extraction kit (MN). The ratio of the three plasmids was 4: 3: 1, and carrying out virus packaging by using a PEI reagent, wherein the mass ratio of PEI to plasmid is 3: 1. the virus supernatants were harvested 48 hours and 72 hours after transfection, centrifuged at 3000rpm at 4 ℃ for 10 minutes, filtered through a 0.45um filter, centrifuged at 8500g at 4 ℃ overnight, and the virus was concentrated two hundred fold before storage at-80 ℃.

2) Preparation of T cells: separating peripheral blood mononuclear cells from peripheral blood, extracting 10ml of human venous blood, diluting the blood with PBS for one time, adding 20ml of ficoll lymphocyte separation liquid to separate the peripheral blood mononuclear cells, culturing the cells with 1640 culture medium, adding 10% fetal bovine serum and 1% streptomycin mixture (P/S) into the 1640 culture medium, and stimulating the proliferation of the peripheral blood mononuclear cells with CD3/CD28 antibodies. 3) Infection of T cells with lentiviruses: after 1million (million) cells were added to 100ul of virus solution and then polybrene (polybrene) was added to continue culturing the cells, CAR expression on T cells was detected.

Example 6

Using several of the CARs in example 3 as an example, several CARs were site-specific inserted into human primary T cells

1. Acquisition of human Primary T cells

Peripheral blood mononuclear cells were isolated from peripheral blood, 10ml of human venous blood was extracted, diluted once with PBS, 20ml of ficoll lymphocyte isolate was added to isolate peripheral blood mononuclear cells, the cells were cultured in 1640 medium, 10% fetal bovine serum and 1% penicillin mixed solution (P/S) were added to the 1640 medium, and proliferation of peripheral blood mononuclear cells was stimulated by CD3/CD28 antibody.

2. Electrotransformation guide RNA and Cas9 protein

Collecting the human primary T cells cultured in the step 1, centrifuging and precipitating the human primary T cells, and then firstly adding 10ugguide RNA in vitro, wherein the sequence of the guide RNA is shown as SEQ ID NO. 14: CAGGGUUCUG GAUAUCUGUG UUUUAGAGCUAGAAAUAGCA AGUUAAAAUA AGGCUAGUCC GUUAUCAACU UGAAAAAGUG GCACCGAGUC GGUGCUUUU are provided.

Combined with 20ug of cas9 protein, mixed with human primary T cells, and electrically transferred by a Celetrix electric transfer system with 20ul of electric transfer system, 0.5-1million of electric transfer cells, electric transfer voltage 620v, pulse time 30 ms. After electrotransformation, the T cells are continuously cultured in 1640 culture medium + 10% fetal calf serum and 1% P/S overnight, and then are added, after electrotransformation for 24h, adeno-associated virus with CAR gene is added, and the fixed-point insertion ratio of CAR is detected by flow type after 3-4 days. After 30min incubation with Anti-mouse FMC63Alex Fluor 647 and Anti CD3 antibody, more than 60% of T cells in TCR knockout T cells were confirmed to reach CAR by flow detection, as shown in FIG. 4.

Example 7

Using the CAR-T cells generated in examples 4 or 5 as an example, the in vitro killing ability of CAR-T cells in coculture with leukemic tumor cells and tumor killing ability assays in a mouse leukemic tumor model were investigated.

In this example, the killing ability of CAR-T cells when encountering the target leukemia tumor cell line NALM6-GFP-Luciferase was investigated.

10000 NALM6-GFP-Luciferase tumor cells were placed in each 96-well round cell culture plate, and CAR-T cells were diluted from 100000 in a gradient manner until 3125, respectively corresponding to the Effector: the Target ratio is 2:1, 1: 1. 1: 2. 1: 4. 1: 8. 1: 16. 1: 32, each set of three replicates each having a volume of about 100ul, after 18 hours of co-incubation, fluorescein substrate was added and fluorescein luminescence was measured using a plate reader, and CAR-T cell killing was calculated from fluorescein luminescence as shown in figure 5, FMC28Z1XX representing tyrosine to proline mutation on the second and third ITAMs and FMC28ZXX3 representing tyrosine to proline mutation on the first and second ITAMs. From the killing effect in vitro, FMC28Z1XX, FMC28Z XX3 have no obvious difference compared with FMC 28Z. However, in vivo animal model tests of mice prove that the antitumor effects of FMC28Z1XX and FMC28Z XX3 in vivo are obviously better than that of FMC28Z, as shown in FIG. 6. The in vivo and in vitro experiment results of the CAR with the intracellular costimulatory signal of 4-1BB and the CAR with the intracellular signal of CD28 are similar to FMC4-1BB1XX and FMC4-1BB XX3, and the antitumor effect in vivo is obviously better than that of FMC4-1 BB.

The above-mentioned embodiments, objects, technical solutions and advantages of the present application are described in further detail, it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present application, and are not intended to limit the scope of the present application, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present application should be included in the scope of the present application.

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile

435 440 445

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

450 455 460

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

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210>7

<211>486

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

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

1 5 1015

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile

435 440 445

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

450 455 460

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

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210>8

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

catttcaggt gtcgtgattc gaattcgccg ccaccatg 38

<210>9

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

tgctcaccat gggtccggga ttctcctc 28

<210>10

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ggcccctcgc ggatctggag caacaaac 28

<210>11

<211>47

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gacgcggtct agagtcgcgg gatccttact tgtacagctc gtccatg 47

<210>12

<211>45

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

gtggaggaga atcccggccc catggcctta ccagtgaccg ccttg 45

<210>13

<211>49

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

caactagaag gcacagtcgc ctagggattt agcgaggggg cagggcctg 49

<210>14

<211>99

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

caggguucug gauaucugug uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60

guuaucaacu ugaaaaagug gcaccgaguc ggugcuuuu 99

Claims

1. A secondary chimeric antigen receptor targeting CD19, comprising: a tumor antigen binding domain, an extracellular hinge and transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain. Wherein the intracellular signal transduction domain comprises a first immunoreceptor tyrosine activation motif, a second immunoreceptor tyrosine activation motif, and a third immunoreceptor tyrosine activation motif, wherein each of the three immunoreceptor tyrosine activation motifs has two tyrosines, and wherein the tyrosine in the second immunoreceptor tyrosine activation motif and the third immunoreceptor tyrosine activation motif is mutated to proline, or wherein the tyrosine in the first immunoreceptor tyrosine activation motif and the second immunoreceptor tyrosine activation motif is mutated to proline.

2. The secondary chimeric antigen receptor of claim 1, wherein the tumor antigen binding domain comprises a single chain antibody targeting CD19, the sequence of said single chain antibody being SEQ ID No. 1.

3. The secondary chimeric antigen receptor according to claim 1 or 2, wherein the intracellular costimulatory signaling domain comprises CD28, and the sequence of the intracellular costimulatory domain is SEQ ID No. 2.

4. The secondary chimeric antigen receptor according to claim 3, wherein tyrosine in the second and third immunoreceptor tyrosine activation motifs is mutated to proline, and the amino acid sequence of the secondary chimeric antigen receptor is SEQ ID No. 3.

5. The secondary chimeric antigen receptor according to claim 3, wherein the tyrosine in the first and second immunoreceptor tyrosine activation motifs is mutated to proline, and the amino acid sequence of the secondary chimeric antigen receptor is SEQ ID No. 4.

6. The secondary chimeric antigen receptor of claim 1 or 2, wherein the intracellular signaling co-stimulatory domain is also 4-1BB, and the tyrosine in the second and third immunoreceptor tyrosine activation motifs in the intracellular signaling domain is mutated to proline. The amino acid sequence of the second generation chimeric antigen receptor is SEQ ID NO. 6.

7. The secondary chimeric antigen receptor of claim 1 or 2, wherein the intracellular signaling co-stimulatory domain is also 4-1BB, the tyrosine in the first and second immunoreceptor tyrosine activation motifs in the intracellular signaling domain is mutated to proline, and the amino acid sequence of the secondary chimeric antigen receptor is SEQ ID No. 7.

8. An expression vector for a secondary chimeric antigen receptor comprising nucleotides encoding the secondary chimeric antigen receptor of any one of claims 1-7.

9. An application of the expression vector of the second generation chimeric antigen receptor in preparing antineoplastic medicines.

10. The use according to claim 8, wherein the anti-neoplastic agent is obtained by expressing the vector on T cells via a second generation chimeric antigen receptor.