CN111187352B

CN111187352B - Chimeric antigen receptor targeting human CD19 and application thereof

Info

Publication number: CN111187352B
Application number: CN202010051900.4A
Authority: CN
Inventors: 李俊; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-11-03
Anticipated expiration: 2040-01-17
Also published as: CN111187352A

Abstract

The invention relates to the field of medical biology, in particular to a chimeric antigen receptor targeting human CD19 and application thereof; the chimeric antigen receptor targeting CD19 comprises an antigen binding domain, a transmembrane region and an intracellular signal domain of an anti-CD 19 humanized antibody, and the invention also provides a T cell modified by the chimeric antigen receptor targeting human CD19, which can be specifically combined with CD19 on the surface of a tumor cell so as to generate a specific killing effect on the tumor cell. The invention also relates to the application of the chimeric receptor targeting the human CD19 and the immune effector cell thereof in the preparation of anti-tumor immunotherapy drugs.

Description

Chimeric antigen receptor targeting human CD19 and application thereof

Technical Field

The invention relates to the field of medical biology, in particular to a chimeric antigen receptor targeting human CD19 and application thereof.

Background

CD19, also known as B4 or Leu-12, is specifically expressed on the surface of normal and malignant B-lymphocyte membranes, as well as on the surface of follicular dendritic cell membranes, and belongs to the immunoglobulin (Ig) superfamily of members, which have a molecular weight of 95kDa, are located on the short arm of chromosome 16, contain 15 exons, and encode a type I transmembrane glycoprotein of 556 amino acids. CD19 is one of the most reliable surface markers for B cells, and it was first expressed in late progenitor B cells and early pre-B cells, occurring upon immunoglobulin gene recombination. CD19 was highly expressed throughout B cell development and maturation until the time of B cell differentiation into plasma cells, the expression was down-regulated, with 3-fold higher expression in mature B cells than in immature cells.

CD19 establishes a B cell signaling threshold by simultaneously regulating B Cell Receptor (BCR) dependent and independent signals, playing an important role in the regulation of B cell development, proliferation and differentiation. CD19, an essential component of the surface multi-molecular complex of mature B cells, forms a complex with the receptors CD21(CD2), CD81(TAPA-1) and CD225, reducing the threshold of antigen concentration required to trigger B cell division and differentiation by modulating endogenous and receptor-induced signals. CD81 acts as a chaperone, provides a molecular docking site for signaling pathways, and regulates the expression of CD 19. CD19 activates Protein Tyrosine Kinases (PTKs) by recruiting and amplifying the activation of Src family protein tyrosine kinases, activating BCR signals. Meanwhile, when the BCR signal is activated, the CD19 can also enhance the BCR signal and promote the proliferation of B cells by activating PI3K and downstream Akt kinase.

CD19 has important regulatory effects on the proliferation and differentiation of B cells. CD19 is widely expressed in almost ALL B cell malignancies, including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), and non-hodgkin lymphoma, among others, and thus CD19 is a specific molecular target for the treatment of B cell malignancies. In recent years, immunotherapeutic strategies targeting CD19, including monoclonal antibodies, bispecific antibodies and chimeric antigen receptor-modified T cells (CAR-T), have been extensively developed in preclinical as well as clinical studies, and have achieved clinical effects significantly superior to conventional small molecule chemotherapeutic regimens, driving the progress of immunotherapy.

The chimeric antigen receptor is mainly composed of two parts, one end is positioned outside cells and can specifically recognize a certain antigen on the surface of a cancer cell, and the other end is positioned in cells and contains a signal activation element (such as a Zeta chain of a T cell receptor) which plays a role in transmitting signals to activate T cells. The hypervariable region sequence of a monoclonal antibody for recognizing a tumor surface specific antigen is recombined and subcloned into a Single-chain antibody fragment (scFv) in vitro, then the scFv is fused with transmembrane protein fragments of other genes and an intracellular signal peptide to form an artificial Chimeric Antigen Receptor (CAR), and the artificial Chimeric antigen receptor is transfected into a T cell to form a Chimeric antigen receptor T cell (CAR-T). CD19-CAR chimeric antigen receptor immunotherapy has changed the traditional approach to hematological malignancy treatment and has achieved compelling success in phase I clinical studies.

Currently, the majority of CD19CART clinical trials use murine CD19 antibody as the extracellular domain. The murine antibody has high immunogenicity, is easy to be eliminated by the immune system of an organism, and influences the survival time of CAR-T cells in vivo.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides a humanized CD19 chimeric antigen receptor with good specificity, higher lethality and lower immunogenicity and application thereof.

The technical scheme is as follows: the invention relates to a chimeric antigen receptor targeting human CD19, which comprises a humanized CD19 antibody binding domain based on a CD19 antigen, a transmembrane region and an intracellular signal domain; wherein, the heavy chain sequence of the CD19 antibody binding domain is shown as SEQ ID NO: 1, and the light chain sequence is shown as SEQ ID NO: 2, respectively.

Further, the amino acid sequence of the binding domain of the CD19 antibody is shown in SEQ ID NO: 3 or a modified amino acid sequence having 85% to 99% identity thereto. Preferably the sequence of SEQ ID NO: 3 has 90-99%, even 95-99% identity.

Further, the transmembrane region is selected from the group consisting of CD4, CD8 α, CD28 and a 4-1BB transmembrane region. Preferably, the transmembrane region structure comprises SEQ ID NO: 6, and the CD8 alpha transmembrane region.

The intracellular signaling domain comprises a signaling domain and/or a co-stimulatory signaling domain selected from one or more of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD 3. CD3zeta, 4-1BB and/or CD28 signaling domains are preferred.

Preferably, the intracellular signaling domain comprises SEQ ID NO: 8, CD3zeta amino acid sequence shown. The intracellular costimulatory signal domain comprises SEQ ID NO: 7, and 4-1BB amino acid sequence.

The chimeric antigen receptor targeting human CD19 further comprises an extracellular signal peptide structure selected from the group consisting of a CD8 α signal peptide, a GM-CSFR α signal peptide, a CD4 signal peptide, and an IL-2 signal peptide. Preferably CD8 α signal peptide, having an amino acid sequence as set forth in SEQ ID NO: 4, respectively.

The chimeric antigen receptor targeting the human CD19 further comprises a hinge region structure for connecting the extracellular region peptide segment and the transmembrane region peptide segment, wherein the hinge region is a CD8 alpha molecule and a CD28 molecule. Preferably a CD8 a molecule having an amino acid sequence as set forth in SEQ ID NO: 5, respectively.

The amino acid sequence of the chimeric antigen receptor targeting human CD19 is shown in SEQ ID NO: 9 or SEQ ID NO: shown at 10.

The invention also discloses a nucleic acid molecule for encoding the chimeric antigen receptor targeting the human CD 19.

Further, the nucleotide coding sequence of the nucleic acid molecule is shown as SEQ ID NO: shown at 11.

The invention also discloses a recombinant expression vector containing the nucleic acid molecule. The recombinant expression vector includes a chimeric antigen receptor expression cassette consisting of a promoter expressible in T cells and a nucleic acid encoding the chimeric antigen receptor downstream of the promoter, and a replication system. The promoter is a cytomegalovirus promoter, an SV40 promoter, an EF1alpha promoter or an RSV promoter.

The invention also discloses a genetically engineered immune cell containing the nucleic acid molecule or the recombinant expression vector. Wherein, the immune cells can be selected from T lymphocytes, NK cells, hematopoietic stem cells, pluripotent stem cells or differentiated immune cells of embryonic stem cell culture. T lymphocytes are further preferred.

Further, the genetically engineered immune cell expresses a chimeric antigen receptor targeting human CD19 comprising an extracellular signal peptide, a humanized antibody binding domain of anti-CD 19, a hinge region, a transmembrane region, and an intracellular signal domain.

The chimeric receptor ligand expressed by the genetically engineered immune cell and targeting human CD19 has the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

The invention also discloses the application of the chimeric antigen receptor targeting the human CD19 in the preparation of antitumor drugs; preferably in the preparation of anti-hematological malignancy drugs, in particular in the preparation of drugs for treating acute myeloid leukemia.

Has the advantages that: the invention provides a chimeric antigen receptor targeting human CD19, which can enable the obtained CAR T cells to effectively and specifically target malignant cells expressing CD19 surface antigen by expressing the chimeric antigen receptor in T cells, thereby providing a more efficient method with fewer side effects and adverse reactions for treating some tumors expressing CD19 surface antigen, such as B lymphocyte leukemia and the like.

Drawings

FIG. 1 is a schematic representation of a chimeric antigen receptor according to the invention;

FIG. 2 is a graph of the results of the lytic killing ability of CD19 CAR-T cells on target cells Nalm 6;

FIG. 3 is a graph of the results of IFN- γ secretion by CD19 CAR-T cells;

FIG. 4 is a graph of the results of IL-2 secretion by CD19 CAR-T cells.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples.

Example A Chimeric Antigen Receptor (CAR) Lentiviral expression vector construction

The intracellular domain of CD137 and the ITAM region of CD3Zeta were used as activation signals, fused with CD19 single-chain antibody, to construct a chimeric antigen receptor expression vector, and subcloned into a PLVX-EF1a (purchased from clontech) vector. The sequence of the combination of the elements in the constructed chimeric antigen receptor lentiviral expression vector is shown in FIG. 1:

the amino acid sequences of all elements in the constructed chimeric antigen receptor are respectively as follows:

signal peptide: SEQ NO.4

MALPVTALLLPLALLLHAARP

Humanized single chain antibody sequence: SEQ NO.3

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTSVTVSS

CD8 hinge region: SEQ NO.5

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 transmembrane region: SEQ NO.6

IYIWAPLAGTCGVLLLSLVITLYC

CD137 endodomain: SEQ NO.7

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

CD3Zeta：SEQ NO.8

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

EXAMPLE two Lentiviral preparation

The specific experimental steps are as follows:

s1, preparing 15cm dish, inoculating 5X 10⁶293T cells (purchased from ATCC) were cultured in complete medium (DMEM high-sugar, 10% FBS, double antibody) at 37 ℃ in 5% CO₂And (5) an incubator for overnight culture.

S2, taking out 100 μ M PEI and lentivirus packaging plasmids (PLVX-EF1a-CAR, pGP, pVSVG) from the refrigerator, thawing at room temperature, and blowing and beating up and down by using a pipette gun to mix completely. Remove PBS or HBSS buffer and warm to room temperature. And (3) adding 10 mu g of Lenti-EF1a-CAR, 4 mu g of pGP and 2 mu g of pVSVG into one hole of a 6-hole plate by taking 2mL of PBS, respectively, blowing and beating the mixture up and down by a pipette, fully and uniformly mixing, adding 18 mu L of 100 mu M PEI, immediately blowing and beating the mixture up and down by a pipette, uniformly mixing, and standing the mixture at room temperature for 10 minutes.

S3, dropwise adding the DNA/PEI complex into a 15cm culture dish, slightly shaking the culture dish, and fully mixing. The culture dish is placed in an incubator with 37 ℃ and 5% CO2, after culturing for 6-8 hours, the culture medium containing the transfection reagent is removed, and replaced by a fresh complete culture medium.

After 48 hours of continuous culture, the virus-containing culture supernatant was collected from the petri dish, filtered through a 0.45 μm filter, transferred to a centrifuge tube, added 20% by volume of PEG6000, allowed to stand at 4 ℃ for 2 and, after trimming, centrifuged at 3000Xg for 0.5 hours at 4 ℃. After centrifugation, the liquid in the centrifuge tube was carefully aspirated in a biosafety cabinet, the pellet was resuspended by adding 500. mu.L of PBS buffer, and the virus was stored at-80 ℃.

EXAMPLE isolation of three Primary T cells

The specific experimental steps are as follows:

s1, the lymphocyte separation solution is inverted several times, and the Lymphoprep reagent is mixed well.

S2, in a biosafety cabinet, 15mL Lymphoprep reagent is added to a 50mL centrifuge tube (or a 15mL centrifuge tube, depending on the volume of the separated blood sample) for use.

S3, blood samples were diluted with an equal volume of PBS + 2% FBS.

And S4, carefully adding the diluted blood sample to the upper layer of the separation reagent along the tube wall by using a pipette gun, and avoiding the mixing of the separation reagent and the blood sample.

S5, setting the centrifuge as 800Xg, setting the rotating speed descending speed as slowest, and centrifuging for 20 minutes at room temperature.

S6, after the centrifugation is finished, collecting the upper layer of light yellow serum into another sterile centrifuge tube, and storing the light yellow serum at-80 ℃.

S7, gently pipette the mononuclear cell layer at the interface between serum and separation reagent into a new centrifuge tube, and wash the cells once with medium.

S8, adjusting the cell density to 1 x 10⁸cells/mL (total volume not exceeding 2.5mL) were resuspended in 5mL round-bottomed tubes.

S9, 100. mu.l/mL of antibody cocktail was added thereto, and the mixture was mixed well and incubated at room temperature for 15 minutes.

S10, taking out the magnetic beads, blowing and beating the magnetic beads for at least 5 times up and down by using a pipette gun, and fully and uniformly mixing the magnetic beads.

S11, 50. mu.l of magnetic beads/mL was pipetted into the sample, and after mixing well, the mixture was incubated at room temperature for 10 minutes.

S12, adding complete culture medium until the total volume in the tube is 2.5mL, inserting the tube (uncapped) into a magnetic pole, and standing for 5 minutes at room temperature.

And S13, after incubation, keeping the tube in the magnetic pole, slightly inverting, and pouring out the cells in the tube.

S14, resuspend cells in X-vivo 15 medium and add 10% FBS, 300U/mL IL-2,5ng/mLIL-15 and 10ng/mL IL-7.

Example activation of four Primary T cells and Lentiviral infection

The specific experimental steps are as follows:

s1, adjusting the cell density to 1 x 10⁶cells/mL, cytokine and antibody complex (final concentration of 300U/mL IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Anti-CD3(OKT3), 2ug/mL Anti-CD28) were added and cultured continuously for 48 hours.

S2, the required amount of virus is calculated according to the MOI of 20. The calculation formula is as follows: required amount of virus (mL) ═ cell number (MOI)/viral titer

S3, taking out the virus from a refrigerator at minus 80 ℃, and quickly melting the virus in a water bath kettle at 37 ℃. The amount of the virus obtained by the above calculation was added to a six-well plate, polybrene was added to a final concentration of 6. mu.g/mL, and after mixing well, the four sides of the six-well plate were sealed with a sealing film, and centrifuged at 800Xg for 1 hour.

S4, tearing off the sealing film after the centrifugation is finished, and placing the six-hole plate at 37 ℃ in 5% CO₂The incubator of (1), the cultivation was continued for 24 hours.

S5, 250Xg centrifugation for 10 minutes, remove virus containing culture medium supernatant, fresh medium heavy suspension cell precipitation, cell transfer to new six-well plate, continued to culture for 3-6 days for use.

Example five CAR-T cells lysis of target cells

The specific experimental steps are as follows:

s1, adjusting the state of the target cells to logarithmic growth phase, and continuously carrying out passage for 2 times before carrying out the experiment;

s2, resuspending the target cells in complete medium, and adjusting the cell density to 5 x 10⁵One new 96-well plate was used to inoculate the target cells at a rate of 100. mu.L/well. The unused wells on the periphery of the 96-well plate were filled with 100. mu.L of sterile water per well to prevent evaporation of water from the middle experimental wells. Place the well plate in 5% CO₂Incubate overnight at 37 ℃.

S3, centrifugally collecting the prepared CAR-T cells, and resuspending the cells in a serum-free 1640 culture medium; remove the 96-well plate from the incubator, completely aspirate the medium from the wells, gently wash the cells with sterile PBSThen, according to the E/T ratio, CAR-T cells are added, and the final volume is supplemented to 100 mu L/hole; maxi lyss and Mini lyss were used to inoculate the same number of target cells, but without the addition of CAR-T cells. Place the well plate in 5% CO₂The cells were cultured at 37 ℃ for 6 hours in an incubator.

S4, after the culture is finished, taking the pore plate out of the incubator, adding the lysate in the LDH detection kit into the Maxi lysis pores, completely lysing the target cells in the lysate, centrifuging the 96 pore plate at 1200Xg room temperature for 5 minutes, gently taking out the plate, transferring 50 mu L of the lysate into another new 96 pore plate from each pore, adding the LDH detection reagent, and reading the OD value by using a microplate reader.

Target cell lysis percentage calculation formula:

s5, the data thus processed are plotted using GraphPad 6.0.

The experimental results are as follows:

using CAR-T cells as effector cells, using lymphoma cell strain Nalm6 cells naturally expressing CD19 as target cells, establishing a co-culture system according to different effective target ratios, namely, in a 96-well plate, the number of target cells fixed in each well is 50000, adding different numbers of CAR-T cells, culturing the co-culture system by using a serum-free culture medium, continuously culturing for 8 hours, taking out the well plate, centrifuging at room temperature of 1200Xg for 10 minutes to ensure that all suspended cells are precipitated to the bottom of the well plate, then taking out 30 microliters of supernatant from each well, and detecting the release amount of LDH in the supernatant of the culture medium to reflect the cracking capacity of the CAR-T cells on the target cells, wherein the result is shown in figure 2, and the effective target ratio is 4: 1, the T cells can be efficiently mediated to kill the tumor cells or the recombinant cells; with the increase of the effective target ratio, the killing effect of CD19 CAR-T cells on target cells Nalm6 is increased, and the highest effect is achieved when the effective target ratio is 8: 1.

Example six CAR-T cytokine secretion level assays

The specific experimental steps are as follows:

s2, resuspending the target cells in complete medium, and adjusting the cell density to 5 x 10⁵One new 96-well plate was used to inoculate the target cells at a rate of 100. mu.L/well. The unused wells on the periphery of the 96-well plate were filled with 100. mu.L of sterile water per well to prevent evaporation of water from the middle experimental wells. The plates were incubated overnight in a 5% CO 237 ℃ incubator.

S3, centrifugally collecting the prepared CAR-T cells, and resuspending the cells in a serum-free 1640 culture medium; removing the 96-well plate from the incubator, completely sucking out the culture medium in the well, gently washing the cells once with sterile PBS, then adding CAR-T cells according to the above E/T ratio, and supplementing the final volume to 100 mu L/well; place the well plate in 5% CO₂The cells were cultured at 37 ℃ for 6 hours in an incubator. A control T cell group was also set.

S4, after the end of the incubation, the plate was removed from the incubator, centrifuged at 1200Xg for 5 minutes in a 96-well plate at room temperature, gently removed, 50. mu.L of the culture supernatant was transferred from each well, and IFN-. gamma.and IL-2 expression was detected using ELISA kit, and OD was read using a microplate reader.

S5, the obtained data are plotted using GraphPad 6.0.

The experimental results are as follows:

using CAR-T cells as effector cells, using lymphoma cell strain Nalm6 cells naturally expressing CD19 as target cells, establishing a co-culture system according to different effective target ratios, namely in a 96-well plate, the number of the target cells fixed in each hole is 50000, adding different numbers of CAR-T cells respectively, culturing the co-culture system by using a serum-free culture medium, continuously culturing for 8 hours, taking out the well plate, centrifuging at room temperature of 1200Xg for 10 minutes to enable all suspended cells to be precipitated to the bottom of the well plate, then taking out 30 microliters of supernatant from each hole, and detecting the expression quantities of IFN-gamma and IL-2 secreted by the CAR-T cells after being activated by the tumor cells in the supernatant of the culture medium by using an ELISA method; as shown in FIGS. 3 and 4, after the CD19-CART cell is combined with the target tumor cell, the primary T cell can be effectively activated, and the secretion expression amount of the cytokine is increased; when the effective target ratio is 2:1, after the CAR-T cells are activated by tumor cells, a large amount of IFN-gamma can be secreted, which is obviously higher than that of control T cells; the secretion reaches the highest value when the effective target ratio is 8: 1.

Sequence listing

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85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser Tyr

115 120125

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

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

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

165 170 175

Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280285

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

290 295 300

Leu Asp Phe Ala Cys Asp Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210>11

<211>1458

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60

cccgacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgaccgcgtg 120

accatcacct gccaggccag ccaggacatc agcaactacc tgaactggta ccagcagaag 180

cccggcaagg cccccaagct gctgatctac gacgccagca acctggagac cggcgtgccc 240

agccgcttca gcggcagcgg cagcggcacc gacttcacct tcaccatcag cagcctgcag 300

cccgaggaca tcgccaccta ctactgccag cagggcaaca ccctgcccta caccttcggc 360

ggcggcacca agctggagat caccggcggc ggcggcagcg gcggcggcgg cagcggcggc 420

ggcggcagcc aggtgcagct gcaggagagc ggccccggcc tggtgaagcc cagccagacc 480

ctgagcctga cctgcaccgt gagcggcggc agcatcagca gcggcgacta ctactggagc 540

tggatccgcc agccccccgg caagggcctg gagtggatcg gctacatcta ctacagcggc 600

agcacctact acaaccccag cctgaagagc cgcgtgacca tcagcgtgga caccagcaag 660

aaccagttca gcctgaagct gagcagcgtg accgccgccg acaccgccgt gtactactgc 720

gcccgccact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 780

gtgaccgtga gcagcaccac cacccccgcc ccccgccccc ccacccccgc ccccaccatc 840

gccagccagc ccctgagcct gcgccccgag gcctgccgcc ccgccgccgg cggcgccgtg 900

cacacccgcg gcctggactt cgcctgcgac atctacatct gggcccccct ggccggcacc 960

tgcggcgtgc tgctgctgag cctggtgatc accctgtact gcaagcgcgg ccgcaagaag 1020

ctgctgtaca tcttcaagca gcccttcatg cgccccgtgc agaccaccca ggaggaggac 1080

ggctgcagct gccgcttccc cgaggaggag ggcggctgcg agctgcgcgt gaagttcagc 1140

cgcagcgccg acgcccccgc ctacaagcag ggccagaacc agctgtacaa cgagctgaac 1200

ctgggccgcc gcgaggagta cgacgtgctg gacaagcgcc gcggccgcga ccccgagatg 1260

ggcggcaagc cccgccgcaa gaacccccag gagggcctgt acaacgagct gcagaaggac 1320

aagatggccg aggcctacag cgagatcggc atgaagggcg agcgccgcgg caagggccac 1380

gacggcctgt accagggcct gagcaccgcc accaaggaca cctacgacgc cctgcacatg 1440

caggccctgc ccccccgc 1458

Claims

1. A chimeric antigen receptor targeting human CD19, wherein the amino acid sequence is as set forth in SEQ ID NO: shown at 10.

2. A nucleic acid molecule encoding the human CD 19-targeted chimeric antigen receptor of claim 1, wherein the nucleotide coding sequence of the nucleic acid molecule is as set forth in SEQ ID NO: shown at 11.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2.

4. A genetically engineered immune cell comprising the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3.

5. The use of the chimeric antigen receptor targeting human CD19 of claim 1 in the preparation of an anti-tumor medicament.