CN111187352B - Chimeric antigen receptor targeting human CD19 and application thereof - Google Patents

Abstract

The present invention relates to the field of medicine and biology, in particular to a chimeric antigen receptor targeting human CD19 and applications thereof; the chimeric antigen receptor targeting CD19 of the present invention comprises an antigen binding domain of an anti-CD19 humanized antibody, The transmembrane region and the intracellular signaling domain, the present invention also provides a chimeric antigen receptor-modified T cell targeting human CD19, which can specifically bind to CD19 on the surface of tumor cells, thereby producing specific effects on tumor cells. killing effect. The invention also relates to the application of the chimeric receptor targeting human CD19 and its immune effector cells in the preparation of anti-tumor immunotherapy drugs.

Description

Chimeric antigen receptor targeting human CD19 and its application

technical field

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

Background technique

CD19, also known as B4 or Leu-12, is specifically expressed on the membrane surface of normal and malignant B lymphocytes, as well as on the membrane surface of follicular dendritic cells. It belongs to the immunoglobulin (Ig) superfamily with a molecular weight of 95kDa and is located on chromosome 16. On the short arm, it contains 15 exons, encoding a type I transmembrane glycoprotein of 556 amino acids. CD19, one of the most reliable surface markers for B cells, is first expressed in late progenitor B cells and early pre B cells, when immunoglobulin genes are recombined. CD19 is highly expressed throughout the development and maturation of B cells, and its expression is down-regulated when B cells differentiate into plasma cells, and its expression in mature B cells is 3 times that of immature cells.

CD19 plays an important role in regulating B cell development, proliferation and differentiation by simultaneously regulating B cell receptor (BCR)-dependent and BCR-independent signaling to establish B cell signaling thresholds. As a major component of the surface multimolecular complex of mature B cells, CD19 forms a complex with the receptors CD21 (CD2), CD81 (TAPA-1) and CD225, and triggers B by regulating endogenous and receptor-induced signal reduction. Threshold of antigen concentration required for cell division and differentiation. CD81 acts as a chaperone, provides a molecular docking site for signaling pathways, and regulates the expression of CD19. CD19 activates protein tyrosine kinases (PTKs) and activates BCR signaling by recruiting and amplifying the activation of Src family protein tyrosine kinases. At the same time, when the BCR signal is activated, CD19 can also enhance the BCR signal and promote the proliferation of B cells by activating PI3K and the downstream Akt kinase.

CD19 plays an important role in regulating 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's lymphoma, etc. Therefore, CD19 has become a specific molecule for the treatment of B-cell malignancies target. In recent years, immunotherapy strategies targeting CD19 have been widely developed in preclinical and clinical studies, including monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor-modified T cells (CAR-T), and have achieved significantly better results than conventional The clinical effect of small molecule chemotherapy regimens has promoted the progress of immunotherapy.

The chimeric antigen receptor is mainly composed of two parts, one end is located outside the cell and can specifically recognize a certain antigen on the surface of the cancer cell, and the other end is located in the cell containing a signal activation element (such as the Zeta chain of the T cell receptor), which transmits the signal. Activate T cells. The hypervariable region sequences of monoclonal antibodies recognizing tumor surface specific antigens were recombined in vitro and subcloned into single chain antibody fragments of variable regions (scFv), which were then combined with transmembrane protein fragments of other genes and intracellular fragments. The signal peptide is fused to form an artificial chimeric antigen receptor (CAR-T), which is transfected into T cells to form chimeric antigen receptor T cells (CAR-T). CD19-CAR chimeric antigen receptor immunotherapy has changed the traditional approach to the treatment of hematological malignancies and achieved impressive results in clinical phase I studies.

At present, the vast majority of CD19CART clinical trials use murine CD19 antibody as the extracellular domain. Mouse-derived antibodies have high immunogenicity and are easily cleared by the body's immune system, affecting the survival time of CAR-T cells in the body.

SUMMARY OF THE INVENTION

Objective of the invention: In view of the above prior art, the present invention provides a humanized CD19 chimeric antigen receptor with good specificity, high lethality, and low immunogenicity and its application.

Technical solution: The chimeric antigen receptor targeting human CD19 according to the present invention comprises a humanized CD19 antibody binding domain, a transmembrane region and an intracellular signal domain based on the CD19 antigen; wherein, the CD19 The heavy chain sequence of the antibody binding domain is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2.

Further, the amino acid sequence of the CD19 antibody binding domain is shown in SEQ ID NO: 3, or an engineered amino acid sequence with 85%-99% identity thereto. It is preferably 90%-99%, even 95%-99% identical to the amino acid sequence shown in SEQ ID NO:3.

Further, the transmembrane region is selected from CD4, CD8α, CD28 or 4-1BB transmembrane region. Preferably, the transmembrane region structure comprises the CD8α transmembrane region amino acid sequence shown in SEQ ID NO:6.

The intracellular signaling domains include signaling domains and/or costimulatory signaling domains, selected from one or more of CD27, CD28, 4-1BB, OX40, CD30, CD40, and CD3. CD3zeta, 4-1BB and/or CD28 signaling domains are preferred.

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

The above-mentioned chimeric antigen receptor targeting human CD19 further comprises an extracellular signal peptide structure selected from CD8α signal peptide, GM-CSFRα signal peptide, CD4 signal peptide or IL-2 signal peptide. The CD8α signal peptide is preferred, and the amino acid sequence is shown in SEQ ID NO:4.

The above-mentioned chimeric antigen receptor targeting human CD19 also includes a hinge region structure for connecting the extracellular region peptide segment and the transmembrane region peptide segment, and the hinge region is CD8α and CD28 molecules. A CD8α molecule is preferred, the amino acid sequence of which is shown in SEQ ID NO:5.

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

The invention also discloses a nucleic acid molecule encoding the chimeric antigen receptor targeting human CD19.

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

The invention also discloses a recombinant expression vector comprising the nucleic acid molecule. The recombinant expression vector includes a chimeric antigen receptor expression cassette and a replication system, the chimeric antigen receptor expression cassette consists of a promoter that can be expressed in T cells and the chimeric antigen encoded downstream of the promoter The nucleic acid composition of the receptor. The promoter is cytomegalovirus promoter, SV40 promoter, EF1alpha promoter or RSV promoter.

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

Further, the chimeric antigen receptor targeting human CD19 expressed by the genetically engineered immune cells includes an extracellular signal peptide, an anti-CD19 humanized antibody binding domain, a hinge region, a transmembrane region and an intracellular signal. domain.

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

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

Beneficial effects: The present invention provides a chimeric antigen receptor targeting human CD19, and by expressing the chimeric antigen receptor in T cells, CAR T cells can effectively and specifically target the surface of CD19 expression Malignant cells of the antigen, thus providing a more efficient method with fewer side effects and adverse reactions for the treatment of some tumors expressing the CD19 surface antigen, such as B lymphocytic leukemia.

Description of drawings

Fig. 1 is the schematic diagram of chimeric antigen receptor in the present invention;

Figure 2 is a graph showing the results of the ability of CD19 CAR-T cells to lyse and kill target cells Nalm6;

Figure 3 is a graph showing the results of CD19 CAR-T cells secreting IFN-γ;

Figure 4 is a graph showing the results of CD19 CAR-T cells secreting IL-2.

Detailed ways

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Example 1 Construction of Chimeric Antigen Receptor (CAR) Lentiviral Expression Vector

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

The amino acid sequences of each element in the constructed chimeric antigen receptor are:

Signal peptide: SEQ NO.4

MALPVTALLLPLALLLHAARP

Humanized single chain antibody sequence: SEQ NO.3

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYGTSVGGSSAMDY

CD8 hinge region: SEQ NO.5

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 transmembrane region: SEQ NO.6

IYIWAPLAGTCGVLLLSLVITLYC

CD137 intracellular domain: SEQ NO.7

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

CD3Zeta: SEQ NO.8

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Example 2 Preparation of lentivirus

The specific experimental steps are as follows:

S1. Prepare a 15cm dish, inoculate 5*10 ⁶ 293T cells (purchased from ATCC), add complete medium (DMEM high glucose, 10% FBS, double antibody), place in a 37°C, 5% CO ₂ incubator overnight nourish.

S2. Take out 100 μM PEI and lentiviral packaging plasmids (PLVX-EF1a-CAR, pGP, pVSVG) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down with a pipette. Remove PBS or HBSS buffer and warm to room temperature. Take 2mL of PBS to a well of a 6-well plate, add 10μg Lenti-EF1a-CAR, 4μg pGP, 2μg pVSVG respectively, pipette up and down to mix well, add 18μL 100μM PEI, and immediately pipette up and down to mix well , let stand for 10 minutes at room temperature.

S3. Add the above DNA/PEI complex dropwise to a 15cm petri dish, shake the petri dish gently, and mix well. The culture dish was placed in a 37°C, 5% CO2 incubator, and after culturing for 6 to 8 hours, the medium containing the transfection reagent was removed and replaced with fresh complete medium.

After 48 hours of continuous culture, collect the virus-containing medium supernatant in the petri dish, filter it with a 0.45 μm filter, transfer it to a centrifuge tube, add 20% volume PEG6000, stand at 4 degrees for 2 and, after balancing, 3000×g 4 Centrifuge for 0.5 hours. After centrifugation, in a biological safety cabinet, carefully aspirate the liquid in the centrifuge tube, add 500 μL of PBS buffer to resuspend the pellet, and store the virus at -80°C.

Example 3 Isolation of primary T cells

The specific experimental steps are as follows:

S1. Invert the lymphocyte separation solution upside down several times, and thoroughly mix the Lymphoprep reagent.

S2. In a biological safety cabinet, add 15 mL of Lymphoprep reagent to a 50 mL centrifuge tube (or 15 mL centrifuge tube, depending on the volume of the separated blood sample), for use.

S3. Dilute the blood sample with an equal volume of PBS+2% FBS.

S4. Use a pipette to slowly add the diluted blood sample along the tube wall to the upper layer of the separation reagent to avoid mixing of the separation reagent and the blood sample.

S5. Set the centrifuge to 800×g, set the rotation speed to the slowest, and centrifuge at room temperature for 20 minutes.

S6. After centrifugation, the upper layer of pale yellow serum was collected into another sterile centrifuge tube and stored at -80°C.

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

S8. Adjust the cell density to 1*10 ⁸ cells/mL (the total volume does not exceed 2.5 mL), and resuspend in a 5 mL round bottom tube.

S9. Add 100 μl/mL antibody cocktail, mix well, and incubate at room temperature for 15 minutes.

S10. Take out the magnetic beads, pipette up and down at least 5 times with a pipette, and mix thoroughly.

S11. Pipette 50 μl of magnetic beads/mL into the above sample, mix well, and incubate at room temperature for 10 minutes.

S12. Add complete culture medium until the total volume in the tube is 2.5 mL, insert the tube (open the cap) into the magnetic pole, and let stand at room temperature for 5 minutes.

S13. After the incubation, the tube continues to remain in the magnetic pole, and is gently inverted to pour out the cells in the tube.

S14. The cells were resuspended in X-vivo 15 medium, and 10% FBS, 300 U/mL IL-2, 5 ng/mL IL-15 and 10 ng/mL IL-7 were added.

Example 4 Activation of primary T cells and lentiviral infection

The specific experimental steps are as follows:

S1. Adjust the cell density to 1*10 ⁶ cells/mL, add cytokines and antibody complexes (IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL with a final concentration of 300U/mL) Anti-CD3 (OKT3), 2ug/mL Anti-CD28), cultured continuously for 48 hours.

S2. According to MOI=20, calculate the required amount of virus. The calculation formula is as follows: required virus amount (mL)=(MOI*cell number)/virus titer

S3. After taking out the virus from the -80°C refrigerator, quickly thaw it in a 37°C water bath. The virus amount calculated above was added to the six-well plate, and polybrene with a final concentration of 6 μg/mL was added. After thorough mixing, the four sides of the six-well plate were sealed with a sealing film, and centrifuged at 800×g for 1 hour.

S4. After the centrifugation, tear off the sealing film, place the six-well plate in a 37°C 5% _CO2 incubator, and continue to culture for 24 hours.

S5, centrifuge at 250 x g for 10 minutes, remove the virus-containing medium supernatant, resuspend the cell pellet with fresh medium, transfer the cells to a new six-well plate, and continue to culture for 3-6 days for later use.

Example 5 Lysis of target cells by CAR-T cells

The specific experimental steps are as follows:

S1. Adjust the state of the target cells to the logarithmic growth phase, and need to be continuously passaged for 2 times before the experiment;

S2. Resuspend the target cells in complete medium, adjust the cell density to 5*10 ⁵ cells/mL, take a new 96-well plate, and inoculate the target cells in an amount of 100 μL/well. To the unused wells around the 96-well plate, add 100 μL of sterile water to each well to prevent evaporation of water in the experimental wells in the middle. The plate was placed in a 5% CO ₂ 37°C incubator overnight.

S3. The CAR-T cells prepared above were collected by centrifugation, and resuspended in serum-free 1640 medium; the 96-well plate was taken out from the incubator, the medium in the wells was completely aspirated, and the cells were gently washed with sterile PBS. , and then add CAR-T cells according to the above E/T ratio, and make up the final volume to 100 μL/well; Maxi lysis and Mini lysis are inoculated with the same number of target cells, but without CAR-T cells. The plate was placed in a 5% CO ₂ 37°C incubator for 6 hours.

S4. After the incubation, take the well plate out of the incubator, add the lysate from the LDH detection kit to the Maxi lysis well, and completely lyse the target cells, centrifuge the 96-well plate at 1200×g for 5 minutes at room temperature, gently gently Take out the plate, transfer 50 μL from each well to another new 96-well plate, add LDH detection reagent, and read the OD value using a microplate reader.

The formula for calculating the percentage of target cell lysis:

S5. Use GraphPad 6.0 to draw the data after the above processing.

Experimental results:

Using CAR-T cells as effector cells and Nalm6 cells, a lymphoma cell line that naturally expresses CD19, as target cells, a co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well For 50,000 cells, different numbers of CAR-T cells were added, and the co-culture system was cultured with serum-free medium. After 8 hours of continuous culture, the well plate was taken out and centrifuged at 1200×g at room temperature for 10 minutes to make all the suspended cells settle into the well plate. At the bottom, 30 microliters of supernatant was removed from each well, and the amount of LDH released in the supernatant of the medium was detected to reflect the ability of CAR-T cells to lyse target cells. The results are shown in Figure 2. When the ratio is 4:1, it can efficiently mediate the killing of T cells to tumor cells or recombinant cells; with the increase of the effect-to-target ratio, the killing effect of CD19 CAR-T cells on the target cell Nalm6 also increases, The highest effect was achieved when the effect-target ratio was 8:1.

Example 6 Detection of CAR-T cytokine secretion level

The specific experimental steps are as follows:

S1. Adjust the state of the target cells to the logarithmic growth phase, and need to be continuously passaged for 2 times before the experiment;

S2. Resuspend the target cells in complete medium, adjust the cell density to 5*10 ⁵ cells/mL, take a new 96-well plate, and inoculate the target cells in an amount of 100 μL/well. To the unused wells around the 96-well plate, add 100 μL of sterile water to each well to prevent evaporation of water in the experimental wells in the middle. The plate was placed in a 5% CO2 37°C incubator overnight.

S3. The CAR-T cells prepared above were collected by centrifugation, and resuspended in serum-free 1640 medium; the 96-well plate was taken out from the incubator, the medium in the wells was completely aspirated, and the cells were gently washed with sterile PBS. , and then add CAR-T cells according to the above E/T ratio, and make up the final volume to 100 μL/well; place the well plate in a 5% CO ₂ 37°C incubator for 6 hours. At the same time, a control T cell group was set up.

S4. After culturing, take the well plate out of the incubator, centrifuge the 96-well plate at 1200×g for 5 minutes at room temperature, gently remove the plate, transfer 50 μL of medium supernatant from each well, and use ELISA kit to detect IFN-γ and For the expression of IL-2, read the OD value using a microplate reader.

S5. Use GraphPad 6.0 to map the data obtained above.

Experimental results:

Using CAR-T cells as effector cells and Nalm6 cells, a lymphoma cell line that naturally expresses CD19, as target cells, a co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well For 50,000 cells, different numbers of CAR-T cells were added, and the co-culture system was cultured with serum-free medium. After 8 hours of continuous culture, the well plate was taken out and centrifuged at 1200×g at room temperature for 10 minutes to make all the suspended cells settle into the well plate. At the bottom, 30 microliters of supernatant was removed from each well, and the expression of IFN-γ and IL-2 secreted by CAR-T cells after being activated by tumor cells in the supernatant of the medium was detected by ELISA; the results are as follows As shown in Figure 3 and Figure 4, CD19-CART cells can effectively activate primary T cells after binding to targeted tumor cells, and cause an increase in cytokine secretion and expression; when the effect-target ratio is 2:1, After being activated by tumor cells, CAR-T cells can secrete a large amount of IFN-γ, which is significantly higher than that of control T cells; the secretion reaches the highest value when the effector-target ratio is 8:1.

sequence listing

<110> Nanjing Landun Biotechnology Co., Ltd.

<120> Chimeric antigen receptor targeting human CD19 and its application

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

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

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

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<213> Artificial Sequence

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr

165 170 175

Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr

180 185 190

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

195 200 205

Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Tyr Tyr

210 215 220

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

225 230 235 240

Thr Val Ser Ser

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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> 5

<211> 45

<212> PRT

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

1 5 10 15

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

20 25 30

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

35 40 45

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

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Ser Leu Val Ile Thr Leu Tyr Cys

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

20 25 30

Pro Glu Glu Glu Gly Gly Cys Glu Leu

35 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 9

<211> 486

<212> PRT

<213> Artificial Sequence

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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 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr 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 Gln

130 135 140

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

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Asp

165 170 175

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

180 185 190

Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

195 200 205

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

210 215 220

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

225 230 235 240

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

245 250 255

Gln Gly Thr Ser Val Thr Val Ser Ser 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

275 280 285

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 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ile Gly Met Lys Gly Glu 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> 10

<211> 462

<212> PRT

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<400> 10

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 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

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

35 40 45

Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro

50 55 60

Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr

65 70 75 80

Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr

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

115 120 125

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 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

275 280 285

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

<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 acctgagac 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 ccccgcccccc 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 (5)

1. A chimeric antigen receptor targeting human CD19, wherein the amino acid sequence is shown in SEQ ID NO: 10. 2 . A nucleic acid molecule encoding the chimeric antigen receptor targeting human CD19 according to claim 1 , wherein the nucleotide coding sequence of the nucleic acid molecule is shown in SEQ ID NO: 11. 3 . 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 application of the chimeric antigen receptor targeting human CD19 according to claim 1 in the preparation of antitumor drugs.

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