CN113527515A

CN113527515A - Chimeric antigen receptor targeting mesothelin and application thereof

Info

Publication number: CN113527515A
Application number: CN202110794199.XA
Authority: CN
Inventors: 李俊; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2021-07-14
Filing date: 2021-07-14
Publication date: 2021-10-22
Also published as: WO2023284053A1

Abstract

The invention discloses a chimeric antigen receptor targeting mesothelin and application thereof, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular structural domain which are sequentially connected; the extracellular region comprises an extracellular signal peptide, an antigen recognition region and a hinge region, wherein the antigen recognition region is fully human anti-mesothelin scFv; the intracellular domain includes an intracellular signaling domain and an intracellular costimulatory signaling domain. The chimeric antigen receptor T cell has strong killing activity on mesothelin positive tumor cells, uses a fully human antibody as scfv, has no immunogenicity, can be repeatedly administered, and enhances the treatment effect of CAR-T cells. The invention also relates to the application of the chimeric receptor targeting human Mesothelin and the immune effector cell thereof in the preparation of drugs for preventing or treating tumors.

Description

Chimeric antigen receptor targeting mesothelin and application thereof

Technical Field

The invention relates to a chimeric antigen receptor, in particular to a chimeric antigen receptor targeting mesothelin and application thereof.

Background

Chimeric Antigen Receptors (CARs) are artificial receptors that mimic the function of T cell receptors, fusing the recognition and binding specificity of antigen and antibody or ligand and receptor and the killing ability of effector T cells to recognized tumor cells. The CAR is formed by sequentially connecting a CD8a leader peptide, an antigen recognition region (ligand or single chain antibody or Fab fragment), a transmembrane region, and a series of signal transduction regions of T cells (CD28, CD3, CD137 intracellular signaling domains). After the T cells are modified, the surface-expressed CAR is firstly combined with the surface antigen of the tumor cells through an antigen recognition area, and then an activation signal is transmitted into the cells through a signal transduction area of the CAR, so that the killing activity of the T cells on the tumor cells is activated in a targeted mode. The DNA sequence expressing the CAR is cloned into a lentivirus expression vector, T cells separated from blood of a patient are infected, the corresponding CAR is expressed on the surface of the T cells, the modified T cells are infused back into the body of the patient, and the modified T cells can kill tumor cells expressing related antigens in a targeted manner in the body of the patient, so that the effect of removing the tumor cells is achieved.

mesothelin is a tumor-associated antigen that is expressed in most malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and some lung cancers, and has a narrow expression range and low expression levels in normal tissues. Therefore, the compound can be used as an immunotherapy target of malignant pleural mesothelioma, pancreatic cancer and ovarian cancer.

The application of the current CAR-T technology is also limited to blood system tumors such as leukemia, myeloma, lymphoma and the like, and the application to solid tumors such as pancreatic cancer is less.

Disclosure of Invention

The purpose of the invention is as follows: the object of the present invention is to provide a chimeric antigen receptor targeting mesothelin; another object of the present invention is to provide the use of a chimeric antigen receptor targeting mesothelin.

The technical scheme is as follows: the invention provides a chimeric antigen receptor targeting mesothelin, which comprises an extracellular region, a transmembrane region and an intracellular domain which are connected in sequence; the extracellular region comprises an extracellular signal peptide, an antigen recognition region and a hinge region, wherein the antigen recognition region is fully human anti-mesothelin scFv; the intracellular domain includes an intracellular signaling domain and an intracellular costimulatory signaling domain.

Preferably, the transmembrane region is selected from one of CD4, CD8a, CD28 and a 4-1BB transmembrane region; the intracellular signaling domain is selected from one of CD27, CD28, 4-1BB, OX40, CD3zeta domain; the intracellular costimulatory signal domain is selected from one of the CD27, CD28, 4-1BB, OX40, CD3zeta domain.

Preferably, the amino acid sequence of the heavy chain of the fully human anti-mesothelin scFv is shown as SEQ NO. 1.

SEQ NO.1：

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQTPEKGLEWIA YIYNSGTTKFNPSLKGRVTISMDASKNQLSMKLSSVTSADTAVYFCARDQGNS PYPDAFDVWGQGTLVTVSS

The light chain amino acid sequence of the fully human anti-mesothelin scFv is shown as SEQ NO. 2.

SEQ NO.2：

QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI YEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNYVFG TGTKVTVL

Preferably, the intracellular signaling domain CD3 ζ is mutated, and the amino acid sequence of the mutated CD3 ζ is as shown in SEQ NO. 3.

SEQ3:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRQNPQEGLYNELQKDKMAEAYSEIGMKGERRRGQGHDGLYQGLSTATKD TYDALHMQALPPR

Preferably, the extracellular signal peptide is a CD8a signal peptide, and the amino acid sequence of the CD8a signal peptide is shown as SEQ number 4.

SEQ4：

MALPVTALLLPLALLLHAARP

Preferably, the hinge region is a CD8a hinge region, and the amino acid sequence of the CD8a hinge region is shown in SEQ NO. 5.

SEQ5：

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

Preferably, the transmembrane region is the CD8a transmembrane region, and the amino acid sequence of the CD8a transmembrane region is shown in SEQ NO. 6.

SEQ6：

IYIWAPLAGTCGVLLLSLVITLYC

Preferably, the intracellular co-stimulatory signaling domain comprises a 4-1BB co-stimulatory signaling region, and the amino acid sequence of the 4-1BB co-stimulatory signaling region is shown in SEQ NO. 7.

SEQ7：

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

In a second aspect, the present invention provides a polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence encoding the chimeric antigen receptor described above; and (2) a complementary sequence to the polynucleotide sequence of the chimeric antigen receptor described above.

Preferably, the polynucleotide sequence is shown in SEQ NO.8 or SEQ NO. 9.

SEQ8：

MALPVTALLLPLALLLHAARPQLQLQESGPGLVKPSETLSLTCTVSGGSIS SSSYYWGWIRQTPEKGLEWIAYIYNSGTTKFNPSLKGRVTISMDASKNQLSM KLSSVTSADTAVYFCARDQGNSPYPDAFDVWGQGTLVTVSSGGGGSGGGGS GGGGSQSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPK LMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNYV FGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRQNPQEGLYNELQKDKMAEAYSEIGMKGERRRGQGHDGLY QGLSTATKDTYDALHMQALPPR

SEQ9:

ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCA 50 CGCCGCCCGCCCCCAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGA 100 AGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATC 150 AGCAGCAGCAGCTACTACTGGGGCTGGATCCGCCAGACCCCCGAGAAGGG 200 CCTGGAGTGGATCGCCTACATCTACAACAGCGGCACCACCAAGTTCAACC 250 CCAGCCTGAAGGGCCGCGTGACCATCAGCATGGACGCCAGCAAGAACCAG 300 CTGAGCATGAAGCTGAGCAGCGTGACCAGCGCCGACACCGCCGTGTACTT 350 CTGCGCCCGCGACCAGGGCAACAGCCCCTACCCCGACGCCTTCGACGTGT 400 GGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGC 450 GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGAGCGCCCTGACCCAGCCCCC 500 CAGCGCCAGCGGCAGCCCCGGCCAGAGCGTGACCATCAGCTGCACCGGCA 550 CCAGCAGCGACGTGGGCGGCTACAACTACGTGAGCTGGTACCAGCAGCAC 600 CCCGGCAAGGCCCCCAAGCTGATGATCTACGAGGTGAGCAAGCGCCCCAG 650 CGGCGTGCCCGACCGCTTCAGCGGCAGCAAGAGCGGCAACACCGCCAGCC 700 TGACCGTGAGCGGCCTGCAGGCCGAGGACGAGGCCGACTACTACTGCAGC 750 AGCTACGCCGGCAGCAACAACTACGTGTTCGGCACCGGCACCAAGGTGAC 800 CGTGCTGACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCA 850 TCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCC 900 GGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACAT 950 CTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGA 1000 TCACCCTGTACTGCAAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAG 1050 CAGCCCTTCATGCGCCCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAG 1100 CTGCCGCTTCCCCGAGGAGGAGGGCGGCTGCGAGCTGCGCGTGAAGTTCA 1150 GCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTAC 1200 AACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCG 1250 CCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCCAGAACCCCC 1300 AGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTAC 1350 AGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCCAGGGCCACGACGG 1400 CCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGC 1450 ACATGCAGGCCCTGCCCCCCCGC

In a third aspect, the invention provides a vector comprising a polynucleotide sequence as described above.

In a fourth aspect, the invention provides a host cell comprising a polynucleotide sequence as described above, or a vector as described above.

The fifth aspect of the present invention provides the use of the chimeric antigen receptor, the polynucleotide sequence, the vector, and the host cell for preparing a drug for preventing or treating tumor.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the chimeric antigen receptor T cell has strong killing activity on mesothelin positive tumor cells, uses a fully human antibody as scfv, has no immunogenicity, can be repeatedly administered, and enhances the treatment effect of CAR-T cells.

Drawings

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

FIG. 2 is a graph showing the comparison of the lytic killing ability of mesothelin-CAR-T cells against BXPC-3 as a target cell;

FIG. 3 is a graph showing the comparison of the lytic killing ability of mesothelin-CAR-T cells against SKOV-3 as a target cell;

FIG. 4 is a graph comparing the IFN-. gamma.and IL-2 secretion ability of mesothelin-CAR-T cells;

FIG. 5 is a graph showing the in vivo efficacy of mesothelin-CAR-T in an animal model of pancreatic cancer;

FIG. 6 is a schematic diagram of the tumor inoculation location of mice.

Detailed Description

The technical solution of the present invention is further explained below.

Example 1 Chimeric Antigen Receptor (CAR) Lentiviral expression vector construction

The intracellular domain of CD137 and the ITAM region of CD3Zeta are used as activation signals to be fused with a single-chain antibody of mesothelin to construct a chimeric antigen receptor expression vector, and the chimeric antigen receptor expression vector is subcloned into a PLVX-EF1a 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

Extracellular antigen binding region heavy chain sequence: SEQ NO.1

Extracellular antigen-binding region light chain sequence: SEQ NO.2

CD8 hinge region: SEQ NO.5

CD8 transmembrane region: SEQ NO.6

CD137 endodomain: SEQ NO.7

CD3ζ(Zeta)：SEQ NO.3

Example 2 Lentiviral preparation

The specific experimental steps are as follows:

s1, preparing 15cm dish, inoculating 5X 10⁶The cells were cultured in complete medium (DMEM high-glucose, 10% FBS, double antibody) at 37 deg.C and 5% CO₂And (5) an incubator for overnight culture.

S2, 100 mu M PEI and lentivirus packaging plasmids (Lenti-EF1a-CAR, pGP, pVSVG) were taken out of the refrigerator, thawed at room temperature, and then blown up and down by a pipette gun to be mixed completely. Remove PBS or HBSS buffer and warm to room temperature. And (3) adding 10 mu g of PLVX-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 from the petri dish was collected, filtered through a 0.45 μm filter, added to a 20% volume 50% PEG6000 solution, incubated at 4 ℃ for 2 hours, transferred to a centrifuge tube, trimmed, and 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 3 isolation of 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 1x10⁸Cells/mL (total volume not exceeding 2.5mL) was 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/mL IL-15, and 10ng/mL IL-7.

Example 4 activation of Primary T cells and Lentiviral infection

The specific experimental steps are as follows:

s1, adjusting the cell density to 1x10⁶cells/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 5 lysis of target cells by CAR-T 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, digesting and suspending the attached target cells in complete culture medium by pancreatin, and adjusting the cell density to 5 x10⁵One 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; 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; 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 pancreatic cancer cell strains and ovarian cancer cell strains which naturally express mesothelin as target cells, establishing a co-culture system according to different effective target ratios, namely setting 50000 target cells fixed in each hole in a 96-well plate, adding different amounts 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 microliter of supernatant from each hole, and detecting the release amount of LDH in the supernatant of the culture medium so as to reflect the cracking capacity of the CAR-T cells on the target cells, wherein the results are shown in figures 2 to 4, and the chimeric antigen receptor T cells which target mesothelin are expressed in an effective target ratio of 1:1, 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 the mesothelin-CAR-T cells on the target cells BXPC3 and SKOV3 is increased, and the highest effect is achieved when the effective target ratio is 8: 1.

Example 6 CAR-T cytokine secretion level assay

The specific experimental steps are as follows:

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 at room temperature in a 96-well plate, gently removed, 50. mu.L of the culture supernatant was transferred from each well, IFN-. gamma.and IL-2 expression was detected using ELISAkit, 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 a pancreatic cancer cell strain bxpc3 naturally expressing mesothelin as target cells, establishing a co-culture system according to different effect-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 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 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 expression quantities of IFN-gamma and IL-2 secreted by the CAR-T cells after being activated by tumor cells in the supernatant by using an ELISA method; the results are shown in fig. 5, after the chimeric antigen receptor T cell targeting mesothelin is combined with the targeted 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 1:1, after the CAR-T cells are activated by tumor cells, a large amount of IL-2 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.

Example 7 CAR-T in vivo efficacy experiments

S1, collecting BxPC3 cells in the logarithmic growth phase, removing the culture solution, washing with PBS twice, and then inoculating (the survival rate of the cells before and after tumor bearing is 97.3%, 94.6%). Tumor cells were inoculated subcutaneously into NCG mice, 1X10 per mouse⁷Each cell per 100 ul. The inoculation site is shown as position 1 in FIG. 6.

S2, divided administration, mouse average tumor volume 100.91mm³Randomized groupings were made according to tumor volume. Each group consisted of 6, 2 and 12 mice. The day of grouping was defined as D0 days, and treatment was administered on the day of grouping.

S3, experimental observations and data collection, effects of tumors on normal behavior of animals were routinely monitored weekly after cell inoculation. The specific contents include the activity of experimental animals, the condition of food intake and water drinking, the condition of weight increase or reduction, eyes, fur and other abnormal conditions.

After the start of the administration, the tumor size was observed and the body weight of the mice was weighed. Tumor volume was calculated as: tumor volume (mm)³) 0.5 × (tumor major diameter × tumor minor diameter)²)。

The experimental results are as follows: the MSLN-CART cell has obvious drug effect in a mouse tumor model.

Sequence listing

<110> Nanjing Landun Biotech Co., Ltd

<120> a chimeric antigen receptor targeting mesothelin and uses thereof

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

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

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Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln Ser

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Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn

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Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

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Ile Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

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

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Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Gly Ser Asn

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Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Thr Thr Thr

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

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

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

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

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

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

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Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 9

<211> 1473

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgc 60

ccccagctgc agctgcagga gagcggcccc ggcctggtga agcccagcga gaccctgagc 120

ctgacctgca ccgtgagcgg cggcagcatc agcagcagca gctactactg gggctggatc 180

cgccagaccc ccgagaaggg cctggagtgg atcgcctaca tctacaacag cggcaccacc 240

aagttcaacc ccagcctgaa gggccgcgtg accatcagca tggacgccag caagaaccag 300

ctgagcatga agctgagcag cgtgaccagc gccgacaccg ccgtgtactt ctgcgcccgc 360

gaccagggca acagccccta ccccgacgcc ttcgacgtgt ggggccaggg caccctggtg 420

accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagccag 480

agcgccctga cccagccccc cagcgccagc ggcagccccg gccagagcgt gaccatcagc 540

tgcaccggca ccagcagcga cgtgggcggc tacaactacg tgagctggta ccagcagcac 600

cccggcaagg cccccaagct gatgatctac gaggtgagca agcgccccag cggcgtgccc 660

gaccgcttca gcggcagcaa gagcggcaac accgccagcc tgaccgtgag cggcctgcag 720

gccgaggacg aggccgacta ctactgcagc agctacgccg gcagcaacaa ctacgtgttc 780

ggcaccggca ccaaggtgac cgtgctgacc accacccccg ccccccgccc ccccaccccc 840

gcccccacca tcgccagcca gcccctgagc ctgcgccccg aggcctgccg ccccgccgcc 900

ggcggcgccg tgcacacccg cggcctggac ttcgcctgcg acatctacat ctgggccccc 960

ctggccggca cctgcggcgt gctgctgctg agcctggtga tcaccctgta ctgcaagcgc 1020

ggccgcaaga agctgctgta catcttcaag cagcccttca tgcgccccgt gcagaccacc 1080

caggaggagg acggctgcag ctgccgcttc cccgaggagg agggcggctg cgagctgcgc 1140

gtgaagttca gccgcagcgc cgacgccccc gcctaccagc agggccagaa ccagctgtac 1200

aacgagctga acctgggccg ccgcgaggag tacgacgtgc tggacaagcg ccgcggccgc 1260

gaccccgaga tgggcggcaa gccccgccgc cagaaccccc aggagggcct gtacaacgag 1320

ctgcagaagg acaagatggc cgaggcctac agcgagatcg gcatgaaggg cgagcgccgc 1380

cgcggccagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa ggacacctac 1440

gacgccctgc acatgcaggc cctgcccccc cgc 1473

Claims

1. A chimeric antigen receptor targeting mesothelin, comprising an extracellular region, a transmembrane region and an intracellular domain connected in sequence; the extracellular region comprises an extracellular signal peptide, an antigen recognition region and a hinge region, wherein the antigen recognition region is fully human anti-mesothelin scFv; the intracellular domain includes an intracellular signaling domain and an intracellular costimulatory signaling domain.

2. The chimeric antigen receptor according to claim 1, wherein the transmembrane region is selected from one of the CD4, CD8a, CD28, and 4-1BB transmembrane region; the intracellular signaling domain is selected from one of CD27, CD28, 4-1BB, OX40, CD3zeta domain; the intracellular costimulatory signal domain is selected from one of the CD27, CD28, 4-1BB, OX40, CD3zeta domain.

3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the heavy chain of the fully human anti-mesothelin scFv is shown as SEQ NO.1, and the amino acid sequence of the light chain of the fully human anti-mesothelin scFv is shown as SEQ NO. 2.

4. The chimeric antigen receptor according to claim 2, wherein the intracellular signaling domain CD3 ζ is mutated, and the amino acid sequence of the mutated CD3 ζ is represented as SEQ No. 3; the transmembrane region is a CD8a transmembrane region, and the amino acid sequence of the CD8a transmembrane region is shown as SEQ NO. 6; the intracellular costimulatory signal domain comprises a 4-1BB costimulatory signal region, and the amino acid sequence of the 4-1BB costimulatory signal region is shown as SEQ NO. 7.

5. The chimeric antigen receptor according to claim 1, wherein the extracellular signal peptide is a CD8a signal peptide, and the amino acid sequence of the CD8a signal peptide is shown as SEQ NO. 4; the hinge region is a CD8a hinge region, and the amino acid sequence of the CD8a hinge region is shown as SEQ NO. 5.

6. A polynucleotide sequence selected from the group consisting of: (1) a polynucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-5; and (2) the complement of the polynucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-5.

7. The polynucleotide sequence of claim 6, wherein the polynucleotide sequence is set forth in SEQ No.8 or SEQ No. 9.

8. A vector comprising the polynucleotide sequence of claims 6-7.

9. A host cell comprising a polynucleotide sequence according to claims 6 to 7 or a vector according to claim 8.

10. Use of the chimeric antigen receptor according to any one of claims 1 to 5, the polynucleotide sequence according to any one of claims 6 to 7, the vector according to claim 8, the host cell according to claim 9 for the preparation of a medicament for the prophylaxis or treatment of a tumor.