CN112608902B

CN112608902B - Fourth-generation CAR-T cell and application thereof

Info

Publication number: CN112608902B
Application number: CN202011521237.6A
Authority: CN
Inventors: 汤朝阳; 请求不公布姓名; 秦乐; 吴迪; 冯世忠; 冯嘉昆; 杨乐旋
Original assignee: Guangdong Zhaotai Cell Biotechnology Co ltd
Current assignee: Guangdong Zhaotai Cell Biotechnology Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2024-07-02
Anticipated expiration: 2040-12-21
Also published as: CN112608902A

Abstract

The invention provides a fourth generation CAR-T cell and application thereof, wherein the fourth generation CAR-T cell expresses chimeric antigen receptor, composite IL-6 and CCL19 which specifically bind to antigen; the complex IL-6 and CCL19 are NFAT regulatory over-expressed. According to the invention, the NFAT regulation promoter is adopted to regulate the expression of T cells on the composite IL-6 and CCL19, and the expression and secretion HIL-6 and CCL19 are carried out after the tumor antigen is identified, so that the proliferation capacity and anti-tumor effect of the CAR-T cells are remarkably improved, systemic or local inflammatory response is avoided, and the side effect of the CAR-T is reduced.

Description

Fourth-generation CAR-T cell and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, relates to a fourth-generation CAR-T cell and application thereof, and particularly relates to a fourth-generation CAR-T cell capable of conditionally over-expressing composite IL-6 and CCL19 and application thereof.

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T cell therapy (CHIMERIC ANTIGEN receptor T-cell immunotherapy, CAR-T) is one of the most promising tumor immunotherapies. Currently, CAR-T cell therapies have been widely used to treat B cell malignancies, and CD 19-targeted CAR-T cells are the precursor of CAR-T therapies to B cell malignancies, providing an effective approach to treating B cell malignancies.

There are still a number of problems with CAR-T cell therapies. In terms of leukemia treatment, CAR-T cell therapy has a problem of high recurrence rate, and promotion of CAR-T cell maintenance and formation of memory T cells may be a technical break to solve the problem; in addition, the clinical treatment research result of the anti-CD 19 CAR-T cells shows that a certain positive correlation exists between the response rate of patients to the treatment of the CAR-T cells and the occurrence of Cytokine Release Syndrome (CRS), and the CRS can possibly cause side effects such as brain disease syndrome (CRES) and the like related to the CAR-T cells, so that the clinical application of the CAR-T cells is severely limited; in the aspect of solid tumor treatment, the CAR-T cell has poor curative effect, and related clinical research breakthrough is not reported at present.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a fourth-generation CAR-T cell and application thereof, and the conditional overexpression of the compound IL-6 and CCL19 not only improves the anti-tumor effect of the CAR-T, but also reduces the side effect of the CAR-T in the treatment process.

To achieve the purpose, the invention adopts the following technical scheme:

In a first aspect, the invention provides a fourth generation CAR-T cell that expresses a chimeric antigen receptor that specifically binds an antigen, composite IL-6, and CCL19;

the complex IL-6 and CCL19 are NFAT regulatory over-expressed.

In the invention, the NFAT regulation promoter is adopted to regulate the expression of T cells on the compound IL-6 and CCL19, when the CAR-T cells are in a resting state, the compound IL-6 and the CCL19 are not expressed, after the CAR-T cells recognize tumor antigens and are activated, the NFAT regulation promoter 5X NFAT-RE-minP starts the transcription and translation of the compound IL-6 and the CCL19, the compound IL-6 and the CCL19 express, the compound IL-6 stimulates the proliferation, the memory differentiation and the enhanced killing effect of the CAR-T cells, the CCL19 recruits peripheral blood immune cells to the inside of tumors to the maximum extent, and the two are mutually matched to realize the targeted apoptosis promoting effect on the tumors.

Preferably, the compound IL-6 is a compound of IL-6 and IL-6Rα, the compound IL-6 (HIL-6) comprises an amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

PPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPARGGGSGGGGSVEPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM.

Preferably, CCL19 includes the amino acid sequence shown in SEQ ID NO. 2;

SEQ ID NO:2：

MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLL IKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS.

Preferably, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and a signaling domain.

Preferably, the antigen binding domain binds to a tumor surface antigen comprising any of CD19, CD20, CD22, CD30, CEA, EGFR, BRAF, HER-2, mesothelin, MUC1, PSCA, GPC3, TERT, PTEN, PD-1, PD-L1 or VEGF, preferably any of CD19, MUC1, GPC3, mesothelin, PSCA or HER2, further preferably Mesothelin.

Preferably, the antigen binding domain is a single chain antibody that targets Mesothelin.

Preferably, the transmembrane domain comprises a CD28 transmembrane domain and/or a CD8 a transmembrane domain, preferably a CD28 transmembrane domain.

Preferably, the signaling domain comprises any one or a combination of at least two of CD28 intracellular domain, cd3ζ, TLR2, 4-1BB, TLR1, CD27, OX40 or DAP10, preferably a combination of CD28 intracellular domain, cd3ζ and TLR 2.

Preferably, the chimeric antigen receptor further comprises a signal peptide.

Preferably, the chimeric antigen receptor consists of a GM-CSF signal peptide, a Mesothelin antigen binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, cd3ζ and TLR2 in tandem.

Preferably, the chimeric antigen receptor comprises the amino acid sequence shown as SEQ ID NO. 3;

SEQ ID NO:3：

MLLLVTSLLLCELPHPAFLLSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSGILEQQGIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS.

In a second aspect, the invention provides an expression vector comprising a chimeric antigen receptor-encoding gene, an NFAT regulatory promoter, a composite IL-6 encoding gene, and a CCL19 encoding gene.

Preferably, the chimeric antigen receptor encoding gene comprises the nucleic acid sequence shown in SEQ ID NO. 4; SEQ ID NO. 4:

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgagccaggtacagctgcagcagtcaggtccaggactcgtgacgccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataacgactatgcagtatctgtgaaaagtcgaatgagcatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactcccgaggacacggctgtgtattactgtgcaagaggaatgatgacttactattacggtatggacgtctggggccaagggaccacggtcaccgtctcctcaggaattctaggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttctcagcctgtgctgactcagtcgtcttccctctctgcatctcctggagcatcagccagtctcacctgcaccttgcgcagtggcatcaatgttggtccctacaggatatactggtaccagcagaagccagggagtcctccccagtatctcctgaactacaaatcagactcagataagcagcagggctctggagtccccagccgcttctctggatccaaagatgcttcggccaatgcaggggttttactcatctctgggctccggtctgaggatgaggctgactattactgtatgatttggcacagcagcgctgctgtgttcggaggaggcacccaactgaccgtcctctccggaattctagaacaacagggtattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgccaggccaaaaggaagcccaggaaagctcccagcaggaacatctgctatgatgcatttgtttcttacagtgagcgggatgcctactgggtggagaaccttatggtccaggagctggagaacttcaatccccccttcaagttgtgtcttcataagcgggacttcattcctggcaagtggatcattgacaatatcattgactccattgaaaagagccacaaaactgtctttgtgctttctgaaaactttgtgaagagtgagtggtgcaagtatgaactggacttctcccatttccgtctttttgatgagaacaatgatgctgccattctcattcttctggagcccattgagaaaaaagccattccccagcgcttctgcaagctgcggaagataatgaacaccaagacctacctggagtggcccatggacgaggctcagcgggaaggattttgggtaaatctgagagctgcgataaagtcc.

preferably, the NFAT regulatory promoter 5 XNFAT-RE-minP comprises the nucleic acid sequence shown as SEQ ID NO. 5;

SEQ ID NO:5：

ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtagatctagactctagagggtatataatggaagctcgaattccagcttggcattccggtactgttggtaaaaagcttggcaatccggtactgttggtaaagccacc.

Preferably, the complex IL-6 (HIL-6) encoding gene comprises the nucleic acid sequence shown in SEQ ID NO. 6;

SEQ ID NO:6：

ccccccgaggagccccagctctcctgcttccggaagagccccctcagcaatgttgtttgtgagtggggtcctcggagcaccccatccctgacgacaaaggctgtgctcttggtgaggaagtttcagaacagtccggccgaagacttccaggagccgtgccagtattcccaggagtcccagaagttctcctgccagttagcagtcccggagggagacagctctttctacatagtgtccatgtgcgtcgccagtagtgtcgggagcaagttcagcaaaactcaaacctttcagggttgtggaatcttgcagcctgatccgcctgccaacatcacagtcactgccgtggccagaaacccccgctggctcagtgtcacctggcaagacccccactcctggaactcatctttctacagactacggtttgagctcagatatcgggctgaacggtcaaagacattcacaacatggatggtcaaggacctccagcatcactgtgtcatccacgacgcctggagcggcctgaggcacgtggtgcagcttcgtgcccaggaggagttcgggcaaggcgagtggagcgagtggagcccggaggccatgggcacgccttggacagaatccaggagtcctccagctcgcggtggtggatcaggaggtggagggtcagtcgagccagtacccccaggagaagattccaaagatgtagccgccccacacagacagccactcacctcttcagaacgaattgacaaacaaattcggtacatcctcgacggcatctcagccctgagaaaggagacatgtaacaagagtaacatgtgtgaaagcagcaaagaggcactggcagaaaacaacctgaaccttccaaagatggctgaaaaagatggatgcttccaatctggattcaatgaggagacttgcctggtgaaaatcatcactggtcttttggagtttgaggtatacctagagtacctccagaacagatttgagagtagtgaggaacaagccagagctgtgcagatgagtacaaaagtcctgatccagttcctgcagaaaaaggcaaagaatctagatgcaataaccacccctgacccaaccacaaatgccagcctgctgacgaagctgcaggcacagaaccagtggctgcaggacatgacaactcatctcattctgcgcagctttaaggagttcctgcagtccagcctgagggctcttcggcaaatg.

Preferably, the CCL19 encoding gene comprises the nucleic acid sequence shown in SEQ ID NO. 7;

SEQ ID NO:7：

atggccctgctactggccctcagcctgctggttctctggacttccccagccccaactctgagtggcaccaatgatgctgaagactgctgcctgtctgtgacccagaaacccatccctgggtacatcgtgaggaacttccactaccttctcatcaaggatggctgcagggtgcctgctgtagtgttcaccacactgaggggccgccagctctgtgcacccccagaccagccctgggtagaacgcatcatccagagactgcagaggacctcagccaagatgaagcgccgcagcagt.

preferably, the expression vector comprises a viral vector.

Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector.

In a third aspect, the invention provides a recombinant lentivirus prepared by co-transfecting mammalian cells with the expression vector of the second aspect and a packaging helper plasmid.

In a fourth aspect, the invention provides a method of preparing a fourth generation CAR-T cell according to the first aspect, the method comprising the step of introducing into a T cell an expression vector according to the second aspect or a recombinant lentivirus according to the third aspect.

In a fifth aspect, the invention provides a pharmaceutical composition comprising a fourth generation CAR-T cell of the first aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a sixth aspect, the invention provides the use of a fourth generation CAR-T cell according to the first aspect, an expression vector according to the second aspect, a recombinant lentivirus according to the third aspect or a pharmaceutical composition according to the fifth aspect in the manufacture of a medicament for the treatment of a tumor.

Preferably, the tumor comprises any one or a combination of at least two of liver cancer, lung cancer, breast cancer, wilms' cell tumor, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor, retinoblastoma, pancreatic cancer, uterine fibroid, cervical cancer or thyroid cancer.

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

(1) According to the invention, CAR-T cells capable of conditionally expressing the composite IL-6 and CCL19 are constructed, the CAR-T expresses and secretes the composite IL-6 and the CCL19 after recognizing tumor antigens, the composite IL-6 enhances the amplification and anti-tumor effects of the CAR-T, the CCL19 recruits peripheral blood immune cells to the inside of tumors to the greatest extent, and the two are matched with each other to realize the targeted apoptosis promoting effect on the tumors;

(2) The CAR-T cell which conditionally expresses IL-6 and CCL19 and is constructed by the invention has stronger tumor inhibition effect, can obviously inhibit the proliferation and volume of tumors, does not cause or aggravate systemic or local inflammatory reaction, and reduces the side effect of the CAR-T.

Drawings

FIG. 1 is a graph showing the results of secretion of HIL-6 and CCL19 by different CAR-T cells;

FIG. 2 shows proliferation of different CAR-T cells;

FIG. 3 is the recruitment ability of different CAR-T cells to T cells;

figure 4 is the killing efficiency of different CAR-T cells.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

EXAMPLE 1 construction of recombinant lentiviral vectors

The following nucleic acid molecules were synthesized by total gene:

① A CAR molecule formed by a tandem of nucleic acid sequences of GM-CSF signal peptide, mesothelin scFv, CD28 transmembrane domain, CD28 intracellular domain, cd3ζ, TLR2 and 2A peptide;

② A conditional molecule formed by tandem of the 5 XNFAT-RE-minP promoter (SEQ ID NO: 5), HIL-6 (SEQ ID NO: 6) and CCL19 (SEQ ID NO: 7);

① is cloned into a lentiviral expression vector pwpxld-eGFP to obtain CAR-pwpxld-eGFP;

① was cloned into lentiviral expression vector pwpxld-tCD19, followed by reverse cloning ② to the end of pwpxld-tCD19, resulting in CAR-HIL-6/CCL19-pwpxld-tCD19.

EXAMPLE 2 lentiviral packaging

The 293T cells are adopted to prepare the recombinant lentivirus, and when the 293T cells are paved at the bottom of a 100mm culture dish plate to 80-90%, the lentivirus is packed:

2 hours before virus packaging, replacing a culture medium with DMEM containing 1% fetal bovine serum, and adding the culture medium into a culture dish with the volume of 6mL/100 mm;

preparing plasmid mixed solution shown in table 1;

TABLE 1

Reagent(s)	Dosage of
		Recombinant lentiviral vectors	4.5μg
pMD2.G	1.5μg
		psPAX2	6μg

Adding 36 μg PEI into another 500 μl opti-MEM culture medium, mixing, and standing at room temperature for 5min;

mixing the plasmid mixed solution shown in the table 1 with PEI, blowing and uniformly mixing, and standing at room temperature for 25-30 min;

Dropwise adding the mixed solution onto 293T cells cultured in a 100mm culture dish;

After 6h of culture, the culture medium is replaced by DMEM containing 1% fetal calf serum, and the addition amount is 7mL/100mm culture dish;

collecting virus supernatant 24h, 48h and 72h after packaging, and simultaneously supplementing culture medium to 293T cells with the addition amount of 7mL/100mm culture dish;

1000g is centrifuged for 10min, and filtered by a 0.45 mu m filter to obtain the recombinant lentivirus, which is stored at 4 ℃ for standby.

EXAMPLE 3T cell activation and lentiviral transfection

Separating mononuclear cells in blood by Ficoll density gradient method, removing erythrocytes by lysing solution of erythrocytes, separating T cells by MACS Pan-T magnetic beads, and diluting with culture medium AIM-V and 5% FBS, 100U/mL penicillin and 0.1mg/mL streptomycin to a concentration of 2.5X10 ⁶/mL; stimulating T cells by using magnetic beads (meitian gentle) coated with CD2, CD3 and CD28 antibodies, wherein the ratio of the magnetic beads to the T cells is 1:2, the T cell density is 5X 10 ⁶/mL/cm ², and the T cells are cultured and stimulated for 48 hours in a 5% CO ₂ incubator at 37 ℃;

Removing magnetic beads in T cells, centrifuging activated T cells at 300g for 5min, removing supernatant, re-suspending with fresh culture medium, respectively adding different recombinant lentivirus, polybrene with the concentration of 8 mug/mL and IL-2 with the concentration of 300IU/mL, culturing in a culture box with the concentration of 5% CO ₂ at the temperature of 37 ℃ for 24h, centrifuging 300g for 5min, removing supernatant, re-suspending cells with fresh culture medium containing IL-2 with the concentration of 300IU/mL, and culturing in a culture box with the concentration of 5% CO ₂ at the temperature of 37 ℃;

Maintaining the density of the CAR-T cells at about 1X 10 ⁶/mL, and carrying out half-volume liquid exchange every 2-3 days; subsequent experiments were performed after 10 days of culture.

The CAR-T cells prepared in this example were Mesothelin CAR-T and HIL-6/CCL19-CAR-T.

EXAMPLE 4 CAR secretion of HIL-6 and CCL19 by T cells

Taking 2×10 ⁶ of two CAR-T cells, culturing with fresh T cell culture medium for 24 hr, and collecting cell supernatant; simultaneously taking 2X 10 ⁶ CAR-HIL-6/CCL19-T, co-culturing with equivalent amount of Mesothelin positive cells K562-Mesothelin-GL for 24 hours, and collecting cell supernatant; each cell was tested for secretion of HIL-6 and CCL19 using ELISA kit (R & D system).

As shown in FIG. 1, the culture supernatant of Mesothelin CAR-T cells did not contain HIL-6 and CCL19, the culture supernatant of HIL-6/CCL19-CAR-T cells did not contain HIL-6 and IL-7, and after co-culture with K562-Mesothelin-GL, CAR-HIL-6/CCL19-T secreted HIL-6 and CCL19, indicating that HIL-6/CCL19-CAR-T cells expressed HIL-6 and CCL19 was under tumor antigen regulation.

Example 5 in vitro proliferation assay of CAR-T cells

Taking 2×10 ⁶ of two CAR-T cells, and culturing with fresh T cell culture medium; two CAR-T cells were simultaneously harvested at 2X 10 ⁶, co-cultured with an equal amount of Mesothelin positive cells K562-Mesothelin-GL, counted daily, and cultured for 7 consecutive days to analyze proliferation of the CAR-T cells.

Results FIG. 2 shows that Mesothelin CAR-T has no proliferation promoting effect on CAR-T cells due to its non-secretion of HIL-6; HIL-6/CCL19-CAR-T does not secrete HIL-6 in the absence of tumor antigen, and does not have proliferation promoting effect on CAR-T cells, but rather has less proliferation capacity than Mesothelin CAR-T due to the influence of HIL-6/CCL 19; HIL-6/CCL19-CAR-T is co-cultured with K562-Mesothelin-GL, and secretion of HIL-6 promotes proliferation of CAR-T cells.

Example 6 CAR in vitro migration experiments of T cells

Two CAR-T cells were each 2X 10 ⁶ and cultured in the lower layer of the Transwell apparatus for 24 hours, 2X 10 ⁶ CAR-T cells and equivalent K562-Mesothelin-GL were simultaneously co-cultured in the lower layer for 24 hours, CFSE-labeled wild T cells were cultured in the upper layer for 5 hours, and the number of CFSE-labeled T cells in the lower layer was detected by flow cytometry.

The statistical results are shown in fig. 3, more T cells can be recruited after the CAR-HIL-6/CCL19-T is co-cultured with tumor cells, which indicates that the CAR-HIL-6/CCL19-T conditionally expresses CCL19, and only after the tumor cells are identified, the CCL19 can be secreted, thereby exerting the recruitment effect and avoiding inflammatory side reactions.

Example 7 in vitro detection of killing function of CAR-T cells on tumor cells

Taking 2X 10 ⁶ of WT, mesothelin CAR-T and HIL-6/CCL19-CAR-T cells, mixing with tumor cells K562-Mesothelin-GL according to the ratio of E: T of 4:1, 2:1, 1:1, 1:2, 1:4, 1:8 and 1:16, adding into a 96-well plate, arranging 3 compound wells in each group, centrifuging for 5min at 250g, and culturing in a 5% CO ₂ incubator at 37 ℃ for 18h;

After 18h, 100 μl/well of Luciferase substrate (1×) was added to the 96-well plate, the cells were resuspended and RLU (RELATIVE LIGHT units) was immediately measured by a multifunctional microplate reader for 1 second, and the killing efficiency evaluation method was quantitatively performed using Luciferase (Luciferase), and the killing effect of WT, mesothelin CAR-T and CAR-HIL-6/CCL19-T on K562-Mesothelin-GL was compared in vitro, and the killing ratio was calculated as follows:

100% × (control well reading-experimental well reading)/control well reading (blank reading without cells can be ignored)

As shown in FIG. 4, the in vitro killing efficiency of the CAR-HIL-6/CCL19-T on K562-Mesothelin-GL is significantly higher than that of the WT and the Mesothelin CAR-T, and the CAR-HIL-6/CCL19-T can also show stronger tumor killing activity under the condition that E: T is very small, i.e. tumor target cells are far larger than effector T cells.

In conclusion, the fourth generation CAR-T cells express and secrete HIL-6 and CCL19 after recognizing tumor antigens, so that the proliferation capacity and anti-tumor effect of the CAR-T cells are remarkably improved, systemic or local inflammatory reaction is avoided, and side effects of the CAR-T are reduced.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

SEQUENCE LISTING

<110> Guangdong Zhaotai in vivo biomedical technology Co., ltd

<120> A fourth generation CAR-T cell and uses thereof

<130> 202012

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 407

<212> PRT

<213> Artificial sequence

<400> 1

Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser

1 5 10 15

Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr

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Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp

35 40 45

Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys

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Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met

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Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe

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Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val

100 105 110

Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp

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Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg

130 135 140

Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp

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

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Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser

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Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser

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Pro Pro Ala Arg Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu Pro

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Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln

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

245 250 255

Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met

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Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro

275 280 285

Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu

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Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr

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Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg

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Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys

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Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala

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

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Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser

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Leu Arg Ala Leu Arg Gln Met

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Met Ala Leu Leu Leu Ala Leu Ser Leu Leu Val Leu Trp Thr Ser Pro

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Ala Pro Thr Leu Ser Gly Thr Asn Asp Ala Glu Asp Cys Cys Leu Ser

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Val Thr Gln Lys Pro Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr

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Leu Leu Ile Lys Asp Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr

50 55 60

Leu Arg Gly Arg Gln Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu

65 70 75 80

Arg Ile Ile Gln Arg Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg

85 90 95

Ser Ser

<210> 3

<211> 665

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Ala Phe Leu Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu

20 25 30

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

35 40 45

Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro

50 55 60

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

65 70 75 80

Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro

85 90 95

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

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr

115 120 125

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

130 135 140

Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

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

165 170 175

Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile

180 185 190

Asn Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser

195 200 205

Pro Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln

210 215 220

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

225 230 235 240

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

245 250 255

Asp Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly

260 265 270

Gly Thr Gln Leu Thr Val Leu Ser Gly Ile Leu Glu Gln Gln Gly Ile

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Ala Leu His Met Gln Ala Leu Pro Pro Arg Gln Ala Lys Arg Lys Pro

500 505 510

Arg Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr

515 520 525

Ser Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu

530 535 540

Glu Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe

545 550 555 560

Ile Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys

565 570 575

Ser His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu

580 585 590

Trp Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu

595 600 605

Asn Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys

610 615 620

Ala Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys

625 630 635 640

Thr Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp

645 650 655

Val Asn Leu Arg Ala Ala Ile Lys Ser

660 665

<210> 4

<211> 1995

<212> DNA

<213> Artificial sequence

<400> 4

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

agccaggtac agctgcagca gtcaggtcca ggactcgtga cgccctcgca gaccctctca 120

ctcacctgtg ccatctccgg ggacagtgtc tctagcaaca gtgctacttg gaactggatc 180

aggcagtccc catcgagagg ccttgagtgg ctgggaagga catactacag gtccaagtgg 240

tataacgact atgcagtatc tgtgaaaagt cgaatgagca tcaacccaga cacatccaag 300

aaccagttct ccctgcagct gaactctgtg actcccgagg acacggctgt gtattactgt 360

gcaagaggaa tgatgactta ctattacggt atggacgtct ggggccaagg gaccacggtc 420

accgtctcct caggaattct aggatccggt ggcggtggca gcggcggtgg tggttccgga 480

ggcggcggtt ctcagcctgt gctgactcag tcgtcttccc tctctgcatc tcctggagca 540

tcagccagtc tcacctgcac cttgcgcagt ggcatcaatg ttggtcccta caggatatac 600

tggtaccagc agaagccagg gagtcctccc cagtatctcc tgaactacaa atcagactca 660

gataagcagc agggctctgg agtccccagc cgcttctctg gatccaaaga tgcttcggcc 720

aatgcagggg ttttactcat ctctgggctc cggtctgagg atgaggctga ctattactgt 780

atgatttggc acagcagcgc tgctgtgttc ggaggaggca cccaactgac cgtcctctcc 840

ggaattctag aacaacaggg tattgaagtt atgtatcctc ctccttacct agacaatgag 900

aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt 960

cccggacctt ctaagccctt ttgggtgctg gtggtggttg ggggagtcct ggcttgctat 1020

agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1080

ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1140

cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1200

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1260

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1320

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1380

atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500

caggccctgc cccctcgcca ggccaaaagg aagcccagga aagctcccag caggaacatc 1560

tgctatgatg catttgtttc ttacagtgag cgggatgcct actgggtgga gaaccttatg 1620

gtccaggagc tggagaactt caatcccccc ttcaagttgt gtcttcataa gcgggacttc 1680

attcctggca agtggatcat tgacaatatc attgactcca ttgaaaagag ccacaaaact 1740

gtctttgtgc tttctgaaaa ctttgtgaag agtgagtggt gcaagtatga actggacttc 1800

tcccatttcc gtctttttga tgagaacaat gatgctgcca ttctcattct tctggagccc 1860

attgagaaaa aagccattcc ccagcgcttc tgcaagctgc ggaagataat gaacaccaag 1920

acctacctgg agtggcccat ggacgaggct cagcgggaag gattttgggt aaatctgaga 1980

gctgcgataa agtcc 1995

<210> 5

<211> 255

<212> DNA

<213> Artificial sequence

<400> 5

ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt 60

ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt 120

ggaggaaaaa ctgtttcata cagaaggcgt agatctagac tctagagggt atataatgga 180

agctcgaatt ccagcttggc attccggtac tgttggtaaa aagcttggca atccggtact 240

gttggtaaag ccacc 255

<210> 6

<211> 1221

<212> DNA

<213> Artificial sequence

<400> 6

ccccccgagg agccccagct ctcctgcttc cggaagagcc ccctcagcaa tgttgtttgt 60

gagtggggtc ctcggagcac cccatccctg acgacaaagg ctgtgctctt ggtgaggaag 120

tttcagaaca gtccggccga agacttccag gagccgtgcc agtattccca ggagtcccag 180

aagttctcct gccagttagc agtcccggag ggagacagct ctttctacat agtgtccatg 240

tgcgtcgcca gtagtgtcgg gagcaagttc agcaaaactc aaacctttca gggttgtgga 300

atcttgcagc ctgatccgcc tgccaacatc acagtcactg ccgtggccag aaacccccgc 360

tggctcagtg tcacctggca agacccccac tcctggaact catctttcta cagactacgg 420

tttgagctca gatatcgggc tgaacggtca aagacattca caacatggat ggtcaaggac 480

ctccagcatc actgtgtcat ccacgacgcc tggagcggcc tgaggcacgt ggtgcagctt 540

cgtgcccagg aggagttcgg gcaaggcgag tggagcgagt ggagcccgga ggccatgggc 600

acgccttgga cagaatccag gagtcctcca gctcgcggtg gtggatcagg aggtggaggg 660

tcagtcgagc cagtaccccc aggagaagat tccaaagatg tagccgcccc acacagacag 720

ccactcacct cttcagaacg aattgacaaa caaattcggt acatcctcga cggcatctca 780

gccctgagaa aggagacatg taacaagagt aacatgtgtg aaagcagcaa agaggcactg 840

gcagaaaaca acctgaacct tccaaagatg gctgaaaaag atggatgctt ccaatctgga 900

ttcaatgagg agacttgcct ggtgaaaatc atcactggtc ttttggagtt tgaggtatac 960

ctagagtacc tccagaacag atttgagagt agtgaggaac aagccagagc tgtgcagatg 1020

agtacaaaag tcctgatcca gttcctgcag aaaaaggcaa agaatctaga tgcaataacc 1080

acccctgacc caaccacaaa tgccagcctg ctgacgaagc tgcaggcaca gaaccagtgg 1140

ctgcaggaca tgacaactca tctcattctg cgcagcttta aggagttcct gcagtccagc 1200

ctgagggctc ttcggcaaat g 1221

<210> 7

<211> 294

<212> DNA

<213> Artificial sequence

<400> 7

atggccctgc tactggccct cagcctgctg gttctctgga cttccccagc cccaactctg 60

agtggcacca atgatgctga agactgctgc ctgtctgtga cccagaaacc catccctggg 120

tacatcgtga ggaacttcca ctaccttctc atcaaggatg gctgcagggt gcctgctgta 180

gtgttcacca cactgagggg ccgccagctc tgtgcacccc cagaccagcc ctgggtagaa 240

cgcatcatcc agagactgca gaggacctca gccaagatga agcgccgcag cagt 294

Claims

1. A fourth generation CAR-T cell, wherein the fourth generation CAR-T cell expresses a chimeric antigen receptor that specifically binds an antigen, a composite IL-6, and CCL19;

the compound IL-6 and CCL19 are NFAT regulation type overexpression;

The compound IL-6 is a compound of IL-6 and IL-6Rα, and the compound IL-6 is an amino acid sequence shown in SEQ ID NO. 1;

the CCL19 is an amino acid sequence shown in SEQ ID NO. 2;

The chimeric antigen receptor includes an antigen binding domain, a transmembrane domain, and a signaling domain;

The antigen binding domain is a single chain antibody targeting Mesothelin;

The single-chain antibody targeting the Mesothelin is amino acids from 21 st to 287 rd in the amino acid sequence shown in SEQ ID NO. 3.

2. The fourth generation CAR-T cell of claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain and/or a CD8 a transmembrane domain.

3. The fourth generation CAR-T cell of claim 2, wherein the transmembrane domain is a CD28 transmembrane domain.

4. The fourth generation CAR-T cell of claim 1, wherein the signaling domain comprises any one or a combination of at least two of a CD28 intracellular domain, cd3ζ, TLR2, 4-1BB, TLR1, CD27, OX40, or DAP 10.

5. The fourth generation CAR-T cell of claim 4, wherein the signaling domain is a combination of CD28 intracellular domain, cd3ζ, and TLR 2.

6. The fourth generation CAR-T cell of claim 1, wherein the chimeric antigen receptor further comprises a signal peptide.

7. The fourth generation CAR-T cell of claim 1, wherein the chimeric antigen receptor consists of GM-CSF signal peptide, a Mesothelin antigen binding domain, a CD28 transmembrane domain, a CD28 intracellular domain, CD3 ζ, and TLR2 in tandem.

8. The fourth generation CAR-T cell of claim 7, wherein the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID No. 3.

9. An expression vector comprising a chimeric antigen receptor-encoding gene, an NFAT regulatory promoter, a composite IL-6-encoding gene, and a CCL 19-encoding gene; the chimeric antigen receptor coding gene is a nucleic acid sequence shown in SEQ ID NO. 4;

The NFAT regulatory promoter is a nucleic acid sequence shown in SEQ ID NO. 5;

the composite IL-6 coding gene is a nucleic acid sequence shown in SEQ ID NO. 6;

The CCL19 coding gene is a nucleic acid sequence shown in SEQ ID NO. 7.

10. The expression vector of claim 9, wherein the expression vector comprises a viral vector.

11. The expression vector of claim 10, wherein the viral vector comprises any one of a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

12. The expression vector of claim 11, wherein the viral vector is a lentiviral vector.

13. A recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of any one of claims 9-12 and a packaging helper plasmid.

14. A method of making a fourth generation CAR-T cell according to any one of claims 1 to 8, comprising the step of introducing the expression vector of any one of claims 9 to 12 or the recombinant lentivirus of claim 13 into a T cell.

15. A pharmaceutical composition comprising the fourth generation CAR-T cell of any one of claims 1-8.

16. The pharmaceutical composition of claim 15, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

17. Use of a fourth generation CAR-T cell according to any one of claims 1 to 8, an expression vector according to any one of claims 9 to 12, a recombinant lentivirus according to claim 14 or a pharmaceutical composition according to claim 15 or 16 in the manufacture of a medicament for the treatment of a tumor.

18. The use of claim 17, wherein the tumor comprises any one or a combination of at least two of liver cancer, lung cancer, breast cancer, wilms' cell tumor, glioma, neuroblastoma, melanoma, nasopharyngeal carcinoma, mesothelioma, islet cell tumor, retinoblastoma, pancreatic cancer, uterine fibroid, cervical cancer, or thyroid cancer.