CN116041546A

CN116041546A - Chimeric antigen receptor, isolated nucleic acid, recombinant vector, CAR-T cell and application thereof

Info

Publication number: CN116041546A
Application number: CN202211674686.3A
Authority: CN
Inventors: 林戈; 罗孝宇; 吴静; 周英; 邵梦思; 王静; 郝建军; 卢光莹; 陈濂生; 卢光琇
Original assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan
Current assignee: Guang Xiu Gao Xin Life Science Co ltd Hunan
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-05-02

Abstract

The invention relates to a chimeric antigen receptor, isolated nucleic acid, recombinant vector, CAR-T cell and application thereof. The chimeric antigen receptor comprises a target binding domain, a hinge region, a transmembrane domain, a BRS of CD3 epsilon, a costimulatory domain and a signal transduction domain which are sequentially connected from an amino terminal to a carboxyl terminal, wherein the costimulatory domain is 4-1BB, and the signal transduction domain is ITAM1 of CD3 zeta. Compared with the traditional CAR-T cells, the CAR-T cells containing the chimeric antigen receptor have stronger killing power on tumor cells, less IL-6 generation, higher cell survival rate, higher proliferation multiple and stronger anti-depletion capability.

Description

Chimeric antigen receptor, isolated nucleic acid, recombinant vector, CAR-T cell and application thereof

Technical Field

The invention relates to the technical field of immunotherapy, in particular to a chimeric antigen receptor, isolated nucleic acid, a recombinant vector, a CAR-T cell and application thereof.

Background

Chimeric Antigen Receptor (CAR) is an artificial fusion protein consisting of a target antigen recognition moiety and a T cell internal signaling domain. Currently, clinical trials of CAR-T cells against the B cell marker CD19 have shown clear efficacy against a variety of hematological malignancies, including Acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), and non-hodgkin's lymphoma, among others. Currently, CAR-T cell therapy has become an important means for treating cancer, is a milestone result in biomedicine, and has huge theoretical, social and economic benefits.

At present, a plurality of CAR-T cell products are marketed worldwide, and although the CAR-T cell therapy has achieved a staged result in treating malignant tumors, the present CAR-T cell therapy has great toxic and side effects. For example, treatment of B-cell acute lymphoblastic leukemia (B-ALL) and non-hodgkin B-cell lymphoma patients with CD19 CAR-T cells, with Cytokine Release Syndrome (CRS) occurring in more than 90% of patients. CRS generally occurs within the first week of CAR-T cell therapy, manifesting as fever, hypotension and respiratory dysfunction, as well as high cytokine levels, among others in patients.

In addition, current CAR-T cells do not survive long in vivo, have poor proliferation capacity, and thus patients cannot be fully relieved, most of the patients who obtain relief show disease recurrence within a few years, and the recurrence rate of B-ALL varies from 21% to 45% and increases with prolonged follow-up time.

Disclosure of Invention

Based on the above, it is necessary to provide a chimeric antigen receptor which has higher killing power to tumor cells, less IL-6 production, higher cell survival rate, higher proliferation factor and higher depletion resistance than conventional CAR-T cells, aiming at the problems of large side effect and short survival time in vivo of conventional CAR-T cells.

A chimeric antigen receptor comprising, in order from amino-terminus to carboxy-terminus, a target binding domain, a hinge region, a transmembrane domain, BRS of CD3 epsilon, a costimulatory domain, and a signaling domain, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 zeta.

The chimeric antigen receptor comprises a target binding domain, a hinge region, a transmembrane domain, a BRS of CD3 epsilon, a costimulatory domain and a signal transduction domain, wherein 4-1BB is used as the costimulatory domain, ITAM1 of CD3 zeta is used as the signal transduction domain, and the BRS of CD3 epsilon is also increased, so that compared with the traditional CAR-T cell, the CAR-T cell containing the chimeric antigen receptor has stronger killing power to the tumor cell and better treatment effect, the generated IL-6 is less and is not easy to cause cytokine release syndrome to be safer, the cell survival rate is higher, the proliferation multiple is higher, the anti-depletion capability is stronger, the duration of treatment effect is longer, and the problems of large side effect and no long in vivo survival time of the traditional CAR-T cell can be improved.

In one aspect, the present application provides a chimeric antigen receptor targeting CD19, comprising, in order from amino terminus to carboxy terminus, a CD 19-targeting antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the costimulatory domain is 4-1BB, and the signaling domain is ITAM1 of cd3ζ.

In one embodiment, the target for which the target binding domain corresponds comprises one or more of the following:

AFP, ANGPT2, AXL, B7H3, BAFFR, BCMA, CAIX, CD a, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD39, CD44, CD46, CD47, CD56, CD64, CD70, CD73, CD79, CD117, CD123, CD126, CD133, CD138, CD147, CD171, CD205, CDH17, CEA, claudin 6, claudin 18.2, CLL1, CS1, CSF-1R, CSPG4, CTLA-4, CXCR4, DLL3, EGFR, EGFRvIII, epCAM, ephA2, FAP, FBP, FGFR, FR, FSHR, GD2, glycolipid F77, GPC2, GPC3, GPRC5D, GPR20, GPR78, GSPG4, GUCY2C, her2, her3, her4, ICAM-1, IGF1R, IL-13R, integrins, L1-CAM, lewis Y, LRB4, LMP1, LIP 2, GEA3, LIC 3, LIOP-2, MAVEGFR 1-35, VEGFR1, VEGFR-3, LIC-3, GLC-35, GL-2, GL-3, and GL-3, TAFR 2, GL-6, GL-VEGFR-3, GLR-XR-3, GL-GR-GRP-3, GL-GRP-3, and GLP-3;

in one embodiment, the hinge region is selected from the group consisting of hinge regions of at least one protein: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

In one embodiment, the transmembrane domain is selected from the transmembrane region of at least one of the following proteins: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, kirs 2, OX40, CD2, CD27, ICOS, GITR, CD, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2rβ, IL2rγ, IL7rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, ceam 1, CRTAM 9, PSGL1, CD100, slamfr 6, SLAM, BLAME, SELPLG, LTBR, PAG/bp, NKp44, NKp46 g2, NKG2, and NKG 2.

In one embodiment, the chimeric antigen receptor further comprises a signal peptide that is amino-terminal and linked to the target binding domain.

In one embodiment, the target binding domain is a CD 19-targeting single-chain antibody, the amino acid sequence of the heavy chain variable region of the CD 19-targeting single-chain antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region of the CD 19-targeting single-chain antibody is shown in SEQ ID No. 2;

the amino acid sequence of SEQ ID NO.1 is:

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO.1)。

the amino acid sequence of SEQ ID NO.2 is:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT(SEQ ID NO.2)。

in one embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 3.

The amino acids of SEQ ID NO.3 are listed as: ^{MALPVTALLLPLALLLHAARP(SEQ} ID NO.3)。

in one embodiment, the hinge region is a CD8 alpha hinge region having the amino acid sequence shown in SEQ ID NO. 4.

The amino acids of SEQ ID NO.4 are listed as:

KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO.4)。

in one embodiment, the transmembrane domain is a CD 8. Alpha. Transmembrane region having the amino acid sequence shown in SEQ ID NO. 5.

The amino acid sequence of SEQ ID NO.5 is: ^{IYIWAPLAGTCGVLLLSLVITLYC(SEQ} ID NO.5)。

in one embodiment, the amino acid sequence of 4-1BB is shown in SEQ ID NO. 6.

The amino acid sequence of SEQ ID NO.6 is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.6)。

in one embodiment, the BRS amino acid sequence of CD3 ε is shown in SEQ ID NO. 7.

The amino acid sequence of SEQ ID NO.7 is: ^KNRKAKAK(SEQ ID NO.7)。

in one embodiment, the amino acid sequence of ITAM1 of CD3 ζ is shown in SEQ ID NO. 8.

The amino acid sequence of SEQ ID NO.8 is:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ(SEQ ID NO.8)。

in one embodiment, the CD 19-targeting chimeric antigen receptor further comprises a signal peptide that is amino-terminal and linked to the CD 19-targeting antigen binding domain.

In one embodiment, the amino acid sequence of the CD 19-targeting chimeric antigen receptor is shown in SEQ ID No. 9.

The amino acid sequence of SEQ ID NO.9 is:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGG

GSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI

IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAKPTTTPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKNRKAKAKKRGRKKL

LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ(SEQ ID NO.9)。

in one embodiment, the chimeric antigen receptor has a nucleotide sequence as set forth in SEQ ID NO. 10.

The nucleotide sequence of SEQ ID NO.10 is:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCaagaatagaaaggccaaggccaagAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGTAA(SEQ ID NO.10)。

in another aspect the present application provides an isolated nucleic acid for expressing a CD19 targeting chimeric antigen receptor according to any one of claims 1 to 5.

In one embodiment, the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence as set forth in SEQ ID NO. 10.

The present application also provides a recombinant vector comprising the isolated nucleic acid of claim 7 or 8.

The present application also provides a CAR-T cell comprising the isolated nucleic acid of claim 7 or 8 or transformed with the recombinant vector of claim 9.

The present application also provides a use in the manufacture of a medicament for the treatment of cancer comprising a CD 19-targeting chimeric antigen receptor according to any one of claims 1 to 5, an isolated nucleic acid according to claim 8, a recombinant vector according to claim 9 or a CAR-T cell according to claim 9.

The present application provides a pharmaceutical composition comprising an expression vector for expressing a CD 19-targeted chimeric antigen receptor according to any one of claims 1 to 6 or a CAR-T cell according to claim 10, and a pharmaceutically acceptable adjuvant.

Drawings

FIG. 1 is a schematic representation of the 1355-CAR and its controls 1326-CAR and 1502-CAR structures of example 1;

FIG. 2 results of killing capacity of CAR-T cells in example 1 without antigen stimulation for D4 days (D0 days prior to antigen stimulation in multiple rounds of antigen stimulation experiments);

FIG. 3 is the results of the killing capacity of CAR-T cells cultured for d21 days with 3 rounds of antigen stimulation in example 1;

FIG. 4 is the secretion levels of granzyme B in supernatant of non-antigen stimulated 1355-CAR-T cells, 1502-CAR-T cells and 1326-CAR-T cells co-cultures with tumor cells in example 1;

FIG. 5 is the secretion levels of granzyme B in the supernatant of example 1 after 3 rounds of antigen stimulation of 1355-CAR-T cells, 1502-CAR-T cells and 1326-CAR-T cells co-culture with tumor cells;

FIG. 6 is a comparison of IL-6 secretion levels in supernatant of 1355-CAR-T cells, 1502-CAR-T cells and 1326-CAR-cells co-culture with tumor cells in example 1;

FIG. 7 is the survival rate of CAR-T cells in example 1;

FIG. 8 is a comparison of the fold proliferation of CAR-T cells d0-d7 days after 1 round of antigen stimulation and d7-d14 days after 2 rounds of antigen stimulation in example 1;

fig. 9 is a comparison of cell depletion resistance after killing tumor cells by 1502-CAR-T cells, 1355-CAR-T cells and 1326-CAR-T cells in example 1.

Detailed Description

The present application will be described more fully hereinafter for the purpose of facilitating an understanding of the present application, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Interpretation of the terms

"T cells" means T lymphocytes (T lymphocytes) which are derived from bone marrow-derived lymphocytes, differentiate in the thymus, develop and mature, and then distribute through the lymph and blood circulation into immune organs and tissues throughout the body to exert immune functions. Mature T cells are distributed to thymus dependent areas of peripheral immune organs through blood flow, and can be recycled through lymphatic vessels, peripheral blood, tissue fluid and the like, thereby playing the functions of cellular immunity, immunoregulation and the like. The recirculation of T cells facilitates extensive exposure to antigenic material into the body, enhancing immune responses and maintaining immune memory for a longer period. T-cells have many different markers on their cell membrane, mainly surface antigens and surface receptors, which are all giant protein molecules that bind to the cell membrane.

The "single chain antibody" is formed by linking the heavy chain variable region (heavy chain variable domain, VH) and the light chain variable region (light chain variable domain, VL) of an antibody via a flexible short peptide (linker) of 10 to 25 amino acids, and is the smallest recombinant antibody form (about 27 kDa). Essentially, scFv is a fusion protein and retains the original immunoglobulin's specificity for an antigen. The scFv has smaller molecular size, brings the advantages of strong penetration in tumor, rapid degradation in blood, small negative feedback in human body and the like, and lays a foundation for the clinical application of scFv.

"and/or" includes any and all combinations of one or more of the associated listed items. "optionally" means by way of example, with or without meaning.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

An embodiment of the present application provides a chimeric antigen receptor comprising a target binding domain, a hinge region, a transmembrane domain, a BRS of CD3 epsilon, a costimulatory domain, and a signaling domain, which are sequentially linked from amino-terminus to carboxy-terminus, the costimulatory domain being 4-1BB and the signaling domain being ITAM1 of CD3 zeta.

CD3 epsilon belongs to a type I transmembrane protein, and the intracellular region contains an Immunoreceptor Tyrosine Activation Motif (ITAM) and a basic amino acid-rich sequence basic residue rich sequence (BRS) consisting of 8 amino acid residues. The intracellular domain of CD3 ζ contains 3 immunoreceptor tyrosine activation motifs (immunoreceptor tyrosine-based activation motif, ITAM), i.e., ITAM1, ITAM2 and ITAM3. The present application uses the first immunoreceptor tyrosine activation motif of CD3 ζ (ITAM 1) as a signaling domain, 4-1BB as a costimulatory domain and introduces the BRS of CD3 ε. It was verified that by matching BRS of 4-1BB, CD3 epsilon with ITAM1 of CD3 zeta, CAR-T cells containing the chimeric antigen receptor have stronger tumor killing ability, less IL-6 production, higher cell survival and proliferation multiple and stronger depletion resistance.

In some embodiments, the target for which the target binding domain corresponds comprises one or more of the following: AFP, ANGPT2, AXL, B7H3, BAFFR, BCMA, CAIX, CD a, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD39, CD44, CD46, CD47, CD56, CD64, CD70, CD73, CD79, CD117, CD123, CD126, CD133, CD138, CD147, CD171, CD205, CDH17, CEA, claudin 6, claudin 18.2, CLL1, CS1, CSF-1R, CSPG4, CTLA-4, CXCR4, DLL3, EGFR, EGFRvIII, epCAM, ephA2 FAP, FBP, FGFR, FR, FSHR, GD, glycolipid F77, GPC2, GPC3, GPRC5D, GPR, GPR78, GSPG4, GUCY2C, her2, her3, her4, ICAM-1, IGF1R, IL-13R, integrins, L1-CAM, lewis Y, LILRB4, LMP1, LMP2, MAGEA3, mesothelin, met, MUC-1, MUC-16, nectin-4, NY-ESO-1, ROR1, PD-L1, PSCA, PSMA, TAG-72, TACI, trop-2, VEGFR1, VEGFR2, VEGFR3 and WT-1.

In some embodiments, the target corresponding to the target binding domain is CD19. Further, the target binding domain is a single chain antibody targeting CD19. In an alternative specific example, the amino acid sequence of the heavy chain variable region of the CD 19-targeting single chain antibody is shown in SEQ ID No.1 and the amino acid sequence of the light chain variable region of the CD 19-targeting single chain antibody is shown in SEQ ID No. 2. It will be appreciated that where the target is CD19, the target binding domain is not limited to the above, but may be other binding domains capable of specifically binding to CD19.

In some embodiments, the hinge region is selected from the hinge region of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154. In an alternative specific example, the hinge region is a CD8 alpha hinge region having the amino acid sequence shown in SEQ ID No. 4. It will be appreciated that in other embodiments, the hinge region is not limited to that described above.

In some embodiments, the transmembrane domain is selected from the transmembrane region of at least one of the following proteins: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, kirs 2, OX40, CD2, CD27, ICOS, GITR, CD, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2rβ, IL2rγ, IL7rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, ceam 1, CRTAM 9, PSGL1, CD100, slamfr 6, SLAM, BLAME, SELPLG, LTBR, PAG/bp, NKp44, NKp46 g2, NKG2, and NKG 2. In an alternative specific example, the transmembrane domain is a CD 8. Alpha. Transmembrane region having the amino acid sequence shown in SEQ ID No. 5.

In some embodiments, the amino acid sequence of 4-1BB is set forth in SEQ ID NO. 6; the amino acid sequence of BRS of CD3 epsilon is shown as SEQ ID NO.7, and the amino acid sequence of ITAM1 of CD3 zeta is shown as SEQ ID NO. 8.

In some embodiments, the chimeric antigen receptor described above further comprises a signal peptide that is amino-terminal and linked to the target binding domain. In an alternative specific example, the amino acid sequence of the signal peptide is shown in SEQ ID No. 3. It will be appreciated that in other embodiments, the signal peptide is not limited to the above, but may be other polypeptides that may be signal peptides.

In some embodiments, the target binding domain is a single chain antibody targeting CD19, the amino acid sequence of the heavy chain variable region of the single chain antibody targeting CD19 is shown as SEQ ID No.1, the amino acid sequence of the light chain variable region of the single chain antibody targeting CD19 is shown as SEQ ID No.2, the amino acid sequence of the signal peptide is shown as SEQ ID No.3, and the hinge region is a CD8 a hinge region having the amino acid sequence shown as SEQ ID No. 4; the transmembrane domain is a CD8 alpha transmembrane region with an amino acid sequence shown as SEQ ID NO.5, and the amino acid sequence of 4-1BB is shown as SEQ ID NO. 6; the amino acid sequence of BRS of CD3 epsilon is shown as SEQ ID NO.7, and the amino acid sequence of ITAM1 of CD3 zeta is shown as SEQ ID NO. 8.

Further, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 9.

It will be appreciated that the individual polypeptides (e.g., signal peptide, single chain antibody, hinge region, transmembrane domain, costimulatory domain, and signal transduction domain) of the present application may be independently selected from the same or different species sources, such as murine (mouse, rat), rabbit, sheep, goat, equine, chicken, bovine, canine, and the like. Further, the species source is a human.

In addition, an embodiment of the present application provides an isolated nucleic acid for expressing the chimeric antigen receptor of any of the above examples.

In some embodiments, the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence as set forth in SEQ ID NO. 10. It will be appreciated that due to the degeneracy of the codons, nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon-optimized nucleic acid sequences for achieving efficient expression of the protein of interest, but the particular sequence form is not limited thereto. It is understood that the nucleic acid may be DNA or RNA.

In addition, an embodiment of the present application also provides a recombinant vector comprising the isolated nucleic acid of any of the above examples.

In some embodiments, the recombinant vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a CRISPR/CAS plasmid. Preferably, the recombinant vector is a lentiviral vector. The slow virus vector can effectively integrate exogenous genes into host chromosomes, thereby achieving the effect of permanently expressing target sequences. Can effectively infect various cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capability, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, non-differentiated cells and the like, the use of the lentiviral vector can greatly improve the transduction efficiency of the target gene, greatly increase the probability of integrating the target gene into the genome of the host cell, and conveniently and rapidly realize the long-term and stable expression of the target gene. It will be appreciated that the type of recombinant vector described above is not limited thereto and may be adapted to the particular needs. In addition, the recombinant vector may further comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, and the like, and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly-U sequences, etc.).

In addition, an embodiment of the present application provides a CAR-T cell comprising the isolated nucleic acid of any of the above examples or transformed with the recombinant vector of any of the above examples.

In some implementations, the type of CAR-T cell described above is any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, and a γδ T cell. Alternatively, the CAR-T cell type is a cd3+ T cell, a cd3+cd4+ T cell, or a cd3+cd8+ T cell.

In addition, an embodiment of the present application also provides the use of any of the above embodiments of the chimeric antigen receptor, any of the above embodiments of the isolated nucleic acid, any of the above embodiments of the recombinant vector, or any of the above embodiments of the CAR-T cell in the manufacture of a medicament for treating cancer.

In addition, an embodiment of the present application further provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises an expression vector for expressing the chimeric antigen receptor of any one of the embodiments or the CAR-T cell of any one of the embodiments.

In some embodiments, the excipients include one or more of diluents, preservatives, buffers, disintegrants, antioxidants, suspending agents, colorants, and excipients.

Optionally, the diluent is selected from one or more of polyethylene glycol, propylene glycol, vegetable oil and mineral oil. In a specific example, the preservative is selected from one or more of sorbic acid, methyl sorbate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, benzyl parahydroxybenzoate, sodium methyl parahydroxybenzoate, benzoic acid, and benzyl alcohol. In a specific example, the buffer is selected from one or more of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, and sodium acetate. In a specific example, the antioxidant is selected from one or more of ethylenediamine tetraacetic acid, disodium ethylenediamine tetraacetic acid, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite.

By pharmaceutically acceptable adjuvant is meant herein a material that is compatible with the other ingredients of the pharmaceutical formulation and is suitable for use in the contact of the tissues or organs of a recipient (e.g., a human or animal). No or little complications of toxicity, irritation, allergic response, immunogenicity, or other problems at the time of use.

In addition, an embodiment of the present application further provides a method for preparing the CAR-T cell, which comprises the following steps: collecting peripheral blood mononuclear cells; sorting T cells (e.g., cd3+ T cells) from the peripheral blood mononuclear cells; and infecting the T cells with a recombinant vector (e.g., virus) comprising the isolated nucleic acid of any of the above examples, followed by expansion culture.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.

Example 1

1. Design of CAR

The scFv region of monoclonal antibody FMC63 (abbreviated as "FMC63 scFv") targeting CD19 from murine, human CD 8. Alpha. Transmembrane region and hinge region, human CD3 zeta chain full length as signaling region, and CAR of 4-1BB sequence as co-stimulatory molecule were designated CD19.BBZCAR.1502, abbreviated as "1502-CAR", and the full length of 1502-CAR was 1502bp. On the basis of 1502-CAR, intracellular CD3 zeta chain is modified, and the first ITAM1 (called as ITAM1 or CD3 zeta ITAM1 for short) in 3 Immunoreceptor Tyrosine Activation Motifs (ITAM) of the CD3 zeta chain is selected, and the sequence with the length of 56 amino acids is formed into a CAR structure sequence with the length of 1326bp, which is named as CD19.BBZCAR.1326 and called as 1326-CAR for short. A basic amino acid motif (BRS) consisting of 8 amino acid residues in the cell of CD3 epsilon (abbreviated as "CD3 epsilon BRS region") is selected on the basis of a 1326-CAR structure and inserted behind the CD8 alpha transmembrane region to form a novel CAR structure, the total length of the sequence of the CAR structure is 1355bp, and the CAR structure is named as CD19 bbzcar.1355, abbreviated as "1355-CAR", and each CAR structure is shown in fig. 1.

In 1502-CAR, 1326-CAR and 1355-CAR, the amino acid sequence of the CD8 alpha signal peptide is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain variable region of FMC63 scFv is shown as SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, the amino acid sequence of the human CD8 alpha hinge region is shown as SEQ ID NO.4, the amino acid sequence of the human CD8 alpha transmembrane region is shown as SEQ ID NO.5, the amino acid sequence of the 4-1BB co-stimulatory molecule is shown as SEQ ID NO.6, the amino acid sequence of the CD3 epsilon BRS region is shown as SEQ ID NO.7, and the amino acid sequence of ITAM1 is shown as SEQ ID NO. 8.

The amino acid sequence of 1355-CAR is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10. The 1326-CAR nucleotide sequence is shown in SEQ ID NO. 11. The 1502-CAR nucleotide sequence is shown as SEQ ID NO. 12.

The 1326-CAR nucleotide sequence is shown in SEQ ID NO. 11:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGTAA(SEQ ID NO.11)

the 1502-CAR nucleotide sequence is shown in SEQ ID NO. 12:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO.12)

2. packaging of lentiviruses

After synthesis of the sequences of 1355-CAR, 1502-CAR and 1326-CAR by Biotechnology (Shanghai) Co., ltd, the sequences were cloned into lentiviral plasmid pSin-EF using EcoR1 and Spe1 restriction endonucleases _α In SOX2-puro (available from Addgene, plasmid # 16577), the original SOX2 fragment was replaced, constituting lentiviral plasmids pSin-EF alpha-1502-puro, pSin-EF alpha-1355-puro and pSin-EF alpha-1326-puro containing the CAR sequence. The expression Plasmid for the CAR and lentiviral packaging plasmids pcmv8.91 (purchased from adedge ne) and pmd.2g Plasmid (purchased from adedge ne, plasmid # 12259) were then transfected into 293FT cells using a calcium phosphate transfection reagent (Invitrogen: K278001) at a ratio of 4:3:1. After 24h of plasmid transfection, the culture medium supernatant containing lentivirus was harvested and finally the collected supernatant was concentrated by centrifugation using ultracentrifugation at a concentration factor of 100. The titer of the concentrated lentivirus reaches 10 by qPCR measurement ⁸ TU/mL above was ready for subsequent use.

Preparation of CAR-T cells

The blood with 10mL of leucocyte enrichment is collected by a menstrual blood cell separator, and is subjected to operation according to instructions by using a kit RosetteSep Human T Cells enrichment cocktail of STEMCELL Techonogies company, and CD3+ T cells with the purity of more than 99% are separated from peripheral whole blood or mechanical blood, and are directly used or frozen by liquid nitrogen.

D0 day: taking a certain amount of CD3 ⁺ T cells, with CAR-T medium (GT-T551 H23+5% CTS ^TM Immune Cell SR+1% non-essential amino acid+1% glutamine+1% Heps+1% sodium pyruvate+1% streptomycin-penicillin+1% GlutaMAX+46-50Unit IL-2, where "%" are all percentages by volume) was cultured and magnetic beads (Dynabeads Human T-Activator CD3/CD28, invitrogen) were added in a 1:1 ratio. Finally, at 37 ℃,5% CO ₂ Is cultured in an incubator for 24 hours.

D1 day: six well plates (overnight at 4 ℃) were coated with a solution of fibronectin from TAKARA. After coating, the supernatant was removed, and the calculated volume of lentivirus was added to the six-well plate and centrifuged at 2000g at 32℃for 2h. After centrifugation, the supernatant was removed and the D1 day harvested CD3 ⁺ T cells together with magnetic beads were added to the six well plate, and then a volume of Polybrene was added to give a final concentration of 4. Mu.g/mL, and finally centrifuged at 32℃for 2h at 1200 g. After centrifugation, the six-well plate was placed in 37℃and 5% CO ₂ The culture was continued for 24 hours.

D2 days: all cells were collected and centrifuged at 350g at room temperature for 5min. Centrifuging, removing supernatant, replenishing fresh CAR-T culture medium to resuspend cells, and inoculating into T25 culture flask to make cell density be 0.5X10 ⁶ /mL. Finally, the cells were placed at 37℃with 5% CO ₂ The culture was continued for 48 hours.

D4 days: harvesting D4 day CAR-T cell supernatant for cytokine detection; CAR-T cells were harvested D4 days for transduction efficiency i.e. (CAR positive rate) and killing capacity assays.

4. Multiple rounds of antigen stimulation experiments

In the study of the viability of CAR-T cells, we performed multiple rounds of antigen stimulation experiments. Taking 4×10D 4 days ⁵ CAR-T cells and RS4;11 acute leukemia cells were mixed at 1:1 ratio for round 1 antigenStimulation, at this time d0 days of multiple rounds of stimulation experiments;

taking 4×10 from d7 days of multiple rounds of antigen stimulation experiments ⁵ CAR-T cells and RS4;11 acute leukemia cells were mixed at 1:1 ratio for round 2 antigen stimulation;

4X 10 days d14 of multiple rounds of antigen stimulation experiments ⁵ CAR-T cells and RS4;11 acute leukemia cells were mixed at 1:1 ratio for round 3 antigen stimulation;

cells were harvested on days d3, d7, d10, d14, and d21, respectively, and subjected to experiments such as killing, proliferation, and depletion.

5. Effect data

(1) The in vitro killing capacity of 1355-CAR-T cells is improved.

The killing ability of tumor cells was determined by taking 3 rounds of antigen-stimulated CAR-T cells on day D21, and CAR-T cells not subjected to antigen stimulation on day D4. Wherein CAR-T cells and RS4 on day d 21; 11 tumor cell number ratio (briefly, target ratio) was co-cultured according to 1:15, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, whereas the target ratio of CAR-T cells without antigen stimulation for D4 days was co-cultured according to 1:5, 1:2, 1:1, 2:1, 5:1, 2×10 per sample collection ⁵ Flow cytometry analysis of cells co-cultured for 72h using CD19 antibody (APC-anti-human-CD 19) accession number (302212) and the results are shown in fig. 2 and 3, wherein fig. 2 is the result of CAR-T cell killing capacity for D4 days prior to antigen stimulation (D0 days prior to antigen stimulation in multiple rounds of antigen stimulation experiments); figure 3 is the results of CAR-T cell killing capacity after 3 rounds of antigen stimulation culture to d21 days.

As can be seen from figures 2 and 3, 1355-CAR-T cells showed comparable tumor cell killing capacity to 1326-CAR-T cells and 1502-CAR-T cells when not subjected to antigen stimulation, i.e. all killed tumor cells at an effective target ratio of 1:5, 1:2, 1:1, 2:1, 5:1. However, after multiple rounds of stimulation, 1355-CAR-T cells showed stronger killing ability at a comparable percentage of CAR19-T cells, and tumor cells could be killed completely at an effective target ratio of 1:15. The killing capacity of 1326-CAR-T cells is not obviously better than that of 1502-CAR-T cells after the multi-round antigen stimulation, which shows that the CAR structure of the first ITAM1 in 3 Immunoreceptor Tyrosine Activation Motifs (ITAMs) of CD3 zeta chains is not obvious in the effect of improving the killing capacity of tumor cells in vitro after the multi-round antigen stimulation, and the killing capacity of tumor cells can be obviously improved after the multi-round stimulation after the addition of the CD3 epsilon-BRS sequences, and the tumor cells can be completely killed when the effective target ratio reaches 1:15. The above demonstrates that ITAM1, which is a combination of the CD3 ε -BRS motif and the CD3 ζ chain, has a promoting effect on tumor killing.

In addition, the killing ability of effector T cells to tumor cells is achieved mainly by secretion of cytokines such as granzyme and perforin. Thus, the culture supernatants of CAR-T cells on day D4 without antigen stimulation and CAR-T cells and tumor cells on day D21 with 3 rounds of antigen stimulation were selected for granzyme assays. 1mL of the cell culture supernatant co-cultured for 72 hours was collected for each sample, and the concentration of granzyme B in the culture supernatant was determined by ELISA. The results are shown in figures 4 and 5, figure 4 is the secretion level of granzyme B in supernatants of non-antigen stimulated 1355-CAR-T cells, 1502-CAR-T cells and 1326-CAR-T cells co-culture with tumor cells; figure 5 is the secretion levels of granzyme B in supernatants of 1355-CAR-T cells, 1502-CAR-T cells and 1326-CAR-T cells co-cultured with tumor cells over 3 rounds of antigen stimulation.

From fig. 4 and 5, it can be seen that the secretion levels of granzyme B were slightly higher for 1355-CAR-T cells and 1326-CAR-T cells than for 1502-CAR-T cells, with or without antigen stimulation. This suggests that 1355-CAR-T cells and 1326-CAR-T cells release more granzyme B, and that 1355-CAR-T cells release granzyme B at best and thus have a stronger ability to kill tumor cells, consistent with their in vitro killing results.

(2) Inflammatory cytokine IL-6 secretion levels by 1355-CAR-T cells were reduced.

Cytokine release syndrome (cytokine releasing syndrome, CRS) is a more severe inflammatory side effect that occurs when CAR-T cells are applied in clinical procedures, with more than 80% of patients exhibiting different levels of CRS responses. Clinical trials have found that this response occurs in association with the presence of a number of inflammatory-related cytokines in the patient's serum, one of which has been shown and recognised by the clinician to be associated with CRS generation is IL-6 (Shannon L et al, N Engl J med.2014). Antibodies against the IL-6 receptor, tocilizumab, have been routinely used clinically to treat CRS during CAR-T therapy. Nonetheless, some patients still die from CRS. It is therefore particularly necessary to conduct an assay for the cytokine IL-6. CAR-T cells and RS 4D 4 days prior to antigen stimulation; after co-culturing 11 tumor cells according to different target ratios, 1mL of cell culture supernatant was collected for co-culture for 72 hours for each sample, and the concentration of IL-6 in the culture supernatant was measured by ELISA, and the results are shown in FIG. 6.

As can be seen from fig. 6, 1355-CAR-T cells released lower levels of IL-6 when co-cultured with target tumor cells than 1502-CAR-T cells (classical second generation CAR-T cells) and control 1326-CAR-T. Thus, 1355-CAR-T cells may reduce the risk of CRS at the time of treatment and thus be safer.

(3) Survival and fold proliferation of 1355-CAR-T cells were improved.

To investigate the viability of CAR-T cells, we counted CAR-T cells on days d3, d7, d10, d14, d17 and d21 of the multi-round stimulation, counted the total cell number, the number of living cells and the number of dead cells of CAR-T, respectively, and then divided the number of living cells by the number of total cells, to obtain the viability of CAR-T cells over different time periods. The CAR-T cell proliferation fold was then calculated for days d0-d7 and d7-d14, and the results are shown in figures 7 and 8. FIG. 7 is a graph showing the survival rate of CAR-T cells, day d0 representing cells not subjected to antigen stimulation in an antigen stimulation experiment; FIG. 8 is a comparison of the fold proliferation of CAR-T cells after 1 round of antigen stimulation for d0-d7 days and the fold proliferation of CAR-T cells after 2 rounds of antigen stimulation for d7-d14 days.

As can be seen from fig. 7 and 8, although the survival rate of 1355-CAR-T cells before antigen stimulation was not as good as that of 1502-CAR-T cells, the survival rate of 1355-CAR-T cells after antigen stimulation was slightly higher than that of 1326-CAR-T cells and 1502-CAR-T cells, and the proliferation rate of 1355-CAR-T cells after 1 round of antigen stimulation was about 1800 times.

(4) The depletion of 1355-CAR-T cells was reduced.

In the multiple round of stimulation experiments, tumor cells (hCD 19) were treated by flow cytometry on the third day (d 3, d10, d17, respectively) after the first (d 0), second (d 7), and third (d 14) antigen stimulation, respectively ⁺ ) Residual detection and CAR-T cell depletion (Tim 3 cell depletion marker, perCP/cyanine5.5anti-human CD366 (Tim-3) anti-body, company: biolegend, cat: 345016 The result of the detection is shown in fig. 9. In FIG. 9, the abscissa indicates 3 rounds of antigen stimulation over d0, d7 and d14 days, cells taken 3 days after each antigen stimulation are taken, and the ordinate indicates complete killing of 4X 10 ⁵ Number of CAR19+ T cells depleted of individual tumor cells.

Tumor cell assays were 0 on days d3, d10 and d17, indicating that CAR-T cells had completely cleared co-cultured tumor cells on the third day after antigen stimulation. From the complete purge of the same number (4X 10 ⁵ ) The depletion resistance of 1355-CAR-T cells on days d3, d10 and d17 was superior to 1326-CAR-T cells and 1502-CAR-T cells in terms of the number of CAR19-T cells depleted by tumor cells, consistent with the previous results of cell survival.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. It should be understood that, based on the technical solutions provided in the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, the description and drawings being presented to the extent that the claims are defined.

Claims

1. A chimeric antigen receptor comprising, in order from amino-terminus to carboxy-terminus, a target binding domain, a hinge region, a transmembrane domain, BRS of CD3 epsilon, a costimulatory domain, and a signaling domain, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 zeta.

2. The chimeric antigen receptor according to claim 1, wherein the target for which the target binding domain corresponds comprises one or more of:

and/or the hinge region is selected from the hinge region of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

and/or the transmembrane domain is selected from the transmembrane region of at least one protein of the group consisting of: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, kirs 2, OX40, CD2, CD27, ICOS, GITR, CD, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2rβ, IL2rγ, IL7rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, LFA-1, ITGAM, CD11b, ITGAX, CD C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, ceam 1, CRTAM 9, PSGL1, CD100, slamfr 6, SLAM, BLAME, SELPLG, LTBR, PAG/bp, NKp44, NKp46 g2, NKG2, and NKG 2.

3. The chimeric antigen receptor according to claim 1 or 2, further comprising a signal peptide, the signal peptide being amino-terminal and linked to the target binding domain.

4. The chimeric antigen receptor according to claim 3, wherein the target binding domain is a single chain antibody targeting CD19, the amino acid sequence of the heavy chain variable region of the single chain antibody targeting CD19 is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region of the single chain antibody targeting CD19 is shown in SEQ ID No. 2;

and/or the amino acid sequence of the signal peptide is shown as SEQ ID NO. 3;

and/or the hinge region is a CD8 alpha hinge region with an amino acid sequence shown as SEQ ID NO. 4;

and/or the transmembrane domain is a CD8 alpha transmembrane region with an amino acid sequence shown as SEQ ID NO. 5;

and/or the amino acid sequence of the 4-1BB is shown as SEQ ID NO. 6;

and/or the BRS amino acid sequence of the CD3 epsilon is shown as SEQ ID NO. 7;

and/or, the amino acid sequence of ITAM1 of CD3 zeta is shown as SEQ ID NO. 8.

5. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 9.

6. An isolated nucleic acid for expressing the chimeric antigen receptor of any one of claims 1 to 5.

7. The isolated nucleic acid of claim 6, wherein the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence set forth in SEQ ID No. 10.

8. A recombinant vector comprising the isolated nucleic acid of claim 7 or 8.

9. A CAR-T cell comprising the isolated nucleic acid of claim 7 or 8 or transformed with the recombinant vector of claim 9.

10. Use of the chimeric antigen receptor of any one of claims 1 to 5, the isolated nucleic acid of claim 6 or 7, the recombinant vector of claim 8 or the CAR-T cell of claim 9 in the manufacture of a medicament for the treatment of cancer.

11. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable adjuvant, the active ingredient comprising an expression vector for expressing the chimeric antigen receptor of any one of claims 1 to 5 or the CAR-T cell of claim 9.