Chimeric antigen receptor with controllable auxin as well as preparation method and application of chimeric antigen receptor

Abstract

The invention provides a chimeric antigen receptor with controllable auxin as well as a preparation method and application thereof, the chimeric antigen receptor comprises at least two subunits, the subunits are mediated by an adaptor, and the adaptor has a switch structure adjusted by the auxin; the invention also provides an immune cell capable of expressing the gene and application of the immune cell. According to the application, a controllable novel CAR-T switch system is constructed by relying on a molecular switch type CAR-T technology and integrating plant hormone response elements, so that the regulation and control of the killing function of CAR-T cells are realized, the toxic and side effects are reduced, the treatment accuracy and controllability are enhanced, and a long-term survival guarantee is provided for tumor patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a chimeric antigen receptor with controllable auxin and a preparation method and application thereof. BACKGROUND

[0002] CAR-T cell is a kind of T cell which is genetically engineered, and its surface expresses chimeric antigen receptor (CAR), which can specifically recognize cancer cell surface antigen, accurately guide T cell to attack cancer cell, and play an anti-tumor role. As an individualized treatment method by reinfusing the patient's own T cells after genetic engineering, it has achieved remarkable success in the treatment of hematological malignancies (such as acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, etc.), and has also been gradually applied in the field of autoimmune diseases. In 2021, 6 CAR-T cell therapy drugs were successively approved for marketing in China, which are used for the treatment of relapsed or refractory multiple myeloma in children / adults.

[0003] CAR-T cell therapy still faces many challenges in the treatment of solid tumors and hematological tumors, such as immunosuppression of tumor microenvironment and exhaustion of CAR-T cells. In order to improve the efficacy and safety, researchers are actively exploring improvement strategies, such as developing new CAR structure, exploring new targets for CAR-T cell therapy, and combining CAR-T therapy with other therapies.

[0004] CAR-T cell technology has two obvious shortcomings in actual treatment cases: toxic side effects of CAR-T cells and non-specific binding of modular CAR structure adapter.

[0005] 1) Toxic side effects of CAR-T cell therapy refer to a series of adverse reactions produced after activated CAR-T cells are reinfused into the human body and continuously kill tumor cells. On the one hand, activated CAR-T cells release a large amount of cytokines, causing inflammatory reactions; on the other hand, a large amount of intracellular substances are released into the blood after tumor cells are rapidly lysed, causing electrolyte imbalance. Under the stimulation of these two factors, some common symptoms such as cytokine release syndrome (CRS) and tumor lysis syndrome (TLS) may occur.

[0006] 2) The adapter of the CAR structure mediated by the adapter may have immunogenicity, which may cause non-specific binding, thereby affecting the targeting, specificity and safety of CAR-T cells. For example, when biotin-streptavidin is used as the adapter, streptavidin and biotin absorbed in the human body may bind, thereby producing side effects. There is also a method of using GCN4 peptide as a target ligand to connect CAR-T cells through anti-GCN4 scFv, which may also have off-target effects, causing non-specific binding.

[0007] Auxin switch system is a technology for conditional gene expression in plant biology, in which the expression of specific genes is controlled by adding or removing auxin, a small molecule plant hormone. Auxin binds to Aux / IAA proteins through TIR1 / AFB receptors, leading to ubiquitination and degradation of Aux / IAA proteins, thereby relieving the inhibition of Auxin Response Factor (ARF) and activating the expression of downstream genes. Currently, the IAA switch system is mainly used to study the functions of various genes, construct biosensors with specific functions, or gene therapy tools. SUMMARY

[0008] In view of the above technical limitations, the present application proposes a new CAR structure, which integrates the auxin switch system into the CAR design, thereby achieving precise regulation of CAR-T activity. Compared with traditional CAR-T cells, this design not only enhances the controllability of T cells, but also improves their killing effect, providing a safer and more efficient strategy for cell therapy.

[0009] To achieve the above object, the following technical solutions are adopted in the present application:

[0010] The application point of the present application is to provide a chimeric antigen receptor, which comprises at least two subunits, the subunits are mediated by an adapter, and the adapter has a switch structure regulated by plant auxin.

[0011] Optionally, the chimeric antigen receptor described above, the plant auxin is selected as Auxin.

[0012] The IAA switch system is an IAA7-TIR1 switch pair, the design structure of the switch pair, and the overall action principle of the chimeric antigen receptor are shown in Figure 1 .

[0013] Optionally, the chimeric antigen receptor described above, at least one subunit (subunit A) of the chimeric antigen receptor comprises a transmembrane domain, an intracellular costimulatory domain and an IAA7 protein domain; at least another subunit (subunit B) comprises a CD3 domain, an intracellular costimulatory domain (CD28 4-1BB domain) and an AFB1 protein domain; the IAA7 protein domain is selected as the amino acid sequence shown in SEQ ID No. 1 or an IAA7 protein domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 1 and having the same or similar function; the AFB1 protein domain is selected as the amino acid sequence shown in SEQ ID No. 2 or an AFB1 protein domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 2 and having the same or similar function.

[0014] The transmembrane domain in subunit A is the CD8a extracellular domain.

[0015] The design structure of subunit is shown as Figure 2 .

[0016] It can be seen from Figure 1 and Figure 2 that auxin binds to Aux / IAA protein through TIR1 / AFB receptor. AFB1 is a natural protein which does not bind to ubiquitinase, so when IAA7 binds to AFB1 protein through Auxin, it is not degraded, thereby causing the target gene connected with AFB1 to be continuously activated.

[0017] The structure of subunit A, the connection sequence is:

[0018] CD8a signal peptide (1) - FMC63 VK (2) - ScFv G4S (3) - FMC63 VH (4) - Hinge (5) - CD8 transmembrane domain (6) - CD28 intra (7) - 4-1BB (8) - IAA7 (Auxin 7 protein) (9);

[0019] The structure of subunit B, the connection sequence is:

[0020] SP1 (1) - DAP10 (2) - CD28 4-1BB co-stimulatory domain (3) - AFB1 protein domain (4) - CD3 intracellular domain (5).

[0021] The IAA7 protein domain, the amino acid sequence is shown as SEQ ID No. 1:

[0022] FSETVDLMLNLQSNKEGSVDLKNVSAVPKEKTTLKDPSKPPAKAQVVG WPPVRNYRKNMMTQQKTSS;

[0023] The AFB1 protein domain, the amino acid sequence is shown as SEQ ID No. 2:

[0024] MGLRFPPKVLEHILSFIDSNEDRNSVSLVCKSWFETERKTRKRVFVGNCYAVSPAAVTRRFPEMRSLTLKGKPHFADYNLVPDGWGGYAWPWIEAMAAKSSSLEEIRMKRMVVTDECLEKIAASFKDFKVLVLTSCEGFSTDGIAAIAATCRNLRVLELRECIVEDLGGDWLSYFPESSTSLVSLDFSCLDSEVKISDLERLVSRSPNLKSLKLNPAVTLDGLVSLLRCAPQLTELGTGSFAAQLKPEAFSKLSEAFSNCKQLQSLSGLWDVLPEYLPALYSVCPGLTSLNLSYATVRMPDLVELLRRCSKLQKLWVMDLIEDKGLEAVASYCKELRELRVFPSEPDLDATNIPLTEQGLVFVSKGCRKLESVLYFCVQFTNAALFTIARKRPNLKCFRLCVIEPFAPDYKTNEPLDKGFKAIAEGCRDLRRLSVSGLLSDKAFKYIGKHAKKVRMLSIAFAGDSDLMLHHLLSGCESLKKLEIRDCPFGDTALLEHAAKLETMRSLWMSSCFVSFGACKLLSQKMPRLNVEVIDEHPPESRPESSPVERIYIYRTVAGPRMDTPEFVWTIHKNPENGVSHLAIK;

[0025] CD8 alpha extracellular domain (CD8 alpha signal peptide) with the murine monoclonal antibody FMC63 VK targeting human CD19 is connected by a short peptide EQKLISEEDL.

[0026] MALPVTALLLPLALLLHAARP;

[0027] The murine monoclonal antibody FMC63 VK targeting human CD19 has the amino acid sequence as shown in SEQ ID No. 4:

[0028] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEIT;

[0029] The CD8 alpha extracellular domain (CD8 alpha signal peptide) is connected with the murine monoclonal antibody FMC63 VK targeting human CD19 by a short peptide EQKLISEEDL.

[0030] ScFv G4S linker, the amino acid sequence of which is shown in SEQ ID No. 5:

[0031] GGGGSGGGGSGGGGS;

[0032] Murine monoclonal antibody FMC63 VH targeting human CD19, the amino acid sequence of which is shown in SEQ ID No. 6:

[0033] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSS;

[0034] Hinge domain, the amino acid sequence of which is shown in SEQ ID No. 7:

[0035] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0036] CD8 transmembrane domain, the amino acid sequence of which is shown in SEQ ID No. 8:

[0037] IYIWAPLAGTCGVLLLSLVITLYC;

[0038] CD28 intracellular domain, the amino acid sequence of which is shown in SEQ ID No. 9:

[0039] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;

[0040] Intracellular signaling domain 4-1BB, the amino acid sequence of which is shown in SEQ ID No. 10:

[0041] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE.

[0042] SP1 (FLAG tag), the amino acid sequence of which is shown in SEQ ID No. 11:

[0043] MALPVTALLLPLSLLLHAARP;

[0044] DAP10, the amino acid sequence of which is shown in SEQ ID No. 12:

[0045] MIHLGHILFLLLLPVAAAQ;

[0046] Spacer sequence, amino acid sequence as shown in SEQ ID No. 13:

[0047] TTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGE;

[0048] CD28 4-1BB costimulatory domain, amino acid sequence as shown in SEQ ID No. 14:

[0049] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEGGCEP;

[0050] CD3 intracellular domain, amino acid sequence as shown in SEQ ID No. 15:

[0051] LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPRG.

[0052] Full-length amino acid sequence of subunit A, amino acid sequence as shown in SEQ ID No. 16:

[0053] MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEFSETVDLMLNLQSNKEGSVDLKNVSAVPKEKTTLKDPSKPPAKAQVVGWPPVRNYRKNMMTQQKTSS;

[0054] Full length amino acid sequence of subunit B, the amino acid sequence is shown as SEQ ID No. 17:

[0055] MALPVTALLLPLSLLLHAARPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEGGCEPMGLRFPPKVLEHILSFIDSNEDRNSVSLVCKSWFETERKTRKRVFVGNCYAVSPAAVTRRFPEMRSLTLKGKPHFADYNLVPDGWGGYAWPWIEAMAAKSSSLEEIRMKRMVVTDECLEKIAASFKDFKVLVLTSCEGFSTDGIAAIAATCRNLRVLELRECIVEDLGGDWLSYFPESSTSLVSLDFSCLDSEVKISDLERLVSRSPNLKSLKLNPAVTLDGLVSLLRCAPQLTELGTGSFAAQLKPEAFSKLSEAFSNCKQLQSLSGLWDVLPEYLPALYSVCPGLTSLNLSYATVRMPDLVELLRRCSKLQKLWVMDLIEDKGLEAVASYCKELRELRVFPSEPDLDATNIPLTEQGLVFVSKGCRKLESVLYFCVQFTNAALFTIARKRPNLKCFRLCVIEPFAPDYKTNEPLDKGFKAIAEGCRDLRRLSVSGLLSDKAFKYIGKHAKKVRMLSIAFAGDSDLMLHHLLSGCESLKKLEIRDCPFGDTALLEHAAKLETMRSLWMSSCFVSFGACKLLSQKMPRLNVEVIDEHPPESRPESSPVERIYIYRTVAGPRMDTPEFVWTIHKNPENGVSHLAIKLRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG.

[0056] A complete chimeric antigen receptor, the amino acid sequence of which is shown as SEQ ID No. 18:

[0057]

[0058] The nucleotide sequences encoding each of the protein domains are shown in SEQ ID No. 19-SEQ ID No. 36, respectively.

[0059] The nucleotide sequence encoding the IAA7 protein domain is shown in SEQ ID No. 19:

[0060] TTCTCTGAGACCGTGGACCTGATGCTGAACCTGCAGTCCAATAAGGAGGGCTCTGTGGATCTGAAGAACGTGAGCGCCGTGCCTAAGGAGAAGACCACACTGAAGGACCCATCCAAGCCCCCTGCCAAGGCACAGGTGGTGGGATGGCCACCCGTGCGGAACTACAGAAAGAATATGATGACCCAGCAGAAGACAAGCTCC;

[0061] The nucleotide sequence encoding the AFB1 protein domain is shown in SEQ ID No. 20:

[0062]

[0063] The nucleotide sequence encoding the extracellular domain of CD8a is shown in SEQ ID No. 21 :

[0064] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG;

[0065] The nucleotide sequence encoding the murine monoclonal antibody FMC63 VK targeting human CD19 is shown in SEQ ID No. 22:

[0066] GATATTCAGATGACCCAGACCACCAGCAGCCTGAGCGCGAGCCTGGGCGATCGCGTGACCATTAGCTGCCGCGCGAGCCAGGATATTAGCAAATATCTGAACTGGTATCAGCAGAAACCGGATGGCACCGTGAAACTGCTGATTTATCATACCAGCCGCCTGCATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTATAGCCTGACCATTAGCAACCTGGAACAGGAAGATATTGCGACCTATTTTTGCCAGCAGGGCAACACCCTGCCGTATACCTTTGGCGGCGGCACCAAACTGGAAATTACC;

[0067] The nucleotide sequence encoding the ScFv G4S linker is shown in SEQ ID No. 23:

[0068] GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC;

[0069] The nucleotide sequence encoding the murine monoclonal antibody FMC63 VH targeting human CD19 is shown in SEQ ID No. 24:

[0070] GAAGTGAAACTGCAGGAAAGCGGCCCGGGCCTGGTGGCGCCGAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCGGATTATGGCGTGAGCTGGATTCGCCAGCCGCCGCGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGGGCAGCGAAACCACCTATTATAACAGCGCGCTGAAAAGCCGCCTGACCATTATTAAAGATAACAGCAAAAGCCAGGTGTTTCTGAAAATGAACAGCCTGCAGACCGATGATACCGCGATTTATTATTGCGCGAAACATTATTATTATGGCGGCAGCTATGCGATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC;

[0071] The nucleotide sequence encoding the hinge domain is shown in SEQ ID No. 25:

[0072] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT;

[0073] The nucleotide sequence encoding the CD8 transmembrane domain is shown in SEQ ID No. 26:

[0074] ATTTATATTTGGGCGCCGCTGGCGGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATTACCCTGTATTGC;

[0075] The CD28 intracellular domain, nucleotide sequence is shown in SEQ ID No. 27:

[0076] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCC;

[0077] A nucleotide sequence encoding an intracellular signaling domain 4-1BB, as set forth in SEQ ID No. 28:

[0078] AAACGCGGCCGCAAAAAACTGCTGTATATTTTTAAACAGCCGTTTATG CGCCCGGTGCAGACCACCCAGGAAGAAGATGGCTGCAGCTGCCGCTTTC CGGAAGAAGAAGAAGGCGGCTGCGAA;

[0079] A nucleotide sequence encoding SP1, as set forth in SEQ ID No. 29:

[0080] ATGGCGCTGCCGGTGACCGCGCTGCTGCTGCCGCTGAGCCTGCTGCT GCATGCGGCGCGCCCG;

[0081] A nucleotide sequence encoding DAP10, as set forth in SEQ ID No. 30:

[0082] ATGATTCATCTGGGCCATATTCTGTTTCTGCTGCTGCTGCCGGTGGCG GCGGCGCAG;

[0083] A nucleotide sequence encoding a spacer sequence, as set forth in SEQ ID No. 31:

[0084] ACCACCCCGGGCGAACGCAGCAGCCTGCCGGCGTTTTATCCGGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCGCTGCTGGCGGGCCTGGTGGCGGCGGATGCGGTGGCGAGCCTGCTGATTGTGGGCGCGGTGTTTCTGTGCGCGCGCCCGCGCCGCAGCCCGGCGCAGGAAGATGGCAAAGTGTATATTAACATGCCGGGCCGCGGCGAA;

[0085] A nucleotide sequence encoding a CD28 4-1BB costimulatory domain, as set forth in SEQ ID No. 32:

[0086] ACCACCACCCCGGCGCCGCGCCCGCCGACCCCGGCGCCGACCATTGCGAGCCAGCCGCTGAGCCTGCGCCCGGAAGCGTGCCGCCCGGCGGCGGGCGGCGCGGTGCATACCCGCGGCCTGGATTTTGCGTGCGATATTTATATTTGGGCGCCGCTGGCGGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATTACCCTGTATTGCCGCAGCAAACGCAGCCGCCTGCTGCATAGCGATTATATGAACATGACCCCGCGCCGCCCGGGCCCGACCCGCAAACATTATCAGCCGTATGCGCCGCCGCGCGATTTTGCGGCGTATCGCAGCAAACGCGGCCGCAAAAAACTGCTGTATATTTTTAAACAGCCGTTTATGCGCCCGGTGCAGACCACCCAGGAAGAAGATGGCTGCAGCTGCCGCTTTCCGGAAGGCGGCTGCGAACCG;

[0087] The nucleotide sequence encoding the CD3 intracellular domain is as shown in SEQ ID No. 33:

[0088] CTGCGCGTGAAATTTAGCCGCAGCGCGGATGCGCCGGCGTATAAACAGGGCCAGAACCAGCTGTATAACGAACTGAACCTGGGCCGCCGCGAAGAATATGATGTGCTGGATAAACGCCGCGGCCGCGATCCGGAAATGGGCGGCAAACCGCGCCGCAAAAACCCGCAGGAAGGCCTGTATAACGAACTGCAGAAAGATAAAATGGCGGAAGCGTATAGCGAAATTGGCATGAAAGGCGAACGCCGCCGCGGCAAAGGCCATGATGGCCTGTATCAGGGCCTGAGCACCGCGACCAAAGATACCTATGATGCGCTGCATATGCAGGCGCTGCCGCCGCGCGGC.

[0089] The nucleotide sequence encoding the subunit A full-length sequence is as shown in SEQ ID No. 34:

[0090]

[0091] The nucleotide sequence encoding the full-length sequence of subunit B is shown as SEQ ID No. 35:

[0092]

[0093] The nucleotide sequence encoding the complete chimeric antigen receptor is shown as SEQ ID No. 36:

[0094]

[0095] Optionally, the chimeric antigen receptor as described above can recognize at least one or more of the following antigens: CD19, CD20, CD22, CD30, CD33, CD99, CD123, CD133, CD138, CD171, Her2.

[0096] Optionally, the chimeric antigen receptor as described above further comprises a CD8 signal peptide, a hinge domain, a CD28 intracellular domain, and a CD3 intracellular domain.

[0097] The second invention point of the present application is to provide an isolated immune cell, which is modified to express the chimeric antigen receptor as described above, the chimeric antigen receptor comprising at least two subunits mediated by an adaptor having a switch structure regulated by auxin; and the immune cell is preferably a T cell.

[0098] Meanwhile, the mesenchymal stem cells MSC can also be modified to express the chimeric antigen receptor as described above comprising at least two subunits mediated by an adaptor having a switch structure regulated by auxin.

[0099] Optionally, the isolated immune cell as described above comprises one or more of the following: T cells, B cells, K cells, NK cells, mast cells, and mononuclear phagocytes; and the immune cell is preferably a T cell.

[0100] The third invention point of the present application is to provide an expression vector encoding the chimeric antigen receptor as described above.

[0101] The fourth invention point of the present application is to provide a host cell comprising the expression vector as described above.

[0102] The fifth invention point of the present application is to provide the use of the chimeric antigen receptor as described above, the isolated immune cell as described above, the expression vector as described above, and the host cell as described above in the preparation of a medicament for treating a tumor disease; and the tumor disease is preferably a hematological malignancy.

[0103] The sixth invention point of the present application is to provide a pharmaceutical composition for treating a tumor disease, which comprises the chimeric antigen receptor as described above, the isolated immune cell as described above, the expression vector as described above, and the host cell as described above.

[0104] The seventh application of the present application is to provide a method for preventing or treating a tumor disease, which is achieved by directly or indirectly administering the above-mentioned chimeric antigen receptor, the above-mentioned isolated immune cell, the above-mentioned expression vector, the above-mentioned host cell, and the above-mentioned pharmaceutical composition to the body to prevent or treat the tumor disease.

[0105] In the prior art, a reversible ON / OFF switch chimeric antigen receptor controlled by lenalidomide is disclosed, which belongs to a CAR structure mediated by an adapter. Lenalidomide belongs to a small molecule adapter, which changes the substrate specificity of an E3 ubiquitin ligase complex by binding to CRBN, thereby leading to ubiquitination and proteasome degradation of certain proteins, producing certain immunomodulatory effects and anti-angiogenic properties in the body, and showing significant efficacy in hematological malignancies such as multiple myeloma and myelodysplastic syndrome. In clinical applications, lenalidomide is associated with various side effects, such as hematological toxicity, thrombosis, teratogenicity, and nervous system side effects.

[0106] Therefore, when using lenalidomide as an adapter-mediated CAR structure activation, on the one hand, the dosage of lenalidomide needs to be closely monitored and adjusted to avoid toxicity and side effects; on the other hand, in order to prevent ubiquitination and inactivation of part of the proteins in the CAR-T cell, the T cell needs to be modified to mutate all the intracellular lysine residues to arginine residues, which increases the complexity of the preparation of CAR-T cells.

[0107] Compared with the prior art, the present application has the following advantages:

[0108] The present application relies on the molecular switch type CAR-T technology, integrates plant hormone response elements, and constructs a controllable new CAR-T switch system, thereby realizing the regulation and control of the killing function of CAR-T cells, reducing toxicity and side effects, enhancing treatment precision and controllability, and providing long-term survival protection for tumor patients.

[0109] The present application uses plant auxins as adapters to mediate the connection of CAR structures. By connecting the auxin co-receptor IAA7 with the E3 plant ubiquitinase mutant AFB1 mutant at the E7 / E10 site, it is not connected with the ubiquitinase of mammals, and will not cause cytotoxicity, greatly reducing the generation of toxicity and the occurrence of side effects; the plant auxin indole acetic acid will not bind to endogenous proteins and cytokines of mammalian cells and produce immunoregulation, thereby ensuring the specificity and safety of CAR-T cells. BRIEF DESCRIPTION OF DRAWINGS

[0110] Figure 1Schematic diagram of the principle of the chimeric antigen receptor loaded with T cells and controlled by plant auxin, which plays a killing role; wherein, the auxin (Auxin) can be combined with the Aux / IAA protein through the TIR1 / AFB receptor; when the AFB1 protein and the IAA7 protein are respectively loaded on the two subunits of the chimeric antigen receptor (CAR), the CAR is in a closed state; when a certain concentration of Auxin is added, the two subunits of the CAR are connected through the Auxin, thereby being activated; and since the AFB1 is a natural protein, it will not be combined with the ubiquitination enzyme, so when the IAA7 and the AFB1 protein are combined through the Auxin, they will not be degraded, thereby causing the target gene connected with the AFB1 to be continuously activated.

[0111] Figure 2 Schematic diagram of the structure of the chimeric antigen receptor Auxin-CAR regulated by plant auxin in an embodiment of the application.

[0112] Figure 3 The in vitro binding diagram of the E7 / E10 site E3 ubiquitinase mutant AFB1 mutant and the auxin co-receptor IAA7 detected by the fluorescence confocal microscope in an embodiment of the application, it can be seen from the diagram that after the addition of auxin induction, single cells can be observed, and the mutant-mcherry fusion protein in the cytoplasm is combined with the IAA7-GFP fusion protein on the cell membrane.

[0113] Figure 4 The combination of the AFB1 mutant-mcherry fusion protein and the IAA7-GFP fusion protein is verified by the fluorescence resonance energy transfer experiment (FRET) in an embodiment of the application; wherein, Figure 4 A is a fluorescence lifetime decay fitting curve, Figure 4 B is a fluorescence lifetime statistical diagram of the IAA7-GFP fusion protein in the presence or absence of auxin (auxin), p<0.01.

[0114] Figure 5 The activation of the auxin receptor AFB1 and the co-receptor IAA7 transduced Jurkat T cells in response to auxin induction is verified by the in vitro killing experiment in an embodiment of the application; wherein, Figure 5 A is the flow cytometry detection result, which compares the response and killing of the Jurkat T cell line under different CAR structures and different auxin concentrations, Figure 5 B is a statistical diagram of the flow cytometry detection result, p<0.001.

[0115] Figure 6As shown in the small animal live imaging experiment in the embodiment of the present application, the therapeutic effects of AUX-CAT-T cells and WT-CAR-T cells were compared in the cell immunity experiment of C-NKG tumor-bearing mice; wherein, the experiment was divided into four groups. The results showed that the therapeutic effects of the AUX CAR NO auxin group and the PBMC group were the same without the induction of auxin; and the therapeutic effect of the AUX CAR with auxin group was significantly improved compared with that of the WT CAR. DETAILED DESCRIPTION

[0116] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail. However, it should be understood that the description herein is only used to explain the present application and is not intended to limit the scope of the present application.

[0117] Unless otherwise defined, 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, and the terms used herein in the specification are only for the purpose of describing the specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are commercially available, and the characterization means involved can be referred to the related description in the prior art, which will not be described herein.

[0118] In order to further understand the present application, the present application will be further described in detail in combination with the best embodiments.

[0119] The sources of the reagents involved in the present application are as follows:

[0120] Auxin: Sigma, 87514.

[0121] Dimethyl sulfoxide (DMSO): Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0122] SYTOX TM Blue: Thermo, MP34857.

[0123] PBS: Biosharp, BL310A.

[0124] In order to more clearly show the detection results, the inventors constructed a fusion protein of IAA7 and green fluorescent protein GFP and a fusion protein of AFB1 and red fluorescent protein mcherry.

[0125] The green fluorescent protein (GFP) constructed into a fusion protein with the IAA7 protein domain, and the amino acid sequence is shown in SEQ ID No. 37:

[0126] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*;

[0127] mCherry, a red fluorescent protein fused with AFB1 protein domain, amino acid sequence as shown in SEQ ID No. 38:

[0128] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0129] The nucleotide sequence encoding green fluorescent protein (GFP) is shown in SEQ ID No. 39:

[0130] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA;

[0131] The nucleotide sequence encoding the red fluorescent protein (mCherry) is shown in SEQ ID No. 40:

[0132] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG.

[0133] Example 1

[0134] Verification of spatial co-localization of AFB1-mcherry fusion protein and IAA7-GFP fusion protein:

[0135] AFB1-mcherry fusion protein and IAA7-GFP fusion protein were transfected into 293T cells simultaneously. After 12 hours, auxin and dimethyl sulfoxide (DMSO) were added for induction for 12 hours, and then the combination was observed by fluorescence confocal microscopy.

[0136] Under the fluorescence confocal microscope, it was observed that the IAA7-GFP fusion protein was expressed on the cell membrane, while the AFB1-mcherry fusion protein was expressed in the cytoplasm. After auxin induction for 12 hours, it was observed that the AFB1-mcherry fusion protein was significantly bound to the cell membrane. While after dimethyl sulfoxide (DMSO) induction for 12 hours, the AFB1-mcherry fusion protein was still expressed in the cytoplasm. Figure 3

[0137] It was verified that the AFB1-mcherry fusion protein and the IAA7-GFP fusion protein could bind to the cell membrane under auxin induction, thereby proving their spatial co-localization.

[0138] Example 2

[0139] Fluorescence resonance energy transfer (FRET) verification of the AFB1-mcherry fusion protein and the IAA7-GFP fusion protein:

[0140] The fusion protein plasmid with IAA7-GFP and the AFB1-mcherry fusion protein plasmid were transfected into 293T cells, and then auxin induction was performed for 12 hours, and the fluorescence lifetime of the IAA7-GFP fusion protein was tested.

[0141] It was found that in the cells co-transfected with the IAA7-GFP fusion protein and the AFB1-mcherry fusion protein, if auxin was added, the fluorescence lifetime of the IAA7-GFP fusion protein decreased, and there was a difference (p<0.001) Figure 4 ). Among them Figure 4 A is the fluorescence lifetime decay fitting curve, Figure 4 B is the statistical result of the fluorescence lifetime of the IAA7-GFP fusion protein with and without auxin.

[0142] The experimental results confirmed that the AFB1-mcherry fusion protein and the IAA7-GFP fusion protein could efficiently bind in cells.

[0143] Example 3

[0144] Auxin receptor AFB1 and co-receptor IAA7 transduced Jurkat T cell line response to auxin induction activation:

[0145] ​After verifying that the AFB1-mcherry fusion protein and the IAA7-GFP fusion protein could bind efficiently in cells under auxin induction, the inventors separated these two proteins into two subunits of the CAR structure. These were transduced into the Jurkat T cell line via lentivirus and co-cultured with CD19 knockout K562 cells and CD19 overexpressing A431 cells at a cell ratio of 3:1 for 16 hours. A WT-CAR-Jurkat T cell group was also established. The proportion of sytox blue cells (damaged cell membrane population) in K562 cells and RAJI cells was then analyzed by flow cytometry.

[0146] WT-CAR-Jurkat T cells were co-cultured with K562 cells and RAJI cells, respectively, while AUX-CAR-Jurkat T cells were co-cultured with RAJI cells, with 0 μM, 0.5 μM, and 1 μM auxin added during co-culture.

[0147] Experiments revealed that SYTOX was detected in RAJI cell populations under different concentrations of auxin. TM The proportion of blue cells increased significantly with increasing auxin concentration. However, in the absence of auxin, the proportion of sytox blue cells in RAJI cells was comparable to that of CD19 knockout K562 cells, demonstrating that AUX-CAT and WT-CAR have the same function in detecting CD19 signaling. Figure 5 ).

[0148] Example 4

[0149] The technology described in this application has been proven feasible through relevant experimental results using primary cells.

[0150] 1. Experimental materials:

[0151] The construction of the human CD19-CAR plasmid was completed by Kingwise Biotechnology Co., Ltd. The lentiviral vector was pCDH, and the packaging plasmids used were psPAX2 (expressing the lentiviral capsid) and Pmd2.G (expressing the lentiviral membrane protein).

[0152] 2. Plasmid design for human CAR-T-CD19:

[0153] Plasmid 1 is subunit A of CAR, and its gene structure consists of 9 parts, connected in the following order:

[0154] CD8a signal peptide (1) - FMC63 VK (2) - ScFv G4S (3) - FMC63 VH (4) - Hinge (5) - CD8 transmembrane domain (6) - CD28 intra (7) - 4-1BB (8) - IAA7 (Integrin alpha 7 protein) (9).

[0155] The protein sequences corresponding to the genes are shown in SEQ ID No. 3 to SEQ ID No. 10 and SEQ ID No. 1, respectively.

[0156] The full-length amino acid sequence of Subunit A is shown in SEQ ID No. 16.

[0157] The specific construction of the plasmid was entrusted to Jinweizhi Biotechnology Co., Ltd.

[0158] 3. Plasmid 2 design of human CAR-T-CD19:

[0159] Plasmid 2 is Subunit B of CAR, and its gene structure includes 5 parts, with the following connection order:

[0160] SP1 (1) - DAP10 (2) - CD28 4-1BB co-stimulatory domain (3) - AFB1 protein domain (4) - CD3 intracellular domain (5).

[0161] The protein sequences corresponding to the genes are shown in SEQ ID No. 11 to SEQ ID No. 15 (including spacer sequence SEQ ID No. 13) and SEQ ID No. 2, respectively.

[0162] The full-length amino acid sequence of Subunit B is shown in SEQ ID No. 17.

[0163] The specific construction of the plasmid was entrusted to Jinweizhi Biotechnology Co., Ltd.

[0164] 4. Preparation of CD19-CAR virus supernatant 1 and supernatant 2:

[0165] PEI transfection was used, and the transfection system was as follows: 4 μg of target plasmid (plasmid 1 or plasmid 2); 3 μg of plasmid psPAX2; 1 μg of Pmd2.G; 32 μg of PEI.

[0166] The above system was mixed with 200 μL DMEM medium (Gibco, C11995500BT) and left for 15 min; then 293T cells with a density of about 60% were added in a 6 cm cell culture dish. After 12 h, 5 mL of fresh culture solution was added. Then the supernatant was collected every 24 h, and 5 mL of fresh culture solution was added. After 3 times, the supernatant was filtered with a 0.45 μm filter, concentrated by ultrafiltration tube, centrifuged at 20000 g, 4 h, 4°C.

[0167] 5. Preparation of human CD19-CAR-T:

[0168] Human peripheral blood was collected after lymphocyte separation medium to collect PBMC, and CD3 + T cells were obtained by magnetic bead sorting, and the T cells were placed in complete culture solution containing CD3 / CD28 antibody (30 ng / mL) and IL-2 cytokine (500 IU / mL). After 24 h, concentrated virus supernatant 1 was added. After 6 h, the virus supernatant 1 was removed by centrifugation, replaced with complete culture solution, and placed in a 37°C cell incubator for culture. After 8 h, the cell precipitate was collected, and concentrated virus supernatant 2 was added. After 6 h, the virus supernatant 2 was removed by centrifugation, replaced with complete culture solution, and placed in a 37°C cell incubator for culture. After 48 h, the expression of CD19-CAR on the surface of T cells was detected by flow cytometry.

[0169] The full-length amino acid sequence of CD19-CAR is shown in SEQ ID No. 41:

[0170] MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGDGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0171] 6、Human primary Aux-CAR-T CD8+ cells and human primary CAR-T CD8 + Cellular immunotherapy of C-NKG tumorigenic mice by human primary Aux-CAR-T CD8+ cells and human primary CAR-T CD8

[0172] 3-week-old C-NKG female mice were used for the experiment after 1-week adaptation. A total of 4 groups of mice were set up, with 3 mice in each group. For each group of mice, 5*10 6The mice were injected with the cells via the tail vein, and the total volume of each injection was 200 μl.

[0173] The mice were injected with the cells via the tail vein, and the total volume of each injection was 200 μl.

[0174] Four groups were set up, including:

[0175] PBMC group: The mice in the group were injected with 3*10 6 human primary PBMC cells;

[0176] AUX CAR NO auxin group: The mice in the group were injected with 3*10 6 human primary Aux-CAR-T CD8 + cells;

[0177] WT CAR group: The mice in the group were injected with 3*10 6 human primary CAR-T CD8 + cells;

[0178] AUX CAR with auxin: The mice in the group were injected with 3*10 6 human primary Aux-CAR-T CD8 + cells, and were given auxin by gavage every 3 days.

[0179] The amount of gavage: 8-12 mg per mouse, preferably 10 mg per mouse.

[0180] The above four groups were imaged once every 7 days. Figure 6 The results show that, without the induction of auxin, the treatment effect of the AUX CAR NO auxin group and the PBMC group is the same; and the treatment effect of the AUX CAR with auxin group is significantly improved compared with the WT CAR.

[0181] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises at least two subunits, which are mediated by an adaptor having a switch structure regulated by plant auxin.

2. The chimeric antigen receptor according to claim 1, characterized in that, The plant growth hormone selected was Auxin.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that, At least one subunit of the chimeric antigen receptor includes a transmembrane domain, an intracellular co-stimulatory domain, and an IAA7 protein domain; at least another subunit includes a CD3 domain, an intracellular co-stimulatory domain, and an AFB1 protein domain; the IAA7 protein domain is selected as the amino acid sequence shown in SEQ ID No. 1 or an IAA7 protein domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 1 and having the same or similar functions; the AFB1 protein domain is selected as the amino acid sequence shown in SEQ ID No. 2 or an AFB1 protein domain having more than 80% homology with the amino acid sequence shown in SEQ ID No. 2 and having the same or similar functions.

4. The chimeric antigen receptor according to claim 3, characterized in that, The chimeric antigen receptor can recognize at least one or more of the following: CD19, CD20, CD22, CD30, CD33, CD99, CD123, CD133, CD138, CD171, and Her2.

5. The chimeric antigen receptor according to claim 4, characterized in that, The chimeric antigen receptor also includes a CD8 signal peptide, a hinge domain, a CD28 intracellular domain, and a CD3 intracellular domain.

6. An isolated immune cell, said isolated immune cell being modified to express the chimeric antigen receptor of any one of claims 1-5, said chimeric antigen receptor comprising at least two subunits mediated by an adaptor, said adaptor having a switch structure regulated by auxin; said immune cell preferably a T cell.

7. The isolated immune cells according to claim 6, characterized in that, The immune cells include, but are not limited to, one or more of T cells, B cells, K cells, NK cells, mast cells, and mononuclear phagocytes; preferably T cells.

8. An expression carrier, characterized in that, The expression vector encodes the chimeric antigen receptor according to any one of claims 1-5.

9. A host cell, characterized in that, The host cell includes the expression vector according to claim 8.

10. The use of the chimeric antigen receptor according to any one of claims 1-5, the isolated immune cell according to claim 6 or 7, the expression vector according to claim 8, and the host cell according to claim 9 in the preparation of a medicament for treating tumor diseases; wherein the tumor disease is preferably a hematologic malignancy.

11. A pharmaceutical composition for treating tumor diseases, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor as described in any one of claims 1-5, the isolated immune cells as described in claim 6 or 7, the expression vector as described in claim 8, and the host cell as described in claim 9.

12. A method for preventing or treating tumor diseases, characterized in that, The prevention or treatment of tumor diseases is achieved by directly or indirectly administering to the body the chimeric antigen receptor as described in any one of claims 1-5, the isolated immune cells as described in claim 6 or 7, the expression vector as described in claim 8, the host cell as described in claim 9, and the pharmaceutical composition as described in claim 11.

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