CONDITION-CONTROLLED SPLICEABLE CHIMERIC ANTIGEN RECEPTOR MOLECULE AND APPLICATION THEREOF

Abstract

A condition-controlled spliceable chimeric antigen receptor molecule and the use thereof.

The spliceable chimeric antigen receptor molecule comprises an antigen recognition unit and a signal transduction unit; the antigen recognition unit comprises an antigen recognition domain, a transmembrane domain, a costimulatory signal domain, an N-terminal splicing domain, and a degrader; and the signal transduction unit comprises a conditional signal response domain, a C-terminal splicing domain, and a signaling domain. Such a condition-controlled spliceable system can achieve splicing of the two units and signaling under a tumor microenvironment signal. The antigen recognition unit can spontaneously/be induced to degrade, thus reducing retention in normal tissues. A signaling unit can respond to a specific condition signal of a tumor microenvironment, and has the characteristics of low expression in a normal tissue environment and high expression in the tumor microenvironment. The condition-controlled spliceable system can achieve preparation of drugs and precise treatment for solid tumors by grafting different functional genes.

Claims

1. A condition-controlled spliceable chimeric antigen receptor molecule, comprising an antigen recognition unit and a signal transduction unit, wherein the antigen recognition unit comprises an antigen recognition domain, a transmembrane domain, a costimulatory signal domain, an N-terminal splicing domain, and a degrader; and the signal transduction unit comprises a conditional signal response domain, a C-terminal splicing domain, and a signaling domain.

2. The chimeric antigen receptor molecule according to claim 1, wherein an antigen to which the antigen recognition domain binds is one or more selected from CD47, AXL, EGFR, CD7, CD24, FAP, CD147, HER2, ROR1, ROR2, CD133, EphA2, CD171,CEA, EpCAM, TAG72, IL-13R, EGFRVIII, GD2, FR, PSCA, PSMA, GPC3, CAIX, Claudin18.2, VEGFR2, PD-L1, MSLN, MUCI, c-Met, B7-H3 or TROP2 antigen; the transmembrane domain is one or more selected from CD3, CD4, CD8 , CD28 or CD137/4-1BB transmembrane domain; more preferably, the transmembrane domain is the CD28 transmembrane domain; the costimulatory domain in the antigen recognition unit is one or more selected from CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,TLR7, TLR8, TLR9, TLR10, TLR11 or Dap10 costimulatory domain; preferably, the N-terminal splicing domain is one or more selected from a protein intron or a SpyTag/SpyCatcher self-assembler; and degrader is one or more selected from a dihydrofolate reductase (DHFR), an estrogen receptor (ER), a Salmonella type III secretion system effector protein (SopE), a plant hormone-inducible protein degrader, an unstable domain (AD) or a photosensitive protein degrader; more preferably, the degrader is one or more selected from an estrogen receptor or a Salmonella type III secretion system effector protein.

3. The chimeric antigen receptor molecule according to claim 1, wherein the conditional signal response domain is one or more selected from an oxygen-dependent degradation domain (ODD), a temperature-sensitive domain, a pH-sensitive domain, a photosensitive domain or an inflammatory cytokine response domain; the C-terminal splicing domain is one or more selected from a protein intron or a SpyTag/SpyCatcher self-assembler; and the signaling domain is one or more selected from a CD3, FcRIII, FcRI or Fc receptor signaling domain or an immunoreceptor tyrosine-based activation motif (ITAM)-carrying signaling molecule.

4. The chimeric antigen receptor molecule according to claim 3, wherein the signal transduction unit further comprises a costimulatory signal domain; and the costimulatory signal domain in the signal transduction unit is one or more selected from CD2, CD27, CD28, CD40, OX40, CD137/4-1BB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11 or Dap10 costimulatory signal domain.

5. The chimeric antigen receptor molecule according to claim 1, wherein an amino acid sequence of the antigen recognition unit is shown in SEQ NO: 1 or SEQ NO: 2; and/or an amino acid sequence of the signal transduction unit is shown in SEQ NO: 3.

6. A nucleic acid molecule coding the chimeric antigen receptor molecule according to claim 1.

7. A vector comprising the nucleic acid molecule according to claim 6, wherein the vector is one or more selected from a plasmid, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia virus vector, a herpes simplex virus vector, a forest encephalitis virus vector, a poliovirus vector, a Newcastle disease virus vector or a transposon.

8. A genetically engineered host cell comprising the exogenous nucleic acid molecule according to claim 6 that is integrated in its chromosome.

9. A method for preparing the genetically engineered host cell comprising: introducing into the host cell the nucleic acid molecule according to claim 6.

10. A method for cellular immunotherapy comprising: introducing into a host cell the nucleic acid molecule according to claim 6, and administrating to a subject in need thereof a therapeutically effective amount of the host cell.

11. The chimeric antigen receptor molecule according to claim 2, wherein the N-terminal splicing domain is a protein intron Int.sup.N.

12. The chimeric antigen receptor molecule according to claim 2, wherein the degrader is a mutant estrogen receptor (ERm).

13. The chimeric antigen receptor molecule according to claim 3, wherein the C-terminal splicing domain is a protein intron Int.sup.C.

14. The nucleic acid molecule according to claim 6, wherein in the nucleic acid molecule, a nucleotide sequence coding the signal transduction unit also comprises a nucleotide sequence coding a conditional signal response element, which is one or more selected from a hypoxia response element (HRE), a temperature-sensitive element, a pH-sensitive element, a photosensitive element or an inflammatory cytokine response element.

15. The nucleic acid molecule according to claim 14, wherein the nucleotide sequence coding the signal transduction unit is shown in SEQ NO: 4.

16. The nucleic acid molecule according to claim 6, wherein a nucleotide sequence of the nucleic acid molecule is shown in SEQ NO: 5 or 6.

17. The genetically engineered host cell according to claim 8, wherein the host cell is one or more selected from an isolated human-derived cell or a genetically engineered immune cell; the isolated human-derived cell is one or more selected from an embryonic stem cell, an umbilical cord blood-derived stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose-derived stem cell, a T cell, an NK cell, an NKT cell or a macrophage; and the genetically engineered immune cell is one or more selected from a genetically engineered T cell, NK cell, NKT cell or macrophage.

18. The genetically engineered host cell according to claim 17, wherein the genetically engineered immune cell is one or more selected from a chimeric antigen receptor T cell (CAR-T cell), a chimeric antigen receptor NK cell (CAR-NK cell), a chimeric antigen receptor NKT cell (CAR-NKT cell), a chimeric antigen receptor macrophage (CAR-m) or a T cell receptor T cell (TCR-T cell).

19. The method for cellular immunotherapy according to claim 10, wherein the method for cellular immunotherapy is useful for treating a hypoxic disease.

20. The method for cellular immunotherapy according to claim 19, wherein the hypoxic disease is a cancer, which is one or more selected from neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer or brain cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. In drawings:

[0060] FIG. 1 shows lentiviral expression plasmid profiles of conventional CD47 CAR (CD47-28BBZ, FIG. 1a), hypoxia-regulated CD47 CAR-ODD fused with an oxygen-dependent degradation domain ODD (CD47-28BBz-ODD, FIG. 1b), an antigen recognition unit 1 (CD47-28BB-Int.sup.N-SopE, FIG. 1c), an antigen recognition unit 2 (CD47-28BB-Int.sup.N-ERm, FIG. 1d), a hypoxia-regulated signal transduction unit (ODD-Int.sup.C-CD3z, FIG. 1e), a two-in-one condition-controlled (hypoxia-regulated) spliceable system 1 (CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z, FIG. 1f) and a two-in-one condition-controlled (hypoxia-regulated) spliceable system 2 (CD47-28BB-Int.sup.N-ERm-HRE-ODD-Intc-CD3z, FIG. 1g).

[0061] FIGS. 2a-c show the spontaneous degradation of the antigen recognition unit 1(CD47-28BB-Int.sup.N-SopE) and the antigen recognition unit 2 (CD47-28BB-Int.sup.N-ERm). FIG. 2a shows a structure and composition of the antigen recognition unit; FIG. 2b shows flow cytometry detection results of CD47 CAR fused with an oxygen-dependent degradation domain ODD alone, an antigen recognition unit 1 fused with a degrader SopE and an antigen recognition unit 2 fused with a degrader ERm on a 293T cell membrane; and FIG. 2c shows a statistical result of flow cytometry detection data in FIG. 2b, wherein the spontaneous degradation capabilities of the degraders SopE and ERm are significantly superior to that of the oxygen-dependent degradation domain ODD, and ERm has the best degradation capability.

[0062] FIGS. 3a-d show splicing activities of the antigen recognition unit 1 (CD47-28BB-Int.sup.N-SopE) and the antigen recognition unit 2 (CD47-28BB-Int.sup.N-ERm). FIG. 3a shows structures and compositions of degrader-fused CAR and a hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z; FIG. 3b shows CAR expression levels of an engineered 293T cell that integrates the CAR and the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z, and an engineered 293T cell that integrates the degrader-fused CAR and the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z under a hypoxic condition, wherein Blank is a flow cytometry detection result of a negative 293T cell, and Mock indicates an engineered 293T cell without the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z, that is, an engineered 293T cell that is transduced with a no-load virus; FIG. 3c shows an induction fold of CAR expression levels after splicing under the hypoxic condition; and FIG. 3d shows results of western blotting detection for three genetically engineered 293T cells which integrate the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z at the same time and are collected after being cultured for 24 h under a normoxic condition (21% O.sub.2) and a hypoxic condition (1% O.sub.2) respectively.

[0063] FIG. 4 shows flow cytometry detection results of CAR molecules on the cell membranes of engineered T cells that transduce the CD47 CAR (CD47-28BBZ, z representing a CD3 signaling domain), the antigen recognition unit 1 (CD47-28BB-Int.sup.N-SopE), the two-in-one condition-controlled (hypoxia-regulated) spliceable system 1 (CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z), the antigen recognition unit 2 (CD47-28BB-Int.sup.N-ERm) and the two-in-one condition-controlled (hypoxia-regulated) system spliceable 2 (CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z). FIG. 4b shows a statistical result of the flow cytometry detection data in FIG. 4a.

[0064] FIG. 5 shows normoxia-and hypoxia-dependent cell killing results of engineered T cells that transduce the CD47 CAR (CD47-28BBz), the antigen recognition unit 1 (CD47-28BB-Int.sup.N-SopE), the two-in-one condition-controlled (hypoxia-regulated) spliceable system 1 (CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z), the antigen recognition unit 2 (CD47-28BB-Int.sup.N-ERm) and the two-in-one condition-controlled (hypoxia-regulated) spliceable system 2 (CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z). Target cells in FIG. 5a are of an ovarian cancer tumor cell line SKOV3; and target cells in FIG. 5b are of a lung cancer tumor cell line NCI-H292.

[0065] FIG. 6 shows in-vivo antitumor activities of the engineered T cells that transduce the two-in-one condition-controlled (hypoxia-regulated) spliceable system 1 (CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z) and the two-in-one condition-controlled (hypoxia-regulated) spliceable system 2 (CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z). FIG. 6a is an experimental flow chart; and FIG. 6b is a statistical result of tumor sizes after infusion of the engineered T cell for 30 days.

MODE OF CARRYING OUT THE INVENTION

[0066] The present invention is further described below in conjunction with specific examples, and the advantages and characteristics of the present invention will be clearer with the description.

[0067] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention.

[0068] Experimental methods used in the following examples were conventional experimental methods in the art unless otherwise specified. Experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent sales companies, wherein: [0069] DMEM medium and RPMI1640 medium were purchased from Corning, and lymphocyte medium X-VIVO 15 was purchased from Lonza. [0070] T cell growth medium, which was composed of a basal medium and cytokines, was prepared with reference to the Chinese invention patent CN201910163391.1. The basal medium was a lymphocyte medium X-VIVO 15, and the cytokines, i.e., 5 ng/ml IL-7, 10 ng/ml IL-15 and 30 ng/ml IL-21 were added. The cytokines IL-7 and IL-15 were purchased from R&D, and IL-21 was purchased from Nearshore Protein Technology Co., Ltd. [0071] Fetal bovine serum was purchased from BI Inc. [0072] A TurboFect Transfection Kit was purchased from Thermo Fisher Scientific. [0073] A Lenti-X lentiviral concentrate reagent was purchased from Takara, Inc. [0074] Gene synthesis was completed by Shanghai Generay Bioengineering Co., Ltd. [0075] Blank lentiviral expression plasmids (pXW-EF1-MCS-P2A-EGFP and pXW-EF1-BFP-MCS1-HRE-MCS2) were derived from a commercial plasmid ABpCCLsin-EF1-MCS purchased from Kanglin Biotechnology (Hangzhou) Co., Ltd. A green fluorescent protein gene and a blue fluorescent protein gene were respectively recombined on this commercial plasmid to obtain the blank lentiviral expression plasmids. A packaging plasmid psPAX2 and an envelope plasmid PMD2.G were purchased from Addgene. [0076] Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. [0077] An endotoxin-free plasmid miniprep kit and an endotoxin-free plasmid midiprep kit were purchased from OMEGA and Macherey Nagel, respectively. [0078] A luciferase substrate was purchased from Promega Biotechnology Co., Ltd. [0079] 293T cells, A549 lung cancer cells, SKOV3 ovarian cancer cells, and NCI-H292 lung cancer cells were purchased from ATCC in the United States. SKOV3-luc and NCI-H292-luc, which stably integrated a firefly luciferase gene respectively, were obtained by transduction of SKOV3 ovarian cancer cells and NCI-H292 lung cancer cells using a lentivirus carrying the firefly luciferase gene. [0080] Severe combined immuno-deficient mice (B-NDG) were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.

Example 1: Composition of Condition-Controlled Spliceable Chimeric Antigen Receptor Molecule

[0081] An amino acid sequence of the antigen recognition unit was shown as SEQ NO: 1 or SEQ NO: 2. SEQ NO: 1 or SEQ NO: 2 was composed of sequences of a CD47 antigen recognition 5 domain, a CD28 transmembrane domain, a CD28 and 4-1BB costimulatory signal domain, an N-terminal splicing domain and a degrader, respectively. The amino acid sequences of the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 and 4-1BB costimulatory signal domain, the N-terminal splicing domain and the degrader were specifically shown in Table 1.

TABLE-US-00001 TABLE1 Aminoacidsequenceofantigenrecognitionunit Name Aminoacidsequence CD47antigen MALPVTALLLPLALLLHAARPDYKDDDDKEVQLVESGGGLVQP recognitiondomain GGSLRLSCAASGFTFSGYGMSWVRQAPGKGLEWVATITSGGTY TYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARSLA GNAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATL SLSPGERATLSCRASQTISDYLHWYQQKPGQAPRLLIKFASQSIS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHGFPRTFGQGT KVEIK(SEQIDNO:12) CD28transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQIDNO:13) domain CD28and4-1BB RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFS costimulatory VVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL signaldomain (SEQIDNO:14) N-terminal TRSGYCLDLKTQVQTPQGMKEISNIQVGDLVLSNTGYNEVLNVF splicingdomain PKSKKKSYKITLEDGKEIICSEEHLFPTQTGEMNISGGLKEGMCL YVKE(SEQIDNO:15) DegraderSopE TKITLSPQNFRIQKQETTLLKEKSTEKNSLAKSILAVKNHFIELRS KLSERFISHKNTESSATHFHRGSASEGRAVLTNKVVKDFMLQTL NDIDIRGSA(SEQIDNO:16) DegraderERm SLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNL ADRELVHMINWAKRVPGFVDLALHDQVHLLECAWMEILMIGLV WRSMEHPGKLLFAPNLLLDRNQGKCVEGGVEIFDMLLATSSRFR MMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDK ITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSSKRMEHL YSMKCKNVVPLSDLLLEMLDAHRL(SEQIDNO:17)

[0082] An amino acid sequence of the signal transduction unit was shown in SEQ NO: 3. SEQ NO: 3 was composed of sequences of a hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), a C-terminal splicing domain and a CD35 signaling domain. The amino acid sequences of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the C-terminal splicing domain and the CD35 signaling domain were specifically shown in FIG. 2.

TABLE-US-00002 TABLE2 Aminoacidsequenceofsignaltransductionunit Name Aminoacidsequence Hypoxiccondition MSEDTSSLFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTE signalresponsedomain TDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETP (oxygen-dependent KPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPS degradationdomainODD) PSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFA EDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLS PLESSSASPESASPQSTVTVFQ(SEQIDNO:18) C-terminalsplicing LKKILKIEELDERELIDIEVSGNHLFYANDILTHNSSSSDV domain (SEQIDNO:19) CD3signalingdomain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQIDNO:20)

[0083] A nucleotide sequence of the signal transduction unit containing a hypoxia response element was shown in SEQ NO: 4. SEQ NO: 4 was composed of sequences of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the C-terminal splicing domain and the CD35 signaling domain. The nucleotide sequences of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the C-terminal splicing domain, the CD35 signaling domain and the hypoxia response element were specifically shown in FIG. 3.

TABLE-US-00003 TABLE3 Nucleotidesequenceofantigentransductionunit Name Nucleotidesequence Hypoxicconditionsignal ATGAGCGAGGACACCAGCAGCCTGTTCGACAAGCTG responsedomain AAGAAGGAGCCCGACGCCCTGACCCTGCTGGCCCCT (oxygen-dependent GCTGCTGGAGACACCATCATCTCCCTGGACTTCGGCA degradationdomainODD) GCAACGACACCGAGACCGACGACCAGCAGCTGGAGG AGGTGCCCCTGTACAACGACGTGATGCTGCCCTCTCC CAACGAAAAACTGCAGAACATCAACCTGGCTATGAG CCCCCTGCCCACCGCCGAAACACCAAAACCCCTGAG ATCCAGCGCCGACCCCGCCCTGAACCAGGAAGTGGC CCTGAAACTGGAACCCAACCCCGAGAGCCTGGAGCT GAGCTTCACCATGCCCCAGATCCAGGACCAGACCCCC AGCCCCAGCGACGGAAGCACCAGACAGAGCAGCCCC GAGCCTAACTCCCCCAGCGAATACTGCTTCTATGTGG ACAGCGACATGGTGAACGAGTTCAAGCTGGAGCTGG TGGAAAAACTGTTCGCCGAGGACACAGAAGCCAAAA ACCCCTTCAGCACCCAGGACACAGACCTGGACCTGG AGATGCTGGCCCCCTACATCCCCATGGACGACGACTT CCAGCTGAGATCCTTCGACCAGCTGAGCCCCCTGGAA AGCAGCAGCGCCTCCCCCGAATCAGCCAGCCCCCAG AGCACCGTGACCGTGTTCCAG(SEQIDNO:21) C-terminalsplicing CTGAAAAAGATCCTGAAGATCGAGGAGCTGGACGAG domain CGGGAACTGATCGACATCGAAGTGTCCGGAAACCAC CTGTTCTACGCCAACGACATCCTGACACACAATAGCA GCAGCAGCGACGTG(SEQIDNO:22) CDsignalingdomain AGAGTGAAATTCAGCAGAAGCGCCGACGCCCCCGCC TACCAGCAGGGACAGAATCAGCTGTACAACGAACTG AACCTGGGCAGAAGGGAGGAATACGACGTGCTGGAC AAGAGGAGAGGAAGGGACCCCGAGATGGGAGGAAA ACCACAGAGAAGAAAGAACCCACAGGAGGGACTGTA CAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTA CAGTGAAATTGGCATGAAGGGAGAGAGAAGAAGAGG AAAGGGACACGACGGCCTGTACCAGGGCCTGAGCAC CGCTACCAAGGACACATACGACGCCCTGCACATGCAG GCCCTGCCACCAAGA(SEQIDNO:23) Hypoxiaresponseelement CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCC ACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCAC AGTGCATACGTGGGCTCCAACAGGTCCTCTTCCACAG TGCATACGTGGGCTCCAACAGGTCCTCTTCCACAGTG CATACGTGGGCTCCAACAGGTCCTCTT(SEQID NO:24)

[0084] A nucleotide sequence of the condition-controlled spliceable chimeric antigen receptor molecule was shown in SEQ NO: 5 or SEQ NO: 6. SEQ NO: 5 was composed of a nucleic acid coding sequence of the CD47 antigen recognition domain, a nucleic acid coding sequence of the CD28 transmembrane domain, a nucleic acid coding sequence of the CD28 and 4-1BB costimulatory signal domain, a nucleic acid coding sequence of the N-terminal splicing domain, a nucleic acid coding sequence of the degrader SopE, the hypoxia response element (HRE), a nucleic acid coding sequence of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), a nucleic acid coding sequence of the C-terminal splicing domain and a nucleic acid coding sequence of the CD3 signaling domain. SEQ NO: 6 was composed of the nucleic acid coding sequence of the CD47 antigen recognition domain, the nucleic acid coding sequence of the CD28 transmembrane domain, the nucleic acid coding sequence of the CD28 and 4-1BB costimulatory signal domain, the nucleic acid coding sequence of the N-terminal splicing domain, a nucleic acid coding sequence of the degrader ERm. the hypoxia response element (HRE), the nucleic acid coding sequence of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the nucleic acid coding sequence of the C-terminal splicing domain and the nucleic acid coding sequence of the CD3 (signaling domain. The nucleic acid coding sequence of the CD47 antigen recognition domain, the nucleic acid coding sequence of the CD28 transmembrane domain, the nucleic acid coding sequence of the CD28 and 4-1BB costimulatory signal domain, the nucleic acid coding sequence of the N-terminal splicing domain. the nucleic acid coding sequence of the degrader SopE/ERm, the hypoxia response element (HRE). the nucleic acid coding sequence of the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the nucleic acid coding sequence of the C-terminal splicing domain and the nucleic acid coding sequence of the CD3 C. signaling domain were specifically shown in FIG. 4.

TABLE-US-00004 TABLE4 Nucleotidesequenceofcondition-controlledspliceablechimeric antigenreceptormolecule Name Nucleotidesequence CD47antigenrecognition ATGGCTCTGCCAGTGACAGCTCTCCTCCTCCCACTC domain GCCCTGCTGCTGCACGCCGCTAGACCTGACTACAAG GACGACGACGACAAGGAGGTGCAGCTGGTGGAGTC TGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTGG CTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGGTGGCCACCATAACTAGTGGTGG AACTTACACCTACTATCCAGACTCTGTGAAGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCACT GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACA CGGCTGTGTATTACTGTGCGAGATCCCTCGCGGGAA ATGCTATGGACTACTGGGGCCAAGGAACCCTGGTCA CCGTCTCCTCAGGCGGAGGCGGCAGTGGCGGGGGC GGGTCCGGCGGAGGCGGGAGCGAAATTGTGTTGAC ACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTAT TAGCGACTACTTACACTGGTACCAACAGAAACCTGG CCAGGCTCCCAGGCTCCTCATCAAATTTGCATCCCAA TCCATTTCTGGCATCCCAGCCAGGTTCAGTGGCAGT GGGTCTGGGACAGACTTCACTCTCACCATCAGCAGC CTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGA ATGGTCACGGCTTTCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAAATCAAA(SEQIDNO:25) CD28transmembranedomain TTTTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCC TGTTACTCCCTGCTGGTGACCGTGGCCTTCATTATCT TCTGGGTG(SEQIDNO:26) CD28and4-1BB AGGAGCAAGAGGAGCAGGCTGCTGCACAGCGACTA costimulatorysignaldomain CATGAACATGACACCCAGGAGACCTGGCCCCACCA GAAAGCACTACCAGCCCTATGCCCCCCCCAGAGACT TTGCCGCCTACAGAAGCAGGTTCAGCGTGGTGAAG AGGGGCAGGAAGAAGCTGCTGTACATCTTCAAGCA GCCCTTCATGAGGCCCGTGCAGACCACCCAGGAGG AGGACGGCTGCAGCTGCAGGTTCCCCGAGGAGGAG GAAGGCGGATGCGAGCTG(SEQIDNO:27) N-terminalsplicingdomain ACCCGGTCTGGCTACTGCCTCGACCTCAAGACCCAG GTGCAGACCCCTCAGGGCATGAAGGAGATTTCTAAC ATTCAGGTGGGCGACCTCGTGCTGAGCAACACCGGC TACAACGAGGTGCTCAACGTGTTCCCAAAGTCTAAG AAGAAGTCTTACAAGATCACACTGGAGGACGGCAA GGAGATTATTTGCTCTGAGGAGCACCTGTTCCCTACC CAGACAGGCGAGATGAACATTTCTGGCGGCCTCAAG GAGGGCATGTGCCTGTACGTGAAGGAG(SEQID NO:28) DegraderSopE ACCAAGATCACCCTGAGCCCCCAGAACTTCCGCATC CAGAAGCAGGAGACCACCCTGCTGAAGGAGAAGAG CACCGAGAAGAACAGCCTGGCCAAGAGCATCCTGG CCGTGAAGAACCACTTCATCGAGCTGCGCAGCAAG CTGAGCGAGCGCTTCATCAGCCACAAGAACACCGA GAGCAGCGCCACCCACTTCCACCGCGGCAGCGCCA GCGAGGGCCGCGCCGTGCTGACCAACAAGGTGGTG AAGGACTTCATGCTGCAGACCCTGAACGACATCGAC ATCCGCGGCAGCGCC(SEQIDNO:29) DegraderERm TCTCTGGCCCTGTCCCTCACAGCCGACCAGATGGTG TCCGCCCTCCTGGACGCCGAGCCACCAATTCTGTAC TCTGAGTACGACCCAACACGCCCTTTCAGCGAGGCC TCTATGATGGGCCTCCTCACAAACCTCGCCGACCGG GAGCTGGTGCACATGATTAACTGGGCCAAGAGAGTG CCCGGCTTCGTGGACCTCGCCCTGCACGACCAGGTG CACCTGCTGGAGTGCGCCTGGATGGAGATCCTCATG ATTGGCCTGGTGTGGCGGTCTATGGAGCACCCAGGC AAGCTGCTGTTCGCCCCTAACCTCCTGCTCGACCGC AACCAGGGCAAGTGCGTGGAGGGCGGCGTGGAGAT TTTCGACATGCTCCTCGCCACATCTAGCCGGTTCCGG ATGATGAACCTCCAAGGCGAGGAGTTCGTGTGCCTG AAGTCTATTATTCTGCTCAACTCTGGCGTGTACACCT TCCTGTCTTCTACACTCAAGTCTCTGGAGGAGAAGG ACCACATTCACCGCGTGCTCGACAAGATTACCGACA CACTCATTCACCTGATGGCCAAGGCCGGCCTCACAC TGCAACAGCAGCACCAGAGACTGGCCCAGCTGCTC CTGATCCTGTCCCACATTAGGCACATGTCTTCTAAGC GCATGGAGCACCTGTACTCTATGAAGTGCAAGAACG TGGTGCCACTGTCTGACCTGCTCCTGGAAATGCTGG ACGCCCACCGGCTG(SEQIDNO:30) Hypoxaresponseelement CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTC CACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCC ACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCA CAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCAC AGTGCATACGTGGGCTCCAACAGGTCCTCTT (SEQIDNO:24) Hypoxicconditionsignal ATGAGCGAGGACACCAGCAGCCTGTTCGACAAGCT responsedomain GAAGAAGGAGCCCGACGCCCTGACCCTGCTGGCCC (oxygen-dependent CTGCTGCTGGAGACACCATCATCTCCCTGGACTTCG degradationdomainODD) GCAGCAACGACACCGAGACCGACGACCAGCAGCTG GAGGAGGTGCCCCTGTACAACGACGTGATGCTGCCC TCTCCCAACGAAAAACTGCAGAACATCAACCTGGCT ATGAGCCCCCTGCCCACCGCCGAAACACCAAAACC CCTGAGATCCAGCGCCGACCCCGCCCTGAACCAGG AAGTGGCCCTGAAACTGGAACCCAACCCCGAGAGC CTGGAGCTGAGCTTCACCATGCCCCAGATCCAGGAC CAGACCCCCAGCCCCAGCGACGGAAGCACCAGACA GAGCAGCCCCGAGCCTAACTCCCCCAGCGAATACTG CTTCTATGTGGACAGCGACATGGTGAACGAGTTCAA GCTGGAGCTGGTGGAAAAACTGTTCGCCGAGGACA CAGAAGCCAAAAACCCCTTCAGCACCCAGGACACA GACCTGGACCTGGAGATGCTGGCCCCCTACATCCCC ATGGACGACGACTTCCAGCTGAGATCCTTCGACCAG CTGAGCCCCCTGGAAAGCAGCAGCGCCTCCCCCGA ATCAGCCAGCCCCCAGAGCACCGTGACCGTGTTCCA G(SEQIDNO:21) C-terminalsplicing CTGAAAAAGATCCTGAAGATCGAGGAGCTGGACGA domain GCGGGAACTGATCGACATCGAAGTGTCCGGAAACC ACCTGTTCTACGCCAACGACATCCTGACACACAATA GCAGCAGCAGCGACGTG(SEQIDNO:22) CD3signalingdomain AGAGTGAAATTCAGCAGAAGCGCCGACGCCCCCGC CTACCAGCAGGGACAGAATCAGCTGTACAACGAACT GAACCTGGGCAGAAGGGAGGAATACGACGTGCTGG ACAAGAGGAGAGGAAGGGACCCCGAGATGGGAGG AAAACCACAGAGAAGAAAGAACCCACAGGAGGGA CTGTACAACGAGCTGCAGAAGGACAAGATGGCCGA GGCCTACAGTGAAATTGGCATGAAGGGAGAGAGAA GAAGAGGAAAGGGACACGACGGCCTGTACCAGGGC CTGAGCACCGCTACCAAGGACACATACGACGCCCTGCA CATGCAGGCCCTGCCACCAAGA(SEQIDNO:23)

Example 2: Construction of Lentiviral Expression Plasmid

[0085] Nucleic acid sequences shown in SEQ ID NOs: 7-9 were synthesized by Shanghai Generay Bioengineering Co., Ltd. and cloned into blank lentiviral expression plasmids respectively to obtain the following plasmids.

[0086] A pXW-EF1-CD47-28BBz-P2A-EGFP recombinant plasmid carrying a nucleic acid sequence SEQ NO: 7, i.e., the traditional CD47 CAR lentiviral expression plasmid (abbreviated as pXW-CD47 CAR) recombined with a CD47-28BBz gene, was obtained by recombination with pXW-EF1-MCS-P2A-EGFP. The nucleotide shown in SEQ ID NO: 7 coded the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 costimulatory signal domain, the 4-1BB costimulatory signal domain, and the CD3 signaling domain (see Tables 1 and 2 for the specific sequences of various components).

[0087] A pXW-EF1-CD47-28BBz-ODD-P2A-EGFP recombinant plasmid carrying a nucleic acid sequence SEQ NO: 8, i.e., a hypoxia-regulated CD47 CAR-ODD lentiviral expression plasmid fused with the oxygen-dependent degradation domain ODD (abbreviated as pXW-CD47 CAR-ODD, recombined with a CD47-28BBz-ODD gene), was obtained by recombination with pXW-EF1-MCS-P2A-EGFP. The nucleotide shown in SEQ ID NO: 8 coded the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 costimulatory signal domain, the 4-1BB costimulatory signal domain, the CD3 signaling domain and the hypoxic condition signal response domain (i.e., oxygen-dependent degradation domain ODD) (see Tables 1 and 2 for the specific sequences of various components).

[0088] A pXW-EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP recombinant plasmid carrying a nucleic acid sequence SEQ NO: 9, i.e., a lentiviral expression plasmid (abbreviated as pXW-CD4 7CAR-Int.sup.N-SopE, recombined with a CD47-28BB-Int.sup.N-SopE gene) of the antigen recognition unit 1, was obtained by recombination with pXW-EF1-MCS-P2A-EGFP. The nucleotide shown in SEQ ID NO: 9 coded the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 costimulatory signal domain, the 4-1BB costimulatory signal domain, the N-terminal splicing domain and the degrader SopE (see Tables 1 and 2 for the specific sequences of various components).

[0089] A pXW-EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP recombinant plasmid carrying a nucleic acid sequence SEQ NO: 10, i.e., a lentiviral expression plasmid (abbreviated as pXW-CD47 CAR-Int.sup.N-ERm, recombined with a CD47-28BB-Int.sup.N-ERm gene) of the antigen recognition unit 2, was obtained by recombination with pXW-EF1-MCS-P2A-EGFP. The nucleotide shown in SEQ ID NO: 10 coded the CD47 antigen recognition domain, the CD28 transmembrane domain, the CD28 costimulatory signal domain, the 4-1BB costimulatory signal domain, the N-terminal splicing domain and the degrader ERm (see Tables 1 and 2 for the specific sequences of various components).

[0090] A pXW-EF1-BFP-HRE-ODD-Int.sup.C-CD3z carrying a nucleic acid sequence SEQ NO: 11, i.e., a lentiviral expression plasmid (abbreviated as pXW-ODD-Int.sup.C-CD3z, recombined with an ODD-Int.sup.C-CD3z gene) of the hypoxia-regulated signal transduction unit ODD-Int.sup.C-CD3z, was obtained by recombination with pXW-EF1-BFP-MCS1-HRE-MCS2. The nucleotide shown in SEQ ID NO: 11 coded the hypoxic condition signal response domain (oxygen-dependent degradation domain ODD), the C-terminal splicing domain and the CD3 signaling domain (see Tables 1 and 2 for the specific sequences of various components).

[0091] A pXW-EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CCD3z carrying a nucleic acid sequence SEQ ID NO: 9 and a nucleic acid sequence SEQ NO: 11 at the same time (abbreviated as pXW-CD47 CAR-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z) was obtained by recombination with pXW-EF1-BFP-MCS1-HRE-MCS2 (see Tables 1, 2 and 3 for the specific sequences of various components).

[0092] A pXW-EF1-BFP-CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z carrying a nucleic acid sequence SEQ NO: 10 and a nucleic acid sequence SEQ NO: 11 at the same time (abbreviated as pXW-CD47 CAR-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z) was obtained by recombination with pXW-EF1-BFP-MCS1-HRE-MCS2 (see Tables 1, 2 and 3 for the specific sequences of various components).

[0093] The profile of each of recombinant plasmids was shown in FIG. 1.

Example 3: Packaging, Concentration and Titer Determination of Lentivirus

1.1 Packaging of Lentivirus

[0094] 293T cell treatment: 24 h before transfection, 293T cells in the logarithmic growth phase were collected, and inoculated in a 10 cm cell culture dish (610.sup.6 to 810.sup.6 cells); and the cells grew in 10 mL of complete DMEM medium, and were cultured in a 37 C., 5% CO.sub.2 cell incubator for 18-24 h, followed by plasmid transfection till the cell density reached 70-90%.

[0095] 293T cell transfection: 1 mL of basal DMEM medium was added to a 15 mL centrifuge tube, and a transfected mixed solution was prepared from seven plasmids prepared in Example 2 according to a mass ratio of lentiviral expression plasmids: packaging plasmids (psPAX2): envelope plasmids (PMD2.G) of 1:3: 1, with a total plasmid amount of 15 g/dish. 30 L of TurboFect transfection reagent was added according to a ratio of plasmids (g): a transfection reagent (L) of 1:2, incubated at room temperature for 15-20 min, then added to a dish plated with 293T cells, and continued to be cultured in a 37 C., 5% CO.sub.2 cell incubator for 48 h; a virus supernatant was then collected, and centrifuged at 1000 g, 4 C. for 10 min; precipitates at the bottom of the tube were discarded; and a virus supernatant was collected.

1.2 Concentration of Lentivirus

[0096] A 0.45 m filter was used to further filter the viral supernatant collected by centrifugation, added with a Lenti-X lentiviral concentration reagent in 1/3 of the volume of the viral supernatant, inverted and mixed uniformly several times, incubated at 4 C. overnight, and centrifuged at 2000 g, 4 C. for 45 min, wherein a white precipitate was seen at the bottom of the centrifuge tube, which was concentrated virus particles. The supernatant was discarded carefully, and the white precipitate was resuspended with a blank RPMI1640 medium in 1/20 of the volume of the original virus supernatant, aliquoted in 250 L and cryopreserved at 80 C. for later use.

1.3 Lentiviral Titer Determination

[0097] Jurkat T cells were inoculated at 110.sup.5 cells/well on a 96-well U-bottom plate, and the collected lentiviral concentrate was diluted in 10-fold increments. 100 L of virus diluent was added to the corresponding wells, added with an infection promoting agent, i.e., protamine sulfate, to adjust the concentration to 10 g/mL, centrifuged at 1000 g, 32 C. and infected for 90 min, cultured overnight followed by replacement with a fresh RPMI1640 complete medium, and continued to be cultured for 48 h; and the proportion of fluorescence-positive cells were detected by a flow cytometer. The virus titer was calculated using the following formula:

[0098] Viral titer (TU/mL) 32 110.sup.5xproportion of fluorescence-positive cells/1001000 corresponding dilution factor.

Example 4: Preparation of Genetically Engineered T Cells

[0099] The following lentiviral vectors were obtained by concentration in Example 3: [0100] LV-EF1-CD47-28BBz-P2A-EGFP (obtained from pXW-EF1-CD47-28BBz-P2A-EGFP); [0101] LV-EF1-CD47-28BBz-ODD-P2A-EGFP (obtained from pXW-EF1-CD47-28BBz-ODD-P2A-EGFP); [0102] LV-EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP (obtained from pXW-EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP); [0103] LV-EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP (obtained from pXW-EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP); [0104] LV-EF1-BFP-MCS1-HRE-MCS2 (obtained from pXW-EF1-BFP-MCS1-HRE-MCS2); [0105] LV-EF1-BFP-HRE-ODD-Int.sup.C-CD3z (obtained from pXW-EF1-BFP-HRE-ODD-Int.sup.C-CD3z); [0106] LV-EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z (obtained from pXW-EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z); and [0107] LV-EF1-BFP-CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z (obtained from pXW-EF1-BFP-CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z).

[0108] The above various lentiviral vectors were added to a 48-well flat-bottom plate plated with 110.sup.6 peripheral blood mononuclear cells, which were preactivated with an equal amount of immunomagnetic beads for three days, at MOI=5 respectively, added with an infection promoting agent, i.e., protamine sulfate, to adjust the working concentration to 10 g/mL, centrifuged at 1000 g, 32 C. and infected for 90 min, cultured overnight followed by replacement with a fresh T cell growth medium, and continued to be cultured.

[0109] The fresh T cell growth medium was added every 2-3 days, and the cell density was adjusted to 0.510.sup.6 to 210.sup.6 cells/mL. 6-7 days after infection, immunomagnetic beads of activated T cells were removed, genetically engineered T cells were continued to be cultured and expanded, and subsequent functional experiments could be performed until the cells were resting (9-14 days after removal of the beads).

Example 5: Detection of the Expression Level of Degrader-Fused CAR Molecules

[0110] In order to verify a spontaneous degradation capability of the degrader of the present invention, a plasmid transfection experiment was performed in 293T cells in this Example.

[0111] The 293T cells were plated in a 48-well plate at 510.sup.4 cells/well and transfected the next day after cell attachment. 0.5 g of recombinant expression plasmids pXW-CD47 CAR, pXW-CD47 CAR-ODD, pXW-CD47 CAR-Int.sup.N-SopE or pXW-CD47 CAR-Int.sup.N-ERm were transfected respectively; a TurboFect transfection reagent was added at a ratio of the plasmids (g): the transfection reagent (L) of 1:2, incubated at room temperature for 15-20 min, then added to a cell culture plate, and cultured in a 37 C., 5% CO.sub.2 cell culture incubator for 48 h; and the expression of CAR molecules on a cell membrane was detected by flow cytometry. The results were shown in FIG. 2.

[0112] FIG. 2a showed a structure of the degrader-fused CAR, in which the degrader SopE or ERm was placed at a C-terminal, and an N-terminal splicing domain Int.sup.N was connected to a signal-deficient CAR molecule and degrader.

[0113] FIG. 2b showed flow cytometry detection results of the expression levels of a positive control CD47 CAR (CD47-28BBz), an oxygen-dependent degradation domain ODD-fused CD47 CAR (CD47 CAR-ODD), and two degrader-fused CARs. The results showed that although there was no significant difference in a CAR positive rate between the groups, the expression level of the degrader SopE or ERm-fused CAR was significantly lower than that of a positive control CD47 CAR and also lower than that of CD47 CAR-ODD.

[0114] The statistical results in FIG. 2c further confirmed that the expression level of the degrader SopE or ERm-fused CAR was only 10% or 7% of that of the positive control CD47 CAR, and a spontaneous degradation rate was as high as 90% or 93%, which was also superior to a 54% degradation rate of the oxygen-dependent degradation domain ODD-fused CD47 CAR.

Example 6: Splicing Activity of Condition-Controlled Spliceable System

[0115] The lentiviral expression plasmid constructed in Example 2 and the preparation method for the lentiviral vector in Example 3 were used to obtain a corresponding lentiviral vector.

[0116] In this Example, a lentiviral vector transduction experiment was performed in 293T cells, and the splicing activity of the condition-controlled spliceable system was tested. The above various recombinant lentiviral vectors and a no-loaded viral vector (LV-) were added individually or mixed at MOI=5 to a 6-well flat-bottom plate plated with 110.sup.6 293T cells, respectively; an infection-promoting reagent (Polybrene) was added and adjusted for a working concentration to g/mL, and cultured overnight; and then a fresh cell growth medium was replaced and 10 continued to be cultured to obtain nine different genetically engineered 293T cells that stably integrate: 1) EF1-CD47-28BBz-P2A-EGFP: 2) EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP: 3) EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP; 4) EF1-CD47-28BBz-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z; 5) EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z; 6) EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z; 7) EF1-CD47-28BBz-P2A-EGFP and EF1-BFP-MCS1-HRE-MCS2 (Mock); 8) EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP and EF1-BFP-MCS1-HRE-MCS2 (Mock); and 9) EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP and EF1-BFP-MCS1-HRE-MCS2 (Mock). The above nine genetically engineered T cells were cultured under a hypoxic condition (1% O.sub.2) for 24 h; the cells were collected after the culture and eluted with FACS buffer once, added with a 2 g/mL flow cytometry antibody PE-anti-DYKDDDDK, incubated at room temperature in the dark for 20min, eluted twice with FACS buffer after the incubation, and resuspended and mixed uniformly with 300 L of FACS buffer; and then, the expression of CAR molecules was detected by a flow cytometer. In addition, the six genetically engineered 293T cells, which stably integrate: 1) EF1-CD47-28BBz-P2A-EGFP, 2) EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP. 3) EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP. 4) EF1-CD47-28BBz-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z, 5) EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z, and 6) EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP and hypoxia-regulated signaling unit EF1-BFP-HRE-ODD-Int.sup.C-CD3z, were cultured under a normoxic condition (21% O.sub.2) and a hypoxic condition (1% O.sub.2) for 24 h, respectively, and after culture, the cells were collected for western blotting. The results were shown in FIG. 3.

[0117] FIG. 3a showed structures and compositions of a degrader-fused CAR and a hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z, wherein ODD-Intc-CD3z was composed of the oxygen-dependent degradation domain ODD, the C-terminal splicing domain Int.sup.C, and the signal domain CD3z.

[0118] The flow cytometry detection result in FIG. 3b showed that the CAR expression levels of engineered cells that integrate both the degrader-fused CAR and the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z were further improved under a hypoxic condition, suggesting that the hypoxia-regulated signaling unit ODD-Int.sup.C-CD3z was enriched under the hypoxic condition and achieving splicing of the degrader-fused CAR to release degraders.

[0119] FIG. 3c showed an induction fold of the CAR expression level after splicing, and the induction fold of the degrader SopE or ERm-fused CAR was 4 or 5-fold respectively, while there was no change in the expression level of a control CD47 CAR.

[0120] A Western Blotting result in FIG. 3d confirmed that the degrader-fused CAR and the signaling unit were successfully spliced with an efficiency of up to 100% under a hypoxic condition, and complete splicing was achieved under a hypoxic condition (1% O.sub.2), thereby obtaining a single band with a molecular weight smaller than that of an unspliced molecule.

Example 7: Expression of Chimeric Antigen Receptor Regulated by Condition-Controlled Spliceable System

[0121] By using the lentiviral expression plasmid constructed in Example 2, the preparation method for the lentiviral vector in Example 3 and the preparation method for the genetically engineered T cells described in Example 4, five engineered T cells, which stably integrate EF1-CD47-28BB-Int.sup.N-SopE-P2A-EGFP, EF1-CD47-28BBz-P2A-EGFP, EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z, EF1-CD47-28BB-Int.sup.N-ERm-P2A-EGFP or EF1-BFP-CD47-28BB-Int.sup.N-ERm-HRE-ODD-Intc-CD3z genes were prepared. The above five genetically engineered T cells were cultured under a normoxic condition (21% O.sub.2) or a hypoxic condition (1% O.sub.2) for 24 h respectively; the cells were collected after the culture and eluted with FACS buffer once, added with a 2 g/mL flow cytometry antibody PE-anti-DYKDDDDK. incubated at room temperature in the dark for 20 min, eluted twice with FACS buffer after the incubation, and resuspended and mixed uniformly with 300 L of FACS buffer; and then, the expression of HER2 CAR molecules was detected by the flow cytometer. The results were shown in FIG. 4.

[0122] The flow cytometry detection results in FIG. 4a showed that CD47 CAR molecules were highly expressed by positive control CD47 CAR-T cells (CD47-28BBz) under both normoxic and hypoxic conditions, while CAR molecules regulated by the condition-controlled spliceable system were lowly expressed in a normoxic environment, but induction-expressed in a hypoxic environment, especially for the engineered T cells stably integrating EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z. The statistical results in FIG. 4b confirmed that a positive rate of the degrader SopE or ERm-fused CAR was 46% or 24% of that of the positive control under a normoxic condition, respectively, and the expression level was reduced to 4% or 2% of that of the control, but was significantly up-regulated under a hypoxic condition; and the induction fold was 9-or 3-fold, respectively, indicating that the condition-controlled spliceable system based on the degrader SopE was more active.

Example 8: Tumor Cell Killing of Chimeric Antigen Receptor T Cells Regulated by Condition-Controlled Spliceable System

[0123] The tumor cell killing efficiency was evaluated by a luciferase-based cytotoxicity assay. First, 110.sup.4 SKOV3-Luc (firefly luciferase gene-modified human ovarian cancer cells) or NCI-H292-Luc (firefly luciferase gene-modified human lung cancer cells) were inoculated on a 96-well flat-bottom black plate with 100 L of medium per well, and cultured in a 37 C., 5% CO.sub.2 cell incubator for 18 h. On the second day, the genetically engineered T cells prepared in Example 7 and non-transduced T cells cultured at the same time were added to the wells containing target cells at a ratio of effector cells: target cells of 1:4 (0.25), 1:2 (0.5), 1:1 (1) and 2:1 (2), and were cultured under a normoxic condition (21% O.sub.2) or a hypoxic condition (1% O.sub.2) for 24 h, respectively; and a luciferase activity value of the target cells was detected by a GloMax96 microplate luminescence detector after co-culture. The results were shown in FIG. 5.

[0124] A formula for calculating a cell killing rate was as follows:

[00001]$\mathrm{Cell}\mathrm{killing}\mathrm{rate}\left(\%\right)=\left(\mathrm{luciferase}\mathrm{activity}\mathrm{value}\mathrm{of}\mathrm{non}-\mathrm{transduced}T\mathrm{cell}\mathrm{group}-\mathrm{luciferase}\mathrm{activity}\mathrm{value}\mathrm{of}\mathrm{experimental}\mathrm{group}\right)/\mathrm{luciferase}\mathrm{activity}\mathrm{value}\mathrm{of}\mathrm{non}-\mathrm{transduced}T\mathrm{cell}\mathrm{group}100.$

[0125] CD47 CAR-T cells (positive control) can effectively kill SKOV3 (FIG. 5a) and NCI-H292 tumor cells (FIG. 5B) under either normoxic or hypoxic condition, and had no selective killing properties.

[0126] The tumor killing activity of the CD47 CAR-T cells regulated by the condition-controlled spliceable system was low under the normoxic condition, and the killing rates of the CD47 CAR-T cells regulated by a SopE-based condition-controlled spliceable system were 3% (SKOV3) and 6% (NCI-H292) at a ratio of effector cells: target cells of 1:1, while SKOV3 tumor cells (84%) and NCI-H292 tumor cells (96%) can be selectively and efficiently killed under the hypoxic condition; and the killing activities against the tumor cells were increased by factors of 28 and 16. The killing rates of the CD47 CAR-T cells regulated by an ERm-based condition-controlled spliceable system were 3% (SKOV3) and 6% (NCI-H292) at a ratio of effector cells: target cells of 1:1, while SKOV3 tumor cells (58%) and NCI-H292 tumor cells (96%) can be selectively and efficiently killed under the hypoxic condition; and the killing activities against the tumor cells were increased by factors of 19 and 16.

Example 9: In-Vivo Antitumor Effects of CAR-T Cells Regulated by Condition-Controlled Spliceable System

[0127] Local hair removal on the back of B-NDG mice raised in a sterile isolator was performed with a hair removal cream or an animal shaver one day in advance, such that the skin of a tumor cell inoculation site was exposed. Each mouse was fixed with the left hand, that is, the head, neck and back skin of the mouse were grasped with the left hand at the same time; and after a part to be hair-removed on the right side of the back was fully exposed by turning its back to the left, this part was disinfected by an alcohol cotton ball with the right hand. A 1 mL insulin syringe was used to blow and mix uniformly the preprepared NCI-H292 tumor cells, and then suck 125 L of cell suspension (510.sup.6 tumor cells), and a tip of a needle was pierced subcutaneously and diagonally into the mouse at an angle of 30-40 from the skin; and the cell suspension was slowly injected to avoid cell spillage. After the injection of 125 L of cell suspension was completed, the needle was left for 2-3 seconds and then quickly withdrawn, and a clearly visible bulge could be seen under the skin at the injection site. After cell inoculation, the tumorigenesis and health status of the mice were observed every 2-3 days, and a baseline tumor volume was measured with a vernier caliper after tumorigenesis, and subsequent experiments were carried out.

[0128] On the fifth and eleventh days after tumor cell inoculation, 510.sup.6 CAR-T cells regulated by the condition-controlled spliceable system and negative control T cells (UTDs) were injected intravenously or intratumorally, respectively; and tumor sizes were measured every 2-3 days, and long and short diameters of tumors were measured with the vernier caliper, respectively. The results were shown in FIG. 6.

[0129] A calculation formula of a tumor volume was as follows: volume=(long diametershort diameter.sup.2)/2.

[0130] The growth of lung cancer can be effectively controlled by intravenous infusion and intratumoral injection of CAR-T cells regulated by condition-controlled spliceable systems.

[0131] Compared with a UTD treatment group, the CAR-T cells (stably integrating EF1-BFP-CD47-28BB-Int.sup.N-SopE-HRE-ODD-Int.sup.C-CD3z or EF1-BFP-CD47-28BB-Int.sup.N-ERm-HRE-ODD-Int.sup.C-CD3z) regulated by a degrader SopE-based condition-controlled spliceable system had a more significant tumor inhibition effect; and the tumor suppression rates were 72.1% (intravenous infusion) and 85.6% (intratumoral injection) at one month after cell reinfusion, respectively.

[0132] The foregoing is only preferred embodiments of the present invention and is not intended to limit the present invention in any form. Although the present invention has been revealed as above with a preferred embodiment, it is not used to limit the present invention. A person skilled in the art, without departing from the scope of the technical solution of the present invention, may make some changes or modifications to obtain the equivalent embodiments of the equivalent changes by using the above disclosed technical content, but any amendments, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.