Universal CAR-T cell and preparation method and use thereof

Abstract

Disclosed are a universal CAR-T cell knocking out one or more of CD3 delta, CD3 gamma, CD3 epsilon and CD3 zeta, and simultaneously introducing the HSV-TK gene. Also disclosed are a method for preparing the above-mentioned CAR-T cell, a preparation comprising the CAR-T cell, and the use of the CAR-T cell.

Claims

1. A universal CAR-T cell in which one or more of CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta is knocked out, wherein said CAR-T cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR), and said CAR includes an extracellular antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular signal transduction domain, wherein said CAR includes an anti-CD19 CAR, and said anti-CD19 CAR comprises the amino acid sequence set forth in SEQ ID NO: 25.

2. A universal CAR-T cell in which one or more of CD3Delta, CD3Gamma, CD3 Epsilon and CD3 zeta is knocked out, wherein said CAR-T cell comprises a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 29.

3. The universal CAR-T cell according to claim 1, wherein said CAR-T cell is derived from cord blood or peripheral blood.

4. A formulation comprising said universal CAR-T cell according to claim 1.

5. A method of treating a CD19 positive tumor in a subject in need thereof, comprising administering said universal CAR-T cell according to claim 1 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also figure and FIG. herein), of which:

(2) FIG. 1A illustrates schematic structural view of the BBZ CAR molecule.

(3) FIG. 1B illustrates a schematic diagram of CD20 targeting CAR constructs. An anti-human CD20 scFv was linked to 41BB and CD3, suicide gene thymidine kinase (TK) was linked to CD3 via a P2A sequence to generate a 20BBZ TK construct.

(4) FIG. 1C illustrates Flow chart of the protocol to generate universal CAR-T (CD3-20uCAR-T) by 20BBZTK lentivirus and Cas9/gRNA lentivirus.

(5) FIG. 1D illustrates schematic view of the results of the phenotypic analysis of the CD3-negative 20BBZ CAR-T cells.

(6) FIG. 1E illustrates schematic diagram of long-term CAR-T population expansion.

(7) FIG. 1F illustrates the knockout efficiency of sgRNA of on CD3 gene.

(8) FIG. 2A illustrates the relative cytotoxicity of CD3.sup.20uCAR-T by analyzing the remaining tumor cells (CD19.sup.+) by flow cytometry.

(9) FIG. 2B illustrates cytokines IFN- and TNF- secreted by CD3.sup.+20CAR-T and CD3.sup.20uCAR-T cells.

(10) FIG. 3A illustrates a schematic diagram of the in vivo xenograft tumor model and CAR-T treatment protocol.

(11) FIG. 3B-3C illustrate Kaplan-Meier curves for overall survival of the mice.

(12) FIG. 3D illustrates analysis of CAR-T cell (mCD45.sup.hCD45.sup.+hCD3.sup.+) persistence and Raji (mCD45.sup.hCD45.sup.+hCD19.sup.+) tumor cell burden

(13) FIG. 4A illustrates schematic view showing the regulation of ganciclovir on the survival of the CD3-negative 20BBZ CAR-T cells and the CD3-negative 19BBZ CAR-T cells in vitro.

(14) FIG. 4B illustrates a schematic diagram of the in vivo xenograft tumor model and CAR-T treatment protocol.

(15) FIG. 4C illustrates analysis of CAR-T cell (mCD45.sup.hCD45.sup.+hCD3.sup.+) persistence and Raji (mCD45.sup.hCD45.sup.+hCD19.sup.+) tumor cell burden.

(16) FIG. 5 illustrates schematic view of the tumor-killing ability of the CD3-negative 20BBZ CAR-T cells and the control CAR-T cells in an embodiment of the present invention;

(17) FIG. 6 illustrates schematic view of the in vivo survival ability of the CD3-negative 20BBZ CAR-T cells and the control CAR-T cell in an embodiment of the present invention.

DETAILED DESCRIPTION

(18) While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

(19) As used herein, the term CRISPR/Cas system generally refers to a group of molecules including RNA-directed nuclease or other effector molecules and gRNA molecules, which can direct and realize the modification of nucleic acid at the site of targeting sequence by RNA-directed nuclease or other effector molecules, e.g., inducing degradation of the target sequence. In some embodiments, the CRISPR system includes gRNA and a Cas protein, e.g., a Cas9 protein. The system including Cas9 or a functional variant thereof is called Cas9 system or CRISPR/Cas9 system in the present application. In some embodiments, the gRNA molecule can be complexed with the Cas molecule to form a ribonucleoprotein (RNP) complex.

(20) As used herein, the terms gRNA molecule or guide RNA, instruction RNA, direct RNA, guide RNA molecule, gRNA can be used interchangeably, and generally refer to a nucleic acid molecule that can promote directing the RNA-directed nuclease or other effector molecules (generally combined with gRNA molecules) specifically to the target sequence. In some embodiments, the directing is achieved by the hybridization of a portion of gRNA with DNA (e.g., via a gRNA guide domain) and the binding of a portion of gRNA molecule with an RNA directed nuclease or other effector molecules (e.g., at least through gRNAtracr). In some embodiments, the gRNA molecule consists of a single continuous polynucleotide molecule, which is herein referred to as a single guide RNA or sgRNA or the like. In other embodiments, the gRNA molecule consists of a plurality of (e.g., two) polynucleotide molecules that can be associated with themselves (generally by hybridization), which is herein referred to as double guide RNA or dgRNA, etc.

(21) As used herein, the term Cas protein generally refers to an enzyme responsible for cutting DNA in the CRISPR/Cas system. It can include enzymes from CRISPR/Cas system Types I, II, and III, e.g., Cas3, Cas9, Cas10.

(22) As used herein, the term Cas9 protein generally refers to an enzyme from the bacterial type II CRISPR/Cas system and responsible for cutting DNA. Cas9 can include the wild-type protein and functional variants thereof.

(23) As used herein, the term chimeric antigen receptor (CAR) generally refers to an antigen receptor fused by fusing an antigen binding region of an antibody which recognizes a tumor associated antigen (TAA) or a binding fragment of other target molecules with an immune receptor tyrosine-based activation motifs (ITAM, typically CD3 or FcRI) of an intracellular signal domain. For example, the basic structure of CAR can include an antigen binding domain of a tumor-associated antigen (TAA) or other target molecules (typically, an scFv originated from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an immunoreceptor tyrosine-based activation motif (ITAM) of an intracellular immune receptor.

(24) As used herein, the term binding domain generally refers to a domain that (specifically) binds to a given target epitope or a given target site of a target molecule (e.g., an antigen), interacts with the given target epitope or the given target site, or recognizes the given target epitope or the given target site.

(25) As used herein, the term specific binding generally refers to a measurable and reproducible interaction, such as, the binding between a target and an antibody, which can determine the presence of a target in the presence of heterogeneous populations of molecules (including biomolecules). For example, antibodies that specifically bind to targets (which can be epitopes) are antibodies that bind the target(s) with greater compatibility, affinity, easiness, and/or duration than other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved in proteins of different species. In another embodiment, the specific binding includes but is not limited to exclusive binding.

(26) As used herein, the term transmembrane domain generally refers to a polypeptide or protein which is encoded at a DNA level by an exon including at least an extracellular region, a transmembrane region, and an intracellular region. The transmembrane domain generally includes three different structural regions: N-terminal extracellular region, middle conserved transmembrane extension region, and C-terminal cytoplasmic region. The transmembrane domain may further include an intracellular region or a cytoplasmic region.

(27) As used herein, the term hinge region generally refers to a region located between the binding domain and the transmembrane domain in the CAR structure. The hinge region usually comes from IgG family, such as IgG1 and IgG4, and some from IgD and CD8. Generally, the hinge region has a certain degree of flexibility, which affects the spatial constraints between the CAR molecule and its specific target, thereby affecting the contact between CAR T cells and tumor cells.

(28) As used herein, the term costimulatory generally refers to a source of the second signal of lymphocyte activation, which is usually generated by an interaction of costimulatory molecules on the surface of immune cells (between T cells/B cells or between antigen presenting cells/T cells) involved in adaptive immunity with their receptors. For example, the complete activation of T cells depends on dual signaling and the action of cytokine. The first signal of T cell activation is derived from the specific binding of its receptors with the antigens, that is, the recognition of T cells to the antigens; and the second signal of T cell activation is derived from the costimulatory molecule, that is, the interaction of the costimulatory molecules of the antigen presenting cells with the corresponding receptors on the surfaces of T cells.

(29) As used herein, the term costimulatory domain generally refers to an intracellular portion of the corresponding receptor of the costimulatory molecule, which can transduce a costimulatory signal (also known as the second signal). For example, in CAR-T cells, the costimulatory domain derived from CD137 (or receptors of other costimulatory molecules) can be activated after the binding of the extracellular binding domain in the CAR structure with the corresponding antigen, thereby transducing a costimulatory signal.

(30) As used herein, the term primary signal transduction domain generally refers to an amino acid sequence within a cell that can generate signals which promote the immune effector function of CAR-containing cells such as CAR-T cells. Examples of the immune effector functions in, e.g., CAR-T cells can include cell lysis activity and auxiliary activity, including cytokine secretion. In some embodiments, the primary signal transduction domain transduces the effector functional signals and directs the cells to perform the specialization function. Although the primary signal transduction domain can be used in its entirety, it is not necessary to use the entire chain in many cases. As for the use of a truncated portion of the primary signal transduction domain, such truncated portion can be used to replace the intact chain, as long as it can transduce the effector functional signals. The term primary signal transduction domain is thus intended to encompass any truncated portion of an intracellular signal transduction domain that is sufficient to transduce the effector functional signals.

(31) As used herein, the term tumor generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor can be a solid tumor or a non-solid tumor. In some embodiments, a visible lump that can be detected by clinical examinations such as, X-ray radiography, CT scanning, B-ultrasound or palpation can be called solid tumor, while a tumor that cannot be seen or touched by X-ray, CT scanning, B-ultrasound and palpation, such as leukemia, can be called non-solid tumor.

(32) As used herein, the term pharmaceutically acceptable diluent or pharmaceutically acceptable excipient generally refers to a pharmaceutically acceptable substance, composition, or vehicle involved in carrying, storing, transferring, or administering a cell preparation, e.g., liquids, semi-solid or solid fillers, diluents, osmotic agents, solvent, or encapsulating substances. The pharmaceutically acceptable diluent or excipient can include a pharmaceutically acceptable salt, wherein the term pharmaceutically acceptable salt includes salts of active compounds prepared by using a relatively nontoxic acid or base, e.g., sodium chloride, depending on the cell nature of the present application. The pharmaceutically acceptable carrier can further include organic acids (e.g., lactic acid), bioactive substances (e.g., polypeptides, antibodies, and the like) and antibiotics (e.g., penicillin, streptomycin), etc. The pharmaceutically acceptable carrier can further include a hydrogel, such as, a hydrogel containing polyacrylamide. The pharmaceutically acceptable diluent or excipient can include storage solution, cryopreservation solution, injection, etc., which can be used for cells. In general, the pharmaceutically acceptable diluent or excipient can maintain the activity of the cells carried by the carrier without hindering its therapeutic efficacy. The pharmaceutically acceptable diluent or excipient can also contribute to the storage, transportation, proliferation and migration of cells, and is suitable for clinical application.

(33) As used herein, the term allogeneic therapy generally refers to a therapy of administering organs, tissues, cells, etc. which do not come from the subject or patient to achieve a therapeutic purpose.

(34) As used herein, the term subject generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey. In some embodiments, said subject is a human.

(35) As used herein, the term include/including or comprise/comprising generally refers to encompassing clearly specified features, but does not exclude other elements.

EXAMPLES

(36) The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1 Preparation of CD3-Negative 20BBZ CAR-T Cells

(37) 1.1 Construction of Lentiviral Vector pLenti-CrisprV2-sgRNA and Production of Virus

(38) A sgRNA for CD3Delta, CD3Gamma, CD3 Epsilon, CD3 zeta is designed by using crispr.mit.edu (Table 1), and cloned into pLenti-CrisprV2.

(39) The correctly sequenced clones were extracted with the endotoxin removal kit, and pLenti-CrisprV2 containing different clones (KO CD3Delta sgRNA, KO CD3Gamma sgRNA, KO CD3 Epsilon sgRNA or KO CD3 zeta sgRNA) and lentivirus packaging plasmids (VSV-g, pMD Gag/Pol and RSV-REV) were co-transfected with 293X, supernatants were collected after 48 and 72 hours, filtered at 0.45 uM and the viruses were concentrated by centrifugation at 25,000 RPM for 2 hours using a Beckman ultracentrifuge and SW28 head to obtain plenti-CRISPRV2-sgRNA viruses for subsequent CAR-T cell production.

(40) TABLE-US-00001 TABLE1 SEQ ID LABEL SEQ NO: KOCD3Delta GAACATAGCA 1 sgRNA1(D1) CGTTTCTCTC KOCD3Delta CCCCTTCAAG 2 sgRNA2(D2) ATACCTATAG KOCD3Gamma GGCTATCATT 3 sgRNA1(G1) CTTCTTCAAG KOCD3Gamma CTTGGTTAAG 4 sgRNA2(G2) GTGTATGACT KOCD3Gamma GTAATGCCAA 5 sgRNA3(G3) GGACCCTCGA KOCD3Epsilon GGGCACTCAC 6 sgRNA1(E1) TGGAGAGTTC KOCD3Epsilon TTGACATGCC 7 sgRNA2(E2) CTCAGTATCC KOCD3zeta GTGGAAGGCG 8 sgRNA1(Z1) CTTTTCACCG KOCD3zeta TTTCACCGCG 9 sgRNA2(Z2) GCCATCCTGC KOCD3zeta GATGGAATCC 10 sgRNA3(Z3) TCTTCATCTA
1.2 sgRNA Knockdown Efficiency Assay

(41) 210.sup.5 Jurkat cells were transiently transfected with 0.6 g pLenti-CrisprV2-hCD3E1/E2/D1/D2/Z1/Z2/G1/G2/G3 plasmids and cells were harvested at 48 h. Anti-TCR antibody was used for flow detection and untransfected plasmids were used as negative control.

(42) FIG. 1F showed that CD3 is not expressed when the corresponding sequence is knocked down with sgRNA. The box in the figure represented the knockdown efficiency, where Z1 has the highest knockdown efficiency (about 81.2%).

(43) 1.3 Construction of 20BBZ CAR

(44) The BBZ (its structure was shown in FIG. 1A, and the antibody extracellular antigen recognition domain used therein is anti-CD20 antibody), HSV-TK was added to its middle by overlap PCR to form the gene encoding the fusion protein, and the lentiviral vector was cloned by adding the enzyme cut site at both ends; the correctly sequenced clones were extracted with the endotoxin removal kit, and the lentiviral packaging plasmid were co-transfected, and the supernatant was collected at a predetermined time, and the virus was concentrated by filtration and centrifugation to obtain the 20BBZ-2A-TK (its structure was shown in FIG. 1B, and the sequence is shown in Table 2) virus.

(45) The 19BBZ CAR (the sequence is shown in Table 2) can also be constructed according to the above method.

(46) TABLE-US-00002 TABLE2 SEQ ID LABEL SEQ NO: anti-CD20 QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPW 11 scFv IYATSNLASGVPVRFSGSGSGTSYSLTISRVEAE DAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGG GGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTA DKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNV WGAGTTVTVS CD8a TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 12 Hinge DFACD CD8a IYIWAPLAGTCGVLLLSLVITLYC 13 trans- membrane domain 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG 14 CEL CD3zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 15 RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR thekappa METDTLLLWVLLLWVPGSTGTG 16 leader sequence 2A GSGATNFSLLKQAGDVEENPGP 19 TK MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEAT 20 EVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRD DIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAG DAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHA PPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFV ALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAM LAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVP PQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNV FAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTS GMVQTHVTTPGSIPTICDLARTFAREMGEAN 20BBZ- METDTLLLWVLLLWVPGSTGTGQIVLSQSPAILSASPGE 21 2A-TK KVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASG (anti- VPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPP CD20 TFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELV scFv- KPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIG hinge-TM- AIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS 41BBICD- EDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAAAA CD3Z-2A- TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL TK) DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFS LLKQAGDVEENPGPRTMASYPCHQHASAFDQAARSRGH SNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGM GKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIA NIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTD AVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAAR YLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDR LAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGS WWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLF RAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYD QSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFA REMGEAN anti-CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK 24 scFv PDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLE QEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNS KSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS 19BBZ- METDTLLLWVLLLWVPGSTGTGDIQMTQTTSSLSASLG 26 2A-TK DRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH (anti- SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP CD19 YTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGL scFv- VAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGV hinge-TM- IWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD 41BBICD- TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAATT CD3Z-2A- TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF TK) ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLL KQAGDVEENPGPRTMASYPCHQHASAFDQAARSRGHS NRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMG KTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANI YTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDA VLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARY LMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRL AKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGS WWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLF RAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYD QSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFA REMGEAN
1.4 Preparation of CD3-Negative 20BBZ CAR-T Cells

(47) Human PBMC were purified by Stemcell T cell isolation kit (negative selection) and inoculated into anti-hCD3 and anti-hCD28-coated 96-well culture plates, and after 2 days, the cells were infected with 20BBZ-2A-TK virus according to MOI=10-20 and pLentiCRISPRv2-hCD3Z1 virus, the cell culture was continued with the medium changed after 1 day, and artificial antigen-presenting cells were used according to every 6 days, and the cells were removed from CD3-positive cells with the Stemcell T cell positive selection kit, i.e., CD3-negative 20BBZ CAR-T cells (CD3-U-CAR-T, referred to as U-CAR-T cells) were obtained for subsequent experiments and phenotypic analysis were shown in FIG. 1D. As can be seen from the figure, the resulting U-CAR-T cells were CAR-positive and CD3-negative.

(48) 1.5 The Characteristic of CD3 20uCAR-T

(49) T cells were infected with 20BBZ TK lentivirus and electroporated with Cas9/gRNA which stimulated by coculture with irradiated (IR) Raji cells once every 6 days. The characteristic of CD3+20CAR-T and CD320uCAR-T were analyzed on day 4 after each stimulation. Overall expansion of CAR-T cells in CD3+20CAR-T and CD320uCAR-T. Relative cell proliferation was calculated by dividing the cell number of Day 1. Experiments were repeated with four different donor-derived T cells (n=4/group). Arrows indicate stimulation time point.

(50) FIG. 1E shows that CAR-T cell expansion was not significantly affected by knockdown of CD3 and still had good amplification.

Example 2 The Cytotoxicity of CD3 20uCAR-T In Vitro

(51) CD3.sup.+20CAR-T and CD3.sup.20uCAR-T cells constructed from three different donor were co-culture with Raji cells at the different effector:target (E:T) ratio for 24 h. Relative cytotoxicity was calculated by analyzing the remaining tumor cells (CD19.sup.+) by flow cytometry. Cytokines IFN- and TNF- secreted by CD3.sup.+20CAR-T and CD3.sup.20uCAR-T cells were determined by Cytometric Bead Array (CBA) assay. Representative results of one from three (FIG. 2A-2B) repeated experiment are shown. *P<0.05, **P<0.01, ***P<0.001; NS: Not Significant

(52) FIG. 2A-2B show that after knockdown of CD3, CD3.sup. UCAR-T still had promising tumor killing effect as well as a higher level of cytokine (IFN-, TNF-) secretion.

Example 3 CD3.SUP..20u CAR-T Cells Show Better Effectively Controlling Tumor In Vivo

(53) NSG mice (n=6/group) were intravenously inoculated with 510.sup.5 Raji (FIG. 3B) or daudi (FIG. 3C) tumor cell. Seven days later, tumor bearing mice were treated with 110.sup.7 CD3.sup.+20CAR-T cells or CD3.sup.20uCAR-T cells or PBS. Kaplan-Meier curves for overall survival of the mice are shown. Statistical significance was determined by Mantel-Cox test, presented **P<0.01, ***P<0.001; NS: Not Significant.

(54) Same as in FIG. 3B, 7 days after the treatment, bone marrow, spleen and peripheral blood were collected for analysis of CAR-T cell (mCD45.sup.hCD45.sup.+hCD3.sup.+) persistence and Raji (mCD45.sup.hCD45.sup.+hCD19.sup.+) tumor cell burden (FIG. 3D). Statistical significance was determined by Mann-Whitney U test and presented by *P<0.05, **P<0.01, ***P<0.001; NS: Not Significant.

(55) FIG. 3D shows that CD3-CAR-T has a lower tumor cell burden and higher CAR-T cell persistence compared to control or CD3.sup.+ CAR-T, indicating that CD3-UCAR-T has better efficacy.

Example 4 the Survival of U-CAR-T Cell is Regulated by Ganciclovir

(56) 4.1 the Survival of U-CAR-T Cell is Regulated by Ganciclovir In Vitro

(57) The anti-CD20 U-CAR-T cells obtained in Example 1, anti-CD19 U-CAR-T cells (prepared according to the method in Example 1) and the control CAR-T (without knockout of CD3) were inoculated into 96-well plates, and the number of surviving CAR-Ts was compared after 48 hours by adding the indicated concentration of ganciclovir (0, 0.1 g/mL, 0.3 g/mL, 1 g/mL, 3 g/mL), and the results are shown in FIG. 4A. From the figure, it can be seen that ganciclovir can regulate the survival of U-CAR-T, which can rapidly remove U-CAR-T from the body under the condition that U-CAR-T causes side effects and improves safety.

(58) 4.2 the Survival of U-CAR-T Cell is Regulated by Ganciclovir In Vivo

(59) NSG mice (n=6/group) were injected intravenously with 510.sup.5 Raji, which was followed by the administration of 110.sup.7 CD3.sup.20uCAR-T cells on day 7 or two hundred micrograms of Ganciclovir were administered on day 7, 10 and 13. 7 days after the treatment, bone marrow, spleen and peripheral blood were collected for analysis of CAR-T cell (mCD45.sup.hCD45.sup.+hCD3.sup.+) persistence and Raji (mCD45.sup.hCD45.sup.+hCD19.sup.+) tumor cell burden (FIG. 4C). Statistical significance was determined by Mann-Whitney U test and presented by *P<0.05, **P<0.01, ***P<0.001; NS: Not Significant. Statistical significance was determined by unpaired t test. Statistical significance was presented by *P<0.05, ***P<0.001.

(60) FIG. 4C shows that ganciclovir effectively cleared CD3.sup. UCAR-T cells from various organs in vivo compared to CD3.sup.+ CAR-T (control is a group without CAR-T cells). As CD3.sup. UCAR-T cells were cleared, there was an elevated tumor load compared to CD3.sup.+ CAR-T.

Example 5 Comparison of Tumor-Killing Ability of U-CAR-T and Control CAR-T

(61) The anti-CD20 U-CAR-T cells obtained in EXAMPLE 1 and the control CAR-T cells (without knockout of CD3) were inoculated into 96-well plates, and Raji tumor cells were added at a CAR-T:tumor cells ratio of 1:1. After 24 and 48 hours, the survival rates of the tumor cells were compared, and the results are shown in FIG. 5. It can be seen from the figure that the U-CAR-T has a similar tumor killing ability to that of the control CAR-T.

Example 6 Comparison of In Vivo Survival Ability of U-CAR-T and Control CAR-T

(62) 10.sup.6 Raji tumor cells were intravenously inoculated into B-NDG mice. After 6 days, the mice were treated with 10.sup.7 U-CAR-T or the control CAR-T, and observed for their survival rate. The results are shown in FIG. 6. It can be seen from the figure that both the U-CAR-T and the control CAR-T result in the prolonged survival time of the mice.

(63) It can be seen from the aforesaid examples that, the universal CAR-T constructed by knockout of CD3 in the present invention exhibits a low graft-versus-host response (GVHD), and greatly enhances and expands the convenience of CAR-T cell therapy. Meanwhile, an HSV-TK is introduced, so that the U-CAR-T can be rapidly cleared from the body by the regulation of ganciclovir, thereby further improving the safety of the universal CAR-T.

(64) While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.