Split type chimeric antigen receptor T cell as well as construction method and application thereof

Abstract

The invention discloses a split type chimeric antigen receptor T cell as well as a construction method and application thereof. According to the invention, an efficient chimeric antigen screening platform is established based on a split type CAR design strategy. Compared with a traditional fixed CAR design, the split type CAR modular design uses UniCAR-T cells with the same positive rate in the same batch to screen different antibodies, engineering modification does not need to be carried out on the T cells again, suitable target spots and antibodies can be efficiently screened out, the difference between the antibodies can be accurately compared under the same background, and the detection accuracy is improved. The time and capital cost is greatly reduced, and the development process of a new target is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to a split chimeric antigen receptor T cell and its construction method and application. Background Art

[0002] The T cell (CAR-T) immunotherapy expressing chimeric antigen receptor (CAR) has shown great therapeutic potential as a revolutionary tumor treatment method. CAR consists of an antigen-specific single-chain variable fragment (scFv) and the intracellular signaling domains of the T cell receptor (TCR) (such as CD3ζ and co-stimulatory domains) (Guedan et al., 2019). The CAR-encoding gene is delivered into T cells through a viral vector. The scFv can recognize the antigen on the surface of tumor cells, and the intracellular signaling domain activates the cytotoxic response. CAR-T cell therapy is hailed as a "living drug" due to its sensitivity, target specificity, self-amplification, and sustained response characteristics (Sadelain et al., 2013). In clinical practice, CAR-T cells targeting CD19 have achieved remarkable curative effects in patients with relapsed and refractory B-cell leukemia and lymphoma (Brentjens et al., 2013, Davila et al., 2014, Maude et al., 2014).

[0003] However, human tumors are heterogeneous in the expression of cell surface antigens, which requires the redirection of the specificity of CAR through the engineering of the extracellular scFv domain to target multiple malignancies (Tamada et al., 2012, Newick et al., 2017). At the same time, for multiple antibodies against the same tumor surface antigen, after being constructed into CAR-T cells, the tumor-targeting killing ability is also different (Panowski et al., 2022). The traditional CAR molecular design is usually fixed, including the scFv for antigen targeting and each part of CAR signal transduction. If multiple different antigens need to be targeted or the effectiveness of different candidate scFvs needs to be verified, it is necessary to design and construct the CAR vector from scratch and engineer T cells, which not only consumes huge time and economic costs but also limits the rapid development of new targets. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel split chimeric antigen receptor T cell and its construction method and application.

[0005] The inventive concept of the present invention is as follows: The present invention provides a split CAR design, which divides the antigen-binding domain and activation domain of CAR into two separate operating units, consisting of a universal chimeric antigen receptor (UniCAR) expressed on T cells as the activation domain and an antigen-specific targeting module of various antigen-specific molecules (such as scFv or mAb) fused with a tag protein. In short, SpyCatcher / SnoopCatcher is fused with the intracellular signal transduction domain to form UniCAR, while SpyTag / SnoopTag is fused with the antigen-specific molecule (such as scFv or mAb) to generate the antigen-specific targeting module. When assembling the complete CAR, the two parts of the split CAR are connected by the high-affinity binding between SpyCatcher / SnoopCatcher and SpyTag / SnoopTag. In addition, to facilitate the detection of the binding between UniCAR and the antigen-specific targeting module, the fluorescent protein is divided into two active parts and fused to the two modules of the split CAR respectively. Compared with the traditional fixed CAR design, this modular design of the split CAR allows the use of the same batch of T cells expressing UniCAR to screen different targets and different targeting modules for the same target without re-engineering the T cells, which is conducive to the rapid preclinical screening of target-specific molecules and more accurate comparison of therapeutic effects, greatly reducing the time cost and capital cost and accelerating the development process of new targets.

[0006] To achieve the object of the present invention, in the first aspect, the present invention provides a universal chimeric antigen receptor (UniCAR) for activating T cells, which sequentially comprises the following elements from the 5′-3′ direction:

[0007] 1) Signal peptide region 1;

[0008] 2) SpyCatcher or SnoopCatcher;

[0009] 3) GFP1-10 region;

[0010] 4) Hinge region;

[0011] 5) Transmembrane region;

[0012] 6) Intracellular signal co-stimulatory region; and

[0013] 7) Intracellular signal stimulation region.

[0014] Wherein, the hinge region can be the hinge region of human CD8α or human CD28, preferably the hinge region of human CD8α.

[0015] The transmembrane region may be the transmembrane region of human CD8α or human CD28, preferably the transmembrane region of human CD8α.

[0016] The amino acid sequence of the SpyCatcher is as shown in any one of SEQ ID NO: 4-13.

[0017] The amino acid sequence of the SnoopCatcher is as shown in SEQ ID No: 14.

[0018] The signal peptide region 1 may be the signal peptide of any mammalian cell surface protein, preferably the signal peptide of human albumin, the signal peptide of human insulin or the signal peptide of murine IgG kappa, more preferably the signal peptide of human albumin, and its amino acid sequence is as shown in SEQ ID NO: 1.

[0019] The amino acid sequence of the GFP1-10 region is as shown in SEQ ID NO: 15.

[0020] The amino acid sequence of the intracellular signal co-stimulation region is as shown in SEQ ID NO: 18.

[0021] The amino acid sequence of the intracellular signal stimulation region is as shown in SEQ ID NO: 19.

[0022] In a second aspect, the present invention provides a nucleic acid molecule encoding the UniCAR or a biological material containing the nucleic acid molecule.

[0023] The biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0024] In a third aspect, the present invention provides a recombinant lentivirus comprising a nucleic acid molecule encoding the UniCAR.

[0025] In a fourth aspect, the present invention provides a method for preparing the recombinant lentivirus, which is prepared by constructing a nucleic acid molecule encoding the UniCAR onto a lentiviral vector to obtain a recombinant lentiviral vector, and then co-infecting a host cell with a lentiviral packaging helper plasmid.

[0026] In a fifth aspect, the present invention provides an antigen-specific targeting module, which is used in combination with the UniCAR;

[0027] Its structure is: from the 5'-3' direction, in sequence are signal peptide 2 - antigen-specific targeting antibody - tag domain SpyTag or SnoopTag - fluorescence detection binding domain - purification tag; from the 5'-3' direction, the antigen-specific targeting module sequentially comprises the following elements:

[0028] 1) Signal peptide region 2;

[0029] 2) Antigen-targeting antibody region;

[0030] 3) SpyTag or SnoopTag;

[0031] 4) Linker;

[0032] 5) GFP-11 region; and

[0033] 6) Protein purification tag.

[0034] Wherein, the amino acid sequence of the SpyTag is shown as any one of SEQ ID NO: 24-26.

[0035] The amino acid sequence of the SnoopTag is shown as SEQ ID NO: 27.

[0036] The signal peptide region 2 can be a signal peptide of a prokaryotic expression system, preferably the signal peptide of OmpA, OmpF, LamB, STII, PhoA, MBP, DsbA or PelB, more preferably the PelB signal peptide, and its amino acid sequence is shown as SEQ ID NO: 20.

[0037] The antigen-targeting antibody region can be any single-chain antibody or nanobody to be screened; preferably an antibody targeting BCMA or an antibody targeting CD38; more preferably a single-chain antibody against BCMA, the amino acid sequence of its heavy chain region is shown as SEQ ID NO: 21, the amino acid sequence of its light chain region is shown as SEQ ID NO: 23, and the amino acid sequence of the linker between the heavy chain and the light chain is shown as SEQ ID NO: 22; more preferably a nanobody against CD38, and its amino acid sequence is shown as any one of SEQ ID NO: 29-38.

[0038] The amino acid sequence of the GFP-11 region is shown as SEQ ID NO: 28.

[0039] The protein purification tag can be any protein purification tag of a prokaryotic expression system, preferably His tag, Strep tag, Flag tag or HA tag, etc., more preferably His tag, and its amino acid sequence is shown as SEQ ID NO: 39.

[0040] In a sixth aspect, the present invention provides a method for constructing a split chimeric antigen receptor T cell, the method comprising: infecting T cells with the recombinant lentivirus to obtain recombinant T cells expressing UniCAR, and then mixing and incubating with the antigen-specific targeting module.

[0041] In a seventh aspect, the present invention provides a split chimeric antigen receptor T cell constructed according to the described method.

[0042] In an eighth aspect, the present invention provides the use of the split chimeric antigen receptor T cell in screening antigen-specific antibodies.

[0043] Specifically, a killing experiment is performed on cells expressing the antigen corresponding to the antibody using the split chimeric antigen receptor T cell, which facilitates the rapid screening of antibodies with better therapeutic effects.

[0044] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0045] (1) The present invention provides a split CAR design, which divides the antigen-binding domain and the activation domain of CAR into two separate operating units, improving the flexibility of screening.

[0046] (2) The present invention designs a universal chimeric antigen receptor (UniCAR), which can bind to a variety of antigen-specific molecules (such as scFv or mAb, etc.), without the need for repeated engineering modification of T cells, greatly simplifying the experimental operation and saving time and cost.

[0047] (3) The present invention applies the SpyCatcher-SpyTag or SnoopCatcher-SnoopTag system to the split CAR, and realizes the rapid binding of UniCAR and the targeting module through high-affinity binding.

[0048] (4) The present invention fuses two active parts of a fluorescent protein to two components of the split CAR respectively, as a detection tool for whether UniCAR and the targeting module bind, facilitating the detection of the targeting module.

[0049] (5) The split CAR modular design provided by the present invention allows simultaneous screening of different antibodies against the same target or antibodies against different targets, without the need for re-engineering of T cells, which is beneficial to the rapid preclinical screening of antigen-specific molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic structural diagram of the UniCAR module and the antigen-specific targeting module of the present invention, wherein (A) represents the primary sequence diagram of UniCAR and the antigen-specific targeting module, and (B) represents the three-dimensional structural diagram after UniCAR binds to the antigen-specific targeting module.

[0051] Figure 2 It is the GFP positive rate and BCMA positive rate results of UniCAR-T cells, BCMA-UniCAR-T cells and BCMA-CAR-T cells in a preferred embodiment of the present invention.

[0052] Figure 3 Comparison of the 24-hour in vitro killing results of BCMA-UniCAR-T cells, BCMA-CAR-T cells and T cells against MM.1S-mCherry target cells in the preferred embodiment of the present invention. The horizontal axis is the effector-to-target ratio, and the vertical axis is the number of remaining target cells.

[0053] Figure 4 Comparison of the 24-hour in vitro killing results of BCMA-UniCAR-T cells, BCMA-CAR-T cells and T cells against MM.1S-mCherry target cells in the preferred embodiment of the present invention. The horizontal axis is the effector-to-target ratio, and the vertical axis is the killing rate.

[0054] Figure 5 Purification electrophoresis pattern of 10 nanobodies for testing CD38 in the preferred embodiment of the present invention.

[0055] Figure 6 Comparison of the 24-hour in vitro killing results of 10 CD38-UniCAR-Ts against MM.1S-mCherry target cells tested in the preferred embodiment of the present invention. The horizontal axis is the effector-to-target ratio, and the vertical axis is the killing rate. Detailed implementation mode

[0056] In view of the problem in the prior art that the use of a fixed CAR design cannot quickly and simply screen multiple antigen-targeted receptors, the present invention proposes a split CAR design, which can quickly and simply screen multiple antigen-specific targeting modules at the same time, greatly saving time and funds. Moreover, the screening of antigen-specific targeting modules using the platform of the present invention is carried out in experiments with the same CAR positive rate and the same T cells, which can better and more accurately compare each antigen-specific targeting module.

[0057] The present invention adopts the following technical solutions:

[0058] In a first aspect, the present invention provides a universal chimeric antigen receptor (UniCAR), which comprises the following elements:

[0059] 1) Signal peptide region 1; 2) SpyCatcher / SnoopCatcher; 3) GFP1-10 region; 4) Hinge region; 5) Transmembrane region; 6) Intracellular signal co-stimulation region; and 7) Intracellular signal stimulation region.

[0060] The amino acid sequence of SpyCatcher / SnoopCatcher is shown as any one of SEQ ID NO: 4-14.

[0061] In an embodiment of the present invention, the signal peptide region can be the signal peptide of any mammalian cell surface protein, such as the human albumin signal peptide, the human insulin signal peptide, or the murine IgGkappa signal peptide. Preferably, the signal peptide is the human albumin signal peptide, and its amino acid sequence is shown in SEQ ID NO:1.

[0062] In an embodiment of the present invention, the amino acid sequence of the GFP1-10 region is shown in SEQ ID NO:15.

[0063] In an embodiment of the present invention, the hinge region can be the hinge region of human CD8α or human CD28. Preferably, the hinge region is the hinge region of human CD8α, and its amino acid sequence is shown in SEQ ID NO:16.

[0064] In an embodiment of the present invention, the transmembrane region can be the transmembrane region of human CD8α or human CD28. Preferably, the transmembrane region is the transmembrane region of human CD8α, and its amino acid sequence is shown in SEQ ID NO:17.

[0065] In an embodiment of the present invention, the amino acid sequence of the intracellular signal co-stimulatory region is shown in SEQ ID NO:18.

[0066] In an embodiment of the present invention, the amino acid sequence of the intracellular signal stimulation region is shown in SEQ ID NO:19.

[0067] In a second aspect, the present invention provides an antigen-specific targeting module, which comprises the following elements:

[0068] 1) Signal peptide region 2; 2) Antigen-targeting antibody region; 3) SpyTag / SnoopTag; 4) Linker; 5) GFP-11 region; and 6) Protein purification tag.

[0069] Wherein the amino acid sequence of SpyTag / SnoopTag is shown in any one of SEQ ID NO:24-27.

[0070] In an embodiment of the present invention, the signal peptide region 2 can be any signal peptide commonly used in prokaryotic expression systems, such as OmpA, OmpF, LamB, STⅡ, PhoA, MBP, DsbA, or PelB. Preferably, the signal peptide is the PelB signal peptide, and its amino acid sequence is shown in SEQ ID NO:20.

[0071] In an embodiment of the present invention, the antigen-targeting antibody region can be any single-chain antibody or nanobody to be screened. In the examples of the present invention, single-chain antibodies against BCMA and nanobodies against CD38 were tested.

[0072] Among them, the amino acid sequence of the anti-BCMA heavy chain region is shown as SEQ ID NO: 21, the amino acid sequence of the anti-BCMA light chain region is shown as SEQ ID NO: 23, the amino acid sequence of the linker between the heavy chain and the light chain of the anti-BCMA antibody is shown as SEQ ID NO: 22, and the amino acid sequence of the anti-CD38 nanobody is shown as any one of SEQ ID NOs: 29-38.

[0073] In an embodiment of the present invention, the amino acid sequence of the GFP-11 region is shown as SEQ ID NO: 28.

[0074] The protein purification tag can be any protein purification tag commonly used in prokaryotic expression systems, such as His tag, Streptag, Flag tag, or HA tag, etc. Preferably, the protein purification tag is His tag, and its amino acid sequence is shown as SEQ ID NO: 39.

[0075] In a third aspect, the present invention provides a nucleic acid molecule encoding UniCAR.

[0076] In a fourth aspect, the present invention provides a nucleic acid molecule encoding an antigen-specific targeting module.

[0077] In a fifth aspect, the present invention provides an expression vector, which contains a nucleic acid molecule encoding UniCAR.

[0078] In a sixth aspect, the present invention provides an expression vector, which contains a nucleic acid molecule encoding an antigen-specific targeting module.

[0079] In a seventh aspect, the present invention provides a recombinant lentivirus, which contains the nucleic acid molecule of the third aspect or is prepared by the expression vector of the fifth aspect.

[0080] In an eighth aspect, the present invention provides a method for preparing a recombinant lentivirus, including co-transfecting mammalian cells with the expression vector of the fifth aspect and an auxiliary plasmid to obtain a recombinant lentivirus.

[0081] In an embodiment of the eighth aspect, the auxiliary plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0082] In an embodiment of the eighth aspect, the mammalian host cell can be but is not limited to HEK293 cells, human PER.C6 cells, human Hela cells, and murine CHO cells, etc., and preferably HEK293 cells.

[0083] In a ninth aspect, the present invention provides UniCAR-T cells, which express the universal chimeric antigen receptor (UniCAR) of the first aspect of the present invention.

[0084] In a tenth aspect, the present invention provides a method for preparing the UniCAR-T cells of the ninth aspect, comprising transfecting T cells with the recombinant lentivirus of the seventh aspect.

[0085] In a specific embodiment of the tenth aspect of the present invention, the method for preparing the UniCAR-T cells of the ninth aspect comprises the following steps:

[0086] (1) Constructing a recombinant lentiviral expression vector carrying a nucleotide sequence encoding UniCAR;

[0087] (2) Transfecting a host cell with the above recombinant lentiviral expression vector and an auxiliary plasmid to prepare a recombinant lentivirus capable of infecting T cells;

[0088] (3) Isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood provided by a donor, and separating and activating T cells using magnetic beads;

[0089] (4) Infecting the T cells with the recombinant lentivirus obtained in step (2) to generate UniCAR-T cells expressing UniCAR.

[0090] In a further embodiment, the method may further comprise the following steps after step (4):

[0091] (5) Culturing the cells obtained in step (4) in vitro;

[0092] (6) Amplifying the cells obtained in step (5) in large quantities; and

[0093] (7) Collecting the UniCAR-T cells.

[0094] In an eleventh aspect, the present invention provides a method for expressing and purifying an antigen-specific targeting module, comprising transforming a prokaryotic expression cell with the expression vector of the sixth aspect of the present invention, inducing protein expression, and purifying the protein.

[0095] In an embodiment of the eleventh aspect, the prokaryotic expression cell may be, but is not limited to, BL21 cells, BL21(DE3) cells, BL21(DE3)PlyS cells, etc., and preferably BL21(DE3) cells.

[0096] In a twelfth aspect, the present invention provides an antigen-specific targeting module, which is obtained by the preparation method of the eleventh aspect.

[0097] In a thirteenth aspect, the present invention provides a method for preparing a complete CAR-T cell comprising an antigen-specific targeting module and UniCAR, comprising mixing the antigen-specific targeting module of the twelfth aspect of the present invention and the UniCAR-T cells of the tenth aspect.

[0098] In a fourteenth aspect, the present invention provides a complete CAR-T cell comprising an antigen-specific targeting module and a UniCAR, and the CAR-T cell is prepared by the method of the thirteenth aspect of the present invention.

[0099] In a fifteenth aspect, the present invention provides a method for detecting the positive rate of an antigen-specific targeting module on a complete CAR-T cell, comprising detecting the signal of the GFP fluorescent protein in the CAR-T described in the fourteenth aspect of the present invention by flow cytometry.

[0100] In a sixteenth aspect, the present invention provides a method for screening an antigen-specific targeting module, comprising performing a killing experiment on target cells with the above-mentioned complete CAR-T cell.

[0101] In an embodiment of the present invention, CAR represents a chimeric antigen receptor, BCMA-CAR represents a chimeric antigen receptor targeting BCMA, UniCAR represents a universal chimeric antigen receptor without an antigen-specific targeting module, CAR-T cell represents a chimeric antigen receptor T cell, BCMA-CAR-T cell represents a chimeric antigen receptor T cell targeting BCMA, and UniCAR-T cell represents a T cell expressing a universal chimeric antigen receptor without an antigen-specific targeting module.

[0102] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0103] Example 1 Construction and Application of Split Chimeric Antigen Receptor T Cells

[0104] 1. Design and Construction of Plasmids Encoding Universal Chimeric Antigen Receptor (UniCAR) and BCMA-CAR

[0105] The plasmid expressing encoded UniCAR was synthesized by Nanjing Biorade Biotech Co., Ltd. This plasmid contains nucleotide sequences encoding the following elements in sequence from the 5′ to 3′ direction of the UniCAR structure: signal peptide region 1 (SEQ ID NO:1, 2 or 3), SpyCatcher (SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12 or 13) or SnoopCatcher (SEQ ID NO:14), GFP1-10 (SEQ ID NO:15), hinge region (SEQ ID NO:16), transmembrane region (SEQ ID NO:17), intracellular signal co-stimulatory region (SEQ ID NO:18), and intracellular signal stimulatory region (SEQ ID NO:19). This plasmid was cloned into the lentiviral backbone plasmid pMSGV-IRES (Nanjing NewBio Biotech Co., Ltd., catalog number: V000092) to obtain the plasmid expressing UniCAR, which is called pMSGV-IRES-UniCAR-SpyCatcher or pMSGV-IRES-UniCAR-SnoopCatcher, and is a recombinant lentiviral expression vector for virus packaging. The schematic diagram of the constructed UniCAR structure is as Figure 1 shown.

[0106] To test the effectiveness of the UniCAR-T cells formed by the assembled UniCAR we constructed, we also prepared BCMA-CAR-T cells expressing BCMA-CAR for comparison with BCMA-UniCAR-T. The design and construction of the plasmid expressing BCMA-CAR are as described below:

[0107] It was synthesized by Nanjing Biorade Biotech Co., Ltd. This plasmid contains nucleotide sequences encoding the following elements in sequence from the 5′ to 3′ direction of the CAR structure: signal peptide region 1 (SEQ ID NO:1), anti-BCMA heavy chain region (amino acid SEQ ID NO:21), linker (amino acid SEQ ID NO:22), anti-BCMA light chain region (amino acid SEQ ID NO:23), hinge region (amino acid SEQ ID NO:16), transmembrane region (SEQ ID NO:17), intracellular signal co-stimulatory region (SEQ ID NO:18), and intracellular signal stimulatory region (SEQ ID NO:19). This plasmid was cloned into the lentiviral backbone plasmid pMSGV-IRES to obtain the plasmid encoding BCMA-CAR, which is called pMSGV-IRES-BCMA and is a recombinant lentiviral expression vector for virus packaging.

[0108] 2. Preparation of recombinant lentivirus

[0109] Transfect mammalian cells with the main plasmid pMSGV-IRES-UniCAR-SpyCatcher or pMSGV-IRES-UniCAR-SnoopCatcher or pMSGV-IRES-BCMA together with three auxiliary plasmids respectively, and recombinant lentiviruses capable of infecting T cells can be prepared. The specific steps are as follows:

[0110] 2.1. Prepare a cell culture flask with a culture area of 525 cm 2 , inoculate 6×10 7 WayneLVPro HEK293 cells (Suspension Adapted) (Zhongshan Kangsheng Biotechnology Co., Ltd., product number A23109, hereinafter referred to as 293T cells), add the complete medium of DMEM (GE Healthcare Life Sciences, product number: SH30243.01) and 10% FBS (GE Healthcare Life Sciences, product number: SH30256.01), and place it in a 37°C, 5% CO 2 cell culture incubator and culture overnight.

[0111] 2.2. Take 576 μL of PEI with a concentration of 1 mg / mL (Merck Biotechnology Co., Ltd., product number: 937762), add 4 mL of OPTI-MEM (Thermo Fisher Scientific, product number: 11058021), pipette and mix well, and let it stand at room temperature for 20 min.

[0112] 2.3. Add 54 μg of the main plasmid pMSGV-IRES-UniCAR-SpyCatcher or pMSGV-IRES-UniCAR-SnoopCatcher or pMSGV-IRES-BCMA and 16 μg of pMD2G-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0648), 16 μg of pRSV-REV-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0370) and 48 μg of pMDLG-pRRE-Kana (Changsha Abiway Biotechnology Co., Ltd., product number: HG-VMA0374) three auxiliary plasmids into 4 mL of OPTI-MEM and mix well.

[0113] 2.4. Add the PEI and OPTI-MEM mixed solution in step 2.2 to the solution in step 2.3, mix well, and let it stand at room temperature for 20 min.

[0114] 2.5. Take 32 mL of DMEM + 10% FBS complete medium and add it to the solution in step 2.4, and mix well.

[0115] 2.6. Aspirate the medium in the 293T cell culture flask and add the medium from step 2.5.

[0116] 2.7. After culturing the 293T cells in an incubator at 37°C and 5% CO 2 for 6 h, change the medium (DMEM + 10% FBS + 0.5% sodium butyrate (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: 303410)) and continue culturing for 48 h.

[0117] 2.8. Centrifuge the above medium to obtain the supernatant, filter it through a 0.22-μm filter membrane, and place it at -20°C for 30 min.

[0118] 2.9. Centrifuge the supernatant from step 2.8 at 1800 g at 4°C for 2 h, discard the supernatant, resuspend the virus particles with 500 μL of KBM581 serum-free medium (Corning Life Sciences (Wujiang) Co., Ltd., catalog number: 88-581-CM), and store at -80°C.

[0119] 3. Detection of recombinant lentivirus titer

[0120] 3.1. Take Jurkat cells (purchased from ATCC, catalog number: TIB-152) after resuscitation and culturing for 2 passages, and centrifuge at 400 g for 10 min.

[0121] 3.2. Discard the supernatant, resuspend the cells with RPMI1640 (Cytiva Biosciences (Hangzhou) Co., Ltd., catalog number: SH30809-01) medium to 2×10 6 cells / mL, seed into a 48-well plate, 2×10 5 cells / well.

[0122] 3.3. Take 100 μL of the recombinant lentivirus prepared in step 2 diluted 200 times with RPMI 1640, add it to the seeded cells, and mix well.

[0123] 3.4. After 6 h, supplement with 800 μL of RPMI 1640 + 10% FBS complete medium and culture in an incubator at 37°C and 5% CO 2 in the cell culture incubator.

[0124] 3.5. Perform flow cytometry detection after 48 h.

[0125] 4. T cell isolation and activation

[0126] 4.1. Under sterile conditions, take 15 mL of peripheral blood from healthy human donors, let it stand at room temperature for 30 min; centrifuge at 400 g for 30 min, and aspirate the plasma for later use.

[0127] 4.2. Dilute the lower layer of blood cells with PBS at a ratio of 1:1 and mix well. Carefully add the upper layer of human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biological Products Co., Ltd., product number: LTS1077) at a ratio of 1:2, and centrifuge at 300 g for 10 min.

[0128] 4.3. After centrifugation, aspirate the white film layer above the separation solution into a new centrifuge tube, add PBS to wash the white film layer, centrifuge at 300 g for 10 min, and discard the supernatant to obtain PBMC (Peripheral Blood Mononuclear Cell).

[0129] 4.4. Use the magnetic bead sorting method of CD3 Microbeads (Human) (Miltenyi Biotec GmbH, product number: 130-050-101) to separate T lymphocytes and perform cell counting.

[0130] 4.5. Take the sorted CD3 + T cells, centrifuge at 300 g for 10 min at 4°C.

[0131] 4.6. Discard the supernatant, add X-VIVO TM 15 serum-free medium (Lonza (China) Investment Co., Ltd., product number: DL-102) containing 40 IU / mL human IL-2 (Suzhou Novoprotein Science & Technology Co., Ltd., product number: GMP-CD66), and adjust the cell concentration to 1×10 6 cells / mL.

[0132] 4.7. Activate T cells according to the instructions of Dynabeads TM Mouse T-Activator CD3 / CD28 for T-Cell Expansion and Activation (Thermo Fisher Scientific Inc., product number: 11452D), and inoculate them in a 24-well cell culture plate at an inoculation concentration of 1×10 6 cells / mL, 1 mL / well; culture in a cell incubator for 48 h to obtain activated unmodified T cells.

[0133] 5. Preparation of UniCAR-T cells and BCMA-CAR-T cells

[0134] 5.1. Take a 24-well plate. After 48 h of T cell separation and activation, add 30 μL of 1 mg / mL protamine (Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: P4020) to each well and pipette to mix well.

[0135] 5.2. Calculate the required virus amount according to MOI = 3. The calculation formula is as follows: Required virus amount (mL) = (MOI × number of cells) / virus titer. According to the calculation results, add the UniCAR lentivirus or BCMA-CAR lentivirus prepared in step 2 into a 24-well plate and mix well.

[0136] 5.3. Seal the four sides of the 24-well plate with sealing film, centrifuge at 1000 g for 30 min at 37 °C, and culture in a 5% CO 2 cell incubator.

[0137] 5.4. Stain BCMA-CAR-T cells with PE-BCMA (Beijing Protein Innovation Co., Ltd., product number: 10620-H02H) antigen, and detect the expression of BCMA-CAR by flow cytometry.

[0138] 6. Preparation of BCMA and CD38 targeting modules

[0139] The plasmid expressing the BCMA single-chain antibody was synthesized by Nanjing Biorad Biotechnology Co., Ltd. The nucleotide sequence encoding the following elements is included in this plasmid in sequence from the 5' to 3' direction: signal peptide region 2 (SEQ ID NO: 20), anti-BCMA heavy chain region (SEQ ID NO: 21), linker (SEQ ID NO: 22), anti-BCMA light chain region (SEQ ID NO: 23) gene, SpyTag (SEQ ID NO: 24, 25 or 26) or SnoopTag (SEQ ID NO: 27), linker (SEQ ID NO: 22) and GFP-11 (SEQ ID NO: 28). Connect and insert the above fragments between the Nco I and Xho I restriction sites of the prokaryotic expression vector pET22b (Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number: 69744), so that the fusion protein encoded by the above sequence has an N-terminal signal peptide and a C-terminal His6 purification tag. This plasmid is called pET22b-SpyTag-BCMA, and the plasmid structure schematic diagram is as Figure 1 (A) shown.

[0140] Then, use the Escherichia coli expression system for protein expression, and purify the recombinant protein by Ni-NTA affinity chromatography. The specific steps are as follows:

[0141] 6.1. Take 1 μL of the pET22b-SpyTag-BCMA plasmid at a concentration of 1 μg / μL and transform it into 100 μL of Escherichia coli competent cells BL21(DE3) (Thermo Fisher Scientific, catalog number EC0114) according to the routine operations of molecular biology. Incubate on ice for 5 min, heat shock at 42 °C for 90 s, add 900 μL of LB medium, and culture in a shaker at 37 °C and 100 rpm for 1 h. Then, take 50 μL of the bacterial solution and spread it on an LB solid culture dish with ampicillin resistance in a laminar flow hood, and incubate it upside down at 37 °C overnight.

[0142] 6.2. Pick a single colony and add it to 200 mL of LB medium containing 100 μg / mL ampicillin, and culture overnight at 37 °C and 240 rpm.

[0143] 6.3. Measure the OD600 of the overnight culture bacterial solution and scale up the culture to 4 L of LB medium containing 100 μg / mL ampicillin (four 2-L culture flasks, 1 L per flask). Adjust the amount of the added bacterial solution so that the OD600 of the scaled-up bacterial solution is between 0.03 and 0.1, and continue to culture at 37 °C and 240 rpm until the OD600 is between 0.6 and 0.8.

[0144] 6.4. Add 1 mL of IPTG at a concentration of 0.5 M (Merck Biotechnology Co., catalog number I6758) to each culture flask, lower the culture temperature to 16 °C, and culture overnight (about 16 h) at 240 rpm.

[0145] 6.5. Transfer the overnight culture bacterial solution into a 1000-mL centrifuge bottle, centrifuge at 4 °C and 9000 g for 20 min, and collect the cell pellet.

[0146] 6.6. After ultrasonic disruption or high-pressure disruption of the cell pellet, centrifuge at 4 °C and 18000 g for 30 min, and collect the supernatant. Filter the supernatant of the bacterial solution through a 0.22-μm filter membrane.

[0147] 6.7. Connect the Ni-NTA pre-packed column (5 mL) to the protein purification instrument, first equilibrate the column with 5 column volumes of deionized water, and then with 5 column volumes of buffer A (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl).

[0148] 6.8. Load the filtered supernatant of the bacterial solution onto the Ni-NTA column at a flow rate of 1 mL / min.

[0149] 6.9. Wash the Ni-NTA column with 10 - 20 column volumes of buffer A at a flow rate of 3 - 5 mL / min.

[0150] 6.10. Set up a concentration gradient and linearly increase from 100% buffer A to 100% buffer B (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 250 mM imidazole) within 20 column volumes, and collect the elution peaks using a fraction collector. Then wash the column with 5 column volumes of buffer B and equilibrate the column with 5 column volumes of buffer A for the next batch of purification.

[0151] 6.11. Run the above elution peaks on SDS-PAGE electrophoresis to detect their purity. Combine, concentrate, and desalt the elution peaks with better purity to buffer C (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl).

[0152] 6.12. Detect the protein concentration using the BCA method or the 280 nm light absorption method, and then aliquot and store at -80 °C for later use.

[0153] The construction and purification of the CD38 targeting module are similar to those of the BCMA targeting module, except that the antibody targeting BCMA is replaced with a nanobody targeting CD38.

[0154] The details are as follows: Ligate the nucleotide fragments encoding signal peptide region 2 (SEQ ID NO: 20), anti-CD38 nanobody (SEQ ID NO: 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38), SpyTag (SEQ ID NO: 24, 25 or 26) or SnoopTag (SEQ ID NO: 27), linker (SEQ ID NO: 22) and GFP-11 (amino acid SEQ ID NO: 28), and insert them between the Nco I and Xho I restriction sites of the prokaryotic expression vector pET22b, so that the fusion protein encoded by the above nucleotide sequences has an N-terminal signal peptide and a C-terminal His6 purification tag. This plasmid was synthesized by Nanjing Baode Biotechnology Co., Ltd. and is called pET22b-SpyTag-CD38 or pET22b-SnoopTag-CD38. The "schematic diagram of plasmid structure" is as Figure 1 (A) shown.

[0155] Taking the combination of 10 nanobodies with SpyTag1 (SEQ ID NO: 24) as an example, the construction, expression and purification are described.

[0156] 7. Preparation of recombinant BCMA-UniCAR-T cells and CD38-UniCAR-T cells

[0157] The UniCAR lentivirus-transfected T cells obtained in step 5 do not yet have specific antigen recognition ability. Add the targeting module obtained in step 6 to the UniCAR-T cells at a ratio of 1:10,000 of the number of cells to the number of targeting modules to obtain BCMA-UniCAR-T cells specifically targeting the BCMA antigen and CD38-UniCAR-T cells specifically targeting the CD38 antigen. Then remove the targeting modules that have not bound to UniCAR by the method of centrifugation and medium replacement. At this time, detect the binding situation of UniCAR and the targeting module by detecting the GFP fluorescence signal with a flow cytometer. In addition, stain the BCMA-UniCAR-T cells with the PE-BCMA antigen and detect the positive rate of BCMA-CAR by flow cytometry, and compare them with BCMA-CAR-T cells, UniCAR-T cells and unmodified T cells respectively.

[0158] 8. Recombinant T cell in vitro killing experiment

[0159] Introduce the mCherry protein into MM.1S cells (Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-5017) to transform them into MM.1S-mCherry cells as target cells. Use BCMA-UniCAR-T cells as the effector cells to be tested, and use BCMA-CAR-T cells and unmodified T cells (the T cells obtained in step 4) as positive control and negative control effector cells respectively for killing activity determination. The specific steps are as follows:

[0160] 8.1. Adjust the target cell state to the logarithmic growth phase and passage continuously 2 times before the experiment.

[0161] 8.2. Adjust the target cell density to 1×10 6 cells / mL and inoculate 2 mL per well into a 6-well plate.

[0162] 8.3. According to the ratios of effector cells to target cells being 0.2 and 0.4 respectively, adjust the effector cell concentration to 1×10 6 cells / mL and add them to the target cell wells for co-culture respectively.

[0163] 8.4. Adjust the concentration of unmodified T cells to 1×10 6 cells / mL. According to the amount of effector cells added in the experimental group, add unmodified T cells to the target cell wells as negative controls, and the target cell wells without added effector cells as blank controls.

[0164] 8.5. Incubate the above 6-well plate in a 37°C, 5% CO 2 cell incubator for 24 h, and then calculate the volume of the medium in the wells and the cell concentration respectively.

[0165] 8.6. All samples were detected by flow cytometry, and the positive rate of target cells was detected through the PE channel.

[0166] The killing experiment of BCMA was similar. For the in vitro killing experiment of target cells by CD38-UniCAR-T cells, only BCMA-UniCAR-T cells need to be replaced with CD38-UniCAR-T cells, and other operations are the same.

[0167] Results and Discussion:

[0168] According to the differences in the SpyCatcher / SnoopCatcher and SpyTag / SnoopTag sequences, we constructed 11 kinds of UniCAR-T cells and 4 kinds of BCMA targeting modules. The UniCAR-T cells and BCMA targeting modules were mixed separately, and then the unbound targeting modules on UniCAR were removed by the method of centrifugal fluid replacement. Finally, the GFP positive rate was detected by flow cytometry, and the binding efficiency of UniCAR and the targeting module was judged accordingly. The results are shown in Table 1. The GFP positive rates of the 11 combinations are all higher than 95%, and there is no significant difference. Therefore, all the following experiments were carried out with the combination of SpyCatcher (SEQ ID NO: 4) and SpyTag (SEQ ID NO: 24).

[0169] Table 1 Binding efficiency of different combinations of SpyCatcher / SnoopCatcher and SpyTag / SnoopTag

[0170]

[0171]

[0172] UniCAR-T cells, BCMA-UniCAR-T cells and BCMA-CAR-T cells were constructed according to the above method, and the GFP and BCMA-CAR positive rates were detected by flow cytometry respectively. The results are as Figure 2 shown. The GFP positive rate of BCMA-UniCAR-T cells is as high as 99.3%, indicating that UniCAR and the targeting module can bind well. The results of PE-BCMA antigen staining show that the BCMA-CAR positive rates of BCMA-UniCAR-T cells and BCMA-CAR-T cells are comparable (96.8% and 97.5% respectively).

[0173] In the killing experiments of BCMA-UniCAR-T cells and BCMA-CAR-T cells on target cells, the results are as Figure 3 and Figure 4As shown, when the effector-to-target ratio was 0.2, both BCMA-UniCAR-T cells and BCMA-CAR-T cells still had obvious killing effects on MM.1s-mCherry target cells. When the effector-to-target ratio was 0.4, the killing rates of BCMA-UniCAR-T cells and BCMA-CAR-T cells on MM.1s-mCherry target cells were both close to 80%, with no obvious difference.

[0174] The BCMA single-chain antibody was used as a targeting module to assemble BCMA-UniCAR-T cells with UniCAR-T cells, which had a comparable BCMA-CAR positive rate to traditional BCMA-CAR-T cells and no difference in the killing ability against target cells. Therefore, this assembled UniCAR-T cells could be used to screen 10 CD38 nanobodies ( Figure 5 Purification electrophoresis diagram of 10 nanobodies against CD38 for testing). The results were as Figure 6 shown. Although the UniCAR-T cells combined with each of the 10 nanobodies had certain killing activities against target cells, there were still obvious differences among them. For example, the 7-CD38-UniCAR-T cells formed by combining the 7th CD38 nanobody with UniCAR-T cells showed the strongest killing activity, with a killing rate of over 90% when the effector-to-target ratio was 0.2; while the killing activity of the 3-CD38-UniCAR-T cells formed by combining the 3rd CD38 nanobody with UniCAR-T cells was relatively weak, with a killing rate of only 20% when the effector-to-target ratio was 0.4. By comparing the killing activities under the same CAR positive rate, it was possible to accurately identify which antibody-combined CAR-T might have better therapeutic effects.

[0175] The present invention provides a platform for simply and quickly screening chimeric antigen receptors (CARs), and its construction and application. The design and screening of traditional CARs require redesigning and constructing vectors and cell lines for each new target and different antibodies against the same target, resulting in a long R & D cycle and high costs. The present invention provides a split CAR design, which divides the antigen-binding domain and T cell activation domain of CAR into two operating units, namely, the antigen-specific targeting module and the universal chimeric antigen receptor (UniCAR), such that the T cell activation domain (i.e., UniCAR) can combine with multiple antigen-binding domains (antigen-specific targeting modules) without the need for re-engineering of the vector, improving the flexibility and throughput of screening and saving time and financial costs. At the same time, the present invention divides the fluorescent protein into two parts, which are respectively fused to the antigen-specific targeting module and UniCAR of CAR, facilitating the detection of the formation of the complete CAR and the positive rate of CAR on cells. This split CAR design allows for batch screening of different antibodies against the same target or antibodies against different targets without the need for re-engineering of T cells, which is beneficial for the rapid preclinical screening of antigen-specific molecules.

[0176] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

[0177] References

[0178] 1. BRENTJENS, R.J., DAVILA, M.L., RIVIERE, I., PARK, J., WANG, X., COWELL, L.G., BARTIDO, S., STEFANSKI, J., TAYLOR, C., OLSZEWSKA, M., BORQUEZ - OJEDA, O., QU, J., WASIELEWSKA, T., HE, Q., BERNAL, Y., RIJO, I.V., HEDVAT, C., KOBOS, R., CURRAN, K., STEINHERZ, P., JURCIC, J., ROSENBLAT, T., MASLAK, P., FRATTINI, M. & SADELAIN, M. (2013), "CD19 - targeted T cells rapidly induce molecular remissions in adults with chemotherapy - refractory acute lymphoblastic leukemia.", Science translational medicine, Vol. 5 No. 177, pp. 177ra38.

[0179] 2. DAVILA, M.L., RIVIERE, I., WANG, X., BARTIDO, S., PARK, J., CURRAN, K., CHUNG, S.S., STEFANSKI, J., BORQUEZ - OJEDA, O., OLSZEWSKA, M., QU, J., WASIELEWSKA, T., HE, Q., FINK, M., SHINGLOT, H., YOUSSIF, M., SATTER, M., WANG, Y., HOSEY, J., QUINTANILLA, H., HALTON, E., BERNAL, Y., BOUHASSIRA, D.C.G., ARCILA, M.E., GONEN, M., ROBOZ, G.J., MASLAK, P., DOUER, D., FRATTINI, M.G., GIRALT, S., SADELAIN, M. & BRENTJENS, R. (2014), "Efficacy and toxicity management of 19 - 28z CAR T cell therapy in B cell acute lymphoblastic leukemia.", Science translational medicine, Vol. 6 No. 224, pp. 224ra25. 3. GUEDAN, S., CALDERON, H., POSEY, A.D.J. & MAUS, M.V. (2019), "Engineering and Design of Chimeric Antigen Receptors.", Molecular therapy. Methods & clinical development, Vol. 12, pp. 145 - 156.

[0180] 4. MAUDE, S.L., FREY, N., SHAW, P.A., APLENC, R., BARRETT, D.M., BUNIN, N.J., CHEW, A., GONZALEZ, V.E., ZHENG, Z., LACEY, S.F., MAHNKE, Y.D., MELENHORST, J.J., RHEINGOLD, S.R., SHEN, A., TEACHEY, D.T., LEVINE, B.L., JUNE, C.H., PORTER, D.L. & GRUPP, S.A. (2014), "Chimeric antigen receptor T cells for sustained remissions in leukemia.", The New England journal of medicine, Vol. 371 No. 16, pp. 1507 - 17. 5. NEWICK, K., O'BRIEN, S., MOON, E. & ALBELDA, S.M. (2017), "CAR T Cell Therapy for Solid Tumors.", Annual review of medicine, Vol. 68 139 - 152.

[0181] 6. PANOWSKI, S.H., SRINIVASAN, S., TAN, N., TACHEVA - GRIGOROVA, S.K., SMITH, B., MAK, Y.S.L., NING, H., VILLANUEVA, J., WIJEWARNASURIYA, D., LANG, S., MELTON, Z., GHOSH, A., DUSSEAUX, M., GALETTO, R., HEYEN, J.R., SAI, T., Van BLARCOM, T., CHAPARRO - RIGGERS, J. & SASU, B.J. (2022), "Preclinical Development and Evaluation of Allogeneic CAR T Cells Targeting CD70 for the Treatment of Renal Cell Carcinoma.", Cancer research, Vol. 82 No. 14, pp. 2610 - 2624.

[0182] 7. SADELAIN, M., BRENTJENS, R. & I. (2013), "The basic principles of chimeric antigen receptor design.", Cancer discovery, Vol. 3 No. 4, pp. 388 - 98.

[0183] 8. TAMADA, K., GENG, D., SAKODA, Y., BANSAL, N., SRIVASTAVA, R., LI, Z. & DAVILA, E. (2012), "Redirecting gene - modified T cells toward various cancer types using tagged antibodies.", Clinical cancer research: an official journal of the American Association for Cancer Research, Vol. 18 No. 23, pp. 6436 - 45.

Claims

1. A universal chimeric antigen receptor (UniCAR) for activating T cells, characterized in that From 5' to 3', the UniCAR contains the following elements: 1) Signal peptide region 1; 2) SpyCatcher or SnoopCatcher; 3) GFP1-10 region; 4) Hinge region; 5) transmembrane region; 6) Intracellular signaling co-stimulatory region; and 7) Intracellular signal stimulation area; Wherein, the hinge region is the hinge region of human CD8α or human CD28, preferably the hinge region of human CD8α; The transmembrane region is the transmembrane region of human CD8α or human CD28, preferably the transmembrane region of human CD8α; The amino acid sequence of the SpyCatcher is shown in any one of SEQ ID NOs: 4-13; The amino acid sequence of SnoopCatcher is shown in SEQ ID No:

14.

2. The UniCAR according to claim 1, characterized in that The signal peptide region 1 is a signal peptide of any mammalian cell surface protein, preferably a human albumin signal peptide, a human insulin signal peptide or a mouse IgG kappa signal peptide, more preferably a human albumin signal peptide, and its amino acid sequence is shown in SEQ ID NO: 1; The amino acid sequence of the GFP1-10 region is shown in SEQ ID NO: 15; The amino acid sequence of the intracellular signal co-stimulatory region is shown in SEQ ID NO: 18; The amino acid sequence of the intracellular signal stimulation region is shown in SEQ ID NO:

19.

3. A nucleic acid molecule encoding the UniCAR according to claim 1 or 2 or a biological material containing the nucleic acid molecule; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

4. A recombinant lentivirus, characterized in that: It comprises a nucleic acid molecule encoding the UniCAR according to claim 1 or 2.

5. The method for preparing the recombinant lentivirus according to claim 4, characterized in that: The nucleic acid molecule encoding the UniCAR according to claim 1 or 2 is constructed into a lentiviral vector to obtain a recombinant lentiviral vector, which is then co-infected with a lentiviral packaging helper plasmid to infect a host cell.

6. An antigen-specific targeting module, characterized in that The antigen-specific targeting module is used in conjunction with the UniCAR according to claim 1 or 2; from the 5′-3′ direction, the antigen-specific targeting module comprises the following elements in sequence: 1) Signal peptide region 2; 2) Antigen-targeting antibody region; 3) SpyTag or SnoopTag; 4) Linker; 5) GFP-11 region; and 6) Protein purification tags; Wherein, the amino acid sequence of the SpyTag is shown in any one of SEQ ID NOs: 24-26; The amino acid sequence of the SnoopTag is shown in SEQ ID NO:

27.

7. The antigen-specific targeting module according to claim 6, characterized in that: The signal peptide region 2 is a signal peptide of a prokaryotic expression system, preferably OmpA, OmpF, LamB, STⅡ, PhoA, MBP, DsbA or PelB signal peptide, more preferably PelB signal peptide, and its amino acid sequence is shown in SEQ ID NO: 20; The antigen-targeting antibody region is any single-chain antibody or nanobody to be screened; preferably an antibody targeting BCMA or an antibody targeting CD38; more preferably an anti-BCMA single-chain antibody, the amino acid sequence of its heavy chain region is shown in SEQ ID NO: 21, the amino acid sequence of its light chain region is shown in SEQ ID NO: 23, and the amino acid sequence of the linker between the heavy chain and the light chain is shown in SEQ ID NO: 22; more preferably an anti-CD38 nanobody, the amino acid sequence of which is shown in any one of SEQ ID NOs: 29-38; The amino acid sequence of the GFP-11 region is shown in SEQ ID NO:28; The protein purification tag is any protein purification tag of a prokaryotic expression system, preferably a His tag, a Strep tag, a Flag tag or a HAtag, more preferably a His tag, and its amino acid sequence is shown in SEQ ID NO:

39.

8. A method for constructing a split chimeric antigen receptor T cell, characterized in that: The method comprises: infecting T cells with the recombinant lentivirus of claim 4 to obtain recombinant T cells expressing UniCAR, and then mixing and incubating with the antigen-specific targeting module of claim 6 or 7.

9. The split chimeric antigen receptor T cell constructed according to the method of claim 8.

10. Use of the split chimeric antigen receptor T cell according to claim 9 in screening antigen-specific antibodies.

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