CD7 endoplasmic reticulum retention signal, CD7 CAR recombinant vector and application of CD7 CAR recombinant vector in preparation of CD7 antigen positive hematologic tumor targeting immunotherapy drug

Abstract

The invention provides a CD7 endoplasmic reticulum retention signal, a CD7 CAR recombinant vector and application of the CD7 endoplasmic reticulum retention signal and the CD7 CAR recombinant vector in preparation of an immunotherapy drug for targeting CD7 antigen positive hematologic tumors, and belongs to the technical field of tumor immunotherapy. The CD7 endoplasmic reticulum retention signal disclosed by the invention comprises the following structure: P-S-CD7SCFV light chain-linker-CD7SCFV heavy chain-KDEL, wherein '-' is a connecting peptide or a peptide bond; p is a promoter; s is a signal peptide; the light chain of the CD7SCFV and the heavy chain of the CD7SCFV are connected through a linker with 10 amino acids. In the invention, the CD7 endoplasmic reticulum retention signal can be used for retarding expression of CD7 antigen protein on the surface of a vector T cell, and has a good retarding effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy, and in particular relates to a CD7 endoplasmic reticulum retention signal, a CD7 CAR recombinant vector and applications thereof in the preparation of immunotherapy drugs targeting CD7 antigen-positive blood tumors. Background Art

[0002] Hematologic malignancies include acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). For both AML and ALL patients, achieving complete remission (CR) again in the refractory / relapsed state is essential for prolonging survival.

[0003] Chimeric antigen receptor (CAR) T cell therapy is one of the most mature cellular immunotherapies. The more mature CARs are the second-generation CARs that contain scFv antigen binding domains, hinge / transmembrane domains, TCRζ and CD28 or 4-1BB co-stimulatory molecule domains. Compared with the first generation, they can continue to proliferate in vivo and have stronger tumor killing.

[0004] T cell tumors have the same or very similar membrane antigen expression as normal T cells. Therefore, the most important thing for CART treatment is to find specific targets for T cell tumors. During the development process, the antigen expression of T cells is mainly positive expression of TdT, cCD3, CD2, and CD7 with CD5+ / -. During the entire process of T cell maturation, it is accompanied by positive expression of CD2 / 5 / 7. Among them, CD7 is a co-stimulatory and cell adhesion molecule expressed on T cells, dendritic cells, and NK cells. It is expressed in about 95% of T-ALL. In addition, CD7 expression is also detected in about 30% of AML cases. Therefore, targeting CD7 antigen has become the most promising treatment for CD7-positive AML. However, in T cell tumors, the target antibody principle should only be expressed on tumor cells, but not on normal T cells, but most target antigens are expressed on both normal T cells and tumor T cells, such as CD7. Therefore, the preparation of CD7-CAR-T also needs to be uniquely designed to avoid the problem of mutual killing between normal T cells, tumor T cells, and CAR-T cells caused by shared antigens.

[0005] At present, by blocking the expression of CD7 antigen protein on the surface of carrier T cells, CART cells can be prevented from killing other CART cells expressing CD7, but the effect of blocking the expression of CD7 antigen protein on the surface of carrier T cells needs to be further improved. Summary of the invention

[0006] The purpose of the present invention is to provide a CD7 endoplasmic reticulum retention signal, a recombinant vector and their use in the preparation of immunotherapy drugs targeting CD7 antigen-positive blood tumors. The CD7 endoplasmic reticulum retention signal of the present invention can effectively block the expression of CD7 antigen protein on the surface of carrier T cells.

[0007] The present invention provides a CD7 endoplasmic reticulum retention signal, comprising the following structure: PS-CD7 SCFV light chain-linker-CD7 SCFV heavy chain-KDEL, wherein "-" is a connecting peptide or a peptide bond; P is a promoter; S is a signal peptide; the amino acid sequence of the CD7 SCFV light chain is shown in SEQ ID NO.1; the amino acid sequence of the CD7 SCFV heavy chain is shown in SEQ ID NO.2; the CD7 SCFV light chain and the CD7 SCFV heavy chain are connected by a linker having 10 amino acids; the amino acid sequence of the KDEL is shown in SEQ ID NO.3.

[0008] Preferably, the P includes hPGK promoter; the S includes CD8 SP; the amino acid sequence of the CD8 SP is shown in SEQ ID NO.4; and the amino acid sequence of the linker is shown in SEQ ID NO.5.

[0009] The present invention also provides a nucleic acid molecule encoding the CD7 endoplasmic reticulum retention signal described in the above scheme.

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.6.

[0011] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted.

[0012] The present invention also provides a CD7 CAR recombinant vector, into which the nucleic acid molecule described in the above scheme and the nucleic acid molecule encoding the chimeric antigen receptor targeting the CD7 antigen are inserted; the chimeric antigen receptor targeting the CD7 antigen comprises the following structure: P1-KOZAK sequence-S1-CD7 SCFV-CD8 hinge region-CD8 transmembrane region-4-1BB intracellular region-CD3 zeta, wherein P1 is a promoter and S1 is a signal peptide; except for the "-" in the "4-1BB intracellular region", the remaining "-" are connecting peptides or peptide bonds; the amino acid sequence of the CD7 SCFV is shown in SEQ ID NO.7.

[0013] Preferably, the P1 includes EF1α; the S1 includes CD8 SP; the amino acid sequence of the CD8 hinge region is shown as SEQ ID NO.8; the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO.9; the amino acid sequence of the 4-1BB intracellular region is shown as SEQ ID NO.10; and the amino acid sequence of CD3 zeta is shown as SEQ ID NO.11.

[0014] Preferably, the backbone vector of the recombinant vector includes a lentiviral expression vector; the lentiviral expression vector includes pCDH-CMV-MCS-EF1αHTLV-puro; the insertion site of the nucleic acid molecule according to claim 4 is between EcoR I and Spe I; the insertion site of the nucleic acid molecule encoding the chimeric antigen receptor is between BamHI and SalI.

[0015] The present invention also provides an engineered cell, comprising the recombinant vector described in the above scheme or the CD7 CAR recombinant vector.

[0016] The present invention also provides the use of the CD7 endoplasmic reticulum retention signal, the nucleic acid molecule, the recombinant vector, the CD7 CAR recombinant vector or the engineered cell in the above scheme in the preparation of anti-tumor drugs; the anti-tumor includes CD7 antigen-positive blood tumors or CD7 antigen-positive myeloid cell tumors.

[0017] The present invention provides a CD7 endoplasmic reticulum retention signal, comprising the following structure: PS-CD7 SCFV light chain-linker-CD7 SCFV heavy chain-KDEL, wherein "-" is a connecting peptide or a peptide bond; P is a promoter; S is a signal peptide; and the number of amino acids in the linker is 10. In the present invention, the CD7 SCFV light chain and the CD7 SCFV heavy chain are connected by a linker having 10 amino acids, which can be used to block the expression of CD7 antigen protein on the surface of carrier T cells, and has a good blocking effect.

[0018] The present invention also provides a CD7 CAR recombinant vector, into which a nucleic acid molecule encoding a CD7 endoplasmic reticulum retention signal and a nucleic acid molecule encoding a chimeric antigen receptor targeting the CD7 antigen are inserted. The CD7 CAR recombinant vector of the present invention uses an endoplasmic reticulum binding peptide to retain CD7 in the cytoplasm, inhibiting the expression of CD7 on the surface of the cell membrane of the carrier T cell; and CD7 chimeric antigen receptor CART cells are prepared using T cells with negative expression of CD7 surface antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 Designed the CD7 ER retention signal and CD7 CAR molecule;

[0021] Figure 2 The figure shows the results of restriction enzyme analysis of pCDH-CMV-MCS-EF1αHTLV-CD8 SP-CD7 CAR vector, where 1: vector is not digested, 2: vector is digested with BamHI and SalI; M: DNA marker;

[0022] Figure 3 The figure shows the results of restriction enzyme analysis of pCDH-hPGK-CD8 SP-CD7 SCFV light chain-linker-CD7 SCFV heavy chain-KDEL-EF1αHTLV-CD8SP-CD7 CAR vector, where 1. the vector was not digested with enzymes, 2. the vector was double-digested with Spe I+EcoR I; M: DNA marker;

[0023] Figure 4 The figure is a flow cytometric graph of a blank control cell group without concentrated virus; A, B, and C are flow cytometric graphs of live cells in the tested samples; D is a flow cytometric graph for detecting the positive rate of CAR transfection; and Figure E is the proportion of cell populations in each flow cytometric graph;

[0024] Figure 5 The figure is a flow cytometric graph of a cell group with 1 μL of concentrated virus added using flow cytometry; A, B, and C are flow cytometric graphs of live cells in the tested samples; D is a flow cytometric graph for detecting the positive rate of CAR transfection; and E is the proportion of cell groups in each flow cytometric graph;

[0025] Figure 6 The figure is a flow cytometric graph of a cell group with 5 μL of concentrated virus added using flow cytometry; A, B, and C are flow cytometric graphs of live cells in the tested samples; D is a flow cytometric graph for detecting the positive rate of CAR transfection; and E is the proportion of cell groups in each flow cytometric graph;

[0026] Figure 7In order to detect the infection of CD7 KDEL-CD7 CAR lentivirus (MOI=3) by flow cytometry, the expression levels of CD7 and CD3 on the cell membrane surface were detected 48 hours later; A1 is the side scattered light (SSC) and forward scattered light (FSC) of blank control cells, and A2 is the expression of CD3 and CD7 in blank control cells; B1 is the side scattered light (SSC) and forward scattered light (FSC) of cells containing 15 linkers, and B2 is the expression of CD3 and CD7 in cells containing 15 linkers; C1 is the side scattered light (SSC) and forward scattered light (FSC) of cells containing 10 linkers, and C2 is the expression of CD3 and CD7 in cells containing 10 linkers;

[0027] Figure 8 This is a flow cytometry image of T cells after lentivirus transfection; A is the live cell gate; B is a flow cytometry image of T cells not transfected with the virus using a CAR-labeled antibody staining image; C is a flow cytometry image of CAR-labeled antibody staining after CAR virus transfection; D and E are CD4+ and CD8+ CART cell populations in the CART cell population;

[0028] Fig. 9 This is a growth trend chart of T cells and CART cells expanded by in vitro culture;

[0029] Fig.10 The expression of surface inhibitory antigens before T cells were transfected with CAR virus; A, B, and C are blank controls; D, E, and F are expression flow cytometry graphs of PD1, TIM3, and LAG3;

[0030] Fig.11 The results of surface inhibitory antigen expression of CART cells on the 10th day after virus transfection; A is the expression of CAR marked by the APC channel; B is the expression of CAR marked by the PE channel; C, D, and E are the expression flow cytometry diagrams of PD1, TIM3, and LAG3, respectively;

[0031] Fig.12 The CD7 antigen expression detection of CCRF-CEM cell line; A represents the live cell gate of blank CCRF-CEM cells; B represents the blank control of CCRF-CEM cell CD7 expression; C represents the proportion of CD7 positive cells in CCRF-CEM cells;

[0032] Fig.13 The figure is a flow cytometry image of CD7-overexpressing Raji-luc cells; A represents the live cell gate of CD7-Raji cells; B represents the blank control of CD7 expression in CD7-Raji cells; C represents the CD7-positive cell ratio of CD7-Raji cells;

[0033] Fig.14The killing efficiency of CD7-targeted CART on CCRF-CEM cells at different effector-target ratios;

[0034] Fig.15 The specific killing efficiency of CD7-targeted CART on CD7+Raji cells and Raji cells that do not express CD7 at different effector-target ratios;

[0035] Fig.16 This is a schematic diagram of the grouping of in vivo experiments in mice;

[0036] Fig.17 This is a diagram of the operation plan for the in vivo experiment in mice;

[0037] Fig.18 This is a flowchart of the actual operation of the in vivo experiment in mice;

[0038] Fig.19 These are bioluminescent images of tumors in different groups of mice on the 7th, 12th and 17th days. DETAILED DESCRIPTION

[0039] The present invention provides a CD7 endoplasmic reticulum retention signal CD7 KDEL, comprising the following structure: PS-CD7 SCFV light chain-linker-CD7 SCFV heavy chain-KDEL, wherein "-" is a connecting peptide or a peptide bond; P is a promoter; S is a signal peptide; the amino acid sequence of the CD7 SCFV light chain is as shown in SEQ ID NO.1, specifically: AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQRPDGTVKLLIYYTSSLHSGVPSRF SGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR; the amino acid sequence of the CD7 SCFV heavy chain is as shown in SEQ ID NO.2, specifically: EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS; the CD7 SCFV light chain and the CD7 SCFV heavy chain are connected by a linker with 10 amino acids; the amino acid sequence of the KDEL is shown in SEQ ID NO.3, specifically: EQKLISEEDLKDEL.

[0040] In the present invention, the light chain of CD7 SCFV and the heavy chain of CD7 SCFV are connected by a linker having 10 amino acids, and the CD7 SCFV is used in the CD7 KDEL sequence to effectively block the expression of CD7 on the cell membrane surface of the carrier T cell.

[0041] In the specific implementation of the present invention, the P includes the hPGK promoter; the S includes the CD8 SP; the amino acid sequence of the CD8 SP is as shown in SEQ ID NO.4, specifically: MALPVTALLLPLALVLHAARP; the amino acid sequence of the linker is as shown in SEQ ID NO.5, specifically: GGGGSGGGGS.

[0042] In the specific implementation of the present invention, a KOZAK sequence with a nucleotide sequence of GCCACC is preferably inserted between the promoter and the signal peptide to enhance protein translation efficiency.

[0043] The present invention also provides a nucleic acid molecule encoding the CD7 endoplasmic reticulum retention signal described in the above scheme.

[0044] In the specific implementation of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the CD7 endoplasmic reticulum retention signal described in the above scheme is shown in SEQ ID NO.6, specifically:

[0045]

[0046] Among them, the single straight line underline sequence is the hPGK promoter nucleotide sequence; the bold sequence is the KOZAK sequence, which is used to enhance the protein translation efficiency; the double straight line underline sequence is the CD8 signal peptide nucleotide sequence; the single wavy line underline sequence is the CD7 SCFV light chain nucleotide sequence; the double wavy line underline sequence is the 10 amino acid linker nucleotide sequence; the dotted underline sequence is the CD7SCFV heavy chain nucleotide sequence; the dotted underline sequence is the KDEL nucleotide sequence; the bold and italic sequences are the restriction endonuclease Spe I recognition site and the restriction endonuclease EcoR I recognition site.

[0047] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted.

[0048] The present invention also provides a CD7 CAR recombinant vector, into which the nucleic acid molecule described in the above scheme and the nucleic acid molecule encoding the chimeric antigen receptor (CD7 CAR) targeting the CD7 antigen are inserted; the chimeric antigen receptor encoding the targeting CD7 antigen comprises the following structure: P1-KOZAK sequence-S1-CD7 SCFV-CD8 hinge region-CD8 transmembrane region-4-1BB intracellular region-CD3zeta, wherein P1 is a promoter and S1 is a signal peptide; except for the "-" in the "4-1BB intracellular region", the remaining "-" are connecting peptides or peptide bonds; the amino acid sequence of the CD7 SCFV is as shown in SEQ ID As shown in NO.7, specifically: AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGGGGS GGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISSRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS.

[0049] In the specific implementation of the present invention, the P1 is EF1αHTLV; the S1 is CD8 SP; the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO.8, specifically: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD; the amino acid sequence of the CD8 transmembrane region is as shown in SEQ ID NO.9, specifically: IYIWAPLAGTCGVLLLSLVITLYC; the amino acid sequence of the 4-1BB intracellular region is as shown in SEQ ID NO.10, specifically: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; the amino acid sequence of CD3 zeta is as shown in SEQID NO.11, specifically: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0050] In the specific implementation of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor targeting CD7 antigen is shown in SEQ ID NO.12, specifically:

[0051]

[0052]

[0053] Among them, the single straight line underlined sequence is the nucleotide sequence of the EF1αHTLV promoter; the bold underlined sequence is the KOZAK nucleotide sequence, which is used to enhance the protein translation efficiency; the double straight line underlined sequence is the CD8 SP (signal peptide) nucleotide sequence; the single wavy line underlined sequence is the CD7 SCFV nucleotide sequence; the double wavy line underlined sequence is the CD8 hinge region (CD8 hinge) nucleotide sequence; the dotted underlined sequence is the CD8 transmembrane region (CD8 transmembrane) nucleotide sequence; the dotted underlined sequence is the 4-1BB intracellular region (4-1BB intracellular) nucleotide sequence; the dot-dash underlined sequence is the CD3 zeta nucleotide sequence; the bold and italicized sequences are the restriction endonuclease BamHI recognition site and the restriction endonuclease Sal I recognition site.

[0054] In the present invention, the backbone vector of the recombinant vector preferably includes a lentiviral expression vector; in the specific implementation of the present invention, the lentiviral expression vector includes pCDH-CMV-MCS-EF1αHTLV-puro. pCDH-CMV-MCS-EF1αHTLV-puro is a third-generation lentiviral expression vector, which has higher safety and transduction efficiency than ordinary lentiviral expression vectors; compared with non-lentiviral expression vectors, due to the lower T cell transfection efficiency, the use of lentiviral infection methods can achieve long-term and efficient expression of the target gene in T cells.

[0055] In the present invention, the insertion site of the nucleic acid molecule encoding the CD7 endoplasmic reticulum retention signal in the above scheme is between EcoR I and Spe I; the insertion site of the nucleic acid molecule encoding the chimeric antigen receptor is between BamHI and Sal I. These two sites are just located upstream of the vector EF1αHTLV promoter and downstream of the coGFP sequence, so that the unnecessary coGFP fragment can be removed and the EF1αHTLV-CD8 SP-CD7 SCFV-CD8 hinge-transmembrane region-4-1BB intracellular region-CD3Zeta chain DNA fragment can be inserted directionally.

[0056] The present invention also provides an engineered cell, comprising the recombinant vector described in the above scheme or the CD7 CAR recombinant vector.

[0057] In the present invention, the original bacteria of the engineered cells include Escherichia coli; the Escherichia coli includes stbl3.

[0058] In the present invention, the engineered cells are characterized in that the immune cells express the CD7 endoplasmic reticulum retention signal and the chimeric antigen receptor targeting the CD7 antigen as described in the above scheme. In the present invention, the immune cells include T cells, NK cells, and macrophages.

[0059] The present invention also provides the use of the CD7 endoplasmic reticulum retention signal, the nucleic acid molecule, the recombinant vector or the engineered cell in the above scheme in the preparation of anti-tumor drugs; the anti-tumor includes CD7 antigen-positive blood tumors or CD7 antigen-positive myeloid cell tumors.

[0060] In the present invention, the anti-tumor effect preferably includes killing tumors and / or inhibiting tumor growth.

[0061] To further illustrate the present invention, a CD7 endoplasmic reticulum retention signal, a CD7 CAR recombinant vector and its application in the preparation of an immunotherapy drug targeting CD7 antigen-positive blood tumors provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] 1. CD7 endoplasmic reticulum retention signal and CD7 CAR molecule design and construction

[0064] CD7 endoplasmic reticulum retention signal and CD7 CAR molecule design Figure 1 As shown, in the pCDH-CMV-MCS-EF1αHTLV-puro vector, the nucleotide sequence of EF1αHTLV-KOZ AK sequence-CD8 SP-CD7 SCFV-CD8 hinge-transmembrane region-4-1BB intracellular region-CD3 Zeta chain DNA sequence shown in SEQ ID NO.16 was cloned and inserted into the site. The restriction enzyme analysis results are shown in Figure 2 .Depend on Figure 2 It can be seen that the inserted EF1αHTLV-KOZAK sequence-CD8SP-CD7 SCFV-CD8 hinge-transmembrane region-4-1BB intracellular region-CD3 Zeta chain DNA sequence contains 2019bp, with Bam HI and Sal I sites at both ends. After the successful cloned vector was cut with these two enzymes, agarose gel electrophoresis showed a 7kb vector fragment and a 2000bp insert fragment, indicating that the vector was successfully constructed.

[0065] The recombinant vector was then double-digested with EcoR I+Spe I, and the nucleotide sequence hPGK-CD8 SP-CD7 SCFV light chain-linker (nucleotide sequence length 30 bp)-CD7 SCFV heavy chain-KDEL sequence shown in SEQ ID NO.15 was ligated thereto. The restriction enzyme digestion analysis results are shown in Figure 3 The results showed that after the recombinant vector was digested with Spe I and EcoR I, a 7.2 kb vector fragment and a 1343 bp insert fragment appeared, indicating that the CD7 CAR recombinant vector was successfully constructed.

[0066] The target plasmid was transformed into DH5a competent cells, incubated briefly and inoculated into LB medium. After picking a single clone, it was placed in liquid LB medium for further activation and amplification. After 16 hours, a large amount of plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0067] 2. Preparation of CD7-KDEL-CD7-CAR Lentivirus

[0068] The 3rd to 5th passage HEK293T cells in the logarithmic growth phase were trypsinized and cultured in DMEM medium. The target plasmid, control plasmid, transfection reagent, and 293T cells were co-cultured for transfection. After 48 hours, the viral supernatant was collected and concentrated. After transfection of 293T cells, they were stained with FAB antibodies and the lentivirus titer was determined according to the positive rate.

[0069] Specific experimental process:

[0070] 1. 293T cell recovery

[0071] Preparation: a box of 1mL pipette tips, DMEM+FBS culture medium, 15mL centrifuge tubes, water bath (37°C)

[0072] (1) Take out one tube of 293T cells from the working cell bank from the liquid nitrogen tank, place it in a water bath at 37°C for rapid thawing, and add the cell suspension in the cryopreserved tube to the 4 mL culture medium in a centrifuge tube, 1500 rpm, centrifuge for 5 minutes.

[0073] (2) After centrifugation, discard the supernatant and add the prepared DMEM medium 5 mL was added to the centrifuge tube, and the bottom sediment was blown off with a pipette to resuspend it;

[0074] (3) Add the cell suspension to a 100 mm cell culture dish and add culture medium until 10 mL, shake gently, examine under a microscope, and culture in a box.

[0075] 3. Cell passaging

[0076] (1) When the cells proliferate and become confluent to about 90% after 48 hours of culture, the original culture medium is removed and the cells are subcultured. 5 Wash once with mLPBS;

[0077] (2) Digest with 3 mL of 0.05% pancreatin for 3 min. When all cells become round, add 4 mL of culture medium for termination;

[0078] (3) Add it to a 50 mL centrifuge tube and centrifuge at 1200 rpm for 5 min. Discard the supernatant and add the prepared DMEM medium. 10 mL was added to the centrifuge tube and the bottom sediment was blown away (gently blown); the cell suspension was evenly divided into two 100 mm culture dishes, and the final volume of the culture medium was 10 mL (cell number 6×10 6 / dish), make sure to spot the samples evenly and shake them gently, examine under a microscope, and put them into a culture box.

[0079] (4) Gelatin pretreatment: 1% gelatin was diluted to a concentration of 0.1% with PBS. After adding gelatin to cover the bottom of each dish, the dish was placed in an incubator for 2 h. The gelatin was taken out and aspirated. The dish was half-opened and placed on a clean bench to dry the gelatin for later use.

[0080] (5) The 293T cells from two dishes that had proliferated and fused to about 85% after being cultured for 48 hours were digested, resuspended and centrifuged, and resuspended in 10 mL of pre-prepared DMEM medium in a 50 mL centrifuge tube. The cell suspension was counted and the 6 ×10 6 Transfer each dish to 8 gelatin-coated 100mm cell culture dishes. Be careful to spot the samples evenly and shake them gently. Check under a microscope and put them into the culture box for culture.

[0081] 4. Plasmid transfection and virus collection

[0082] When the cells were cultured for 24 hours and the fusion rate reached about 85%, 293T cells were transfected by calcium phosphate precipitation method.

[0083] (1) pMD2G: 3 μg / dish, psPAX2: 9 μg / dish, CD7-CAR: 12 μg / dish, three plasmids were mixed, 63 μL 2M CaCL2 / dish was added, and 0.1×TE was added to make up to 500 μL / dish, and a total of 8 small dishes were mixed;

[0084] (2) Slowly add the mixture of 0.1×TE and plasmid into the 2×HEBS dish, mixing slowly;

[0085] See Table 1 for actual operation.

[0086] Table 1 Formula of virus packaging components

[0087]

[0088] (3) The liquid turns milky white after mixing. Let it stand for 15 minutes, then gently add 1 mL of the mixture to a dish, making sure to apply the mixture evenly and mix gently.

[0089] (4) After 6 hours, gently replace the culture medium in the culture dish with 6 mL of new culture medium. Note: The new culture medium is DMEM + 10% FBS (the liquid is preheated to 37°C), add 1 mL of new culture medium per 100 mm culture dish. 36 μL sodium butyrate.

[0090] (5) Collect the liquid at 24, 36, and 48 hours. Collect the supernatant of the virus liquid and store it at 4°C each time. Replace the culture medium each time. (After mixing all the supernatant, take 2 mL for virus titer determination)

[0091] (6) Then collect all the liquid supernatant and mix them evenly, centrifuge at 3500 rpm, 15 min, 4°C to discard the precipitate, take 700 μL of the virus supernatant for titer measurement. The remaining supernatant is mixed with PEG at a ratio of 4:1 and stored at 4°C overnight.

[0092] Actual operation: 140mL supernatant + 35mL PEG

[0093] (7) Centrifuge the overnight liquid at 3000 rpm, 30 min, 4°C, discard the supernatant, remove as much residual liquid as possible from the tube (you can tilt the tube downward and use a 50 mL centrifuge tube to wash away the residual liquid on the tube wall), and re-select the precipitate into an appropriate volume of IMDM medium (operate on ice).

[0094] Actual operation: 400μL.

[0095] (8) The resuspended virus concentrate was dispensed into 1.5 mL EP tubes, 50 μL per tube. (10 tubes)

[0096] 5. Titer detection

[0097] (1) After resuscitation, HT1080 cells were plated in 6-well plates at 3 x 10 cells per well. 5 cells. The next day 5.8x10 5 / well, add the virus supernatant to HT1080 cells at 100μL / well and 500μL / well of virus supernatant, 1μL / well and 5μL / well of concentrate, and 4μL / well of polybrene. Shake slowly, place in the incubator, and measure flow cytometry for 48h;

[0098] (2) Digestion: Wash twice with PBS, resuspend in 500 μL PBS, add 2 μL primary antibody CD7 protein, incubate at 4°C in dark for 20 min, wash twice, resuspend in 500 μL PBS, add 2 μL secondary antibody Steptavidin-APC, incubate at 4°C in dark for 20 min, wash twice, centrifuge at 1200 rpm for 5 min, resuspend in 500 μL PBS, and add 2 μL 7AD to label the cells;

[0099] (3) Analysis of flow cytometry results was calculated according to the formula "ln(1-a%)*b / c", where a% is the positive percentage determined by flow cytometry; b is the count value of one of the wells in the 6-well plate before infection; and c is the amount of infection.

[0100] CD7 virus titer: 3.8×10 7 TU / mL, 10 vials, 50μL / vial.

[0101] The lentiviral titer obtained was 3.8×10 7 TU / mL. Results are shown in Figures 4 to 6 .

[0102] Test Example 1

[0103] 1. Detection of CD7-negative T cells

[0104] T cells were sorted from PBMCs at one time and activated and expanded with CD3 / CD28 magnetic beads. The proportion of CD7 negative cells in the peripheral blood of healthy donors was about 90%. After T cells were infected with CD7-KDEL-CD7-CAR lentivirus (MOI=3) for 48 hours, CD7 antibody (PE labeled) and CD3 antibody (FITC labeled) were used to stain and detect the expression levels of CD3 and CD7 on the cell membrane surface.KB( Figure 7 A1 and A2 in the figure represent the non-transfected virus control; CAR15+12-2 ( Figure 7 B1 and B2 in the figure indicate T cells infected with lentivirus containing 15 amino acids in the linker of CD7 KDEL sequence; CAR15+12-3( Figure 7 C1 and C2 in the figure indicate T cells infected with lentivirus containing 10 amino acids in the linker of CD7 KDEL sequence.

[0105] The proportion of CD3+CD7+ double positive cells in the negative control was 89.53% ( Figure 7 A2 in the figure); after infection with a lentivirus containing the 15-amino acid linker KDEL sequence, the proportion of CD3+CD7+ double-positive cells was 72.14% ( Figure 7 B2 in the figure); however, the proportion of CD3+CD7+ double positive cells after infection with lentivirus containing the 10 amino acid linker KDEL sequence was 23.27% ( Figure 7C2 in the figure), indicating that the CD7 KDEL sequence of the 10-amino acid linker can inhibit the expression of CD7 cell membrane surface more efficiently.

[0106] The nucleotide sequence of the 15 amino acid linker is shown in SEQ ID NO.13, specifically: 5'-GGCGGCGGGGGCAGCGGCGGCGGCAGCGGCGGCGGCGGCAGC-3', and the amino acid sequence is shown in SEQ ID NO.14, specifically: GGGGSGGGGSGGGGS.

[0107] The nucleotide sequence of the 10 amino acid linker is specifically: 5'-GGCGGCGGCGGCAGCGGCGGCGGCGGCAGC-3' (SEQ ID NO.15), and the amino acid sequence is specifically: GGGGSGGGGS (SEQ ID NO.5).

[0108] 2. Viral infection and expansion of T cells

[0109] After CD7-T cells were stimulated and activated for 24 hours, they were co-cultured with CD7-CAR lentivirus. After 24 hours, new culture medium was added. The culture medium was changed every 2 days. The infection efficiency was detected after culturing for 14 days. The proportion of CD7-CART cells was detected by flow cytometry. The transfection efficiency of CD7-CART cells transfected with lentivirus was 33% to 40%. When CART was harvested, the amplification multiple of CART was 18 times. Figure 8 and Fig. 9 .

[0110] Specific experimental process:

[0111] 1) CD7-CAR transfection was performed 24 hours after CD7-T cells were stimulated and activated. A 24-well plate was used with 5×10 cells per well. 5 The CD7-CAR lentivirus titer was 3.8×10 7 TU / mL, the required virus amount for well A is 33 μL according to MOI=2.5, virus amount (μL)=MOI*number of cells / virus titer (TU / mL)×1000.

[0112] 2) Centrifuge the cells, discard most of the supernatant, leaving only about 50 μL of supernatant, resuspend the cells, add the corresponding volume of virus, mix well, and place at room temperature for 20 minutes (the time should not exceed 30 minutes, and gently blow once every 10 minutes); plate the 24-well plate, three wells A, B, and C, 500 μL of culture medium in each well, transfer the cell virus suspension to the well plate and continue to culture in the incubator. After 6 hours, aspirate the supernatant, add T cell culture medium to 2 mL, and continue to culture in the incubator.

[0113] 3) On the second day, 1 mL of culture medium was added and the cells were in good condition.

[0114] 4) On November 13, cell counting was performed, and 1×10 cells were counted in the 20 μL virus group. 6 The cell count in 30 μL is 9.8×10 4 The cells in each well were transferred to two wells of a six-well plate, and fresh culture medium was added for continued culture.

[0115] 5) On November 15, the cell count was 20 μL and the virus count was 2×10 7 The cell count in 30 μL is 1.2×10 6 Flow cytometry was performed, and 4 flow cytometry tubes were taken and marked as blank group KB respectively. The test result showed that the infection rate of CAR was 33%. The CART cells were transferred to T75 bottles for further culture and expansion.

[0116] 6) On the 10th day of CART amplification, the cell count was 6.1×10 7 pcs, 1×10 per tube 7 The cells were frozen, 1 mL per tube, and 5 tubes were frozen. The remaining CART cells were cultured and expanded for subsequent in vitro cell killing activity testing.

[0117] 7) CART cell freezing: The cell suspension is prepared in a ratio of immune cell culture medium: 20% human albumin = 11:5, and the cell freezing solution is prepared in a ratio of fetal bovine serum: DMSO = 4:1. The freezing volume uses the two liquids in a ratio of 1:1.

[0118] 8) Take 22mL of immune cell culture medium and add 10mL of human albumin to prepare a cell suspension, take 12mL of fetal bovine serum and add 3mL of DMSO to prepare a cell freezing solution. The total freezing volume is 5mL. Centrifuge the cells, discard the supernatant, take 2.5mL of the suspension to resuspend the cells, add 2.5mL of freezing solution, mix gently, divide into 5 cryotubes, place in a program cooling box, place in a -80℃ refrigerator overnight, and then transfer to a liquid nitrogen tank.

[0119] 9) Flow cytometry:

[0120] (1) Take 1x10 CART cells after infection 5 The cells were placed in flow cytometry tubes, marked as blank tube CART-KB, detection tubes PD1-APC, TIM3-PE, and LAG3-FITC, respectively, washed with 2 mL PBS and centrifuged at 1200 rpm for 5 min.

[0121] (2) Gently pour off the supernatant, resuspend the cells in 200 μL PBS, add the corresponding antibodies to the flow cytometry tubes according to the label, and incubate on ice for 30 min.

[0122] (3) After incubation, add 2 mL of PBS to the flow cytometry tube to wash the cells, centrifuge at 1200 rpm for 5 min, and then resuspend the cells with 200 μL of PBS. The cells in each tube are tested separately.

[0123] The results showed that the inhibitory receptors on the surface of CART cells on DAY10 showed no significant changes compared with the expression at the time of transfection. Fig.10 and Fig.11 .

[0124] 10) CD7-Raji-luc cells

[0125] (1) Raji-Luc cell plating

[0126] Take the cultured Raji-Luc cells, centrifuge at 1200RPM for 5min, discard the supernatant, resuspend the cells in 1mL culture medium, and count the cells;

[0127] The number of cells required for the experiment is 3×10 4 ,

[0128] Use a 24-well plate, with 500 μL of culture medium per well, and the number of cells per well is 2×10 4 .

[0129] (2) Preparation of lentivirus samples

[0130] According to MOI = 15, the required virus volume is 0.006mL, and the CD7 lentivirus titer is 5.5×10 7 , add 6μL and 7.5μL of virus to each well of experimental group a and b; no virus is added to control wells 1 and 2. (Calculate the amount of virus to be added to each well according to the set MOI, the amount of virus added to each well (μL) = MOI* number of cells / virus titer (TU / mL) × 1000)

[0131] (3) Add virus samples to the corresponding 24-well plate according to the table

[0132] First, take 100 μL of cell suspension and add it to the well, add culture medium to 250 μL, add 6 μL and 7.5 μL of virus, and then culture in a 37°C incubator for 6 hours.

[0133] (4) After 6 h, add culture medium to 500 μL and continue culturing in the incubator.

[0134] (5) After 24 h, the transfection effect was observed under a fluorescence microscope, and the culture medium was replenished to 2 mL to reduce the effect of the virus on cell viability and continue to be cultured in the incubator.

[0135] (6) Because Raji cells have been transfected with Luc-Puro virus, multiple screening with puromycin cannot be continued, and a second infection is planned to increase the positive rate.

[0136] (7) Green fluorescence can be observed in cells in both wells. Take 1 mL of cells from well b and transfer to well c. Add 6 μL CD7 virus again and continue culturing. Repeat step (5) for well C. Label the transfected cells with CD7-PE antibody and place them in a flow cytometer to sort CD7-Raji-luc cells.

[0137] The results showed that: CCRF-CEM cells expressing CD7 were constructed and CD7-positive tumor cells were constructed, and the targeted killing function of CART against CD7 was further verified. The results showed that the CD7 positive rate of CCRF-CEM cells was over 98%, and Raji cells could express CD7 after lentiviral transfection, with a purity of over 95%. Fig.12 and Fig.13 .

[0138] Test Example 2

[0139] 1. CD7-CART targeted killing CCRF experiment

[0140] (1) Preparation of CCRF cells: Prepare CCRF cells and count the cells at 2×10 per well. 4 5 E:T concentration groups and 1 CCRF control group were set up, with 3 replicates in each group. The required CCRF cell amount was 2×10 4 / hole × 4 holes × 6 groups = 4.8 × 10 5 indivual.

[0141] (2) Preparation of CD7-CAR-T cells: Cell recovery, thaw CART cells in a 37°C water bath, resuspend in T cell culture medium overnight, and count CART live cells on the second day. Set the E:T gradient to 4:1, 2:1, 1:1, 1:2, 1:4, and the required number of CART cells is 1.395×10 6 indivual.

[0142] (3) The culture volume of each well was 100 μL, and the test was performed after 48 h of mixed culture.

[0143] (4) CCK8 assay: 48 h after plating, add 10 μL of CCK8 solution to each well, incubate in a dark incubator for 1 h, and then measure the absorbance at 450 nm using a microplate reader.

[0144] (5) Calculation method of killing efficiency: [1-(mean OD of group ①-mean OD of group ②) / mean OD of group ③] × 100%.

[0145] The test results are shown in Table 2:

[0146] Table 2 Evaluation of CD7-CART targeting CCRF killing effect for 48h (CCK8 method)

[0147]

[0148] 2. CD7CART targeted killing CD7-Raji-luc experiment

[0149] (1) Preparation of CD7-Raji-Luc cells: Plate the cells one day in advance and count the cells at 1×10 per well. 4 Calculate the number of cells, set up three E:T concentration groups of 8:1, 4:1, 1:1 and one CD7-Raji-Luc control group, set up 3 replicates in each group, and the required CD7-Raji-Luc cell amount was 1×10 4 / hole × 5 holes × 4 groups = 2 × 10 5 indivual.

[0150] Take the cultured CD7-Raji-Luc cells and count the cells: 3×10 5 According to the design, 2×10 5 cells, add culture medium to 1 mL (5 wells × 4 groups × 50 μL), each well contains 1×10 4 cell.

[0151] (2) Preparation of Raji-Luc cells: Plate the cells one day in advance and count the cells at 1×10 per well. 4 1×10 cells were calculated, and three E:T concentration groups (8:1, 4:1, 1:1) and one HT1080 control group were set up. Three replicate wells were set up in each group. The required amount of HT1080 cells was 1×10 4 / hole × 5 holes × 4 groups = 2 × 10 5 indivual.

[0152] Take the cultured Raji-Luc cells and count the cells: 6×10 5 According to the design, 2×10 5 cells, add culture medium to 1 mL (5 wells × 4 groups × 50 μL), each well contains 1×10 4 cell.

[0153] (3) CD7-CAR-T cell preparation: Cell recovery, take CART cells and melt them in a 37°C water bath, add T cell culture medium and resuspend overnight, and count CART live cells on the second day. Set the E:T gradient to 8:1, 4:1, 1:1, and the required number of CART cells is: 8:1 group: 10 wells × 8 × 10 4 ; 4: 1 group: 10 holes × 4 × 10 4 ; 1: 1 group: 10 holes × 1 × 10 4, blank control group 4 wells × 1 × 10 4 , a total of 1.34×10 6 indivual.

[0154] (4) The culture volume of each well was 100 μL, and the test was performed after 48 h of mixed culture.

[0155] (5) Calculation method of killing efficiency

[0156] Killing efficiency % = (Kmin-K) / (Kmin-Kmax) × 100%

[0157] Kmin: Cell luciferase activity value detected without adding CAR-T cells to the culture system

[0158] K: The mixed cells in the culture system are centrifuged and precipitated, and the cells are resuspended in culture medium, and then a luciferase substrate is added to detect the luciferase activity value.

[0159] Kmax: Adding cell lysis solution to the culture medium containing target cells will lead to the maximum concentration of target cell killing, and the luciferase activity value of the cells will be detected.

[0160] Results Fig.14 and Fig.15 The results showed that the killing effect of CD7-CART cells targeting CD7-positive cells was detected in vitro, and the results showed that CART produced effective killing under different effector-target ratios.

[0161] Test Example 3

[0162] Mouse experiments to verify the killing effect of CD7-CART in vivo, the experiment was designed as follows Fig.16 and Fig.17 shown.

[0163] Experimental process:

[0164] Preparation and use of test / control substances

[0165] 3.1 Test substance 1: CD7-CAR-T cells (CAR positive T cell ratio 40%). Manufacturer: Beijing Sanyou Likang Cell Technology Co., Ltd.; Batch number: to be determined; CD7 CAR-T cells were prepared into 2.5×10 7 The solution was prepared at a concentration of 1 mL / mL (1.4 mL in total) and stored in an ice box away from light.

[0166] 3.2 Test substance 2: T cells not transfected with CD7-CAR (derived from PBMC of healthy donors), batch number: to be determined; when used, the T cells not transfected with CD7-CAR were prepared into 2.5×10 7The solution (1.4 mL) was stored in an ice box away from light.

[0167] 3.3 Control (solvent): The solvent in this experiment was PBS (pH 7.2-7.4), provided by Beijing Solebow Biotechnology Co., Ltd.

[0168] 3.4 Use: The test personnel shall obtain the test substance from the personnel who prepare the test substance and perform the operation according to the test protocol.

[0169] 4. Laboratory Animal Breeding and Quarantine

[0170] 20 SPF female NSG mice (including 3 spare animals), 6-8 weeks old, weighing 15-20 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (Animal Production License No.: SCXK (Su) 2019-0010). The animals were kept in the A110 breeding room of Beijing Boreishian Technology Co., Ltd. (Experimental Animal Use License No.: SYXK (Beijing) 2022-0025), 3-5 mice per cage, and quarantined and adapted to the environment for at least 3 days (including the day of receipt) before the experiment, and the weight reached 21-25 g. The experimental animal breeding environment and facilities meet the relevant requirements of GB14925-2023.

[0171] 5 Test methods

[0172] 5.1 Tumor cell preparation and inoculation

[0173] CD7-Raji-GFP-Luc human Burkitt's lymphoma cells were cultured in RPMI-1640 medium (containing 10% FBS, 100U / mL penicillin, 100μg / mL streptomycin) in a 37°C, 5% CO2 incubator. The medium was usually replaced every 2 to 3 days. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and fresh medium was added for subculture.

[0174] CD7-Raji-GFP-Luc cells in the logarithmic growth phase were centrifuged, washed and resuspended in PBS to a final cell concentration of 7.5×10 6 cells / mL, a total of 3.6 mL, slowly injected intravenously at 0.2 mL / animal (shaved the injection site before injection, disinfected with alcohol cotton balls), after the injection, press the injection site with dry cotton balls to stop bleeding, and then return the animal to the breeding cage for breeding.

[0175] 5.2 Route, cycle and frequency of administration of the test substance

[0176] Administration route and method: In this study, the test CD7-CAR-T cells or T cells not transfected with CD7-CAR were administered by intraperitoneal injection. The first administration time was the third day after the injection of CD7-Raji-GFP-Luc cells.

[0177] Dosing cycle and frequency: 1 time / day, dosing cycle 1 day.

[0178] Dosage: The test substance was administered according to the client's requirements, and the normal control group was administered with an equal volume of solvent.

[0179] Dosage design reason: Determined according to the client's requirements

[0180] Administration volume: The injection volume of each group was 0.2 mL / animal (1 mL syringe, accuracy 0.01 mL).

[0181] 5.3 Animal grouping and identification

[0182] On the third day (Day 3) after CD7-Raji-GFP-Luc cell injection (the day of injection is Day 0), the fluorescence intensity of tumor cells was measured using a small animal in vivo imaging system. When the fluorescence signal reached 1×10 6 ~1×10 7 p / s, the mice were randomly divided into 3 groups according to the intensity of tumor fluorescence signal and combined with body weight, with 6 mice in each group. After grouping, the solvent or test substance was given; the average body weight difference between groups after the animals were grouped did not exceed ±20%. The groups were distinguished by animal cage cards, and the tail numbers were used to identify the animals during the quarantine period and the test period after grouping. After the grouping and the end of the test, the animals that did not enter the test were handled according to the relevant standard operating procedures, and the "Experimental Animal Transfer Record" was filled in.

[0183] The animal grouping and dosage information are shown in Table 3.

[0184] Table 3 Animal groups and dosage

[0185]

[0186] Note: Refers to the number of CD7 CAR-positive cells.

[0187] 5.4 Observation indicators and methods

[0188] 5.4.1 Clinical symptom observation

[0189] Observe at least once a day, record whether there are any abnormalities in the animal's appearance, nutrition, fur, skin, mucous membrane, mental behavior, head, breathing, digestion, urogenital, and other aspects. If there are any abnormalities, increase the number of observations. Record the animal's survival status every day until the end of the experiment, and draw an animal survival curve.

[0190] 5.4.2 Body weight measurement

[0191] Weigh on Day 3, 7, 12, 17, 22, 24, 27 and 31.

[0192] 5.4.3 Determination of tumor cell fluorescence signal intensity

[0193] CD7-Raji-GFP-Luc cells were inoculated on Day 3, Day 7, Day 12, Day 17, Day 22, Day 27, and Day 31. Each animal was intraperitoneally injected with 0.2 mL of luciferase substrate (15 mg / mL). After isoflurane inhalation anesthesia, in vivo fluorescence imaging was performed using a small animal imaging system to measure the fluorescence signal intensity ( Fig.18 ).

[0194] 5.4.4 CD7 CAR-T cells and T lymphocyte ratio in peripheral blood (data for this group were tested externally after specimen collection)

[0195] On Day 10, Day 17, Day 24, and Day 31 after CD7-Raji-GFP-Luc cells were inoculated, 100 μL of anticoagulated blood (EDTA-Na2 anticoagulation) was collected from the orbital venous plexus. After lysing the red blood cells, 50 μL was used for flow cytometry to detect CD7-CAR-T cells in peripheral blood (G2 group: CD3-PercP, BiotinlytedCD7-protein+Anti Biotin-PE, CD4-PE-Cy, CD8-APC-Cy, PD1-APC, LAG3-FITC or CD3-PercP, CD7antibody+Anti Biotin-APC, CD4-PE-Cy, CD8-APC-Cy, CCR7-PE, CD 45RA-FITC, or CD3-FITC, CD7antibody+Anti Biotin-APC, CD4-percp, CD8-PE) and the proportion of T lymphocytes. The proportion of CD7-CAR-T cells to total T cells, the proportion of CAR-T CD4+ and CAR-T CD8+ in total CAR-T cells, and the proportions of TE, TEM, TCM, and TNB were analyzed in the samples. Another 50 μL was used for flow cytometry detection of CD7-Raji-GFP-Luc cells and the absolute number of tumor cells was calculated.

[0196] 5.4.5 Number of T lymphocytes and CD7-Raji-GFP-Luc in peripheral blood

[0197] CD7-Raji-GFP-Luc cells were inoculated on Day 28 (when most of them died, the cut-off time was D31, and when most of them were still alive, the cut-off time was postponed to D30) or Day 30. The surviving animals in each group were treated with xylazine hydrochloride (dose 12-25 mg / kg BW) combined with sildenafil. 50 (dose 15-50mg / kg BW, can be adjusted appropriately according to the veterinarian's opinion) intraperitoneal injection anesthesia, 300-400μL anticoagulated blood (EDTA-Na2 anticoagulation) was collected from the abdominal vena cava, and then the spleen and bone marrow were taken (the spleen and bone marrow were prepared into single cell suspensions respectively). A part of the anticoagulated blood, spleen cell suspension, and bone marrow cell suspension were used for blood cell counter to detect the number of lymphocytes, and another part of the anticoagulated blood, spleen cell suspension, and bone marrow cell suspension was used to lyse the red blood cells, and the flow cytometry was used to detect the T lymphocytes (anti human CD3-PercP) and CD7-Raji-GFP-Luc cell ratio (GFP / CD7-PE) in the peripheral blood. Or after lysing the red blood cells, the flow counting microspheres were used to count the T lymphocytes (anti humanCD3-PercP) and CD7-Raji-GFP-Luc cells (GFP / CD7-PE) in the peripheral blood, spleen cell suspension, and bone marrow cell suspension.

[0198] The results showed that CART cells can effectively kill tumors in mice. Fig.19 .

[0199] In summary, the above embodiments and test examples verify the correctness of the CD7 sequence, verify the availability of the CD7-CAR sequence, and improve the construction and preparation of CART through transformation experiments, plasmid extraction experiments, lentiviral coating, lentiviral transfection, cell amplification and other technologies, and obtain active CD7-CART cells. Then, by targeting CD7-positive tumor cell killing, the specificity of CD7-CART cells killing CD7-positive cells is fully demonstrated, and CD7-CART cells in mice can be effectively amplified and tumor growth can be inhibited. The technical solution of the present invention has effectively demonstrated the effectiveness of CD7-CART, and further confirmed that in addition to CD7-positive blood tumors that can be treated with the CART, the same CD7-positive myeloid cell tumors can also be treated with the CD7-CART.

[0200] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A CD7 endoplasmic reticulum retention signal, characterized in that It includes the following structure: PS-CD7 SCFV light chain-linker-CD7SCFV heavy chain-KDEL, wherein "-" is a connecting peptide or peptide bond; P is a promoter; S is a signal peptide; The amino acid sequence of the CD7 SCFV light chain is shown in SEQ ID NO.1; the amino acid sequence of the CD7 SCFV heavy chain is shown in SEQ ID NO.2; and the amino acid sequence of KDEL is shown in SEQ ID NO.

3.

2. The CD7 endoplasmic reticulum retention signal according to claim 1, characterized in that The P includes hPGK promoter; the S includes CD8 SP; the amino acid sequence of the CD8 SP is shown in SEQ ID NO.4; The amino acid sequence of the linker is shown in SEQ ID NO.

5.

3. A nucleic acid molecule encoding the CD7 endoplasmic reticulum retention signal according to any one of claims 1 to 3.

4. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

6.

5. A recombinant vector, characterized in that: Inserted with the nucleic acid molecule according to claim 3 or 4.

6. A CD7 CAR recombinant vector, characterized in that: Inserted with the nucleic acid molecule of claim 3 or 4 and a nucleic acid molecule encoding a chimeric antigen receptor targeting CD7 antigen; the chimeric antigen receptor targeting CD7 antigen comprises the following structure: P1-KOZAK sequence-S1-CD7 SCFV-CD8 hinge region-CD8 transmembrane region-4-1BB intracellular region-CD3 zeta, wherein, except for the "-" in "4-1BB intracellular region", the remaining "-" are connecting peptides or peptide bonds; P1 is the promoter, and S1 is the signal peptide; the amino acid sequence of the CD7 SCFV is shown in SEQ ID NO.

7.

7. The CD7 CAR recombinant vector according to claim 6, characterized in that The P1 includes EF1α; the S1 includes CD8 SP; the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.8; the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.9; the amino acid sequence of the 4-1BB intracellular region is shown in SEQ ID NO.10; and the amino acid sequence of CD3 zeta is shown in SEQ ID NO.

11.

8. The CD7 CAR recombinant vector according to claim 6 or 7, characterized in that The backbone vector of the recombinant vector includes a lentiviral expression vector; the lentiviral expression vector includes pCDH-CMV-MCS-EF1αHTLV-puro; the insertion site of the nucleic acid molecule of claim 4 is between EcoR I and Spe I; the insertion site of the nucleic acid molecule encoding the chimeric antigen receptor is between BamHI and SalI.

9. An engineered cell, characterized in that: Comprising the recombinant vector of claim 5 or the CD7 CAR recombinant vector of any one of claims 6 to 8.

10. Use of the CD7 endoplasmic reticulum retention signal according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the recombinant vector according to claim 5, the CD7 CAR recombinant vector according to any one of claims 6 to 8, or the engineered cell according to claim 9 in the preparation of anti-tumor drugs; the anti-tumor includes CD7 antigen-positive blood tumors or CD7 antigen-positive myeloid cell tumors.

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