Construction method and application of chimeric antigen receptor T cell interfered by Rc3h1 gene

Abstract

The invention discloses a construction method and application of Rc3h1 gene interfered chimeric antigen receptor T cells, and relates to the technical field of CAR-T cell therapy. The construction method of the chimeric antigen receptor T cell interfered by the Rc3h1 gene comprises the following steps: (1) designing an RNAi target sequence; (2) designing an interference sequence according to the RNAi target sequence; (3) constructing a pLL3.7 NKG2D CAR-Rc3h1 shRNA (short hairpin Ribonucleic Acid) plasmid; (4) carrying out plasmid transfection, and carrying out lentivirus packaging; and (5) constructing the shRc3h1NKG2D CAR-T cell. By adopting the construction method and application of the Rc3h1 gene interfered chimeric antigen receptor T cell, the problems that the existing CAR-T is limited in proliferation and activation in solid tumor treatment, is seriously depleted and is poor in effectiveness and persistence of an anti-tumor effect can be solved.

Description

Technical Field

[0001] The present invention relates to the technical field of CAR-T cell therapy, and in particular to a construction method and application of Rc3h1 gene-interfered chimeric antigen receptor T cells. Background Art

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy refers to adoptive cell therapy that enables specific killing by endowing T cells with the ability to recognize specific tumor cell surface antigens through in vitro genetic engineering. The main structure of CAR includes the following parts: single-chain antibody variable region (scFv) or T cell / NK cell receptor binding domain that specifically recognizes tumor antigens, linker, transmembrane region, and co-stimulatory molecules and CD3ζ fusion intracellular segment. The dual-signal design enables CAR-T cells to recognize and kill tumor cells in an MHC-unrestricted manner. CAR-T is relatively mature in the treatment of malignant hematological tumors, and there are many FDA-approved CAR-T cell products; although there are many related studies on CAR-T treatment of solid tumors, and good results have been achieved in some solid tumors, the overall response rate is still unsatisfactory. The main dilemma of CAR-T in the treatment of solid tumors is that CAR-T cells are difficult to infiltrate in the immunosuppressive tumor microenvironment, proliferation and activation are limited, and exhaustion is serious, and they cannot continuously exert anti-tumor effects. Therefore, optimizing CAR-T performance with the goal of enhancing the anti-tumor activity of CAR-T cells and prolonging their survival time in the body is a potential research and development direction.

[0003] Roquin1 is an RNA binding protein encoded by the Rc3h1 (Ring finger and CCCH-type domains 1) gene, which plays an important role in post-transcriptional regulation. In adaptive immune cells, Roquin1 is closely related to cell homeostasis, activation and differentiation fate. Its downstream target mRNAs such as Icos, Tnfa, Ox40, etc. are mostly related to T cell activation. Some studies have suggested that Rc3h1 is a regulator of CD8 + Important genes for T cell expansion and demonstrated that Rc3h1-deficient CD8 + T cell proliferation and anti-tumor activity were enhanced, and this phenotype may be related to the upregulation of IRF4 expression caused by the loss of Rc3h1.

[0004] Roquin1 is located in the cytoplasm and is enriched in P bodies, but relocates to stress granules when cells are stressed. The protein contains an RNA binding domain called ROQ, which mainly recognizes constitutive decay elements located in the 3'-untranslated region of the target mRNA, and can also bind to the "pyrimidine-purine-pyrimidine" sequence in the U-rich linear structure with lower affinity. The ROQ domain can also bind to alternative decay elements with a "GUUYUA" six-base loop or a U-rich loop hairpin structure with variable length. Roquin-1 also includes a HEPN domain and a CCCH-type zinc finger domain (Znf), which play a role in maintaining the stability of the interaction between Roquin-1 and double-stranded RNA and mediating the binding of the mRNA destabilizing protein TTP (Tristetraprolin) to the mRNA AU-rich elements.

[0005] This invention uses RNA interference technology on NKG2D CAR-T for the first time to reduce the expression of Rc3h1 gene, with the purpose of enhancing the performance of CAR-T in treating solid tumors. The experimental results show that NKG2D CAR-T cells interfering with Rc3h1 gene were constructed on T cells derived from peripheral blood mononuclear cells. The successful construction of CAR-T cells was confirmed by flow cytometry, and the effectiveness of interference sequence on Rc3h1 gene interference in CAR-T was demonstrated at the mRNA and protein levels. The modified NKG2D CAR-T cells showed stronger anti-tumor effects than before the modification, whether co-incubated with tumor cell lines in vitro or used for the treatment of NSG mouse subcutaneous tumor-bearing models. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing and using a chimeric antigen receptor T cell with Rc3h1 gene interference, which can improve the problems of limited proliferation and activation, severe exhaustion, and poor effectiveness and sustainability of anti-tumor effects of existing CAR-T in the treatment of solid tumors. The chimeric antigen receptor T cell with Rc3h1 gene interference constructed by the present invention has better anti-tumor activity and longer survival time in the body.

[0007] To achieve the above object, the present invention provides an Rc3h1 interference sequence, which is composed of a sense sequence and an antisense sequence, a spacer "TTCAAGAGA" connecting the two, and a poly T termination signal at the 3' end, including one of the following:

[0008] Rc3h1-shRNA-1, the sense sequence of which is shown in SEQ ID NO.1, and the antisense sequence of which is shown in SEQ ID NO.2;

[0009] Rc3h1-shRNA-2, the sense sequence of which is shown in SEQ ID NO.3, and the antisense sequence of which is shown in SEQ ID NO.4;

[0010] Rc3h1-shRNA-3, its sense sequence is shown in SEQ ID NO.5, and its antisense sequence is shown in SEQ ID NO.6.

[0011] Furthermore, the target sequence Rc3h1-siRNA-1 of Rc3h1-shRNA-1 is shown as SEQ ID NO.7, the target sequence Rc3h1-siRNA-2 of Rc3h1-shRNA-2 is shown as SEQ ID NO.8, and the target sequence Rc3h1-siRNA-3 of Rc3h1-shRNA-3 is shown as SEQ ID NO.9.

[0012] The present invention also provides a method for constructing a chimeric antigen receptor T cell with Rc3h1 gene interference, comprising the following steps:

[0013] (1) Design RNAi target sequence;

[0014] (2) designing an interfering sequence according to the RNAi target sequence, wherein the interfering sequence is the above-mentioned Rc3h1 interfering sequence;

[0015] (3) Construction of pLL3.7 NKG2D CAR-Rc3h1 shRNA plasmid;

[0016] (4) Plasmid transfection and lentiviral packaging;

[0017] (5) Construction of shRc3h1 NKG2D CAR-T cells.

[0018] Furthermore, the specific method of step (3) is as follows: adding 20 base homology arms at both ends of the interference sequence to synthesize two full-length oligonucleotide sequences, the two chains are annealed and complementary to form a double-stranded chain, and then connected to the U6 promoter of the pLL3.7 NKG2D CAR plasmid by homologous recombination, and then identified by EcoRI single enzyme digestion and DNA sequencing to successfully construct the plasmid.

[0019] Furthermore, the specific method of step (4) is: selecting HEK-293T cells with good growth status for transfection, using a lentiviral three-plasmid system for lentiviral packaging, and packaging three viruses: LV-puro (Mock), LV-NKG2D CAR), and LV-shRc3h1-1-NKG2D CAR.

[0020] Furthermore, the specific method of step (5) is as follows: construct shRc3h1 NKG2D CAR-T by infecting PBMC-derived T cells at a virus infection multiplicity of 15, and adding the virus at a volume of 1×10 6179 μl LV-shRc3h1-1-NKG2D CAR virus concentrate was added to each cell, an equal volume of LV-Puro virus concentrate was added to the Mock group, and 326 μl LV-NKG2D CAR virus concentrate was added to the non-interference group. The culture medium was changed after 24 hours, and the cells were collected after another 24 hours of culture, stained with APCAnti-Human-CD314 (NKG2D) antibody, and the positive rate was detected by flow cytometry.

[0021] The present invention also provides a chimeric antigen receptor T cell with Rc3h1 gene interference constructed by the above construction method.

[0022] The present invention also provides the use of the above-mentioned Rc3h1 gene interfered chimeric antigen receptor T cells in the preparation of anti-tumor drugs.

[0023] The advantages and positive effects of the construction method and application of the Rc3h1 gene-interfered chimeric antigen receptor T cells of the present invention are:

[0024] 1. The Rc3h1 interference sequence in the present invention can significantly reduce the mRNA expression and protein expression of Rc3h1, thereby promoting CD8 + T cell proliferation and antitumor activity.

[0025] 2. The Rc3h1 interference sequence in the present invention can significantly reduce the expression of the Rc3h1 gene, promote the killing efficiency of CAR-T cells on tumor cells in vivo and in vitro, and promote the anti-tumor function of CAR-T cells.

[0026] 3. The Rc3h1 interference sequence in the present invention can significantly reduce the expression of the Rc3h1 gene, effectively promote the activation of CAR-T cells, reduce the exhaustion level of CAR-T cells, and promote the anti-tumor function of CAR-T cells.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a plasmid structure diagram in Example 1 of the present invention;

[0029] Figure 2 For the restriction enzyme digestion and identification of the pLL3.7-NKG2D-Rc3h1 shRNA plasmid in the present invention;

[0030] Figure 3 For the detection of the positive rate of NKG2D CAR-J cells in each group of the present invention;

[0031] Figure 4The real-time fluorescence quantitative PCR and reverse transcription PCR in the present invention compare the interference efficiency of the three interference sequences in NKG2D CAR-J, wherein A is the real-time fluorescence quantitative PCR result and B is the reverse transcription PCR result;

[0032] Figure 5 Detection of the LV-shRc3h1-1-NKG2D CAR virus titer in the present invention;

[0033] Figure 6 For the detection of the positive rate of shRc3h1 NKG2D CAR-T cells in the present invention;

[0034] Figure 7 The interference efficiency of Rc3h1-shRNA-1 in NKG2D CAR-T detected by real-time fluorescence quantitative PCR and Western blot in the present invention, wherein A is the interference efficiency of Rc3h1-shRNA-1 in NKG2D CAR-T detected by real-time fluorescence quantitative PCR, and B is the interference efficiency of Rc3h1-shRNA-1 in NKG2D CAR-T detected by Western blot;

[0035] Figure 8 is the killing level of shRc3h1 NKG2D CAR-T on CAPAN2 and PANC1 in the present invention, wherein A is the killing level of shRc3h1 NKG2D CAR-T on CAPAN2, and B is the killing level of shRc3h1NKG2D CAR-T on PANC1;

[0036] Fig. 9 It is the detection of activation indexes after co-incubation of shRc3h1 NKG2D CAR-T with PANC1 in the present invention;

[0037] Fig.10 This is the exhaustion level detection of shRc3h1 NKG2D CAR-T in the present invention after co-incubation with PANC1 for one week;

[0038] Fig.11 The changes of PANC1 tumor volume over time in each group of mice after adoptive cell therapy in the present invention;

[0039] Fig.12 This is the IVIS tumor imaging of each group of mice after adoptive cell therapy in the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below through the accompanying drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0041] Example 1 Construction of pLL3.7-NKG2D CAR-Rc3h1 shRNA plasmid

[0042] 1.1 RNAi target sequence design

[0043] The transcript sequence of human Rc3h1 gene was searched in the Nucleotide library of NCBI (Accession: NM_172071); RNAi sequence design was performed using the RNAi design tool website. The results are shown in Table 1.

[0044] Table 1 Rc3h1 gene RNAi target sequence

[0045]

[0046] 1.2 Design interference sequence according to target sequence

[0047] The shRNA sequence was designed based on the above siRNA sequence. The shRNA consists of a sense sequence and an antisense sequence, a spacer "TTCAAGAGA" connecting the two, and a poly T termination signal at the 3' end. To facilitate subsequent identification, an EcoRI restriction site and a protective base were added to the 3' end of the sense chain. The results are shown in Table 2.

[0048] Table 2 Oligonucleotide sequences designed based on the Rc3h1 gene RNAi target sequence

[0049]

[0050] 1.3 Construction of pLL3.7 NKG2D CAR-Rc3h1 shRNA plasmid

[0051] Homologous arms of about 20 bases were added to both ends of the designed shRNA, and two full-length oligonucleotide sequences were synthesized by Anshengda Life Science Technology Co., Ltd. After the two chains were annealed and complementary to form double strands, they were connected to the back of the U6 promoter of the pLL3.7 NKG2D CAR plasmid by homologous recombination. After EcoRI single restriction enzyme digestion and DNA sequencing, the plasmid was successfully constructed. The plasmid structures of pLL3.7 NKG2D CAR-Rc3h1shRNA-1, pLL3.7 NKG2D CAR-Rc3h1 shRNA-2, pLL3.7NKG2D CAR-Rc3h1shRNA-3 and the control plasmids pLL3.7 Puro and pLL3.7 NKG2D CAR used in subsequent experiments are shown in the following figure. Figure 1 The results of plasmid sequencing and enzyme digestion identification are shown in Figure 2 shown.

[0052] Example 2pLL3.7-NKG2D CAR-Rc3h1 shRNA lentiviral packaging

[0053] 2.1 Plasmid transfection

[0054] The lentiviral three-plasmid system was used for lentiviral packaging. HEK-293T cells with good growth status were selected for transfection.

[0055] Packaged viruses: LV-puro (Mock), LV-NKG2D CAR, LV-shRc3h1-1-NKG2D CAR, LV-shRc3h1-2-NKG2D CAR, LV-shRc3h1-3-NKG2D CAR.

[0056] The main steps are as follows:

[0057] (1) Transfection was performed when the density of HEK-293T cells reached about 80%;

[0058] (2) According to the amount of cells to be transfected, prepare transfection system A and system B in centrifuge tubes according to the formula shown in Table 3, and let stand for 5 minutes;

[0059] (3) Add system A to system B, vortex mix, and continue to stand at room temperature for 20 min;

[0060] (4) Add 1 ml of the mixed system evenly and dropwise to each dish of cells, place the cells in an incubator and continue culturing for 6-8 hours, then discard the supernatant and replace with 10 ml of DMEM complete medium for continued culturing.

[0061] Table 3 Transfection system

[0062]

[0063]

[0064] 2.2 Lentivirus harvesting and concentration

[0065] After transfection and medium replacement, 293T cells were cultured for 48 hours, and the culture supernatant (containing virus) was collected into a 50 ml centrifuge tube and temporarily stored at 4°C. 5-6 ml of DMEM complete medium was added along the side wall of the cell culture dish and cultured for 24 hours, and the culture supernatant was collected again into a 50 ml centrifuge tube.

[0066] The harvested lentivirus was named LV-shRc3h1-1-NKG2D CAR, LV-shRc3h1-2-NKG2D CAR, and LV-shRc3h1-3-NKG2D CAR. The virus solution was collected into a centrifuge tube, centrifuged to remove cell debris, and the supernatant was filtered through a 0.22μm filter membrane and ultracentrifuged for 2h to allow the virus to settle at the bottom of the tube. The supernatant was discarded, and 1% of the original volume of X-VIVO medium was added to fully dissolve the virus at 4°C overnight. The next day, the virus concentrate was mixed and collected into a cryopreservation tube and stored at -80°C.

[0067] 2.3 Comparison of the interference efficiency of Rc3h1-shRNA-1, Rc3h1-shRNA-2 and Rc3h1-shRNA-3 in Jurkat cells

[0068] (1) CAR-J was constructed using LV-NKG2D CAR, LV-shRc3h1-1-NKG2D CAR, LV-shRc3h1-2-NKG2D CAR, and LV-shRc3h1-3-NKG2D CAR, respectively. Jurkat cell line was infected at a virus infection multiplicity (MOI) of 15, and the volume of virus added was calculated according to Eq (2).

[0069]

[0070] After 24 hours, the culture medium was changed and the cells were collected after another 24 hours of culture. The expression of NKG2D molecules was detected by flow cytometry to confirm the successful construction of CAR-J cells. Figure 3 As shown, the results showed that NKG2D CAR-J cells in each group were successfully constructed, and the positive rate was above 98%.

[0071] (2) Take about 5×10 CAR-J cells that have been successfully constructed and are in good condition. 5 Cell RNA was extracted by guanidine isothiocyanate / phenol method and cDNA was prepared by reverse transcription. Real-time fluorescence quantitative PCR was performed using qPCR SYBR Green Master Mix kit (with housekeeping gene GAPDH as internal reference) and 2 -△△Ct The experimental results were analyzed by reverse transcription PCR and agarose gel electrophoresis using cDNA to further confirm the interference efficiency of Rc3h1-shRNA-1. Figure 4 As shown, the results showed that Rc3h1-shRNA-1 had the most obvious interference effect and the highest interference efficiency compared with Rc3h1-shRNA-2 and Rc3h1-shRNA-3.

[0072] Therefore, the following experiments take Rc3h1-shRNA-1 as an example.

[0073] 2.4 Virus titer detection

[0074] (1) HEK-293T cells were digested, centrifuged, resuspended and counted. 2×10 5 10 cells / well, add 0.5μl, 1μl, 2μl, 4μl of virus concentrate to each well, fill up to 500μl per well with DMEM complete medium, mix thoroughly. Place the 24-well plate in a CO2 incubator for culture.

[0075] (2) After 48 hours of incubation, remove the 24-well plate from the incubator, digest the cells, collect them by centrifugation, and discard the supernatant. Resuspend each sample in 1 ml of Staining buffer, collect them in a 1.5 ml centrifuge tube, and label them.

[0076] (3) Collect cells by centrifugation, stain with APC Anti-Human-CD314 (NKG2D) antibody, and detect by flow cytometry. Figure 5 shown.

[0077] Data analysis was performed using FlowJo V10.8.1 software. The percentage of APC-positive cells in each group was counted. The groups with a percentage of 10-30% were selected to calculate the virus titer. The calculation formula is shown in Eq (1). The titer of LV-shRc3h1-1-NKG2D CAR was 8.36×10 7 TU / ml (4.60×10 in the control group LV-NKG2D CAR 7 TU / ml). The calculation formula is:

[0078]

[0079] (*TU: transducing units, which refers to the number of viral genomes that can infect and enter target cells, TU / ml: refers to the number of biologically active viral particles contained in each milliliter.)

[0080] Example 3 Construction of shRc3h1 NKG2D CAR-T and detection of interference efficiency

[0081] 3.1 Construction of shRc3h1 NKG2D CAR-T

[0082] PBMC-derived T cells were infected with the virus at a multiplicity of infection (MOI) of 15 to construct shRc3h1 NKG2D CAR-T. The volume of virus added was calculated according to Eq (2), i.e., for every 1×10 6179 μl LV-shRc3h1-1-NKG2D CAR virus concentrate was added to each cell (the Mock group was added with an equal volume of LV-Puro virus concentrate, and the non-interference group was added with 326 μl LV-NKG2D CAR virus concentrate according to the calculated results).

[0083]

[0084] After 24 hours, the culture medium was changed, and the cells were collected after another 24 hours of culture. They were stained with APC Anti-Human-CD314 (NKG2D) antibody, and the positive rate was detected by flow cytometry. Figure 6 As shown, the NKG2D positivity rates of Mock T, NKG2D CAR-T, and shRc3h1 NKG2DCAR-T were 45.4%, 96.5%, and 94.9%, respectively.

[0085] 3.2 Verification of the interference efficiency of Rc3h1-shRNA-1 on NKG2D CAR-T

[0086] 1. Real-time fluorescence quantitative PCR

[0087] Each group took about 5×10 CAR-T cells that were successfully constructed and in good condition. 5 Cell RNA was extracted by guanidine isothiocyanate / phenol method, cDNA was prepared by reverse transcription, and real-time fluorescence quantitative PCR was performed using qPCR SYBR Green Master Mix Kit (with housekeeping gene GAPDH as internal reference). -△△Ct The interference efficiency of Rc3h1-shRNA-1 interference sequence in NKG2D CAR-T is as follows Figure 7 As shown in A, the expression level of Rc3h1 mRNA in the CAR-T group after interference decreased to less than 30% of that before interference.

[0088] 2. Western blot

[0089] Each group took about 1×10 CAR-T cells that were successfully constructed and in good condition. 7The total cell protein was extracted by RIPA lysis buffer, quantified by BCA kit, and the total protein amount between groups was adjusted according to the quantitative results, and loading buffer was added for Western blot detection. 100 μg of total protein was added to each well of SDS-PAGE gel for electrophoresis. After the loading buffer reached the end point, the protein was transferred from the gel to the PVDF membrane using a membrane transfer instrument. After the PVDF membrane was blocked with 5% (w / v) milk-PBS for 1 hour, the residual milk was washed with PBS, and the PVDF membrane was incubated with Mouse Anti-human Rc3h1 and Mouse Anti-humanGAPDH primary antibodies overnight. The unbound primary antibodies were washed with PBST, and then Rabbit anti-Mouse secondary antibodies were added for incubation for color development, and the results were obtained by scanning the membrane. The Western blot results are shown in the figure. Figure 7 As shown in B, the results showed that the expression of Rc3h1 at the protein level was also significantly downregulated after RNA interference.

[0090] Example 4: Validation experiment on knocking down Rc3h1 to promote the killing efficiency of CAR-T cells against tumors in vitro

[0091] This experiment was conducted using two pancreatic cancer cell lines, CAPAN2 and PANC1, which were in logarithmic growth phase and in good condition. In order to distinguish tumor cells from CAR-T cells, tumor cells were labeled with CFSE dye, and after labeling, the cells were resuspended in a certain amount of X-VIVO medium and the cell density was adjusted.

[0092] Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T were resuspended in X-VIVO medium and the cell density was adjusted. The labeled tumor cells were spread into 96-well low adsorption plates with three types of T cells according to a certain effector cell: target cell ratio (E:T), and each well was filled with X-VIVO basal medium to a total volume of 200μl. After thorough mixing, it was placed in an incubator and continued to be cultured for 13h. A separate culture group of tumor cells that were not co-incubated with T cells was set up to exclude tumor cell apoptosis caused by interfering factors such as experimental operations.

[0093] After 13 hours of co-incubation, the cells were stained with Annexin V-APC and PI, and then immediately tested. + The apoptosis rate of tumor cells was analyzed in the cell group (i.e., the CFSE-stained tumor cell group). The killing rates of Mock T, NKG2DCAR-T, and shRc3h1 NKG2D CAR-T on tumor cells at different E:T levels were as follows: Figure 8The results showed that the killing effect of shRc3h1 NKG2D CAR-T on CAPAN2 and PANC1 was significantly better than that of the NKG2D CAR-T group.

[0094] Example 5 Validation experiment of knocking down Rc3h1 to promote CAR-T cell activation

[0095] (1) Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T cells were co-incubated with CFSE-labeled Panc1 cells at an E:T ratio of 3:1, and the Golgi inhibitor Golgiplug was added and cultured for 4 h;

[0096] (2) Collect cells and stain with APC Anti-human CD69 antibody;

[0097] (3) The cells that have completed surface staining were fixed with 4% (w / v, PBS) paraformaldehyde in the dark for 15 min, and the paraformaldehyde was washed away;

[0098] (4) Resuspend in cell membrane permeabilization buffer, incubate at 4°C in the dark for 20 min, then add PE-Cy7 Anti-human GzmB antibody directly to the permeabilization buffer for light-proof staining. After fully washing the unbound antibody, resuspend the cells in a volume of not less than 200 μl of PBS for flow cytometry detection. The expression levels of CD69 and GzmB in Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T after tumor cell stimulation are shown in Figure 2. Fig. 9 The results showed that the activation level of the shRc3h1 NKG2D CAR-T group was significantly higher than that of the NKG2D CAR-T group.

[0099] Example 6 Validation experiment of knocking down Rc3h1 to reduce the level of CAR-T cell exhaustion

[0100] (1) Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T cells were co-cultured with CFSE-labeled Panc1 cells at an E:T ratio of 5:1 for one week;

[0101] (2) Cells were collected and stained with PD-1PE and TIM3 APC antibodies in the dark;

[0102] (3) After fully washing the unbound antibodies, resuspend the cells in a volume of no less than 200 μl of PBS and perform flow cytometry. The expression levels of PD-1 and TIM3 in Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T after continuous stimulation of tumor cells are shown in Figure 2. Fig.10The results showed that under long-term antigen stimulation, the exhaustion level of shRc3h1 NKG2D CAR-T was significantly lower than that of NKG2D CAR-T, thus being able to exert a longer-lasting effect.

[0103] Example 7 Validation experiment of knocking down Rc3h1 to enhance the anti-tumor function of CAR-T cells in vivo

[0104] 7.1 Construction of subcutaneous tumor-bearing model in NSG mice and CAR-T therapy injection

[0105] The PANC1-Luc PBS cell suspension was mixed with Matrigel at a volume ratio of 1:1 (so that the final concentration of PANC1-Luc cells was 1×10 7 All operations involving live mice were performed in an SPF animal room. Female NSG mice aged 6-8 weeks were shaved and 50 μl (i.e., 5×10 5 cells) and injected subcutaneously into the right back. When the tumor volume reached about 100 mm 3 The mice were divided into groups (n=7) and treated by tail vein injection.

[0106] Prepare sufficient amounts of Mock T, NKG2D CAR-T, and shRc3h1 NKG2D CAR-T cells in advance, resuspend each group of T cells in PBS, and adjust the density to 5×10 7 Each mouse was injected with 100 μl of CAR-T cell suspension through the tail vein and returned to the cage for further culture.

[0107] 7.2 Tumor volume recording

[0108] The tumor volume was measured with a vernier caliper every 4 days from the day of treatment, and mice were imaged weekly by IVIS (if the tumor volume exceeded 2000 mm 3 The mice were killed. The tumor volume of mice was calculated according to Eq (3). The tumor volume-time curve is shown in Fig.11 shown.

[0109]

[0110] 7.3IVIS In Vivo Imaging

[0111] The IVIS living imager should be turned on and precooled before use. It can be operated when the instrument's imaging element cools down to -82°C. Each mouse is intraperitoneally injected with 100 μl of fluorescent enzyme substrate (D-Luciferase, 15 mg / ml). After about 2 minutes of reaction, the mouse is anesthetized with isoflurane. The completely unconscious mouse is fixed in the corresponding position in the darkroom of the instrument for imaging. Adjust the exposure time and record the results. Analyze the results using Livingimage software. After the experiment, all mice were killed by cervical dislocation and the tumors were removed. The IVIS image results are shown in the figure below. Fig.12 The experimental results showed that although NKG2D CAR-T cells also had a certain therapeutic effect on subcutaneous tumors in mice, the restriction on tumors was very limited. In contrast, the tumor volume of mice in the shRc3h1NKG2D CAR-T treatment group was significantly reduced after three weeks of treatment.

[0112] Therefore, the present invention adopts the above-mentioned Rc3h1 gene interference chimeric antigen receptor T cell construction method and application, can improve the CAR-T in the treatment of solid tumors in the limited proliferation and activation, severe exhaustion, anti-tumor effect of the effectiveness and sustainability of the problem, the Rc3h1 gene interference chimeric antigen receptor T cell constructed by the present invention has better anti-tumor activity, longer survival time in vivo.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Rc3h1 interference sequence, consisting of a sense sequence, an antisense sequence, and a spacer connecting the two "TTCAAGAGA" and a poly T termination signal at the 3' end, characterized in that Includes one of the following: Rc3h1-shRNA-1, the sense sequence of which is shown in SEQ ID NO.1, and the antisense sequence of which is shown in SEQ ID NO.2; Rc3h1-shRNA-2, the sense sequence of which is shown in SEQ ID NO.3, and the antisense sequence of which is shown in SEQ ID NO.4; Rc3h1-shRNA-3, its sense sequence is shown in SEQ ID NO.5, and its antisense sequence is shown in SEQ ID NO.

6.

2. The Rc3h1 interference sequence according to claim 1, characterized in that The target sequence of Rc3h1-shRNA-1 is shown in SEQ ID NO.

7. The target sequence of Rc3h1-shRNA-2, Rc3h1-siRNA-2, is shown in SEQ ID NO.8, and the target sequence of Rc3h1-shRNA-3, Rc3h1-siRNA-3, is shown in SEQ ID NO.

9.

3. A method for constructing a chimeric antigen receptor T cell with Rc3h1 gene interference, characterized in that: The following steps are involved: (1) Design RNAi target sequence; (2) designing an interfering sequence according to the RNAi target sequence, wherein the interfering sequence is the Rc3h1 interfering sequence as described in claim 1; (3) Construction of pLL3.7 NKG2D CAR-Rc3h1 shRNA plasmid; (4) Plasmid transfection and lentiviral packaging; (5) Construction of shRc3h1 NKG2D CAR-T cells.

4. The construction method according to claim 3, characterized in that: The specific method of step (3) is as follows: 20 base homology arms are added to both ends of the interference sequence to synthesize two full-length oligonucleotide sequences, the two chains are annealed and complementary to form a double-stranded chain, and the two chains are connected to the U6 promoter of the pLL3.7NKG2D CAR plasmid by homologous recombination, and then the plasmid is successfully constructed by EcoRI single enzyme digestion identification and DNA sequencing.

5. The construction method according to claim 3, characterized in that: The specific method of step (4) is: selecting HEK-293T cells with good growth status for transfection, using a lentiviral three-plasmid system for lentiviral packaging, and packaging three viruses: LV-puro (Mock), LV-NKG2D CAR), and LV-shRc3h1-1-NKG2D CAR.

6. The construction method according to claim 3, characterized in that: The specific method of step (5) is as follows: the PBMC-derived T cells are infected with the virus at a multiplicity of infection of 15 to construct shRc3h1NKG2D CAR-T, and the volume of the added virus is 1×10 6 Add 179 μl per cell LV-shRc3h1-1-NKG2D CAR virus concentrate, the Mock group was added with an equal volume of LV-Puro virus concentrate, and the non-interference group was added with 326μl LV-NKG2D CAR virus concentrate. The culture medium was changed after 24h, and the cells were collected after another 24h of culture, stained with APC Anti-Human-CD314 (NKG2D) antibody, and the positive rate was detected by flow cytometry.

7. A chimeric antigen receptor T cell with Rc3h1 gene interference constructed by the construction method according to any one of claims 3 to 6.

8. Use of the chimeric antigen receptor T cell with Rc3h1 gene interference as claimed in claim 7 in the preparation of anti-tumor drugs.

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