CN111944758A

CN111944758A - Method for enhancing killing capacity of T cells to tumor cells, product and application

Info

Publication number: CN111944758A
Application number: CN202010652645.9A
Authority: CN
Inventors: 江文正; 韩梅梅; 高尧鑫; 何聪; 刘明耀; 席在喜
Original assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Current assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Priority date: 2020-07-08
Filing date: 2020-07-08
Publication date: 2020-11-17
Anticipated expiration: 2040-07-08
Also published as: CN111944758B

Abstract

The application discloses a method, a product and application for enhancing killing capacity of T cells to tumor cells. The method comprises the following steps: reducing or knocking out the mRNA level of β 2adrenergic receptor (ADRB2) in a T cell of interest by siRNA; the target sequence of the siRNA comprises a sequence shown in SEQ ID NO. 1. The invention firstly applies the RNA interference method to knock down the expression of beta 2adrenergic receptor genes in CAR-T cells, increases the proportion of effector CAR-T cells, enhances the release of CAR-T cell effector factors, and thereby enhances the killing capability of the CAR-T cells on tumor cells. The invention can reduce the dosage of CAR-T cells, thereby reducing the treatment cost and the cytokine storm.

Description

Method for enhancing killing capacity of T cells to tumor cells, product and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for enhancing the killing capacity of T cells to tumor cells, a product and application.

Background

The incidence and mortality of cancer continues to rise, becoming the second leading "killer" of death from the disease. In recent years, with the development of scientific and technical and medical means, many methods have appeared for the treatment of cancer. Such as radiotherapy, chemotherapy and surgical resection, however, these treatments have good therapeutic effect only on the initial tumors, and these treatments also cause serious adverse effects to patients themselves. With the development of tumor immunotherapy, the treatment of tumors by chimeric antigen receptor T cells (CAR-T) gradually enters clinical trials, and the chimeric antigen receptor T cells can well treat hematological malignancies, so that new hopes are brought to more cancer patients. However, CAR-T also presents a number of difficulties in treating solid tumors because of the strong immune microenvironment surrounding the solid tumor, thereby reducing the therapeutic efficacy of CAR-T. Furthermore, another obstacle to CAR-T treatment of solid tumors is the lack of specificity. Therefore, finding effective targets to improve CAR-T treatment of solid tumors is of central importance.

NKG2D is a ligand structurally similar to the major histocompatibility complex and is commonly expressed by tumor tissues and tumor cell lines, such as acute and chronic leukemias of lymphoid and myeloid origin, neuroblastoma, colorectal cancer, ovarian cancer, cervical cancer, breast cancer, pancreatic cancer and prostate cancer, while rarely detectable on the surface of healthy cells and tissues. To date, NKG 2D-based chimeric antigen therapy has been used for acute myeloid leukemia and ovarian cancer treatment with high tumor-free survival and minimal side effects in mice. These previous reports show that NKG2D may also be a good target for prostate cancer and may be used with CAR-T therapy.

Cancer patients are able to secrete various hormones (e.g., epinephrine) to accelerate cancer progression, primarily due to activation of the β 2 epinephrine system. Since some cancer cells contain all the enzymes of epinephrine synthesis and are capable of secreting epinephrine upon stimulation, this epinephrine activates the β 2adrenergic receptor (ADRB2) on T cells and inhibits its activation, proliferation and killing ability in a variety of ways. Beta 2-adrenergic signaling inhibition of CD8⁺Production of calcineurin in T cells, thereby reducing IL-2, INF-gamma, IL-4 and TNF-alpha, and also inhibiting CD8⁺T cell co-stimulatory molecules such as 4-1BB and CD28 cause their inactivation.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method, a product and application for enhancing the killing capacity of T cells on tumor cells.

In one aspect, the present invention provides a method for enhancing the killing ability of T cells against tumor cells, comprising: reducing or knocking out mRNA levels of β 2adrenergic receptors (ADRB2) in T cells by siRNA; the target sequence of the siRNA comprises a sequence shown in SEQ ID NO. 1;

can also comprise the sequence shown in SEQ ID NO.8 and/or SEQ ID NO. 9.

In the above method, the T cell comprises a CAR-T cell,

preferably, the CAR comprises: an extracellular domain that binds to a specific target antigen, a transmembrane domain, and an intracellular signaling domain;

more preferably, the antigen targeted by the extracellular domain is selected from one or any of the following: NKG2D ligand, alpha folate receptor, 5T4, alpha v beta 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79B, CD123, CD138, CD171, CEA, CSPG4, EGFR family comprising ErbB2(HER2), EGFRvIII, EGP2, EPCAM, EphA2, EpNYCAM, FAP, fetal AchR, FR alpha, GD2, glypican-3 (GPC 2), PSC-2 + 2, HLA-A2+ 5, HLA-A2, HLA-5 + 5, HLA-2, HLA-A + 2, HLA-2-2-HLA-487-A + 487, HLA-5-2-I-D, HLA-2, HLA-I-D, HLA-I, SSX, survivin, TAG72, TEM, VEGFR2, and WT-1; more preferably, NKG2D ligand;

more preferably, the transmembrane domain is selected from any one or any several of the following transmembrane regions: a T cell receptor alpha or beta chain, CD3, CD3, CD3 gamma, CD3 zeta, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154; more preferably, a CD8 transmembrane domain;

more preferably, the intracellular signaling domain comprises a costimulatory signaling domain and/or a primary signaling domain;

more preferably, the primary signalling domain comprises one or any of FcR γ, FcR β, CD3 γ, CD3, CD3, CD3 ζ, CD22, CD79a, CD79b or CD66 d; more preferably, CD3 ζ;

more preferably, the co-stimulatory signaling domain is selected from one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD278(ICOS), DAP10, LAT, NKD2C, SLP76, TRIM or ZAP70, more preferably 4-1 BB.

In the above method, the extracellular domain is the extracellular segment of NKG2D, preferably, the amino acid sequence of the extracellular segment of NKG2D is shown in 22-157 of SEQ ID No.7, more preferably, the coding sequence of the extracellular segment of NKG2D is shown in 64-471 of SEQ ID No. 6;

preferably, the N end of the extracellular segment of NKG2D is connected with a CD8 signal peptide, more preferably, the amino acid sequence of the CD8 signal peptide is shown as 1-21 in SEQ ID NO.7, and more preferably, the coding gene sequence of the CD8 signal peptide is shown as 1-63 in SEQ ID NO. 6.

In the above method, the T cell is CD3⁺T cells, or CD4⁺T cells and/or CD8⁺A T cell;

and/or, the tumor cells comprise benign tumor cells and malignant tumor cells; preferably, the malignant tumor cells are from any one or more of pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, uterine body tumor and osteosarcoma;

and/or, the siRNA and/or the CAR are produced by transfection into the T cell following in vitro synthesis, or by introduction into the T cell of a recombinant vector; preferably, the recombinant vector comprises: viral vectors, and/or non-viral vectors; the virus vector comprises an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector and/or an oncolytic virus vector, and the non-virus vector comprises a cationic high molecular polymer, a plasmid vector and/or a liposome; more preferably, the recombinant vector comprises a lentiviral vector.

In another aspect, the invention also provides a T cell with enhanced killing ability on tumor cells, which is prepared by any one of the methods.

In another aspect, the present invention also provides an siRNA for reducing or knocking out the expression level of mRNA of a beta 2adrenergic receptor in a T cell, wherein the target sequence of the siRNA comprises the sequence shown in SEQ ID NO.1, and can also comprise the sequence shown in SEQ ID NO.8 and/or SEQ ID NO. 9.

In another aspect, the invention also provides a gene encoding the siRNA.

In another aspect, the present invention further provides a biomaterial, wherein the biomaterial is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell containing the gene encoding the siRNA;

the recombinant vector comprises: viral vectors, and/or non-viral vectors; the virus vector comprises an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector and/or an oncolytic virus vector, and the non-virus vector comprises a cationic high molecular polymer, a plasmid vector and/or a liposome.

Preferably, the recombinant vector, the recombinant bacterium or the recombinant cell further contains a gene and/or a protein encoding the CAR;

the recombinant bacteria can be engineering bacteria and are used for propagating, storing or detecting the genes;

the recombinant cell may be a cell for lentivirus packaging purification, such as a mammalian cell, preferably a 293T cell.

In another aspect, the invention protects the use of the above-mentioned T cell, the siRNA, the gene, and the biomaterial in the preparation of a medicament for the treatment of tumors.

Has the advantages that:

the invention firstly applies the RNA interference method to knock down the expression of beta 2adrenergic receptor genes in CAR-T cells, increases the proportion of effector CAR-T cells, enhances the release of CAR-T cell effector factors, and thereby enhances the killing capability of the CAR-T cells on tumor cells. The invention can reduce the dosage of CAR-T cells, thereby reducing the treatment cost and the cytokine storm.

Drawings

FIG. 1 is a schematic structural diagram of an interference plasmid pLL3.7-shRNA- (A/NC) -EGFP, wherein the plasmids from top to bottom are as follows: no-load pLL3.7 (namely pLL3.7-U6-EGFP), pLL3.7-shRNA-A-EGFP and pLL3.7-shRNA-NC-EGFP, wherein U6promoter is U6promoter gene, EF1 alpha promoter is EF1 alpha promoter gene, and EGFP is enhanced green fluorescent protein gene.

FIG. 2 shows the results of single-restriction enzyme identification of EcoR I of the interfering plasmid, where M1 is DL2000 Marker. 1 is unloaded pLL3.7 (namely pLL3.7-U6-EGFP), 2 is pLL3.7-shRNA-NC-EGFP, and 3 is pLL3.7-shRNA-A-EGFP.

FIG. 3 is a schematic structural diagram of the targeting plasmid pLL3.7-shRNA- (A/NC) -NKG2D-CAR, wherein the plasmids from top to bottom are: pLL3.7-NKG2D-CAR, pLL3.7-shRNA-A-NKG2D-CAR, and pLL3.7-shRNA-NC-NKG2D-CAR, wherein NKG2D is a gene encoding NKG2D extracellular domain, the sequence between NKG2D and 4-1BB is a gene encoding CD8 transmembrane domain, 4-1BB is a gene encoding the intracellular domain of the co-stimulatory receptor for T cell activation 4-1BB, and CD3 ζ is a gene encoding the intracellular domain of the signal CD3 ζ for T cell activation.

FIG. 4 shows the results of single-restriction enzyme identification of EcoR I of the targeting plasmid, wherein M1 is DL2000 Marker. 1 is pLL3.7-shRNA-NC-NKG2D, 2 is pLL3.7-NKG2D-CAR, and 3 is pLL3.7-shRNA-A-NKG 2D.

FIG. 5 shows the relative expression levels of ADRB2mRNA in human T cells infected with different lentiviruses, where 1 is a negative control group, 2 is a lentivirus A group (blank group), 3 is a lentivirus C group (negative interference group), and 4 is a lentivirus B group (interference group).

Fig. 6 shows the ratio change of target cells after mixed culture of target cells and effector cells, wherein 1 is mixed negative control group T cells, 2 is mixed lentivirus a group (blank group) T cells, 3 is mixed lentivirus C group (negative interfering group) T cells, and 4 is mixed lentivirus B group (interfering group) T cells.

FIG. 7 shows the results of single-restriction enzyme identification of EcoR I of the recombinant plasmid, wherein M1 is DL2000 Marker. 1 is unloaded pLL3.7 (namely pLL3.7-U6-EGFP), 2 is pLL3.7-shRNA-NC-EGFP, 3 is pLL3.7-shRNA-A-EGFP, 4 is pLL3.7-shRNA-C1-EGFP, and 5 is pLL3.7-shRNA-C2-EGFP.

FIG. 8 shows the relative expression of ADRB2mRNA in human T cells infected with different lentiviruses. Among them, 1 is a negative control group, 2 is a lentivirus group A (blank group), 3 is a lentivirus group C (interfering group), 4 is a lentivirus group C1 (interfering group), and 5 is a lentivirus group C2 (interfering group).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of pLL3.7-ADRB2-shRNA interference vector

1. RNAi target sequence design

The gene sequence number of a beta 2adrenergic receptor (ADRB2) (Homo sapiens beta 2adrenergic receptor) is found in an NCBI website: AY 136741.1. The RNAi target sequence of ADRB2 gene was designed on the website (https:// rnaidesigner. thermofisher. com/rnainexpress/design. do) according to the gene number, and the results are shown in Table 1.

TABLE 1 RNAi target sequences of ADRB2 genes

2. Design of interfering sequences based on target sequences

Based on the screened target sequences, the interference sequences are designed and determined according to the following principle: the 5' end is started with G, and the content of G + C is set to be 30-50%. According to the requirements of the pLL3.7 vector, (1) adding T at the 5' end of a sense strand to reconstruct T at the l position of the U6 promoter; (2) adding Loop 'TTCAAGAGAGA' after the interference target sequence; (3) adding an inverted complementary sequence and a termination signal 'TTTT'; (4) the EcoR I enzyme cutting site GAATTC is added at the 3' end so as to facilitate the identification; (5) then filling in Xho I enzyme cutting site to synthesize a pair of complementary fragments. The interfering sequences were shuffled to design negative control sequences (NC), each of which is shown in Table 2 below.

TABLE 2 oligonucleotide sequences designed separately for target and NC sequences

3. Construction of interference plasmid pLL3.7-shRNA- (A/NC) -EGFP

Oligonucleotide sequences shown in Table 2 are synthesized into a double-stranded DNA sequence, the unloaded pLL3.7 is subjected to double enzyme digestion by restriction enzymes XhoI and HpaI, and the recombinant plasmid pLL3.7-shRNA- (A/NC) -EGFP is constructed by recombinase ligation, wherein the structure of the recombinant plasmid is shown in figure 1. The recombinant plasmid is identified by EcoR I single enzyme digestion, the result is shown in figure 2, and the 1427bp specific enzyme digestion fragment represents that the construction of the recombinant plasmid is successful.

4. Construction of targeting plasmid pLL3.7-shRNA- (A/NC) -NKG2D

The full-length CDS region of the NKG2D gene is found through a website https:// www.ncbi.nlm.nih.gov/pubmed/and the extracellular region of the NKG2D protein is found at the website https:// www.uniprot.org/and the corresponding extracellular region gene sequence is found in the full-length NKG2D gene sequence and is shown as 64 th to 468 th positions of SEQ ID NO.6, and the amino acid sequence is shown as 22 th to 157 th positions of SEQ ID NO. 7. Since the NKG2D protein is a two-type transmembrane protein, its extracellular segment sequence is located at the N-terminal and does not contain a signal peptide, we add the amino acid sequence of CD8 signal peptide (positions 1-21 in SEQ ID NO. 7) to the front end (N-terminal) of the extracellular segment sequence of NKG2D, and the coding gene sequence is shown in positions 1-63 of SEQ ID NO. 6. Primers (containing a CD8 signal peptide gene sequence) are designed by SnapGene software and synthesized in a company, an extracellular segment of NKG2D is amplified by an RT-PCR method by taking human T cell cDNA as a template, and an RT-PCR product is sequenced to obtain a Sig-NKG2D extracellular segment coding gene sequence (named as a sigNKG2DEX sequence) as follows:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGATGTTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG(SEQ ID NO.6)。

the amino acid sequence is as follows:

MALPVTALLLPLALLLHAARPMLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ ID NO.7)。

sequentially connecting the sigNKG2DEX sequence and the coding sequence of the CD8 transmembrane domain with the coding sequences of 4-1BB and CD3 zeta, and replacing EGFP on pLL3.7 to obtain a pLL3.7-NKG2D-CAR vector; recovering small fragments of the constructed pLL3.7-shRNA- (A/NC) -EGFP vector through XbaI and NheI double-enzyme gel cutting, recovering large fragments of the pLL3.7-NKG2D-CAR vector through XbaI and NheI double-enzyme gel cutting, and connecting the pLL3.7-shRNA- (A/NC) -NKG2D-CAR vector through T4DNA ligase overnight to obtain the recombinant vector pLL3.7-shRNA- (A/NC) -NKG2D-CAR, as shown in figure 3. The results of single enzyme digestion identification by EcoR I are shown in FIG. 4, wherein the 2163bp specific enzyme digestion fragment represents the successful construction of the recombinant vector.

Example 2 viral packaging

1. Plasmid transfected cells

1) Placing plasmids pLL3.7-NKG2D-CAR, pLL3.7-shRNA-A-NKG2D-CAR or pLL3.7-shRNA-NC-NKG2D-CAR, PEI, Opti-MEM medium at room temperature for 5 min;

2) putting 436 μ l of Opti-MEM into a 1.5ml EP tube, adding 64 μ l of PEI, uniformly mixing, and standing at room temperature for 5min to obtain a PEI-Opti-MEM solution;

3) taking 5 mu g of plasmid pLL3.7-NKG2D-CAR, pLL3.7-shRNA-A-NKG2D-CAR or pLL3.7-shRNA-NC-NKG2D-CAR, 3 mu g of psPAX2 and 5 mu g of pMD2.G, adding Opti-MEM to 500 mu l, and standing at room temperature for 5min to obtain Opti-MEM containing the plasmid;

4) adding the PEI-Opti-MEM solution into Opti-MEM containing corresponding plasmids, and standing at room temperature for 20min to obtain a DNA/PEI mixture;

5) transfection: slowly dropping 1ml of DNA/PEI mixture into a 293T culture dish paved the day before, gently mixing, incubating in an incubator at 37 ℃, replacing fresh culture medium after 6-8h, and putting into the incubator at 37 ℃ for further incubation.

2. Virus collection and concentration

1) After plasmid transfection for 48h, collecting supernatant, adding 10ml of fresh culture medium, continuously culturing for 72h, collecting supernatant again, mixing with the supernatant collected for 48h, and placing in a refrigerator at 4 ℃ for later use;

2) centrifuging at 4 deg.C and 4000g for 10min, and removing cell debris to obtain virus supernatant;

3) the resulting virus supernatant was filtered through a 0.45 μm filter;

4) transferring the filtered virus supernatant into an ultracentrifuge tube, centrifuging for 2h at 25000 r, diluting with PBS (1/100) in the volume of the supernatant, repeatedly blowing and beating, and transferring into a sealed centrifuge tube for overnight at 4 ℃ to obtain virus liquid;

5) the virus solution was dispensed to appropriate volumes, stored at-80 ℃ and 200. mu.l of virus solution was titered.

3. Viral titer determination

1) Digesting 293T cells, centrifuging, counting, preparing cell suspension with serum-containing medium, and adjusting cell density to 4 × 10⁵Per ml, 0.5ml of cell suspension was added to each well of a 24-well plate;

2) diluting the virus solution with the whole culture medium according to the following proportion: 1: 3; 1: 9; 1: 27;

3) respectively adding 100 mul of virus liquid (stock solution) and the virus liquid diluted according to different proportions into a 24-pore plate inoculated with cells;

4) after 16h, the infection supernatant was discarded, and 0.5ml of fresh whole medium was added;

5) after 48h, carrying out flow detection on the expression of a target gene NKG2D of the infected cells;

6) the titer, titer 2 × 10, was calculated⁵X infection efficiency x dilution times.

The results are as follows: after the virus is collected and concentrated, the titers of lentiviruses A, B and C obtained by three plasmids of pLL3.7-NKG2D-CAR, pLL3.7-shRNA-A-NKG2D-CAR and pLL3.7-shRNA-NC-NKG2D-CAR are respectively 1.5 multiplied by 10⁷、1.4×10⁷、1.3×10⁷。

Example 3 interference validation test

Human peripheral blood collected from hospital is used for lysing erythrocytes, and then CD3 is obtained by magnetic bead sorting⁺T cells (or separately isolated CD 4)⁺T cells and CD8⁺T cell post 1:1 mix), after 2 days of activation with CD3, CD28 antibodies, the solutions were centrifuged and T cells were seeded in 24-well plates at 2X 10 per well⁶And (4) cells. The method is divided into 4 groups: negative control group without viral infection (CK), lentivirus a group (blank), lentivirus B group (interfering group) and lentivirus C group (negative interfering group), 2 wells each. Each group was spiked with the corresponding volume of virus at the MOI of 10:1 and 10 μ g/ml polybrene was added to facilitate infection. After 24h, the cells were collected, centrifuged at 1000g for 10min, the medium was discarded and fresh medium was added.

Cells (namely CAR-T cells) are harvested 48h after lentivirus infection, and the expression efficiency of the NKG2D extracellular segment, namely the virus infection positive rate, is detected in a flow mode. The infection efficiency of the three lentiviruses A, B and C on T cells was close to 100%.

Groups 4 of T cells were harvested 48h after infection. Cells were lysed by Trizol, total RNA was extracted, reverse transcription was performed, and relative expression of ADRB2mRNA (with GAPDH as an internal reference) was analyzed by Q-PCR, and the results are shown in FIG. 5. The results show that after the T cells are infected by the lentivirus B (corresponding to pLL3.7-shRNA-A-NKG2D-CAR), the transcription of ADRB2mRNA in the T cells can be remarkably inhibited, and the expression level of the ADRB2mRNA is reduced to 20 percent of the original expression level.

Example 4 selection of target cells and CAR-T killing function study

The following experiment was carried out using a prostate cancer cell line PC3 that highly expresses NKG2D ligand as a target cell.

1. Target cell labeling

Preparation of 1X 10 Single cell suspension of prostate cancer cell line PC3⁶And/ml. 2ml of PBS was added and washed twice by centrifugation, and the serum was washed off. Resuspend the cells in PBS, adjust the cell density to 2X 10⁶And/ml. Adding equal volume of 10 mu M eFluor 670 reagent, vortexing the cells, and incubating for 10 minutes at 37 ℃ in the dark; adding 4-5 times volume of pre-cooled complete culture medium of 10% serum, and incubating for 5min on ice; complete medium was washed 3 times. The prostate cancer cell line PC3 was labeled with eFluor 670 dye.

2. Mixed culture of target cells and effector cells

Respectively marking the prostate cancer cell line PC3 marked in the step 1 according to the proportion of 4 multiplied by 10⁴The number of the wells is inoculated into a 96-well plate for culturing the ultra-low adsorption cells; four groups of T cells after 48 hours of virus infection in example 3 are respectively inoculated into target cells according to effective target ratios (E: T) of 1:3, 1:1 and 3:1, and 12 treatments are carried out, wherein each treatment is provided with two repetitions (wells), and each well is replenished with liquid to 200 mu l;

culturing the culture plate with the mixed cells in an incubator at 37 ℃ for 24 hours; after 24h, all cells in each well were collected, transferred to a flow tube, and the target cell ratio was detected by flow cytometry, the results are shown in fig. 6.

3. Analysis of killing efficiency

As shown in fig. 6, compared with the T cells of the untransfected negative control group, the T cells transfected by another three groups of lentiviruses all have a strong killing effect on cancer cells, and compared with the T cells transfected by the lentivirus group a (blank group) and the T cells transfected by the lentivirus group C (negative interference group), the T cells transfected by the lentivirus group B (interference group) have a stronger killing efficiency on cancer cells, and the killing efficiency is improved by 15%, which indicates that the NKG2D extracellular segment can well target prostate cancer cell strains to function, and the interference ADRB2 can significantly enhance the killing efficiency of CAR-T cells on cancer cells.

Comparative example, shRNA-A interference sequence screening

In the process of screening the shRNA-A interference sequence, different interference sequences are designed aiming at a plurality of different target sequences simultaneously, and relevant experiments are carried out. The experimental procedure was the same as in examples 1-3.

Other RNAi target sequences selected are shown in Table 3, and upstream and downstream fragments of the interference sequence corresponding to each target sequence are shown in Table 4, wherein shRNA-A is the original sequence of example 1.

TABLE 3 RNAi target sequences of ADRB2 genes

TABLE 4 oligonucleotide sequences designed separately for the target and negative control sequences

According to the method of step 3 in example 1, the upstream and downstream fragments of the interference sequence shown in Table 4 were synthesized into DNA duplexes and ligated to pLL3.7 vector to obtain four recombinant plasmids pLL3.7-shRNA-A/C1/C2/NC-EGFP, and then EcoR I single-restriction enzyme identification was performed, the results are shown in FIG. 7, wherein the 1427bp specific restriction enzyme fragment represents the successful construction of the recombinant plasmid.

According to the method of example 2, four recombinant plasmids pLL3.7-shRNA-A/C1/C2/NC-EGFP are respectively transformed into 293T cells to obtain four viruses, then the viruses are respectively transfected into Jurkat cells, and the expression amount of ADRB2mRNA in the transfected cells is analyzed, and the result is shown in FIG. 8, wherein the result shows that shRNA-A has the best effect in terms of ADRB2mRNA expression in a plurality of screened interfering RNAs, and the expression amount of ADRB2mRNA in an interfering group of shRNA-C1 and shRNA-C2 is higher than that of shRNA-A group, so that shRNA-A fragments have the best interference effect.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

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Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys

35 40 45

Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser

50 55 60

Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser

65 70 75 80

Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met

85 90 95

Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly

100 105 110

Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly

115 120 125

Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys

130 135 140

Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val

145 150 155

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<223> RNAi target sequence of ADRB2 Gene C1

<400> 8

gcatcgtcat gtctctcatc g 21

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<223> RNAi target sequence of ADRB2 Gene C2

<400> 9

gctccagaag attgacaaat c 21

<210> 10

<211> 65

<212> DNA

<213> Artificial sequence

<223> ADRB2-shRNA-C1-F

<400> 10

tgcatcgtca tgtctctcat cgttcaagag acgatgagag acatgacgat gcttttttga 60

attcc 65

<210> 11

<211> 69

<212> DNA

<213> Artificial sequence

<223> ADRB2-shRNA-C1-R

<400> 11

tcgaggaatt caaaaaagca tcgtcatgtc tctcatcgtc tcttgaacga tgagagacat 60

gacgatgca 69

<210> 12

<211> 65

<212> DNA

<213> Artificial sequence

<223> ADRB2-shRNA-C2-F

<400> 12

tgctccagaa gattgacaaa tcttcaagag agatttgtca atcttctgga gcttttttga 60

attcc 65

<210> 13

<211> 69

<212> DNA

<213> Artificial sequence

<223> ADRB2-shRNA-C2-R

<400> 13

tcgaggaatt caaaaaagct ccagaagatt gacaaatctc tcttgaagat ttgtcaatct 60

tctggagca 69

Claims

1.A method of enhancing the killing ability of T cells against tumor cells, comprising: the method comprises the following steps: reducing or knocking out the mRNA level of the β 2adrenergic receptor in the T cell by the siRNA; the target sequence of the siRNA comprises a sequence shown in SEQ ID NO. 1.

2. The method of claim 1, wherein: the T cells include CAR-T cells,

3. The method of claim 2, wherein: the extracellular domain is an extracellular segment of NKG2D, preferably, the amino acid sequence of the extracellular segment of NKG2D is shown as 22-157 th position of SEQ ID NO.7, more preferably, the coding sequence of the extracellular segment of NKG2D is shown as 64-471 th position of SEQ ID NO. 6;

4. A method according to any one of claims 1-3, characterized in that: the T cell is CD3⁺T cells, or CD4⁺T cells and/or CD8⁺A T cell;

and/or, the tumor cells comprise benign tumor cells and/or malignant tumor cells; preferably, the malignant tumor cells are from any one or more of pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, uterine body tumor and osteosarcoma;

5. A T cell with enhanced killing of tumor cells, comprising: prepared by the process of any one of claims 1 to 4.

6. An siRNA for reducing or knocking out the expression level of β 2 adrenoreceptor mRNA in a T cell, wherein: the target sequence of the siRNA comprises a sequence shown in SEQ ID NO. 1;

preferably, the T cell is a CAR-T cell.

7. A gene, characterized by: the gene encodes the siRNA of claim 6.

8. A biomaterial, characterized by: the biological material is an expression frame, a recombinant vector, a recombinant bacterium or a recombinant cell containing the gene of claim 7;

9. The biomaterial of claim 8, wherein: the recombinant vector, recombinant bacterium or recombinant cell further comprises a gene and/or protein encoding the CAR of claim 2 or 3.

10. Use of the T cell of claim 5, the siRNA of claim 6, the gene of claim 7, the biomaterial of claim 8 or 9 for the preparation of a medicament for the treatment of tumors.