WO2019113745A1

WO2019113745A1 - Application of tipe2 gene as target point in preparation of drugs for treating tumours

Info

Publication number: WO2019113745A1
Application number: PCT/CN2017/115483
Authority: WO
Inventors: 万晓春; 陈有海; 鄢德洪
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2017-12-11
Filing date: 2017-12-11
Publication date: 2019-06-20

Abstract

Provided is an application of the TIPE2 gene as a target point in the preparation of drugs for treating tumours. Provided is a new target point, i.e. the TIPE2 gene, for the treatment of primary and metastasic tumours; inhibiting the TIPE2 gene expression inhibits the growth of primary tumours and can also inhibit tumour cell metastasis. Thus, the TIPE2 gene can be a target point for preparing drugs for the treatment of primary and metastasic tumours, providing a new solution and strategy for the targeted drug treatment of tumours.

Description

Application of TIPE2 gene as a target in the preparation of drugs for treating tumors

Technical field

The present application relates to the field of tumor targeted therapy, and in particular to the use of the TIPE2 gene as a target for the preparation of a medicament for treating tumors.

Background technique

TIPE2, a tumor necrosis factor a-induced protein 8-2, is a member of the TNFAIP8 family. It has been reported that TIPE2 is an immunoregulatory factor that is selectively expressed in the thymus, spleen, lymph nodes and other immune organs and inflammatory tissues. TIPE2 negatively regulates NF-κB and MAPK signaling pathways, inhibits TCR-mediated T cell activation and TLR-mediated innate immunity of macrophages, and plays a key role in inhibiting inflammation and maintaining immune homeostasis. In addition, TIPE2 is also expressed in tumor cells or tumor tissues. Clinical specimens showed low levels of TIPE2 expression in liver cancer, gastric cancer, and non-small cell lung cancer, but TIPE2 was highly expressed in renal cell carcinoma, colorectal cancer, and thyroid cancer. The study found that TIPE2 can play a negative regulatory role in Ras and Rac signaling pathways in tumor cells. TIPE2 binds directly to RalGDS and RacGTPases, inhibiting Ras, Rac-mediated signal transduction and subsequent biological activities.

At present, clinically available tumor immunotherapy can be used effectively and effective therapeutic targets, mainly including immunological checkpoint molecules PD-1, PD-L1, CTLA-4 or tumor antigen markers HER2, VEGF, NY-ESO1, etc. molecule. Due to the limitation of effective targets, the effective range of clinical tumor immunotherapy is only 20% of tumors, which greatly limits the application range of tumor immunotherapy. Therefore, it is urgent to find new tumor antigen markers for clinical treatment of primary hair. Immunotherapy for sexual or metastatic tumors provides new targets and therapeutic strategies.

Summary of the invention

The purpose of the present application is to provide a novel therapeutic target for primary or metastatic tumors, namely the TIPE2 gene, which is targeted for the preparation of a medicament for treating tumors with the TIPE2 gene as a target.

In order to achieve the above objectives, the present application adopts the following technical solutions:

An aspect of the present application discloses a use of the TIPE2 gene as a target for the preparation of a medicament for treating tumors, wherein targeting the TIPE2 gene includes using gene knockout, gene knockdown or chemical drugs to reduce the expression of the TIPE2 gene. The tumor is a primary tumor or a metastatic tumor.

It should be noted that the study found that the TIPE2 gene is deleted, which can not only treat primary tumors, but also inhibit tumor cell metastasis, and can be used for treating metastatic tumors. Therefore, the TIPE2 gene can be used as a target for the preparation of a medicament for treating a primary tumor or a metastatic tumor; The expression of the TIPE2 gene can be used to treat primary tumors or metastatic tumors.

A further aspect of the present application discloses the use of a tumor-infiltrating leukocyte targeting a TIPE2 gene for the preparation of a medicament for treating a tumor, wherein the TIPE2 gene is targeted to include a gene knockout, a gene knockdown or a chemical drug to reduce the TIPE2 gene. Expression; the tumor is a primary tumor or a metastatic tumor.

The application of the present application specifically refers to the treatment of primary tumors or metastatic tumors by immunotherapy based on tumor infiltrating leukocytes.

It should be noted that this study has found that tumor-infiltrating leukocytes with TIPE2 gene can enhance anti-tumor immune response. Therefore, tumor-infiltrating leukocytes with TIPE2 gene as target can be used to treat primary tumors or metastatic tumors. drug.

A further aspect of the present application discloses a TIPE2 gene knockout tumor infiltrating leukocyte.

Another aspect of the present application discloses a method for preparing a TIPE2 gene knockout tumor infiltrating leukocyte of the present application, comprising the following steps,

Tumor cell implantation was performed on mice with TIPE2 gene deletion by subcutaneous injection;

After the tumor cells to be implanted grow into tumor tissue blocks, the tumor tissue blocks are removed from the mice, and the tumor tissue blocks are digested with an enzyme mixture including collagenase I, collagenase IV, hyaluronidase and deoxygenation. Ribozyme; wherein the tumor cells grow into tumor tissue blocks generally within 2-3 weeks, that is, the tumor tissue blocks can be taken within 2-3 weeks after the tumor cells are implanted, and one implementation of the present application is after planting. Tumor tissue blocks collected in 16 days;

After digestion, the digestion was terminated with RPMI 1640 complete medium, and the tumor tissue blocks were placed on a cell sieve to collect the tissue slurry through the cell sieve. The RPMI 1640 complete medium was used to terminate the digestion, which was directly cultured with RPMI 1640. The centrifugation tube can terminate the digestion; in one implementation of the present application, the cell screen is a 70 μm sieve, and after grinding, the tumor cells are all suspended in the tissue slurry through the cell sieve;

The tissue slurry was subjected to low-speed centrifugation at 4 ° C to obtain a cell supernatant, and the precipitated impurities were discarded; the cell supernatant was centrifuged at 4 ° C at high speed to collect precipitated cells; wherein, 4 ° C low-speed centrifugation means lower at 4 ° C. Centrifugation at a rate at which the cells are still suspended in solution, while larger impurities are removed by centrifugation. In one implementation of the present application, the speed of low speed centrifugation is 60 g; 4 °C high speed centrifugation means 4 Centrifugation at a higher speed, at which the cells are pelleted by centrifugation for removal of the supernatant. In one implementation of the present application, the speed of high-speed centrifugation is 1260 g;

The collected precipitated cells were resuspended in RPMI 1640 complete medium, and the collected precipitated cells were isolated using mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit. Specifically, the cells were completely cultured with RPMI 1640. The base weight suspension is added to the liquid surface of the separation liquid prepared according to the kit, centrifuged at 1260 g, 25 ° C, and the two layers of annular milky white cell layers appearing in the centrifuge tube are aspirated and collected by a pipette; In the mouse tumor tissue lymph, mononuclear macrophage, granulocyte separation solution kit, in an implementation manner of the present application, specifically used is the LDS1090Z kit purchased from Tianjin Haoyang Biological Products Technology Co., Ltd. The liquid level or gradient interface of the separation liquid is prepared according to the conventional method disclosed in the specification, and is not specifically limited herein. The centrifugation at 1260 g and 25 ° C is mainly for achieving better separation effect, and the parameter condition can be adjusted within the error range allowed by the test. ;

The collected milky white cell layer was washed with PBS solution, 1260 g, centrifuged at 4 ° C, the supernatant was discarded, and resuspended in PBS to obtain a PBS cell suspension, which is the TIPE2 knockout tumor infiltrating leukocyte of the present application. . In the present application, 1260g, centrifuged at 4 ° C, wherein the centrifuge speed of 1260g is to precipitate the cells without rupture; the centrifugation temperature of 4 ° C is mainly to make the cells at a lower temperature, slowing down their growth and metabolism, so that It can be more stable.

In one implementation of the present application, the TIPE2 gene-deficient mouse is subjected to tumor cell implantation, wherein the tumor cell used is LLC mouse lung cancer cell or B16F10 mouse melanoma tumor cell; that is, planting LLC mouse lung cancer Finally, TIPE2 knockout lung cancer tumor infiltrating leukocytes are obtained. The lung cancer tumor infiltrating leukocytes have a good therapeutic effect on primary or metastatic lung cancer. Planting B16F10 mouse melanoma tumor cells finally obtains TIPE2 gene knockout melanoma tumor infiltration. White blood cells, the melanoma tumor infiltrating leukocytes have a good therapeutic effect on primary or metastatic melanoma tumors.

Preferably, before the digestion of the tumor tissue block by the enzyme mixture, the tumor tissue block is further cut into small pieces and then digested. It can be understood that cutting the tumor tissue block into small pieces can better digest and improve the quality and efficiency of digestion.

Preferably, the enzyme mixture contains 50 mg/mL of collagenase I, 50 mg/mL of collagenase IV, 5 mg/mL of hyaluronidase, and 1 U/mL of deoxyribonuclease.

Preferably, in tumor cell planting, the amount of tumor cells implanted is 2 x 10 ⁶ tumor cells per mouse.

Preferably, the tumor tissue block is removed from the mouse, specifically, the mice that have been sacrificed by cervical dislocation are immersed in 75% alcohol, and then the mouse is placed on a sterile clean bench, and the tumor is removed with surgical scissors and forceps. Organization block.

Still another aspect of the present application discloses the use of the TIPE2 gene knockout tumor infiltrating leukocytes of the present application for the preparation of a medicament for treating a primary tumor or a metastatic tumor.

It can be understood that the TIPE2 knockout tumor infiltrating leukocytes of the present application have a good tumor suppressing effect, can inhibit tumor growth and tumor cell metastasis, and therefore, can completely prepare corresponding drugs for treating primary tumors or metastasis. Sexual tumors. The drug may also include other pharmaceutically acceptable auxiliary ingredients or active ingredients, which are not specifically limited herein.

Due to the adoption of the above technical solutions, the beneficial effects of the present application are:

The present application provides a novel target for the treatment of primary tumors and metastatic tumors, that is, the TIPE2 gene can inhibit the growth of primary tumors by inhibiting the expression of TIPE2 gene, and can inhibit tumor cell metastasis. The TIPE2 gene knockout tumor infiltrating leukocytes of the present application can be used to develop new targeted drugs for preventing or treating primary tumors or metastatic tumors.

DRAWINGS

1 is a graph showing tumor volume increase results of LLC mouse lung cancer cells implanted in wild type mice and TIPE2 gene deletion mice, respectively, in the examples of the present application;

2 is a graph showing tumor volume increase results of B16F10 mouse melanoma tumor cells implanted in wild type mice and TIPE2 gene deletion mice, respectively, in the examples of the present application;

Figure 3 is a diagram showing the results of transfer of B16F10 mouse melanoma tumor cells to the lungs after B16F10 mouse melanoma tumor cells were planted in wild type mice and TIPE2 gene deletion mice, respectively, in the examples of the present application;

Figure 4 is a graph showing the results of testing the effect of TIPE2 gene deletion on the ratio of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells in the examples of the present application;

Figure 5 is a graph showing the results of testing the effect of TIPE2 gene deletion on IFN-γ production by CD45 ⁺ CD3 ⁺ CD8 ⁺ cells in the examples of the present application;

Fig. 6 is a graph showing the results of an ability test for inhibiting the growth of melanin tumors in B16F10 mice by TIPE2 gene-deficient CD45 ⁺ CD3 ⁺ CD8 ⁺ cells in the examples of the present application.

Detailed ways

In the process of studying MDSC, this application found a new therapeutic target, TIPE2 gene. It was confirmed by research that knocking out the TIPE2 gene reduced the expression of TIPE2 gene, which can effectively inhibit tumor growth and inhibit tumor cell metastasis. And enhance the anti-tumor immune response, play the role of prevention and treatment of tumors. It can be understood that as long as the expression of TIPE2 gene can be reduced, tumor growth can be inhibited and the effect of preventing or treating tumor can be achieved; therefore, in addition to gene knockout, gene knockdown or chemical drug can reduce the expression of TIPE2 gene, and can also serve the same. The effect of preventing or treating a tumor.

In addition, it has been confirmed by the application of the present application that after the TIPE2 knockout tumor infiltrating leukocytes are further sorted, the sorting products can inhibit tumor growth and enhance the anti-tumor immune response, thereby preventing or treating tumors; therefore, TIPE2 knockout tumor infiltrating leukocytes can be used to develop new prophylactic or therapeutic tumor targeting drugs.

The present application will be further described in detail below by way of specific embodiments and the accompanying drawings. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.

Example

In this case, LLC mouse lung cancer cells and B16F10 mouse melanoma tumor cells were planted subcutaneously in wild type mice (abbreviated WT) and TIPE2 gene deletion mice (abbreviated Tipe2 ^-/- ), respectively, LLC and B16F10. The cells all carry the normal function of TIPE2 gene, and the TIPE2 gene deletion inhibits the growth of primary tumors. Among them, B16F10 cells carry the normal function TIPE2 gene.

In addition, B16F10 mouse melanoma cells were injected into wild-type mice (WT) and TIPE2 gene-deficient mice (Tipe2 ^-/- ) by tail vein injection to observe the effect of TIPE2 gene deletion on tumor metastasis, among which B16F10 cells Carry the normal function of the TIPE2 gene.

The details are as follows:

Test 1 Effect of TIPE2 gene knockout on primary tumors

LLC mouse lung cancer cells and B16F10 mouse melanoma tumor cells were separately cultured in DMEM complete medium, and a sufficient number of cells, ie, 2×10 ⁶ /mouse dose, were obtained by subcutaneous injection in wild type mice. (WT) and gene-deficient mice TIPE2 (Tipe2 ^{- / -)} dose of 2 × 10 ⁶ cultivation. That is, LLC mouse lung cancer cells were planted with wild type mice (WT) and TIPE2 gene deletion mice (Tipe2 ^-/- ), and B16F10 mouse melanoma tumor cells were planted with wild type mice (WT) and TIPE2 gene deletion type. Rat (Tipe2 ^-/- ). Then, the long diameter and the short diameter of the tumor of the LMC mouse lung cancer cells were continuously recorded by means of a ruler measurement recording for 21 days, and the long diameter and short diameter of the tumor of the B16F10 mouse melanoma tumor cells were continuously recorded for 16 days. Tumor volume was calculated according to the tumor volume formula, and tumor volume changes were counted, and data analysis was performed using GraphPad 6.0 software.

Among them, the tumor volume (mm ³ ) = short diameter ² × long diameter × 1/2.

The LLC mouse lung cancer cells and the B16F10 mouse melanoma tumor cells of this example were purchased from ATCC. DMEM complete medium included high glucose DMEM + 10% fetal bovine serum + 1% penicillin and streptomycin, all purchased from Hyclone. Both the LLC mouse lung cancer cells and the B16F10 mouse melanoma tumor cells carry the normal function TIPE2 gene.

Tumor volume calculations and statistical results are shown in Figure 1 and Figure 2. Figure 1 shows the tumor volume growth of wild-type mice (WT) and TIPE2 gene-deficient mice (Tipe2 ^-/- ) in the experimental mouse lung cancer cells. Curve, Figure 2 is the tumor volume growth curve of wild-type mice (WT) and TIPE2 gene-deficient mice (Tipe2 ^-/- ) implanted in B16F10 mouse melanoma tumor cells. In Figure 1 and Figure 2, the abscissa is The number of days after planting, the ordinate is the tumor volume; the "▲" curve is the tumor volume growth curve of the Tipe2 ^-/- mice, and the "■" curve is the tumor volume growth curve of the WT mice. The results in Figures 1 and 2 show that TIPE2 gene deletion has the effect of inhibiting primary tumor growth in both the LLC and B16F10 tumor models.

Test 2 Inhibition of tumor cell metastasis by TIPE2 knockout

In this example, 2×10 ⁵ B16F10 mouse melanoma tumor cells were injected into wild-type mice (WT) and TIPE2 gene-deficient mice (Tipe2 ^-/- ) by tail vein injection, in which B16F10 cells carried normal functions. The TIPE2 gene was used to record the number of B16F10 tumor cells transferred to B16F10 cells at 14 days of lung metastasis, and data analysis was performed using GraphPad 6.0 software.

The results are shown in Fig. 3. In Fig. 3, the abscissa is WT mouse and Tipe2 ^-/- mice, and the ordinate is the number of B16F10 tumor cells; the results in Fig. 3 show that TIPE2 gene deletion has an inhibitory effect on B16F10 tumor metastasis. .

Test 3 TIPE2 gene knockout enhances anti-tumor immune response

Tumor cell planting

2×10 ⁶ B16F10 mouse melanoma tumor cells were implanted subcutaneously in wild type mice (WT) and TIPE2 gene deletion mice (Tipe2 ^-/- ), wherein B16F10 cells carry the normal function TIPE2 gene. . When the tumor was grown to 16 days, the same number of tumor tissue blocks of WT and Tipe2 ^-/- were taken for extracting tumor infiltrating leukocytes.

2. Tumor infiltration leukocyte extraction

The mice sacrificed by necking were immersed in 75% alcohol for 5 minutes, and then the mice were placed on a sterile clean bench, and the tumor tissue pieces were removed with surgical scissors and forceps and placed in a 10 cm culture dish. Cut the tumor tissue into small pieces with scissors, add 10 mL of the enzyme mixture and resuspend in a 50 mL centrifuge tube, shake for 45-60 minutes in a shaking incubator at 200 rpm, and fill the tube with RPMI 1640 complete medium to terminate digestion. .

The tissue block suspension was placed on a 70 μm cell sieve and repeatedly ground with a 5 mL syringe handle, and the cells were all passed through a sieve into a new 50 ml centrifuge tube. The sieve was discarded, and the polishing liquid was centrifuged at 60 g for 5 min at 4 ° C, and the precipitate was discarded. The supernatant of the cells was centrifuged at 1260 g, centrifuged at 4 ° C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium. Take a 15 mL centrifuge tube and carefully add 3 mL of the separation solution, 2 mL of the separation solution 2, and 1 mL of the separation solution 3 according to the mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit instructions to prepare a volume ratio of 3:2: 1. The total volume is equal to the gradient interface of the RPMI 1640 complete medium resuspended single cell suspension volume. The mouse tumor tissue lymph, mononuclear macrophage, granulocyte separation solution kit was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LDS1090Z.

A sample of the cell suspension was carefully pipetted with a pipette and applied to the liquid surface of the separation solution, and centrifuged at 1260 g for 30 minutes at 25 °C. After centrifugation, two layers of annular milky white cells appeared in the centrifuge tube. Pipette two layers of circular target cells into a new 15 mL centrifuge tube, add 10 mL of PBS to the tube, and mix the cells. After centrifugation at 1260 g for 10 min at 4 ° C, the supernatant was discarded, and the cell pellet was resuspended in PBS for use, that is, the tumor infiltrating leukocyte suspension of this example. In this case, WT tumor infiltrating leukocytes and Tipe2 ^-/- tumor infiltrating leukocytes were extracted.

3.CD45 ⁺ CD3 ⁺ CD8 ⁺ detection

The tumor infiltrating leukocyte suspension prepared in this example was stained with the flow antibody CD45/CD3/CD8 purchased from BioLegend for 30 minutes at 4 ° C, and then WT tumor infiltrating leukocytes and Tipe 2 ^-/- tumor infiltrating leukocytes were detected by flow cytometry. The ratio of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells. The flow cytometer of this example was purchased from BD Bioscience.

4. TIPE2 knockout tumor infiltrating leukocytes enhance anti-tumor immune response test

The tumor infiltrating leukocyte suspension prepared in this example was treated with anti-CD3/CD28 magnetic beads for 48 hours, and 50 ng/mL of phorbol ester (abbreviated PMA) and 1 μg/mL of ions were added for the last 4-6 hours of stimulation. Taxomycin (ie, Ionomycin) and 500 ng/mL of Brefeldin A (abbreviated BFA). The collected cells were stained with the flow antibody CD45/CD3/CD8 purchased from BioLegend for 30 minutes at 4 ° C, and then lysed with BD Cytofix/Cytoperm ^TM reagent to stain the IFN-γ and the corresponding isotype control, and then the upflow cells. The ratio of IFN-γ produced by each of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells in WT tumor infiltrating leukocytes and Tipe 2 ^-/- tumor infiltrating leukocytes was measured.

Among them, anti-CD3/CD28 magnetic beads were purchased from Invitrogen, Inc., number 11456D.

The results of detecting the ratio of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells are shown in Fig. 4, and the ratio of IFN-γ produced by CD45 ⁺ CD3 ⁺ CD8 ⁺ cells is shown in Fig. 5. The results in Figures 4 and 5 show that TIPE2 gene deletion not only increases the infiltration of tumor-killing CD8 ⁺ T cells into tumor tissues, but also up-regulates the number of CD8 ⁺ T cells producing anti-tumor activity cytokine IFN-γ, enhancing the resistance. Tumor immunological activity.

Test 4 TIPE2 gene-deficient CD45 ⁺ CD3 ⁺ CD8 ⁺ cells inhibit the growth of melanoma tumors in B16F10 mice

2×10 ⁶ B16F10 mouse melanoma tumor cells were implanted by subcutaneous injection in TIPE2 gene-deficient mice (Tipe2 ^−/− ), wherein B16F10 cells carried the normal function TIPE2 gene. The three groups were injected intraperitoneally with the isotype control (ie, the Isotype antibody group), the CD8 ⁺ T cells (ie, the Anti-CD8 antibody group), and the CD4 ⁺ T cells (ie, the Anti-CD4 antibody group), and then recorded using a ruler. The means of continuous recording of the long diameter and short diameter of tumors of B16F10 mouse melanoma tumor cells were 13 days. Tumor volume was calculated according to the tumor volume formula, and tumor volume changes were counted, and data analysis was performed using GraphPad 6.0 software.

Tumor volume calculation and statistical results are shown in Figure 6. Figure 6 shows the tumor volume growth curve of TIPE2 gene-deficient mice (Tipe2 ^-/- ) implanted in B16F10 mouse melanoma tumor cells. The abscissa is the number of days after planting. The ordinate is the tumor volume; the "●" curve is the tumor volume growth curve of the Isotype antibody group, the "■" curve is the tumor volume growth curve of the Anti-CD8 antibody group, and the "▲" curve is the tumor volume growth of the Anti-CD4 antibody group. curve. The results in Figure 6 show that the TIPE2 gene-deficient CD45 ⁺ CD3 ⁺ CD8 ⁺ cells have an enhanced inhibitory effect on primary tumor growth in the B16F10 tumor model.

The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. For the ordinary person skilled in the art to which the present invention pertains, a number of simple deductions or substitutions may be made without departing from the spirit of the present application.

Claims

The use of the TIPE2 gene as a target for the preparation of a medicament for treating tumors, wherein targeting the TIPE2 gene comprises using gene knockout, gene knockdown or chemical drugs to reduce the expression of the TIPE2 gene; the tumor is a primary tumor Or metastatic tumors.
The use of tumor infiltrating leukocytes targeting the TIPE2 gene for the preparation of a medicament for treating tumors, wherein targeting the TIPE2 gene comprises using gene knockout, gene knockdown or chemical drugs to reduce the expression of the TIPE2 gene; Primary tumor or metastatic tumor.
The use according to claim 2, wherein the application comprises immunotherapy for treating a primary tumor or a metastatic tumor based on the tumor infiltrating leukocytes.
A TIPE2 knockout tumor infiltrating leukocyte.
The method for preparing a TIPE2 gene knockout tumor infiltrating leukocyte according to claim 4, comprising the steps of:

Tumor cell implantation was performed on mice with TIPE2 gene deletion by subcutaneous injection;

After the tumor cells to be implanted grow into tumor tissue blocks, the tumor tissue pieces are removed from the mice, and the tumor tissue blocks are digested with an enzyme mixture including collagenase I, collagenase IV, and hyaluronidase. And deoxyribonuclease;

After the digestion is completed, the digestion is terminated by using RPMI 1640 complete medium, and the tumor tissue block is placed on a cell sieve to be ground, and the tissue polishing liquid passing through the cell sieve is collected;

The tissue slurry is subjected to low-speed centrifugation at 4 ° C to obtain a cell supernatant, and the precipitated impurities are discarded; the cell supernatant is subjected to high-speed centrifugation at 4 ° C to collect precipitated cells;

The collected precipitated cells were resuspended in RPMI 1640 complete medium, and the collected precipitated cells were isolated using mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit. Specifically, the cells were completely cultured with RPMI 1640. The base weight suspension is added to the liquid surface of the separation liquid prepared according to the kit, centrifuged at 1260 g, 25 ° C, and the two layers of annular milky white cell layers appearing in the centrifuge tube are aspirated and collected by a pipette;

The collected milky white cell layer was washed with PBS solution, 1260 g, centrifuged at 4 ° C, the supernatant was discarded, and resuspended in PBS to obtain a PBS cell suspension, that is, the TIPE2 knockout tumor infiltrating leukocytes.
The preparation method according to claim 5, wherein the TIPE2 gene-deficient mouse is subjected to tumor cell planting, wherein the tumor cell used is LLC mouse lung cancer cell or B16F10 mouse melanoma tumor cell; planting LLC mouse Lung cancer eventually obtained TIPE2 knockout lung cancer tumor infiltrating leukocytes; implanted B16F10 mouse melanoma tumor cells finally obtained TIPE2 knockout melanoma tumor infiltrating leukocytes.
The preparation method according to claim 5, wherein the enzyme mixture contains 50 mg/mL of collagenase I, 50 mg/mL of collagenase IV, 5 mg/mL of hyaluronidase, and 1 U/mL. Deoxyribonuclease.
The preparation method according to any one of claims 5 to 7, wherein in the tumor cell planting, the amount of the tumor cells implanted is 2 × 10 6 tumor cells per mouse.
The preparation method according to any one of claims 5 to 7, wherein the removing the tumor tissue block from the mouse comprises: immersing the mouse that has been subjected to neck removal in 75% alcohol, and then small The rats were placed on a sterile clean bench and the tumor tissue pieces were removed with surgical scissors and forceps.
The TIPE2 gene knockout tumor infiltrating leukocyte according to claim 4 for use in the preparation of a medicament for treating a primary tumor or a metastatic tumor.