CN110862967A - Natural killer cell line SILK-NK independent of cytokine culture - Google Patents

Abstract

The invention discloses a natural killer cell line SILK-NK. which is not dependent on cytokine culture and provides a preparation method of the natural killer cell line SILK-NK which is not dependent on cytokine culture, which comprises the following steps of expressing a protein compound containing IL-15 protein and an IL-15 binding region of IL-15R α on the surface of an NK-92 cell line in a membrane binding mode, and then culturing the cell line by using a culture medium without IL-2 to obtain a cloned cell line, namely the natural killer cell line SILK-NK., wherein the cell line can be stably subcultured and expanded in the culture medium without IL-2, and can keep the killing activity of the NK-92, so that the cell line becomes an engineering cell line with clinical application value and can be used for the adoptive immunotherapy of tumors.

Description

A cytokine-independent natural killer cell line SILK-NK

technical field

The invention relates to the field of adoptive immunotherapy of tumors, in particular to a cytokine-independent natural killer cell line SILK-NK and a preparation method thereof.

Background technique

NK (Nature Killer) cells are an important class of cytotoxic killer cells in the immune system, which can release perforin and cytokines to induce cancer cell apoptosis. The recognition of target cells by NK cells is not MHC-restricted and tumor-associated antigen-dependent, and thus has a broader spectrum of anti-tumor capabilities than T cells. The non-MHC-restricted nature of NK cells allows them to support allogeneic transplantation and to kill cancer cells that evade T-cell killing by reducing the expression of MHC molecules. NK cells do not secrete large amounts of IL-6 and therefore do not cause a strong cytokine storm. However, NK cells only account for about 10% of blood cells, which requires a cumbersome process of separation and enrichment of blood cells, and the low transfection efficiency of NK cells also increases the difficulty of genetic modification.

NK-92 is an NK cell line derived from non-Hodgkin's lymphoma patients, which has the advantages of NK cells and overcomes the shortcomings of NK cells. It not only possesses MHC-unrestricted and tumor-associated antigen-independent broad-spectrum anti-tumor capabilities, but also is very easy to large-scale culture and easier to perform genetic manipulation. More importantly, the clinical phase I data of NK-92 infusion patients have shown high safety, and no serious adverse reactions occurred even at the dose of 1×10 ¹⁰ /m ² .

Cytokines, especially interleukin-2 (IL-2) or IL-15, are required for the proliferation and activation of NK cells. IL-2 can stimulate the proliferation of Treg cells and induce T cell apoptosis, which has certain toxic and side effects in clinical applications. IL-15 shares many functions with IL-2, and IL-15 is an indispensable cytokine responsible for the development, differentiation and maintenance of NK. The NK-92 cell line expressing IL-15 showed stronger proliferation and cancer cell killing ability, but still could not get rid of the dependence on IL-2.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a natural killer cell line SILK-NK cultured independently of cytokines and a preparation method thereof, so as to change the biological characteristics of NK-92 cells, get rid of the dependence on IL-2, and improve the proliferation and killing ability , making NK-92 cells more suitable for clinical application.

In the first aspect, the present invention claims a method for preparing a cytokine-independent natural killer cell line SILK-NK.

The preparation method of the cytokine-independent natural killer cell line SILK-NK provided by the present invention may include the following steps: binding the protein complex containing the IL-15 protein and the IL-15 binding region of IL-15Rα with a membrane It was expressed on the surface of NK-92 cell line in the manner of IL-2, and then cultured in medium without IL-2, and the obtained cloned cell line was the natural killer cell line SILK-NK.

Further, the protein complex is composed of the IL-15 binding region, the transmembrane region and the IL-15 protein of IL-15Rα from the N-terminus to the C-terminus (as shown in Figure 1).

Wherein, the transmembrane region may be, but not limited to, the CD8α transmembrane region. In a specific embodiment of the present invention, the transmembrane region is a CD8α transmembrane region.

Further, the amino acid sequence of the IL-15 binding region of IL-15Rα may be SEQ ID No. 1, or the amino acid sequence of 99% or more, 95% or more, or 90% of the amino acid sequence shown in SEQ ID No. 1. More than 85%, more than 80%, or more than 75% homology, or a sequence with the same function after substitution and/or deletion and/or addition of one or several amino acid residues. The amino acid sequence of the CD8α transmembrane region can be SEQ ID No. 2, or the amino acid sequence shown in SEQ ID No. 2 has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% Or more than 75% homology, or a sequence with the same function after substitution and/or deletion and/or addition of one or several amino acid residues. The amino acid sequence of the IL-15 protein can be SEQ ID No. 3, or the amino acid sequence shown in SEQ ID No. 3 has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% Or more than 75% homology, or a sequence with the same function after substitution and/or deletion and/or addition of one or several amino acid residues.

More specifically, the amino acid sequence of the protein complex can be SEQ ID No. 4, or the amino acid sequence shown in SEQ ID No. 4 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more. % or more or more than 75% homology, or a sequence with the same function after substitution and/or deletion and/or addition of one or several amino acid residues.

Further, the method may include the steps of: inserting the encoding gene of the protein complex into a lentiviral expression vector, and then using the obtained recombinant lentiviral vector to package the lentivirus (for example, by inserting the obtained recombinant lentiviral vector into the lentivirus vector). The lentivirus was prepared by co-transfecting 293T cells with the packaging plasmid), the NK-92 cell line was infected with the lentivirus, cultured in a medium without IL-2 and obtained by limiting dilution. The cloned cell line described above.

In a specific embodiment of the present invention, the IL-2-free medium is specifically GT-T551 containing a final concentration of 2.5% (volume percent) serum substitute (eg, product number: HPCFDCGL50, product of Helios Company). -H3 medium (such as product number: WK593S, product of TAKARA company).

Wherein, in the encoding gene of the protein complex, the nucleotide sequence encoding the IL-15 binding region of the IL-15Rα can be the 1-216th position of SEQ ID No. Positions 1-216 of 5 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more homology and encode the IL-15 binding region of the IL-15Rα sequence. In the encoding gene of the protein complex, the nucleotide sequence encoding the CD8α transmembrane region can be the 217-423 position of SEQ ID No.5, or the nucleotide sequence with the 217-423 position of SEQ ID No.5 More than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology and encoding the CD8α transmembrane region sequence. In the encoding gene of the protein complex, the nucleotide sequence encoding the IL-15 protein may be the 490-831 position of SEQ ID No.5, or the same as the 490-831 position of SEQ ID No.5. A sequence encoding the IL-15 protein having a homology of more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75%.

Specifically, the nucleotide sequence of the encoding gene of the protein complex may be SEQ ID No. 5, or the nucleotide sequence of SEQ ID No. 5 with 99% or more, 95% or more, 90% or more, 85% or more, 80 % or more or more than 75% homology and the sequence encoding the protein complex.

In a second aspect, the present invention claims a cytokine-independent cultured natural killer cell line SILK-NK prepared by the method described above.

The STR phenotype of the natural killer cell line SILK-NK is

Amelogenin: X, Y

CSF1PO: 11,12

D13S317: 9, 12

D16S539: 11,12

D5S818: 12,13

D7S820: 10,11

THO1: 6,9.3

TPOX: 8

vWA: 18

D21S11: 31.2, 32.

The expression of IL-15 and IL-15 binding region of IL-15Rα can be detected by mRNA reverse transcription PCR.

It is in the form of suspended clonal clusters or dispersed cells.

Further, the natural killer cell line SILK-NK is specifically a cell line deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the registration number is CGMCC No. 15583.

The third aspect, the application of the aforementioned natural killer cell line SILK-NK in any of the following also belongs to the protection scope of the present invention:

(A1) Preparation of products for adoptive immunotherapy of tumors;

(A2) Preparation of related products for cell therapy.

For example, the natural killer cell line SILK-NK is genetically modified to express a chimeric antigen receptor, and the targeting of the chimeric antigen receptor is used to mediate the specificity of the cell line to cancer cells expressing the target antigen kill. The natural killer cell line SILK-NK is genetically modified to express a chimeric antigen receptor, and cell therapy is performed by infusing the modified cells into a patient.

The present invention establishes a new type of killer cell called SILK-NK (Specific Interleukin Linked Killer, SILK-NK) by trying to express IL-15 and the IL-15 binding region complex of IL-15Rα in NK-92 at the same time, Using the IL-15 binding domain of IL-15Rα as a super activator of IL-15, the complex composed of IL-15 and the IL-15 binding domain of IL-15Rα was used to activate NK-92 cells and increase the NK-92 cells Cancer cell killing ability. On the one hand, this complex stimulates NK-92 in a membrane-bound form in cis-presented, enabling it to proliferate in the absence of cytokines, and on the other hand, stimulates host autologous T in trans-presented form. The proliferation and activation of cells and NK cells can improve their own anti-tumor ability.

The present invention starts from cytokines that play a regulatory role in the process of NK cell differentiation and development, because 1. IL-15 is expressed in various tissues in the human body, and plays an important role in the process of NK cell differentiation and development; 2. NK-92 It is an undifferentiated mature NK cell, which can express IL-15 receptor on the cell surface; 3. The toxic and side effects of IL-15 are significantly less than that of IL-2; activator. Therefore, the present invention uses a viral vector expressing membrane-bound IL-15 and the IL-15 binding domain complex of IL-15Rα to infect NK-92 cells, in order to obtain IL-15 binding expressing membrane-bound IL-15 and IL-15Rα. NK-92 cells in the region complex, through the trans- and homeopathic activation of IL-15 and the IL-15 binding region complex of IL-15Rα, the culture of NK-92 cells can completely get rid of the dependence on IL-2 and improve the The ability of NK-92 cells to proliferate, kill and reduce toxic and side effects significantly improves their potential for clinical use.

Beneficial effects of the present invention: In the prior art, NK-92 expressing IL-15 can improve the cytotoxicity of NK-92, but its NK-92 cells still cannot get rid of the dependence on IL-2. The present invention stably expresses the complex of membrane-bound IL-15 and the IL-15 binding region of IL-15Rα in NK-92 cells, and utilizes the super agonist effect of the IL-15 binding region of IL-15Rα on IL-15, which can The NK-92 cells can proliferate for a long time without IL-2 and have higher killing activity, which is beneficial to large-scale culture and clinical application.

Preservation Instructions

Suggested nomenclature: Human natural killer cell line

Participating Biomaterials Co., Ltd.: SILK

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Preservation date: April 3, 2018

Deposit Center Registration Number: CGMCC No.15583

Description of drawings

Figure 1 is a structural diagram of the protein complex of the present invention. A: IL-15 binding domain of IL-15Rα, B: transmembrane domain, C: IL-15.

Figure 2 is a schematic diagram of the structure of the nucleic acid sequence encoding the protein complex of the present invention. A: IL-15 binding domain of IL-15Rα, B: transmembrane domain, C: IL-15.

Figure 3 is a map of the lentiviral vector of the present invention loaded with the nucleic acid sequence of IL-15Rα-TM-P2A-IL-15.

Figure 4 is a photograph of the morphology of SILK-NK-D8 cells and NK-92 cells of the present invention. A is NK-92 cells and B is SILK-NK-D8 cells.

Figure 5 is a comparison of the 3-day proliferation folds of SILK-NK-D8 cells of the present invention and NK-92 at different culture time points. A is the 3-day multiplication fold curve graph of NK-92 plus 100U/mL IL-2 culture; B is the 3-day multiplication fold curve graph of SILK-NK-D8 cultured without IL-2.

FIG. 6 is an electrophoresis diagram of the mRNA expression of IL-15 and the IL-15 binding region of IL-15Rα identified by RT-PCR of the present invention. A is NK-92 cells and B is SILK-NK-D8 cells. P1 is primer 1 + primer 2, and P2 is primer 3 + primer 4.

Figure 7 shows the killing experiment of NK-92 and SILK-NK-D8 cells of the present invention on K562 cells expressing GFP. Fluorescence shows K562 cells.

Figure 8 is the killing experiment of NK-92 and SILK-NK-D8 cells of the present invention on K562 cells expressing firefly luciferase. A is NK-92 cells and B is SILK-NK-D8 cells.

Figure 9 shows the results of STR analysis of the SILK-NK-D8 cells of the present invention.

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

NK-92 cells were purchased from Wuhan University China Type Culture Collection Center, the medium was GT-T551-H3 medium (Cat. No.: WK593S, product of TAKARA company) that can be used to culture T lymphocytes and NK cells, and serum replacement ( Product No.: HPCFDCGL50, product of Helios Company)) to its final concentration of 2.5% (volume percentage).

293T medium is DMEM supplemented with 10% (volume percent) fetal bovine serum.

pLTR-abg019: Nanjing Erbin Viola Biotechnology Co., Ltd., product number abg019.

pCMV-dR8.2 plasmid: Addgene, Cat. No. 8455.

pCMV-VSV-G plasmid: Addgene, Cat. No. 8454.

K562 cell line K562-GFP-Luc expressing GFP and luciferase: Nanjing Erbinjin Biotechnology Co., Ltd., Cat. No. 562-GL.

Example 1. Preparation and identification of cytokine-independent natural killer cell line SILK-NK

1. Construction of pLTR-IL15Rα-IL15 lentiviral expression vector

As shown in Figure 2 and Figure 3, the DNA sequence (SEQ ID No. 5) of IL-15Rα-TM-P2A-IL-15 was synthesized from Nanjing GenScript Biotechnology Co., Ltd. with BamHI (5 ') and MluI (3'), the synthesized sequence and the pLTR-abg019 plasmid were double digested with BamH I and Mlu I, respectively, and then ligated and transformed into DH5α competent bacteria, using the upstream of the IL-15 binding region sequence of IL-15Rα. The primer (SEQ ID No. 6) and the downstream primer in the IL-15 sequence, SEQ ID No. 7) were used to identify positive clones by colony PCR, and sequenced to identify the target expression plasmid pLTR-IL15Rα-IL15.

The structure of the recombinant plasmid pLTR-IL15Rα-IL15 is described as a recombinant plasmid obtained by replacing the small fragment between the restriction sites BamHI and Mlu I of the pLTR-abg019 plasmid with the DNA fragment shown in SEQ ID No. 5.

SEQ ID No.5 is the coding gene sequence of IL-15Rα-TM-P2A-IL-15 protein complex, wherein the 1-216th position is the coding gene of the IL-15 binding region of IL-15Rα, and the 217th-423rd position It is the coding gene of CD8α transmembrane region, and positions 490-831 are the coding gene of IL-15 protein. SEQ ID No. 5 encodes the protein shown in SEQ ID No. 4. Wherein, positions 1-72 of SEQ ID No. 4 are the amino acid sequence of the IL-15 binding region of the IL-15Rα (ie, SEQ ID No. 1); positions 73-141 of SEQ ID No. 4 are the CD8α The amino acid sequence of the transmembrane region (ie, SEQ ID No. 2); positions 164-277 of SEQ ID No. 4 are the amino acid sequence of the IL-15 protein (ie, SEQ ID No. 3).

2. pLTR-IL15Rα-IL15 plasmid packaging virus

The recombinant vector pLTR-IL15Rα-IL15 constructed in step 1, the packaging plasmids pCMV-dR8.2 and pCMV-VSV-G were transfected with PEI (1 μg/μl) at a ratio of 15:10:1 (mass ratio) to 293T cells.

The specific implementation is as follows: Passage 8×10 ⁶ cells of 293T cells to T75 culture flask on day 0; on day 1: add three plasmids pLTR-IL15Rα-IL15: pCMV-dR8.2: pCMV-VSV-G to 1 ml Opti-MEM =15μg: 10μg: 1μg, after mixing, add 78μl PEI, shake and mix well, place at room temperature for 15 minutes, then add to T75 culture flask for culturing 293T, shake and mix gently. Change to fresh medium 12-16 hours after transfection. The medium was collected 48 hours after transfection, that is, on the third day, and the cells were removed by filtration with a 0.45 μM filter to obtain the virus stock solution. 1/3 volume of 40% PEG was added to the virus stock solution, mixed and placed at 4°C overnight. Centrifuge the next day, 4°C, 1800g for 45 minutes, discard the supernatant and resuspend the virus pellet with 1/10 volume of the virus stock solution in NK-92 medium (adding a final concentration of 100U/ml IL-2) to obtain a 10-fold Concentrated virus suspension.

3. Infection of NK-92 cells

3×10 ⁵ NK-92 cells were resuspended with 1 mL of the 10-fold concentrated virus prepared in step 2 and added with 8 μg/ml polybrane, transferred to one well of a 24-well plate, centrifuged at 1500g at 32°C for 45 minutes, and then transferred to a carbon dioxide incubator Culture, centrifuge to remove the supernatant after 3 hours, and change to add NK-92 medium with a final concentration of 100U/ml IL-2; repeat the infection the next day and add NK-92 with a final concentration of 100U/ml IL-2. 92 medium, then maintained for 3 days and passaged once. After 2 weeks, the medium was not supplemented with IL-2 during subculture, and the culture was continued for two weeks, and the surviving cells were the obtained target cells SILK-NK (Specific Interleukin Linked Killer). The obtained cells were serially diluted in a 96-well plate, and 5 monoclonal cell lines were identified: A6, A9, D8, F6, F7, and clone D8 (hereinafter referred to as SILK-NK-D8) was used in subsequent examples.

SILK-NK-D8 was deposited on April 3, 2018 in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the registration number is CGMCC No. 15583. The proposed classification is named as human natural killer cell line; Co., Ltd.) is SILK.

4. Cell morphology and proliferation

As shown in Figure 4, SILK-NK-D8 has a similar cell morphology to NK-92, and tends to form loose clonal clusters. As shown in Figure 5, SILK-NK-D8 can reach IL-2 in medium without IL-2. 2-3 days doubled proliferation rate, same as NK-92.

V. Exogenous gene expression identification

Total RNA was extracted from SILK-NK-D8 and NK-92, respectively, and cDNA was obtained by reverse transcription with Oligo(dT)18. The primers for the IL-15 binding region of IL-15Rα (P1 is primer 1 + primer 2, P2 It is primer 3+primer 4) to do PCR, as shown in Figure 6, 1% agarose gel electrophoresis shows that SILK-NK-D8 has a positive band of about 500bp size, but NK-92 does not.

Primer 1: 5'-atgtccgtggaacacgcagac-3' (SEQ ID No. 6);

Primer 2: 5'-cttgaggtcg gagatgacgt tc-3' (SEQ ID No. 7);

Primer 3: 5'-cccagtctcaaatgcattagagacc-3' (SEQ ID No. 8);

Primer 4: 5'-ggtgtcatggatgctggcg-3' (SEQ ID No. 9).

6. Detection of cell killing ability

The K562 cell line K562-GFP-Luc expressing GFP and luciferase was used as the target cell, and SILK-NK-D8 and NK-92 were used as target cells at the ratio of 10:1, 5:1, 1:1 and 1 × 10 ⁴ targets, respectively Cells were incubated in 100 μl system in a 96-well plate, and GFP fluorescence and luciferase were detected after 24 hours. The results are shown in Figure 7 and Figure 8, SILK-NK-D8 and NK-92 have the same killing activity on K562 cells.

SILK-NK-D8 cells were tested for STR. The results are shown in Figure 9. The STR phenotype is

"Amelogenin: X, Y

CSF1PO: 11,12

D13S317: 9, 12

D16S539: 11,12

D5S818: 12,13

D7S820: 10,11

THO1: 6,9.3

TPOX: 8

vWA: 18

D21S11: 31.2, 32".

The expression of IL-15 and IL-15 binding region of IL-15Rα can be detected by mRNA reverse transcription PCR.

Taking the above ideal embodiments according to the present invention as inspiration, and through the above description, relevant personnel can make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content of the above-mentioned specific embodiments, and its technical scope must be determined according to the claims and the scope of the description.

<110> Tianjin Tianrui Biotechnology Co., Ltd.

<120> A cytokine-independent natural killer cell line SILK-NK

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Claims (10)

1. A method for preparing a cytokine-independent natural killer cell line SILK-NK, comprising the steps of: combining a protein complex containing an IL-15 protein and an IL-15 binding region of IL-15Rα in a membrane-bound manner It is expressed on the surface of NK-92 cell line, and then cultured in medium without IL-2, and the obtained cloned cell line is the natural killer cell line SILK-NK. 2 . The method according to claim 1 , wherein the protein complex is sequentially composed of the IL-15 binding region, the transmembrane region and the IL-15 protein of IL-15Rα from the N-terminus to the C-terminus. 3 . 3. The method according to claim 1 or 2, wherein the amino acid sequence of the IL-15 binding region of the IL-15Rα is SEQ ID No.1, or the amino acid sequence shown in SEQ ID No.1 The sequence has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology, or has undergone substitution and/or deletion and/or addition of one or several amino acid residues and has a sequence of the same function; and / or The transmembrane region is CD8α transmembrane region; Further, the amino acid sequence of the CD8α transmembrane region is SEQ ID No.2, or the amino acid sequence shown in SEQ ID No.2 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more. % or more or more than 75% homology, or a sequence with the same function after substitution and/or deletion and/or addition of one or several amino acid residues; and / or The amino acid sequence of the IL-15 protein is SEQ ID No.3, or the amino acid sequence shown in SEQ ID No.3 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or More than 75% homology, or sequences with the same function after substitution and/or deletion and/or addition of one or several amino acid residues. 4. The method according to claim 3, wherein the amino acid sequence of the protein complex is SEQ ID No. 4, or the amino acid sequence shown in SEQ ID No. 4 has 99% or more, 95% or more, More than 90%, more than 85%, more than 80% or more than 75% homology, or sequences with the same function after substitution and/or deletion and/or addition of one or several amino acid residues. 5. The method according to any one of claims 1-4, wherein the method comprises the steps of: inserting the encoding gene of the protein complex into a lentiviral expression vector, and then using the obtained recombinant lentivirus The viral vector packaged the lentivirus, infected the NK-92 cell line with the lentivirus, cultured in IL-2-free medium and obtained the cloned cell line by limiting dilution method. 6 . The method according to claim 5 , wherein in the encoding gene of the protein complex, the nucleotide sequence encoding the IL-15 binding region of the IL-15Rα is the first nucleotide sequence of SEQ ID No. 5. 7 . Positions 1-216, or have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more homology with positions 1-216 of SEQ ID No. 5 and encode the the sequence of the IL-15 binding region of IL-15Rα; and/or In the encoding gene of the protein complex, the nucleotide sequence encoding the CD8α transmembrane region is the 217-423 position of SEQ ID No. 5, or the nucleotide sequence with the 217-423 position of SEQ ID No. More than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75% homology and encoding the CD8α transmembrane region; and/or In the encoding gene of the protein complex, the nucleotide sequence encoding the IL-15 protein is the 490-831 position of SEQ ID No. More than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology and the sequence encoding the IL-15 protein; Further, the nucleotide sequence of the encoding gene of the protein complex is SEQ ID No. 5, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more of SEQ ID No. 5 Or a sequence that is more than 75% homologous and encodes the protein complex. 7. The cytokine-independent natural killer cell line SILK-NK prepared by the method of any one of claims 1-6. 8. The natural killer cell line SILK-NK according to claim 7, wherein the STR phenotype of the natural killer cell line SILK-NK is Amelogenin: X, Y CSF1PO: 11,12 D13S317: 9, 12 D16S539: 11,12 D5S818: 12,13 D7S820: 10,11 THO1: 6,9.3 TPOX: 8 vWA: 18 D21S11: 31.2, 32; The expression of IL-15 and IL-15 binding region of IL-15Rα can be detected by mRNA reverse transcription PCR. 9. The natural killer cell line SILK-NK according to claim 7 or 8, characterized in that: the natural killer cell line SILK-NK is deposited in the General Microorganism Center of the China Microorganism Culture Collection and Administration Commission, and the registration number is It is the cell line of CGMCC No. 15583. 10. Use of the natural killer cell line SILK-NK described in claims 7-9 in any of the following: (A1) Preparation of products for adoptive immunotherapy of tumors; (A2) Preparation of related products for cell therapy.

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