WO2019205403A1 - Genetically engineered natural killer cell product for treating tumor - Google Patents

Abstract

Provided is a genetically engineered natural killer cell product for treating tumors. Said cell product is obtained by stably transferring human IL-2 and HSV-tk genes into natural killer cells (NK) by means of a genetic engineering method.

Description

Genetically engineered natural killer cell products for treating tumors [Technical Field]

The invention belongs to the field of biopharmaceutical products, in particular to genetically engineered natural killer cell products for treating tumors.

【Background technique】

Tumor biotherapy is the fourth major treatment after surgery, radiotherapy, and chemotherapy. Since the development of traditional surgery, radiotherapy and chemotherapy has entered the plateau period, people are paying more and more attention to the biological treatment of tumors. Gene and cellular immunotherapy Cancer is a cutting-edge technology for cancer biotherapy. Compared with traditional treatment methods such as chemotherapy and radiotherapy, it has the advantages of relatively small toxic and side effects and remarkable curative effect. Especially in the past few years, the medical community has shown great interest in cellular immunotherapy for hematological malignancies. Cellular immunotherapy and pre-chemotherapy and radiotherapy have non-intersecting clinical effects with few side effects. It has been observed that in various blood patients undergoing bone marrow transplantation, when the disease recurs, receiving allogeneic leukocyte injection can achieve complete remission of the disease [1-4]. If leukocytes are treated with interleukin-2 (IL-2) prior to injection, the killing ability and killing range of donor leukocytes to the patient's tumor cells will be further enhanced. Natural killer cells (NK) are considered to be an important part of the decisive role of donor leukocytes in killing tumor cells in patients, and are an important component of the innate anti-infective and malignant tumor immune system [5]. Unlike T cells, they kill tumor cells without pre-immunization and their killing activity is not limited by the major histocompatibility complex (MHC). The tumor cell killing activity of NK cells is achieved by a variety of mechanisms, of which the perforated esterase pathway is the most commonly used mechanism. The perforated esterase destroys the tumor cell membrane, which in turn induces apoptotic cell death. The killing activation and killing inhibitory receptors expressed by NK cells play a decisive role in the recognition of tumor cells [6-9]. Killer-activated receptors recognize marker molecules on the surface of tumor cells and virus-infected cells, while killer-inhibitory receptors recognize MHC class I molecules. When the killer-activated receptor and the marker molecule are engaged, a "kill" signal is sent to the NK cell, and in the normal cell, the "kill" signal is inhibited by the killing inhibitor receptor and the class I MHC molecule. Repression, thereby avoiding side effects that should not occur. Malignant tumor cells are often defective in MHC class I expression, no longer interact with killer inhibitory receptors, and do not provide an inhibitory signal and, therefore, can be killed by NK cells. Unfortunately, the NK cell function of patients with a wide variety of malignant tumors is severely damaged, unable to identify the target of malignant tumors, and lose the function of growth and killing cells in vitro [10]. In addition, cancer patients' autologous NK cells are also inhibited by "self" expression of HLA, which in turn loses the function of killing malignant tumors. Compared to autologous NK cells, allogeneic NK cells will be a more promising cellular immunotherapeutic product and offer the potential for large-scale production of cellular immunotherapeutic products.

NK-92 (ATCC CRL-2407) is a pure, allogeneic activated NK cell line. NK-92 cells lack the killer cell inhibitory receptor (KIR) and have a broad spectrum of blood and solid tumor cell killing functions. Compared with LAK (lymphokine-activated killer cells) cells, NK-92 cells have a stronger and broader cancer cell killing function. However, NK-92 cells are IL-2-dependent and cause a lot of inconvenience to large-scale production and clinical applications. In addition, clinical use of allogeneic activated NK cells also carries certain safety risks (eg, graft versus host disease). In order to improve the safety of the product, the NK cell immune product needs to undergo a small amount of radiation treatment before being injected into the patient. Unfortunately, while improving safety, micro-radiation treatment also reduces the immunotherapeutic effect of the product. In order to promote the large-scale production of NK cells under standard GMP conditions, to avoid the clinical use of NK cell products, and to inject patients with recombinant IL-2 protein products, and the potential for graft-versus-host disease, it is very There is a need to develop new, safe and effective genetically engineered NK cell products that are not required to be radiotreated.

literature

1. Porter, D.L., et al., Adoptive immunotherapy with donor mononuclear cell infusions to treat relapse of acute leukemia or myelodysplasia after allogeneic bone marrow transplantation. Bone Marrow Transplantation, 1996. 18(5): p.975-80.

2. Porter, D.L., et al., Induction of graft-versus-host disease as immunotherapy for relapsed chronic myeloid leukemia. New England Journal of Medicine, 1994. 330(2): p.100-6.

3. Collins, R.H., Jr., et al., Donor leukocyte infusions in 140 patientss with relapsed malignancy after allogeneic bone marrow transplantation. [see comment]. Journal of Clinical Oncology, 1997. 15(2): p.433-44.

4. Kolb, HJ, et al., Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. [see comment]. Blood, 1995.86(5): p .2041-50.

5.Zeis, M., et al., Allogeneic MHC-mismatched activated natural killer cells administered after bone marrow transplantation provides a strong graft-versusleukemia effect in mice. British Journal of Haematology, 1997. 96(4): p.757-61.

6. Moretta, A., et al., Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes. Immunological Reviews, 1997. 155: p. 105-17.

7. Lanier, L.L., NK cell receptors. Annual Review of Immunology, 1998. 16: p. 359-93.

8. Smyth, M.J., et al., New aspects of natural-killer-cell surveillance and therapy of cancer. Nature Reviews. Cancer, 2002.2(11): p.850-61.

9. Chiorean, E.G. and J.S. Miller, The biology of natural killer cells and implications for therapy of human disease. Journal of Hematotherapy & Stem Cell Research, 2001. 10(4): p.451-63.

10. Whiteside, T.L., N.L. Vujanovic, and R.B. Herberman, Natural killer cells and tumor therapy. Current Topics in Microbiology & Immunology, 1998. 230: p. 221-44.

[Summary of the Invention]

The object of the present invention is to develop a novel genetically engineered natural killer cell product for treating tumors for clinical treatment of tumors.

The present invention is achieved by the following technical means, wherein the genetically engineered natural killer cell (NK) product is capable of stably expressing human interleukins and cell suicide factors by genetic modification by natural killer cells.

Further, the human interleukin factor can be human IL-2 or IL-15.

Further, the cell suicide factor may be an HSV-tk or a cytosine deaminase gene.

Further, the natural killer cell can be any of the human natural killer cell lines.

Further, the natural killer cells are modified with a gene stabilized by the T2A fusion of the IL-2 gene and the HSV-tk gene.

Further, genetically engineered natural killer cells are used to prepare gene cell drug products for treating various malignant tumors.

Further, it is produced under large-scale serum-free suspension culture technology under GMP conditions.

Further, it can be used for intravenous injection, intraarterial injection, intratumoral injection, subcutaneous injection, organ injection, intrathoracic injection or intra-abdominal injection.

A beneficial effect of the technical solution of the present invention is that the present invention discloses a method of constructing a genetically engineered natural killer cell product for treating a tumor. The genetically engineered natural killer cells, NK-SBN cells, have good anti-cancer function and safety and can be used for clinical cancer treatment.

[Description of the Drawings]

Figure 1 shows the expression of IL-2 in genetically engineered NK92 cloned cells.

Figure 2 shows the expression of TK in genetically engineered NK92 cloned cells.

Figure 3 shows the results of TK sensitivity detection of genetically engineered NK92 cloned cells.

Figure 4 shows the anti-cancer effect of NK-SBN cells in vitro

Figure 5 shows the anticancer effect of NK-SBN cells in animals.

Figure 6 shows the co-culture of NK-SBN cells and healthy peripheral blood mononuclear cells (PBMC).

【detailed description】

The technical solution of the present invention will be further specifically described below with reference to the accompanying drawings and embodiments.

1. Construction and expression of IL-2-T2A-HSV-tk vector gene engineering plasmid vector

A cloned expression plasmid for the synthesis of the IL-2 cDNA and HSV-tk cDNA gene fragments was ordered from Life Technologies, USA. The expression plasmid was lyophilized. The IL-2 expression plasmid vector was constructed by cloning the synthesized IL-2 cDNA into the pcDNA3.1/Neo (Invitrogen V79020) vector. The synthetic HSV-tk cDNA gene fragment was cloned into the pcDNA3.1/Hygro (Invitrogen V870-20) vector to construct a suicide gene plasmid vector. The IL-2 cDNA and HSV-tk cDNA gene fragments were sequenced. For the IL-2 cDNA gene sequence, please refer to the gene sequence table 1. For the HSV-tk cDNA gene sequence, please refer to the gene sequence table 2. The sequencing results confirmed that the synthesized IL-2 and HSV-tk gene sequences and the theoretical reference sequence were 100% identical.

The synthetic IL-2 cDNA pcDNA3.1(+) vector and the HSV-tk cDNA pcDNA3.1(+) vector were purchased from Life Technologies and transformed into Stbl3 E.coli (Life Technologies, C7373-03), respectively, using LB. The culture medium was used to culture the genetically engineered plasmid-transformed Stbl3 E. coli overnight in a 37 ° C shaker to amplify the DNA of the genetically engineered plasmid vector. The amplified IL-2 and HSV-tk genetically engineered plasmid vectors were purified the following day using a Qiagen plasmid purification column (QIAprep Spin Miniprep Kit, 27104). The HSV-tk gene plasmid and the pcDNA3.1(+)-IL-2 vector were simultaneously cleaved with EcoRI (New England Biolabs, R0101S) and NotI (New England Biolabs, R0189S) specific endonuclease, and reacted in a 37 ° C water bath. One hour, the HSV-tk gene was cloned into the pcDNA3.1(+)-IL-2 vector to construct a pcDNA3.1(+)-IL-2-HSV-tk genetically engineered plasmid vector. The constructed pcDNA3.1(+)-IL-2-HSV-tk genetically engineered plasmid was transformed into Stbl3 E.coli (Life Technologies, C7373-03), and the gene was cultured overnight in a 375 medium shaker using LB medium. The plasmid-transformed Stbl3 E. coli was used to amplify the DNA of the genetically engineered plasmid vector. The amplified pcDNA3.1(+)-IL-2-HSV-tk genetically engineered plasmid vector was purified the next day using a Qiagen plasmid purification column (QIAprep Spin Miniprep Kit, 27104).

A T2A double-stranded DNA gene fragment from Thomasaasigna virus was synthesized by PCR amplification. For the T2A DNA gene sequence, please refer to the gene sequence table 3. During the PCR amplification, a 14 bp sequence overlapping the ppn3.1(+)-IL-2-HSV-tk plasmid with Kpn1 specific endonuclease was added to both ends of the T2A DNA sequence. For the gene sequence, please refer to Table 4 and Table 5 of the gene sequence. The synthetic T2A double-stranded DNA gene fragment was denatured and renatured, and the synthetic T2A double-stranded DNA fragment and pcDNA3.1(+)-IL-2-HSV-tk plasmid DNA were simultaneously cleaved with Kpn1-specific endonuclease, and In -Fusion HD cloning method (Clontech, 638909) cloned the T2A sequence into pcDNA3.1(+)-IL-2-HSV-tk vector to construct pcDNA3.1(+)-IL-2-T2A-HSV-tk gene Engineering plasmid vector.

The constructed pcDNA3.1(+)-IL-2-T2A-HSV-tk genetically engineered plasmid was transformed into Stbl3 E.coli (Life Technologies, C7373-03), and incubated in a LB medium at 37 ° C overnight. The genetically engineered plasmid-transformed Stbl3 E. coli was cultured to amplify the DNA of the genetically engineered plasmid vector. The amplified pcDNA3.1(+)-IL-2-T2A-HSV-tk genetically engineered plasmid vector was purified the next day using a Qiagen plasmid purification column (QIAprep Spin Miniprep Kit, 27104). The purified genetically engineered plasmid was stored in a -20 degree refrigerator for use. Sequence analysis was performed on the constructed pcDNA3.1(+)IL-2-T2A-HSV-tk genetically engineered plasmid vector. For the gene sequence (7036 bp), please refer to the gene sequence table 6. The sequencing results confirmed that the constructed pcDNA3.1(+)IL-2-T2A-HSV-tk gene sequence and the theoretical reference sequence were 100% fully consistent.

2. Construction of genetically engineered NK cells

NK cells, such as NK-92 (ATCC CRL-2407) cells, are cultured and expanded using conventional cell culture methods. NK cells were transfected with a plasmid vector that simultaneously expressed the IL-2 and HSV-tk suicide genes. Plasmid vector transfection was performed according to the recommended method of Life Technology's Lipofectamine reagent (#11668-019). After transfection, NK cells stably expressing IL-2 and HSV-tk suicide genes were screened with neomycin-containing culture medium to establish an initial cell population. Cloning and Purification of Cell Populations Finally, genetically engineered NK clonal cells were established and stored in liquid nitrogen for further research and production.

NK-92 (ATCC CRL-2407) cells were cultured in AMEM medium supplemented with 12% bovine serum and 150 U/mL recombinant IL-2. The pcDNA3.1(+)IL-2-T2A-HSV-tk plasmid vector was simultaneously transfected into NK-92 cells using Lipofectamine reagent (Life Technology, #11668-019). After 48 hours of transfection, the culture broth was converted to a neomycin-containing medium to screen for NK-92 cells stably expressing the IL-2 and HSV-tk suicide genes. After 30 days of continuous culture, the viable cells were diluted and transferred to a 96-well plate to continue the culture. Dilute to only one cell in each well.

The growth of the cells was observed under a microscope every day, and the cell culture medium was changed as necessary. After more than three months of culture, the cells were expanded from the wells of a 96-well plate into wells of a 24-well plate, and 400 μl of the culture solution was added to each well to continue the culture and expansion. The culture medium was changed once every 3-5 days. The growth of NK92 cells was relatively slow. After more than 2 months of culture, there was a certain amount of cells in the 24-well plate for amplification into 6-well plates. Add 2 ml of the culture solution to each well to continue the culture amplification. The culture medium was changed once every 3-5 days. After more than one month of culture, the cells were expanded from a 6-well plate to a 5 mL T-25 cell culture flask to allow a sufficient amount of cells to be cultured for analysis and liquid nitrogen refrigeration. A total of 23 T-25 cell culture flasks, each using a T-25 cell culture flask alone. The culture medium was changed once every 3-5 days as before. After more than one month of culture, the concentration of cells in each T-25 cell culture flask reached 1.1-1.5x106/mL. 1 mL of each T-25 cell culture flask was transferred to a new T-25 cell culture flask for further culture for cell detection. The remaining cells were centrifuged at 1000 rpm for 5 minutes, and the resulting cell pellet was suspended in 2 ml of liquid nitrogen refrigerated buffer. The liquid nitrogen cryopreservation buffer was prepared by adding 10% DMSO to the whole growth medium of genetically engineered NK92 cells. 1 mL of the cell suspension was added to a 2 ml cryoline, ie, two cryotubes were cloned per genetically engineered NK92 cell clone. The cryotube containing the cells was placed in an alcohol-filled Mr.Frosty freezer, placed in a -80 C cryostat overnight, and the cells were frozen. The next day, the frozen cells were stored in a liquid nitrogen refrigerated tank for a long period of time. A total of 23 cloned cells of genetically engineered NK92 were cryopreserved.

3. Detection of genetically engineered NK92 cell clones

The structure, copy number and stability of the introduced foreign gene are a very important quality indicator for genetically engineered NK cells. After establishing genetically engineered NK cell clones, conventional molecular biology methods were used to analyze and study the establishment of foreign gene structures in genetically engineered NK cell clones. The genomic DNA of the cells is extracted. PCR primers were designed according to the DNA sequence of the pcDNA3.1(+)-IL-2-T2A-HSV-tk plasmid, and the intracellular copy number of the IL-2 and HSV-tk genes was detected and determined by qPCR. Restriction endonuclease profiling was used to confirm the integrity of the introduced foreign gene. To confirm the stability of the genetically engineered NK cell foreign gene, the cells were cultured and serially passaged for 30 to 40 generations. The molecular biological analysis methods described above were used to detect and determine the integrity of the IL-2 and HSV-tk genes and the number of copies in the cells to confirm the stability of the introduced foreign gene.

The expanded cells were collected from each T-25 cell culture flask. The cell culture was added to a centrifuge tube and centrifuged at 1500 rpm, 4 C for 5-7 minutes. The supernatant was removed and the cells were washed by adding ice-cold PBS buffer to the cells at the bottom of the tube. Centrifuge again at 1500 rpm, 4 C for 5-7 minutes. The supernatant was removed and the cells at the bottom of the tube were added with ice-cold RIPA (radioimmunoprecipitation assay buffer) cell lysate. Stir for 30 minutes at 4C. It was then centrifuged at 16000 g, 4 C for 20 minutes. The supernatant was collected, placed on ice, and the protein concentration was determined by a conventional protein concentration assay. Used for protein expression detection.

·Detection of IL-2 protein expression

The protein is separated by conventional gel electrophoresis. The isolated protein was transferred to a Western blot membrane. IL-2 expression was detected using an anti-human IL-2 polyclonal antibody (Thermo Scientific, Cat #710146). See Figure 1 for the expression level of IL-2 in genetically engineered NK92 cloned cells. Lanes 1-23 in the figure are clone numbers of genetically engineered NK92 cells, lane 24 is a negative control (original NK92 cells), and lane 25 is a positive control (recombinant human IL-2 protein, Life Technologies, Cat# PHC0026, 0.1 μg). All genetically engineered NK92 cloned cells express IL-2 protein at different levels. This is consistent with the phenomenon that cloned cells can grow in IL-2-free medium. Relatively speaking, clones 4, 7, and 11 have higher IL-2 expression levels.

·Expression of HSV-tk protein

The protein is separated by conventional gel electrophoresis. The isolated protein was transferred to a Western blot membrane. The expression of TK was detected using an anti-HSV-TK monoclonal antibody (Thermo Scientific, Cat# MA1-21542). See Figure 2 for the expression level of TK in genetically engineered NK92 cloned cells. Lanes 1-23 in the figure are clone numbers of genetically engineered NK92 cells, lane 24 is a negative control (original NK92 cells), and lane 25 is a positive control (MOLT4 cell lysate, 10 μg, Abcam, Cat#ab7912).

All genetically engineered NK92 cloned cells expressed TK protein at different levels. Relatively speaking, clones 7, 13, and 23 have higher TK expression levels.

·TK sensitivity detection

Based on the expression levels of IL-2 and TK of the genetically engineered NK92 cloned cells, cell clone 7 was selected and further TK sensitivity detection was performed. ATCC NK-92 cells were used as controls. Clone 7 and ATCC NK-92 cells were seeded into corresponding 12-well plates, 1 mL per well, cell concentration 1×10 ⁵ cells/mL, one 12-well plate for cloning 7 cells, and another 12-well plate for ATCC NK -92 cells. 150 units/ml of recombinant interleukin-2 was added to the culture medium of ATCC NK-92 cells. After 24 hours of culture, 0, 5, 10, 50, 75, and 100 μM (final concentration) of ganciclovir (Ganciclovir, GCV, Sigma, Catalog #G2536) were added to the cultured cells, respectively. There are two wells per GCV concentration. Continue to train for 96 hours. Samples were taken and cell viability was determined by trypan blue staining. See Figure 3 for the TK sensitivity test results of genetically engineered NK92 cloned cells.

Clone 7 cells have significant sensitivity to GCV, and most cells die after 96 hours at GCV above 10 μM. The activity of the TK gene into which the gene was transferred was confirmed. In contrast, ATCC NK-92 cells that did not contain the TK gene showed no significant sensitivity to GCV.

· Copy number of IL-2 and HSV-TK genes in cell clones

Genomic DNA was extracted from genetically engineered NK92 clone 7 cells. The IL-2 and HSV-TK suicide gene copy numbers were detected and determined by quantitative PCR. The RNase P gene was used as a standard for quantitative PCR gene copy number calculation.

For the IL-2 gene quantitative PCR detection method, please refer to the gene sequence table 7 for the left primer sequence, the gene sequence table for the right primer sequence, and the gene sequence table 9 for the probe sequence.

For the quantitative PCR detection of TK gene, please refer to the sequence of the left primer for the sequence of the gene 10, the sequence of the right primer is shown in Table 11 of the gene sequence, and the sequence of the probe is shown in Table 12 of the gene sequence.

PCR cycle method

Denaturation/activation: 95 ° C, 5 minutes, once

Denaturation: 95 ° C, 15 seconds

Annealing / extension: 60 ° C, 1 minute

Cycle 40 times

Table 1 Copy number of IL-2 and HSV-TK genes in cell clones

clone	Copy number of IL-2 gene in cells	Copy number of HSV-TK gene in cells
7	1.3	1.2

There is approximately 1 copy of the IL-2 and HSV-TK genes in each of the genetically engineered NK92 clone 7 cells. The cloned cells were named NK-SBN cells.

4, NK-SBN anti-cancer effect in vitro

NK-SBN cells and different tumor cells, such as HL, are mixed and cultured under different ratios of NK-SBN cells and tumor cells, such as 50:1, 20:1, 10:1, 5:1 and 1:1. -60, K562 and Jurkat cells, the lytic activity of NK-SBN cells against tumor cells was determined by Europium cytotoxicity assay (DELFIA, PerkinElmer) to confirm the anti-tumor cell effect of NK-SBN cells. Peripheral blood mononuclear cells (PBMC) of healthy individuals activated by IL-2 were used as controls in the test, cancer cells were treated with detergent (100% cancer cell lysis), and Europium cytotoxicity assay was calibrated.

Tumor cells (HL60, Raji, K562 and Jurkat cells) were cultured in RPMI medium. The cells were centrifuged and the cells were washed with PBS. The cells were labeled with DELFIA BATDA reagent and incubated at 37 ° C for 30 minutes. The labeled cells were washed with PBS and diluted to 1 x 10 ⁵ /mL with RPMI medium. Place 5000 cells in the wells of a 96-well plate (50 microliters). The NK-SBN cells in the logarithmic growth phase were taken, the cells were centrifuged, and the cells were washed with PBS. The washed cells were suspended in a culture medium at a concentration of 1E6/ml. Different ratios of NK-SBN cells (eg, 50:1, 20:1, 10:1, 5:1, and 1:1) were added to 96-well plates containing tumor cells. The total volume is 100 microliters/well. The 96-well plate was centrifuged at 200 g for 4 minutes to ensure sufficient contact between the NK-SBN cells and the tumor cells. The 96-well plate was incubated for 2 hours in a 37 ° C / 5% CO 2 cell incubator. Mix the liquid in each well 5-10 times with a multichannel pipette. The 96-well plate was centrifuged at 500 g for 5 minutes. Twenty microliters were removed from each well and placed in a new 96-well plate for fluorescence detection. Add 200 microliters of DELFIA Europium test solution to each well. The 96-well plate was wrapped in an aluminum film and placed on an orbital shaker for 15 minutes. 100 microliters of Europium standard solution was added to the wells of the two wells in a 96-well plate. Fluorescence intensity was measured using a fluorescent microplate reader (Synergy 2, Biotek) at different time intervals. The measurement setting conditions are,

Excitation wavelength: 330nm

Emission wavelength: 615nm

Time lag: 400μs

Measuring gap: 1000μs

Measuring height: 365 mm

NK-SBN cell cancer cell killing rate (%) = NK-SBN cell-treated cancer cell fluorescence intensity / detergent-treated cancer cell fluorescence intensity.

See Figure 4 for the in vitro anti-cancer effect of NK-SBN cells. NK-SBN genetically engineered NK cells have significant killing power against a variety of tumor cells. In contrast, normal human PBMC showed no killing power against cancer cells.

5. Anti-cancer effects of NK-SBN in animals

On the basis of verifying the results of in vitro anti-tumor cells, the anti-tumor effect of genetically engineered NK cells in animals was further verified. Since genetically engineered NK cells are human cells, animals in vivo are tested with severe combined immunodeficiency (SCID) mice. First, by intraperitoneal severe combined immunodeficiency (SCID) mice into 5x10 ⁶ human leukemia cell, TA27, after five days, and then by intraperitoneal injection of 2x10 ⁷ NK-SBN cells, injected once every other day, 5 times in total. The control mice were injected with saline. The health status, tumor stage and survival time of the mice were observed. Genetically engineered NK cell therapy should exhibit significant anti-tumor effects and prolong survival in mice.

Severe combined immunodeficiency (SCID) mice were used to perform anti-cancer effects in vivo in NK-SBN cells. First, the cultured human leukemia cells (cell line TA27) were inoculated into the mouse, and TA27 was cultured with RPMI + 10% fetal bovine serum. Cells in logarithmic growth phase were collected for animal experiments. Four days later, they received NK-SBN cell therapy. A total of 30 mice were shared. Divided into 3 groups of 10 mice each.

Group 1: injected 5x10 ⁶ TA27 cells within the first day of each mouse peritoneal. TA27 cells were suspended in PBS buffer prior to injection. This group of mice did not receive NK-SBN cell therapy (negative control group), received saline injection, and injected every other day for a total of 5 times. The health and weight of the mice were observed daily.

Group 2: injections 5x10 ⁶ TA27 cells within the first day of each mouse peritoneal. TA27 cells were suspended in PBS buffer prior to injection. On the fifth day, injections 2x10 ⁷ NK-SBN cells per mouse intraperitoneally. Inject once every other day for a total of 5 times. The health and weight of the mice were observed daily.

Group 3: This group of mice did not receive injections of human leukemia cells.

On the fifth day, mice were injected with 2x10 ⁷ cells each (positive control group) NK-SBN intraperitoneally. The mice were observed every other day for a total of 5 times, and the health and weight of the mice were observed every day. Figure 5 shows the survival rate of mice relative to the observation time (days). NK-SBN cells showed significant anti-tumor effects in animals, effectively prolonging the survival of mice.

6, NK-SBN cell safety

Peripheral blood mononuclear cells (PBMC) of NK-SBN cells and activated healthy individuals were cultured in a 1:1 co-mix for 4 days, and then PBMC cells were seeded in methylcellulose culture medium, and the number of red blood cell colonies was counted ( BFU-E), granulocyte and macrophage colony number (CFU-GEMM) and other cell colony counts (CFU-C), using peripheral blood mononuclear cells (PBMC) not co-cultured with NK-SBN cells as a control . Peripheral blood mononuclear cells from three healthy individuals were used in the experiment. See Figure 6 for the co-culture results of NK-SBN cells and healthy peripheral blood mononuclear cells (PBMC). The results showed that NK-SBN cells had no adverse effects on the growth of healthy peripheral blood mononuclear cells (PBMC).

[Industrial Applicability]

The present invention discloses a genetically engineered natural killer cell product for treating tumors, which is constructed by genetically engineering a human IL-2 and HSV-tk gene stably into natural killer cells (NK). The cell product is an allogeneic cell product with good stability, anti-tumor effect and safety. Large-scale production can be carried out without adding IL-2. Using the genetically engineered natural killer cell (NK) product of the present invention, a clinical grade gene cell therapeutic article can be prepared for treating and preventing various malignant tumors in humans. Methods of constructing genetically engineered natural killer cell products for treating tumors are also disclosed. The genetically engineered natural killer cells, NK-SBN cells, have good anti-cancer function and safety, and can be used for clinical cancer treatment, and have industrial applicability.

Claims (8)

1. A genetically engineered natural killer cell product for treating tumors, characterized in that the genetically engineered natural killer cell product is genetically modified to enable natural killer cells to stably express human interleukins and cell suicide factors.
2. The genetically engineered natural killer cell product for treating a tumor according to claim 1, wherein the human interleukin factor is human IL-2 or human IL-15.
3. The genetically engineered natural killer cell product for treating a tumor according to claim 1, wherein the cell suicide factor is HSV-tk or cytosine deaminase.
4. The genetically engineered natural killer cell product for treating a tumor according to claim 1, wherein the natural killer cell is any one of a human natural killer cell line.
5. The genetically engineered natural killer cell product for treating a tumor according to claim 1, wherein the natural killer cell is modified with a gene stabilized by the T2A fusion of the IL-2 gene and the HSV-tk gene.
6. The genetically engineered natural killer cell product for treating a tumor according to claim 1, which is useful for preparing a gene cell drug product for treating various malignant tumors.
7. The gene cell drug product according to claim 6, which is produced under a GMP condition using a large-scale serum-free suspension culture technique.
8. The genetically engineered natural killer cell product for treating a tumor according to claim 1, which can be used for intravenous injection, intraarterial injection, intratumoral injection, subcutaneous injection, organ injection, intrathoracic injection or intra-abdominal injection.

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