Promoting sustainable proliferation of NK cells by up-regulating IL2 and IL12 through CRISPR-CAS9Promoting sustainable proliferation of NK cells by up-regulating IL2 and IL12 through CRISPR-CAS9

NK cells are an important part of human immunity and are often used in clinical antitumor adoptive cell immunotherapy.However, the small number and low activity of NK cells in the tumor microenvironment limit its clinical ecacy.Studies have found that cytokines IL2 and IL12 play key roles in the proliferation of NK cells and the improvement of the activity of killing tumor cells, the toxic and side effects, however, are relatively large when the systemic application reaches the effective concentration. To study the effect of autocrine secretion of IL2 and IL12 on proliferation and anti-tumor activity of NK92 cell lines by editing NK cells' genes, and to construct NK92 cells with sustainable proliferation. Lentivirus was transfected into NK92 cells through the synergistic effect of crispr/cas9 localization gene editing technology and VP64 transcriptional activation domain to construct NK92-IL2-IL12 cell lines. The expression levels of IL2 and IL12 in the cell lines were detected by qPCR and ELISA.NK92 cell lines were inoculated into mouse tumor and the proliferation and anti-tumor effect of NK cells in tumor were observed. The vector plasmids IL2 and IL12 were successfully transferred into NK92 cells, which activated the up-regulated expression of IL2/IL12 in NK92, signicantly increased the activity and lethality of NK92 cells, and sustained the proliferation of NK92 cells in mouse tumor.IL2 and IL12 can signicantly promote the proliferation and killing activity of NK92 cell lines through autocrine, which have stronger anti-tumor activity than the wild-type ones, with continuous proliferation in the tumor. two cytokines IL2 and IL12, which play key roles in cell proliferation and killing activity, were up-regulated in NK92 cells. They act on NK92 cells through cell autocrine or paracrine, enhance the cytotoxicity and activity of NK92 cells, and achieve self-sustainable proliferation and activity enhancement. In this study, the lentiviral expression plasmid of gRNA was obtained by lentivirus packaging infection, and conrmed by sequencing. After infection and integration, the resistance screening of puromycin was observed by uorescence microscope to obtain cells with a purity of more than 80%. The qPCR and ELISA detection of NK92 cells in each group proved that cytokines IL2 and IL12 were successfully overexpressed in NK92 cells and secreted to extracellular. It is proved that the target cell line of NK92-IL2-IL12 was successfully constructed in this study, which can maintain the sustained and stable overexpression and release of IL2 and IL12. Here, we used CCK-8 method to detect that the proliferation function of this cell increased by 1.75 times. Through the detection of in vivo imaging system and tumor immunohistochemistry in mice for 4 weeks, it was conrmed that the NK92-IL2-IL12 cell line continued to proliferate and enhance its activity in the tumor-bearing mice, so that the general condition of the experimental mice was improved and the survival time was prolonged. gene editing NK92 consistently and IL12 while IL2 and IL12 cytokines on peripheral cells and autologous cells through autocrine the and anti-tumor effect of NK92 cells. In this study, we explored the function of NK92 cells with overexpression of two kinds of cytokines, and found that NK92 cells had stronger anti-tumor activity on the basis of their proliferation ability enhanced greatly. cells and signicantly


Introduction
NK cells are an important part of human immunity and have strong innate immunity. Studies have found that the number of NK cells that re ect the immune function of the body is closely related to the prognosis of patients, but with the progression of tumor, the number of NK cells in patients often decreases signi cantly [1]. NK cells can recognize and kill malignant cells without pre-sensitization [2,3]; adoptive transferred NK cells seem to have the lowest targeted toxicity spectrum [4], avoiding dangerous cytokine recruitment, which usually occurs in many T cell-based immunotherapy [4]. No obvious side effects were found in the clinical application of allogeneic NK cells in non-patients. Therefore, it is often used in clinical anti-tumor adoptive cellular immunotherapy, and has become an effective means to enhance the traditional treatment of cancer [5]. For example, it has been successfully used in the treatment of hematological diseases such as leukemia [6][7][8]. And the treatment of solid tumors such as melanoma and [2]metastatic neuroblastoma [9,10]. However, the therapeutic effect on other solid tumors such as colorectal cancer is less impressive [3]. The possible reason is that the small number and low activity of NK cells in the tumor microenvironment restrict its clinical curative effect, and the related research focuses on promoting its in vivo survival, killing activity and the e ciency of migration to the tumor site [3,4,1,6]. These strategies are important advances in the emerging eld of clinical NK cellular immunotherapy [4,11]. Among them, the most important thing is the sustained steady-state proliferation and enhanced activity in the tumor, which is rarely reported at present. Therefore, the sustainable expansion and enhanced activity of NK cells in tumor are highly attractive as a means to inhibit or eliminate the growth of unconstrained tumor cells [12]. A great deal of evidence shows that how to improve the killing activity of NK cells in tumor microenvironment and the stable and sustainable proliferation of NK cells in tumor is the key link that restricts the curative effect.
IL12 is a cytokine with direct inhibitory effect on tumor, which is composed of two subunits of p35 and p40. At the same time, it can make NK cells proliferate continuously and be in a state of cellular immune response that can effectively play an anti-tumor effect [13]. The proliferation of NK cells also depends on IL-2 stimulators. Studies have found that cytokines IL2 and IL12 play a key role in the proliferation of NK cells and improve the activity of killing tumor cells, but the toxicity and side effects become more serious and intolerable when these cytokines are used in the whole body at an effective concentration. Therefore, NK cells are usually isolated and cultured in vitro and then infused in vivo to treat tumors. However, recent studies have shown that NK cells activated by cytokines and expanded in vitro lack or inadequately express homing receptors; such adoptive natural NK cells rarely enter and expand in tumors.
The CRISPR/Cas9 system is a genome editing technology that has been widely used in humans [20][21], mice [22], zebra sh [23][24] and other different species using RNA-guided nuclease to modify genomic DNA. , can achieve targeted and precise genetic modi cation in any animal. It is widely used in gene knockout, gene insertion, gene site-directed mutation, gene function inhibition, animal breed breeding and human disease animal model creation, etc., providing a new direction for gene therapy.In 2012, Jinek et al [12] transformed the CRISPR/Cas9 system, which contains two non-coding RNA crRNA and tracrRNA, into a single-directed RNA (Single-guide RNA, sgRNA), which can guide Cas9 proteins to carry out targeted double-strand cleavage of speci c DNA sequences (directly to the upstream 3 bp of DNA double-strand containing PAM (Protospacer adjacent motif) NGG); to form a (Double strand breaks,DSBs with a blunt end DNA double-strand break at a speci c location). It was found that the localization function of the CRISPR/Cas9 system combined with the fusion VP16 tetramer activator VP64 could directly up-regulate the transcriptional level of endogenous genes [14]. In this study, through the localization function of CRISPR/Cas9 in the genome, the dCas9-VP64 fusion protein was constructed, and the expression of the target gene was up-regulated under the guidance of sgRNA localization [15,16]. In this study, we used this gene editing technique to make NK92 cell line constantly up-regulate the overexpression of cytokines IL2 and IL12, on NK92 cells by autocrine or paracrine, which could not only improve the cytotoxic activity of NK92 cells, but also promote its continuous The NK-92 cell lines used in this study were donated by Professor Park Zhenghao of Hangzhou normal University (purchased from ATCC); 293T and K562 cell lines and preserved in the laboratory; Ecoli-DH5 α competent cells were purchased by Shanghai Biological Engineering Co., Ltd.; lentivirus packaging plasmids ps PAX2 and PMD2.G laboratory provided. The core plasmids dcas9-vp64 (LW415) and grnaIL2/IL12 (LW593) were synthesized by Beijing Hesheng Genetics Company.

Design of gRNA primers
The 5KB DNA fragments upstream of transcriptional initiation site of IL-2 target gene (GenBank number: NM_000586), IL-12A target gene (GenBank number: NM_000882) and IL-12B target gene (GenBank number: NM_002187) were obtained from the database of (NCBI) of the National Information Technology Center of the United States. SgRNA, selected the activated gRNA sequence with good e cacy and speci city from the website (http://crisprera.Stanford.edu/). The miss effect is predicted through the website, and the sequences of gRNA:IL2 gRNA, IL12A gRNA and IL12B gRNA with the lowest miss effect are shown in Table 1, respectively. The AA base only plays a terminating role through two A base intervals, and its gene sequence is: GGATCTGGCGCCACCAACTTCTCTCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCAGGCCCA;. Beijing Hesheng Gene Company was entrusted to synthesize IL2-IL12A-IL12B gRNA;PCR and amplify double-stranded DNA, to form double-stranded DNA,. The PUC57vetcor, was digested by PacI,AgeI restriction enzyme and then combined with linear skeleton PUC57vetcor by T4 DNA ligase to construct lentiviral vector (pLV-U6-gRNA1-U6-gRNA2-U6-gRNA3-CMV-EGFP). Among them, pLV is lentiviral plasmid, U6 is U6 promoter sequence, gRNA1, gRNA2, gRNA3 represent IL2 gRNA, IL12A gRNA, IL12B gRNA,CMV represent CMV promoter sequence, EGFP represents green uorescent protein sequence. The recombinant vector was used to transform DH5a competent cells, and the clones were selected, and the plasmids were extracted and sequenced. High purity extracted plasmid, SSA report vector (skeleton PUC57 1) Design gRNA primers (Table 1). On the basis of previous experiments, refer to the website http://crispr-era.Stanford. Edu/, selected gRNA with better speci city and e cacy.
2) Entrusting Syngenics Company to synthesize viral vector plasmids dcas9-vp64 (LW415) and gRNA IL2 / IL12 (LW593)according to gRNA synthesis. The generation of lentiviral vectors is schematically presented in Fig 1a- 3) The two plasmids were synthesized and transferred to DH5-α competent cells, then centrifuged at 37 ℃ for 3 hours, 100ul coated plate was taken, incubator was incubated overnight at 37 ℃, larger strains were selected, and the larger strains were ampli ed by 150Met 200rpm / min at 37 ℃.

4)
Plasmid extraction kit, DNA concentration was measured and stored at-20 °.

5)
Identi cation of positive clones: three or four single bacteria were selected and the plasmid DNA was extracted and sequenced. The primer for sequencing was CAGGAAGAGGGCCTATTTCCC.
6) The identi ed plasmid is used as the target plasmid for subsequent transfection.

lentivirus plasmid packaging and virus concentration.
293T cells were cultured in 6-well plate (DMEM medium, 10% FBSpl 1% penicillin streptomycin), the cell density reached 70% 80%, and the adhesion was uniform, and the growth state was good; Lipofectamine 3000 transfection reagent, according to A solution: lipo3000 3.75ul, α-MEM medium (serum-free) 121.25ul liquid B: α-MEM medium, psPAX2 1.5ug, PMD2.G 0.5ug, LW415 2ug, LW593 2ug P3000 12ul (2ul/ug) ratio con guration mixture; After 5 minutes, liquid B was added to liquid A, and after 20 minutes, 293T cell culture medium was added evenly, and the uorescence effect was observed under uorescence microscope after 48 hours and 96 hours. 293T cells and their culture medium were collected and centrifuged at 4 ℃ 3000g for 20 minutes. The supernatant containing lentivirus was taken. The virus supernatant was collected and ltered by 0.45um, and 5x PEG8000 mother liquid after 7.5ml sterilization was added to each 30ml, and the virus solution was mixed with each 30min for 5 times, standing overnight at 4 ℃, centrifuging for 20 minutes at 4 ℃, and dissolving the virus solution with a certain amount of PBS. The virus suspension was packed into 50 ul each and stored at-80 ℃.
The day before detection, 293T cells were passaged, 1 × 104 cells were added to each well in 96-well plate and cultured at 37 ℃ for 24 hours. Before transduction, the cells digested in one hole were counted and the cell quantity was obtained. the supernatant of 100ul virus was mixed in the medium of 100ul (DMEM+10%FBS). After mixing, it was labeled as 10murl, and then diluted with 10 times gradient until diluted to 10mur8. (6 wells in each group), take 1 ul virus concentrate and 200ul medium (DMEM+10%FBS), after mixing, mark it as 10 Mel 1, then dilute it with 10 times gradient until diluted to 10 mi 8. (6 holes in each group), select the desired cell pores, absorb the culture medium from the culture pores, add the gradient diluted virus solution 190 ul, and gently mix it, and then culture it in the 5%CO2 incubator at 37 ℃. After 24 hours, the number of uorescent cells in each well was observed under uorescence microscope and counted (the culture medium was absorbed and washed twice by PBS before taking pictures were taken and changed to complete medium 5ml. The cell infection e ciency was observed under uorescence microscope after 48h and 72h. In order to further improve the transfection e ciency, 1RV 1000 was added to 5ul polybrene to improve the infection e ciency, and the infection could be repeated at intervals of two days. After repeated screening with 5ul puromycin (concentration of 2mg/ml) added to the culture medium, the cells without infection were killed and the positive cells proliferated stably for 24 hours each time. The results showed that the green uorescence expression level of 293T cells was higher after lentivirus transfection, and the transfection rate was more than 99%. After 48 hours of infection, the green uorescence expression level of NK92 cells was found, and the infection rate was about 20%. After repeated infection and repeated screening with puromycin, the positive rate was about 80-90%( Figure 1C).
NK92 cells in logarithmic phase lentivirus transfection group and wild type group (α-MEM culture medium, 12.5% FBScr 12.5% horse serum, 1% penicillin streptomycin, 100U/ml IL2 (1000pg/ml)), count 10 ^ 7, added to T25 culture bottle, xed volume to 5ml, were placed at 37 ℃ and 5%CO2 incubator for 48 hours, then centrifuged to take culture medium, IL2 and IL12 protein expression was detected according to ELISA kit protocol, 3 pairs of holes were set in each group. They were divided into blank medium group, factor modi cation group and wild type NK92 group. The cells of factor modi ed group and wild type NK92 group were extracted from RNA,Takara kit by Trizol method and reverse transcribed into CDNA,qPCR for quantitative detection of IL2 and IL12 gene expression. Factor modi ed group and wild type NK92 cell group were set up. Three groups of parallel holes were set in each group and repeated three times. QPCR results were used to observe the size of ampli ed fragments by gel electrophoresis and compare the size of genes. determine the OD value of the 450nm wavelength of each well with an enzyme meter. The calculation method of cytotoxic activity: the average value of three parallel holes was calculated, and the cytotoxic activity of NK was calculated according to the following method, which was expressed by killing rate (%). Cytotoxic activity = [1-(effector cell acting pore OD value-effector cell pore OD value) / target cell pore OD value × 100%.

GAPDH-PF GAAGGTCGGTGTGAACGGAT
Ltd.). ) they were randomly divided into three groups: PBS group, experimental group NK92 group and control group NK92 cell group, with 8 rats in each group, and were fed in the Animal Experimental Center of Hangzhou normal University with standardized pathogen-free feeding conditions; all the research procedures involved in animals met the animal ethical standards of this research institution. 200 μ l cells containing 2 × 10 6 HT-29 cells labeled with red uorescent protein were injected subcutaneously on the right side of each group to form tumor. These labeled HT29 cells expressed red uorescent protein. As mentioned earlier, the model of SCID mice bearing colon tumor was established and fed routinely. The tumor size was measured twice a week. The tumor volume was calculated according to the formula V = 0.52xL × W2). "L" represents the longest diameter of the tumor, and "W" is the transverse diameter perpendicular to the longest diameter. After the tumor volume reached about 8mm3, the experimental group was injected with 4 × 10 6 NK92-IL-2-IL-12 cells, while the control group was injected with 4 × 10 6 wild NK92 cells, both of which were washed with PBS before injection. The blank control group was only injected with 100 μ l PbS;. NK92 cells were injected into the tumor of mice every 48 hours in 2 weeks. Within 8 hours after each injection, the uorescence distribution of NK92 cells in the tumor of the two groups was observed by using the in vivo imaging system (IVIS) (Xenogen,Caliper Life Sciences,Hopkinton,MA). The carestream MI software (Shanghai) was used to draw a constant target area on the tumor area, the uorescence signal intensity was measured, and the experiment of unit total light intensity / cm2 / sr (p / s / cm2 / sr), was repeated twice. Four weeks later, three mice in each group were killed, and the tumor tissue of one meter in the center of the tumor was taken. Rabbit anti-human CD56 antibody and sheep anti-rabbit IgG antibody (cy3 red uorescence) labeled NK92 cells, and DAPI staining nucleus (blue uorescence) was detected by immuno uorescence. The number of NK cells was compared in 5 visual elds under 400x magni cation. The rest of the tumor-bearing mice died of natural disease, and the survival time was recorded.

Statistical analyses
The data was showed as mean ± SD ( x ± s). One-way and two-way analysis of variance analyses were applied to compare the sample means of test groups and control groups. The results of the CCK8l-assay and results of the in-vivo migration were analyzed using Student's t-test. A log-rank test was used for analyses of the survival data. P< 0.05 was considered statistically signi cant. All statistical analysis was carried out with Prism software version 7.0 (GraphPad Software Inc., San Diego, USA).

lentiviral vector plasmids of IL2 and IL12 were successfully constructed.
Plasmid vector sequencing: plasmid DNA was extracted and sequenced. The sequencing primer is: CAGGAAGAGGGCCTATTTCCC. The partial sequencing results of the skeleton of PUC57, are as follows, and the sequences in the three boxes are the gRNA target sequences of IL2, IL12A and IL12B in turn. The alignment results show that the lentiviral vector plasmids of IL2 and IL12 are constructed successfully.

ACC
The green uorescence expression level of 293T cells was higher after lentivirus transfection, and the transfection rate was more than 99% ( Figure 1c). 2) the transcriptional expression level of IL2/IL12 mRNA in NK92 cells detected by IL2/IL12 mRNA uorescence quantitative PCR was signi cantly higher than that in wild-type NK92 cells after 48 hours of lentivirus transfection (Figure 2b), and the fragment size was con rmed to be the target gene by gel electrophoresis ( Figure 2c).
2.3The expression of IL2/IL12 protein in NK92 was detected by ElISA. The expression of IL2/IL12 protein in NK92 cells of modi ed group was signi cantly higher than that of wild type NK92 (Fig. 2d).  Fig 2c.The size of qPCR products is about 200bp by gel electrophoresis experiment , which is consistent with the size of PCR products designed by primers Fig.2d The left gure The NK cells in the experimental group had a higher expression of IL2 than the control NK cells and the complete medium measured by ELISA. The data was measured repeatedly for three times and the average value was obtained. Results are expressed as means of triple experiments per treatment.
The right gure:The NK cells in the experimental group had a higher expression of IL 12 than the control NK cells and the complete medium measured by ELISA. Wild type NK92 control group and complte medium group could not detect the content of IL-12 protein.Results are expressed as means of triple experiments per treatment.

Killing effect of NK92 cells on tumor target cells.
In the experimental group, the killing activity of NK92 cells to K562 tumor target cells was signi cantly stronger than that of ordinary NK92 cells. the killing activity of NK cells to K562 cells in group A was higher than that in group B, and the killing activity of NK92 cells against K562 cells increased gradually with the effector-target ratio from 1.25 to 10 (P < 0. 05). The result is shown in Figure 3a.
2.5 Comparison of proliferative activity of NK92. The result is shown in Figure3b.   Fig3a. Comparison of the killing activity of NK92 cells against K562 cells showed that the killing activity of the IL2/IL12 modi ed group was signi cantly higher than that of the wild group, and was proportional to the effective target ratio *p 0.05 .Results are expressed as means of triple experiments per treatment. Fig.3b Comparison of proliferative activity of NK92. Compared with the control group, the test group has stronger NK cell proliferation activity.It gradually increased with incubation time, most ampli ed at 48 hours, about 1.75 times that of the control group. (The test group is the NK cell group overexpressing IL-2-IL-12, and the control is the wild type NK cell group.

2.6
In the experimental group, NK92 continued to proliferate and its activity increased in tumor-bearing mice.
2.6.1 After 4 weeks, the expression of green uorescence in tumor was signi cantly higher than that at 2 weeks (at the end of injection of NK92-IL2-IL12 cells) (Figure 4), indicating that NK92 cells (NK92-IL2-IL12) with overexpression of IL-2 and IL-12 edited by adoptive transfer genes in ltrated and proliferated in the tumor, and SCID mice were injected with HT29 cells labeled with HT29. There is a red uorescent protein on the right wing. When the tumor grew to about 0.8 cm, NK92-IL-2-IL-12 cells carrying green uorescent protein were injected into the tumor. The uorescence imaging system of mice showed that the in ltration of NK cells in the tumor of SCID mice was signi cantly increased (Fig. 4a). The uorescence intensity of NK in tumor-bearing mice in the experimental group was also signi cantly higher than that in the control group (Fig. 4b). According to the oval volume of the tumor (vault 0.52 × L × W2), the size of the tumor in the NK92-IL-2-IL-12 cell group was signi cantly smaller than that in the control group (Fig.4c). Combined with the results of gure 6 (increased cytotoxicity of NK92 cells in the experimental group), it could be seen that the killing activity of NK92-IL-2-IL-12 cells in the tumor was signi cantly stronger than that in the control group, and the survival time of the experimental group was signi cantly prolonged (Fig.4d). It can be seen that the increase in the number and activity of NK92 cells reduced the tumor load and prolonged the survival time of tumor-bearing mice. Color represents the intensity of the GFP protein carried by NK92 cells, red indicates the strongest.Compared with the control group, the test group had more NK cells in ltrated into the tumor.In addition, the number of NK cells in ltrating in the tumor of the test group was signi cantly more than four weeks after injection than two weeks, indicating that the experimental group had stronger NK cell proliferation activity in vivo.  2.6.2 the red uorescence of rabbit anti-human CD56 and sheep anti-rabbit cy3 labeled NK92 cells in lentivirus transfection group was more obvious than that in wild type tumor, and continued to express green uorescence ( Figure 5). while the expression of MHC class I molecules on the surface of tumor cells decreased or lost, and NK cells showed killing effect due to the dominant effect of KAR. Therefore, NK cells can directly recognize and kill tumor cells without pre-sensitization [19], and the anti-tumor immunity of NK cells is not only e cient, but also has the unique advantages of broad anti-tumor spectrum, individual versatility and immediacy, and can become an important part of general, rapid and e cient cellular immune agents and comprehensive therapy [20,21]. NK92 cells belong to CD56+ CD16-NK cells, which have cytotoxicity and are not involved in antibody-dependent cell-mediated cytotoxicity ((ADCC)). Because of its safe and effective anti-tumor therapy in vivo, it has been used in the clinical study of adoptive immunotherapy of tumor, and has entered phaseclinical trials, and has been successfully used in clinical treatment of lung cancer, glioma and other clinical treatments (https://clinicaltrials.gov/). Studies have found that the ne number of NK in ltrated in the tumor [22][23][24][25] and the improvement of tumor killing activity are very important for the curative effect [26,27]. In this study, NK92 can proliferate sustainably and enhance its anti-tumor activity by up-regulating the autocrine or paracrine of IL-2, IL-12 and other factors. Crispr/cas9 gene editing technology has been widely used in the eld of gene editing in recent years, and has been successfully applied in the elds of upregulating gene expression in mammalian cells [16,28], gene therapy for genetic diseases and infectious diseases [14] and so on. In this paper, through the construction of transcriptional activation domain dcas9-vp64, the sgRNA genes of IL2 and IL12 were overlapped by T2A to obtain the gRNA sequence of IL2-IL12, and the lentiviral expression plasmid of gRNA was constructed. after transfection of NK92, the inactivated dCas9-VP64 was located in the upstream of IL2 and IL12 expression genes of nuclear DNA, and the two cytokines IL2 and IL12, which play key roles in cell proliferation and killing activity, were upregulated in NK92 cells. They act on NK92 cells through cell autocrine or paracrine, enhance the cytotoxicity and activity of NK92 cells, and achieve selfsustainable proliferation and activity enhancement. In this study, the lentiviral expression plasmid of gRNA was obtained by lentivirus packaging infection, and con rmed by sequencing. After infection and integration, the resistance screening of puromycin was observed by uorescence microscope to obtain cells with a purity of more than 80%. The qPCR and ELISA detection of NK92 cells in each group proved that cytokines IL2 and IL12 were successfully overexpressed in NK92 cells and secreted to extracellular. It is proved that the target cell line of NK92-IL2-IL12 was successfully constructed in this study, which can maintain the sustained and stable overexpression and release of IL2 and IL12. Here, we used CCK-8 method to detect that the proliferation function of this cell increased by 1.75 times. Through the detection of in vivo imaging system and tumor immunohistochemistry in mice for 4 weeks, it was con rmed that the NK92-IL2-IL12 cell line continued to proliferate and enhance its activity in the tumor-bearing mice, so that the general condition of the experimental mice was improved and the survival time was prolonged.
In short, through gene editing technique, NK92 consistently overexpressed IL2 and IL12 cytokines, while IL2 and IL12 cytokines act on peripheral cells and autologous cells through autocrine to promote the proliferation and anti-tumor effect of NK92 cells. In this study, we explored the function of NK92 cells with overexpression of two kinds of cytokines, and found that NK92 cells had stronger anti-tumor activity on the basis of their proliferation ability enhanced greatly.
As a result, a new type of NK cells with sustained proliferation and signi cantly enhanced anti-tumor activity was successfully developed.    Fig3a. Comparison of the killing activity of NK92 cells against K562 cells showed that the killing activity of the IL2/IL12 modi ed group was signi cantly higher than that of the wild group, and was proportional to the effective target ratio *p 0.05 .Results are expressed as means of triple experiments per treatment. Fig.3b Comparison of proliferative activity of NK92. Compared with the control group, the test group has stronger NK cell proliferation activity.It gradually increased with incubation time, most ampli ed at 48 hours, about 1.75 times that of the control group. (The test group is the NK cell group overexpressing IL-2-IL-12, and the control is the wild type NK cell group.   Fig.4a shows the in ltration region of NK92 cells carrying GFP at different times (LW593 NK92 for test Scid mice,LW415-NK92 for control Scid mice.). Color represents the intensity of the GFP protein carried by NK92 cells, red indicates the strongest. Compared with the control group, the test group had more NK cells in ltrated into the tumor. In addition, the number of NK cells in ltrating in the tumor of the test group was signi cantly more than four weeks after injection than two weeks, indicating that the experimental group had stronger NK cell proliferation activity in vivo. Fig.4b Compared with the control NK92 group the uorescen intensity of tumor area was signi cantly increased in the test NK92 group. p<0.0001 Fig4c-d.Reduced tumor burden and prolonged survival in mice bearing tumors treated with adoptively transferred ex vivo LW590-nk92 cells.(Test group: LW590-nk92 group, Control group:LW415-nk92 group) Fig.4c shows the tumor size of SCID mice injected with nk92 cells intravenously from the tail, which proves that compared with the pbs group and the control nk92 group, the tumor burden of mice in the test nk92 group is signi cantly reduced.(* p < 0.05). Fig.4d shows the survival period of tumor-bearing mice in the test group is signi cantly longer than that in the pbs group and control group.(P 0.01)