Characterization And Anti-tumor Activity Of A New Human NK Cell Line

Natural killer (NK) cells as part of the innate immune system are important players in the first line of defense against malignancies. The use of NK cells in immunotherapy of human cancers has been proposed and more recently trailed in a clinical context. NK cell-based cellular immunotherapy includes activation of endogenous NK cells, alloreactive NK cells, adoptive transfer of ex vivo expanded autologous NK cells or donor-derived NK cells, and adoptive transfer of NK cell line. By comparison, there are some limitations as to the efficacy of the reinfusion of ex vivo expanded autologous or allogeneic NK cells. Clinical use of human permanent NK cell lines would overcome some of the limitations, which are more cytotoxic and can be easily expanded and maintained in vitro without contamination by other lymphocytes. Until now, NK-92 is the only NK cell line that has entered clinical trials and proved to be safe and has generated anti-tumor effects. However, it was proposed that the NK cell line established in Western countries such as NK-92 might not be suitable for Chinese patients. Missing expression of donor KIR ligand(s) (HLA) in the recipient induces the function of NK cells, which would be a possible beneficial effector for the NK cell line immunotherapy; but the significant differences in HLA molecules among the races might induce HLA-antibodies in the recipient to inhibit the immune effects. So, it is important to use a NK cell line established in China for Chinese cancer patients.In addition, tumor diagnosis and staging is critical to the clinical decision-making for tumor therapy. Lung cancer as one of the leading causes of cancer death has increasingly been an urgent problem in the world, with high frequency of tumor metastases and recurrence. Development of sensitive and specific detection methods for circulating cancer cells in peripheral blood may have important implications.In this study, flow cytometry analysis was used to determine the phenotypical and functional characteristics of a new huaman NK cell line NKG cells. [3H] thymidine riboside incorporation was used for proliferation assay of irradiated NKG cells. Cytotoxicity assay (4 h 51Cr release) was used to detect the anti-tumor activity of NKG cells. Immunotherapy of human ovarian cancer with irradiated NKG cells was determined in xenograft mouse model. By the WAVE Bioreactor, the large-scale NKG cell culture procedure was established, and then master cell bank and working cell bank were established. The cell identification test, sterility test, exogenous factor detection, tumorigenicity and acute toxicity test were done according to (partⅢ,2005 version). Additionally, quantitative real-time RT-PCR was used to evaluate the molecular markers including LunX mRNA et al. for their specificity for lung cancer clinically. Our major findings are shown as followed:1. Phenotypical and functional characteristics of NKG cellNKG cell is a permanent NK cell line established by the 863 research group of Tian's lab in 2003. NKG cells as suspension cells grew with cell aggregator forming potential, and expanded for one generation in 2-3 days. The NKG cells were phenotypically identified as CD56+CD16-CD27-CD3-αβTCR-γδTCR-CD4-CD8-CD19-CD161-CD45+, and with high levels of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, CD11c) and co-stimulatory receptor CD48, an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C) and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, IFN-y), although inhibitory receptors including CD94/NKG2A, CD158b and CD85j were detected. NKG cells displayed characteristics of activated NK cells.2. The anti-tumor activity and its molecular mechanisms of NKG cellNKG cells showed high cytotoxicity to several kinds of tumor cells. In particular, the cytotoxicity against the Ho-8910 and SGC7901 cells was more than 80% at the E/T ratio of 20/1. Compared with NK-92, NKL and YT cells, NKG cells were the most cytotoxic against Ho-8910 cells. High cytotoxicity of NKG cells was absolutely dependent on the recognition of NKG2D and NKp30. Irradiated at the dose up to 8 Gy, NKG cells were still with high cytotoxicity against tumor cells in 48 h, but not with proliferation. Further, the irradiated-NKG cells were demonstrated with strong cytotoxicity (75%-45%, E/T=20/1) against human primary ovarian cancer cells in vitro.3. Immunotherapy of human ovarian cancer with irradiated NKG cells in xenograft mouse modelHo-8910 cells were injected i.p into the nude mice to establish human ovarian tumor model. When the irradiated NKG cells were adoptively transferred into the mice together with Ho-8910 cells with ratio of 5/1, the tumorgenesis (%) was significantly decreased from 100%to 50%, and the palpable tumor developed time was delayed from 28～44 days to 90～135 days, the abdomen circumference (AC) was significantly decreased. Although 50% mice with NKG cell treatment were dead with tumor progress, the mean survival time was obviously prolonged from 61.5 to 210.2 days. These results demonstrated that the irradiated NKG cells owned the high cytotoxicity against the ovarian tumor cells in vivo.After the mice were challenged with Ho-8910 cells for 21 days, NKG cell treatment could significantly inhibit the tumorgenesis (from 100% to 62.5%) with the delayed time of palpable tumor development (from 28～38 days to 56～66 days). The severity of ovarian cancer was attenuated as indicated by the significantly decreased AC. The mean survival time was obviously prolonged from 64.6 to119.6 days. Further, after 35 days of Ho-8910 cell challenge, the mice with palpable tumor were selected for NKG cell treatment, which could also attenuate the severity of ovarian cancer, shown by the decreased AC in the early stage of treatment and obviously prolonged mean survival time (from 64.2 to 93.6 days) although all the mice were dead with tumor progress ultimately. These results demonstrate the immunotherapy of human ovarian cancer with irradiated NKG cells.4. Establishment of the large-scale NKG cell culture procedureBy the WAVE Bioreactor, the large-scale NKG cell culture procedure was established. Inoculation density:2.0×104cells/ml; culture medium:a-MEM complete medium (a-MEM medium contain 10% new-born calf serum and 10% horse serum) with100U/ml rhIL-2; Inoculation volume:500ml, and 500ml complete medium containing rhIL-2 (final concentration 100U/ml) were added at 2,4 and 6 days of cell inoculation respectively. Max culture volume was less than 2000ml. Culture condition was 37℃,5.0% CO2 with flow rate of 0.15L/min,7rpm/7°. The number, purity and activity of the NKG cells cultured by this procedure well meet the clinical requirements.5. Acute toxicity test of NKG cellThe C57BL/6 mice were i.p. injected 800cGy irradiated NKG cells at a dose of 150 fold of clinical application (1x108cells/mouse). No toxic reaction, allergic reaction and local stimulation reaction was observed in successive 7 days after NKG cell transfer. There is no significance difference in the body weight between the NKG cell transferred group and control group. NKG cells were demonstrated with no acute toxicity. 6. NKG cell banks:the establishment and identificationThe master cell bank and working cell bank were established by the large-scale NKG cell culture procedure. The biological assay of the working cell bank indicated that the NKG cell phenotype is identical to the primary seed cells, and with no bacterial, fungus and mycoplasma contamination, no exogenous virokine and no tumorigenicity in vitro and in vivo.7. Diagnostic utility of LunX mRNA in early micrometastasis and therapeutic assessment for patients with primary non-small cell lung cancerQuantitative real-time RT-PCR was used to determine LunX, CK19, CEA, VEGF-C and hnRNP A2/B1 mRNA levels in peripheral blood and pleural fluid from NSCLC patients, compared with those from patients with other epithelial cancer (esophagus cancer and breast cancer), benign lung disease (pneumonia and tuberculo pleurisy) and from healthy volunteers. In peripheral blood LunX mRNA was detectable in 75.0%(33/44) of patients with NSCLC, but not in patients with other epithelial cancer, benign lung disease or in healthy volunteers. In contrast, all other genetic markers were detected in patients with either NSCLC, other epithelia cancer or benign lung disease, and in healthy volunteers. Furthermore, LunX mRNA was detected in 92.9%(13/14) of malignant pleural fluid samples and was the only marker whose expression level was significantly different between malignant and benign pleural fluid. The expression level and positive rate of LunX mRNA in peripheral blood correlated with the pathologic stage of NSCLC. Additionally, expression of LunX mRNA and CK19 mRNA in the peripheral blood of NSCLC patients decreased shortly after clinical treatment. As for the expression of CK19 mRNA in the peripheral blood from the other epithelia cancer patients, LunX mRNA is the most specific gene marker for lung cancer and has potential diagnostic utility when measured in the peripheral blood and pleural fluid of NSCLC patients.Conclusion:With the characteristics of activated NK cells, NKG cells were highly cytotoxic against a series of tumor cell lines. Immunotherapy of human ovarian cancer with irradiated NKG cells was firstly demonstrated in xenograft mouse model, even for the ovarian tumor in the advance stage. Use of the NKG cell line would represent a novel immunotherapy for the treatment of human cancer in clinic. The master cell bank and working cell bank were established by the established large-scale NKG cell culture procedure, which was the base for its clinical use. Additionally, it was demonstrated that LunX mRNA is the most specific gene marker for lung cancer and has potential diagnostic utility in early micrometastasis and therapeutic assessment when measured in the peripheral blood and pleural fluid of NSCLC patients.