| Objective:The aim of this study was to measure dose and purity of natural killer(NK) cells after amplification in vitro, and evaluate the efficacy and safety of therapy of patients with metastatic castration resistant prostate cancer(mCRPC) using NK cells.Methods:(1) Eight patients with mCRPC were enrolled in this study from Jan 2013 to Jun 2014. The situation of patients have been evaluated including chest x-ray and bone scan, computed tomography(CT) or magnetic resonance imaging(MRI), Numerical rating scale(NRS).About 50 ml peripheral blood in patients with PCA have been obtained every time.(2) The peripheral blood mononuclear cells(PBMCs) were isolated by Ficoll density centrifugation. CD137L(5 ug/mL×1mL) was used in T75 flask coating the night previously and stored at 4 oC. Following the NK cell seeding, the INFγ(1000 U/mL) was added in and that day was designated as d0. In the following day(d1), 1000× concentrated IL-1β(1000 U/ml), IL-7(1000 U/ml), IL-12(1000 U/ml), IL-18(1000 U/ml), IL-21(1000 U/ml) were added into medium(complete medium). When the pH of medium droped to 7.0-7.2, depending upon the cell growth, the cultured NK cells suspension were divided into two coated-T75 flasks with half fresh complete medium. Then the NK cells suspension was transferred into a 1.0 L culture bag for further proliferation. One vial of GT-T551(1 mL) containing IL-15(2ng/L final concentration) was supplemented into complete medium. Thereafter, the medium was supplemented with GT-T551 + 1000 IU IL-2 + 10% autologous serum(or plasma) before transfusion.(3) NK cells were cultured and amplified in vitro. The cell growth was observed and the purity of NK cells was detected by flow cytometry. The cytotoxicity of NK cells to PC-3 cells was analyzed by Calcein-AM assay. The Potential capacity of NK cells in the treatment of prostate cancer was identified. The quality inspection of the cell is passed by the quality of the sterility, endotoxin and Mycoplasma before transfusion.(4)All patients received infusions of autologous activated NK cells.Four patients received three infusions. The other patients received four infusions. Clinical and Immunologic outcomes were evaluated after infusion.Results:(1) NK cells were isolated from peripheral blood of 50 ml. The total cell number increased about 1000 times. The average total cell number reached 4.39×109, and the average purity of CD3-CD16/56+NK cells was 84.3%.(2) Expanded NK cells have demonstrated cytotoxicity against K562 cells and PC-3 cells. NK cells exhibited highest cytotoxicity at E:T ratio of 10:1, the result is 78% for PC-3 and 82% for K562.(3) Five patients indicated decrease of prostate-specific antigen(PSA). Two patients showed increase of PSA. There was no obvious change in one patient whose PSA was above 10000ng/ml. The percentage of CD3-CD56+NK cells increased in seven patients. The percentage of CD8+T cells increased in six patients.(4) According to Kamofsky and NRS, The quality of life were improved,meanwhile,ostealgia subsided in different degrees in all patients. No adverse events and side effects were found.Conclusion:(1) After culture, the total cell number of NK cells increased about 1000 times. The average total cell number reached 4.39×109, and the average purity of CD3-CD16/56+ NK cells was 84.3%.(2) The therapy of infusion of NK cells was tolerable and safe. Some encouraging outcomes were seen in mCRPC. Treatment of NK cells shoud have a good future for patients with mCRPC.(3) There were no obvious increase of the proportion of peripheral blood IL2, IL12, IFN and TNF in all patients.The immunotherapy of NK cells could evoke cellular immune response against PCA.(4)PSA was decreased in patients with a Gleason score under 9 compared with the patients with Gleason score of 9. The higher the Gleason score, the treatment effect of NK cells was not good. |
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