Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

민보경,최하나,허정현,정미영,김효진,정미영,이은경,조성유,황유경,신의철 대한면역학회 2018 Immune Network Vol.18 No.4

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3+ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

Min, Bokyung,Choi, Hana,Her, Jung Hyun,Jung, Mi Young,Kim, Hyo-Jin,Jung, Mi-young,Lee, Eun-Kyoung,Cho, Sung Yoo,Hwang, Yu Kyeong,Shin, Eui-Cheol 한국조명·전기설비학회 2018 한국조명·전기설비학회 학술대회논문집 Vol. No.

<P>Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3<SUP>+</SUP> T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. <I>In vivo</I> antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.</P>

Expression of Gpnmb in NK Cell Development from Hematopoietic Stem Cells

최인표,김태돈,정진웅,윤석란,이석형,김미선,정미라,이지원,신나라 대한면역학회 2008 Immune Network Vol.8 No.2

Background: Molecular mechanisms of natural killer (NK) cell development from hematopoietic stem cells (HSCs) have not been clearly elucidated, although the roles of some genes in NK cell development have been reported previously. Thus, searching for molecules and genes related NK cell developmental stage is important to understand the molecular events of NK cell development. Methods: From our previous SAGE data-base, Gpnmb (Glycoprotein non-metastatic melanoma protein B) was selected for further analysis. We confirmed the level of mRNA and protein of Gpnmb through RT-PCR, quantitative PCR, and FACS analysis. Then we performed cell-based ELISA and FACS analysis, to know whether there are some molecules which can bind to Gpnmb. Using neutralizing antibody, we blocked the interaction between NK cells and OP9 cells, and checked IFN-γ production by ELISA kit. Results: Gpnmb expression was elevated during in vitro developmental stage and bound to OP9 cells, but not to NK precursor cells. In addition, we confirmed that the levels of Gpnmb were increased at NK precursor stage in vivo. We confirmed syndecan4 as a candidate of Gpnmb's binding molecule. When the interaction between NK cells and OP9 cells were inhibited in vitro, IFN-γ production from NK cells were reduced. Conclusion: Based on these observations, it is concluded that Gpnmb has a potential role in NK cell development from HSCs. Background: Molecular mechanisms of natural killer (NK) cell development from hematopoietic stem cells (HSCs) have not been clearly elucidated, although the roles of some genes in NK cell development have been reported previously. Thus, searching for molecules and genes related NK cell developmental stage is important to understand the molecular events of NK cell development. Methods: From our previous SAGE data-base, Gpnmb (Glycoprotein non-metastatic melanoma protein B) was selected for further analysis. We confirmed the level of mRNA and protein of Gpnmb through RT-PCR, quantitative PCR, and FACS analysis. Then we performed cell-based ELISA and FACS analysis, to know whether there are some molecules which can bind to Gpnmb. Using neutralizing antibody, we blocked the interaction between NK cells and OP9 cells, and checked IFN-γ production by ELISA kit. Results: Gpnmb expression was elevated during in vitro developmental stage and bound to OP9 cells, but not to NK precursor cells. In addition, we confirmed that the levels of Gpnmb were increased at NK precursor stage in vivo. We confirmed syndecan4 as a candidate of Gpnmb's binding molecule. When the interaction between NK cells and OP9 cells were inhibited in vitro, IFN-γ production from NK cells were reduced. Conclusion: Based on these observations, it is concluded that Gpnmb has a potential role in NK cell development from HSCs.

인체신세포암에서 자연살해세포의 활성화

류현열,Eschenbach, Andrew C. von 고신대학교 의학부 1995 高神大學校 醫學部 論文集 Vol.10 No.2

Natural killer (NK) cells that had infilterated renal cell carcinoma(RCC) proliferated vigorously in culture which activated interleukin-2(IL-2) and lysed autologous tumor cells. We studied the susceptibility of RCC cells to NK-cell lysis and their ability to stimulate proliferation and function of NK cells. Cells from primary culture of RCC(p-RCC cells) were significantly more susceptible to lysis mediated by human NK3.3 clones than were cells from primary culture of metastatic melanomas. RCC cells clones was also susceptible to lysis by NK3.3 clones and IL-2 activated peripheral blood lymphocytes(PBLs). Incubation of NK3.3 clones with p-RCC cells in the absence of IL-2 induced proliferation of NK3.3 clones, whereas incubation with cells from primary culture of metastatic melanomas, K562 cells tested did not. The p-RCC cells from earlier passages were more potent inducers of NK-cell proliferation than were those from older passages. Cell-free culture supernatants of p-RCC cells with or without NK3.3 clones failed to induce NK-cell proliferation. Incubation of NK cells purified from PBLs with p-RCC cells induced higher proliferation of the NK cells only in the presence of IL-2, whereas incubation with cells from primary culture of metastatic melanomas did not. In summary, these results suggest that RCC cells are able to activate NK cells, potentially through cell-to-cell interaction.

인체신세포암에서 자연살해세포의 활성화

류현열,Andrew C. von Eschenbach 고신대학교(의대) 고신대학교 의과대학 학술지 1995 고신대학교 의과대학 학술지 Vol.10 No.2

-Abstract- Natural killer (NK) cells that had infilterated renal cell carcinoma (RCC) proliferated vigorously in culture which activated interleukin-2 (IL-2) and lysed autologous tumor cells. We studied the susceptibility of RCC cells to NK cell lysis and their ability to stimulate proliferation and function of NK cells. Cells from primary culture of RCC (p-RCC cells) were significantly more susceptible to lysis mediated by human NK 3.3 clones than were cells from primary culture of metastatic melanomas. RCC cells clones was also susceptible to lysis by NK 3.3 clones and IL-2 activated peripheral blood lymphocytes (PBLs). Incubation of NK 3.3 clones with p-RCC cells in the absence of IL-2 induced proliferation of NK 3.3 clones, whereas incubation with cells from primary culture of metastatic melanomas, K 562 cells tested did not. The p-RCC cells from earlier passages were more potent inducers of NK-cell proliferation than were those from older passages. Cell-free culture supernatants of p-RCC cells with or without NK 3.3 clones failed to induce NK-cell proliferation. Incubation of NK cells purified from PBLs with p-RCC cells induced higher proliferation of the NK cells only in the presence of IL-2, whereas incubation with cells from primary culture of metastatic melanomas did not. In summary, these results suggest that RCC cells are able to activate NK cells, potentially through cell-to-cell interaction.

Expression of Gpnmb in NK Cell Development from Hematopoietic Stem Cells

Shin, Na-Ra,Lee, Ji-Won,Lee, Ji-Won,Jeong, Mi-Ra,Kim, Mi-Sun,Lee, Suk-Hyung,Yoon, Suk-Ran,Chung, Jin-Woong,Kim, Tae-Don,Choi, In-Pyo The Korean Association of Immunobiologists 2008 Immune Network Vol.8 No.2

Background: Molecular mechanisms of natural killer (NK) cell development from hematopoietic stem cells (HSCs) have not been clearly elucidated, although the roles of some genes in NK cell development have been reported previously. Thus, searching for molecules and genes related NK cell developmental stage is important to understand the molecular events of NK cell development. Methods: From our previous SAGE data-base, Gpnmb (Glycoprotein non-metastatic melanoma protein B) was selected for further analysis. We confirmed the level of mRNA and protein of Gpnmb through RT-PCR, quantitative PCR, and FACS analysis. Then we performed cell-based ELISA and FACS analysis, to know whether there are some molecules which can bind to Gpnmb. Using neutralizing antibody, we blocked the interaction between NK cells and OP9 cells, and checked IFN-${\gamma}$ production by ELISA kit. Results: Gpnmb expression was elevated during in vitro developmental stage and bound to OP9 cells, but not to NK precursor cells. In addition, we confirmed that the levels of Gpnmb were increased at NK precursor stage in vivo. We confirmed syndecan4 as a candidate of Gpnmb's binding molecule. When the interaction between NK cells and OP9 cells were inhibited in vitro, IFN-${\gamma}$ production from NK cells were reduced. Conclusion: Based on these observations, it is concluded that Gpnmb has a potential role in NK cell development from HSCs.

Effects of Electrical Stimulation on Enhancing the Cytotoxic Activity and Half-life of Natural Killer Cells

Minseon LEE,Soonjo KWON 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Natural killer (NK) cells are responsible for the effector function against tumors in innate immune system, predominantly mediated by the secretion of lytic granules containing granzyme and perforin at immunological synapses. However, strategies for NK cell-immunotherapy still should be developed due to the existing immune evasion of tumors by protecting cells from granule uptake and being resistant to NK cell-mediated killing. In this study, we designed a direct current electrical stimulation (ES) system using platinum electrodes as a strategy for enhancing cytotoxic activity and half-life of NK cells for efficient immunotherapy. ES effects have been studied in tissue engineering for bone healing and stem cells by targeting calcium signaling on cell differentiation and migration. Exposure to electrical field could induce changes in calcium flux and mediated pathway, which could be also indispensable for exocytosis of cytotoxic enzyme and tumor killing in NK cells. Cultured KHYG-1 cells were exposed to 1hr direct current ES (voltage range from 0.2 to 1.0V/㎝) and effect of stimulation was analyzed by cell viability, NK cell-mediated cytotoxicity, and gene expression level of related genes. Gene expression level of GZMB was 1.36-fold and 1.58-fold higher without any effects on cell viability, following exposure to ES with 0.5V/cm and 1.0V/㎝, respectively. These findings provide the useful insight regarding the strategies for improving NK cell-mediated cytotoxicity through calcium-dependent exocytosis of lytic granules without any genetic modification, which need to be further studied as one of the new immunotherapy methods.

XRP44X Enhances the Cytotoxic Activity of Natural Killer Cells by Activating the c-JUN N-Terminal Kinase Signaling Pathway

Kwang-Soo Kim,박경순 한국발생생물학회 2020 발생과 생식 Vol.24 No.1

Natural killer (NK) cells are innate lymphocytes that play an essential role in preventing cancer development by performing immune surveillance to eradicate abnormal cells. Since ex vivo expanded NK cells have cytotoxic activity against various cancers, including breast cancers, their clinical potential as immune-oncogenic therapeutics has been widely investigated. Here, we report that the pyrazole chemical XRP44X, an inhibitor of Ras/ ERK activation of ELK3, stimulates NK-92MI cells to enhance cytotoxic activity against breast cancer cells. Under XRP44X stimulation, NK cells did not show notable apoptosis or impaired cell cycle progression. We demonstrated that XRP44X enhanced interferon gamma expression in NK-92MI cells. We also elucidated that potentiation of the cytotoxic activity of NK-92MI cells by XRP44X is induced by activation of the c-JUN N-terminal kinase (JNK) signaling pathway. Our data provide insight into the evaluation of XRP44X as an immune stimulant and that XRP44X is a potential candidate compound for the therapeutic development of NK cells.

Distribution of Lymphocyte Subpopulation and Natural Cytotoxicity of Peripheral Blood from Colon Cancer Patient

Park, Il Young,Park, Jang Sang,Chang, Suk Kyun,Lee, Jae Hak CATHOLIC MEDICAL CENTER 1990 Bulletin of the Clinical Research Institute Vol.18 No.1

Depression of general immune reactivity has been well documented in patients with colorectal cancer as well as in patient with other types of solid tumors. The main effector of immune surveillance against cancer have been considered to be sensitized T lymphocyte and activated macrophages. Recently there has been increasing recognition that natural cell-mediated cytotoxicity is potentially an important antitumor mechanism especially in recurrence, metastasis and prognosis. Authors investigated to study the relation between subpopulation, stimulated or non-stimulated natural cytotoxicity of lymphocyte and stage of disease, serum CEA levels, or differentiation of cancer. The results were as follows; 1. The peripheral blood lymphocyte count was significantly decreased only in group 4 as compared with control. 2. The natural cytotoxicity against K_562 cells were significantly decreased in group 2 and group 4 than that of control. But except for group 3, there were no significant differences in natural cytotoxicity against SBA cells. 3. The natural cytotoxieity against K_562 cells of interleukin-2 stimulated lymphocyte was significantly increased than that of non-stimulated lymphocytes in both of control and stage Ⅳ patients group. 4. The distributions of lymphocyte subpopulation (CD_4, CD_8, CD_16) were slightly decreased in group 3 and decrease of CD_4, CD_16 with increase of CD_8 in group 4 without statistical significancy. But in group 3, CD_4 cells were significantly increased than that of control and CD_8 cells were significantly increased as compared with control. 5. The ratio of CD_4 to CD_8 was significantly decreased in group 3 than that of control, but no significant differences in group 2 and 4. 6. The serum CEA was positive (>5 ng/ml) in 43% of patients and the level was increased with advance of diseases. But there were no correlation between serum CEA level, tumor cell differentiation and natural cytotoxicity, CD_4 to CD_8 ratio or K_562 cytotoxicity to CD_15 cells. With above results, authors insist that the natural cytotoxicity by natural killer (NK) cells is decreased but not by cytotoxic T-cell in colon cancer patients and these cytotoxic activities can be enhenced by interleukin-2 stimulation. The NK cells was slightly increased in all stage of disease, but the decrease of helper T cell and increase of suppressor T-cell were significant in patients with lymph node metastasis.

정신분열증과 주요 우울장애에서 Natural Killer Cell 수와 백분율 변화NK cell

윤장봉,이재우,박두병 大韓神經精神醫學會 1996 신경정신의학 Vol.35 No.5

The purpose of this study was to compare the number and percentage of natural killer cell in schizophrenic and major depressive patients with those in normal healthy subjects 15 Schizophrenic patients, 15 major depressive patients and 16 healthy subjects were included in this study There was no statistical difference in the age and sex between patients and control subjects. All of these patients were drug-free for at lest two weeks before this study. The results of this study were as follows : 1) In schizophrenic patients, the number and percentage of natural killer cell of the group with below-mean score of general psychopathology were significantly lower than those of the group with above-mean score(p<.05). The number but not the percentage of natural killer cell of the group with below-mean BPRS score was significantly lower than that of the group with above-mean BPRS score(p<.05). 2) The number of natural killer cell showed statistically negative correlation with generalpsychopathology score or BPRS score(p<0.05). In conclusion, these findings suggest that the number and percentage of natural killer cell is not significantly changed in schizophrenia and major depressive disorder, but in schizophrenic patients, severe psychopathology in the rating scales is associated with suppression of the number and percentage of natural killer cell.