• Molecules and Cells
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• 제41권8호
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• pp.717-723
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• 2018

Chimeric antigen receptor (CAR) T-cell therapy, an emerging immunotherapy, has demonstrated promising clinical results in hematological malignancies including B-cell malignancies. However, accessibility to this transformative medicine is highly limited due to the complex process of manufacturing, limited options for target antigens, and insufficient anti-tumor responses against solid tumors. Advances in gene-editing technologies, such as the development of Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), have provided novel engineering strategies to address these limitations. Development of next-generation CAR-T cells using gene-editing technologies would enhance the therapeutic potential of CAR-T cell treatment for both hematologic and solid tumors. Here we summarize the unmet medical needs of current CAR-T cell therapies and gene-editing strategies to resolve these challenges as well as safety concerns of gene-edited CAR-T therapies.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.257-260
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• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• Journal of Microbiology and Biotechnology
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• 제16권5호
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• pp.695-703
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• 2006

The dissolved oxygen level of any cell culture environment has a critical effect on cellular metabolism. Specifically, hypoxia condition decreases cell viability and recombinant protein productivity. In this work, to develop CHO cells producing recombinant protein with high productivity, mammalian expression vectors containing a human tissue-type plasminogen activator (t-PA) gene with hypoxia response element (HRE) were constructed and stably transfected into CHO cells. CHO/2HRE-t-PA cells produced 2-folds higher recombinant t-PA production than CHO/t-PA cells in a $Ba^{2+}-alginate$ immobilized culture, and 16.8-folds in a repeated batch culture. In a non-aerated batch culture of suspension-adapted cells, t-PA productivity of CHO/2HRE/t-PA cells was 4.2-folds higher than that of CHO/t-PA cells. Our results indicate that HRE is a useful tool for the enhancement of protein productivity in mammalian cell cultures.

• Tuberculosis and Respiratory Diseases
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• 제70권5호
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• pp.405-415
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• 2011

Background: In an effort to find alternative therapeutic agents to prevent excessive fibrosis as a sequela to complicated parapneumonic effusion or empyema, we examined the effect of tissue plasminogen activator (tPA) as a fibrinolytic agent combined with talc or transforming growth factor (TGF)-${\beta}1$ in a human pleural mesothelial cell line, MeT-5A. Methods: MeT-5A cells were stimulated with various doses of talc, doxycycline or TGF-${\beta}1$ for 24 h and then were treated with tPA for an additional 24 h. Cell viability was measured by MTT assay. The production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in the culture supernatants was measured by ELISA. Real-time PCR was carried out for measurement of type I collagen mRNA. Results: MeT-5A cells treated with talc showed a dose-dependent increase in production of IL-8. Talc also increased production of type I collagen mRNA at low doses, but talc did not influence the induction of VEGF. Addition of tPA to talc-stimulated cells showed further increases in the production of IL-8, but tPA did not influence the production of VEGF or type I collagen mRNA. TGF-${\beta}1$ increased the production of both VEGF and collagen type I mRNA, both of which were effectively inhibited by additional tPA treatment in MeT-5A cells. Conclusion: TGF-${\beta}1$ is a potent inducer of collagen synthesis without induction of IL-8 in MeT-5A cells. Addition of tPA after TGF-${\beta}1$ stimulation inhibited further fibrosis by direct inhibition of collagen mRNA synthesis as well as by inhibition of VEGF production.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.457-462
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• 2002

Low Electromagnetic Field (EMF) intensity in the range of $1{\mu}T\;to\;10{\mu}T$(Tesla) was found to enhance the growth of CHO cells and the production of tPA in batch and perfusion cultivations. At $1{\mu}T\;intensity,\;1.3{\times}10^7$ viable cells/ml of maximum cell density and 80 mg/l of maximum tPA production were obtained in batch cultivation, compared to $2.8{\times}10^6$ viable cells/ml and 59 mg tPA/1 in unexposed case (control). A similar trend was observed in the perfusion process, where it was possible to obtain $1.2{\times}10^7$ viable cells/ml of maximum cell density and 81 mg tPA/l of maximum tPA production by more than 80 days of cultivation. However, there was not much difference between $1{\mu}T\;and\;10{\mu}T$ in perfusion cultivation, possibly due to better environmental growth conditions being maintained by continuous feeding of fresh medium into the reactor. On the contrary, both cell growth and tPA production were severely inhibited at higher than 1 mT intensity, showing no growth at 10 mT exposure. Specific growth rate was linearly correlated to specific tPA production rate at $1{\mu}T$EMF intensity, which represents a partially growth-related relationship. It was also found that a large amount of $Ca^2+$ was released at low EMF intensity, even though the cell growth was not much affected. Low EMF intensity significantly improved both cell growth and tPA production, and tPA production seemed to be more affected than the cell growth, possibly due to the changes of cell membrane characteristics. It can be concluded that the elaboration of EMF intensity less than $10{\mu}T$ could improve cell growth and tPA production, but mainly tPA secretion through batch or perfusion process in a bioreactor.

• Tuberculosis and Respiratory Diseases
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• 제44권3호
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• pp.516-524
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• 1997

목 적 : 악성 종양에 있어서 단백질 분해 효소에 의한 세포외기질의 분해는 종양 침습 및 전이에 있어서 중요한 역할을 한다. Urokinase-type plasminogen activator(u-PA)는 이러한 단백질 분해효소 중 하나이며, u-PA의 길항제인 plasminogen activator inhibitor(PAI-1, PAI-2)도 u-PA에 의한 종양조직 자체의 분해를 방어하여 종양의 성장, 침습 및 혈관신생을 촉진하는 역할을 한다. u-PA와 PAI-1은 폐암 등의 종양 조직내의 양이 증가하며, 침습도 및 예후 둥과 유의하게 상관되는 것으로 보고되고 있다. 저자 등은 폐암환자의 혈장 u-PA와 PAI-1의 농도를 측정하여 조직형 및 병기와의 관련성을 조사해 보고자 한다. 방 법 : 폐암 환자 37예, 여러 가지 양성 폐질환 환자 21예 및 유사연령의 정상 대조군 24예에서 혈장 u-PA 및 PAI-1 항원의 농도를 ELISA법으로 측정하였다. 결 과 : 혈장 u-PA 항원 농도는 정상 대조군에서 $1.0{\pm}0.3ng/mL$, 양성 폐질환군에서 $1.0{\pm}0.3ng/mL$, 폐암환자군에서 $0.9{\pm}0.3ng/mL$로 유의한 차이가 없었다. 혈장 PAI-1 항원 농도는 정상 대조군에서 $14.2{\pm}6.7ng/mL$, 양성 폐질환군에서 $14.9{\pm}6.3ng/mL$, 폐암 환자군에서 $22.1{\pm}9.8ng/mL$로 폐암 환자에서 정상 대조군 및 양성 폐질환군에 비하여 유의하게 높았다. 혈장 u-PA 항원 농도는 편평상피암에서 $0.7{\pm}0.4ng/mL$, 선암에서 $0.8{\pm}0.3ng/mL$, 대세포암에서 0.9ng/mL, 소세포암에서 $1.1{\pm}0.7ng/mL$로 유의한 차이가 없었다. 혈장 PAI-1 항원 농도는 편평상피암에서 $22.3{\pm}7.2ng/mL$, 선암에서 $22.6{\pm}9.9ng/mL$, 대세포암에서 42ng/mL, 소세포암에서 $16.0{\pm}14.2ng/mL$로 유의한 차이가 없었다. u-PA 항원 농도는 stage I에서 0.74ng/mL, stage II에서 $1.2{\pm}0.6ng/mL$, stage IIIA에서 $0.7{\pm}0.4ng/mL$, stage IIIB에서 $0.7{\pm}0.4ng/mL$, stage IV에서 $0.7{\pm}0.3ng/mL$였고, 혈장 PAI-1 항원 농도는 stage I에서 21.8ng/mL, stage II에서 $22.7{\pm}8.7ng/mL$, stage IIIA에서 $18.4{\pm}4.9ng/mL$, stage IIIB에서 $25.3{\pm}9.0ng/mL$, stage IV에서 $21.5{\pm}10.8ng/mL$로 유의한 차이가 없었다. 폐암 환자에서 T기를 T1-3 와 T4로 나누어서 비교를 한 결과, 혈장 u-PA 항원 농도는 T1-3에서 $0.8{\pm}0.4ng/mL$였고 T4에서 $0.7{\pm}0.4ng/mL$로 차이가 없었으나, 혈장 PAI-1 항원 농도는 T1-3에서 $17.9{\pm}5.6ng/mL$였고 T4에서 $26.1{\pm}9.1ng/mL$로 T4에서 유의하게 높았다. 혈장 u-PA 항원 농도는 M0에서 $0.8{\pm}0.4ng/mL$, M1에서 $0.7{\pm}0.3ng/mL$였고, 혈장 PAI-1 항원 농도는 M0에서 $23.6{\pm}8.3ng/mL$, M1에서 $21.5{\pm}10.8ng/mL$로 유의한 차이가 없었다. 결 론 : 이상의 결과에서 폐암 환자에서 혈장 PAI-1은 정상대조군 및 양성 폐질환에 비하여 특이적으로 증가하였고, 또한 폐암의 국소적 침습 정도와의 관련성을 보여 주었다.

• KSBB Journal
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• 제9권1호
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• pp.48-54
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• 1994

HEK 세포 배양액 중에 존재하는 scu-PA와 tc-PA의 보다 정확한 측정을 위해 fibrin plate법과 기존의 amidolytic 방법을 변형시킨 측정법을 이용했다. 1%의 저혈청 배지를 이용한 T-flask 배양에서 $1.65{\times}10^6$(viable cells/ml)의 최대 세포수에 도달하여 1670(IU/ml)의 scu-PA 생산량을 보였으며 평균 10% 미만이 변환율을 보였다. 또한 Spinner vessel에서의 회분배양시 최대 세포수가 $4.43{\times}10^6(total cells/ml)$ 와 1560(IU/ml)의 scu-PA 생산량을 나타냈으며 평균 11.4%의 변환율을 보였다. 연속배양에서는 0.449(1/day)의 비생육속도와 $3.13{\times}10^{-4}(IU/cell)$의 비생산속도를 보였으며 평균 10.18% 정도의 전환율을 보였다. 이는 회분식 및 유가식 배양 결과와 큰 차이가 없으며 배양공정에 관계없이 약 90% 이상의 회수율이 가능하다는 것을 의미한다.

• IMMUNE NETWORK
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• 제5권3호
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• pp.172-178
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• 2005

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and trans activator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. Methods: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-${\gamma}$ producing T lymphocytes were measured. Results: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-${\gamma}$ secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+T cell to $CD4^+$ cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. Conclusion: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.126-131
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• 1996

HL-60 was cultivated to produce tPA (tissue-type plasminogen activator) and study the mechanism of cell death. Maximum cell density and tPA production were obtained as $5.27{\times}10^6$ cells/ml and 324ng/ml, respectively under perfusion cultivation. tPA production was enhanced to 420ng/ml in adding 160nM of phorbol ester. The cells were gradually differentiated to granulocytes rather than proliferation. By Fluorescent microscope, apoptosis was prevailed except the death phase and in high agitation speed, but necrosis was prevailed in thawed cells and during the latter periods of the cultivation. It was also proved that tPA was most produced in apoptosis. To obtain higher tPA productivity, the cells must be maintained in apoptosis, not necrosis phase when the cells were dying.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.203-212
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• 2020

Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.