• KSBB Journal
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• 제26권5호
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• pp.386-392
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• 2011

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2035-2038
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• 2014

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an important protein involved in the regulation of the immune system. The +49 G/A polymorphism is the only genetic variation in the CTLA-4 gene that causes an amino acid change in the resulting protein. It is therefore the most extensively studied polymorphism among all CTLA-4 genetic variants and contributions to increasing the likelihood of developing cancer are well known in various populations, especially Asians. However, there have hiterto been no data with respect to the effect of this polymorphism on breast cancer susceptibility in our North Indian population. We therefore assayed genomic DNA of 250 breast cancer subjects and an equal number of age-, sex- and ethnicity-matched healthy controls for the CTLA-4 +49 G/A polymorphism but no significant differences in either the gene or allele frequency were found. Thus the CTLA-4 +49 G/A polymorphism may be associated with breast cancer in other Asians, but it appears to have no such effect in North Indians. The study also highlights the importance of conducting genetic association studies in different ethnic populations.

• KSBB Journal
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• 제32권3호
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• Pediatric Infection and Vaccine
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• 제30권3호
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• pp.129-138
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• 2023

목적: 선천면역장애 환자들은 감염에 취약할 뿐만 아니라, 면역이 정상인 사람들에 비해 암 발생률도 높은 것으로 알려져 있다. 본 연구는 단일 기관에서 추적 중인 선천면역장애 환자들에서의 암 발생을 조사하여 보고자 하였다. 방법: 1994년 11월부터 2023년 9월까지 삼성서울병원에서 선천면역장애 진단 하에 추적하는 환자를 대상으로 후향적으로 의무기록을 리뷰하였다. 선천면역장애 환자 중에서 암으로 진단된 환자를 확인하였다. 결과: 총 194명의 선천면역장애 환자 중, 7명(3.6%)의 환자에서 암이 진단되었다. 5명의 환자가 림프종으로 진단받았으며 그 중 4명의 환자는 Epstein-Barr 바이러스 연관 림프종이었다. 나머지 암은 위암과 다발 골수종이었다. 암 진단 당시 나이는 중앙값 18세 (범위, 1세–75세)였다. 암이 발생한 환자들의 면역결핍 질환은 X-linked lymphoproliferative disorder-1 (XLP-1) 3명, activated phosphoinositide 3-kinase delta disease (APDS) 2명, cytotoxic T-lymphocyte antigen 4 (CTLA-4) haplo-insufficiency 2명이었다. 개별 질환별로 분석하였을 때, XLP-1 환자의 75.0%, APDS 환자의 40.0%, CTLA-4 환자의 50.0%에서 암이 발생하였다. XLP-1 환자는 APDS 및 CTLA-4 haplo-insufficiency 환자에 비해 더 이른 나이에 암이 발생하였다 (중앙연령 5세, P<0.001). 한 명은 조혈모세포 이식 치료 중 사망하였다. 결론: 국내 단일 기관에서 진료받는 선천면역장애 환자들의 3.6%에서 암이 발생하였다. 선천면역장애 환자들을 진료하는 의료진들은 이들 환자에서 감염이나 염증 등의 문제외에도 암 발생의 가능성, 특히 Epstein-Barr 바이러스 감염과 연관된 암의 비중이 높은 것에 대한 인식을 갖는 것이 중요하다.

• Biomolecules & Therapeutics
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• 제30권6호
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• pp.529-539
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• 2022

Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-bead-assisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.

• IMMUNE NETWORK
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• 제5권1호
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• pp.36-44
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• 2005

Background: The anti-tumor therapeutic effect of autologous tumor cell lysate pulseddendritic cells (DCs) was studied for non-immunogenic and immune suppressive lung cancer model. To test the possibility as an adjuvant therapy, minimal residual disease model was considered in mouse in vivo experiments. Methods: Syngeneic 3LL lung cancer cells were inoculated intravenously into the C57BL/6 mouse. Autologous tumor cell (3LL) or allogeneic leukemia cell (WEHI-3) lysate pulsed-DCs were injected twice in two weeks. Intraperitoneal DC injection was started one day (MRD model) after tumor cell inoculation. Two weeks after the final DC injection, tumor formation in the lung and the tumor-specific systemic immunity were observed. Tumor-specific lymphocyte proliferation and the IFN-${\gamma}$ secretion were analyzed for the immune monitoring. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 for 7 days and pulsed with tumor cell lysate for 18 hrs. Results: Compared to the saline treated group, tumor formation was suppressed in 3LL tumor cell lysate pulsed-DC treated group, while 3LL-specific immune stimulation was minimum. WEHI-3-specific immune stimulation occurred in WEHI-3 lysate-pulsed DC treated group, which had no correlation with tumor regression. Conclusion: The data suggest the possible anti-tumor effect of cultured DCs as an adjuvant therapy for minimal residual disease state of lung cancer. The significance of immune modulation in DC therapy including the possible involvement of NK cell as well as antigen-specific cytotoxic T cell activity induction was discussed.

• KSBB Journal
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• 제31권4호
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• pp.277-283
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• 2016

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

• KSBB Journal
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• 제20권4호
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• pp.278-284
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• 2005

식물세포의 느린 생장과 낮은 생산량의 이유로 지금까지 는 주로 미생물이나 동물세포에서 유전자 재조합 단백질을 생산하여 왔다. 그러나 저렴한 배지 가격, 동물 유래 바이러스 감염 위험성으로부터의 안정성, glycosylation 등의 post-translational modification이 가능하다는 장점들로 인하여 최근 들어 식물세포배양은 생물학적 활성을 가진 고부가가치의 단백질을 생산하는데 많이 이용되고 있다. 본 연구에서는 생장배지에 첨가했던 sucrose의 소비와 induction 배지로의 교환에서 오는 배지내의 삼투압을 조절하여 hCTLA4-Ig의 생산성을 높이고자 하였다. 다양한 삼투압 조절제 첨가 실험을 통해 sorbitol을 선별하고, 40 mM의 sorbitol 첨가에서 상대적으로 높은 생존도와 induction 후 7일째 대조구보다 1.7배 높은 생산성을 확인하였다. 또한, 저농도의 glucose 첨가를 통한 생산성 증대에 있어서는 8 mM glucose에서 induction 이후에도 높은 세포농도를 유지하면서 최대 37.3 mg/L까지 hCTLA4-Ig 생산량을 증가시켰다. 5-L bioreactor에서 회분식 배양과 induction시의 hCTLA4-Ig 생산량을 비교한 결과 induction시 배양 18일째 최고 45.3 mg/L까지 높일 수 있었으며, 회분식 배양에 비해 2.1배 증가됨을 확인하였다.

• Journal of Chest Surgery
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• 제37권12호
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• pp.967-977
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• 2004

배경: DNA 메칠화란 유전자의 Promoter에 있는 CpG dinucleotide의 cytosine기에 메칠기가 붙는 현상을 말한다. CpG dinucleotide에 과메틸화가 일어나면 일부 유전자의 발현이 감소되며, 그 반대로 CpG dinucleotide의 메칠화가 억제되면 유전자 발현이 증가된다. DNA 메칠화 억제제인 5-aza-2'- deoxycytidine (ADC)을 폐암세포에 처치했을 때 암항원 유전자의 발현 유무와 이를 위한 최적 조건을 조사하고, 아울러 MHC와 B7의 발현과 세포 성장에 미치는 영향을 조사하여 암치료 백신에 ADC를 임상적으로 이용할 수 있는 지를 연구하였다. 대상 및 방법: 4개의 사람 폐암세포주 (NCIH1703, NCIH522, MRC-5 및 A549)에 ADC를 1 uM 농도로 처치한 후 48시간 뒤에 MAGE family, GAGE, NY-ESO-1, PSMA, CEA 및 SCC항원 유전자에 대한 RT-PCR을 실시하였고, 폐암세포에서 암항원의 발현을 증가시키는 최적의 ADC처치 조건을 규명하기 위하여 ADC농도와 처치 시간을 다양하게 하여 암세포를 자극한 후 암항원 유전자 발현성을 분석하였다. 또한 ADC 처리가 폐암 세포주의 MHC와 B7 발현을 증가시키는 가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시하였고, ADC가 세포성장에 미치는 영향을 알아보기 위하여, ADC를 0.2, 1 및 5 uM 농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 조사하였다. 결과: 세포주에 따라 차이는 있으나 MAGE, GAGE, NY-ESO-1 및 PSMA의 발현이 유도되었으며, MAGE아형 중에는 MAGE-1, -2, -3, -4, -6으로 나타났다. 그러나 비암항원인 CEA발현은 변화가 없었으며 SCC항원 유전자의 발현은 오히려 ADC처치에 의해 감소되었다. ADC 처치 후 24∼48 시간이 지난 뒤부터 암항원 유전자의 발현이 증가하였으며 ADC처리에 의해 유도된 유전자의 발현성은 ABC처치 후 최소 14일까지 유지되었다. 또 ADC를 0.2, 1, 5 uN 농도로 첨가하여 48시간 배양한 후 암항원 유전자 발현성을 측정한 결과 세포주에 따라 다소 차이는 있으나 대개 0.2 uM농도에서도 유전자 발현이 유도되었으며 1, 5 uM농도에서 매우 강하게 유도되었다. ADC 처리가 페암세포주의 MHC와 B7 발현을 증가시키는가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시한 결과 4개의 페암세포주에서 MHC 및 B7분자의 발현은 유도되지 않았다. 또 ADC농도가 세포성장에 미치는 영향을 알아보기 위하여 ADC를 0.2, 1, 5 uM농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 알아본 결과 ADC 처치 농도가 증가함에 따라 세포의 성장은 매우 감소하였다. 결론: 폐암세포주에서 ADC처치는 MAGE, GAGE 및 NY-ESO-1과 같은 세포독성 T 림프구 반응을 유도할 수 있는 암항원의 발현을 증가시킬 수 있으며, ADC의 세포독성과 항원 발현 유발시간을 분석할 때 1 uM 농도에서 48시간 처치한 후 ADC가 없는 배지에서 수일간 배양하는 것이 가장 효과적이라고 생각된다. 그러나, ADC를 처치하여도 MHC 및 B7의 발현의 변화는 없었으므로 ADC를 처치한 폐암세포를 암백신으로 사용하기 위해서는 MHC나 B7 및 cytokine의 발현을 증가시키는 추가적인 처치가 필요하다고 생각된다.