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

• KSBB Journal
• /
• 제31권4호
• /
• pp.284-290
• /
• 2016

In this research, recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

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

• Journal of Microbiology and Biotechnology
• /
• 제17권12호
• /
• pp.1944-1948
• /
• 2007

Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at $60^{\circ}C$ for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SH-added medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.

• 미생물학회지
• /
• 제49권3호
• /
• pp.221-227
• /
• 2013

인간면역결핍바이러스(Human immunodeficiency virus; HIV), B형 간염 바이러스(Hepatitis B virus; HBV), 그리고 C형 간염 바이러스(Hepatitis C virus; HCV)는 만성 감염질환을 일으키는 대표적인 바이러스들이다. 인체내 감염시 임상적 진행경과에 따른 바이러스 특이 T림프구의 항바이러스 기능변화 및 바이러스의 체내 지속성과 T림프구에 발현되는 다양한 면역인자(e.g., CD28, CD25, FoxP3, PD-1, CTLA-4)들과의 구체적인 상관관계는 최근 많은 국내외 연구진들을 통해 연구되고 있다. 그 중 FoxP3 (forkhead box P3), PD-1 (programmed death-1) 그리고 CTLA-4 (cytotoxic T lymphocyte-associated antigen 4)는 T림프구에서 발현되는 면역조절인자로 만성 바이러스성 감염시 그 발현이 증가되는 것으로 관찰되었으며, 항바이러스 작용을 가지는 T림프구의 기능결핍과 밀접한 상관관계가 있는 것으로 알려져 있다. 본 총설에서는 만성적인 HIV, HBV, 그리고 HCV 감염에서 바이러스 특이 T림프구에서 발현되는 FoxP3, PD1, 그리고 CTLA-4의 발현변화와 각 질환의 임상적 진행경과와의 상관성, 그리고 이들 발현이 T림프구의 항바이러스 기능에 미치는 영향 등을 중심으로 기술하였다.

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

• Journal of Microbiology and Biotechnology
• /
• 제19권8호
• /
• pp.781-786
• /
• 2009

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

• KSBB Journal
• /
• 제28권4호
• /
• pp.260-268
• /
• 2013

Transgenic plant cell cultures are an attractive expression system for the production of industrial and pharmaceutical proteins because of their advantages in safety and low production cost. Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced and secreted when sugar was depleted in culture medium by transgenic rice cell lines (Oryza sativa L.) using RAmy3D promoter. Due to the production of the target protein by sugar depletion, concomitant occurrence of cell death is inevitable. For that reason, inhibition of cell death for enhancing productivity was necessary for the production period without energy sources. Supplementation of 0.1 mM sodium nitroprusside improved cell viability by 1.4-fold and maximum hCTLA4Ig production by 1.3-fold compared to those of control. Addition of 1 and 10 mM glutathione, N-acetylcysteine (NAC), and nicotinamide inhibited apoptotic-like programmed cell death by decreasing the activity of reactive oxygen species. Production hCTLA4Ig was enhanced 1.4-, 1.25-, and 1.15-fold with 10 mM NAC, 1 mM NAC, and 1 mM glutathione, respectively. In addition, it was found that the supplementation of NAC enhanced the cell viability.

• IMMUNE NETWORK
• /
• 제2권1호
• /
• pp.35-40
• /
• 2002

Background: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail. Methods: In this study CD152 expression in both $CD4^+$ and $CD8^+$ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex. Results: Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were $CD152^+$ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in $CD8^+$ cells and about 60% of $CD8^+$ cells were $CD152^+$ at this stage. High molecular weight (>350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non-reducing condition. Conclusion: It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.

• 생물정신의학
• /
• 제10권1호
• /
• pp.80-84
• /
• 2003

Objective : Bipolar disorder is known to have strong genetic background and cellular immune activation. Based on the hypothesis that abnormalities of normal inhibitory control of T cell immunity can contribute to the pathophysiology of bipolar disorder, we investigated the relationship between the first exon at position +49(A/G) polymorphism of cytotoxic T lymphocyte antigen 4(CTLA4) gene and bipolar disorder. Method : Among the Korean patients diagnosed as bipolar disorder according to DSM-IV, 90 patients without serious medical illness, neurologic illness, hormonal disorder, or concomitant mental illness were selected. The normal control group consisted of 149 age-and sex-matched subjects without current or past history of autoimmune diseases or mental disorder. DNA was extracted from whole blood and the exon 1 region of CTLA-4 gene was amplified by polymerase chain reaction. Gene typing was performed using single strand conformation polymorphism. Results : There were no significant differences in genotype frequencies of G/G, G/A, and A/A between the patients with bipolar disorder and the control group(48.9% vs 46.3%, 44.4% vs 39.6%, and 6.7% vs 14.1%, respectively). There were no significant differences in allelic frequencies of G and A between the patients with bipolar disorder and the control group(71.1% vs 66.1%, and 28.9% vs 33.9%, respectively). Conclusion : This study did not show the association of exon 1 polymorphism of CTLA-4 gene with bipolar disorder.