• Journal of Korean Neurosurgical Society
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• v.29 no.6
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• pp.731-737
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• 2000

Objective : Growth factor receptors on the tumor cells are known to be expressed highly allowing the tumor cells to bind growth factors to stimulate cellular division. Immunotoxin therapy is one of the novel approaches to the primary malignant brain tumor, and expression of cell-surface receptor is essential for the immunotoxin to have specific anti-tumor activity. Despite promising cytotoxic activity of immunotoxin, tumor responses are not curative on clinical trials, and additional studies are needed regarding various factors influencing the efficacy of the immunotoxin. The purpose of this study is to detect the expression of various growth factor receptors on brain tumor cell lines which are going to be used in these studies. Materials and Methods : The authors detected transferrin receptor(TR), insulin-like growth factor-1 receptor(IGF-1R), and interleukin-4 receptor(IL-4R) on medulloblastoma cell line(Daoy) and glioblastoma cell lines(U373 MG and T98 G) by flow cytometric analysis. Results : TR was expressed on Daoy, U373 MG, and T98 G. IGF-1R was expressed on Daoy and U373 MG, but not on T98 G. IL-4R was expressed on all cell lines tested. Conclusion : The transferrin and interleukin-4 receptors might be good targets for immunotoxin therapy. The results should be considered in additional in vitro and in vivo studies regarding immunotoxin and in establishing the proper treatment model of the immunotoxin therapy including selection of the adequate immunotoxin.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.459-463
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• 1999

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-2-pyridyldithio)toluamido]toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.

• The Journal of Korean Society of Virology
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• v.28 no.4
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• pp.383-391
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• 1998

A murine monoclonal antibody (mAb) specific for the envelope glycoprotein gp120 of human immunodeficiency virus type-I (HIV -1) was chemically coupled to pokeweed antiviral protein (PAP) from Phytolacca americana. The immunotoxin was purified by FPLC using S200 colum. The purified immunotoxin efficiently bound to HIV-infected T cells as evidenced by fluorescenceactivated cell sorter analysis. The immunotoxin selectively killed human T lymphoid lines infected with $HIV-1_{IIIB}$ at less than 250 pM of the immunotoxin cells, while PAP or mAb alone did not have any significant effect on infected cells. The uninfected control T cell lines were not affected. Human cells infected with HIV-2 or other HIV-1 strains were not killed, suggesting that the killing depends completely on the antibody used for coupling. These in vitro results suggest that the PAP-mAb conjugate may be used to selectively remove cells expressing viral antigens from individuals infected with HIV.

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.210-210
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• 1994

천연자원으로부터 얻어진 항암 또는 세포독성물질로는 주로 alkaloid, lignan, terpenoid, macrolide 등에 속하는 물질들이고 그 중 sesquiterpene lactone계열 화합물에서 세포독성 및 항종양 활성이 있는 물질이 다수 얻어졌으며 그 대부분은 국화과의 식물에서 분리 보고되고 있다. Achillea 속 식물은 국화과에 속하는 다년생초본으로 남한에는 톱풀과 산톱풀 2종만이 자생한다. screening 결과 이 두 식물에는 segquiterpene lactone 화합물과 Peroxide등 다수의 활성성분이 함유되어 있을 것으로 기대되어 본 연구에 착수하였다. 두 식물의 각 분획에 대하여 SNU-1(위암세포주)과 SNU-354(간암세포주)를 이용해 검색한 결과 두 식물 모두 MC ext.와 MeOH ext.에서 현저한 세포독성을 보였으며 현재 MC ext. 로부터 세포독성물질의 분리를 수행하고 있다. EtOAc ext.는 세포독성은 거의 나타내지 않았으나 flavonoid 성분이 다수 존재함을 확인하였으며 면역활성 및 enzyme inhibition등의 활성을 검토하고자 화합물을 분리하였다. 그 결과 톱풀로 부터는 luteolin-7-0-glucoside와 apigenin-7-0-glucoside를 그리고 산톱풀로부터는 luteolin-7-0-glucoside, apigenin-7-0-glucoside, luteolin, apigenin을 분리 확인 동정하였다.

• Toxicological Research
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• v.11 no.2
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• pp.193-197
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• 1995

Antibody-toxin conjugates, termed immunotoxins, are currently being evaluated as potential new anticancer agents and one of the most extensively studied toxins for construction of immunotoxin is ricin which exists in the seeds of castor bean, Ricinus communis. Another toxic lectin from castor bean is RCA (Ricinus communis agglutinin). Both toxins are very homologous. We reported the puriffcation procedure and biological properties of ricin from the Korean castor bean in another place and here we report those of RCA. The purified RCA shows three bands on denatured SDS PAGE while ricin shows two bands. On cultured $K_{562}$ cells ricin and RCA both inhibit the multiplication of cells extensively. $30{\mu}g/ml$ of ricin shows 73% of inhibition rate at day 4 compared to 68% in same condition of RCA. The inhibition of multiplication of cells are directly proportional to the concentration of toxins and the incubation period. In every case ricin was more toxic than RCA. The $LD_{50}$ dose of ricin on ICR mice was 60 ng at day 3 but that of RCA was $10{\mu}g$.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.136-136
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• 1993

immunotoxin으로 쓰일 수 있는 리보솜 불활성화 단백질 (RIP, ribosome-inactivating protein)을 보리 씨앗에서 분리하였다 이 단백질은 분자량이 약 30,000 kDa 정도되고, pl가 9.0 보다 높다. 이러한 성질을 이용하여 Na-phosphate 완충용액으로 추출하고, 황산암모늄 60-80％ 포화로 분획화하였다. 다음 CM-cellulose를 이용한 이온 교환 크로마토그래피, Sephacryl S-200 HR 컬럼을 이용한 gel filtration을 하여 순수히 분리하였고, 이를 전기영동하여 확인하였다.

• Bulletin of the Korean Chemical Society
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• v.22 no.12
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• pp.1361-1365
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• 2001

Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudo monas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5977-5981
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• 2015

Background: Worldwide, breast cancer is the most common cancer diagnosed among women and a leading cause of cancer deaths. The age of onset in Iran has become reduced by a decade for unknown reasons. Herceptin, a humanized monoclonal antibody, is a target therapy for breast cancer cells with over expression of HER2-neu receptors, but it is an expensive drug with only 20% beneficial rate of survival. This study introduces a novel approach to enhance the efficacy of this drug through immunoconjugation of the antibody to botulinum toxin. Decreasing the cost and adverse effects of the antibody were secondary goals of this study. Materials and Methods: Botulinum toxin was conjugated with Herceptin using heterobifunctional cross linkers, succinimidyl acetylthiopropionate (SATP) and sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the supplier's guidelines and tested on two breast cancer cell lines: SK-BR-3 and BT-474. Toxin and Herceptin were also used separately as controls. The cytotoxicity assay was also performed using the new bioconjugate on cultured cells with Alamar blue and a fluorescence plate reader. Results: Herceptin-Toxin bioconjugation significantly improved Herceptin efficacy on both breast cancer cell lines when compared to the control group. Conclusions: Toxin-Herceptin bioconjugation can be a potential candidate with increased efficiency for treating breast cancer patients with over expression of the HER2 receptor.

• Molecules and Cells
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• v.46 no.12
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• pp.764-777
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• 2023

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC₅₀ values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.