• BMB Reports
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• 제38권4호
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.121-124
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• 2002

This work was initiated to develope a recombinant oral poliovaccine (OPV), which is highly advanced in safety (minimizing VAPP) by introducing Type 2,3 poliovirus epitopes into our RPS-Vax system. We have introduced several potential vaccine epitopes of poliovirus Type 2, and 3 into RPS-Vax system, resulting in production of recombinant polioviruses. Any of these chimeric viruses, however, were not detected for their foreign gene expression by serotype-specific mouse antiserum. We have designed several folding units to stabilize the introduced vaccine protein and attached short epitope-concatamer or epitope-multimer to them, followed by production of chimeric viruses. Only those who have an HIV-1 Tat-mediated folding unit were nicely detected for the introduced foreign proteins by anti-Tat antiserum and type-specific peptide-induced antisera. Nevertheless, introduced epitopes were not detected in Western blot experiment with each serotype-specific antiserum. None of the mice inoculated with these chimeric viruses showed preventative immunity when challenged with Lansing and Leon wildtype 2 and 3 poliovirus, and the antiserum did not show neutralizing capacity in vitro. Conformational epitope covering B/C loop region of type 2 and 3 were newly designed by computer modeling, and introduced into the RPS-Vax vector system, followed by production of chimeric viruses. Introduced epitope regions were nicely detected by anti-Tag23 mAb or peptide antibody, but still not detected by poliovirus antiserum. Nevertheless, neutralizing antibody was detected in the Tg-PVR mice even when inoculated once with these chimeric viruses. Also, the immunized mice showed perfect preventative immunity against the wild Type poliovirus Lancing or Leon. When boosted appropriately, those chimeric virus-inoculated Tg-PVR mice produced equivalent amounts of neutralizing antibody to those in Sabin 2/3-immunized mice. These data strongly suggest that our recombinant poliovirus (RPS-PV2 and RPS-PV3) can be used as a safe and effective rec-OPV instead of any preexisting poliovaccine.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.304-316
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• 2021

Vaccination is the most effective way to prevent influenza virus infections. However, conventional vaccines based on hemagglutinin (HA) have to be annually updated because the HA of influenza viruses constantly mutates. In this study, we produced a 3M2e-3HA2-NP chimeric protein as a vaccine antigen candidate using an Escherichia coli expression system. The vaccination of chimeric protein (15 ㎍) conferred complete protection against A/Puerto Rico/8/1934 (H1N1; PR8) in mice. It strongly induced influenza virus-specific antibody responses, cytotoxic T lymphocyte activity, and antibody-dependent cellular cytotoxicity. To spare the dose and enhance the cross-reactivity of the chimeric, we used a complex of poly-γ-glutamic acid and alum (PGA/alum) as an adjuvant. PGA/alum-adjuvanted, low-dose chimeric protein (1 or 5 ㎍) exhibited higher cross-protective effects against influenza A viruses (PR8, CA04, and H3N2) compared with those of chimeric alone or alum-adjuvanted proteins in vaccinated mice. Moreover, the depletion of CD4+ T, CD8+ T, and NK cells reduced the survival rate and efficacy of the PGA/alum-adjuvanted chimeric protein. Collectively, the vaccination of PGA/alum-adjuvanted chimeric protein induced strong protection efficacy against homologous and heterologous influenza viruses in mice, which suggests that it may be a promising universal influenza vaccine candidate.

• 식물조직배양학회지
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• 제21권6호
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• pp.353-356
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• 1994

사람 B형 간염 바이러스의 pre-S2 표면항원에 결합하는 키메라 항체 유전자(카파 및 감마사슬의 cDNA클론)를 식물체에서 발현시키기 위해 식물체 발현벡터인 pBKS-1에 XbaI 자리를 이용하여 클로닝하였다. 이들 유전자를 포함하는 대장균의 플라스미드 핵산을 추출하여 아그로박테리움에 형질전환 시켰다. 다음 담배의 조직절편과 아그로박테리움을 공동배양함으로써 담배의 형질 전환을 시도하였다. 카나마이신이 포함된 신초유기배지에서 나온 신초를 시료로 하여 Western blot을 실시함으로써 이들 유전자가 형질전환 담배에서 안정하게 발현됨을 확인하였다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1998년도 Proceedings of UNESCO-internetwork Cooperative Regional Seminar and Workshop on Bioassay Guided Isolation of Bioactive Substances from Natural Products and Microbial Products
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• pp.150-151
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• 1998

Drug delivery to the brain is facilitated by the synthesis of chimeric peptides, where in neuropharmaceuticals are linked to a vector such as an antibody to the transferrin receptor that mediates transcytosis through the blood-brain barrier (BBB). When disulfide linkers are used in the chimeric peptide, it is crucial that the S-S bridge is stable during transit and that cleavage does not occur prematurely within endothelial cells, as the peptide drug moiety would then be sequestered by the BBB instead of passing through it. The present study addressed that problem. As a model drug a metabolically stable opioid peptide, [$^3$H]DALDA (Tyr-dArg-Phe-Lys-NH$_2$), was used. It was monobiotinylated with NHS-SS-biotin to yield bio-[$^3$H]DALDA. The biotinylated peptide was bound to the vector OX26-SA which is a covalent conjugate of OX26 and streptavidin (molar ratio = 1: 1). In vitro treatment of the chimeric peptide, bio-[$^3$H]DALDA/OX26-SA, with a reducing agent, dithiothreitol, released the labeled peptide from the vector by conversion of bio-[$^3$H]DALDA to the desbiotinylated derivative, desbio-[$^3$H]DALDA.

• IMMUNE NETWORK
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• 제22권1호
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• pp.4.1-4.22
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• 2022

In the era of immunotherapeutic control of cancers, many advances in biotechnology, especially in Ab engineering, have provided multiple new candidates as therapeutic immuno-oncology modalities. Bispecific Abs (BsAbs) that recognize 2 different antigens in one molecule are promising drug candidates and have inspired an upsurge in research in both academia and the pharmaceutical industry. Among several BsAbs, T cell engaging BsAb (TCEB), a new class of therapeutic agents designed to simultaneously bind to T cells and tumor cells via tumor cell specific antigens in immunotherapy, is the most promising BsAb. Herein, we are providing an overview of the current status of the development of TCEBs. The diverse formats and characteristics of TCEBs, in addition to the functional mechanisms of BsAbs are discussed. Several aspects of a new TCEB-Blinatumomab-are reviewed, including the current clinical data, challenges of patient treatment, drawbacks regarding toxicities, and resistance of TCEB therapy. Development of the next generation of TCEBs is also discussed in addition to the comparison of TCEB with current chimeric antigen receptor-T therapy.

• Nuclear Medicine and Molecular Imaging
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• 제40권2호
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• pp.66-73
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• 2006

Monoclonal antibodies are designed to bind specifically to certain antigen, give therapeutic effect to the target and to be produced in large scale with homogeneity. The monoclonal antibodies conjugated with radionuclide can deliver therapeutic irradiation to the target, and showed successful results in certain malignancies, which is known as radioimmunotherapy. The target-to-background ratio depends on the antigen expression in the target and normal tissues, which is related to the therapeutic efficacy and toxicity in radioimmunotherapy. For the solid tumor beta-ray energy should be high, but lower beta energy is better for the hematological malignancies. I-l31 is widely used in thyroid cancer with low cost and high availability. Labeling monoclonal antibody with I-131 is relatively simple and reproducible. Some preclinical data for the I-131 labeled monoclonal antibodies including acute toxicity and efficacy are available from already published literatures in KIRAMS, physician sponsored clinical trial protocols using Rituximab, KFDA approved anti-CD20 chimeric monoclonal antibody and I-131 were approved by KFDA and currently are ongoing.

• Clinical and Experimental Vaccine Research
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• 제12권4호
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• pp.304-312
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• 2023

Purpose: Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Materials and Methods: Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe₃O₄ NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. Results: The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. Conclusion: The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1104-1110
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• 2006

Recombinant antibody-toxin is a bifunctional protein that binds and kills a target cell expressing a specific antigen on the surface of the cell, and its structure is chimeric, in which a toxin is fused to an antigen-binding domain such as scFv or Fab. Divalent antibody-toxin molecules showed higher cytotoxicities against cancer cell lines than monovalent molecules. However, the yields of the divalent molecules were very low. In this study, we introduced the CH2, CH3, or CH2-CH3 (=Fc) domain of antibody in the middle of the Fab-toxin between the hinge region of human IgG1 and the toxin domain to increase the yield. The covalently bonded dimer could be formed by three disulfide bridges from cysteine residues in the hinge region. The molecule with the CH3 domain showed about 3-fold higher dimerization yield than previously constructed Fab-toxin molecules, while maintaining the cytotoxic activity comparable to that of scFv-toxin. However, the introduction of CH2 or Fc domain to the same position showed little effect on the dimerization yield. We also observed that the introduction of the CH3 region made it possible to form noncovalently associated dimer molecules.

• BMB Reports
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• 제37권5호
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.