• YAKHAK HOEJI
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• v.54 no.6
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• pp.500-506
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• 2010

TNF-${\alpha}$ and VCAM-1 play a pivotal role in the pathogenesis of rheumatoid arthritis, and the development of drugs targeting these molecules has extended the therapeutical approaches to rheumatoid arthritis patients. Bispecific antibodies combine the antigen-binding sites of two antibodies within a single molecule and thus they are able to bind to two different epitopes simultaneously. A specific bispecific antibody format termed "Di-diabody" was made for the efficient approach to anti-inflammation. In this study, the DNA vector construct of Di-diabody was built up against two antigens, VCAM-1 and TNF-${\alpha}$. For evaluating this Di-diabody as a bispecific antibody on the efficacy of anti-inflammation, the proteins were analyzed according to each antigen binding affinity and cell based assay related separate molecules. The 7H/Humira Di-diabody produced in this study interacted with its ligands, VCAM-1 and TNF-${\alpha}$, respectively. Also, this antibody exhibited the similar functional activities as compared to 7H-IgG in respect to inhibition of hVCAM-1-induced cell adhesion and Humira-IgG in respect to inhibition of TNF-${\alpha}$ induced cytotoxicity. Further study to elucidate the pharmacological significance of the Di-diabody is warranted using experimental animals.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.418-426
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• 2022

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1⁺ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8⁺ T cells having CD8⁺CD44⁺CD62L^low phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.

• Biomedical Science Letters
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• v.26 no.3
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• pp.136-148
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• 2020

A bispecific antibody (BsAb) is an artificial protein containing two kinds of specific antigen binding sites. BsAb can connect target cells to functional cells or molecules, and thus stimulate a directed immune response. Last several decades a wide variety of bsAb formats and production technologies have been developed. BsAbs are constructed either chemically or biologically, exploiting techniques like cell fusion and recombinant DNA technologies. There are over 100 different formats of bsAb so far developed, but they could be classified into the two main categories such as Fc-based (with a Fc region) bsAbs and fragment-based (without a Fc region) bsAbs. BsAb has a broad application prospect in tumor immunotherapy and drug delivery. Here, we present a brief introduction to the structure of antibody, pharmacological mechanisms of antibodies and the trend in the production technologies of therapeutic antibodies. In addition, we address a review on the current status of various bsAb format development and their production technologies together with global situation in the clinical studies of bsAb.

• BMB Reports
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• v.47 no.3
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• pp.130-134
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• 2014

The combination of a high-affinity antibody to a hapten, and hapten-conjugated compounds, can provide an alternative to the direct chemical cross-linking of the antibody and compounds. An optimal hapten for in vitro use is one that is absent in biological systems. For in vivo applications, additional characteristics such as pharmacological safety and physiological inertness would be beneficial. Additionally, methods for cross-linking the hapten to various chemical compounds should be available. Cotinine, a major metabolite of nicotine, is considered advantageous in these aspects. A high-affinity anti-cotinine recombinant antibody has recently become available, and can be converted into various formats, including a bispecific antibody. The bispecific anti-cotinine antibody was successfully applied to immunoblot, enzyme immunoassay, immunoaffinity purification, and pre-targeted in vivo radioimmunoimaging. The anti-cotinine IgG molecule could be complexed with aptamers to form a novel affinity unit, and extended the in vivo half-life of aptamers, opening up the possibility of applying the same strategy to therapeutic peptides and chemical compounds.

• BMB Reports
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• v.53 no.10
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• pp.533-538
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• 2020

Notch signaling has been identified as a critical pathway in gastric cancer (GC) progression and metastasis, and inhibition of Delta-like ligand 4 (DLL4), a Notch ligand, is suggested as a potent therapeutic approach for GC. Expression of both DLL4 and vascular endothelial growth factor receptor 2 (VEGFR2) was similar in the malignant tissues of GC patients. We focused on vascular endothelial growth factor (VEGF), a known angiogenesis regulator and activator of DLL4. Here, we used ABL001, a DLL4/VEGF bispecific therapeutic antibody, and investigated its therapeutic effect in GC. Treatment with human DLL4 therapeutic antibody (anti-hDLL4) or ABL001 slightly reduced GC cell growth in monolayer culture; however, they significantly inhibited cell growth in 3D-culture, suggesting a reduction in the cancer stem cell population. Treatment with anti-hDLL4 or ABL001 also decreased GC cell migration and invasion. Moreover, the combined treatment of irinotecan with anti-hDLL4 or ABL001 showed synergistic antitumor activity. Both combination treatments further reduced cell growth in 3D-culture as well as cell invasion. Interestingly, the combination treatment of ABL001 with irinotecan synergistically reduced the GC burden in both xenograft and orthotopic mouse models. Collectively, DLL4 inhibition significantly decreased cell motility and stem-like phenotype and the combination treatment of DLL4/VEGF bispecific therapeutic antibody with irinotecan synergistically reduced the GC burden in mouse models. Our data suggest that ABL001 potentially represents a potent agent in GC therapy. Further biochemical and pre-clinical studies are needed for its application in the clinic.

• Biomolecules & Therapeutics
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• v.30 no.4
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• pp.299-308
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• 2022

T cells are attractive targets for the development of immunotherapy to treat cancer due to their biological features, capacity of cytotoxicity, and antigen-specific binding of receptors. Novel strategies that can modulate T cell functions or receptor reactivity provide effective therapies, including checkpoint inhibitor, bispecific antibody, and adoptive transfer of T cells transduced with tumor antigen-specific receptors. T cell-based therapies have presented successful pre-clinical/clinical outcomes despite their common immune-related adverse effects. Ongoing studies will allow us to advance current T cell therapies and develop innovative personalized T cell therapies. This review summarizes immunotherapeutic approaches with a focus on T cells. Anti-cancer T cell therapies are also discussed regarding their biological perspectives, efficacy, toxicity, challenges, and opportunities.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2795-2798
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• 2012

Objective: This study is conducted to evaluate the effects of anti-HER-2${\times}$anti-CD3 bi-specific antibodies(BsAb) on HER-2/neuover-expressing human colorectal carcinoma cells. Methods: Growth was assessed by MTT assays after exposure of HCT-116 cells to Herceptin, anti-CD3 and BsAb antibodies. Immunocytochemistry was applied to test the HER-2 level of HCT-116. In a nude mouse model, HER-2${\times}$CD3 BsAb was combined with effector cells (peripheral blood lymph cells from normal human being) for observations on in Vivo growth of tumors. Results: Compared with the control group, using effector cells combined with anti-CD3 McAb, Herceptin or HER2${\times}$CD3 BsAb, tumor cell growth in vitro and in vivo was significantly inhibited (P<0.05), most remarkably in the HER2${\times}$CD3 BsAb case. The growth of xenografts with HER2${\times}$CD3 BsAb combined with effector cells was also significantly inhibited when compared with the anti-CD3 McAb or Herceptin groups (P<0.05). Conclusion: HER-2/neu might be a useful target for immunotherapy in colorectal carcinoma, anti-HER2${\times}$anti-CD3 BsAb exerting clear anti-tumor effects.