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Cmyc tag 펩타이드에 특이적인 9E10 VHH 나노바디의 개발
김진규,한승희,김수현,홍선나 한국미생물학회 2022 미생물학회지 Vol.58 No.3
Based on structure of murine 9E10 antibody bound to cmyc tag peptide antigen, It was found that the complementarity determining region 3 of the heavy chain variable region of the 9E10 antibody consists of 18 long amino acids, which dominantly involves in cmyc tag peptide binding. This finding led us to engineer heavy chain variable domain of original mouse-derived 9E10 antibody into a camelid 9E10 nanobody. Since the camelid antibody consists only of a heavy chain variable domain with a size of only about 15 kDa and high solubility, it is called as 9E10 VHH nanobody. To produce 9E10 VHH nanobody, the cDNA for the heavy chain variable domain of murine 9E10 antibody was extracted from the hybridoma 9E10 cell line and its translated amino acids sequences was aligned with the camelid heavy chain variable domain sequences to replace mismatched murine amino acids with camelid amino acids. After completion of cloning of each gene into pUC119 expression vector, both original murine 9E10 heavy chain variable domain and 9E10 VHH nanobody were expressed and purified as a soluble protein in Escherichia coli. As expected, only camelid 9E10 VHH nanobody was expressed as a soluble protein. It showed the cmyc tag peptide binding activity in both enzyme-linked immunosorbent assay and Western blot. In addition, the range of dissociation constant for the interaction with the cmyc tag peptide was determined from 4.43 × 10-7 to 4.77 × 10-6 M by using bio-layer interferometry.
Neutralization of Human Papillomavirus by Specific Nanobodies Against Major Capsid Protein L1
( Minaeian Sara ),( Fatemeh Rahbarizadeh ),( Sayyed Hamid Zarkesh Esfahani ),( Davoud Ahmadvand ),( Oliver Jay Broom ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.5
The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigenbinding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a Ni+-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.
CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin
Jingbo Ma,Xiaolong Xu,Chunjin Fu,Peng Xia,Ming Tian,Liuhai Zheng,Kun Chen,Xiaolian Liu,Yilei Li,Le Yu,Qinchang Zhu,Yangyang Yu,Rongrong Fan,Haibo Jiang,Zhifen Li,Chuanbin Yang,Chengchao Xu,Ying Long,J 한국생체재료학회 2022 생체재료학회지 Vol.26 No.4
Background: It is highly desirable to develop new therapeutic strategies for gastric cancer given the low survival rate despite improvement in the past decades. Cadherin 17 (CDH17) is a membrane protein highly expressed in cancers of digestive system. Nanobody represents a novel antibody format for cancer targeted imaging and drug delivery. Nanobody targeting CHD17 as an imaging probe and a delivery vehicle of toxin remains to be explored for its theragnostic potential in gastric cancer. Methods: Naïve nanobody phage library was screened against CDH17 Domain 1-3 and identified nanobodies were extensively characterized with various assays. Nanobodies labeled with imaging probe were tested in vitro and in vivo for gastric cancer detection. A CDH17 Nanobody fused with toxin PE38 was evaluated for gastric cancer inhibition in vitro and in vivo. Results: Two nanobodies (A1 and E8) against human CDH17 with high affinity and high specificity were successfully obtained. These nanobodies could specifically bind to CDH17 protein and CDH17-positive gastric cancer cells. E8 nanobody as a lead was extensively determined for tumor imaging and drug delivery. It could efficiently co-localize with CDH17-positive gastric cancer cells in zebrafish embryos and rapidly visualize the tumor mass in mice within 3 h when conjugated with imaging dyes. E8 nanobody fused with toxin PE38 showed excellent anti-tumor effect and remarkably improved the mice survival in cell-derived (CDX) and patient-derived xenograft (PDX) models. The immunotoxin also enhanced the anti-tumor effect of clinical drug 5-Fluorouracil. Conclusions: The study presents a novel imaging and drug delivery strategy by targeting CDH17. CDH17 nanobodybased immunotoxin is potentially a promising therapeutic modality for clinical translation against gastric cancer.
Han Choe,Minh Quan Nguyen,Do Hyung Kim,Hye Ji Shim,Huynh Kim Khanh Ta,Thi Luong Vu,Thi Kieu Oanh Nguyen,Jung Chae Lim 한국분자세포생물학회 2023 Molecules and cells Vol.46 No.12
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.
Application of Single-Domain Antibodies (“Nanobodies”) to Laboratory Diagnosis
Pillay Tahir S.,Muyldermans Serge 대한진단검사의학회 2021 Annals of Laboratory Medicine Vol.41 No.6
Antibodies have proven to be central in the development of diagnostic methods over decades, moving from polyclonal antibodies to the milestone development of monoclonal antibodies. Although monoclonal antibodies play a valuable role in diagnosis, their production is technically demanding and can be expensive. The large size of monoclonal antibodies (150 kDa) makes their re-engineering using recombinant methods a challenge. Single-domain antibodies, such as “nanobodies,” are a relatively new class of diagnostic probes that originated serendipitously during the assay of camel serum. The immune system of the camelid family (camels, llamas, and alpacas) has evolved uniquely to produce heavy-chain antibodies that contain a single monomeric variable antibody domain in a smaller functional unit of 12–15 kDa. Interestingly, the same biological phenomenon is observed in sharks. Since a single-domain antibody molecule is smaller than a conventional mammalian antibody, recombinant engineering and protein expression in vitro using bacterial production systems are much simpler. The entire gene encoding such an antibody can be cloned and expressed in vitro. Single-domain antibodies are very stable and heat-resistant, and hence do not require cold storage, especially when incorporated into a diagnostic kit. Their simple genetic structure allows easy re-engineering of the protein to introduce new antigen-binding characteristics or attach labels. Here, we review the applications of single-domain antibodies in laboratory diagnosis and discuss the future potential in this area.