- Guided Selection of Human Antibody Light Chains against TAG-72 Using a Phage Display Chain Shuffling Approach
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Sang Jick Kim , Hyo Jeong Hong
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J. Microbiol. 2007;45(6):572-577.
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DOI: https://doi.org/2606 [pii]
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To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.
- Generation and Characterization of a Monoclonal Antibody with Specificity for Mycoplasma arginini
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Yeon Sung Son , Hyo Jeong Hong
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J. Microbiol. 2007;45(6):547-552.
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DOI: https://doi.org/2610 [pii]
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Previously, we generated monoclonal antibodies (MAbs) that bound to the surface of human embryonic stem cells (hESCs) in an attempt to discover new hESC-specific surface markers. In this study, MAb 47-235 (IgG1, κ) was selected for further characterization. The MAb bound to the surface of undifferentiated hESCs but did not bind to mouse ESCs or mouse embryonic fibroblast cells in flow cytometric analysis. The antibody immunoprecipitated a 47 kDa protein from the lysates of cell surface-biotinylated hESCs. Identification of the protein by quadrupole time of flight tandem mass spectrometry revealed that 47-235 binds to Ag 243-5 protein of Mycoplasma arginini. BM-Cyclin treatment of the hESCs that reacted with 47-235 resulted in loss of mycoplasma DNA and the reactivity to 47-235. Nevertheless, the hESCs that were reactive to 47-235 maintained self-renewal and pluripotency and thus could be differentiated into three embryonic germ layers.
- Affinity Maturation of an Anti-Hepatitis B Virus PreS1 Humanized Antibody by Phage Display
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Gi-Hyeok Yang , Sun Ok Yoon , Myung Hee Jang , Hyo Jeong Hong
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J. Microbiol. 2007;45(6):528-533.
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DOI: https://doi.org/2640 [pii]
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In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementarydetermining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.
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