title: Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen.
abstract: To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.|Graduate Program of Bioengineering, Seoul National University, Seoul 151-742, Korea.
authors.forename: Seung Kee
authors.initials: SK
authors.lastname: Moon
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: So Ra
authors.initials: SR
authors.lastname: Park
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: Ami
authors.initials: A
authors.lastname: Park
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: Hyun Mi
authors.initials: HM
authors.lastname: Oh
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: Hyun Jung
authors.initials: HJ
authors.lastname: Shin
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Jeon
authors.affiliation: Bio Medicine Lab., CKD Research Institute, ChongKunDang Pharm., Yongin 446-916, Korea.
authors.forename: Seiwhan
authors.initials: S
authors.lastname: Kim
authors.affiliation: Graduate Program of Bioengineering, Seoul National University, Seoul 151-742, Korea.
authors.forename: Hyun June
authors.initials: HJ
authors.lastname: Park
authors.affiliation: The Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea.
authors.forename: Young Joo
authors.initials: YJ
authors.lastname: Yeon
authors.affiliation: Graduate Program of Bioengineering, Seoul National University, Seoul 151-742, Korea.
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
mesh_terms: D000915:Antibody Affinity; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D022801:Complementarity Determining Regions / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D010766:Phosphorylation / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D022801:Complementarity Determining Regions; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human
keywords: HER2; anti-proliferative activity; antibody optimization; binding affinity; phage display; random mutagenesis