Publications

Detailed Information

Improvement in anti-cancer activity and binding affinity of the therapeutic anti-HER2 monoclonal antibody and bioprocess development

Cited 0 time in Web of Science Cited 0 time in Scopus
Authors

문승기

Advisor
유영제
Major
공과대학 협동과정
Issue Date
2016-02
Publisher
서울대학교 대학원
Keywords
Antibody engineeringAntibody optimizationHER2Phage displayRandom mutagenesisBioprocess optimization
Description
학위논문 (박사)-- 서울대학교 대학원 : 협동과정 생물화학공학전공, 2016. 2. 유영제.
Abstract
To generate a bio-better which has improved therapeutic activity than that of hu4D5 (Herceptin), the hu4D5 antibody was used as a model system. scFv libraries were constructed by random mutagenesis of several resides of CDR-H3, L3 and L2 of hu4D5, and the scFv clones isolated from the phage display libraries using the stringent panning, and their anti-proliferative activity as IgG1 against breast cancer cells was evaluated as a primary selection criterion.
Among 139 variants variant, AH06 was the best candidate as a bio-better antibody that has an increase by 7.2-fold in anti-proliferative activity (IC50 : 0.81 nM) against gastric cancer cell NCI-N87 and by 7.4-fold in binding affinity (KD : 60 pM) to HER2 compared to hu4D5, respectively.
AH06 specifically bound to domain IV of HER2 and did not have cross-reactivity with other receptor tyrosine kinases (RTK) except HER2, and decreased the level of phosphorylation of HER2 and AKT to a similar extent, but most of all, highly increased the overall level of p27 in gastric cancer cell NCI-N82 as compared to hu4D5.
Binding energy calculation indicated that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2. And, molecular modeling stimulation indicated that the direct hydrophobic interactions between the aromatic ring of W98 within AH06 and the aliphatic group of I613 within antigen HER2 domain IV, and the inter-chain hydrophobic interactions between the phenyl ring of F100c in CDR-H3 and the hydrophobic groups that consist of Y36, P44 and F98 located in VL could have synergistic effects on improvement of binding affinity of AH06 to HER2.
The expression vector for producing AH16 was constructed, and the AH16 producing AH16F1-3-14-80-26 clone whose antibody growth rate and productivity had been consistently maintained during 1~15 passages was finally screened as a stable producer cell line. One basal media and 3 additives were selected, and the final productivity of AH16 using the optimized media mixture was approximately 1.3g/L in flask culture.
The three chromatography steps using Protein A resin column (1st step) and two steps of ion-exchange chromatography provided the yield of 93.8% and the purity of 99.9%, and non-Protein A-based cation-exchange chromatography provided the yield of 99% and the purity of 97.5%.
And, analysis of physico-chemical, biological and immunological characteristics of AH16 verified that it meets the standards set based on the Specifications and Analytical Procedures.
Through this study, the upstream antibody engineering technology required to discover and improve biological efficacy of various therapeutic lead antibodies and the downstream bioprocess technology required to produce antibody were able to be established and applied.
Language
English
URI
https://hdl.handle.net/10371/134965
Files in This Item:
Appears in Collections:

Altmetrics

Item View & Download Count

  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Share