Identification of Highly Reactive Cysteine Residues at Less Exposed Positions in the Fab Constant Region for Site-Specific Conjugation

详细信息    查看全文

• 作者：Yasuhisa Shiraishi ; Takashige Muramoto ; Kazutaka Nagatomo ; Daisuke Shinmi ; Emiko Honma ; Kazuhiro Masuda ; Motoo Yamasaki
• 刊名：Bioconjugate Chemistry
• 出版年：2015
• 出版时间：June 17, 2015
• 年：2015
• 卷：26
• 期：6
• 页码：1032-1040
• 全文大小：333K
• ISSN：1520-4812

文摘

Engineered cysteine residues are currently used for the site-specific conjugation of antibody鈥揹rug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.