High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers

• DOI: 10.1021/acs.bioconjchem.7b00791
• Publication Date: 2018-02-09T19:42:23Z
• Authors: Rachana Ohri; Sunil Bhakta; Aimee Fourie-O’Do...; Josefa dela Cruz-...; Siao Ping Tsai; Ryan Cook; Binqing Wei; Carl Ng; Athena W. Wong; Aaron B. Bos; Farzam Farahi; Jiten Bhakta; Thomas H. Pillow; Helga Raab; Richard Vandlen; Paul Polakis; Yichin Liu; Hans Erickson; Jagath R. Junutula; Katherine R. Kozak
• Keywords: Conjugate; Disulfide Linkage; Succinimides; Maleimide

THIOMAB antibody technology utilizes cysteine residues engineered onto an to allow for site-specific conjugation. The has enabled the exploration of different attachment sites on in combination with small molecules, peptides, or proteins yield conjugates unique properties. As reported previously ( Shen , B. Q. et al. 2012 ) Nat. Biotechnol. 30 184 - 189 ; Pillow T. H. 2017 Chem. Sci. 8 366 370 ), specific location site conjugation can impact stability linkage both thio-succinimide and disulfide bonds. High is usually desired maximize delivery cargo intended target. In current study, cysteines were individually substituted into every position anti-HER2 (trastuzumab), stabilities drug conjugations at those evaluated. We screened a total 648 antibody-drug conjugates, each generated from trastuzamab prepared by sequentially mutating non-cysteine amino acids light heavy chains cysteine. Each variant was conjugated either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) pyridyl monomethyl (PDS-MMAE) using high-throughput, on-bead purification method. Greater than 50% variants successfully MMAE derivatives ratio (DAR) >0.5 <50% aggregation. relative vitro plasma approximately 750 assessed enzyme-linked immunosorbent assays, stable confirmed affinity-capture LC/MS-based detection methods. Highly two types identified chains. Although maleimide shown be greater many that both. Furthermore, selected translated across cytotoxic payloads target antibodies as well vivo stability.