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

化学 半胱氨酸 结合 共轭体系 抗体 二硫键 丁二酰亚胺 组合化学 免疫球蛋白轻链 马来酰亚胺 立体化学 生物化学 有机化学 聚合物 免疫学 数学分析 生物 数学
作者
Rachana Ohri,Sunil Bhakta,Aimee Fourie-O’Donohue,Josefa dela Cruz-Chuh,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 K. Erickson,Jagath R. Junutula,Katherine R. Kozak
出处
期刊:Bioconjugate Chemistry [American Chemical Society]
卷期号:29 (2): 473-485 被引量:106
标识
DOI:10.1021/acs.bioconjchem.7b00791
摘要

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