Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues

Abstract

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

Shifting the Antibody–Drug Conjugate Paradigm: A Trastuzumab-Gold-Based Conjugate Demonstrates High Efficacy against Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer Mouse Model

“…Therefore, bioengineered antibodies were developed with added cysteine residues on the light and heavy chains that provided reactive thiols for conjugation while maintaining antibody assembly and antigen binding . These engineered mAbs based on trastuzumabcalled THIOMAB antibody technologyhave been used in several studies to yield highly potent site-specific ADCs targeting HER2-positive cancers. – …”

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins

“…There are many different sites in Fab and Fc regions that have been engineered to introduce single unpaired Cys residues for site-specific conjugation [ 15 , 45 , 46 , 47 , 48 , 49 ]. Recently, antibody engineering by introducing double and triple unpaired Cys has also been described leading to conjugates with more payloads (DAR of more than two) per antibody [ 22 , 50 , 51 , 52 , 53 ].…”

Resistance to antibody‐drug conjugates in breast cancer: mechanisms and solutions

“…Currently, this conjugation technology is being revised for the production of site-specific ADCs with higher DARs. Refinements such as engineering multiple unpaired cysteines in the antibody or utilizing branched linkers are under development [113,114].…”