One-Step Conjugation Method for Site-Specific Antibody–Drug Conjugates through Reactive Cysteine-Engineered Antibodies

Abstract

Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.

Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction

“…However, surface Cys can undergo competing disulfide bond formation and, in the case of tetramers such as streptavidin, multimerization through disulfides can quickly lead to precipitation. Furthermore, maleimide conjugates can re-arrange, hydrolyze, or exchange in the presence of other thiols ( Shinmi et al., 2016 ). There is a wide literature on the use of NHS-dye conjugates, with some examples where labeling interferes with binding properties and examples of excess dye labeling reducing overall fluorescence ( Vira et al., 2010 , Zanetti-Domingues et al., 2013 ).…”

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

“…The unreacted RNase A (1 a) is recovered and product is immobilized on resin selectively through K7 with excellent efficiency (12, > 95 %). Subsequently, the resin is distributed in four tubes for transoximization with O-hydroxylamine (5) and its derivatives 9 a-c (step 3). The resin (11) is recycled and centrifugal spin concentration delivers analytically pure K7tagged RNase A with hydroxylamine (6 a) and three different probes .…”

“…[1] Over the past few decades, the pre-engineered protein driven selective labeling allowed the investigation of several pathways through reporters, toxins, biophysical probes, and imaging probes. [2] Here, the unnatural inserts, [3] enzymatic protocols, [4] engineered Cys, [5] or a Cys in p-clamp [6] have been the prime enablers. Though these methods are remarkable, they do not extend the precision biophysical and biochemical tools to a vast repository of native proteins.…”

Homogeneous antibody-drug conjugates: DAR 2 anti-HER2 obtained by conjugation on isolated light chain followed by mAb assembly

“…19 Badescu and coworkers used a bis-sulfone linker designed to react with two antibody cysteines to conjugate an auristatin payload (monomethyl auristatin E (MMAE)) to trastuzumab and its antigen-binding fragments (Fabs). 20 The approaches described above are time consuming and specific for each construct, as they often require extensive antibody engineering to identify the optimal conjugation sites 9,21 where unique side chains can be introduced for conjugation, or they require complex linker and payload modifications that have not been clinically validated. 8 As a result, they are not suitable for converting existing antibodies directly into ADCs.…”