Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Abstract

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

“…[1][2][3] Thes elective introduction of bioorthogonal functionalities into ap rotein represents the main challenge in this research area. [4][5][6][7][8][9][10][11] Enzymatic modification avoids complex manipulations of the translational machinery and only requires as hort amino acid sequence in the protein of interest, which is recognized by the enzyme to incorporate abioorthogonal functionality or to connect the payload to the protein. [12,13] Formylglycine-generating enzyme (FGE) is av aluable tool for bioconjugation.…”

Modular Assembly of Reversible Multivalent Cancer‐Cell‐Targeting Drug Conjugates

“…enzymes, pH, GSH) release of the cargo at the target . Hence, linkers often exhibit structures with a high functional density, and their construction involves a series of complex and costly synthetic steps that are typically unsuitable for straightforward structural diversification (Scheme A) ,. Therefore, the engineering of composite multifunctional molecules that enable the tuning of the construct properties by simple variation of the individual properties of the components is highly desirable for the expedient discovery of new TDCs with therapeutic usefulness ,…”

Of Thiols and Disulfides: Methods for Chemoselective Formation of Asymmetric Disulfides in Synthetic Peptides and Polymers

“…The trimethoxyphenylthio group ( S ‐Tmp) is another thiol‐labile alternative, since S ‐ t Bu removal proves to be occasionally difficult (Scheme A). A similar approach relies on thiol‐free reducing agents such as tris(2‐carboxyethyl)phosphine (TCEP) which liberates the free cysteine thiol for further coupling reactions from an S ‐ i Pr protecting group . The advantage of the pathways outlined above is the orthogonal reactivity to most cysteine protecting groups, the compatibility with the Fmoc strategy and relatively mild deprotection procedures.…”

“…As imilara pproach relies on thiol-free reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) which liberates the free cysteine thiol for furtherc oupling reactions from an S-iPr protecting group. [133] The advantage of the pathways outlined above is the orthogonal reactivity to most cysteine protecting groups, the compatibility with the Fmoc strategy and relativelym ild deprotection procedures.H owever,t he thiol reactivity of these groups is limited to the thiol moieties employed in the deprotection and they do not mediate directed disulfide formation.…”