Tuning the Diels–Alder Reaction for Bioconjugation to Maleimide Drug-Linkers

The thiol–maleimide linkage is widely used for antibody–drug conjugate (ADC) production; however, conjugation of maleimide–drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels–Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol–maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates

Structure–Cytotoxicity Relationships of Analogues of <i>N</i>¹⁴-Desacetoxytubulysin H

Herein we report structure–cytotoxicity relationships for analogues of <i>N</i>¹⁴-desacetoxytubulyisn H <b>1</b>. A novel synthetic approach toward <b>1</b> enabled the discovery of compounds with a range of activity. Calculated basicity of the <i>N</i>-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of <b>1</b> upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer

Straightforward Glycoengineering Approach to Site-Specific Antibody–Pyrrolobenzodiazepine Conjugates

Antibody–drug conjugates (ADCs) have become a powerful platform to deliver cytotoxic agents selectively to cancer cells. ADCs have traditionally been prepared by stochastic conjugation of a cytotoxic drug using an antibody’s native cysteine or lysine residues. Through strategic selection of the mammalian expression host, we were able to introduce azide-functionalized glycans onto a homogeneously glycosylated anti-EphA2 monoclonal antibody in one step. Conjugation with an alkyne-bearing pyrrolobenzodiazepine dimer payload (SG3364) using copper-catalyzed click chemistry yielded a site-specific ADC with a drug-to-antibody ratio (DAR) of four. This ADC was compared with a glycoengineered DAR two site-specific ADC, and both were found to be highly potent against EphA2-positive human prostate cancer cells in both an <i>in vitro</i> cytotoxicity assay and a murine tumor xenograft model