Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies

As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolismprocesses that are critical to ADC efficacy and toxicityimaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a “clickable” ADC bearing an azide-functionalized linker-payload (LP) poised for “click” reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems

Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS⁴)

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6­(9<i>H</i>)-one (<b>1</b>), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by <b>1</b> in a labile peptide–heme–peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS⁴) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that <b>1</b> was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of <b>1</b>. The final orientation of <b>1</b> perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of <b>1</b> by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain

Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation

Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic

Optimization of Tubulysin Antibody–Drug Conjugates: A Case Study in Addressing ADC Metabolism

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody–drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs