Impact of cathepsin B-sensitive triggers and hydrophilic linkers onin vitroefficacy of novel site-specific antibody–drug conjugates

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Abstract

Promising anti-HER2 site-specific ADCs with an in vitroefficacy equivalent to Kadcyla®.

Abstract

Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody–drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed viathe conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character viaapplication of a PEG ₁₂spacer. Through evaluation of physical properties, in vitrocytotoxicity, and receptor affinity of the resulting antibody–drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitrocytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitropotency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

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Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index.

Robert P Lyon, Tim Bovee, Svetlana Doronina … (2015)

The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.

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Current ADC Linker Chemistry

Nareshkumar Jain, Sean W Smith, Sanjeevani A. Ghone … (2015)

The list of ADCs in the clinic continues to grow, bolstered by the success of first two marketed ADCs: ADCETRIS® and Kadcyla®. Currently, there are 40 ADCs in various phases of clinical development. However, only 34 of these have published their structures. Of the 34 disclosed structures, 24 of them use a linkage to the thiol of cysteines on the monoclonal antibody. The remaining 10 candidates utilize chemistry to surface lysines of the antibody. Due to the inherent heterogeneity of conjugation to the multiple lysines or cysteines found in mAbs, significant research efforts are now being directed toward the production of discrete, homogeneous ADC products, via site-specific conjugation. These site-specific conjugations may involve genetic engineering of the mAb to introduce discrete, available cysteines or non-natural amino acids with an orthogonally-reactive functional group handle such as an aldehyde, ketone, azido, or alkynyl tag. These site-specific approaches not only increase the homogeneity of ADCs but also enable novel bio-orthogonal chemistries that utilize reactive moieties other than thiol or amine. This broadens the diversity of linkers that can be utilized which will lead to better linker design in future generations of ADCs. Electronic supplementary material The online version of this article (doi:10.1007/s11095-015-1657-7) contains supplementary material, which is available to authorized users.