Oligonucleotide‐based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. Here we introduce bivalent peptide–DNA conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toehold‐mediated strand displacement reactions. Employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nM concentrations of peptide–DNA locks and oligonucleotide displacer strands. Introduction of two different toehold strands on the peptide–DNA lock allowed signal integration of two different inputs, yielding logic OR‐ and AND‐gates. The range of molecular inputs could be further extended to protein‐based triggers by using protein‐binding aptamers.
Despite tremendous efforts in the field of targeted cancer therapy with antibody-drug conjugates (ADCs), attrition rates have been high. Historically, the priority in ADC development has been the selection of target, antibody, and toxin, with little focus on the nature of the linker. We show here that a short and polar sulfamide spacer (HydraSpace™, Oss, The Netherlands) positively impacts ADC properties in various ways: (a) efficiency of conjugation; (b) stability; and (c) therapeutic index. Different ADC formats are explored in terms of drug-to-antibody ratios (DAR2, DAR4) and we describe the generation of a DAR4 ADC by site-specific attachment of a bivalent linker-payload construct to a single conjugation site in the antibody. A head-to-head comparison of HydraSpace™-containing DAR4 ADCs to marketed drugs, derived from the same antibody and toxic payload components, indicated a significant improvement in both the efficacy and safety of several vivo models, corroborated by in-depth pharmacokinetic analysis. Taken together, HydraSpace™ technology based on a polar sulfamide spacer provides significant improvement in manufacturability, stability, and ADC design, and is a powerful platform to enable next-generation ADCs with enhanced therapeutic index.
Antibody-drug conjugates (ADCs) are increasingly powerful medicines for targeted cancer therapy. Inspired by the trend to further improve their therapeutic index by generation of homogenous ADCs, we report here how the clinical-stage GlycoConnect™ technology uses the globally conserved
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-glycosylation site to generate stable and site-specific ADCs based on enzymatic remodeling and metal-free click chemistry. We demonstrate how an engineered endoglycosidase and a native glycosyl transferase enable highly efficient, one-pot glycan remodeling, incorporating a novel sugar substrate 6-azidoGalNAc. Metal-free click attachment of an array of cytotoxic payloads was highly optimized, in particular by inclusion of anionic surfactants. The therapeutic potential of GlycoConnect™, in combination with HydraSpace™ polar spacer technology, was compared to that of Kadcyla® (ado-trastuzumab emtansine), showing significantly improved efficacy and tolerability.
We have found that conjugation of toxic payloads to the native glycan of a monoclonal antibody by chemoenzymatic remodeling (GlycoConnect™ technology) consistently provides antibody-drug conjugates (ADCs) with enhanced therapeutic index (TI) increases versus those of ADCs prepared by mainstream clinical technologies. For example, head-to-head comparison of GlycoConnect™ ADCs -based on the same antibody and payload components- with the marketed drugs Adcetris and Kadcyla revealed an improvement in both efficacy and safety, which is further enhanced by use of a polar HydraSpace™ technology. Importantly, a similar improvement in TI was noted for comparison of a GlycoConnect™ ADC versus or a site-specific ADC derived from a cysteine-engineered antibody, the most important emerging technology in the clinic. With the aim to better understand the superiority of ADCs based on combined GlycoConnect™ and HydraSpace™ technologies, we have performed in-depth in vitro and in vivo investigation into factors contributing to the overall performance of ADCs prepared by glycan conjugation with a polar spacer. For example, it is found that stability is significantly improved versus mainstream technologies in terms of aggregation and linker stability. Rapid aggregation was observed for both Kadcyla and Adcetris, as well as ADC decomposition due to retro-Michael reaction resulting in free payload (for Adcetris) or albumin-conjugated DM1 (for Kadcyla), as indicated by LC-MS and immunoassay analysis. The latter image is confirmed by in vivo pharmacokinetic analysis, showing a dramatic difference in total antibody versus total conjugated antibody by premature release of payload. Finally, analysis with a solid 3D tumoroid model indicates enhanced penetration of site-specific versus randomly conjugated ADC, which may also contribute to better efficacy in solid tumors. The data generated further underline the importance of conjugation and linker technology as critical quality attributes for next-generation ADCs with higher therapeutic index.
Citation Format: Floris van Delft, Brian Janssen, Remon van Geel, Marloes Wijdeven, Jorge Verkade, Sander van Berkel. Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3815.
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