Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate

Abstract

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

Site-Specific Bioconjugation and Multi-Bioorthogonal Labeling via Rapid Formation of a Boron–Nitrogen Heterocycle

“…For example, high loaded ADCs generated using branched linkers to couple multiple drug molecules to a specific site on the antibody have demonstrated enhanced potency. 27,28 Other recent work has shown that the dual delivery of two different cytotoxic payload classes may offer opportunities for improved efficacy. 29,30 Likewise, bispecific ADCs are gaining interests, as they have shown improved internalization and lysosomal trafficking as compared to mono-targeted ADCs.…”

A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates

“…Taken together, the carbamoyl sulfamide technology (HydraSpace™) described herein not only improves the manufacturability of ADCs through efficient conjugation of a toxic payload to an azido-modified monoclonal antibody [23], but also has the potential to provide next-generation ADCs with enhanced therapeutic index. Interestingly, metal-free click conjugation of branched payload constructs, most recently also applied for the preparation of a DAR4 ADCs by attachment of a branched azido-modified peptide carrying two auristatin or thailanstatin payloads, [35] has the potential to be extended to formats carrying two payloads with different mode-of-action, which may further boost the potential of the GlycoConnect™ technology in the generation of “dual warhead” ADCs [36,37,38]. Finally, we most recently corroborated that an ADC based on GlycoConnect and HydraSpace™ technologies also displays a significantly increased therapeutic index versus an ADC based on a cysteine-engineered antibody (unpublished data).…”

Design and Validation of Linkers for Site-Specific Preparation of Antibody–Drug Conjugates Carrying Multiple Drug Copies Per Cysteine Conjugation Site

“…The conjugation of the engineered antibody with branched linkers was carried out at elevated pH (8-8.5) resulting in antibody-linker conjugate that could be used for generating high-DAR ADCs. It should be noted that the iodoacetyl-based conjugation with a cysteine-inserted antibody is thiol-specific at the specified pH range [14,15]. The antibody concentration employed for conjugating branched linkers ranged from 2 to 4 mg/mL (Supplementary Information).…”

“…One way to overcome the instability of thiol–maleimide conjugation is to use alternative conjugation methods leading to plasma stable thioethers at cysteine in a rapid and efficient manner. The reaction of thiol with the iodoacetyl group to form a stable thioether bond is one such conjugation technique and has been previously employed for making ADCs [ 12 , 13 , 14 , 15 ]. The design of branched heterofunctional linkers for obtaining high-DAR ADCs is shown in Figure 2 .…”