Abstract 337: Highly potent, bispecific ADCs by application of clinical stage GlycoConnect® technology to bYlok® antibodies

Abstract Bispecific antibodies (bsAbs), with the ability to bind selectively to two different tumor-associated antigens in concerted fashion, have been increasingly successful for the treatment of cancer patients, delivering increased therapeutic potency compared to traditional mAbs, whilst also contributing to a reduction in the development of clinical resistance. Unfortunately, production of bsAbs is often plagued by HC-LC mispairing or by reduced titers, binding affinity, thermal stability or immunogenicity. Recently, we demonstrated that bsAbs generated using Lonza’s bYlok® modification display a high percentage (>95%) of correct HC-LC pairing and can be purified to high levels using standard downstream purification (https://www.lonza.com/biologics/expression-technologies/bYlok). Another increasing successful modality for anti-cancer therapy is the antibody-drug conjugate (ADC). Although typically based on random conjugation to cysteine or lysine, we have shown that the native glycan of monoclonal antibodies is a privileged site for attachment of cytotoxic payloads1, while the therapeutic index is further elevated by a highly polar spacer technology (HydraSpace®)2. Complemented by a broad choice of payloads with various mode-of-action (toxSYN® platform), these joint technologies have been applied in multiple clinical programs up to phase 3 (see: https://synaffix.com/partnered-pipeline/). Here we will demonstrate how bYlok® and GlycoConnect® technology are seamlessly combined to generate bispecific ADCs (bsADCs) with high yield and purity. Various bsADCs, featuring cytotoxic payloads with multiple MOA, including topoisomerase 1 inhibitors and tubulin binders, were fully analytically characterized and functionally evaluated, showing compelling tumor regression in multiple in vivo xenograft models. Combination of the technologies was further expanded to provide first-in-class bispecific ADCs with dual mode-of-action (dual warhead bsADCs). 1 (a) van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved NGlycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. (b) Wijdeven et al. Enzymatic glycan remodeling–metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. MAbs 2022, DOI: 10.1080/19420862.2022.2078466.2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Floris van Delft, Lianne Lelieveldt, Jasper van de Sande, Anette Sommer. Highly potent, bispecific ADCs by application of clinical stage GlycoConnect® technology to bYlok® antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 337.