Enzymatic glycan remodeling–metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering

Abstract

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 N -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.

A glyco-engineering approach for site-specific conjugation to Fab glycans

“…However, other studies have found benefit to the reduction or inactivation of interaction with Fc-γ-receptors and/or mannose receptors to potentially reduce off-target uptake and cytotoxicity. 17 It remains to be determined which therapeutic applications would benefit the most from having intact Fc glycans on mAbs carrying cytotoxic (or other types of) payloads.…”

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins

“…The chemically introduced thiol on Q295 could be selectively conjugated using maleimide chemistry with improved plasma stability. In another report, Wijdeven et al described an improved method from enzymatic glycan remodeling followed by metal-free click chemistry [ 35 ]. An engineered endoglycosidase and a native glycosyltransferase were selected for chemoenzymatic reaction using a novel azido sugar, generating ADCs with improved efficacy.…”

“…Among many different methods as described, the site-specific conjugations through engineered Cys, unnatural amino acid, and enzymatic glycan remodeling–metal-free click chemistry have been performed at big scale and the produced conjugates are being tested in clinical trials [ 11 , 35 ]. Besides ADCs, site-specific antibody conjugation methods have also been applied for coupling other payloads as described in following sections ( Figure 1 ).…”

Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology

“…With optimal conjugation conditions and a preferred linker-drug design established for MMAE, we shifted focus to a variety of ultrapotent payloads for which a DAR1 format could be highly beneficial in terms of enhancing the MTD in patients. Based on the DAR1-linker design of 7, three linker-drugs were synthesized (SI 2.3), attaching either a PBD dimer (11), PNU-159,682 (12), or our proprietary calicheamicin variant (13) (see Figure 5). Having LDs 11−13 in hand, conjugation to azido-trastuzumab was performed following the optimized protocol for the generation of DAR1 ADCs (AP-remodeled azido-mAb (5 mg/mL), sodium deoxycholate (11 mM), 3 equiv of LD, and either DMF or PG as a co-solvent at RT for 16 h).…”

“…Figure 5. Structures of LDs 11−13 comprising a bis-BCN spacer through the central nitrogen atom to various payloads, containing a va-PABC-PBD dimer unit(11), a vc-PABC-PNU-159,682 unit(12), or vc-PABC-calicheamicin D(13). Linker-drug 14 is a linear BCN-HS-PEG 2 -va-PABC-PBD dimer variant used in the preparation of a benchmark DAR2 ADC analog of Trast-11.…”