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.

Keywords: Antibody-drug conjugates (ADCs); chemoenzymatic; glycan remodeling; metal-free click chemistry; non-genetic; therapeutic index.

Graphical abstract

Figure 1.

General scheme for enzymatic remodeling of antibody glycan (a⟶b) followed by metal-free click chemistry conjugation of payload (b⟶c). The drug-to-antibody ratio (DAR) can be tailored (DAR2 or DAR4) by using a linear of branched BCN-linker-drug construct (y = 1 or 2).

Figure 2.

Endoglycosidase trimming of various antibody glycoforms to core GlcNAc attached to Asn²⁹⁷. Potential substitution of the core GlcNAc with a (1⟶6)-α-fucosyl moiety [▼] does not affect the endoglycosidase efficiency.

Figure 3.

Various azidosugars 1–3 for glycosyl transfer to core GlcNAc.

Figure 4.

Stability of antibody-drug-conjugates based on azidosugar 1 (GalNAz) or 3 (6-azidoGalNAc). (a) Structures of BCN-HydraSpace™-linker-drugs 4 and 5. (b) Aggregation levels of ADCs derived from brentuximab (red lines) or trastuzumab (blue lines), remodeled with azidosugar 1 (solid lines) or 3 (dashed lines). Both azidosugar-remodeled derivatives of brentuximab were conjugated to linker-drug 5 (⟶DAR4 ADC), while trastuzumab azidosugar derivatives were conjugated to linker-drug 4 (⟶DAR2 ADC).

Scheme 1.

Synthetic preparation of UDP 6-azido-6-deoxy-GalNAc (UDP-3). Reagents and conditions: a) 1. SOCl₂, Et₃N, CH₂Cl₂, 0°C. 2. RuO₄, NaIO₄, CH₂Cl₂, CH₃CN, water, 83% over 2 steps; b) 1. NaN₃, DMF, rt, 2. H₂SO₄, THF, water; c) pyridine, Ac₂O, 80% over two steps; d) 1-propylamine, THF; e) 5-(ethylthio)-1 H-tetrazole, bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite, CH₂Cl₂, MeCN; f) m-CPBA, 54% over three steps; g) Et₃N, MeOH, H₂O, 50°C, quantitative; h) sodium UMP-imidazolide (13), MgCl₂, DMF, 52% yield. A chemical scheme showing how N-acetyl-D-galactosamine can first be converted into its 6-azido derivative and finally into UDP 6-azidoGalNAc, compound UDP-3.

Figure 5.

Optimization of metal-free click conjugation in the presence of surfactants. (a) Conjugation of branched MMAE-based linker-drug 5 for generation of DAR4 ADC. (b) Conjugation of linear calicheamicin-based linker-drug 15 for generation of DAR2 ADC. Surfactant concentrations: sodium deoxycholate (11 mM), sodium decanoate (37.5 mM), CHAPS (12 mM).

Figure 6.

Structure of branched BCN-HydraSpace™-vc-PABC-Ahx-maytansine 19 (SYNtansine™) for the preparation of DAR4 ADC.

Figure 7.

Tumor volume over time of mouse PDX T226 treated with Kadcyla® or trastuzumab-SYNtansine™ at low or high dose (3 and 9 mg/kg, respectively).

Figure 8.

Monitoring of body weight over time of Sprague-Dawley rats treated with (a) Kadcyla® or (b) GlycoConnect™ ADC with SYNtansine™ at 20–35–50–60 mg/kg.