Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates

Abstract

A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

Antibody-Drug Conjugates in Head and Neck Cancer

“…One of the latest technologies based on this strategy is the GlycoConnect technology developed by van Delft and coworkers (van Geel et al, 2015) (Fig. 6D).…”

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…Analogous to the above described technologies, they then attached galactose derivatives to the core GlcNAc by using the same mutant β1,4-galactosyltransferase. To demonstrate the versatility and power of their platform, they used different antibodies, different galactose derivatives, linkers and toxins to generate homogeneous ADCs [109].…”

Site‐Selective Cysteine–Cyclooctyne Conjugation

“…[4,5] One such approach (Figure 1a) introduces azides that react with cyclooctyne reagents through strain-promoted azide-alkyne cycloaddition (SPAAC). [6] SPAAC has been widely used for selectively labelling cell surface glycans [7,8] , membrane proteins [9] , and antibodies [10,11] . Azides are not genetically encoded and therefore need to be introduced into proteins via metabolic glycoengineering [7,8] , incorporation of unnatural amino acids [9] , or enzyme-catalyzed reactions [11] .…”

“…Several groups have reported using azide-containing antibodies for site-selective conjugation to DBCO reagents. [11,35,36] However, genetic code expansion or glycoengineering are required to produce azide-containing antibodies. The DBCO tag is composed of natural amino acids; therefore, we reasoned that the DBCO-tag-mediated conjugation could be a complementary and potentially advantageous approach to azide-based methods for selectively conjugating DBCO reagents to antibodies.…”