Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios

Abstract

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug-to-antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This four-step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

Keywords: antibodies; antibody-drug conjugates; biocatalysis; glycoengineering; medicinal chemistry.

Figure 1

Glycoengineering strategy to introduce four aldehyde groups into Trastuzumab 1 for subsequent site‐specific functionalization. In the first biocatalytic step, β‐1,4‐galactosyltransferase 1 (β4‐GalT1) transfers D‐galactose units from UDP‐Gal to each biantennary N‐glycan chain of native 1. Subsequently, C6‐hydroxy groups of galactose units are oxidized to aldehyde with galactose oxidase (GOase M₁).

Figure 2

HILIC/MS and Fc/2 analyses of N‐glycosylation patterns of (a) native 1, (b) galactosylated 2, and (c) oxidized 3 Trastuzumab. (d) HILIC/MS analysis of GOase‐oxidized Trastuzumab [²H₄]‐3 after reduction of 3 with sodium borodeuteride. See the Supporting Information for details (Figures S2–S24) and experimental procedures.

Figure 3

(a) Functionalization of oxidized Trastuzumab 3 with 8 azido groups via TKM. The bifunctional azido‐pyrazolone linker 4 was synthesized in a single step from commercially available starting material (Supporting Information). Fc/2 analysis of the functionalized antibody 5 (b) before and (c) after treatment with PNGase F shows functionalization of the oxidized N‐glycan chain with four azido‐pyrazolone linkers being specific.

Figure 4

Conjugation of azido‐Trastuzumab 5 to DBCO‐TAMRA by SPAAC.