Synthesis and Evaluation of Three Azide-Modified Disaccharide Oxazolines as Enzyme Substrates for Single-Step Fc Glycan-Mediated Antibody-Drug Conjugation

Abstract

Antibody-drug conjugates (ADCs) hold great promise for targeted cancer cell killing. Site-specific antibody-drug conjugation is highly desirable for synthesizing homogeneous ADCs with optimal safety profiles and high efficacy. We have recently reported that azide-functionalized disaccharide oxazolines of the Manβ1,4GlcNAc core were an efficient substrate of wild-type endoglycosidase Endo-S2 for Fc glycan remodeling and conjugation. In this paper, we report the synthesis and evaluation of new disaccharide oxazolines as enzyme substrates for examining the scope of the site-specific conjugation. Thus, azide-functionalized disaccharide oxazolines derived from Manβ1,4GlcNAc, Glcβ1,4GlcNAc, and Galβ1,4GlcNAc (LacNAc) were synthesized. Enzymatic evaluation revealed that wild-type Endo-S2 demonstrated highly relaxed substrate specificity and could accommodate all the three types of disaccharide derivatives for transglycosylation to provide site-specific azide-tagged antibodies, which were readily clicked with a payload to generate homogeneous ADCs. Moreover, we also found that Endo-S2 was able to accommodate drug-preloaded minimal disaccharide oxazolines as donor substrates for efficient glycan transfer, enabling a single-step and site-specific antibody-drug conjugation without the need of an antibody click reaction. The ability of Endo-S2 to accommodate simpler and more easily synthesized disaccharide oxazoline derivatives for Fc glycan remodeling further expanded the scope of this bioconjugation method for constructing homogeneous antibody-drug conjugates in a single-step manner. Finally, cell-based assays indicated that the synthetic homogeneous ADCs demonstrated potent targeted cancer cell killing.

Hiding Payload Inside the IgG Fc Cavity Significantly Enhances the Therapeutic Index of Antibody–Drug Conjugates

“…However, either for the GTase-based or for the ENGase-based one, these approaches are restricted in substrate synthesis and dependent on two or more enzymes, a quite complicated and challenging procedure. To solve this issue, Wang et al, as well as our group, reported the one-step strategy for gsADCs assembly from native antibodies by Endo-S2 catalyzed glycoengineering. In this strategy, Endo-S2 acts as either an endoglycosidase to release the heterogeneous glycans from IgG or a glycosynthase possessing the capability to recognize a disaccharide-linker-drug combination as a substrate and transfer it onto the deglycosylated IgG, thus achieving gsADCs in a one-step manner.…”

“…However, either for the GTase-based or for the ENGase-based one, these approaches are restricted in substrate synthesis and dependent on two or more enzymes, a quite complicated and challenging procedure. To solve this issue, Wang et al, 17 as well as our group, 18 reported the one-step strategy for gsADCs assembly from native antibodies by Endo-S2 catalyzed glycoengineering.…”

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins

“…Recently, there were advances from site-specific antibody conjugation using transglycosylation. A one-pot reaction for deglycosylation and transglycosylation in antibody Fc was described using wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) [ 54 , 55 ]. The enzyme was shown to efficiently introduce the functionalized disaccharide oxazolines carrying site-selectively modified azide in varied numbers, resulting in ADCs with a precise control of DAR ranging from 2 to 12 via a copper-free strain-promoted click chemistry.…”

Tag‐Free Enzymatic Modification for Antibody−Drug Conjugate Production

“…However, the established technology requires using two different enzymes, which may complicate CMC and production, including the separation of ADCs from enzymes. [93] To overcome this issue, several promising approaches were published: Wang's group [94,95] and Huang's group [96] reported remodeling glycans in one step directly from native mAbs by disaccharide LacNAc oxazolines, respectively. These approaches enable simplifying the preparation of glycan-mediated antibody conjugates and have applications in related manufacturing.…”