INVESTIGATION OF GLYCOENZYME SUBSTRATE SPECIFICITY FOR GLYCOENGINEERING OF THERAPEUTIC ANTIBODIES

Abstract

Immunoglobulin G (IgG) antibodies have been exploited as drug candidates due to their target specificity and ability to elicit host cell effector functions. These Y-shaped glycoproteins possess two antigen-binding (Fab) regions for selective binding to epitopes and one constant (Fc) region for mediating effector functions. As glycoproteins, these antibodies are also N-glycosylated at position N297, where the Fc glycans modulate the stability and effector functions of the antibodies. However, due to the non-template synthesis of these antibody glycans, the resulting oligosaccharide composition and structures are highly heterogeneous. My work aims to leverage the substrate specificities of various glycosyltransferase enzymes to generate antibody preparations with homogenous glycan structures. For my first project, I performed a comparative study on three sialyltransferases, including two bacterial sialyltransferases from Helicobacter cetorum (HcST-22) and from Photobacterium damselae (Pd2,6ST), respectively, and the human -2,6-sialyltransferase (ST6Gal1), to determine which is more efficient at sialylating intact antibody Fc glycans. We found that HcST-22 was more efficient than Pd2,6ST and ST6Gal-1 at sialylating intact Fc glycans. For my second project, I contributed to a collaborative effort in generating intravenous immunoglobulin (IVIg) glycoforms. I incorporated two sialic acid derivatives, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) and 9-azido-9-deoxy-Neu5Ac (9AzNeu5Ac), onto free glycan before transferring these glycans onto deglycosylated IVIg. The resulting azide-tagged IVIg was then further elaborated by the click addition of sialocomplex-type glycan asparagine with a DBCO tag (SCT-Asn-DBCO) to yield a final glycoform that extended the Fc glycan and increased the valency of the terminal native sialic acid. For my third research project, I designed a novel Fc-glycosylated site-specific antibody drug conjugate (ADC) design with a drug to antibody ratio (DAR) of 6. I achieved this by glycoengineering the Fc glycan of recombinant trastuzumab to generate a triantennary agalactosylated complex type glycan at the N297 position. Then, using a mutant N-acetylgalatosamine (GalNAc) transferase enzyme β1,4-GalT Y289L I transferred N-azidoacetylgalactosamine (GalNAz) to each glycan terminus of the triantenary Herceptin, then click-conjugated a cytotoxic payload onto each azide on the antibody Fc region, generating a trastuzumab ADC with a DAR of 6. Through a cell viability assay, we determined that this homogeneously glycosylated DAR-6 ADC showed potent anti-cancer activity.