Metabolic exploitation of the sialic acid biosynthetic pathway to generate site-specifically labeled antibodies

Abstract

Lack of a universal site-specific conjugation methodology for antibodies limits their potential to be developed as tumor-specific imaging agents or targeted therapeutics. A potential mechanism for site-specific conjugation involves utilization of the conserved N-glycosylation site in the CH2 domain. We sought to develop an antibody with an altered azido-sugar at this site whereby site-specific label could be added. The HB8059 hybridoma was cultured with peracetylated N-azidoacetlymannosamine (Ac4ManNAz). The resulting azido-sugar antibody was conjugated to phosphine-polyethylene glycol (PEG3)-biotin via a modified Staudinger reaction. Biochemical and functional characterization of the biotinylated antibody was performed. The azido-sugar antibody was also labeled with DyLight-650-Phosphine and injected into mice harboring pancreatic cancer xenografts. The tumors were dissected and imaged utilizing an IVIS fluorescent camera. The antibody was successfully produced in 100 μM Ac4ManNAz. The biotinylated antibody demonstrated a 50 kDa heavy and 25 kDa light chain on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but demonstrated a single band at 50 kDa on western blot. Treatment with a N-linked glycosidase extinguished the band. Flow cytometry demonstrated antigen-specific binding of CA19-9-positive cells and the antibody localized to the antigen-positive tumor in vivo. We successfully produced an antibody with an azido-sugar at the conserved CH2 glycosylation site. We were able to utilize this azide to label the antibody with biotin or fluorescent label and demonstrate that the label is added in a site-specific manner to the heavy chain, N-linked glycosylation site. Finally, we demonstrated functionality of our antibody for in vitro and in vivo targeting of pancreatic cancer cells.

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

“…This approach is therefore amenable to the glycans in the biotherapeutic industry, where bioengineered immunoglobulins are increasingly used to treat a variety of diseases and also offer the potential for antibody drug conjugates [39]. As both fucosylation and sialylation are known as important regulators of protein bioactivity, modification of sialic acids with an azido group provides a unique opportunity to systematically study the occurrence of these residues by MS in mAbs expressed under conditions known to promote high sialylation levels [41].…”

“…In vivo method using TZMm2 In an alternative approach, Ac 4 ManNAz was synthesized as a sialic acid biosynthetic pathway precursor for azide incorporation [39,40]. Ac 4 ManNAz was analyzed by 13 C NMR and ESI-MS prior to metabolic incorporation.…”

“…Currently, the analysis of azido glycans by mass spectrometry (MS) has been limited [37,38], as researchers have rather used fluorescent click labels for detection (e.g., [39,40]). In one published MS study [38], N-azidoacetylgalactosamine was fed to cells, and expressed glycoproteins were bound to dibenzocyclooctyne-sulfo-biotin.…”

“…In this report, peracetylated N-azidoacetylmannosamine (Ac 4 ManNAz) was prepared [40], and its metabolic incorporation into a double mutant trastuzumab monoclonal antibody (TZMm2 mAb) [41] was investigated systematically by varyi n g i t s c o n c e n tra t io n [39]. The presence of Nazidoacetylneuraminic acid (SiaNAz) on TZMm2 mAbs expressed in Chinese hamster ovary (CHO) cells ( Fig.…”

“…Alternatively, c TZMm2 mAbs were cultured and expressed under normal conditions, then desialylated and reincubated with ST6Gal1 and CMP-SiaNAz to promote sialylation by the bioorthogonal SiaNAz. Adapted from [40] and [39] Laughlin-Bertozzi et al [40]. CHO3E7 cells were grown in suspension in F17 medium supplemented with 4 mM glutamine and 0.1 % Kolliphor to a cell density of 1.5×10 6 cells/ mL (>99 % viability) in ventilated flasks (SF) shaken at 120 rpm in a humidified incubator at 37°C with 5 % CO 2 .…”