Impact of Acetylated and Non-Acetylated Fucose Analogues on IgG Glycosylation

Abstract

The biological activity of therapeutic antibodies is highly influenced by their glycosylation profile. A valuable method for increasing the cytotoxic efficacy of antibodies, which are used, for example, in cancer treatment, is the reduction of core fucosylation, as this enhances the elimination of target cells through antibody-dependent cell-mediated cytotoxicity. Development of fucose analogues is currently the most promising strategy to reduce core fucosylation without cell line engineering. Since peracetylated sugars display enhanced cell permeability over the highly polar free hydroxy sugars, this work sought to compare the efficacy of peracetylated sugars to their unprotected forms. Two potent fucose analogues, 2-deoxy-2-fluorofucose and 5-alkynylfucose, and their acetylated forms were compared for their effects on fucosylation. 5-alkynylfucose proved to be more potent than 2-deoxy-2-fluorofucose at reducing core fucosylation but was associated with a significant higher incorporation of the alkynylated fucose analogue. Acetylation of the sugar yielded only slightly lower fucosylation levels suggesting that acetylation has a minor impact on cellular entry. Even though the efficacy of all tested components was confirmed, results presented in this study also show a significant incorporation of unnatural fucose analogues into the glycosylation pattern of the produced IgG, with unknown effect on safety and potency of the monoclonal antibody.

Use of 5‐Thio‐L‐Fucose to modulate binding affinity of therapeutic proteins

“…Fucosylation is key for ADCC activity of IgG, which can be modulated by various fucose analogues by inhibition of FUT8 in combination with a substrate feedback inhibition (Allen et al, 2014, 2016; Kizuka et al, 2017; McKenzie et al, 2018; Okeley, Toki, et al, 2013; Rillahan et al, 2012; Zhou et al, 2017; Zimmermann et al, 2019). As the commercial use of these fucose analogues is restricted by patents, our study aimed at developing other derivatives able to increase ADCC.…”

“…Fucosylation is key for ADCC activity of IgG, which can be modulated by various fucose analogues by inhibition of FUT8 in combination with a substrate feedback inhibition (Allen et al, 2014(Allen et al, , 2016Kizuka et al, 2017;McKenzie et al, 2018;Rillahan et al, 2012;Zhou et al, 2017;Zimmermann et al, 2019). As the commercial use of these fucose analogues is restricted by patents, FcγR unveiled the highest affinity for FcγRI and lower affinities for FcγRIIIa V176, FcγRIIIa F176, and finally FcγRIIb, whereby the order of descending affinity was also described elsewhere (Bruhns et al, 2009).…”

“…The relative quantitative analysis of the N‐linked glycans was performed after glycan cleavage from the antibody and derivatization using the GlycoWorks™ RapiFluor‐MS™ N ‐Glycan Kit (Waters 176003713) and with ultra‐performance liquid chromatography coupled to a mass spectrometer (UPLC‐MS). UPLC analysis was performed according to the manufacturer's protocol using an ACQUITY UPLC Glycan BEH Amide Column (300 Å, 1.7 µm, 2.1 × 150 mm 2 ) coupled to an ACQUITY UPLC® FLR Detector (Ex: 265 nm, Em: 425 nm) as described in previous studies (Zimmermann et al, 2019). Identification of the glycan structures was performed using a dextran calibration ladder (Waters 186007982) to detect the specific retention time and MS spectra by calculating the mass to charge ratio with an electrospray ionization (ESI) source in positive mode (Synapt G1 HDMS, Waters).…”

“…Since modulation of Fc fucosylation is a powerful strategy to enhance the potency of therapeutic antibodies, several fucose analogues or acetylated derivatives have been reported with respect to their ability to reduce core‐fucosylation by acting as competitive inhibitors of fucosyltransferase 8 (FUT8) and triggering substrate feedback inhibition (Okeley, Alley, et al, 2013; Zimmermann et al, 2019). Some of these have also been applied in cell culture to successfully reduce core‐fucosylation: 2‐deoxy‐2‐fluorofucose (2F‐Fucose; Rillahan et al, 2012; Zhou et al, 2017; Zimmermann et al, 2019), 5‐Alkynyl‐Fucose (Kizuka et al, 2017; Okeley, Toki, et al, 2013; Zimmermann et al, 2019), 6,6,6‐trifluorofucose (McKenzie et al, 2018), and l ‐fucose phosphonate (Allen et al, 2014, 2016). These publications focus on removal, changed orientation, or substitution of hydroxyl groups with various functional groups to inhibit enzymes in either the de novo or the salvage pathway required for core‐fucosylation.…”

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media

“…The cell culture medium (CCM) formulation is critical for the culture longevity and viability as well as quality attributes of the end product (D. Bruhlmann et al, 2015 ; David Brühlmann et al, 2017 ; Ehret et al, 2019 ; Zimmermann et al, 2019 ). To grow and produce high amounts of antibodies or other therapeutic proteins, CHO cells need regular supplementation with essential amino acids (AA) through feeding (Carrillo‐Cocom et al, 2015 ; Sellick et al, 2011 ).…”