HDAC Inhibitors as Epigenetic Regulators

The reason for this slight discrepancy between theoretical and actual CE measured values for G0-F is not clear, but could be attributed to variations in the efficiency of PNGase F digestion or the quantum efficiency of the fluorophore used for CE, which was conjugated in close proximity to the fucose. Table?1. and IIb, and substantially increased binding to FcRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcRIIIa genotype of the effector cells. Keywords: afucosylated antibody, antibody-dependent cellular cytotoxicity, FcRIIIA, fucosylation, glycoform variants, Glycosylation, monoclonal antibody Introduction The glycans attached to the asparagine at the 297 position (N297) of the Fc region of IgG play a critical role on the effector functions of antibodies.1-3 These N-linked glycans are situated within a cleft VE-821 formed by the paired heavy chains in the CH2 domain of IgGs such that they may undergo extensive non-covalent interactions with the adjacent protein surface.4-6 There is evidence that interactions between the IgG Fc region and the effector ligands (Fc receptors and C1q) are critically dependent on IgG Fc proteinCglycan interactions.7,8 Both the conformation and functionality of antibodies can be modulated by manipulation of these oligosaccharides.9,10 Antibodies depleted of N-linked glycans at Asn-297 behave similarly to normal antibodies with respect to antigen binding and Protein A binding capacity. However, they are defective in binding to Fc receptors, activating complement and inducing ADCC.4,11-13 Structural and thermodynamic data have shown that the precise structure of the IgG-Fc N-linked glycans helps to determine the binding affinity of the IgG to Fc receptors and thus the effector functions of the antibodies.14,15 Specifically, the N-glycans stabilize particular conformations of the CH2 domains and act as spacers, holding the CH2 domains apart to provide an open state of the horseshoe-shaped IgG-Fc fragment, allowing increased accessibility and tighter binding to Fc receptors.7,16-18 The majority of human IgG-Fc N-linked glycans are based on a common core structure of biantennary heptapolysaccharide containing GlcNAc and mannose.19,20 Further modification of the core carbohydrate structure through the addition of fucose, as well as bisecting GlcNAc, galactose and sialic acid, substantially increases structural heterogeneity, with more than 30 variant forms possible.21 For both serum-derived endogenous human IgGs and IgG produced from engineered mammalian cell lines, the majority of Fc N-linked glycans carry different degrees of terminal galactosylation resulting in a G0 glycoform, a G1 glycoform and a G2 glycoform. Whereas these glycans are predominantly fucosylated, i.e., contain a fucose attached to the innermost GlcNAc residue in the core structure, small amounts of naturally occurring glycoforms that lack the core fucose have been observed in both human serum-derived and CHO cell produced IgG. It is well-documented that the absence of core fucose in IgG results in higher affinity binding to the FcRIIIa receptor (both the F158 and V158 allotypes of this receptor) and increased ADCC activity.22-27 In 2002, Shields et al. first reported that recombinant human IgG1 produced from the CHO-Lec13 cell line showed enhanced FcRIIIa binding and ADCC activity compared with IgGs produced by regular CHO cells. The CHO-Lec13 cell line is deficient in its ability to add fucose to glycans, but produces IgGs with oligosaccharides VE-821 that are otherwise similar to those found in normal CHO cell lines.22 Similar results were later reported by other groups using afucosylated antibodies produced from engineered CHO cell lines in which -1,6-fucosyltransferase (FUT8) was either downregulated by RNA Rabbit Polyclonal to PDK1 (phospho-Tyr9) interference technology or genetically knocked out.26,27 In fact, enhancement of ADCC activity by fucose removal has been demonstrated with all four subclasses of IgGs, as well as with Fc fusion proteins in both in vitro assays and in vivo animal models.23,25,28-32 Moreover, the antigen density required to induce efficient ADCC activity is lower when the IgG has low, rather than high, fucose content.24 Based on these data, afucosylated forms of therapeutic antibodies are VE-821 expected to have clinical advantages over the fucosylated forms because of the increased efficiency of ADCC induction and its ability to better compete with endogenous IgGs for binding with FcRIIIa on effector cells. To date, several antibodies with no or low levels of fucose have been produced from genetically engineered cell lines and are currently in clinical studies as candidate therapeutics for various indications. ADCC is a critical effector function that has been implicated in the clinical efficacy of a number of therapeutic antibodies.33,34 In ADCC, target cells bound by the Fab regions of antibodies are lysed by activated effector cells following.