Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood.</p> <p>Methods</p> <p>Using a human <it>ex vivo </it>B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcγR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood.</p> <p>Results</p> <p>In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcγRIIIa binding activity to overcome competition from plasma IgG for binding to FcγRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcγRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20.</p> <p>Conclusion</p> <p>Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcγRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).</p

Evolutional and clinical implications of the epigenetic regulation of protein glycosylation

Protein N glycosylation is an ancient posttranslational modification that enriches protein structure and function. The addition of one or more complex oligosaccharides (glycans) to the backbones of the majority of eukaryotic proteins makes the glycoproteome several orders of magnitude more complex than the proteome itself. Contrary to polypeptides, which are defined by a sequence of nucleotides in the corresponding genes, glycan parts of glycoproteins are synthesized by the activity of hundreds of factors forming a complex dynamic network. These are defined by both the DNA sequence and the modes of regulating gene expression levels of all the genes involved in N glycosylation. Due to the absence of a direct genetic template, glycans are particularly versatile and apparently a large part of human variation derives from differences in protein glycosylation. However, composition of the individual glycome is temporally very constant, indicating the existence of stable regulatory mechanisms. Studies of epigenetic mechanisms involved in protein glycosylation are still scarce, but the results suggest that they might not only be important for the maintenance of a particular glycophenotype through cell division and potentially across generations but also for the introduction of changes during the adaptive evolution

Cellular and complement-dependent cytotoxicity of Ep-CAM-specific monoclonal antibody MT201 against breast cancer cell lines

MT201 is a fully human monoclonal IgG1 antibody with moderate affinity for epithelial cell adhesion molecule (Ep-CAM) being clinically developed for the treatment of carcinomas. Like many other clinically validated IgG1 monoclonal antibodies, MT201 primarily acts by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Here, we analysed ADCC and CDC induced by MT201 and, as reference, trastuzumab against a panel of nine human breast cancer cell lines expressing distinct surface levels of Ep-CAM and human epithelial growth factor receptor type 2 antigen. Maximal cell lysis by ADCC by MT201 and trastuzumab in the presence of peripheral mononuclear cells did not significantly differ when averaged over the nine cell lines, but showed marked differences with respect to individual cell lines. The extent of cell lysis at intermediate surface target density was highly variable, suggesting a dominant influence of other susceptibility factors. Only one breast cancer cell line was eliminated via CDC, but only by MT201. Resistance to CDC appeared to correlate with high expression levels of complement resistance factors. Our present data as well as recent data on the prevalence and prognostic relevance of Ep-CAM expression in metastatic breast cancer suggest that Ep-CAM-specific monoclonal IgG1 antibodies may have a significant therapeutic potential in the treatment of breast cancer

Inflammatory bowel disease associates with proinflammatory potential of the immunoglobulin g glycome

BACKGROUND: Glycobiology is an underexplored research area in inflammatory bowel disease (IBD), and glycans are relevant to many etiological mechanisms described in IBD. Alterations in N-glycans attached to the immunoglobulin G (IgG) Fc fragment can affect molecular structure and immunological function. Recent genome-wide association studies reveal pleiotropy between IBD and IgG glycosylation. This study aims to explore IgG glycan changes in ulcerative colitis (UC) and Crohn's disease (CD). METHODS: IgG glycome composition in patients with UC (n = 507), CD (n = 287), and controls (n = 320) was analyzed by ultra performance liquid chromatography. RESULTS: Statistically significant differences in IgG glycome composition between patients with UC or CD, compared with controls, were observed. Both UC and CD were associated with significantly decreased IgG galactosylation (digalactosylation, UC: odds ratio [OR] = 0.71; 95% confidence interval [CI], 0.5–0.9; P = 0.01; CD: OR = 0.41; CI, 0.3–0.6; P = 1.4 × 10(−9)) and significant decrease in the proportion of sialylated structures in CD (OR = 0.46, CI, 0.3–0.6, P = 8.4 × 10(−8)). Logistic regression models incorporating measured IgG glycan traits were able to distinguish UC and CD from controls (UC: P = 2.13 × 10(−6) and CD: P = 2.20 × 10(−16)), with receiver–operator characteristic curves demonstrating better performance of the CD model (area under curve [AUC] = 0.77) over the UC model (AUC = 0.72) (P = 0.026). The ratio of the presence to absence of bisecting GlcNAc in monogalactosylated structures was increased in patients with UC undergoing colectomy compared with no colectomy (FDR-adjusted, P = 0.05). CONCLUSIONS: The observed differences indicate significantly increased inflammatory potential of IgG in IBD. Changes in IgG glycosylation may contribute to IBD pathogenesis and could alter monoclonal antibody therapeutic efficacy. IgG glycan profiles have translational potential as IBD biomarkers

In Response to: ‘Impact of Glycosylation on Effector Functions of Therapeutic IgG ’ (Pharmaceuticals 2010, 3, 146–157)

pharmaceutical

In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment

In our study, a novel, fully human, recombinant monoclonal antibody of the IgG1 isotype, called MT201, was characterized for its binding properties, complement-dependent (CDC) and antibody-dependent cellular cytotoxicity (ADCC), as well as for its in vivo antitumor activity in a nude mouse model. MT201 was found to bind its target, the epithelial cell adhesion molecule (Ep-CAM; also called 17-1A antigen, KSA, EGP-2, GA733-2), with low affinity in a range similar to that of the clinically validated, murine monoclonal IgG2a antibody edrecolomab (Panorex®). MT201 exhibited Ep-CAM-specific CDC with a potency similar to that of edrecolomab. However, the efficacy of ADCC of MT201, as mediated by human immune effector cells, was by 2 orders of magnitude higher than that of edrecolomab. Addition of human serum reduced the ADCC of MT201 while it essentially abolished ADCC of edrecolomab within the concentration range tested. In a nude mouse xenograft model, growth of tumors derived from the human colon carcinoma line HT-29 was significantly and comparably suppressed by MT201 and edrecolomab. The fully human nature and the improved ADCC of MT201 with human effector cells will make MT201 a promising candidate for the clinical development of a novel pan-carcinoma antibody that is superior to edrecolomab

Reduction of IgG in nonhuman primates by a peptide antagonist of the neonatal Fc receptor FcRn

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG–hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases