Immunochemical Determination of Hemoglobin-A1c Utilizing a Glycated Peptide as Hemoglobin-A1c Analogon

We describe the development of a heterogeneous affinity-matrix based immunoassay for the determination of HbA1c which could in future be applicable to analytical devices. We developed an immunoenzymometric assay (IEMA) where the glycated pentapeptide Val-His-Leu-Thr-Pro (VHLTP) as HbA1c analogon is immobilized either to the surface of a microtiter plate by adsorption or to an amino-modified cellulose membrane by covalent linkage. The immobilized analogon competes together with the HbA1c in the sample for the antigen binding sites of the anti-HbA1c antibodies. Glucose oxidase-labeled antibodies have been used to indicate the antigen-antibody reaction indirectly and enzyme activity was detected optically. Calibration curves for HbA1c were obtained with a linear range of 1,5-10 µg ml-1 (23-155 nM). In a mixture of non-glycated and glycated hemoglobin with a total hemoglobin concentration of 30 µg ml-1 (465 nM) a linear range was obtained between 5-50 % HbA1c. Since the glycated peptide shows a high affinity for the anti-HbA1c antibody (Kd = 0,3 nM) only a low contact time (< 1 min) between the modified solid support and the preincubated mixture of HbA1c and anti-HbA1c antibody was required. Regeneration of the affinity-matrix was carried out with 10 mM HCl for 3 min without loss of antibody binding activity

Therapeutic antibody glycosylation impacts antigen recognition and immunogenicity.

In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. While non-human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc-associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies. We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. Furthermore, we show that DC-uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti-drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco-engineering may help to decrease clinical immunogenicity and ADA-associated adverse events such as hypersensitivity reactions

Therapeutic antibody glycosylation impacts antigen recognition and immunogenicity.

In this study we show that glycosylation is relevant for immune recognition of therapeutic antibodies, and that defined glycan structures can modulate immunogenicity. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. While non-human glycosylation is thought to cause hypersensitivity reactions and immunogenicity, less is known about effects of Fc-associated glycan structures on immune cell responses. We postulated that glycosylation influences antigen recognition and subsequently humoral responses to therapeutic antibodies by modulating 1) recognition and uptake by dendritic cells (DCs), and 2) antigen routing, processing and presentation. Here, we compared different glycosylation variants of the antibody rituximab (RTX) in in vitro assays using human DCs and T cells as well as in in vivo studies. We found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. Furthermore, we show that DC-uptake of RTX increases or decreases if glycosylation is selectively modified to recognize activating (by mannosylation) or inhibitory lectin receptors (by sialylation). Moreover, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, we demonstrate that antibody glycosylation impacts anti-drug antibody (ADA) responses to RTX in vivo. Hence, defined glycan structures can modulate immune recognition and alter ADA responses. Glyco-engineering may help to decrease clinical immunogenicity and ADA-associated adverse events such as hypersensitivity reactions