Fc-specific Deglycosylation of IgG

Deglycosylation of IgG using Endoglycosidases

 

Monoclonal antibodies are a very important class of therapeutics with a broad range of clinical applications. While the antigen binding Fab regions determine an antibody’s specificity, the effector functions that the mAb elicits and therefore its mode-of-action is guided by the Fc region. Antibodies can elicit their function in a plethora of ways: From simply blocking or sequestering crucial components in a pathway by binding to them to specific activation of the immune system. This can happen through interaction with Fcg receptors (FcgRs) and activation of cellular immunity such as antibody-dependent cellular cytotoxicity (ADCC) or activation of the complement system. Both the subclass of the antibody and nature of the conserved Fc glycan play an important role in this process.

 

Genovis IgG specific endoglycosidases GlycINATOR (EndoS2) and IgGZERO (EndoS) provide a fast and easy way to remove the conserved glycans to “silence” the Fc effector functions such as binding to FcgRs or the complement system. This allows for studies of the mode of action of a mAb and provides a way to reduce non-specific interactions of antibodies in imaging applications.

 

Deglycosylation with GlycINATOR abolishes ADCC

The iLite ADCC reporter assay is a cell-based luciferase assay measuring CD16 signaling in a reporter cell upon incubation with a mAb and a cell line expressing the antibody´s and target. This allows for a easy read-out on a mAbs ability to induce CD16 siganliing in immune cells and therefore elicit ADCC (Figure 1).

Deglycosylation - GlycINATOR

Figure 1. Schematic illustration of the iLite Reporter gene ADCC Effector cell and positive Target Cell.

The interaction between the Fc domain of an IgG antibody and the CD16 receptor is dependent on the Fc glycan and its structure. Lack of core fucosylation greatly increases the interaction and in turn ADCC while antibodies missing the Fc glycan altogether are unable o interact with the receptor. GlycINATOR specifically removes the Fc glcyans from therapeutic mAbs. This abolishes their ability to induce ADCC as shown with the iLite assay (Figure 2) and allows for the study of a mAbs mode-of-action.

 

ADCC - Deglycosylation

 

Figure 2. Study of ADCC of native and deglycosylated antibodies. The effect of rituximab (b) was ana- lyzed using iLite ADCC Effector (V) Assay Ready Cells (BM4001) and iLite ADCC Target CD20 (+) Assay Ready Cells (BM4010) while the effect of trastuzumab (a) was analyzed using iLite ADCC Effector (V) Assay Ready Cells (BM4001) and iLite ADCC Target HER2 (+) Assay Ready Cells (BM4011). Effector and Target cells were diluted and mixed with the antibody solutions. Incubation was performed at 37°C and 5% CO2. Luminescence reading was performed after 4 h (rituximab) or 6 h (trastuzumab).

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