Impact of Core Fucosylation on Antibody Effector Functions using TransGLYCIT®

Application

Functional comparison of antibody glycoforms by combining glycoengineering with ADCC and ADCP reporter assays to assess Fc effector activity.

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) effector functions represent the mode-of-action of many therapeutic antibodies. ADCC and ADCP rely on the binding of the antibody Fc domain to specific Fc-gamma receptors on the effector cells which activate a number of downstream pathways.

In collaboration with Svar Life Science, we generated glycoengineered trastuzumab containing homogeneous G0F glycans and core afucosylated G0 glycans using TransGLYCIT. This allows a direct comparison between the performance of the fucosylated and afucosylated glycoforms in reporter gene bioassays where specific target and effector cells were used to determine the ADCC and the ADCP of the modified antibodies. Following FabRICATOR digestion, the deconvoluted mass spectra of the glycoengineered trastuzumab scFc domain demonstrates the highly efficient generation of the G0 and G0F glycoforms using TransGLYCIT.

Efficient generation of glycoengineered antibodies with homogeneous glycan profiles

ADCC and ADCP functional assays (iLite®, SVAR) were carried out using the glycoengineered trastuzumab to determine the impact of core fucosylation on antibody effector function. In the ADCC reporter assay, we can see a clear shift to the left in the afucosylated antibody which indicates a clear and considerable increase in the ADCC potency, and in this instance, there was a calculated 10-fold decrease in EC50 following core afucosylation. In the ADCP assay we observe that there was not significant horizontal shift, which indicates that ADCP potency is similar between the two samples, however, the clear vertical shift that we observe following afucosylation indicates a considerable increase in ADCP efficacy

Core afucosylation increases ADCC potency and ADCP efficacy

The use of TransGLYCIT to remodel antibody glycan profiles allows a direct functional assessment of specific glycoforms to be performed using bioassays of this type which can support product development and increase functional understanding.

Determination of relationship between Fc N-glycan structure and antibody effector function

Fc N-glycan core afucosylation increases the ADCC potency and ADCP efficacy considerably. Deconvoluted mass spectra of the scFc fragment of trastuzumab carrying the G0 glycoform with and without core fucose. The antibody was digested with FabRICATOR and the subunits analyzed by reversed-phase LC-MS on an Agilent LC system coupled to a Bruker Impact II ESI-QTOF MS (top). ADCC and ADCP difference in potency (shift horizontally) and efficacy (shift vertically) of trastuzumab with the corresponding glycoforms using functional reporter assays (iLite®, SVAR). In accordance with previous studies, core afucosylation resulted in an increase in ADCC potency (here, an approx. 10-fold decrease in EC50 value). The ADCP potency, in contrast, remained similar upon core afucosylation, whereas the ADCP efficacy increased considerably

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