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Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with Fc?RIII Binding and ADCC.

ABSTRACT: Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Aleuria aurantia lectin (AAL) were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (Fc?RIII; receptor for the constant region of an antibody)-whose affinity is mediated through core fucosylation-and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

SUBMITTER: Roucka M

PROVIDER: S-EPMC5374361 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC.

Roucka Markus M Zimmermann Klaus K Fido Markus M Nechansky Andreas A

Microarrays (Basel, Switzerland) 20161224 1

Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (<i>Physcomitrella patens</i>) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins <i>Aspergillus oryzae ...[more]

PMID: 28029136

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Project description:?-Glutamyl transpeptidase 1 (GGT1) is an N-glycosylated membrane protein that catabolizes extracellular glutathione and other ?-glutamyl-containing substrates. In a variety of disease states, including tumor formation, the enzyme is shed from the surface of the cell and can be detected in serum. The structures of the N-glycans on human GGT1 (hGGT1) have been shown to be tissue-specific. Tumor-specific changes in the glycans have also been observed, suggesting that the N-glycans on hGGT1 would be an important biomarker for detecting tumors and monitoring their progression during treatment. However, the large quantities of purified protein required to fully characterize the carbohydrate content poses a significant challenge for biomarker development. Herein, we investigated a new antibody-lectin sandwich array (ALSA) platform to determine whether this microanalytical technique could be applied to the characterization of N-glycan content of hGGT1 in complex biological samples.Our data show that hGGT1 can be isolated from detergent extracted membrane proteins by binding to the ALSA platform. Probing hGGT1 with lectins enables characterization of the N-glycans. We probed hGGT1 from normal human liver tissue, normal human kidney tissue, and hGGT1 expressed in the yeast Pichia pastoris. The lectin binding patterns obtained with the ALSA platform are consistent with the hGGT1 N-glycan composition obtained from previous large-scale hGGT1 N-glycan characterizations from these sources. We also validate the implementation of the Microcystis aeruginosa lectin, microvirin, in this platform and provide refined evidence for its efficacy in specifically recognizing high-mannose-type N-glycans, a class of carbohydrate modification that is distinctive of hGGT1 expressed by many tumors.Using this microanalytical approach, we provide proof-of-concept for the implementation of ALSA in conducting high-throughput studies aimed at investigating disease-related changes in the glycosylation patterns on hGGT1 with the goal of enhancing clinical diagnoses and targeted treatment regimens.

Dataset Information

Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with Fc?RIII Binding and ADCC.

Publications

Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC.

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