Afucosylated antibodies increase activation of FcγRIIIa-dependent signaling components to intensify processes promoting ADCC.

Scot D Liu,Cecile Chalouni,John B Lowe,Judy C Young,Teemu T Junttila,Mark X Sliwkowski

doi:10.1158/2326-6066.cir-14-0125

Scot D Liu, Cecile Chalouni + Show 4 more

Open Access

https://doi.org/10.1158/2326-6066.cir-14-0125

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Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.

Highlights

Rituximab is a chimeric therapeutic antibody used for treating individuals with CD20þ hematologic cancers [1], whereas trastuzumab is a humanized therapeutic antibody used for treating individuals with HER2þ cancers [2, 3]
Rituximab and trastuzumab mobilize the innate immune system to attack tumors by promoting the interaction of immune cells and cancer cells expressing CD20 or overexpressing HER2, respectively. This method of cytotoxicity is mediated at the cellular level through the interaction of FcgRIIIa expressed on natural killer (NK) cells or macrophages and the Fc portion of the tumor-bound antibody
We demonstrate here that human NK cells stimulated with afucosylated trastuzumab or obinutuzmab exhibit an increase in activation of proximal FcgRIIIa signaling components and downstream signaling pathways, including the Vav-1, MAPK, and PI3K pathways

Summary

Introduction

Rituximab is a chimeric therapeutic antibody used for treating individuals with CD20þ hematologic cancers [1], whereas trastuzumab is a humanized therapeutic antibody used for treating individuals with HER2þ cancers [2, 3]. Along with the ability to disrupt survival and growth signaling in cancerous cells, these antibodies can facilitate antibody-dependent cellular cytotoxicity (ADCC) to destroy tumor cells [3, 4] In this context, rituximab and trastuzumab mobilize the innate immune system to attack tumors by promoting the interaction of immune cells and cancer cells expressing CD20 or overexpressing HER2, respectively. Rituximab and trastuzumab mobilize the innate immune system to attack tumors by promoting the interaction of immune cells and cancer cells expressing CD20 or overexpressing HER2, respectively This method of cytotoxicity is mediated at the cellular level through the interaction of FcgRIIIa expressed on natural killer (NK) cells or macrophages and the Fc portion of the tumor-bound antibody.

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