FucoFree - afucosylated antibody with enhanced antibody-dependent cellular cytotoxicity

Introduction

Recent advancements in the development of therapeutic monoclonal antibodies (mAbs) have led to effective treatment of a variety of different types of cancer and inflammatory disorders. It is well known that monoclonal antibodies forge an important defense barricade against many types of threats, from environmental pathogens, foreign objects, to oncogenic entities in situ. A monoclonal antibody distinguishes its antigen via highly specific intermolecular interactions, and subsequently recruits serum immune cells to eliminate targets via antibody-associated effector functions, namely antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Detailed studies have recently focused on ADCC and how it may be enhanced for therapeutic applications. For example, a mAb unleashes the power of effector cells to destroy its target upon the interaction between the mAb Fc domain and the FcvRs on the surface of effector cells. The ADCC of a mAb can be modulated with alterations to the Fc region and one of such approaches is through Fc glycan chain engineering. Said engineering is a unique and innovative way to both improve and target mAb therapeutic action. Given these studies, ADCC is now one of the major modes of action for many current therapeutic antibodies. It is the primary mechanism which determines and drives the efficacy of an antibody and is therefore a crucial factor for its therapeutic potential.

As mentioned above, the structure and composition of the mAb glycan content can influence its ADCC activity. Particularly, an absence of core fucose residues on the Fc N-glycan moiety has been shown to increase IgG1 Fc binding affinity to CD16 (Fc gamma receptor IIIa). This receptor is present on immune effector cells such as natural killer cells and said binding by IgG1 leads to enhanced ADCC activity. The elimination of the fucose residues was initially achieved via a combination of chemical and enzymatic treatment of the mAb, a laborious process with high operational cost. Recently, the discovery of the gene responsible for the addition of the fucose residues, fucosyltransferase 8 (FUT8), along with advancements in gene manipulation techniques, render it possible to create antibody-expressing cell lines with anFUT8 gene knockout for the production of afucosylated mAbs (Figure 1).


Figure 1. The removal of glycan core fucose residues is achieved by knockout of the host cell fucosyltransferase-8 (FUT8) gene.

Establishment of FucoFree System

Given the importance and impact of glycan engineering enhancement of mAbs for therapeutic use, researchers at Sino Biological have used such gene editing techniques to establish a proprietary “FucoFree” eukaryotic cell culture system for the custom expression of afucosylated mAbs. These FucoFree cell lines have been rigorously tested both at the genetic level to verify the exhaustive knockout of the FUT8 gene and at the protein level to confirm the absence of fucose residues in the antibody glycan chain. Host cells in the FucoFree system include a FUT8 knockout (FUT-/-) CHO cell line and a FUT-/- HEK293 cell line, each individually engineered to satisfy the demands of various projects and scale of production (Figure 2).




Figure 2. High-quality Antibodies Produced in “FucoFree” Cell Lines. A) Recombinant antibody demonstrating >95% purity and >98% monomer distribution. (B) LC-MS analysis of a mAb expressed in a conventional (two top panels in B) and the corresponding“FucoFree” (two bottom panels in B) cell line. A difference in the heavy chain molecular weight of 145 Da was observed, corresponding well with the MW of a fucose residue.

The FucoFree system is a superior antibody/recombinant protein expression platform in many ways including:

1. Well-established gene knockout technology
2. Afucosylated antibody
3. High-yield and high-throughput antibody expression
4. Multi-scale antibody production
5. Full-spectrum antibody characterization
6. Excellent developability and licensing opportunities.

Through the application of the FucoFree system, Sino Biological offers a comprehensive yet flexible service package designated to address:

1. mAb expression using custom sequences
2. mAb glycan profiling
3. mAb ADCC assessment
4. ADCC-enhanced mAb candidate screening
5. Stable expression cell line establishment

By Molecule Initial