Emma Zhao discusses how high-yield and high-throughput afucosylated mAb expression benefits therapeutic antibody efficacy
An absence of fucose residues on the antibody Immunoglobulin γ (IgG) constant (Fc) region N-glycan moiety has been demonstrated to increase the binding affinity to IgG-Fc-receptor (FcγR) IIIa. It results in increased antibody-dependent cellular cytotoxicity (ADCC) since this receptor is found on immune effector cells like natural killer cells. Sino Biological has established a proprietary FucoFree eukaryotic cell culture system for the custom expression of afucosylated mAbs. The company provides high-yield and high-throughput antibody expression with >95% purity and low endotoxin level (<1 EU/mg).
How Does The Antibody Work To Eliminate The Antigens?
Targets are eliminated by immune cells recruited by antibodies that specifically bind to their antigen. Through their effector functions, IgG antibodies mediate immunity and inflammation. Interactions between complement proteins and FcγR on myeloid and Natural Killer (NK) cells are mediated by the Fc region of the IgG antibody. The next step is antibody-dependent cellular cytotoxicity (ADCC), in which the NK cells lyse the target cells with antigens presented on the membrane surface. It is one of the major Fc-dependent effector functions of IgG. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the FcγRIIIa.
Targeted monoclonal antibody (mAb) therapy is a promising therapeutic approach for many cancer types. The majority of mAbs that are approved for use in oncology treatments trigger ADCC. Patients with metastatic breast cancer who were given trastuzumab and a taxane responded better as FcgRIIIa antibody binding increased. One way to improve the binding is to design and engineer the Fc region on antibodies, such as afucosylation.
Fucosyltransferase (FUT), which regulates the fucosylation of O- and N-glycans, is crucial for numerous physiological and pathological processes, including infection, cancer, and inflammation. The N-glycans on the Fc portion of human IgG are typically heavily fucosylated (~90%). Low- to non-fucosylated monoclonal antibodies improves Fc_IIIa binding. Obinutuzumab, an afucosylated anti-CD20 antibody, has received approval to treat people with chronic lymphocytic leukemia because clinical trials have demonstrated its better efficacy.
How Can The Fucosylation On Fc Be Lowered?
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 costs. α-1,6-fucosyltransferase (FUT8) is the gene responsible for the addition of the core fucose residues that catalyses the transfer of fucose from GDP-fucose to N-linked type complex glycopeptides (Fig. 2). The key enzyme in N-glycan core fucosylation is FUT8. Due to advancements in gene editing techniques, it is now possible to create endogenous FUT8 knockout cell lines to generate fucose-free antibodies. Humanised IgG1 antibodies expressed in fucose-free CHO cells have been shown to bind human FcgRIIIa with a 50-fold increased affinity compared to those produced in wild-type CHO cells.
A Novel Expression System
Given the significance and impact of glycan engineering enhancement of mAbs for therapeutic use, Sino Biological has developed the FucoFree eukaryotic cell culture system using gene editing techniques. These FucoFree cell lines have undergone extensive testing, both at the genetic and protein levels, to confirm the exhaustive knockout of the FUT8 gene and 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. Starting from amino acid sequences of antibody provided by customers, Sino Biological will deliver the afucosylated mAbs with its glycan profiling (Fig. 3B). The company provides high-yield and high-throughput antibody expression with >95% purity and >98% monomer distribution (Fig. 3A). It can also assess its ADCC response and perform ADCC-enhanced mAb candidate screening. The production scale can range from milligram to gram level. Afucosylated mAb expression is also included in the firm’s HTP services, rendering faster turnaround time and lower costs. A stable and high afucosylated mAb expression cell line can also be established.
Currently, at least 35 glycoengineered antibodies, with their Fc fucose partially or entirely removed, have been investigated in animal models or clinical trials. Sino Biological anticipates that its services with the expression of afucosylated mAb will be helpful in the research and development community and open doors for more afucosylated mAb to enter clinical trials and subsequently be approved for use in humans.
Dr Emma Zhao is with Sino Biological