Figure 1: Mechanisms of Antibody Action
We use advanced gene-editing technology to knock out the fucosyltransferase (Fut8) alleles in the genome of the self-engineered CHOK1BN cells. Fut8 is an enzyme that catalyzes the formation of α-1,6 fucosyl glycosidic linkages. By targeting and eliminating the gene responsible for this enzyme's expression in the CHO cell genome, the cells become incapable of performing normal fucosylation on secreted antibodies. This approach effectively achieves the objective of eliminating fucosylation.1
Fucose-free host cell CHOK1BN-Fut8KO can be used for industrial production after antibody characterization and functional verification. Compared to the unmodified CHOK1BN cell line, the antibody produced by the modified cell line not only achieves afucosylation but also maintains consistency in terms of expression quantity and quality.
Figure 2: FUT8 Gene's Role in Fucosylation Modulation
Results:
Glycosylation profiling was performed on two types of cells. Fucose was not detected on modified cells in the N-glycan LC-MS – indicating that the CHOK1BN-Fut8KO cell can successfully express afucosylated antibodies. (C2193xxxx-1 is the standard CHOK1BN, C2193xxxx-2 is the CHOK1BN-Fut8KO).
Results:
High purity for antibodies expressed by CHOK1BN and CHOK1BN-Fut8KO. SDS-PAGE and SEC-HPLC verified that both antibody aggregation and purity met the >95% standard.
Results:
Comparing the ADCC effect of antibodies (Rituximab) expressed by the afucosylated host cell CHOK1BN-Fut8KO and antibodies expressed by CHOK1BN (as a control) on target cells through ADCC testing, the antibodies expressed by CHOK1BN-Fut8KO have significantly increased NK cell-mediated killing of target cells compared to antibodies from CHOK1BN. In addition, ADCC EC50 values were significantly improved.
Biacore affinity detection results
Results:
Using Biacore to measure the affinity between the antibody and the antigen FcγRIIIa, the afucosylated antibody expressed by CHOK1BN-Fut8KO can significantly increase its affinity for binding to the receptor, thereby enhancing the ADCC effect.
Afucosylation has been demonstrated to increase the effectiveness of antibody-dependent cellular cytotoxicity (ADCC). ADCC is regulated by N-linked glycosylation in the Fc region of the antibody.
Importantly, in therapeutic antibodies such as CD20, Her2, and EGFR, absence of core fucose on the Fc N-glycan enhances IgG1 Fc binding affinity to FcγRIIIa on immune effector cells such as natural killer cells. This results in increased ADCC activity, and thus can improve therapeutic efficacy.2
Chinese hamster ovary (CHO) cell lines are derived from the ovary of adult, female Chinese hamsters. CHO cells were first established in 1957 by T. Puck, and were subsequently multiplied and optimized in vitro, allowing it to be cultured indefinitely. The CHO-K1 cell line was derived as a subclone from that parental CHO cell line. They are typically the preferred host expression system for recombinant antibodies due to their advantages in producing complex therapeutics, manufacturing adaptability, and glycosylation features similar to human IgG.3
However, the Fc domain of the anti-tumor antibody drugs produced by wild-type CHO cells carries fucose sugar residues on both of its two biantennary complex polysaccharide chains. The presence of fucose residues can hinder the binding between the antibody and Fc receptor, thereby affecting the antibody's ADCC activity and anti-cancer efficacy.
Although YB2/0 hybridoma cells and plant cells can be used as alternatives to CHO cells for producing antibody molecules, they lack the stability and scalability advantages of CHO cells.
ADCC causes the natural killer (NK) cells to release cytokines and cytolytic agents, eventually killing the target cell. This is triggered by the engagement of immune complexes with FcγRIIIa present on NK cells, when the Fc binds with the Fc-γ receptor. This binding is significantly influenced by N-glycans in the CH2 structural domain.2
Afucosylated antibodies are monoclonal antibodies which have been modified. The N-glycan (oligosaccharide and sialylation) residues in the antibody’s Fc region do not have core fucose sugar units, which can increase the effector function of ADCC.
Several factors, including cell line characteristics, process control parameters, and cell culture medium components, may affect glycosylation and, consequently, biological activity, efficacy, stability, immunogenicity, clearance rate, and ADCC.2
1.Ma, M., Fu, Y., Zhou, X., Guan, F., Wang, Y., & Li, X. (2019). Functional roles of fucosylated and O-glycosylated cadherins during carcinogenesis and metastasis. Cellular Signalling, 63, 109365. https://doi.org/10.1016/j.cellsig.2019.109365
2.Pereira, N. A., Chan, K. F., Lin, P. C., & Song, Z. (2018). The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. mAbs, 10(5), 693–711. https://doi.org/10.1080/19420862.2018.1466767
3.Fischer, S., Handrick, R. and Otte, K. (2015) ‘The art of cho cell engineering: A comprehensive retrospect and future perspectives’, Biotechnology Advances, 33(8), pp. 1878–1896. doi:10.1016/j.biotechadv.2015.10.015.
