Biointron Announces the Official Launch of Afucosylated Antibody Expression Platform!

Biointron is excited to announce our newly launched CHOK1-Fut8KO expression platform for afucosylated antibodies. Combined with our high-throughput recombinant antibody expression platform and cell culture technology, afucosylated antibodies can be rapidly produced.

In July 2023, Osemitamab (TST001) was developed by Transcenta and received approval from the China CDE (Center for Drug Evaluation) and South Korea MFDS (Ministry of Food and Drug Safety). It demonstrated anti-tumor activities in preclinical models with a broad range of CLDN18.2 expression. Osemitamab will be tested in a TranStar 301 Global Phase III Pivotal Trial, in combination with Nivolumab and chemotherapy for the first-line treatment of patients with HER2 negative CLDN18.2, expressing locally advanced or metastatic gastric or gastroesophageal (G/GEJ) adenocarcinoma. Additionally, they are in the process of EU and FDA regulatory interaction.

01: The mechanism of therapeutic antibodies

Monoclonal antibody drugs that have been widely approved for cancer treatment primarily belong to the IgG1 subtype. The central mechanism by which these antibodies function is through the ADCC (antibody-dependent cell-mediated cytotoxicity) effect. The constant region (Fc) of IgG1 mediates antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis by binding to the FcγRIIIa receptor on the surface of cells, thus targeting and killing cancer cells 1.

02: Associations between fucose modification and antibody ADCC

The mechanism of action of monoclonal antibodies, glycosylation sites, types of glycans, and their abundance can all impact the effectiveness, safety, and stability of the product. The glycosylation level of the Fc region of the monoclonal antibody can also directly influence the efficiency of ADCC and CDC in killing cancer cells.

ADCC is triggered after the Fc binds with the Fc-γ receptor (FcγR). This binding is significantly influenced by N-glycans in the CH2 structural domain. Studies have shown that reducing or removing fucose in the core glycan structure can enhance ADCC activity. In therapeutic antibodies such as CD20, Her2, and EGFR, removal or reduction of core fucose in N-glycans results in stronger affinity for FcγRIIIa, enhanced ADCC activity, and better therapeutic effects in animal models or clinical settings. Furthermore, research has shown that IgG antibodies expressed by mutant strains with fucose removed exhibit markedly improved binding capacity to FcγRIIIa and enhanced ADCC effects2.

03: The importance of removing fucose modification

Chinese hamster ovary (CHO-K1) host cells have been widely used in the development of various biological antibody drugs, such as the development and production of therapeutic proteins. CHO-K1 cells have significant advantages in producing complex formulations, such as antibodies with glycosylation features similar to human IgG. 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 antibody-dependent cell toxicity (ADCC).

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. Therefore, CHO cells remain the first choice for biopharmaceutical companies.

Currently, there are many methods to reduce or remove fucose, such as genetic engineering, enzymatic deglycosylation, regulation of cellular metabolic pathways, or chemical modification. However, finding a more efficient method to remove fucose is still an urgent need.

04: Biointron’s platform advantages

Biointron uses gene-editing technology to knock out fucosyltransferase (Fut8) genes in independently modified CHOK1BN cells. Fut8 is an enzyme that catalyzes the formation of α-1,6 fucosyl glycosidic bonds. By knocking out the gene that expresses this enzyme in the CHO cell genome, the cells are unable to perform normal fucose modification on secreted antibodies, thus achieving the goal of afucosylation.

Biointron’s fucose-free host cell CHOK1BN-Fut8KO can be used for industrial production after antibody characterization and functional verification. Compared to the unmodified CHOK1BN, the antibody produced by the modified cell line not only achieves the removal of fucose but also maintains consistency in terms of expression quantity and quality.

• Fast antibody delivery cycle for purified antibodies

• Host cell line with fucose removed

• High purity, low endotoxin antibodies

Case Study

1)Glycosylation profiling

Glycosylation profiling was performed on two types of cells, and the results showed that the modified cells did not detect fucose modification in 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).

2)Purity verification of the antibody expressed by CHOK1BN-Fut8KO

Results showed 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.

3)Antibody ADCC activity

When 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, results showed that the antibodies expressed by CHOK1BN-Fut8KO have significantly increased NK cell-mediated killing of target cells compared to antibodies from CHOK1BN. Also, their ADCC EC50 values were significantly improved.

4)Antibody affinity measuring

When using Biacore to measure the affinity between the antibody and the antigen FcγRIIIa, the results showed that the afucosylated antibody expressed by CHOK1BN-Fut8KO can significantly increase its affinity for binding to the receptor, thereby enhancing the ADCC effect.

The construction of Biointron’s afucosylated CHOK1BN-Fut8KO cell line eliminates the need for additional processes to remove fucose, reducing production costs while also improving product uniformity and purity. Biointron will continue to optimize the afucosylated antibody expression platform, ensuring the best quality and service to support early research on antibody drugs.

References

[1] Nitin Agarwal etal., N-Glycosylation of IgG and IgG-Like Recombinant Therapeutic Proteins: Why Is It Important and How Can We Control It? ; Annu. Rev. Chem. Biomol. Eng. 2020. 11:13.1-13.28.

[2] Shields RL et al.，Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem. 2002; 277:26733-40.