ADCC Enhancement Technology

ADCC-Enhancement-Technology

ADCC Enhancement Technology

Antibody-dependent cellular cytotoxicity (ADCC) is an important immune mechanism for antibodies. ADCC-enhanced antibodies have been shown to exhibit anti-cancer effects in vivo. Current research is focusing on using identification and construction of therapeutic antibodies to enhance their affinity to FcR and thereby, improve the ADCC process. Being an innate immune defense mechanism and now proved to be highly customizable tools for us to fight against cancer, ADCC is believed to be the new milestone!

Creative Biolabs has a team of professional scientists that, with their profound knowledge of ADCC enhancement technologies, can help you target various diseases of interests and accelerate your research.

ADCC Mechanism

ADCC is a simple but yet vital mechanism that happens when an immune system reacting to diseased or infected cells. The ADCC effect is triggered by interaction between the Fc region of an antibody to the Fc receptor (FcR) on the effector cells (including natural killer (NK) cells, macrophages, and dendritic cells) resulting in effector cell degranulation on Interferon-gamma (IFNγ), granzymes or other cytotoxic compounds cleave-target cells. This process is highly dependent on the glycosylation profile on Fc portion of an Ab and the polymorphism of the Fc gamma receptor.

1-1-ADCC-Enhancement-Technology-11.png
1-1-ADCC-Enhancement-Technology-12.png
1-1-ADCC-Enhancement-Technology-13.png
1-1-ADCC-Enhancement-Technology-14.png

Versatile Platforms

Afuco™ Platform
Glymax™ Platform
Fucoanalog™ Platform
Fc Protein-Engineering Platform
Cross-Isotype Engineering Platform
CD16A Bispecific Antibody Engineering Platform
Glyco- and Fc Protein Dual Engineering Platform

Afuco™ Platform

The oligosaccharide from the amino acid asparagine-297 (N297), existing in the IgG domain, is critical for the binding between FcγR and complement factor C1q. Currently, several glycoengineered antibody treatments, anti-CD20 mAb (obinutuzumab) and the anti-CCR4 mAb (mogamulizumab), have been approved. Sufficient rationale is studied to prove enhancing effect of glycoengineering-based ADCC. Creative Biolabs has established an advanced Afuco™ platform for the glycoengineered therapeutic antibody production.

Afuco™ technology platform Fig.1 Our Afuco™ technology platform: Development of defucosylated antibody drugs by FUT8 gene knockout.

Using our DEFUCO™ platform, the de-fucose antibodies developed exhibit a significant ADCC activity increase in vitro compared to unmodified antibodies:

Anti-CD39 5F2 Exerts ADCC against Mouse and Human CD39 Fig.2 Anti-CD39 5F2 Exerts ADCC against Mouse and Human CD39.

● Glymax™ Platform

Instead of directly eliminating any genes, Creative Biolabs' proprietary Glymax™ technology redirects fucose synthesis through a heterologous enzyme, which could deplete fucose pool inside the cell. For any successfully established stable CHO cell lines, it could produce defucosylated antibodies with up to 100-fold ADCC activity against tumor cells or other virus-infected cells.

Antibodies produced by GlymaxX engineered cells show an increased ADCC response Fig.3 Antibodies produced by GlymaxX engineered cells show an increased ADCC response.

We use Glymax for any novel antibody projects and biomarkers (ADCC enhanced originator mAbs) or biosimilars projects if adjusting for specific fucose levels is critical.

● Fucoanalog™ Platform

On the contrary with genetic engineering at the cellular level, Creative Biolabs has made a breakthrough in reducing or removing the fucose content on the antibody of interest. This is achieved by adding a fucose analog to the medium to regulate the metabolic pathway of the cell. A significant increase in the activity of the monoclonal antibody ADCC is therefore achieved.

Fc Protein-Engineering Platform

The rationale for developing Fc protein-engineered ADCC is to swap amino acids directly in the FcγR / FcγRIIIa binding site within the Fc domain of that antibody. For example, modifactions on the following five sites of Margetuximab, F243L/R292P/Y300L/L235V/P396L in the Fc region, can reduce its binding ability to CD32B and hence, improve the ADCC effect. By exchanging all solvent amino acids in the human IgG1 Fc domain with alanine, our approach can identify various Fc domain variants with different FcγR / FcγRIIIa binding profiles. Using this approach, we identified a triple variant that shows a high affinity for FcγR / FcγRIIIa and thus, stronger ADCC activity.

Fc-optimized antibody shows an increased ADCC response Fig.4 Fc-optimized antibody shows an increased ADCC response.

Cross-Isotype Engineering

Creative Biolabs developed a “cross-isotype” Fc domain – IgGA, using advanced protein engineering strategies to produce ADCC-enhanced antibodies via a hybrid IgG1/IgA Fc domain. These engineered IgGA antibodies can significantly increase FcγRIIIa (CD16a) binding capacity. In vitro and animal experiments have shown that IgA exerts an efficient and stronger cell-killing effect than IgG1, who goes through the FcaRI receptor by the surface of effector cells.

MOA of hybrid IgG1/IgA antibody Fig.5 MOA of hybrid IgG1/IgA antibody

Hybrid IgG1/IgA design and SPR evaluation Fig.6 Hybrid IgG1/IgA design and SPR evaluation.

CD16A Bispecific Antibody Engineering

Creative Biolabs’ ADCC antibody development team provides a novel way to target NK and DC cells through high-affinity CD16A bispecific antibodies, and then driving activated NK/DC cells to exert ADCC effects on tumor cells. A kind notice that our antibody customization team would be able to help you design and co-develop CD16A bispecific antibodies, specifically in combination with other highly expressed antigens on the surface of tumor cells, such as EGFP, BCMA, etc. A variety of published studies have demonstrated that CD16A BiAbs with enhanced ADCC modification have better in vitro killing effects.

MOA of CD16A biAbs in tumor immune microenvironment Fig.7 MOA of CD16A biAbs in tumor immune microenvironment.

CD16A BiAb (AFM24) potently kills HCT-116 cells with higher efficacy and potency than cetuximab Fig.8 CD16A BiAb (AFM24) potently kills HCT-116 cells with higher efficacy and potency than cetuximab.

Glyco- and Fc Protein Dual Engineering

ADCC-optimized amino acid exchanged Fc variants, when working with glycoengineered engineered variants, show improved NK cell-mediated ADCC activity. Creative Biolabs’ scientists have developed combined Fc engineering approaches based on the latest research. We are committed to achieving greater success with you together.

Schematic diagram of a dual engineering strategy for glyco- and Fc proteins Fig.9 Schematic diagram of a dual engineering strategy for glyco- and Fc proteins.

The RTX-DE(+fu) antibody was glyco-optimized for ADCC and harbored an amino acid to exchange S239D-I332E against CD20. Enhanced FcγRIIIa binding and ADCC activity were observed. Fig.10 The RTX-DE(+fu) antibody was glyco-optimized for ADCC and harbored an amino acid to exchange S239D-I332E against CD20. Enhanced FcγRIIIa binding and ADCC activity were observed.


For more details about our ADCC enhancement technology service, please do not hesitate to contact us.

References

  1. Kanazawa T.; et al. Anti-CCR4 Monoclonal Antibody Mogamulizumab for the Treatment of EBV-Associated T- and NK-Cell Lymphoproliferative Diseases. Clinical Cancer Research. 2014, 20(19):5075-5084.
  2. Junttila T T.; et al. Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer. Cancer Research. 2010, 70(11):4481-4489.
  3. Kelton W.; et al. IgGA: A “Cross-Isotype” Engineered Human Fc Antibody Domain that Displays Both IgG-like and IgA-like Effector Functions. Chemistry & Biology. 2014, 21(12):1603-1609.
  4. Kellner, C.; et al. Fc Glyco- and Fc Protein-Engineering: Design of Antibody Variants with Improved ADCC and CDC Activity. Methods in Molecular Biology. 2018, 381–397.

RESOURCES

Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details. 

All products and services are for Research Use Only. Do Not use in humans.

CASE STUDY

ADCC Assay WT vs AfucoTM Mabs  Visit

Resource

Antibody Fc Engineering: Towards Better Therapeutics  Visit

ONLINE INQUIRY

Creative Biolabs has established a team of customer support scientists ready to discuss ADCC/CDC optimization strategies, antibody production, bioinformatics analysis and other molecular biology/biotechnology issues.

  • *
  • *
  • *