Effector Function Improvement via Antibody Skeleton Engineering

Rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Approaches in Fc protein engineering have been intensively evaluated by scientists at Creative Biolabs. As an acknowledged antibody service provider, Creative Biolabs can offer antibody functional assay services to help our customers obtain ideal laboratory test results contributing to your research and projects successfully. Our professional technical scientists can not only save your valuable time but also provide highly sensitive and qualified services for our clients all over the world.

Fc Protein Engineering

There are mainly two Fc protein engineering approaches, amino acid substitution and exchanging larger amino acid stretches between different isotypes. Exchanging amino acids directly in the FcγR binding site within the antibody’s Fc domain was a logical strategy to manipulate Fc/FcR interactions. Shields et al. (2001) exchanged all solvent-exposed amino acids in the Fc domain of human IgG1 to alanine and thereby identified various Fc domain variants with altered FcγR binding profiles. In this pioneering approach, a triple variant (S298A-E333A-K334A) was identified, which exerted a higher affinity to FcγRIIIa but showed diminished binding to FcγRIIa and FcγRIIb. This modification resulted in enhanced antibody-dependent cellular cytotoxicity (ADCC) activity with NK cells.

While many Fc protein engineering approaches are based on the substitution of selected individual amino acids, alternative strategies have been developed by exchanging larger amino acid stretches between different isotypes, demonstrating interesting profiles of effector functions. For instance, Kelton et al. (2014) reported the engineering of an IgGA ‘cross-isotype’ antibody which combines selected effector functions of both IgG and IgA. IgGA binds to FcαRI, FcγRI and FcγRIIa, while no binding to FcγRIIIa was observed.

Fig.1 Fc protein-engineering strategies. (Kellner, 2017)

Combining Glyco- and Protein Engineering

Most engineering strategies focus on either introducing amino acid exchanges in the protein backbone or in modifying the Fc bound glycosylation profile. Recently, combined Fc engineering approaches have been illustrated. For example, the ADCC-optimized S298A-E333A-K334A Fc variant was glycoengineered by expression in Lec13 cells to reduce fucose content. This double-engineered variant displayed improved NK cell-mediated ADCC activity.

Fig.2 Effects of IgG glycosylation on antigen recognition and effector functions. (Seeling, 2017)

Protein engineering could also be applied to improve antibodies’ capacity to trigger complement dependent cytotoxicity (CDC). Most CDC-optimized Fc variants were designed to more potently trigger CDC while maintaining FcγR-mediated ADCC activity. With novel technology platforms and professional experiment services, Creative Biolabs gets ready to provide you with the best protein engineering services. Please feel free to contact us for more details.

References

1. Shields, R. L.; et al. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. Journal of Biological Chemistry. 2001, 276(9): 6591-6604.
2. 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.
3. Kellner, C.; et al. Modulating cytotoxic effector functions by Fc engineering to improve cancer therapy. Transfusion Medicine and Hemotherapy. 2017, 44(5): 327-336.
4. Seeling, M.; et al. Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity? Nature Reviews Rheumatology. 2017, 13(10): 621.

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