Fc Engineering Service for Complement Activation

Therapeutic monoclonal antibody is one of the most effective biotherapeutics so far. An important aspect of antibodies is their ability to bind antigens while at the same time recruiting immune effector functions. Most of the approved recombinant monoclonal antibody therapies belong to the human IgG1 subclass, which can participate in both the humoral and cellular components of the immune system. The wealth of information about antibodies provides researchers with the ability to molecularly engineer antibodies to modulate effector functions. Activation of the complement cascade acts as an early step in complement activation. Antibodies interact with the complement cascade through C1q binding to the Fc domain in the immune complex. This is an affinity-driven (very low affinity) interaction and is therefore suitable for Fc engineering to produce more effective antibodies.

Creative Biolabs focuses on the recent progress in therapeutic Fc engineering-associated effector functions (ADCC, ADCP, and CDC), and are committed to developing the optimal Fc mutation strategy to support the innovative drugs currently being studied in clinical trials. These Fc mutation methods have been validated and can fine-tune antibodies to improve the interaction with immune components, and finally provide clinical research examples as next-generation antibodies or biobetters.

Fc Mutation Strategies for Enhancing C1q Binding

The interaction of antibodies with FcγR and C1q depends on the amino acid sequence of the hinge and proximal CH2, as well as the glycosylation of the conserved amino acid N297 in the CH2 region. Several approved antibodies have shown effective in vitro CDC activity, such as anti-CD20 mAb rituximab and ofatumumab, and a variety of Fc engineering methods have been validated to enhance complement-based CDC effector functions. Studies have shown that antibodies produced by Fc engineering technology have a 50-fold higher affinity for C1q, so that a single monomer antibody can effectively activate complement-dependent cytotoxicity (CDC) against cancer cell lines.

The interaction between Fc and C1q is a key step in triggering the CDC effect. Because the affinity between Fc and C1q is weak, more efficient antibodies can be obtained through Fc modification. The increase in CDC activity is closely related to the increase in affinity for C1q in a series of Fc mutations, of which various combinations of S267E, H268F and S324T are the most effective. Idusogie et al. found that K326W and E333S can increase the binding of C1q by 3 times and 2 times, respectively. Fc region of Rituximab has been modified by K326W/E333S, which can increase C1q binding capacity by 5 times and CDC activity by 2.3 times. Moore et al. found that the modification of S267E/H268F/S324T can also increase the C1q binding capacity by 47 times and the CDC activity by 6.9 times. These mutated Fc domains will have a wide range of uses, and can improve complement activation through therapeutic antibodies.

Fc Engineering Isotype Intended Function Result
K326W/E333S Glyco-IgG1 Increased C1q binding Enhance CDC
S267E/H268F/S324T Glyco-IgG1 Increased C1q binding
IgG1/IgG3 cross subclass Glyco-IgG1 Increased C1q binding
E345R/E430G/S440Y Glyco-IgG1 Hexamerization

Fig.1 Fc modification for enhancing CDC.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

References

  1. Sondermann, P., & Szymkowski, D. E. (2016). Harnessing Fc receptor biology in the design of therapeutic antibodies. Current opinion in immunology, 40, 78-87.
  2. Wang, X., Mathieu, M., & Brezski, R. J. (2018). IgG Fc engineering to modulate antibody effector functions. Protein & cell, 9(1), 63-73.

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.

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