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Engineered Fc-glycosylation switch to eliminate antibody effector function
Qun Zhou1, Julie Jaworski1, Yanfeng Zhou1
1Biologics Research, Sanofi , Framingham, MA, USA.
Mabs
|September 7, 2020
Summary
Engineered antibodies with altered glycosylation at Asn298 eliminate effector functions like ADCC and CDC. This novel strategy avoids issues associated with traditional mutagenesis, offering a promising approach for safer therapeutic antibodies.
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- Antibodies mediate effector functions via Fcγ receptor (FcγR) interactions and complement activation.
- These effector functions are often undesirable for therapeutic antibodies, necessitating their elimination.
- Current methods for effector elimination via mutagenesis can cause manufacturability and immunogenicity problems.
Purpose of the Study:
- To develop a novel strategy for eliminating antibody effector functions by engineering glycosylation.
- To create alternative antibody glycosylation variants by switching the native glycosylation site to position 298.
- To assess the impact of this glycosylation switch on antibody binding, effector functions, and in vivo behavior.
Main Methods:
- Engineered antibody variants with a glycosylation site at Asn298.
- Confirmed engineered glycosylation site using SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB: 6X3I).
- Assessed FcγR binding via surface plasmon resonance, and effector functions (ADCC, CDC) in vitro. Evaluated in vivo T and B cell depletion in transgenic mice.
Main Results:
- The NNAS mutant (S298N/T299A/Y300S) showed no detectable binding to mouse or human FcγRs.
- Complete elimination of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was observed.
- In vivo studies showed no T or B cell depletion with the NNAS mutant, unlike the wild-type antibody.
- Structural analysis confirmed glycosylation at Asn298, predicted to clash with FcγRs.
- NNAS mutants retained antigen and neonatal Fc receptor binding, with comparable stability, half-life, and purification yields.
Conclusions:
- Switching antibody glycosylation to Asn298 is a novel and effective strategy to eliminate effector functions.
- This approach avoids potential manufacturability and immunogenicity issues associated with mutagenesis.
- Engineered antibodies devoid of ADCC and CDC activities demonstrate potential for safer therapeutic applications.
