A variant human IgG1-Fc mediates improved ADCC

Area of Science:

• Immunology
• Protein Engineering
• Biochemistry

Background:

• The Fc region of immunoglobulin G (IgG1) interacts with FcγRIIIa on natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC).
• Improving FcγRIIIa binding enhances ADCC, a crucial mechanism for therapeutic antibodies.

Purpose of the Study:

• To engineer improved human IgG1 Fc variants with enhanced binding to human FcγRIIIa using ribosome display.
• To characterize the functional impact of these variants on ADCC activity.

Main Methods:

• Ribosome display was employed to select IgG1 Fc variants with improved binding to FcγRIIIa from a library generated by error-prone PCR.
• Binding affinities were assessed using radioimmunoassay and AlphaScreen assays.
• Functional ADCC activity of engineered antibodies was evaluated in cell-based assays.

Main Results:

• Several IgG1 Fc variants demonstrated improved binding to FcγRIIIa compared to wild-type.
• A specific variant, 125_B01, exhibited significantly enhanced ADCC activity, with a four-fold reduced EC50 and increased maximal lysis.
• The enhanced function of 125_B01 was attributed to a Phe243Leu substitution in the CH2 domain, which also influenced glycosylation patterns.

Conclusions:

• Engineered IgG1 Fc variants can significantly enhance FcγRIIIa binding and ADCC activity.
• The Phe243Leu substitution is a key driver of improved FcγRIIIa binding and ADCC, potentially through altered glycosylation.
• These findings offer a strategy for developing more potent therapeutic antibodies targeting FcγRIIIa-expressing cells.