Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability.

Area of Science:

• Biochemistry
• Immunology
• Protein Engineering

Background:

• Therapeutic antibody development requires careful isotype selection due to differential Fcγ receptor and complement binding.
• IgG2 and IgG4 isotypes exhibit lower Fcγ receptor affinity, but IgG2 retains some effector function and IgG4 can undergo Fab arm exchange.
• Existing IgG1 scaffolds often possess effector functions that may compromise safety and efficacy.

Purpose of the Study:

• To engineer an IgG1-based scaffold with eliminated effector function while maintaining or improving stability.
• To minimize mutations for achieving both stability and lack of effector function.
• To develop a versatile scaffold for Fc-containing therapeutics needing reduced Fcγ receptor binding.

Main Methods:

• Engineered IgG1 variants by mutating the Asn²⁹⁷ glycosylation site, focusing on N297G, N297Q, and N297A.
• Introduced a novel disulfide bond in the CH2 domain of the N297G variant to enhance stability.
• Conducted extensive biophysical analyses and pharmacokinetic (PK) studies in multiple species.

Main Results:

• The N297G variant demonstrated superior stability and developability compared to N297Q and N297A mutants.
• The engineered disulfide bond significantly improved the stability of the N297G scaffold beyond parental IgG1 levels.
• PK studies confirmed the scaffold's developability and suitability for therapeutic applications.

Conclusions:

• A novel IgG1 scaffold with engineered lack of effector function and enhanced stability was successfully developed.
• This scaffold, featuring the N297G mutation and a novel disulfide bond, offers a viable alternative to other IgG isotypes.
• The engineered scaffold holds potential for broad application in therapeutic antibodies, bispecific antibodies, and Fc fusions requiring attenuated effector functions.