Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

Area of Science:

• Immunology
• Biotechnology
• Pharmacology

Background:

• The Fc region of antibodies is crucial for cytotoxic activity and serum half-life.
• Therapeutic antibodies often require attenuated effector functions to avoid immune activation and safety liabilities.
• Previous studies focused on human Fc-γ receptor interactions, leaving murine antibody Fc function less understood.

Purpose of the Study:

• To evaluate the efficacy of known Fc region variants in reducing effector functions in murine antibodies.
• To identify novel Fc variants that can be translated across species for preclinical studies.
• To assess the impact of Fc variants on antibody pharmacokinetics, thermostability, and bispecific antibody production.

Main Methods:

• Testing N297G (NG) and D265A, N297G (DANG) variants for complement activation and Fc-γ receptor-mediated cytotoxicity in murine models.
• Introducing L234A, L235A, P329G (LALA-PG) substitutions into murine IgG2a and human IgG1 antibodies.
• Evaluating LALA-PG variant effects on complement binding, Fc-γ receptor-dependent cytotoxicity, pharmacokinetics, and thermostability in rodents.
• Assessing LALA-PG variant suitability for knobs-into-holes bispecific antibody production.

Main Results:

• NG and DANG variants retained potent complement activation in mice, causing safety issues.
• The LALA-PG variant eliminated complement fixation and Fc-γ receptor-mediated cytotoxicity in both murine and human antibody formats.
• LALA-PG variant antibodies exhibited normal pharmacokinetics in rodents and maintained thermostability.
• The LALA-PG variant facilitated efficient production of effector-attenuated bispecific antibodies.

Conclusions:

• Standard Fc variants like NG and DANG are unsuitable for reducing effector function in murine models due to retained complement activation.
• The LALA-PG variant provides a reliable method for generating effectorless antibodies that translate effectively between murine and primate systems.
• LALA-PG variant antibodies are suitable for preclinical development, offering typical pharmacokinetics and enabling the production of attenuated bispecific antibodies.