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

Abstract

Edited by Peter CresswellThe antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-␥ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-␥-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

Regulation of antibody-mediated complement-dependent cytotoxicity by modulating the intrinsic affinity and binding valency of IgG for target antigen

“…A possible solution to decouple Fc-mediated effector functions from the intended mechanisms of action of the bispecific antibody can be achieved by engineering the Fc with a set of mutations that abrogate immune effector mechanisms. [62][63][64] In conclusion, the results of our study provide a deeper understanding of factors that regulate CDC and underline the pivotal roles of antibody's intrinsic affinity and binding valency to the target antigen in the capacity to recruit C1q and mediate complement activation. We suggest a careful examination of these key design parameters when developing clinically relevant antibody and bispecific antibody therapeutics.…”

Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies

“…The blocking anti-C1q antibody was produced using the variable domain sequences of the previously described anti-C1q M1 antibody (Hong et al, 2016a) on a mouse IgG2a backbone with the effector-attenuating substitutions L234A, L235A, and P329G (Lo et al, 2017). 9-months-old female mice were anesthetized with 3% isoflurane and placed on a stereotaxic apparatus (Stoelting) for surgery.…”

TIGIT-Fc Promote Immune Tolerance at the Feto-maternal Interface

“…The LALA-PG Fc variant was constructed as previously described (24). All fusion proteins were obtained via the FreeStyle 293 expression system (Invitrogen) according to previously reported methods (25,26) and subsequently purified using protein A-sepharose from the harvested cell culture supernatant.…”