Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro

Abstract

Edited by Peter CresswellThe stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fc␥ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem. 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey Fc␥Rs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus Fc␥Rs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20؉ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro. These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.Monoclonal antibodies (mAbs) are the largest class of biopharmaceuticals and have diverse clinical applications (1). The choice of therapeutic mAb isotype to develop (IgG1, IgG2, or IgG4) is dependent on the target (cell surface versus soluble), desired biology, safety (risk of immunogenicity and undesired immunological effects), and manufacturability (expression, formulation, and stability). More than 80% of approved therapeutic mAbs are IgG1 isotypes that target cell surface receptors and are effective for oncology indications (2). For these therapeutic approaches, mAb isotypes that can induce cell killing such as complement-dependent cytotoxicity (CDC) 2 and antibody-dependent cell-mediated cytotoxicity (ADCC) are often desirable (3, 4). However, for non-oncologic indications, therapeutic mAbs without cytotoxic effector function may be more appropriate because cell killing may not be a goal of therapy.The Fc portion of IgG has interaction sites for the effector ligands, including Fc␥ receptors (Fc␥RI, Fc␥RII, and Fc␥RIII), C1q complement, and the neonatal Fc receptor (FcRn). IgG isotypes differentially engage Fc␥ receptors and C1q binding to recruit immune effector functions and initiate cytotoxic effector functions, (either ADCC or CDC (5)). Historically, IgG2 or IgG4 isotypes were thought to have minimal cytotoxic effector function, and have been selected for applications where cytotoxic effector function is not required or desirable (5). However, recent evidence suggests that IgG2 is...

Engineering of Fc Fragments with Optimized Physicochemical Properties Implying Improvement of Clinical Potentials for Fc-Based Therapeutics

“…It has been shown that the Tm of CH2 with mutations in L242C and L334C in IgG1 is 8.7°C higher than that in wtIgG1 ( 79 ). In another research ( 80 ), to improve the stability of an IgG1 variant with mutation of N297G (mAbW.IgG1, an effector function silenced IgG1), an additional disulfide bond according to the design of m01 was introduced between position L242 and K334 ( 81 ). As expected, the stability of this IgG1 variant (mAbW.SEFL2.0) with mutations on L242C/N297G/K334C is improved particular in the thermal stability, but it shows faster clearance in the rat in pharmacokinetics study.…”

“…A major concern is that deglycosylation will lead to instability and aggregation. Hence, the IgG1 scaffold with stable effector functions missing ( 81 ) needs to be further optimized. Based on the computational modeling, two variants mAbW.SEFL2.1 and mAbW.SEFL2.2 with tri-mutations on A287C/N297G/L306C and R292C/N297G/V302C, respectively, as mentioned earlier showed improved stability, decreased clearance rate, and longer half-life ( 80 ), which could be used for development of therapeutic mAbs without effector functions.…”

Next generation Fc scaffold for multispecific antibodies

“…Although SEFL2.2 is an efficient strategy to reduce Fc binding to FcγRI, FcγRIIA and to FcgRIIIA receptors ( Jacobsen et al., 2017 ; Liu et al., 2017 ), little is known about its mechanism of action. To unveil the molecular basis that mediates the loss of Fcγ receptor activity for SEFL2.2, we compared this structure with the co-crystal structures of 3 Fcγ receptors [FcγRI (PDB: 4X4M ), FcγRIIA (PDB: 3RY6 ) and FcγRIIIA (PDB: 5XJE )], each determined in complex with WT Fc ( Figure 2 A) ( Lu et al., 2015 ; Ramsland et al., 2011 ; Sakae et al., 2017 ).…”

Immune Complex Formation Is Associated With Loss of Tolerance and an Antibody Response to Both Drug and Target

“…The charge pair mutations create a heteroimmunoglobulin molecule with two distinct antigen-binding fragments (Fabs). By virtue of the absence of an Fc glycan, AMG 966 is a stable effector functionless (SEFL) antibody, which lacks the ability to interact with Fcγ receptors ( 11 ).…”