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

Abstract

The 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-γ 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-γ-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.

Keywords: Fc receptor; Fc-γ receptor; aglycosylation; antibody engineering; antibody-dependent, cell-mediated cytotoxicity (ADCC); bispecific antibody; complement; complement-dependent cytotoxicity (CDC); protein stability; recombinant protein expression.

FIGURE 1.

Illustration of the location of the effector reduction substitutions on the antibody Fc lower hinge region. A, structure of the antibody Fc-γRIII receptor complex (PDB code 1E4K). The two chains of the antibody Fc are shown in teal and green. The Fc-γ receptor is shown in pink, and residues on the Fc within 5 Å are colored gray in the surface rendering. The location of the Asp-265 and Asn-297 variants (DANG) that eliminate glycosylation and reduce effector functions are indicated in red. The locations of the Leu-234, Leu-235, and Pro-329 positions are highlighted in yellow. B, alignment of relevant regions of the hinge (gray fill) and C_H2 domain (cyan fill) of murine IgG2a and human IgG. The naturally effector-attenuated human IgG4 sequence is also included for reference. The positions of the LALA-PG and DANG variants are indicated by yellow and red, respectively. The numbering of the C_H2 residues for human IgG1 is according to Eu convention.

FIGURE 2.

LALA variants improve half-antibody expression and bispecific antibody assembly compared with NG variants. A, knob (blue) and hole (red) murine IgG2a half-antibodies were expressed in CHO, followed by MabSelect SuRe affinity purification. Recoveries were normalized to wild-type knob and hole half-antibodies. A significant impact on expression is observed for the aglycosylated NG and DANG variants. All glycosylated variants, including LALA and LALA-PG, show expression profiles similar to the wild-type antibody. B) Assembly efficiency of murine IgG2a bispecific antibodies from knob and hole half-antibodies with substitution in the C_H2 domain. Representative S.E. for three or more experimental replicates is shown for wild-type and DANG antibody variants.

FIGURE 3.

LALA-PG antibodies retain the stability of wild-type antibodies. A–D, differential scanning calorimetry isotherms of wild-type murine IgG2a antibody (A; experimental data are shown in blue, and calculated isotherms are shown in orange), aglycosylated (NG) murine IgG2a (B; experimental data are shown in black, and calculated isotherms are shown in orange), murine IgG2a monospecific WT (blue) bispecific LALA-PG (red) antibodies (C), and human IgG1 and bispecific WT (blue) bispecific LALA-PG (red) antibodies (D). E, summary of the first and second thermal melt transition for murine (m) and human (h) Fc fragments and full-length antibodies.

FIGURE 4.

Murine C1q binding and C3 fixation are significantly reduced by LALA-PG substitutions. A–D, ELISAs of serum murine C1q binding (A), serum murine C3 fixation (B), isolated murine C1q binding (C), and tabulated EC₅₀ values for antibody complement fixation and binding (D). WT, blue; NG, black; DANG, green; LALA-PG, red. LALA-PG substitutions show a significant reduction in complement binding and fixation relative to NG and DANG substitutions. The data shown are representative of multiple experimental replicates. Error bars represent standard error. The data include two or more experimental replicates in A and B. All experiments include two or more technical replicates.

FIGURE 5.

Murine Fc-γ binding is significantly reduced by LALA-PG substitutions. A–D, ELISAs of representative mIgG2a antibody binding to Fc-γRI (A), Fc-γRII (B), Fc-γRIII (C), and Fc-γRIV (D). Tabulated binding was estimated by dividing the equivalent nanograms per milliliter wild type concentration at 25,000 ng/ml variant by 25000 ng/ml. The tabulated binding ratio was calculated as WT/variant at 25,000 ng/ml. The color code is as in Fig. 4. LALA-PG substitutions show a significant reduction in Fc-γ binding. In all cases, the LALA-PG substitutions demonstrate greater reduction in Fc-γ binding than N297G alone and equivalent or greater reduction in Fc-γ binding than DANG, except Fc-γRIII. Error bars represent standard deviation for two technical replicates.

FIGURE 6.

Anti-TfR LALA-PG bispecific antibodies do not deplete reticulocytes. A, no statistically significant reticulocyte depletion was observed with mIgG2a LALA-PG at 24 h post-dose. B, antibody levels are within the expected historical range. Some increase in antibody clearance is observed with anti-TfR^m, a murinized version of anti-TfR^D. C, pharmacokinetics analysis of non-targeting murine IgG2a LALA-PG (red) antibodies is similar to historic values (gray) for other non-targeting antibodies with wild-type effector regions.