Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity

Abstract

The effector activity of antibodies is dependent on engagement with Fcγ receptors (FcγRs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with FcγRs is hampered by substantial structural and functional FcγR diversity among species. In this report, we used mice expressing only human FcγRs to evaluate the contribution of FcγR-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon FcγR engagement, with minimal protection against anthrax toxin observed in FcγR-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human FcγRs and assessed their activity in FcγR-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating FcγRs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.

Figure 1. Generation and characterization of mouse-human chimeric anti-PA mAb.

(A) Representation of the mouse-human chimeric anti-PA (19D9) mAb. The constant regions of the heavy (CH1-3) and light chain (Cκ) of mouse IgG1, κ (parental mAb) were replaced with those of hIgG1 and Cκ, respectively. Recombinant 19D9 hIgG1 wild-type and Fc domain variants were generated. No difference in their affinity and specificity for PA was evident among the various Fc domain variants, as assessed by (B) ELISA and (C) surface plasmon resonance (affinity values are shown in Supplemental Table 2).

Figure 2. FcγR requirement for the neutralizing activity of the anti-PA mouse-human chimeric mAb.

(A) The protective activity of 19D9 hIgG1 against anthrax LeTx was compared in BMDMs obtained either from humanized FcγR (wild-type) or FcγR-deficient (FcγRα^null) mice. n = 4; ***P < 0.0001; n = 4. (B) Human FcγR expression profile of BMDMs from humanized FcγR (solid black line) or FcγRα^null (solid gray filled) mice (isotype control; dotted line). (C) Comparison of the protective activity of 19D9 hIgG1 (750 μg i.p.) in humanized FcγR and FcγRα^null mice following challenge with B. anthracis Sterne strain. n = 5 per group; **P = 0.005, FcγRα^null vs. humanized FcγR.

Figure 3. Enhancement of the neutralization activity of anti-PA hIgG1 mAb through Fc domain engineering.

Fc domain variants of 19D9 hIgG1 with differential binding capacity for the various classes of human FcγRs were generated and their neutralization activity was assessed both (A) in vitro and (B and C) in vivo. (A) LeTx-induced cytotoxicity was assessed in BMDMs in the presence of the different Fc domain variants of 19D9 hIgG1. n = 2; *P < 0.05, **P < 0.01, ***P < 0.001, vs. wild-type IgG1. (B) Enhanced in vivo protective activity of the G236A/S239D/A330L/I332E 19D9 hIgG1 variant in FcγR-humanized mice challenged with B. anthracis. Mice received the indicated mAb variant (350 μg) or PBS i.p. 3 hours prior to challenge. n = 11–12 per group; *P = 0.0094, compared with wild-type hIgG1 group. (C) The neutralization activity of 19D9 hIgG1 Fc domain variants (750 μg i.p.) with differential FcγR binding capacity was compared in humanized FcγR mice following challenge with B. anthracis. n = 10–12 per group; *P = 0.005, **P = 0.002, compared with N297A group.