FcRn, but not FcγRs, drives maternal-fetal transplacental transport of human IgG antibodies

Abstract

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.

Keywords: Fc domain; FcRn; IgG; immunoglobulin; placental transfer.

Fig. 1.

Relative abundance of IgG Fc glycans in paired maternal and cord bloods in the Ugandan cohort. Total or antimalaria CSP IgGs were purified from maternal or venous cord blood, and the relative abundance of the following Fc glycoforms were characterized for each IgG category: G0, G1, G2, G0F, G1F, G2F, total galactosylation (G), total fucosylation (F), total bisection (N), and total sialylation (S). Significance was assessed by two-way ANOVA with Bonferroni’s multiple comparisons test where P < 0.0125 was considered significant, *P < 0.0125, **P < 0.0025, and ***P < 0.00025. Figure is related to SI Appendix, Tables S1 and S2.

Fig. 2.

Relative abundance of IgG Fc glycans in paired maternal and cord blood in the Nicaraguan cohort. Total or anti-Zika virus envelope (E) IgGs were purified from maternal or venous cord blood, and the relative abundance of the following Fc glycoforms were characterized for each IgG category: G0, G1, G2, G0F, G1F, G2F, total galactosylation (G), total fucosylation (F), total bisection (N), and total sialylation (S). Significance was assessed by two-way ANOVA with Bonferroni’s multiple comparisons test where P < 0.0125 was considered significant, *P < 0.0125, **P < 0.0025, and ***P < 0.00025. Figure is related to SI Appendix, Tables S1 and S2.

Fig. 3.

IgG subclass distribution in paired maternal and cord bloods. (A and B) Total or antimalaria CSP IgG from the Ugandan cohort. (C and D) Total or anti-Zika virus envelope (E) IgG from the Nicaraguan cohort. Data from Fig. 3 are quantified in SI Appendix, Table S2. The abundance of specific IgG subclasses is shown. Significance was assessed by paired t test with Bonferroni’s correction. ****P < 0.0001. Figure related to SI Appendix, Tables S1 and S2.

Fig. 4.

Effect of IgG1 core fucosylation and galactosylation on FcγR engagement. (A) The affinity of glycoengineered mouse-human chimeric IgG1 of anti-gpIIb (6A6) mAb for human FcγRIIIa^158F, FcγRIIIa^158V, and FcRn ectodomains was determined by SPR analysis, and representative SPR sensorgrams are presented. Data correspond to one experiment per interaction tested, which is representative of three independent experiments that gave similar results. (B) The equilibrium dissociation constant (K_D, M) and the SD are indicated for each tested interaction. Significance was assessed by one-way ANOVA followed by Bonferroni multiple comparison test. ns: not significant; ***P < 0.005; ****P < 0.0001. Figure is related to SI Appendix, Figs. S1–S3.

Fig. 5.

Effect of IgG2 core fucosylation and galactosylation on FcγR engagement. (A) The affinity of glycoengineered mouse-human chimeric IgG2 of anti-gpIIb (6A6) mAb for human FcγRIIIa^158F, FcγRIIIa^158V, and FcRn ectodomains was determined by SPR analysis, and representative SPR sensorgrams are presented. Data correspond to one experiment per interaction tested, which is representative of two or three independent experiments that gave similar results. (B) The equilibrium dissociation constant (K_D [M]) and the SD are indicated for each tested interaction. Significance was assessed by unpaired t test; ns: not significant. Figure is related to SI Appendix, Figs. S1–S3.

Fig. 6.

Evaluation of the in vivo Fc effector function and half-life of human IgG antibodies in FcγR and FcγR/FcRn humanized mice. (A) The cytotoxic activity of glycoengineered mAbs was evaluated in FcγR humanized mice. Time-course analysis of platelet counts following administration of glycoengineered anti-gpIIb (6A6) IgG1 (Upper) and IgG2 (Lower) mAb (2 mg/kg) is presented as the mean ± SEM from three to eight mice/group. The Fc domain variant N297A, lacking Fc glycosylation and therefore the FcγR binding ability, is used as negative control. Significance was assessed by two-way ANOVA followed by Bonferroni multiple comparison test; *P < 0.05 G0 vs. G0F and G2 vs. G2F. (B and C) The in vivo half-life of glycoengineered antihemagglutinin (FI6) IgG1 (Upper) and IgG2 (Lower) mAb (50 µg/mouse) was evaluated in FcRn/FcγR humanized mice. Results are presented as the mean ± SD from four to five mice/group. Significance was assessed by unpaired t test. ns: not significant. Figure is related to SI Appendix, Figs. S4 and S5.

Fig. 7.

Evaluation of transplacental transfer of human IgG antibodies in FcRn/FcγR humanized mice. (A) Pregnant FcRn/FcγR humanized mice were injected i.v. with human IgG1 mAb (3BNC117; 400 μg) 1 d prior to delivery, and the serum levels of human IgG1 were evaluated in the pups at different time points postpartum. (B) Experimental overview to differentiate placental- from GI-tract–mediated maternal-to-fetal transfer of human IgG in FcRn/FcγR humanized mice involved the use of surrogate mothers that were injected or not with human IgG antibodies. (C) To assess the contribution of placental-mediated IgG transfer, pups born to mothers that were injected with human mAbs (3BNC117 hIgG1; 100 μg i.v.) 1 d prior to delivery remained either with their biological mother (nonsurrogate) or transferred to naive surrogate mothers. Human IgG levels in the serum of pups were quantified at different time points following birth. n = 4/group; ****P < 0.0001; **P = 0.006 surrogate vs. nonsurrogate. (D) Comparison of placental- (red boxes) and GI-tract–mediated (gray boxes) transfer of human IgG antibodies from maternal to fetal circulation. n = 4–5 mice/group; **P = 0.001, ****P < 0.0001. (E) FcγR and FcRn binding profile of Fc domain mutants with differential receptor affinity that were generated to study the contribution of human FcRn and FcγRs to the transplacental transfer of human IgG antibodies. (F) Experimental strategy to evaluate the maternal-to-fetal transport efficacy of human IgG antibodies in FcRn/FcγR humanized mice. (G) Comparison of the human IgG levels (expressed as pg/mg of fetal tissue) in fetuses born to mothers previously injected (1 d prior to delivery) with Fc domain variants of a human IgG1 mAb (3BNC117; 100 μg i.v.) n = 3–9/group; ns: not significant vs. WT; *P < 0.02 vs. WT or GAALIE; **P = 0.003 vs. GRLR. Figure is related to SI Appendix, Figs. S4 and S5.