Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 provide both effective pre-exposure prophylaxis and treatment of HIV-1 infection in murine and nonhuman primate models, suggesting their potential use in humans. Although much is known about the role of variable domains in the neutralization breadth and potency of these bNAbs, the contribution of Fc domains to their activities is, by contrast, poorly characterized. Assessment of the in vivo activity of several bNAbs revealed that FcγR-mediated effector function contributes substantially to their capacity to block viral entry, suppress viremia, and confer therapeutic activity. Enhanced in vivo potency of anti-HIV-1 bNAbs was associated with preferential engagement of activating, but not inhibitory FcγRs, and Fc domain-engineered bNAb variants with selective binding capacity for activating FcγRs displayed augmented protective activity. These findings reveal key roles for Fc effector function in the in vivo activity of anti-HIV-1 bNAbs and provide strategies for generating bNAbs with improved efficacy.

Figure 1. Generation and characterization of mouse-human chimeric anti-HIV-1 mAbs with differential FcγR binding profile

Mouse-human anti-HIV-1 mAbs were generated by replacing the constant region of the heavy chain (CH1–3) of human IgG1 (parental IgG subclass) with that of mouse IgG1 or mouse IgG2a. A null FcγR binding variant of mouse IgG1 (mIgG1 D265A) was also generated. (A) Relative FcγR binding profile (as assessed by SPR analysis (Table S1)) for the various mouse IgG Fc subclass variants. The affinity of mouse-human chimeric Fc domain variants of anti-HIV-1 mAbs for recombinant gp140^YU-2 was determined by SPR analysis and representative SPR sensograms for 1–74 mAb (B) are presented (gp140 concentration ranging from 31.25–1000 nM). Affinity kinetics measurements for all the other anti-HIV mAbs are presented in Table S2. (C) The capacity of Fc domain variants of mouse-human anti- HIV-1 mAbs to bind to HEK293T cells expressing gp160Δct^YU2 was assessed by flow cytometry. GFP-expressing HEK293T cells (GFP) were used to determine background mAb binding. Data represent the geometric mean fluorescence intensity (Geo MFI) from three independent experiments. See also related Figure S1 (D) In vitro neutralization activity of mouse-human anti- HIV-1 mAbs against HIV-1^YU-2 was determined by a standardized TZM-bl assay (Montefiori, 2005) and IC₅₀ values are presented. (see also Table S3).

Figure 2. Mouse IgG2a subclass variants of 3BNC117 exhibit improved in vivo activity

The in vivo activity of mouse-human chimeric Fc variants of 3BNC117 was assessed in a mouse model for HIV entry (Pietzsch et al., 2012) using luciferase reporter mice. (A) Infection with HIV-1^YU-2 Cre pseudovirus was accomplished by adenoviral-mediated expression of human CD4 and CCR5. (B-D) Enhanced in vivo activity was observed for mouse IgG2a subclass variants of 3BNC117, compared to mouse IgG1 and mouse IgG1 D265A (B: representative in vivo luminescence image; C: 3BNC117 variants administered at different doses (0.1–100 µg/ml, i.p.), data are presented as the mean ± SEM, n=5/group, *p<0.05 mouse IgG2a vs. mouse IgG1 D265A; D: 3BNC117 administered at a single concentration (50 µg, i.p.) n=5–6/group, ***p<0.001 compared to PBS-treated group. (E) Isotype subclass variants of a non-HIV-1 reactive mAb (mGO53, 500 µg) displayed no protective activity, n=5–6/group. (F-G) No significant difference in the in vivo activity was observed between mouse IgG2a and mouse IgG1 D265A (FcγR null binding) variants of 3BNC117 (50 µg, i.p.) in two strains of FcγR deficient mice (FFcγRα ^null;GFcer1g^−/−; Fcgr2b^−/−) NS: not significant mouse IgG2a vs. PBS or mouse IgG1 D265A. (H–I) Enhanced in vivo activity of mouse IgG2a 3BNC117 was accompanied by improved virus clearance. Serum pseudovirus content (1 (H) and 3 (I) days following HIV-1^YU-2 Cre pseudovirus infection) was determined using a qRT-PCR-based assay specific for Cre. Data are presented as the mean ± SEM. *p<0.05; **p<0.01 compared to PBS/mIgG1 D265A groups; n=4 mice/group.

Figure 3. Enhanced in vivo activity of mouse IgG2a Fc variants of anti-HIV-1 mAbs is not restricted by epitope specificity or neutralization potency

Mouse Fc domain variants of anti-HIV-1 mAbs were administered to luciferase reporter mice (i.p. 3BNC60, 3BNC117: 50 µg; 1–74: 500 µg; 1–79: 350 µg; 3BNC176: 150 µg; PGT121, PG16: 200 µg; mGO53: 500 µg) 24 hrs prior to HIV-1^YU-2 Cre pseudovirus infection and their in vivo activity was assessed by whole body imaging. (A–B) Significant enhancement in the in vivo activity of mouse IgG2a subclass variants of 1–74 was observed in (A) wild-type, but not in (B) FcγR-deficient mice, n=5–6/group, *p<0.05 compared to PBS-treated group; NS: not significant. (C–E) Increased in vivo potency of the mouse IgG2a variant was also evident for mAbs targeting different epitopes. (C) PGT121, n=6/group, **p<0.01 compared to PBS; (D) Overview of the in vivo activity of the different mouse IgG Fc subclass variants of the various anti-HIV-1 mAbs. Maximal infection obtained from PBS-injected mice was used to calculate % infection; data are presented as the mean ± SEM; n=5–7 mice/group, *p<0.05, **p<0.01 compared to the respective mouse IgG1 D265A mAb variant. (E) Quantitative comparison of infection (infection index: averaged % infection of mouse IgG2a- or mouse IgG1-treated over mouse IgG1 D265A) between the Fc domain variants standardized to the mouse IgG1 D265A variant of each respective mAb. See also Figure S2.

Figure 4. Generation and characterization of Fc domain variants of anti-HIV-1 mAbs with selective FcγR binding capacity

Fc domain variants (GRLR and GASDALIE) of hIgG1 anti-HIV-1 mAbs with differential FcγR affinity (A–B; see also Table S4 (A: IgG Fc domain variant affinity against different classes and common allelic variants of human and mouse FcγRs; B: Fold enhancement in the affinity of Fc domain variants compared to wild-type human IgG1 for human FcγRs. n.b.: no binding)) were generated and characterized in terms of (C) antigen specificity (see also related Figure S3) and (D) in vitro neutralization activity. (C) Binding of anti-HIV-1 mAb hIgG1 Fc domain variants to gp160Δct^YU-2- or GFP-expressing HEK293T cells was assessed by flow cytometry. Data represent the geometric mean fluorescence intensity (Geo MFI) from three independent experiments. (D) In vitro neutralization activity (against HIV-1^YU-2) of human Fc domain variants of anti-HIV-1 mAbs was determined by a standardized TZM-bl assay (Montefiori, 2005)(see also Tables S5 and S6).

Figure 5. Enhancement of the in vivo activity of anti-HIV-1 mAbs through Fc domain engineering for selective FcγR engagement

(A–C) The in vivo activity of 3BNC117 Fc variants with differential FcγRs binding capacity was assessed in luciferase reporter mice crossed to FcγR humanized mice. Wild-type (WT) IgG1, GRLR, or GASDALIE variants of 3BNC117 were administered (i.p.) to mice (A: low dose, 20 µg, **p<0.01 compared to WT; ***p<0.001 compared to GRLR; B: high dose, 100 µg, ***p<0.001/**p<0.01 compared to GRLR; C: Isotype control (mGO53), 100 µg) 24 h prior to infection with HIV-1 Cre pseudovirus. Maximal infection obtained from PBS-injected mice was used to calculate % infection and data are presented as the mean ± SEM. n=5–6.group (D) Fc domain variants (GRLR and GASDALIE) of 10–1074 mAb were administered (200 µg, i.v.) to NRG mice prior to transfer of in vitro HIV-1^YU-2-infected human CD4⁺ T cells (2×10⁷, i.v.). Measurement of plasma viral load (24 h post inoculation of infected cells) revealed a substantial reduction in viremia in mice that have previously received 10–1074 GASDALIE. n=3–5 mice/group, **p<0.01 compared to PBS-treated mice. NS: not significant. See also Figure S4.

Figure 6. In vivo therapeutic activity of anti-HIV-1 bNAbs requires FcγR effector function in HIV-1-infected humanized mice

Humanized NRG mice were infected with HIV-1^YU-2 and 3 weeks post-infection (viral load >10⁴ copies/ml) were treated for 6 weeks (red-shaded area) with a combination of 3BNC117, 10–1074, and PG16 mAb Fc domain variants exhibiting either enhanced binding capacity for activating FcγRs (GASDALIE; A) or diminished binding to all classes of FcγRs (GRLR; B). Plasma viral load was quantified at weekly intervals to assess the capacity of anti-HIV-1 bNAb Fc variants to suppress viremia. Each line represents an individual mouse and dotted lines represent mice with evidence of recurrent mutations in mAb-targeting epitopes of gp120 (Figure S5). Quantitation of serum bNAb concentration (right axis, red line) following cessation of antibody therapy revealed sustained viremia suppression in GASDALIE-treated mice at limiting anti-HIV-1 bNAb serum levels. (C–D) Comparison of their in vivo therapeutic activity revealed that GASDALIE mAb variants induced a significant and faster reduction in viral load (C) resulting in substantially lower proportion of mice with viremia levels above the detection limit (>800 copies/ml)(D); n=12/group **p<0.05, *** p<0.01 GASDALIE vs. GRLR-treated groups. See also Figure S5.