Fc-engineered antibody therapeutics with improved efficacy against COVID-19

Abstract

Monoclonal antibodies (mAbs) with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefit in cases of mild to moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these mAbs with limited efficacy in preventing disease complications or mortality among hospitalized COVID-19 patients5. Here we report the development and evaluation of Fc-optimized anti-SARS-CoV-2 mAbs with superior potency to prevent or treat COVID-19 disease. In several animal models of COVID-19 disease6,7, we demonstrate that selective engagement of activating FcγRs results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection upon SARS-CoV-2 challenge and treatment of pre-infected animals. Our results highlight the importance of FcγR pathways in driving antibody-mediated antiviral immunity, while excluding any pathogenic or disease-enhancing effects of FcγR engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered mAbs with optimal Fc effector function and improved clinical efficacy against COVID-19 disease.

Extended Data Fig. 1:. Cloning and characterization of the IgG binding activity of hamster FcγRs.

a, Syrian hamster FcγRs were cloned, and their sequences were determined. The FcγR ectodomains are underlined. b-e, The affinity of human IgG1 and Fc variants (b, e, SPR sensorgrams), as well as of mouse (c) and hamster (d) IgG subclass variants for the various classes of hamster FcγRs was determined by surface plasmon resonance (SPR), using soluble hamster FcγR ectodomains. n.d.b., no detectable binding.

Extended Data Fig. 2:. Comparison of the FcγR expression levels in the various effector leukocyte populations between young and older FcγR humanized mice.

FcγR expression was assessed by flow cytometry in peripheral blood leukocyte populations from young (6–7 weeks old; orange) and older (17 weeks old; blue) FcγR humanized mice. a, Gating strategy to identify the various leukocyte populations, b, Representative histogram overlay plots of FcγR expression in young and older FcγR humanized mice. Corresponding isotype controls are indicated in lighter shade. c, Quantitation of FcγR expression (MFI, median fluorescence intensity subtracted from the respective isotype control) in various leukocyte populations. Results are from 4 or 5 mice per group for young and older mice, respectively.

Extended Data Fig. 3:. Histopathological analysis of lung tissue from SARS-CoV-2-infected FcγR humanized mice.

Lungs from SARS-CoV-2-infected (MA10 strain, 10⁴ pfu, i.n.) FcγR humanized mice (16–22 weeks old) were harvested on day 4 post-infection and evaluated histologically to assess the pathological changes associated with SARS-CoV-2 infection. a, Uninfected mice were characterized by clear alveolar spaces and absence of inflammatory cell infiltrates (low magnification (left panel, 200x); high magnification (center and right panels, 400x). b, In contrast, SARS-CoV-2 infection was associated with perivascular and peribronchial mononuclear leukocyte infiltration (400x, left and center panels), as well as the presence of macrophages and neutrophils in alveoli and necrotic cellular debris in alveolar spaces (600x, right panel). c, In addition, SARS-CoV-2-infected mice exhibited perivenular mixed neutrophilic, histiocytic, and lymphocytic inflammation, reactive endothelium and extravasation of leukocytes (left panel, 400x), as well as foci of interstitial neutrophilic and macrophage inflammation with hemorrhage and single cell necrosis (center and right panels, 400x). Images are representative of one uninfected and six infected mice.

Extended Data Fig. 4:. High-dose treatment of SARS-CoV-2-infected FcγR humanized mice with anti-SARS-CoV-2 mAbs Fc variants enhanced for activating FcγR binding is not associated with enhanced disease.

Following the experimental strategy in Fig. 1b, SARS-CoV-2-infected (MA10, 10⁴ pfu, i.n.) FcγR humanized mice (n=3 for PBS and n=5 for mAb-treated groups in one experiment) were treated (i.v.) with 40 mg/kg REGN mAb cocktail expressed as Fc variants with diminished (GRLR) or enhanced activating FcγR binding (GAALIE). Comparison of a, Weight loss (mean ± s.e.m.) and b, survival was compared between GRLR and GAALIE-treated groups by two-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons) and log-rank (Mantel–Cox) test, respectively. NS, not significant.

Extended Data Fig. 5:. In vitro neutralization activity and antigenic specificity of Fc variants of anti-SARS-CoV-2 mAbs.

To confirm that changes in the Fc domain have no effect on the neutralization activity and Fab-mediated functions of anti-SARS-CoV-2 mAb, Fc domain variants were characterized in (a-e) in vitro neutralization assays using SARS-CoV-2 (MA10) pseudotyped reporter viruses and (f, g) in ELISA assays using SARS-CoV-2 RBD. n= 1 experiment performed in duplicates. a, b, In vitro neutralization curves and IC₅₀ values of REGN (a) and BMS/RU (b) mAbs against SARS-CoV-2 MA10. (c, f) REGN and (d, g) BMS/RU mAb cocktails were expressed as Fc variants and their in vitro neutralization activity (c, d, e, IC₅₀ values) and antigenic specificity (f, g) was compared among Fc variants.

Extended Data Fig. 6:. In vivo half-life of Fc variants of anti-SARS-CoV-2 mAbs.

Fc variants of the REGN mAb cocktail were administered (i.v.; 50 μg) to FcγR humanized mice and antibody serum levels were determined by ELISA at various time points after antibody administration. n=3 mice per group in two independent experiments. Data are mean ± s.e.m.

Fig. 1:. Contribution of Fc effector function to the protective activity of neutralizing anti-SARS-CoV-2 mAbs in hamster infection models.

a, Overview of the FcγR locus organization in humans, mice, and Syrian hamsters. b, Fc variants of human IgG1 were evaluated for their affinity for hamster FcγRs. Numbers indicate the fold-change in affinity compared to wild-type human IgG1. n.d.b., no detectable binding. c, d, Wild-type and FcR null (GRLR) variants of REGN mAb cocktail (c) or S309 mAb (d) were administered i.v. (5 mg/kg) to Syrian hamsters one day before (prevention model, c) or after (therapy model, d) i.n. challenge with SARS-CoV-2 (NYC isolate, 10⁵ pfu) (n=9–10 hamsters per group from two independent experiments for c and n=6 hamsters per group from two independent experiments for d). Hamsters were monitored for weight loss (left; mean ± s.e.m.) and lung viral titers (right, analyzed on day 7 (c) or 6 (d) post-infection) were compared between treatment groups by one-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons). P values are indicated. e-g, SARS-CoV-2-infected hamsters (10⁵ pfu, NYC isolate) were treated on day 1 post-infection with Fc variants of the REGN mAb cocktail (5 mg/kg, i.v.) exhibit differential hamster FcγR binding affinity and A/I ratio (calculated based on FcγRIV/FcγRIIb affinity). Weight loss (e, plotted over time (mean ± s.e.m.) or f, as max change) and lung viral titers (g, assessed on day 6 post-infection) were compared by one-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons). P values are indicated. n=5–9 hamsters per group from two independent experiments.

Fig. 2:. Fc-FcγR interactions are required for the therapeutic activity of neutralizing anti-SARS-CoV-2 mAbs in mouse infection models.

a, b, FcγR humanized mice were infected with mouse-adapted SARS-CoV-2 (MA10 strain, 10⁴ pfu, i.n.) and weight loss (mean ± s.e.m.) was compared in (a) young (7 weeks old) and older (18 weeks old) mice, as well as in (b) mice (16–19 weeks old) challenged with the indicated inoculum dose. n=4–5 mice per group in two independent experiments. c, d, The therapeutic activity of REGN mAb cocktail (expressed as human IgG1 and administered at 5 mg/kg one day post-infection) was evaluated in FcγR humanized and FcγR deficient (FcγR_null) mouse strains challenged with SARS-CoV-2 (MA10 strain, 10⁴ pfu i.n.). n=5 mice per group for FcγR_null and n=11–12 mice per group for FcγR humanized mice. e, f, SARS-CoV-2-infected FcγR humanized mice (MA10 strain, 10⁴ pfu i.n.) were treated with wild-type human IgG1 or GRLR variants of REGN mAb cocktail one day post-infection. n=5–8 mice per group in two independent experiments. Weight loss (c, e; mean ± s.e.m.) and survival curves (d, f) were compared to the corresponding PBS-treated group by two-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons) and log-rank (Mantel–Cox) test, respectively. P values are indicated. NS, not significant.

Fig. 3:. Selective engagement of activating FcγRs improves the therapeutic activity of anti-SARS-CoV-2 mAbs.

a, Human IgG1 Fc variants with differential affinity for specific classes of human FcγRs were generated for anti-SARS-CoV-2 mAbs. Numbers indicate the fold-change in affinity compared to wild-type human IgG1. b-g, Following the experimental strategy in panel b, SARS-CoV-2-infected FcγR humanized mice were treated (i.v.) at the indicated dose with REGN (c-e) or BMS/RU (f-g) mAb cocktail expressed as wild-type human IgG1 or as Fc variants with differential affinity for human FcγRs. Weight loss (mean ± s.e.m.) (d and g, left panels; e, curves from individual mice) and survival curves (c, f and d, g, right panels) of antibody-treated mice were compared with the corresponding PBS-treated group by two-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons) and log-rank (Mantel–Cox) test, respectively. P values are indicated. NS, not significant. c, n=6 mice per group in two independent experiments; d, e, n=9–11 mice per group in four independent experiments; f, n=5–6 mice per group in one independent experiment; g, n=7–10 mice per group in two independent experiments.

Fig. 4:. Prophylactic activity of anti-SARS-CoV-2 mAbs is enhanced by selective engagement of activating FcγRs.

In a model of mAb-mediated prophylaxis of SARS-CoV-2 infection (a), the activity of wild-type and GAALIE variants of the REGN mAb cocktail was assessed. FcγR humanized mice were treated (i.v.) at the indicated dose with REGN mAb cocktail expressed as wild-type human IgG1 or as GAALIE variant one day prior to challenge with SARS-CoV-2 (MA10, 10⁴ pfu i.n.). Weight loss (mean ± s.e.m.) (c, left panel) and survival curves (b and c, right panel) of antibody-treated mice were compared with the PBS-treated group by two-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons) and log-rank (Mantel–Cox) test, respectively. P values are indicated. NS, not significant. b, n=3 mice per group in one experiment; c, n=7–9 mice per group in three independent experiments.