A monoclonal anti-hemagglutinin stem antibody modified with zanamivir protects against both influenza A and B viruses

Abstract

Influenza remains a significant public health threat. Both monoclonal antibodies and small-molecule inhibitors can target the influenza surface glycoproteins hemagglutinin (HA) or neuraminidase (NA) for prevention and treatment of influenza. Here, we combine the strengths of anti-influenza antibodies and small molecules by site-specific conjugation of the NA inhibitor zanamivir to MEDI8852, an HA-specific fully human monoclonal antibody. MEDI8852 targets the conserved stem region of HA and inhibits HA-mediated fusion of the viral and host cell membranes. Elimination of virus-infected cells involves Fcγ receptor-mediated effector functions. The efficacy of MEDI8852 is limited to influenza A viruses. Zanamivir, on the other hand, binds to the active site of NA in both influenza A and B viruses to inhibit NA activity and virus release. However, because of its small size, zanamivir has a short half-life and requires repeated dosing at high concentrations. We produced a MEDI8852-zanamivir antibody-drug conjugate (ADC) that engages Fc-mediated effector functions and benefits from neonatal Fc receptor (FcRn)-mediated recycling. The MEDI8852-zanamivir conjugate extends the circulatory half-life of zanamivir, targets both influenza HA and NA, and shows enhanced antibody-dependent cellular cytotoxicity (ADCC) compared to MEDI8852 alone. The MEDI8852-zanamivir conjugate protected mice from a lethal (10 × LD₅₀) challenge with influenza A and B viruses at a dose similar to that required for broadly neutralizing anti-NA antibodies, with the added advantage of simultaneously targeting NA (influenza A and B) and HA (influenza A).

Keywords: anti-hemagglutinin monoclonal antibody; antibody–drug conjugate; antiviral therapy; influenza; neuraminidase inhibitor.

Fig. 1.

Modification of a monoclonal anti-HA stem antibody MEDI8852 using a sortase reaction. (A) Synthesis of the MEDI8852–zanamivir conjugate. The C termini of the MEDI8852 heavy and light chains were modified with a sortase recognition motif (LPETGGH₆) and conjugated with triglycine-modified zanamivir through a sortase-catalyzed transpeptidation reaction. (B) Synthesis of the MEDI8852–Cy5 conjugate. The attachment of Cy5 on both heavy and light chains of MEDI8852, showing quantitative conversion of both heavy and light chains based on fluorescence intensity, was confirmed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Blue: Cy5 signal. (C) Saturation binding curves of the MEDI8852–zanamivir conjugate and wild-type MEDI8852 (lacking a sortase recognition motif) to influenza virus HAs and NA. (D) In vitro dose-NA inhibition curves for the MEDI8852–zanamivir conjugate and wild-type MEDI8852.

Fig. 2.

The MEDI8852–zanamivir conjugate protects mice from a lethal infection caused by influenza A and B viruses. Balb/c mice were infected with the 10 × LD₅₀ of influenza virus as indicated. Mice were given the antibody or antibody conjugate intraperitoneally 1 h before infection. Mice were killed when they lost more than 25% of their initial body weight or appeared moribund. (A) Body weight change curves for mice (n = 5) infected with influenza virus A/Puerto Rico/8/1934 (H1N1) and treated with the MEDI8852–zanamivir conjugate, a combination of the MEDI8852–triglycine conjugate (MEDI8852-GGG) and free zanamivir in equimolar amounts, or a combination of MEDI8852 lacking a sortase recognition motif and free zanamivir in equimolar amounts. (B) Body weight change and survival curves [analyzed using log-rank (Mantel–Cox) test] for mice (n = 5) infected with influenza virus B/Florida/4/2006 and treated with the MEDI8852–zanamivir conjugate or a combination of MEDI8852 lacking a sortase recognition motif and free zanamivir in equimolar amounts.

Fig. 3.

Comparison of the antiviral activities of the MEDI8852–zanamivir conjugate with monoclonal antibodies that target the active site of influenza virus NA. Balb/c mice were infected with the 10 × LD₅₀ of influenza virus as indicated. Mice were given the antibody or antibody conjugate intraperitoneally 1 h before infection. Mice were killed when they lost more than 25% of their initial body weight or appeared moribund. (A) Body weight change and survival curves [analyzed using log-rank (Mantel–Cox) test] for mice (n = 5) infected with influenza virus A/Puerto Rico/8/1934 (H1N1) and treated with 1 mg/kg of MEDI8852–zanamivir conjugate, FNI9, 1G01, or DA03E17. (B) Body weight change and survival curves [analyzed using log-rank (Mantel–Cox) test] for mice (n =5) infected with influenza virus B/Florida/4/2006 and treated with 3 mg/kg of MEDI8852–zanamivir conjugate, FNI9, 1G01, or DA03E17.

Fig. 4.

The contribution of Fcγ receptor–mediated effector functions to the antiviral efficacy of MEDI8852–zanamivir. (A) ADCC reporter assay (human Fcγ receptors) for MEDI8852-GGG, MEDI8852–zanamivir, and their “LALA” mutants, designed to disrupt interactions with Fcγ receptors. The Fc portion of the antibody conjugate triggered luciferase expression in effector cells through engagement of surface-expressed human FcγRIIIa when coincubated with virus-infected MDCK cells. The level of ADCC induced by the antibody conjugate was quantified as the fold induction in luminescence intensity compared to the control group without antibody. (B) Body weight change and survival curves [analyzed using log-rank (Mantel–Cox) test] for mice (n = 5) infected with influenza virus A/Puerto Rico/8/1934 (H1N1) and treated with MEDI8852-GGG or its “LALA” variant. (C) Body weight change and survival curves [analyzed using log-rank (Mantel–Cox) test] for mice (n = 5) infected with influenza virus A/Puerto Rico/8/1934 (H1N1) and treated with MEDI8852–zanamivir conjugate or its “LALA” variant. Balb/c mice were infected with the 10 × LD₅₀ of influenza virus A/Puerto Rico/8/1934 (H1N1). Mice were given the antibody or antibody conjugate intraperitoneally 1 h before infection. Mice were killed when they lost more than 25% of their initial body weight or appeared moribund.