Drug-Fc conjugate CD388 targets influenza virus neuraminidase and is broadly protective in mice

Abstract

The ability of influenza virus to undergo rapid antigenic shift to elude humoral immunity highlights the need for effective broad-spectrum influenza antivirals for treatment, prophylaxis and pandemic preparedness. Strategies providing durable, universal influenza protection in healthy and high-risk populations are urgently needed. Here we describe the design and preclinical characterization of CD388, a first-in-class antiviral drug-Fc conjugate (DFC), in mice and cynomolgus macaques. CD388 comprises a multivalent conjugate of the influenza virus neuraminidase inhibitor zanamivir, linked to a CH1-Fc hybrid domain of human IgG1 engineered for extended half-life. CD388 improves the antiviral activity of zanamivir, demonstrating potent, universal activity across influenza A and B viruses, including high pathogenicity and neuraminidase inhibitor resistant strains, a low potential for resistance development and potent efficacy in lethal mouse infection models. These results suggest that CD388 has the potential for universal prevention of influenza A and B in healthy and high-risk populations.

Fig. 1. Structure of CD388 and universal activity against influenza A and B in cell-based CPE and microneutralization assays.

a, Molecular surface representation of the full-length hIgG1 (PDB code 1HZH) structure redacted to show only the Fc construct boundaries used in CD388. Positions of lysine residues that were preferentially conjugated in CD388 are shown in green (based on proteolytic digestion and peptide mapping using a conjugate with wild-type hIgG1; Methods). CD388 is a multivalent conjugate of a ZAN dimer (shown in blowup) stably conjugated to lysines on the N-terminal extended hIgG1 Fc with an average drug:antibody ratio (DAR) of 4.5:1. The spatial disposition of conjugated lysine residues on the Fc domain surface and the length and flexibility of the PEG linkers result in ZAN dimer constellations on CD388 where sufficient separation (>90 Å) exists between ZAN dimers to allow simultaneous engagement of a single CD388 molecule with multiple active sites within an NA tetramer, between neighbouring NA tetramers on the same virus particle, or between NA tetramers on different virus particles. b–f, EC₅₀ values [nM] of CD388, OST, ZAN or BXA against a panel of influenza strains A/H1N1 (b), A/H3N2 (c), B in a cell-based cytopathic effect assay (d), and A/H5N1 (e) and A/H7N9 (f) in a cell-based microneutralization assay. In b–f, data points in box and whiskers plots represent mean EC₅₀ values for different influenza strains derived from dose-response curves measured in duplicate. The boxes bound the 25th and 75th percentile, with median values shown in the boxes. Whiskers highlight strains with the highest and lowest EC₅₀s. Source data

Fig. 2. Universal efficacy of CD388 in lethal mouse models of influenza A and B infection.

a–e, Efficacy of CD388 dose response on survival (a) and % body weight change (b), and the effect of CD388 on lung viral burden (c) (P values from left to right: >0.05, 0.0014, 0.0004, 0.0007, 0.0002, 0.0002) and pro-inflammatory lung cytokine levels of IL-6. bld, below levels that are detectable. (d) (P values from left to right: >0.05, <0.0001 for remaining columns) and MCP-1 (e) (P values from left to right: >0.05, <0.0001 for remaining columns) at 4 days post infection following lethal challenge with mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) in BALB/c mice. f–j, Efficacy of OST on survival (f) and % body weight change (g) in a lethal mouse model with mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) in BALB/c mice (n = 5 per group), and effect of OST on lung viral burden (h) (P values from left to right: 0.0041, >0.05) and pro-inflammatory lung cytokine levels of IL-6 (i) (P values from left to right: <0.0001 for all columns) and MCP-1 (j) (P values from left to right: <0.0001 for all columns) at 4 days post infection. For all viral burden and cytokine analyses, plots show mean ± s.d.; n = 5 per group. k–m, Efficacy of CD388 in lethal mouse models of A/Hong Kong/1/1968 (H3N2) (k), B/Florida/4/2006 (Yamagata) (l) and B/Malaysia/2506/2004 (Victoria) (m). n, Efficacy of CD388 against mouse-adapted A/Puerto Rico/8/1934 (H1N1) in immune-compromised BALB/c SCID mice (n = 5 per group). Statistical analyses were performed using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001; NS, not statistically significant) in GraphPad Prism v.9. Data distribution was assumed to be normal, but this was not formally tested. Source data

Fig. 3. Mouse pharmacokinetics and prophylactic efficacy of CD388 in lethal mouse models of influenza A and B infection.

a, Plasma levels of CD388 at doses of 0.3–30 mg kg⁻¹ following i.m. administration in mice (mean; n = 2 per group) by NA capture. b, CD388 levels in plasma and lung epithelial lining fluid (ELF) of mice after i.m. dosing (mean ± s.d.; n = 6 per group) by NA capture. c–f, Protection provided by CD388 dosed as indicated i.m. 7 days before lethal challenge against influenza A/Puerto Rico/8/1934 (H1N1) (c), A/Hong Kong/1/1968 (H3N2) (d), B/Florida/4/2006 (Yamagata) (e) and B/Malaysia/2506/2004 (Victoria) (f) in BALB/c mice (n = 5 per group). Source data

Fig. 4. CD388 efficacy in lethal mouse models of influenza A and B harbouring NA mutations that confer reduced susceptibility to other NAIs.

a–c, Efficacy of CD388 administered i.m. at the indicated doses at 2 h post infection against influenza A/Texas/23/2012 (H1N1)pdm09 NA H275Y variant (a), ZAN-susceptible influenza B/Laos/0080/2016 (Victoria) (b) and ZAN-resistant B/Laos/0654/2016 NA H134N variant (Victoria) (c) in BALB/c mice (n = 5 per group). d,e, Comparisons of dose responses of ZAN administered intranasally starting at 2 h post infection for 5 days and CD388 administered i.m. once at 2 h post infection at 0.3 mg kg⁻¹ to ZAN-susceptible influenza B/Laos/0080/2016 (d) and ZAN-resistant B/Laos/0654/2016 NA H134N (e) variants in BALB/c mice (n = 5 per group). Source data

Fig. 5. Cross-resistance of CD388-selected NA variants to OST and ZAN.

a,b, Fold change of a plaque-purified clone isolated at passage 10 versus parental strains by EC₅₀ [nM] to CD388-selected NA variants S231R (S247R) in A/WSN/1933 (H1N1) (a) or A246V in combination with HA N113H/T453I in A/Victoria/3/75 (H3N2) (b) for CD388, OST and ZAN in cell-based PRA. EC₅₀ values were derived from 3 independent PRA replicates. Source data

Extended Data Fig. 1. The NA tetramer with tetrameric ZAN.

Crystal structure of tetrameric ZAN (green) complexed to an NA tetramer (PDB code 3TI5) from A/California/04/2009 H1N1. The C7 position on ZAN that was chosen for attachment to the Fc carrier (via an NHS ester) in the work presented here is highlighted. C7 on ZAN is solvent exposed and unencumbered sterically, allowing conjugation to the C7 position with minimal influence on binding affinity to NA.

Extended Data Fig. 2. Universal activity of CD388 against influenza A and B in lethal mouse models.

CD388 efficacy against lethal challenge with influenza in BALB/c mice. Shown is dose-response % body weight change in response to CD388 ranging from 0.03 – 1 mg/kg administered IM at 2 h post-infection against lethal challenge with influenza (a) A/Hong Kong/1/1968 (H3N2), (b) B/Florida/4/2006 (Yamagata), (c) B/Malaysia/2506/2004 (Victoria), and % body weight change and survival against (d) A/WSN/1933 (H1N1), (e) A/California/07/2009 (H1N1)pdm, (f) A/California/12/2012 (H1N1)pdm09, (g) A/North Carolina/04/2014 (H1N1)pdm09, (h) A/Hawaii/70/2019 (H1N1)pdm09, and (i) B/Colorado/6/2017 (Victoria) in BALB/c mice. Source data

Extended Data Fig. 3. CD388 prophylactic activity – body weight change.

% body weight change in response to CD388 administered IM at 7 days prior to lethal challenge against influenza (a) A/Puerto Rico/8/1934 (H1N1), (b) A/Hong Kong/1/1968 (H3N2), (c) B/Florida/4/2006 (Yamagata) and (d) B/Malaysia/2506/2004 (Victoria). Source data

Extended Data Fig. 4. CD388 prophylactic activity – Survival and body weight change.

Survival and % body weight change in response to CD388 administered IM at 7 days prior to lethal challenge against influenza (a) A/WSN/1933 (H1N1), (b) A/California/07/2009 (H1N1)pdm, (c) A/California/12/2012 (H1N1)pdm09, (d) A/North Carolina/04/2014 (H1N1)pdm09, (e) A/Hawaii/70/2019 (H1N1)pdm09, and (f) B/Colorado/6/2017 (Victoria) in BALB/c mice. Source data