Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Developing a dry powder formulation biparatopic-Fc nanobody for inhaled delivery with broad neutralisation against SARS-CoV-2 variants of concern. (#412)

Phillip Pymm 1 , Amy Adair 1 , Lynn Tan 1 , Kenneth Margo 2 , Francesca Mordant 3 , Charley Mackenzie-Kludas 3 , Srija Mukherjee 4 , Purba Pahari 4 , Leo Lee 3 , Anupriya Aggarwal 5 , Dibyendu K Das 4 , Stuart Turville 5 , Michelle McIntosh 2 , Kanta Subbarao 3 , Wai-Hong Tham 1
  1. Infectious Diseases and Immune Defences, WEHI, Parkville, Vic, Australia
  2. Medicines manufacturing centre, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia
  3. Peter Docherty Institute, University of Melbourne, Parkville, Victoria, Australia
  4. 4Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh, India
  5. Kirby Institute, University of New South Wales, Kensington, NSW, Australia

The rapid emergence of SARS-CoV, MERS-CoV and SARS-CoV-2 over 20 years highlight coronaviruses as critically important human pathogens with future pandemic potential. Therapeutics capable of broad neutralisation amenable to scalable production and stable formulation will be essential to improve patient outcomes for evolving SARS-CoV-2 variants of concern (VoC) and future novel coronavirus outbreaks. Nanobodies, single chain antigen-recognition domains, have great potential as therapeutics for respiratory diseases due to their high affinity antigen binding, pH and temperature stability, ease of modular assembly and suitability for delivery as inhaled formulations.

Two lead candidate nanobodies were identified from our WT-SARS-CoV-2 receptor binding domain (RBD) immunised alpaca library, binding with picomolar affinity and protecting mice from SARS-CoV-2 WT infection at sub-nanomolar concentrations in vivo, though were unable to neutralise Omicron lineage VoC.

Structural characterisation of their interaction with Spike by X-ray Crystallography and Cryo-EM allowed rational design of a tetravalent biparatopic-Fc nanobody construct which demonstrated broad Omicron lineage recognition and neutralisation in vitro. Cryo-EM of the biparatopic-Fc-Omicron BA.1 Spike complex revealed the impact of BA.1 mutations on nanobody-antigen interactions.

A spray dried formulation of the biparatopic-Fc was successfully developed for inhaled delivery to the upper and lower respiratory tract. Particle size was assessed by scanning electron microscopy and aerosolization properties from a dry powder inhaler were assessed using a next generation impacter. Intranasal delivery of this formulation protected mice in vivo in models of Omicron BA.1 and BA.5 SARS-CoV-2 infection.

These studies demonstrate that a rationally designed biparatopic-Fc nanobody format can improve both potency and breadth of neutralisation against SARS-CoV-2 variants. Intra-nasal delivery of a dry powder formulation of the biparatopic-Fc shows protection in mouse models of Omicron infection, indicating its potential for development into an inhalable therapeutic and demonstrating the utility of this nanobody format as a potential therapeutic for respiratory infections.