Tung Yep, Andrew David; (2023) Increasing the breadth and potency of a neutralising single domain antibody against the influenza haemagglutinin stem. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Among influenza A group 1 viruses, those with the haemagglutinin (HA) subtypes H1, H2, H5 and H9 are considered to pose the greatest pandemic risk. The alpaca-derived single domain antibody R1a-B6 binds to a conserved epitope on the influenza HA stem, resulting in neutralisation by membrane fusion inhibition. R1a-B6 has demonstrated in vivo efficacy against H1 and H5 viruses but lacks potency against H2 and H9 viruses, tested in vitro. We created a rationally designed library of millions of R1a-B6 variants displayed on the surface of yeast by incorporating amino acid variation from clonally related antibodies. These cells were sorted by flow cytometry in successive rounds according to their binding to H9 HA, resulting in enrichment of high-affinity H9 binding variants. R1a-B6 variant WC21 demonstrated dramatic improvements to affinity, ELISA EC50 and pseudovirus neutralisation titre against H9 and H2 antigens and maintained effectiveness against H1 and H5 antigens. The improvements were mediated by one CDR1 substitution (R31I) and two CDR3 substitutions (D95N and Y102S). R1a-B6 single substitution variants R31I, D95N and Y102S each slowed dissociation from H2 and H9 antigens and these three substitutions in combination demonstrated the same affinity improvements as WC21. We used saturation mutagenesis and deep mutational scanning to identify HA substitutions which led to loss of antibody binding. We demonstrated that WC21 has a more compact epitope footprint than R1a-B6 and speculate that this may represent a higher genetic barrier for viral escape from WC21. Furthermore, this experiment highlighted an H2-specific HA polymorphism, HA2 I45F, which abrogated R1a-B6 binding but not WC21 binding. Overcoming the barrier to binding posed by HA2 I45F is suggested to be a primary reason for the dramatic increase in H2 potency. Our approach informs strategies which may be broadly applicable to the molecular evolution of monoclonal antibodies against antigenically variable pathogens.

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