Trivalency of a Nanobody Specific for the Human Respiratory Syncytial Virus Fusion Glycoprotein Drastically Enhances Virus Neutralization and Impacts Escape Mutant Selection

Abstract

ALX-0171 is a trivalent Nanobody derived from monovalent Nb017 that binds to antigenic site II of the human respiratory syncytial virus (hRSV) fusion (F) glycoprotein. ALX-0171 is about 6,000 to 10,000 times more potent than Nb017 in neutralization tests with strains of hRSV antigenic groups A and B. To explore the effect of this enhanced neutralization on escape mutant selection, viruses resistant to either ALX-0171 or Nb017 were isolated after serial passage of the hRSV Long strain in the presence of suboptimal concentrations of the respective Nanobodies. Resistant viruses emerged notably faster with Nb017 than with ALX-0171 and in both cases contained amino acid changes in antigenic site II of hRSV F. Detailed binding and neutralization analyses of these escape mutants as well as previously described mutants resistant to certain monoclonal antibodies (MAbs) offered a comprehensive description of site II mutations which are relevant for neutralization by MAbs and Nanobodies. Notably, ALX-0171 showed a sizeable neutralization potency with most escape mutants, even with some of those selected with the Nanobody, and these findings make ALX-0171 an attractive antiviral for treatment of hRSV infections.

Antigenic and sequence variability of the human respiratory syncytial virus F glycoprotein compared to related viruses in a comprehensive dataset

“…2 ), explaining the capacity of site II specific mAbs to bind to both forms of hRSV F. A number of escape mutants have been isolated by different groups with mAbs that bind to antigenic site II. Most laboratory mutants were selected with mAbs of murine origin [20] , [21] , [54] but also with Nanobodies [55] or with bovine mAbs [18] . Some of these mutations, specifically those at residues 272 and 275, have been also found in sequences of hRSV F obtained from nasopharyngeal secretions of children infected with hRSV that were treated prophylactically with either palivizumab (Pz) or Mz [25] , [26] .…”

Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2

“…Affinity maturation can improve this, although this extends the development timeline. Instead, generating linked multivalent or multi-paratopic VH binders could be a more rapid approach to utilize avidity to boost affinity and efficacy 23 . Linking VH domains into such homo-and hetero-bifunctional formats is more straightforward than preparing similar multifunctional antibodies, because the latter requires correct heavy-and light-chain pairing to maintain binding affinity, whereas multifunctional VH domains have no such requirements 11 .…”

Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody

“…Nanobodies have been used previously with the same approach to treat syncytial virus infection (RSV). Successful preclinical and clinical trials have been performed indicating that 6mg/kg has been a safe and efficient dose for RSV (66)(67)(68)(69)(70). In this case, a monomeric Nanobody called Nb017 with a Kd of ~ 17.88 nM was trimerized to a drug called ALX-0171 increasing the binding affinity to RSV to a Kd of ~ 0.113 nM.…”