A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Abstract

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. Thus, there is a need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we isolated a nanobody that potently neutralizes SARS-CoV-2, including the B.1.351 variant, and cross-neutralizes SARS-CoV. We demonstrate the therapeutic potential of the nanobody in a human ACE2 transgenic mouse model. Using biochemistry and electron cryomicroscopy we show that this nanobody simultaneously interacts with two RBDs from different spike trimers, rapidly inducing the formation of spike trimer-dimers. This naturally elicited bispecific monomeric nanobody establishes a novel strategy for potent immobilization of viral antigens.

Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies

“…Neutralizing antibody titers of purified nanobodies were highly correlated with the preliminary periplasm screens, with nanobodies C7 and E2 displaying exceptional potency with IC50s in the range of 0.01 µg/ml (Fig 4A). Note that E4 is identical to the "Fu2" nanobody that was isolated via more traditional colony picking from the same immunized animal, and is extensively described elsewhere 19 . Two of the selected non-RBD-specific nanobodies (C11, D9) were also capable of neutralization, albeit weakly and for D9 plateauing at approximately 50 A subset of nanobodies are broadly neutralizing.…”

“…Nanobodies do not need to bind the RBD or block ACE2 receptor interaction to neutralize SARS-CoV-2. Nanobodies have been shown to neutralize SARS-CoV-2 by various mechanisms, including direct competition with ACE2 6,7 , locking of RBDs in an ACE2-inaccessible conformation 24 , dimerizing spikes and agglutinating virions 19 , or triggering the post-fusion conformation and shedding of S1 8 .…”

“…The alpaca nanobody library used in this study was already described and used here 19 . In brief, one adult female alpaca (Funny) at PreClinics, Germany, was immunized four times in a 60-day immunization schedule.…”

“…6 data from 50% of the untreated, challenged animals are historical controls from previous experiments performed under identical conditions. This challenge experiment was run at the same time as that from Hanke et al (2021) 19 , and the control mice were shared between both.…”

Nanobodies as powerful pulmonary targeted biotherapeutics against SARS-CoV-2, pharmaceutical point of view

“… Aerosol-mediated delivery by neutralizer directly to the airway epithelia. [ 52 ] 6 Monovalent (Fu2) Monovalent Fc conjugated (Fu2-Fc) Dimers Immunized Alpaca with spike protein and RBD Fu2: E. coli Fu2-Fc: Mammalian cell RBD A PRNT using the B.1.351 (501Y·V2)1 variant showed that Fu2 very potently neutralized this novel variant and that the Fu2 dimer and Fu2-Fc improved neutralization efficiency Prophylactic and therapeutic efficacy in transgenic mice that express human ACE2 as a model was done and the data shows that the Fu2-Ty1 dimer suppresses SARS-CoV-2 in vivo [ 53 ] 7 Monovalent (Nb 12, 15,17,19, 30, 56) Bivalent Trivalent Immunized Llama and Transgenic mice (nanomice) with spike protein and RBD Unknown RBD Dissociation constants more than 30 nM The Nb monomers displayed nM and sub-nM half-maximal inhibitory concentration (IC50) in in vitro lentiviral particles pseudotyped with the SARS-CoV-2 spike Some Nbs are ineffective against viruses that carry substitutions in the RBD, but combining the two Nb classes into heterotrimeric constructs might further improve their efficacy against wild and mutant type of virus. Nbs were thermostable and can be aerosolized with commercially available mesh nebulizers without losing neutralization activity.…”