Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Morro, Ainhoa Moliner; Sheward, Daniel J.; Karl, Vivien; Vidakovics, Laura Perez; Murrell, Ben; McInerney, Gerald M.; Hanke, Leo

doi:10.3390/biom10121661

Cited by 35 publications

(47 citation statements)

References 47 publications

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“…Phage display screens with immobilized RBD identified one promising nanobody, called Fu2. Since multimerization of nanobodies can substantially improve virus neutralization potency 3,17 , we created three different dimers (illustrated in Fig. 1A): An Fu2-Fc fusion expressed in mammalian cells, a chemically linked Fu2 homodimer, and thirdly, a heterodimer of Fu2 with a previously described RBD-specific neutralizing nanobody Ty1 14 .…”

Section: Isolation and Characterization Of A Potent Rbd-specific Nanobodymentioning

confidence: 99%

“…1A): An Fu2-Fc fusion expressed in mammalian cells, a chemically linked Fu2 homodimer, and thirdly, a heterodimer of Fu2 with a previously described RBD-specific neutralizing nanobody Ty1 14 . Chemically-linked constructs were generated using a combination of sortase A labelling and Cu-free click-chemistry as described in detail previously 3 .…”

Section: Isolation and Characterization Of A Potent Rbd-specific Nanobodymentioning

confidence: 99%

“…To generate homo-and hetero-dimers, the different nanobodies were first site-specifically functionalized on the C-terminus using sortase A with either an azide or a dibenzocyclooctine (DBCO) as described in detail here 3 for 3 h at 25 °C. In both reactions, unreacted nanobody, sortase A and excess nucleophile were removed using Ni-NTA resin and PD-10 columns or size-exclusion chromatography.…”

Section: Dimer Generation Using Click Chemistrymentioning

confidence: 99%

“…These so-called 'nanobodies' not only bind their antigen with very high specificity and affinity, but they can also be easily produced to large quantities in E. coli 2 and show favorable biochemical characteristics, including good thermal stability. Importantly, they can easily be combined to form homo-and heterodimers or other multimers to increase potency and target multiple epitopes simultaneously [3][4][5] . This strategy allows the neutralization of multiple known or unknown virus variants and reduces the risk for viral escape 5 .…”

Section: Introductionmentioning

confidence: 99%

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

Hanke

Das

Sheward

et al. 2021

Preprint

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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.

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Section: Isolation and Characterization Of A Potent Rbd-specific Nanobodymentioning

confidence: 99%

Section: Isolation and Characterization Of A Potent Rbd-specific Nanobodymentioning

confidence: 99%

Section: Dimer Generation Using Click Chemistrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Hanke

Das

Sheward

et al. 2021

Preprint

Self Cite

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“…Nanobody dimerization can significantly increase potency 36,37 . Combining nanobodies with distinct specificity can in addition limit viral escape 8 .…”

Section: Rapid Click-chemistry Based Dimerization To Identify Potent Homo-and Heterodimer Combinationsmentioning

confidence: 99%

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

Hanke

Sheward

Pankow

et al. 2021

Preprint

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Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 μg/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

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Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS‐CoV‐2 Variants

Zupancic

Schardt

Desai

et al. 2021

Advanced Therapeutics

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The COVID-19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS-CoV-2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS-CoV-2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS-CoV-2 cross-reactive nanobody (VHH-72) initially generated against SARS-CoV. This synergy is epitope specific and is not observed for a second high-affinity nanobody against a non-conserved epitope in the receptor-binding domain. Importantly, a hexavalent VHH-72 nanobody retains binding to spike proteins from multiple highly transmissible SARS-CoV-2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH-72 nanobodies also display drug-like biophysical properties, including high stability, high solubility, and low levels of non-specific binding. The unique neutralizing and biophysical properties of VHH-72 multivalent nanobodies make them attractive as therapeutics against SARS-CoV-2 variants.

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Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Cited by 35 publications

References 47 publications

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

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

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

Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS‐CoV‐2 Variants

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