A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

AbstractSARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

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A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

10.21203/rs.3.rs-548968/v1 ◽

2021 ◽

Author(s):

Raymond Owens ◽

Jiandong Huo ◽

Halina Mikolajek ◽

Audrey Le Bas ◽

Jordan Clark ◽

...

Keyword(s):

Therapeutic Efficacy ◽

Unmet Need ◽

Spike Protein ◽

Protein Crystal ◽

Hamster Model ◽

Cryo Electron Microscopy ◽

Effective Prophylaxis ◽

Escape Mutants ◽

Single Domain Antibodies ◽

Global Threat

Abstract SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

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Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Science ◽

10.1126/science.abe6230 ◽

2021 ◽

Vol 371 (6530) ◽

pp. eabe6230 ◽

Cited By ~ 5

Author(s):

Paul-Albert Koenig ◽

Hrishikesh Das ◽

Hejun Liu ◽

Beate M. Kümmerer ◽

Florian N. Gohr ◽

...

Keyword(s):

Electron Microscopy ◽

Receptor Binding ◽

Conformational Changes ◽

Passive Immunization ◽

Spike Protein ◽

Receptor Binding Domain ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Escape Mutants

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

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The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

10.1101/2020.08.25.266775 ◽

2020 ◽

Cited By ~ 8

Author(s):

Yunkai Zhu ◽

Fei Feng ◽

Gaowei Hu ◽

Yuyan Wang ◽

Yin Yu ◽

...

Keyword(s):

Critical Role ◽

Surface Expression ◽

Spike Protein ◽

Hamster Model ◽

Entry Pathway ◽

Genome Wide ◽

A Genome ◽

Model Animal ◽

Public Health Challenges

SUMMARYThe global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

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Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants

10.1101/2022.01.12.22269023 ◽

2022 ◽

Author(s):

Zhaochun Chen ◽

Peng Zhang ◽

Yumiko Matsuoka ◽

Yaroslav Tsybovsky ◽

Kamille West ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Structural Basis ◽

Hamster Model ◽

Golden Syrian Hamster ◽

Phage Display Libraries ◽

Antibody Phage ◽

Antibody Phage Display ◽

Early Isolate

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity1. This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies1,2. Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe3. Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

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Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail

Science ◽

10.1126/science.abd0827 ◽

2020 ◽

Vol 369 (6506) ◽

pp. 1010-1014 ◽

Cited By ~ 115

Author(s):

Johanna Hansen ◽

Alina Baum ◽

Kristen E. Pascal ◽

Vincenzo Russo ◽

Stephanie Giordano ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Selective Pressure ◽

Three Dimensional ◽

Spike Protein ◽

Humanized Mice ◽

Dimensional Structure ◽

Antibody Treatment ◽

Virus Escape ◽

Escape Mutants ◽

Antibody Cocktail

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola—one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

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Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

Scientific Reports ◽

10.1038/s41598-020-74761-y ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Jianbo Dong ◽

Betty Huang ◽

Bo Wang ◽

Allison Titong ◽

Sachith Gallolu Kankanamalage ◽

...

Keyword(s):

Computer Aided Design ◽

Treatment Options ◽

Unmet Need ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Human Igg1 ◽

Vhh Antibodies ◽

Phage Libraries ◽

Aided Design ◽

Potent Neutralization

Abstract SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.

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Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Science ◽

10.1126/science.abe3354 ◽

2020 ◽

Vol 370 (6519) ◽

pp. 950-957 ◽

Cited By ~ 21

Author(s):

M. Alejandra Tortorici ◽

Martina Beltramello ◽

Florian A. Lempp ◽

Dora Pinto ◽

Ha V. Dang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Viral Escape ◽

Angiotensin Converting Enzyme 2 ◽

Effector Functions ◽

Human Antibodies ◽

Binding Domains ◽

Isolation And Characterization ◽

Cryo Electron Microscopy ◽

Escape Mutants

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo–electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

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De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

Science ◽

10.1126/science.abe0075 ◽

2020 ◽

Vol 370 (6521) ◽

pp. 1208-1214 ◽

Cited By ~ 4

Author(s):

Thomas W. Linsky ◽

Renan Vergara ◽

Nuria Codina ◽

Jorgen W. Nelson ◽

Matthew J. Walker ◽

...

Keyword(s):

Protein Design ◽

De Novo ◽

High Specificity ◽

Spike Protein ◽

Host Proteins ◽

Angiotensin Converting Enzyme 2 ◽

Cryo Electron Microscopy ◽

De Novo Protein Design ◽

Single Spike

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo–electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

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A Delphic consensus assessment: imaging and biomarkers in gastroenteropancreatic neuroendocrine tumor disease management

Endocrine Connections ◽

10.1530/ec-16-0043 ◽

2016 ◽

Vol 5 (5) ◽

pp. 174-187 ◽

Cited By ~ 40

Author(s):

Kjell Oberg ◽

Eric Krenning ◽

Anders Sundin ◽

Lisa Bodei ◽

Mark Kidd ◽

...

Keyword(s):

Therapeutic Efficacy ◽

Unmet Need ◽

Evaluation Criteria ◽

Neuron Specific Enolase ◽

Disease Status ◽

Diagnostic Value ◽

Imaging Modalities ◽

Delphi Consensus ◽

Predictive Capacity ◽

Biological Tool

The complexity of the clinical management of neuroendocrine neoplasia (NEN) is exacerbated by limitations in imaging modalities and a paucity of clinically useful biomarkers. Limitations in currently available imaging modalities reflect difficulties in measuring an intrinsically indolent disease, resolution inadequacies and inter-/intra-facility device variability and that RECIST (Response Evaluation Criteria in Solid Tumors) criteria are not optimal for NEN. Limitations of currently used biomarkers are that they are secretory biomarkers (chromogranin A, serotonin, neuron-specific enolase and pancreastatin); monoanalyte measurements; and lack sensitivity, specificity and predictive capacity. None of them meet the NIH metrics for clinical usage. A multinational, multidisciplinary Delphi consensus meeting of NEN experts (n = 33) assessed current imaging strategies and biomarkers in NEN management. Consensus (>75%) was achieved for 78% of the 142 questions. The panel concluded that morphological imaging has a diagnostic value. However, both imaging and current single-analyte biomarkers exhibit substantial limitations in measuring the disease status and predicting the therapeutic efficacy. RECIST remains suboptimal as a metric. A critical unmet need is the development of a clinico-biological tool to provide enhanced information regarding precise disease status and treatment response. The group considered that circulating RNA was better than current general NEN biomarkers and preliminary clinical data were considered promising. It was resolved that circulating multianalyte mRNA (NETest) had clinical utility in both diagnosis and monitoring disease status and therapeutic efficacy. Overall, it was concluded that a combination of tumor spatial and functional imaging with circulating transcripts (mRNA) would represent the future strategy for real-time monitoring of disease progress and therapeutic efficacy.

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