Therapeutic antibodies and fusion inhibitors targeting the spike protein of SARS-CoV-2

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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Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

International Journal of Molecular Sciences ◽

10.3390/ijms19020487 ◽

2018 ◽

Vol 19 (2) ◽

pp. 487 ◽

Cited By ~ 20

Author(s):

Shuai Xia ◽

Wei Xu ◽

Qian Wang ◽

Cong Wang ◽

Chen Hua ◽

...

Keyword(s):

Membrane Fusion ◽

Spike Protein ◽

Fusion Inhibitors

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RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

10.1101/2020.10.31.363044 ◽

2020 ◽

Author(s):

Yan Ni ◽

Bas J.H.M. Rosier ◽

Eva A. van Aalen ◽

Eva T.L. Hanckmann ◽

Lieuwe Biewenga ◽

...

Keyword(s):

Target Recognition ◽

Point Of Care ◽

Low Cost ◽

Digital Camera ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Protein Biomarkers ◽

External Calibration ◽

Quantitative Measurements ◽

Clinical Laboratories

AbstractHeterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a “mix-and-measure” homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

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Key substitutions in the spike protein of SARS-CoV-2 variants can predict resistance to monoclonal antibodies, but other substitutions can modify the effects

10.1101/2021.07.16.452748 ◽

2021 ◽

Author(s):

Sabrina Lusvarghi ◽

Wei Wang ◽

Rachel Herrup ◽

Sabari Nath Neerukonda ◽

Russell Vassell ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Receptor Binding ◽

Clinical Stage ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Therapeutic Antibodies ◽

Epistatic Interactions ◽

Neutralization Activity ◽

Virus Susceptibility

Mutations in the spike protein of SARS-CoV-2 variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), A.23.1 and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside of the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies.

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SARS-CoV-2 Delta Variant Displays Moderate Resistance to Neutralizing Antibodies and Spike Protein Properties of Higher Soluble ACE2 Sensitivity, Enhanced Cleavage and Fusogenic Activity

Viruses ◽

10.3390/v13122485 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2485

Author(s):

Sabari Nath Neerukonda ◽

Russell Vassell ◽

Sabrina Lusvarghi ◽

Richard Wang ◽

Fernando Echegaray ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Assay ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Partial Loss ◽

Fusogenic Activity ◽

Fold Reduction ◽

Protein Properties ◽

Moderate Resistance ◽

Second Wave

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants’ spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

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Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors

The Lancet ◽

10.1016/s0140-6736(04)15788-7 ◽

2004 ◽

Vol 363 (9413) ◽

pp. 938-947 ◽

Cited By ~ 256

Author(s):

Shuwen Liu ◽

Gengfu Xiao ◽

Yibang Chen ◽

Yuxian He ◽

Jinkui Niu ◽

...

Keyword(s):

Spike Protein ◽

Heptad Repeat ◽

Fusion Inhibitors

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SARS-CoV-2 spike therapeutic antibodies in the age of variants

Journal of Experimental Medicine ◽

10.1084/jem.20210198 ◽

2021 ◽

Vol 218 (5) ◽

Author(s):

Alina Baum ◽

Christos A. Kyratsous

Keyword(s):

Infectious Diseases ◽

Viral Vector ◽

Vice President ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Antibody Therapeutics ◽

Associate Director

Christos Kyratsous, Vice President of Research, Infectious Diseases, and Viral Vector Technologies at Regeneron Pharmaceuticals, and Alina Baum, Associate Director, Infectious Diseases Associate at Regeneron Pharmaceuticals, discuss the development of antibody therapeutics targeting the spike protein of SARS-CoV-2.

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Key substitutions in the spike protein of SARS-CoV-2 variants can predict resistance to monoclonal antibodies, but other substitutions can modify the effects

Journal of Virology ◽

10.1128/jvi.01110-21 ◽

2021 ◽

Author(s):

Sabrina Lusvarghi ◽

Wei Wang ◽

Rachel Herrup ◽

Sabari Nath Neerukonda ◽

Russell Vassell ◽

...

Keyword(s):

Clinical Stage ◽

Severe Disease ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Epistatic Interactions ◽

Neutralization Activity ◽

Complex Interactions ◽

Large Panel ◽

Resistance Patterns ◽

Late Stages

Mutations in the spike protein of SARS-CoV-2 variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), A.23.1 and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside of the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. Importance Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant, but that other substitutions can affect the degree of resistance in unpredictable ways. These finding highlight complex interactions among substitutions in the spike protein affecting virus neutralization and potentially virus entry into cells.

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Application of a Method for Engineering Multivalent Antibodies to Substantially Enhance Functional affinity of Clinical Trial Anti-SARS-CoV-2 Antibodies

10.21203/rs.3.rs-259484/v1 ◽

2021 ◽

Author(s):

Adam Leach ◽

Ami Miller ◽

Emma Bentley ◽

Giada Mattiuzzo ◽

Jemima Thomas ◽

...

Keyword(s):

Clinical Trial ◽

Clinical Trials ◽

Severe Acute Respiratory Syndrome ◽

Viral Infection ◽

Clinical Investigation ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Methodological Development ◽

Tetramerization Domain ◽

Spike Proteins

Abstract Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed Quads, that retain efficient binding to the SARS-CoV-2 spike protein and show up to two orders of magnitude enhancement in neutralization of pseudovirus infection. The tetramerization method is simple and general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

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