Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape

The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape. Rational combinations of these nanobodies that bind to distinct sites within and between spike subunits exhibit extraordinary synergy and suggest multiple tailored therapeutic and prophylactic strategies.

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Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

10.1101/2021.04.06.438709 ◽

2021 ◽

Author(s):

Tyler N Starr ◽

Nadine Czudnochowski ◽

Fabrizia Zatta ◽

Young-Jun Park ◽

Zhuoming Liu ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Binding Domain ◽

Viral Escape ◽

Binding Motif ◽

Receptor Binding Domain ◽

Viral Neutralization ◽

Parental Antibody

An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

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480 Preliminary evaluation of a novel coronavirus vaccine (CORVax) using electroporation of plasmid DNA encoding a stabilized prefusion SARS-CoV-2 spike protein alone or with transfection of plasmid IL-12

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0480 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A514-A514

Author(s):

Shawn Jensen ◽

Christopher Twitty ◽

Christopher Paustian ◽

Madelein Laws ◽

Glenna McDonnell ◽

...

Keyword(s):

Immune Responses ◽

Receptor Binding ◽

Binding Domain ◽

Tumor Rejection ◽

Spike Protein ◽

Preliminary Evaluation ◽

List Type ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Viral Neutralization

BackgroundSARS-CoV-2 (CoV2) has precipitated a global pandemic and the effectiveness of standard vaccine strategies to induce potent and persistent immunity to CoV2 is in question, particularly for the elderly. This problem is not dissimilar to what we have struggled with in our quest to induce immunity to cancer antigens, where vaccine-induced anti-cancer immune responses can be weak. Here, we describe a novel vaccine approach which leverages electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax). As IL-12 has been shown to augment the efficacy of immunotherapy in aged mice,1 we have initiated studies to evaluate if plasmid IL-12 (TAVO™) can similarly augment anti-CoV2 immune responses in young mice and have planned studies in aged animals.MethodsA prefusion stabilized CoV2 spike plasmid expression vector was constructed, a master cell bank generated and clinical-grade plasmid manufactured. C57BL/6 and BALB/c were vaccinated via intramuscular (IM) and/or intradermal (ID) injection followed immediately by EP of plasmids encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12 on days 1 and 14 or 21. Mice were followed for >120 days to assess safety. Splenocytes and serum were harvested at different time points to interrogate virus-specific cellular responses as well anti-spike IgG1/IgG2 antibody titers. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble hACE2R binding to immobilized CoV2 spike.ResultsPreliminary data shows that EP of CORVax alone or combined with IL-12 was safe. EP of CORVax was able to elicit anti-Spike IgG antibodies (IC50 = 1/2112), as well as IgG antibodies targeting the receptor binding domain of the Spike protein (IC50 = 1/965) approximately 40 days after the booster vaccination. In 2 of 2 experiments, CORVax combined with IL-12 significantly (P<0.0001) increased the sVNT titers at 2 months, but this benefit was lost by 3 months.ConclusionsEarly preclinical data shows that EP of CORVax can induce IgG responses to CoV2 Spike and the receptor binding domain (RBD) as well as apparent viral neutralizing activity. The addition of IL-12, at least transiently, increased sVNT titer. We plan to investigate alternate vaccine boosting strategies while extending these studies into aged animals and initiate a clinical trial in the near future.ReferencesRuby CE, Weinberg AD. OX40-Enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol2009;182:1481–9. https://doi.org/10.4049/jimmunol.182.3.1481.

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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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Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants

10.1101/2021.02.25.432969 ◽

2021 ◽

Cited By ~ 4

Author(s):

Lingshu Wang ◽

Tongqing Zhou ◽

Yi Zhang ◽

Eun Sung Yang ◽

Chaim A. Schramm ◽

...

Keyword(s):

Receptor Binding ◽

Binding Domain ◽

Receptor Binding Domain ◽

Therapeutic Antibodies ◽

Resistance Development ◽

Neutralizing Activity ◽

Escape Mutants

AbstractThe emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 μg/mL; IC80 < 0.0006 to 0.0251 μg/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.One Sentence SummaryUltrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.

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Highly potent bispecific sybodies neutralize SARS-CoV-2

10.1101/2020.11.10.376822 ◽

2020 ◽

Cited By ~ 1

Author(s):

Justin D. Walter ◽

Cedric A.J. Hutter ◽

Alisa A. Garaeva ◽

Melanie Scherer ◽

Iwan Zimmermann ◽

...

Keyword(s):

Receptor Binding ◽

Binding Sites ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Conformational States ◽

Global Crisis ◽

Therapeutic Applications ◽

Symmetric Complex

ABSTRACTThe COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications.

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Durability of Viral Neutralization in Asymptomatic Coronavirus Disease 2019 for at Least 60 Days

The Journal of Infectious Diseases ◽

10.1093/infdis/jiab140 ◽

2021 ◽

Author(s):

Amanda Haymond ◽

Abdulla A Damluji ◽

Aarthi Narayanan ◽

Claudius Mueller ◽

Alex Reeder ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Immunoglobulin G ◽

Neutralizing Antibodies ◽

Healthcare Workers ◽

Binding Domain ◽

Receptor Binding Domain ◽

Serum Samples ◽

Viral Neutralization ◽

Time Period

Abstract A cohort consisting of asymptomatic healthcare workers donated temporal serum samples after infection with severe acute respiratory syndrome coronavirus 2. Analysis shows that all asymptomatic healthcare workers had neutralizing antibodies, that these antibodies persist for ≥60 days, and that anti-spike receptor-binding domain immunoglobulin G levels were correspondingly durable over the same time period.

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Advances in MERS-CoV Vaccines and Therapeutics Based on the Receptor-Binding Domain

Viruses ◽

10.3390/v11010060 ◽

2019 ◽

Vol 11 (1) ◽

pp. 60 ◽

Cited By ~ 48

Author(s):

Yusen Zhou ◽

Yang Yang ◽

Jingwei Huang ◽

Shibo Jiang ◽

Lanying Du

Keyword(s):

Middle East ◽

Viral Infection ◽

Receptor Binding ◽

Infectious Virus ◽

Binding Domain ◽

Structural Proteins ◽

Receptor Binding Domain ◽

Therapeutic Antibodies ◽

S Protein ◽

Important Target

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is an infectious virus that was first reported in 2012. The MERS-CoV genome encodes four major structural proteins, among which the spike (S) protein has a key role in viral infection and pathogenesis. The receptor-binding domain (RBD) of the S protein contains a critical neutralizing domain and is an important target for development of MERS vaccines and therapeutics. In this review, we describe the relevant features of the MERS-CoV S-protein RBD, summarize recent advances in the development of MERS-CoV RBD-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy.

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Vaccine monitoring shows that focused immunization with SARS-CoV-2 receptor-binding domain provides a better neutralizing antibody response than full-length spike protein

10.21203/rs.3.rs-134388/v1 ◽

2021 ◽

Author(s):

Rafael Bayarri-Olmos ◽

Manja Idorn ◽

Anne Rosbjerg ◽

Laura Pérez-Alós ◽

Cecilie Hansen ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralization Test ◽

Neutralizing Antibody ◽

Binding Domain ◽

Full Length ◽

Receptor Binding Domain ◽

Vaccine Response ◽

Viral Neutralization ◽

Neutralization Capacity

Abstract Effective tools to monitor SARS-CoV-2 transmission and humoral immune responses are highly needed. Protective humoral immunity involves neutralizing antibodies and will be a hallmark for the evaluation of a vaccine response efficacy. Here we present a sensitive, fast and simple neutralization ELISA method to determine the levels of antibody-mediated virus neutralization. We can show that it is strongly correlated with the more elaborate plaque reduction neutralization test (PRNT) (ρ = 0.9231, p < 0.0001). Furthermore, we present pre-clinical vaccine models using recombinant receptor binding domain (RBD) and full-length spike antigen as immunogens showing a profound antibody neutralization capacity that exceeds the highest neutralization titers from convalescent individuals. Using a panel of novel high-affinity murine monoclonal antibodies (mAbs) we also show that majority of the RBD-raised mAbs have inhibitory properties while only a few of the spike-raised mAbs do. In conclusion, the ELISA-based viral neutralization test offers a time- and cost-effective alternative to the PRNT. The immunization results indicate that vaccine strategies focused only on the RBD region may have major advantages over those based on the full spike sequence.

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A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-CoV-2 Spike Glycoprotein

10.1101/2020.03.15.992883 ◽

2020 ◽

Cited By ~ 8

Author(s):

M. Gordon Joyce ◽

Rajeshwer S. Sankhala ◽

Wei-Hung Chen ◽

Misook Choe ◽

Hongjun Bai ◽

...

Keyword(s):

Receptor Binding ◽

Cross Reactivity ◽

Binding Domain ◽

Cell Entry ◽

Viral Escape ◽

Human Antibody ◽

Receptor Binding Domain ◽

Diagnostic Strategies ◽

High Resolution Structure ◽

Molecular Information

SUMMARYSARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease—COVID-19— within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronavirus’s surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 Å. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the “closed” prefusion S structure, but is accessible in “open” conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses.HIGHLIGHTSHigh resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD).Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies.Structure of the SARS-COV-2 RBD in complex with antibody CR3022.Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.

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