scholarly journals Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism

2021 ◽  
Author(s):  
Dianfan Li ◽  
Tingting Li ◽  
Bingjie Zhou ◽  
Zhipu Luo ◽  
Yanling Lai ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Motif ◽  
Original Strain ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Direct Competition ◽  
Therapeutic Development ◽  
Alpha Variant

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV-2 strain with IC50 of 86 ng mL-1 (DL4) and 385 ng mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 414 ng mL-1 for Alpha, 1060 ng mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

scholarly journals A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

2021 ◽  
Vol 17 (3) ◽  
pp. e1009328
Author(s):  
Hebang Yao ◽  
Hongmin Cai ◽  
Tingting Li ◽  
Bingjie Zhou ◽  
Wenming Qin ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Motif ◽  
Fusion Partner ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Neutralization Activity ◽  
Effective Strategies ◽  
High Affinity ◽  
Considerable Research

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

scholarly journals A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

2020 ◽  
Author(s):  
Hebang Yao ◽  
Hongmin Cai ◽  
Tingting Li ◽  
Bingjie Zhou ◽  
Wenming Qin ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Binding Motif ◽  
Fusion Partner ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Neutralization Activity ◽  
Effective Strategies ◽  
High Affinity ◽  
Considerable Research

ABSTRACTA key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

scholarly journals Development and Characterization of Nanobodies Targeting the Kupffer Cell

2021 ◽  
Vol 12 ◽  
Author(s):  
Fang Zheng ◽  
Jinhong Zhou ◽  
Zhenlin Ouyang ◽  
Jiaxin Zhang ◽  
Xinyi Wang ◽  
...  
Keyword(s):  
Kupffer Cell ◽  
Cell Receptor ◽  
Therapeutic Agents ◽  
Membrane Receptors ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Functional Investigation ◽  
And Function ◽  
Distinct Features

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

scholarly journals Generation and characterization of anti-α-enolase single-chain antibodies in chicken

2010 ◽  
Vol 137 (3-4) ◽  
pp. 251-260 ◽  
Author(s):  
Sy-Jye Leu ◽  
Yu-Ching Lee ◽  
Neng-Yao Shih ◽  
I-Jen Huang ◽  
Ko-Jiunn Liu ◽  
...  
Keyword(s):  
Single Chain ◽  
Single Chain Antibodies

scholarly journals Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0170162 ◽  
Author(s):  
Amy M. Hurwitz ◽  
Wanzhi Huang ◽  
Baijun Kou ◽  
Mary K. Estes ◽  
Robert L. Atmar ◽  
...  
Keyword(s):  
Single Chain ◽  
Single Chain Antibodies ◽  
Identification And Characterization

scholarly journals Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes

PLoS Biology ◽  
2021 ◽  
Vol 19 (5) ◽  
pp. e3001209
Author(s):  
Dan Fu ◽  
Guangshun Zhang ◽  
Yuhui Wang ◽  
Zheng Zhang ◽  
Hengrui Hu ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Therapeutic Interventions ◽  
Structural Basis ◽  
Binding Motif ◽  
Global Public Health ◽  
Variable Regions ◽  
Treatment Groups ◽  
X Ray Crystallography ◽  
Antibody Discovery

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

scholarly journals Human single-chain antibodies neutralize SARS-CoV-2 variants by engaging an essential epitope of the spike: a new weapon against COVID-19

2021 ◽  
Author(s):  
Olga Minenkova ◽  
Daniela Santapaola ◽  
Ferdinando Maria Milazzo ◽  
Annamaria Anastasi ◽  
Gianfranco Battistuzzi ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Therapeutic Interventions ◽  
Binding Motif ◽  
Single Chain ◽  
Drug Candidates ◽  
Treatment And Prevention ◽  
Variable Domains ◽  
Phage Libraries ◽  
Syncytia Formation

As of June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global emergency and effective therapeutic interventions for the treatment and prevention of coronavirus disease 2019 (COVID-19) are urgently needed. SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) represent a promising approach to COVID-19 therapy. However, the recently described accumulating mutations in the SARS-CoV-2 spike protein are challenging the efficacy of approved and investigational mAbs, whose widespread use is also hampered by their significant costs and possible side effects, including Antibody-Dependent Enhancement (ADE). Here we describe a cluster of SARS-CoV-2 neutralizing human single chain variable fragment antibodies, identified by phage display, sharing a common VH CDR3 sequence. Phage libraries were built by amplifying variable domains of immunoglobulin genes from cDNA derived from lymphocytes of COVID-19 convalescent subjects living in Bergamo, Italy. The scFv76-cluster antibodies (scFv76-cl Abs) exhibit high affinity for the spike receptor binding domain (RBD) of Wuhan strain and emerging variants, leading to inhibition of RBD/human ACE2 interaction. The antigenic epitope recognized by scFv76 was mapped in the receptor binding motif (RBM) of RBD at residues L455, F456, Y473, N487 and Y489. None of these residues has been to date listed among the RBD mutations of SARS-CoV-2 variants of concern (VOCs), suggesting an important role of such epitope in viral infectivity. Treatment with scFv76-cl Abs is effective against SARS-CoV-2, as determined by in vitro experiments of viral infection, replication, cytopathogenicity and spike-mediated syncytia formation. Moreover, their intranasal administration is shown to counteract infection in two independent animal models. Overall, the biochemical and biological characteristics of scFv76-cl Abs are compatible with their clinical use for COVID-19 therapy by intranasal or aerosol administration. To our knowledge, this is the first example of promising human anti-SARS-CoV-2 scFv antibodies as drug candidates for COVID-19 therapy.

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