Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

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Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016093117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28046-28055 ◽

Cited By ~ 7

Author(s):

Anum Glasgow ◽

Jeff Glasgow ◽

Daniel Limonta ◽

Paige Solomon ◽

Irene Lui ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Surface Display ◽

Random Mutagenesis ◽

Yeast Surface Display ◽

Host Cells ◽

Spike Protein ◽

Receptor Protein ◽

Yeast Surface

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

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Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

10.1101/2020.07.31.231746 ◽

2020 ◽

Cited By ~ 7

Author(s):

Anum Glasgow ◽

Jeff Glasgow ◽

Daniel Limonta ◽

Paige Solomon ◽

Irene Lui ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Surface Display ◽

Random Mutagenesis ◽

Yeast Surface Display ◽

Host Cells ◽

Spike Protein ◽

Receptor Protein ◽

Yeast Surface ◽

Flexible Protein

AbstractAn essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

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Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

BioMed Research International ◽

10.1155/2019/6051870 ◽

2019 ◽

Vol 2019 ◽

pp. 1-22 ◽

Cited By ~ 1

Author(s):

Biancamaria Cembrola ◽

Valentino Ruzza ◽

Fulvia Troise ◽

Maria Luisa Esposito ◽

Emanuele Sasso ◽

...

Keyword(s):

High Throughput Sequencing ◽

Surface Display ◽

Affinity Maturation ◽

Yeast Surface Display ◽

Antigen Concentration ◽

Selection Scheme ◽

High Affinity ◽

Novel Approach ◽

Yeast Surface

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

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Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

Journal of Biological Chemistry ◽

10.1074/jbc.m116.756718 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3481-3495 ◽

Cited By ~ 30

Author(s):

Valeria Arkadash ◽

Gal Yosef ◽

Jason Shirian ◽

Itay Cohen ◽

Yuval Horev ◽

...

Keyword(s):

Directed Evolution ◽

Surface Display ◽

High Specificity ◽

Computational Design ◽

Computational Method ◽

Yeast Surface Display ◽

Mmp Inhibitor ◽

Starting Point ◽

Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

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Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library

10.1101/2021.01.07.425806 ◽

2021 ◽

Author(s):

Benjamin Nikola Bell ◽

Abigail E. Powell ◽

Carlos Rodriguez ◽

Jennifer R Cochran ◽

Peter S. Kim

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

In Vitro Selection ◽

Surface Display ◽

Binding Domain ◽

Yeast Surface Display ◽

Surface Glycoprotein ◽

Human Antibody ◽

Receptor Binding Domain

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naive antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naive antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naive antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

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