A cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies

AbstractAntibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.

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A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

10.1101/2020.10.29.361287 ◽

2020 ◽

Author(s):

Xun Chen ◽

Matteo Gentili ◽

Nir Hacohen ◽

Aviv Regev

Keyword(s):

Neutralizing Antibodies ◽

Affinity Maturation ◽

Antibody Engineering ◽

Pseudotyped Virus ◽

Free Antibody ◽

A Cell ◽

Heavy Chain Antibody ◽

Rapid Generation ◽

Unique Capability

AbstractAntibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

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Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins

Toxins ◽

10.3390/toxins13010018 ◽

2020 ◽

Vol 13 (1) ◽

pp. 18

Author(s):

Janne Leivo ◽

Markus Vehniäinen ◽

Urpo Lamminmäki

Keyword(s):

In Vitro Selection ◽

Specific Binding ◽

Binding Properties ◽

Phage Displays ◽

Synthetic Antibody ◽

Wide Range ◽

Antibody Libraries ◽

Rapid Generation ◽

Selection Of

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

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In vitro ribosome synthesis and evolution through ribosome display

10.1101/692111 ◽

2019 ◽

Cited By ~ 1

Author(s):

Michael J. Hammerling ◽

Brian R. Fritz ◽

Danielle J. Yoesep ◽

Do Soon Kim ◽

Erik D. Carlson ◽

...

Keyword(s):

Cell Viability ◽

Directed Evolution ◽

Ribosome Display ◽

Highly Active ◽

Integrated Strategy ◽

A Cell ◽

Rapid Generation ◽

Free Environment ◽

Selection Of

ABSTRACTDirected evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

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Faculty Opinions recommendation of In vitro selection of a deoxyribozyme that can utilize multiple substrates.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004319.49956 ◽

2002 ◽

Author(s):

Niles Lehman

Keyword(s):

In Vitro Selection ◽

Multiple Substrates ◽

Selection Of

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Faculty Opinions recommendation of CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1017658.206556 ◽

2004 ◽

Author(s):

Sachdev Sidhu

Keyword(s):

In Vitro Selection ◽

Dna Complexes ◽

Selection Of

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The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

Antibiotics and Chemotherapy ◽

10.37489/0235-2990-2020-65-9-10-3-7 ◽

2020 ◽

Vol 65 (9-10) ◽

pp. 3-7

Author(s):

V. V. Gostev ◽

Yu. V. Sopova ◽

O. S. Kalinogorskaya ◽

M. E. Velizhanina ◽

I. V. Lazareva ◽

...

Keyword(s):

Staphylococcus Aureus ◽

In Vitro Selection ◽

Methicillin Resistant Staphylococcus Aureus ◽

High Concentration ◽

Short Term ◽

High Concentrations ◽

Selection Of ◽

Selection Of Resistance ◽

Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

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