Affinity Maturation of Antibodies: Optimized Methods to Generate High-Quality ScFv Libraries and Isolate IgG Candidates by High-Throughput Screening

Directed Evolution of Aptamer Discovery Technologies

10.1101/2021.11.23.469732 ◽

2021 ◽

Author(s):

Diana Wu ◽

Chelsea Gordon ◽

John Shin ◽

Michael Eisenstein ◽

Hyongsok Tom Soh

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Click Reaction ◽

Clinical Diagnostics ◽

High Quality ◽

Affinity Reagents ◽

Wide Range ◽

Target Binding ◽

Modified Nucleobases ◽

Parallel Fashion

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

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High-Throughput Screening of Antisolvents for the Deposition of High-Quality Perovskite Thin Films

ACS Applied Materials & Interfaces ◽

10.1021/acsami.0c06110 ◽

2020 ◽

Vol 12 (23) ◽

pp. 26026-26032

Author(s):

Joseph G. Manion ◽

Andrew H. Proppe ◽

Garion E. J. Hicks ◽

Edward H. Sargent ◽

Dwight S. Seferos

Keyword(s):

Thin Films ◽

High Throughput ◽

High Throughput Screening ◽

High Quality

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Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400104 ◽

1999 ◽

Vol 4 (1) ◽

pp. 15-25 ◽

Cited By ~ 35

Author(s):

Ingrid Schmid ◽

Isabel Sattler ◽

Susanne Grabley ◽

Ralf Thiericke

Keyword(s):

Drug Discovery ◽

Sample Preparation ◽

High Throughput ◽

High Throughput Screening ◽

Solid Phase ◽

Structural Diversity ◽

High Quality ◽

Synthetic Compound ◽

Natural Origin ◽

Compound Libraries

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

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Drug Discovery (Lead Identification and High Throughput Screening)

Research Journal of Pharmacology and Pharmacodynamics ◽

10.52711/2321-5836.2021.00010 ◽

2021 ◽

pp. 46-50

Author(s):

Ravi Kumar

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Clinical Development ◽

High Quality ◽

New Drug ◽

Lead Identification ◽

Pharmaceutical Industries

In this review we will discuss about the Lead identification, the lead identification is mostly used for the discovery of successful clinical development compound, and it is an essential site for drug discovery. Various important factors that required for discovery a quality leads, such as- Physicochemical, ADME, Biological and PK parameters. These all parameters are required for the identification of high-quality leads. The Combinational chemistry is mostly used for the generation of many compounds in only one process from a mixture. The high throughput screening is suitable for new drug in pharmaceutical industries and it’s mostly used from last two decades.

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High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107301320 ◽

2007 ◽

Vol 12 (5) ◽

pp. 628-634 ◽

Cited By ~ 31

Author(s):

Chris D. Forbes ◽

Joshuaine G. Toth ◽

Can C. Özbal ◽

William A. Lamarr ◽

Jennifer A. Pendleton ◽

...

Keyword(s):

Mass Spectrometry ◽

Catalytic Activity ◽

High Throughput ◽

High Throughput Screening ◽

Lipid Synthesis ◽

High Quality ◽

Compound Libraries ◽

Z Value ◽

Large Compound ◽

Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFire™ platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z′ value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target. ( Journal of Biomolecular Screening 2007:628-634)

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High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

10.1101/285015 ◽

2018 ◽

Cited By ~ 2

Author(s):

Derek M Mason ◽

Cédric R Weber ◽

Cristina Parola ◽

Simon M Meng ◽

Victor Greiff ◽

...

Keyword(s):

Directed Evolution ◽

High Throughput ◽

High Throughput Screening ◽

Mammalian Cells ◽

Affinity Maturation ◽

Antibody Engineering ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Variant Discovery ◽

Novel Variant

ABSTRACTAntibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.

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