Antibody selection using clonal cocultivation of
            <i>Escherichia coli</i>
            and eukaryotic cells in miniecosystems

Zheng, Tianqing; Xie, Jia; Yang, Zhuo; Tao, Pingdong; Shi, Bingbing; Douthit, Lacey; Wu, Peng; Lerner, Richard A.

doi:10.1073/pnas.1806718115

Cited by 9 publications

(4 citation statements)

References 17 publications

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“…The combinatorial antibody library technology allows the same evolutionary process to be performed in vitro as it restores the "fossil record" of an individual's antibody response in a test tube (17). Random coupling of VH and VL sequences in scFv libraries greatly expands diversity, thereby allowing for selection of novel antibodies with high binding affinity and neutralization efficacy (18)(19)(20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Qiang

et al. 2020

Preprint

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Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.

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Section: Introductionmentioning

confidence: 99%

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Qiang

et al. 2020

Preprint

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“…Direct antibody screening for the desired function (addressing known targets or agnostic of those in pure phenotypic screens 7,[38][39][40] ) in a high throughput manner could increase the likelihood for best-in-class antibody discovery. Pioneering early works mainly using autocrine, i.e., self-labeling, one cell systems [41][42][43][44] or mammalian display, with focus on antibody developability such as affinity maturation 45 and beyond, 5 are all compatible with flow cytometry for high throughput variant screening. The advent of microfluidics broadened opportunities for functional screening of secreted antibodies for internalization and intracellular signaling, 20 virus neutralization, 29 optimal agonistic properties, 28 and further biotherapeutics 23 when combined with suitable, mostly fluorescence-based cellular reporter assays.…”

Section: Discussionmentioning

confidence: 99%

Versatile and rapid microfluidics-assisted antibody discovery

Gaa

Menang-Ndi

Pratapa³

et al. 2021

mAbs

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Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

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“…www.nature.com/scientificreports www.nature.com/scientificreports/ medium-containing aqueous droplets and were able to detect reporter cell activation by agonist antibody fragment (scFv) presented on the phage surface 50 . This work expands the compatibility of droplet-based functional screening towards one of the best-established screening approaches, phage display 51 .…”

Section: Discussionmentioning

confidence: 99%

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

Yanakieva

Elter

Bratsch

et al. 2020

Sci Rep

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in this study, we present a straightforward approach for functional cell-based screening by coencapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarosecontaining microdroplets. our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and fAcS. this indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins. The importance of biopharmaceuticals (biologics) in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 2025 1. Frequently applied biologics comprise substances such as cytokines, monoclonal antibodies, hormones, soluble receptors, recombinant DNAs, enzymes, and synthetic vaccines. While biologics in targeted therapies often demonstrate remarkable safety and specificity, especially in case of autoimmune diseases 2 and cancer 3 , the discovery of novel molecules and the necessary functional validation still represent a bottleneck in the development of novel biopharmaceuticals. To overcome these shortcomings, powerful display technologies such as phage display 4 and yeast surface display 5 have been developed which allow for the isolation of specific high-affinity molecules and respective genes from complex variant libraries. However, identified binders frequently show poor physiological activity in a biological context. Thus, extensive secondary functional screens are necessary for identification of hit molecules with the desired functional activity. Furthermore, those screens require elaborate subcloning of the surface-displayed hits into soluble expression formats and outcoming clones frequently demand further optimization of physicochemical and pharmacokinetic properties 6. Consequently, implementing functional assays and phenotypic screens in an earlier selection phase appears highly beneficial for the discovery of new potent biologic drugs or even first-in-class medicines with novel molecular mechanisms of action 7. In this context, a major limitation is represented by the relatively low throughput of classical phenotypic screens, falling far behind the performance of high-diversity library-based approaches resting on affinity-driven selection protocols 8. Complex p...

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Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems

Cited by 9 publications

References 17 publications

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Versatile and rapid microfluidics-assisted antibody discovery

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

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