Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins

Fang, Yongliang; Chu, Thach H.; Ackerman, Margaret E.; Griswold, Karl E.

doi:10.1080/19420862.2017.1381812

Cited by 17 publications

(19 citation statements)

References 56 publications

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“…We expect native V H :V L antibodies to show superior selectivity and biophysical properties compared to randomly paired V H and V L antibodies isolated using other display platforms 9 , 10 . Native antibody libraries displayed on yeast can also be screened for antigens that bind to B cell surface ligands (e.g., sialic acid 26 or CR2 27 ) and are therefore not suitable for single B cell sorting, and separately to discover antibodies targeting insoluble antigens, including membrane proteins 28 – 30 .…”

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confidence: 99%

Functional interrogation and mining of natively paired human VH:VL antibody repertoires

et al. 2018

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We present a technology to screen natively-paired human antibody repertoires from millions of B cells. Libraries of natively-paired variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

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confidence: 99%

Functional interrogation and mining of natively paired human VH:VL antibody repertoires

et al. 2018

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“…In comparison, several similar screening approaches have been published in recent years, which once again emphasizes the high demand in this field. In 2017, Fang et al proposed an affinity-based antibody screening platform, based on co-encapsulation of antibody-secreting Pichia pastoris and fixed mammalian target cells in agarose microdroplets 48 . This study demonstrated that GMD-FACS is a reliable selection method and classical antibody staining procedures can be applied for the specific fluorescent detection of target-bound antibodies in the agarose microbeads.…”

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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“…In fact, our method of cytoplasmic IgG expression circumvents membrane translocation of these large macromolecules altogether, which is important because traversing tightly sealed biological membranes is a rate limiting and energy intensive step that can serve as a potential source of selection bias in these previous IgG screening methods. While other membrane-less IgG screening strategies exist, in particular methods for encapsulating single IgG antibody secreting cells in water-in-oil droplets 43,44 or gel microdroplets [45][46][47][48] , construction of such drop-based secretor cell libraries is non-trivial, often involving microfluidics, and screening must typically be performed in conjunction with FACS, which introduces additional challenges as discussed above.…”

Section: Discussionmentioning

confidence: 99%

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm ofEscherichia coli

Robinson

Cox

et al. 2020

Preprint

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1We describe a facile and robust genetic selection for isolating full-length IgG antibodies from 2 combinatorial libraries expressed in the cytoplasm of the genetically engineered Escherichia coli 3 strain, SHuffle. The method is based on the transport of a bifunctional substrate comprised of an 4 antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial 5 cells co-expressing cytoplasmic IgGs called 'cyclonals' that specifically capture the chimeric 6 antigen and sequester the antibiotic resistance marker in the cytoplasm. The selective power of 7 this approach was demonstrated by facile isolation of novel complementarity-determining regions 8 for a cyclonal that specifically recognized the basic-region leucine zipper domain of the yeast 9 transcriptional activator protein Gcn4. 10 11 10-12 , yeast 13-15 , and mammalian cells 16 , effectively circumventing the reformatting issue. 32Nonetheless, screening methods such as these require each library member to be individually 33 evaluated, which necessitates specialized equipment (e.g., flow cytometer) to access meaningful

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Going native: Direct high throughput screening of secreted full-length IgG antibodies against cell membrane proteins

Cited by 17 publications

References 56 publications

Functional interrogation and mining of natively paired human VH:VL antibody repertoires

Functional interrogation and mining of natively paired human VH:VL antibody repertoires

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

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm ofEscherichia coli

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