Retrocyte Display® technology: Generation and screening of a high diversity cellular antibody library

Breous-Nystrom, Ekaterina; Schultze, Kornelia; Meier, Marco; Flueck, Lukas; Holzer, Christina; Boll, Melanie; Seibert, Volker; Schuster, Andrea; Blanusa, Milan; Schaefer, Verena; Grawunder, Ulf; Martin‐Parras, Luis; Dijk, Marc A. van

doi:10.1016/j.ymeth.2013.09.003

Cited by 23 publications

(14 citation statements)

References 29 publications

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“…51 Yet another approach is used in a mammalian display platform called Retrocyte Display developed by 4-Antibody, and is based on irreversible, cre-recombinase mediated deletion of TM exons. 16 It would be interesting to determine whether our technology can be transferred to other widely established cell lines such as Chinese hamster ovary (CHO) or HEK293T, and to investigate the extent to which different host cell lines would be beneficial in terms of the library sizes that could be generated and screened, the ratio of membrane-bound vs. secreted IgG that could be achieved with these non-B cell lines, and finally how the properties of the isolated antibodies compare with antibodies isolated from the B cell host cell line employed here. Importantly, PiggyBac seems to be active in a wide range of mammalian cell lines, 45,52,53 and thus could in principle be applied to other cell systems of choice.…”

Section: Discussionmentioning

confidence: 99%

“…11,12 In order to perform library screening under the most physiological conditions possible, numerous mammalian cell-based antibody expression platforms have been developed, involving simple transient transfection of antibody libraries 13 or their viral transduction using vaccinia, 14 sindbis, 15 or retrovirus vectors. 16 While these approaches are believed to deliver mAbs with favorable biophysical properties that have a higher potential in drug development and therapeutic use, mammalian cell-based screening platforms are currently also associated with a number of limitations, which has led to a slow adoption of these technologies in academia and industry.…”

Section: Introductionmentioning

confidence: 99%

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Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Waldmeier

Hellmann

Gutknecht

et al. 2016

mAbs

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In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Waldmeier

Hellmann

Gutknecht

et al. 2016

mAbs

Self Cite

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“…Both antibodies are derived from a proprietary mammalian display technology, Retrocyte Display™. 102 Balstilimab is being evaluated as monotherapy in a Phase 1/2 study (NCT03104699) and in combination with zalifrelimab in Phase 1/2 (NCT03495882) and Phase 2 (NCT03894215) studies. Phase 1/2 study results could potentially support BLA submissions as early as 2020 under FDA's accelerated approval pathway, according to projections by Agenus.…”

Section: Loncastuximab Tesirine (Adc Therapeutics Sa)mentioning

confidence: 99%

Antibodies to watch in 2020

Kaplon

Muralidharan²,

Schneider

et al. 2019

mAbs

395

296

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This 2020 installment of the annual 'Antibodies to Watch' series documents the antibody therapeutics approved in 2019 and in regulatory review in the United States or European Union, as well as those in late-stage clinical studies, as of November 2019*. At this time, a total of 5 novel antibody therapeutics (romosozumab, risankizumab, polatuzumab vedotin, brolucizumab, and crizanlizumab) had been granted a first approval in either the US or EU, and marketing applications for 13 novel antibody therapeutics (eptinezumab, teprotumumab, enfortumab vedotin, isatuximab, [fam-]trastuzumab deruxtecan, inebilizumab, leronlimab, sacituzumab govitecan, satralizumab, narsoplimab, tafasitamab, REGNEB3 and naxituximab) were undergoing review in these regions, which represent the major markets for antibody therapeutics. Also as of November 2019, 79 novel antibodies were undergoing evaluation in late-stage clinical studies. Of the 79 antibodies, 39 were undergoing evaluation in late-stage studies for non-cancer indications, with 2 of these (ublituximab, pamrevlumab) also in late-stage studies for cancer indications. Companies developing 7 (tanezumab, aducanumab, evinacumab, etrolizumab, sutimlimab, anifrolumab, and teplizumab) of the 39 drugs have indicated that they may submit a marketing application in either the US or EU in 2020. Of the 79 antibodies in late-stage studies, 40 were undergoing evaluation as treatments for cancer, and potentially 9 of these (belantamab mafodotin, oportuzumab monatox, margetuximab, dostarlimab, spartalizumab, 131I-omburtamab, loncastuximab tesirine, balstilimab, and zalifrelimab) may enter regulatory review in late 2019 or in 2020. Overall, the biopharmaceutical industry's clinical pipeline of antibody therapeutics is robust, and should provide a continuous supply of innovative products for patients in the future. *Note on key updates through December 18, 2019: 1) the US Food and Drug Administration granted accelerated approval to enfortumab vedotin-ejfv (Padcev) on December 18, 2019, bringing the total number of novel antibody therapeutics granted a first approval in either the US or EU during 2019 to 6; 2) the European Commission approved romosozumab on December 9, 2019; 3) the European Medicines Agency issued a positive opinion for brolucizumab; 4) Sesen Bio initiated a rolling biologics license application (BLA) on December 6, 2019; 5) GlaxoSmithKline submitted a BLA for belantamab mafodotin; and 6) the status of the Phase 3 study (NCT04128696) of GSK3359609, a humanized IgG4 anti-ICOS antibody, in patients with head and neck squamous cell carcinoma was updated to recruiting from not yet recruiting. ARTICLE HISTORY

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“…A number of approaches have been described to introduce single antibody genes into each cell, including viral-based systems and transposons. [5][6][7][8] A disadvantage of these approaches is that single copy integration is controlled by limited infection or transfection, requiring a compromise between library size and single gene insertion. In addition, integration within the genome is random, leading to potential variation in transcription level based on the transcriptional activity of the integration locus.…”

Section: Introductionmentioning

confidence: 99%

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

Parthiban¹,

Perera²,

Sattar³

et al. 2019

mAbs

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The construction of large libraries in mammalian cells allows the direct screening of millions of molecular variants for binding properties in a cell type relevant for screening or production. We have created mammalian cell libraries of up to 10 million clones displaying a repertoire of IgG-formatted antibodies on the cell surface. TALE nucleases or CRISPR/Cas9 were used to direct the integration of the antibody genes into a single genomic locus, thereby rapidly achieving stable expression and transcriptional normalization. The utility of the system is illustrated by the affinity maturation of a PD-1-blocking antibody through the systematic mutation and functional survey of 4-mer variants within a 16 amino acid paratope region. Mutating VH CDR3 only, we identified a dominant "solution" involving substitution of a central tyrosine to histidine. This appears to be a local affinity maximum, and this variant was surpassed by a lysine substitution when light chain variants were introduced. We achieve this comprehensive and quantitative interrogation of sequence space by combining high-throughput oligonucleotide synthesis with mammalian display and flow cytometry operating at the multi-million scale.

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Retrocyte Display® technology: Generation and screening of a high diversity cellular antibody library

Cited by 23 publications

References 29 publications

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

Antibodies to watch in 2020

A comprehensive search of functional sequence space using large mammalian display libraries created by gene editing

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