Simultaneous Expression of Displayed and Secreted Antibodies for Antibody Screen

Zhou, Yuanping; Wang, Junjie; Zhou, Ivan; Lou, Haibo; Li, Changzheng; Chen, Zhen-Rui; Zhang, Zhehuan; Liu, Shuwen; Wu, Shuai; Tan, Wanlong; Jiang, Shibo; Zhou, Chen

doi:10.1371/journal.pone.0080005

Cited by 10 publications

(11 citation statements)

References 19 publications

(19 reference statements)

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“…Secretion in this system is achieved by a Furin cleavage site located between the PDGFR-TM and the HC. 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.…”

Section: Discussionmentioning

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

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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%

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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show abstract

“…22 Another strategy used the introduction of enzymatic cleavage sequences. 23 For example, Zhou et al 24 inserted a furin cleavage site between the CH3 constant domain and a transmembrane domain to produce cell surface-displayed and soluble antibodies. However, the relative levels of either form remained constant and did not switch according to B-cell maturation.…”

Section: Discussionmentioning

confidence: 99%

A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status

et al. 2015

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The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.

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“…Another approach used the RIRR sequence that can be recognized and partially cleaved by the furin enzyme, leading to simultaneous secretion and display of antibodies (Zhou et al, 2013). Yet another approach is based on alternative splicing, thus allowing titration of the secretion to display ratio (Aebischer-Gumy et al, 2020;Horlick et al, 2013).…”

Section: Antibody Screening With Mammalian Displaymentioning

confidence: 99%

“…One approach used Cre recombination sites flanking the transmembrane domain, which facilitated Cre-mediated deletion of the transmembrane domain and therefore switching from antibody display to antibody secretion ( Tomimatsu et al., 2013 ). Another approach used the RIRR sequence that can be recognized and partially cleaved by the furin enzyme, leading to simultaneous secretion and display of antibodies ( Zhou et al., 2013 ). Yet another approach is based on alternative splicing, thus allowing titration of the secretion to display ratio ( Aebischer-Gumy et al., 2020 ; Horlick et al., 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

Csepregi¹,

Ehling²,

Wagner³

et al. 2020

iScience

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Advances in reading, writing, and editing DNA are providing unprecedented insights into the complexity of immunological systems. This combination of systems and synthetic biology methods is enabling the quantitative and precise understanding of molecular recognition in adaptive immunity, thus providing a framework for reprogramming immune responses for translational medicine. In this review, we will highlight state-of-the-art methods such as immune repertoire sequencing, immunoinformatics, and immunogenomic engineering and their application toward adaptive immunity. We showcase novel and interdisciplinary approaches that have the promise of transforming the design and breadth of molecular and cellular immunotherapies.

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Simultaneous Expression of Displayed and Secreted Antibodies for Antibody Screen

Cited by 10 publications

References 19 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

A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status

Immune Literacy: Reading, Writing, and Editing Adaptive Immunity

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