Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin

Cloutier, Sylvain M.; Couty, S.; Terskikh, Alexey V.; Marguerat, L.; Crivelli, Valérie; Pugnières, Martine; Mani, Jean‐Claude; Leisinger, H.-J.; Mach, Jean‐Pierre; Deperthes, David

doi:10.1016/s0161-5890(01)00023-2

Cited by 69 publications

(58 citation statements)

References 27 publications

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“…By ELISA assays, anti-HE4 biobody affinity appeared to be increased after preincubation with anti-V5, consistent with a previous report by Wang and colleagues (Wang et al, 2004), but was dramatically improved after preincubation with streptavidin, consistent with previous reports of tetramerization of biotinylated recombinant antibodies in presence of streptavidin (Muhlrad et al, 1992;Saviranta et al, 1998;Cloutier et al, 2000). In addition, the affinity of five anti-HE4 mBb increased when they were preincubated with streptavidin together instead of separately, which suggests a cooperative effect of the biobodies.…”

Section: Discussionsupporting

confidence: 90%

“…Specific biotinylation of proteins and recombinant antibodies has been obtained in E.coli in vivo (Kipriyanov et al, 1996;Sibler et al, 1999;Santala and Lamminmaki, 2004;Warren et al, 2005) and in vitro with purified biotin ligase (Saviranta et al, 1998;Cloutier et al, 2000). Several groups have demonstrated independently that biotinylated recombinant antibodies tetramerize in presence of streptavidin and that tetramers show both higher affinity constants and lower non-specific binding than monomeric single chains (Muhlrad et al, 1992;Saviranta et al, 1998;Cloutier et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…Several groups have demonstrated independently that biotinylated recombinant antibodies tetramerize in presence of streptavidin and that tetramers show both higher affinity constants and lower non-specific binding than monomeric single chains (Muhlrad et al, 1992;Saviranta et al, 1998;Cloutier et al, 2000). However, current in vivo biotinylation protocols can only label one protein at a time through lengthy molecular engineering that is not compatible with high-throughput approaches.…”

Section: Introductionmentioning

confidence: 99%

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Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

Scholler

Garvik

Quarles

et al. 2006

Journal of Immunological Methods

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We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. Coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K d = 4.8×10 -9 M and K d =5.1×10 -9 M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

Scholler

Garvik

Quarles

et al. 2006

Journal of Immunological Methods

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“…This approach was used previously to increase the binding avidity of peptide-MHC complexes to the TCR or to increase the sensitivity of recombinant Ab molecules (35). Another advantage of using fluorescently labeled tetramers is that only a single staining step is required, whereas monomeric unlabeled Fabs require a fluorescently labeled secondary Ab.…”

Section: Detection Of Complexes On Pulsed Apcsmentioning

confidence: 99%

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Cohen

Sarig

Yamano

et al. 2003

The Journal of Immunology

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The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

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“…Construct A7:IAH was designed to match a previously published tag that had been used for in vivo biotinylation. 30,31 The streptavidin-HRP protein gel blot shows that in vivo biotinylation of the Avitag does happen in the antibody located in the pellet. No in vivo biotinylation could be detected in antibody purified from the supernatant (Fig.…”

Section: Discussionmentioning

confidence: 99%

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

Lykkemark

Mandrup

Friis

et al. 2014

mAbs

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Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin

Cited by 69 publications

References 27 publications

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

Degradation of C-terminal tag sequences on domain antibodies purified fromE. colisupernatant

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