A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Philibert, Pascal; Stoessel, Audrey; Wang, Wei; Sibler, Annie-Paule; Bec, Nicole; Larroque, Christian; Saven, Jeffery G.; Courtête, Jérôme; Weiss, Étienne; Martineau, Pierre

doi:10.1186/1472-6750-7-81

Cited by 71 publications

(59 citation statements)

References 66 publications

(101 reference statements)

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“…Libraries of camelid-derived sdAb are available (75), and such libraries can be panned against any target cell or tissue to identify specific sdAb. Alternatively, scFv libraries that have enhanced stability in the reducing environment of the cytoplasm, which would circumvent the need for ER-associated expression of this class of molecule, have been developed (54). Thus, Ads that express a wide array of polypeptides for specific targeting of a variety of cells and tissues can be developed.…”

Section: Discussionmentioning

confidence: 99%

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Poulin

Lanthier

Smith

et al. 2010

J Virol

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Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

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Section: Discussionmentioning

confidence: 99%

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Poulin

Lanthier

Smith

et al. 2010

J Virol

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“…The use of peptides instead of antibodies to functionalize SPION, as in the present work, could furthermore contribute to cost reductions in diagnosis and in patient care. The outstanding development of biosynthetic technologies nowadays (35,36) is susceptible to render the expense of peptide synthesis more profitable, with production yields that are much improved over the classical chemical routes. The biosynthetic technologies were even exploited in the synthesis of particulate inorganic material, which is produced by various mono-cellular organisms, the magnetotactic bacteria being able to synthesize magnetite (37).…”

Section: Discussionmentioning

confidence: 99%

In vitro biomedical applications of functionalized iron oxide nanoparticles, including those not related to magnetic properties

Burtéa

Laurent

Mahieu

et al. 2010

Contrast Media & Molecular

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Superparamagnetic iron oxide nanoparticles (SPION) are very promising contrast media, especially for molecular imaging, due to their superior NMR efficacy. They even have wider biomedical applications such as in drug and gene delivery, tissue engineering and bioseparation, or as sensitive biological nanosensors. By coupling them to affinity ligands, SPION can bind to drugs, proteins, enzymes, antibodies or nucleotides. For in vitro biomedical applications, the detection of molecular interaction is possible by using a diversity of systems capable of sensing the magnetic properties of these materials. The goal of the present work was to develop and validate various in vitro biomedical applications of ultrasmall superparamagnetic particles of iron oxide (USPIO), including some that are not related to their magnetic properties. USPIO coated with dextran, starch or bisphosphonate exposing carboxylate groups were synthesized and some of them were functionalized by conjugating various biomolecules, such as biotin, streptavidin and apoptosis, or VCAM-1 specific peptides. The in vitro biomedical applications assessed in the present work included: (1) the relaxometric measurement of antibody concentration, cell receptor expression, molecular interaction, and enzymatic activity in aqueous suspensions; (2) MRI visualization of cells and detection of molecular interaction in an ELISA system; (3) ELISA applications of USPIO derivatives; and (4) detection of specific biomolecules by histochemistry. Our results confirm that rapid and simple in vitro detection of a diversity of functionalized SPION with relevance in medicine is possible by the existing NMR techniques and by chemical staining reactions. The protocols can be applied to minimally prepared biological samples (e.g. whole blood, blood plasma or serum, cell suspensions, biopsies, histological preparations, etc.), and often do not need complicated systems of signal amplification. The use of SPION labeled compounds could furthermore contribute to cost reductions in the diagnosis and in patient care.

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“…Alternatively, antibodies compatible with expression in the cytoplasm have been isolated from libraries based on highly diverse or hyperstable scFvs, which were then screened by conventional phage display. 13,18,19,20 However, screening is carried out under conditions where disulfide bonds are formed, so that only a fraction of the antibodies isolated are found to be compatible with cytoplasmic expression (e.g., Ref. 20).…”

Section: Discussionmentioning

confidence: 99%

“…17 Alternatively, phage display has been employed successfully for the directed evolution of hyperstable antibody frameworks that in some cases can withstand expression in the reducing environment of the cytoplasm. 1 In turn, natural or engineered hyperstable antibody frameworks have been used as scaffolds for the creation of large synthetic libraries containing randomized CDRs 13,[18][19][20] enabling the isolation of scFvs that are folded in the absence of disulfides. Alternatively, MBP-scFv fusions have been shown to exhibit significant activity when expressed in the cytoplasm of Escherichia coli or mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Engineering antibody fragments to fold in the absence of disulfide bonds

et al. 2009

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Disulfide bonds play a critical role in the stabilization of the immunoglobulin b-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.

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A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Cited by 71 publications

References 66 publications

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

In vitro biomedical applications of functionalized iron oxide nanoparticles, including those not related to magnetic properties

Engineering antibody fragments to fold in the absence of disulfide bonds

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