An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv

Pihkala, Päivi; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

doi:10.1016/j.bbrc.2004.09.178

Cited by 8 publications

(3 citation statements)

References 28 publications

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“…Plasmids encoding the ER-targeted versions of pIX-MR1 produced proteins slightly larger than those produced in the cytoplasm ( Fig. 2A), possibly due to posttranslational modification(s) (55). As expected, pIX-GFP and pIX-MR1 did not bind the EGFRvIII epitope.…”

Section: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitsupporting

confidence: 56%

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: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitsupporting

confidence: 56%

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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“…We made constructs containing the D1 or D2 domain, the whole extracellular D1D2 domain, or nothing, as the extracellular domain. Since our previous studies always used constructs containing only the D2 domain (Kawahara et al, 2002(Kawahara et al, , 2004aPihkala et al, 2004), this is the first report to compare a series of antibody/ receptor chimeras with different scaffolds in the extracellular domain. The chimeric receptors containing the whole extracellular D1D2 domain (SD1D2g) or lacking any EpoR extracellular domain (Sg) showed strict ligand dependency without any ligand-independent cell proliferation ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Construction of a fluorescein‐responsive chimeric receptor with strict ligand dependency

Liu

Kawahara

Ueda

et al. 2008

Biotech & Bioengineering

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Since many cell functions are regulated by members of the cytokine receptor superfamily, the artificial mimicry of the cytokine receptor system would be attractive for cellular engineering. We previously showed that an antibody/cytokine receptor chimera can transduce a growth signal in response to non-natural ligands, such as fluorescein-conjugated BSA. However, considerable background of cell proliferation was observed without antigen. Therefore, we redesigned chimeric receptor constructs with different combinations of domains containing anti-fluorescein single chain Fv (ScFv), extracellular D1/D2 as well as transmembrane domains of erythropoietin receptor (EpoR), and the intracellular domain of glycoprotein 130 (gp130), to obtain strictly fluorescein-dependent chimeric receptors. When interleukin-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding individual chimeric receptors, the chimeras either with both D1 and D2 domains or without any EpoR extracellular domain attained a strict ligand-dependent ON/OFF regulation.

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“…The construction of plasmids pMK‐SX e g‐IG‐X e HM, pBS‐X e HM and pMX‐NLS‐Cre was previously described (Pihkala et al, ). To obtain a Sfms chimera gene, pMK‐S‐fms‐IG (Tanaka et al, ) was digested with Bam HI and Bsp EI, and subcloned into pMK‐SX e g‐IG‐X e HM digested with the same enzymes to create pMK‐SX e f‐IG‐X e HM.…”

Section: Methodsmentioning

confidence: 99%

Cell fate conversion by conditionally switching the signal‐transducing domain of signalobodies

Tone

Kawahara

Hayashi

et al. 2013

Biotech & Bioengineering

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Conditionally and strictly controlling cell fates is important for biomedical applications including cell therapies. Although previous studies have been based on regulating the expression or activation of signaling molecules, the techniques therein require improvement in terms of reducing leakiness and complexity. In this study, we propose a novel cell fate converting system using our previously developed antibody/receptor chimeras named "signalobodies" in combination with a Cre/loxP recombination system. We designed a "switch vector" where a growth signalobody gene was flanked by two loxP sites and a death signalobody gene was placed downstream of the floxed cassette. Cells transduced with the switch vector showed superior growth activity in the presence of a specific antigen. Subsequent expression of Cre induced the death signalobody, leading to conditional cell death. This technology could be applicable for other cell fate conversion systems including differentiation and migration, by using appropriate signal-transducing domains.

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An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv

Cited by 8 publications

References 28 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

Construction of a fluorescein‐responsive chimeric receptor with strict ligand dependency

Cell fate conversion by conditionally switching the signal‐transducing domain of signalobodies

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