Direct Phage to Intrabody Screening (DPIS): Demonstration by Isolation of Cytosolic Intrabodies Against the TES1 Site of Epstein Barr Virus Latent Membrane Protein 1 (LMP1) that Block NF-κB Transactivation

Gennari, Francesca; Mehta, Smita; Wang, Yan; Tallarico, Aimée St. Clair; Palù, Giorgio; Marasco, Wayne A.

doi:10.1016/j.jmb.2003.09.073

Cited by 31 publications

(21 citation statements)

References 78 publications

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“…A key problem arises from the conditions under which antibodies against intracellular targets are isolated and engineered. With the exception of the yeast two-hybrid approach to intrabody isolation (5), antibodies are isolated and engineered under oxidizing conditions by yeast or phage display (1,6,7), where stabilizing disulfide bonds form; however, disulfide bonds do not form as readily in the reducing environment of the cytoplasm, where intrabodies are intended to function. Lead optimization or incremental improvement of intrabody function has not been reported to date with a yeast two-hybrid approach, perhaps due to the qualitative nature of that screening system.…”

mentioning

confidence: 99%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

145

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Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets

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mentioning

confidence: 99%

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Colby

Chu

Cassady

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

169

145

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“…1) was generated to express the A3H5 intrabody. A3H5 targets the 34 amino acid transformation effector site-1 motif of the cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus (Gennari et al, 2004). MLV-based retroviral vectors, based on the LZRS system (Kinsella and Nolan, 1996), were shuttle-packaged through Phoenix (amphotropic) packaging cells into the GaLV-pseudotyped packaging cells lines PG13 to generate high-titer producer cell lines with titers ranging from 1.0 · 10 5 to 2.6 · 10 5 TU/ml of cell supernatant.…”

Section: In Vitro Transduction Of Cd4mentioning

confidence: 99%

In VivoSelection of CD4⁺T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

et al. 2012

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“…A recent report states that CTAR-1-specific LMP1 intrabodies are capable of reducing NF-B activation in cells [35] . The present study goes further by demonstrating that the intrabody H3 not only has the ability to interact with the LMP1 CTAR-1 region in vivo, but also inhibits MDCK-LMP1 cells transmigration ability.…”

Section: Discussionmentioning

confidence: 99%

Modulation of Epstein-Barr Virus Latent Membrane Protein 1 Activity by Intrabodies

et al. 2007

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Objective: The Epstein-Barr virus (EBV) has been implicated in the development of many human neoplasias including B lymphoma and nasopharyngeal carcinoma. EBV latent membrane protein 1 (LMP1) is essential to virus-induced B cell immortalization and the downregulation of cell adhesion molecules that increases cell motility. Therefore, identifying LMP1 activity modulation methods may lead to the development of new therapies for LMP1-positive tumors. Methods: This study uses a phage display single-chain variable fragments (scFvs) library to screen recombinant antibodies specific to the LMP1 C terminal region. A total of 45 individual clones were obtained, and these scFvs were cloned as intrabodies and transfected into LMP1-positive cells. Results: One of the scFv clones, designated H3, was capable of reducing LMP1-mediated NF-ĸB activation in HEK293 cells. Immunofluorescence and co-immunoprecipitation studies show that scFv H3 could interact with LMP1 in vivo. In addition, expression of scFv H3 intrabody could reduce cell motility in MDCK-LMP1 cells in the transwell migration assay. Conclusion: These data indicate that scFv H3 intrabody can inhibit LMP1 functions in epithelial cells and may be useful for attenuating the LMP1 function in LMP1-positive tumors.

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Direct Phage to Intrabody Screening (DPIS): Demonstration by Isolation of Cytosolic Intrabodies Against the TES1 Site of Epstein Barr Virus Latent Membrane Protein 1 (LMP1) that Block NF-κB Transactivation

Cited by 31 publications

References 78 publications

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

In VivoSelection of CD4⁺T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

Modulation of Epstein-Barr Virus Latent Membrane Protein 1 Activity by Intrabodies

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Direct Phage to Intrabody Screening (DPIS): Demonstration by Isolation of Cytosolic Intrabodies Against the TES1 Site of Epstein Barr Virus Latent Membrane Protein 1 (LMP1) that Block NF-κB Transactivation

Cited by 31 publications

References 78 publications

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

In VivoSelection of CD4+T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

Modulation of Epstein-Barr Virus Latent Membrane Protein 1 Activity by Intrabodies

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In VivoSelection of CD4⁺T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat