A universal strategy for stable intracellular antibodies

Shaki-Loewenstein, Shelly; Zfania, Rahely; Hyland, Stephen; Wels, Winfried S.; Benhar, Itai

doi:10.1016/j.jim.2005.05.004

Cited by 41 publications

(41 citation statements)

References 76 publications

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“…A similar plasmid for the expression of the heavy chain of the anti EGFR antibody 225 in E. coli was constructed as follows: the VH variable domain was recovered by PCR using plasmid pCMV/H6myc/cyto-225(Fv) 14 as template with primers 225VH-NdeI-FOR and 225VH-NheI-REV. The PCR product was digested with NdeI and NheI and cloned into a pHAK-IgH-T427 vector (described above) that was linearized using the same enzymes.…”

Section: Materials and Methods: Construction Of Vectors For Expressiomentioning

confidence: 99%

“Inclonals”

Hakim

Benhar

2009

mAbs

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Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of shake flask culture) of highly purified (>90%) full-length antibodies and antibody-toxin fusions were obtained. The bacterially produced antibodies, named "Inclonals," equaled the performance of the same IgGs that were produced using conventional mammalian cell culture in binding properties as well as in cell killing potency. The rapid and cost effective IgG production process and the high quality of the resultant product may make the bacterial production of full-length IgG and IgG-drug fusion proteins an attractive option for antibody production and a significant contribution to recombinant antibody technology.

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Section: Materials and Methods: Construction Of Vectors For Expressiomentioning

confidence: 99%

“Inclonals”

Hakim

Benhar

2009

mAbs

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“…37 In contrast to the approach that focuses on selection of antibodies with specific "intrabody properties," attempts have been made to convert arbitrary antibodies into intrabodies by fusion to another protein because the approach could be more generally applied. 38 To enhance cytosolic expression, antibodies were fused to a Cκ domain, 39,40 to N utilization substance A (NusA) 41 or to maltose binding protein (MBP). 38 Fusion of a Cκ domain to an anti-p53 scFv led to increased expression levels in the cytoplasm of mammalian cells compared to the anti-p53 scFv alone, which can thus be performed using a single standardized subcloning step after selection of a scFv antibody or Fab from a phage display library.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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“…10,13,27,28 Thereby autocrine secretion of EGFRspecific scFv(225) antibody blocked ligand-induced EGFR activation on the cell surface and EGF-induced growth of transformed cells. 10,11,29 Here the membrane-anchored monospecific derivative 225.TM displayed similar activities, localizing together with EGFR mainly on the cell surface and inhibiting ligand-induced receptor activation and tumor cell growth. Despite comparable overall protein levels, surface expression of monospecific TM.30 and bispecific 225.TM.30 molecules was much lower than that of 225.TM.…”

Section: Discussionmentioning

confidence: 86%

“…9 Experimentally, ligand-induced EGFR activation could be prevented in an autocrine fashion, and anchorage-independent growth could be inhibited by expression of scFv(225) as a secreted molecule directly in EGFR-transformed tumor cells. 10,11 Antibodies like scFv(225) expressed as secreted or ERresident intrabodies have proven very useful to interfere with target proteins located at the cell surface or within the secretory compartment. 12 In contrast, functional intrabodies that bind to cytoplasmic target proteins are rare.…”

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confidence: 99%

A bispecific transmembrane antibody simultaneously targeting intra‐ and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

Müller

Hartmann

Genßler

et al. 2013

Intl Journal of Cancer

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Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR-specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv (225) Epidermal growth factor receptor (EGFR, ErbB) belongs to the family of ErbB receptor tyrosine kinases that also includes the closely related ErbB2 (HER2/Neu), ErbB3 (HER3) and ErbB4 (HER4) molecules.1 These type I transmembrane proteins share a common molecular architecture characterized by a glycosylated extracellular domain to which peptide ligands bind, a single a-helical transmembrane region and a cytosolic domain with tyrosine kinase activity. The EGFR extracellular domain is structurally separated into four subdomains. Subdomains I and III together provide the binding interface for ligands of the EGF-like growth factor family. Ligand binding stabilizes a dimerization-competent state of the receptor by preventing an inhibitory intramolecular contact between subdomains II and IV, thereby exposing a dimerization interface in subdomain II that facilitates the formation of receptor homodimers or heterodimers with other ErbB family members.2 Receptor dimerization stimulates the intrinsic tyrosine kinase activity, which results in autophosphorylation of specific tyrosine residues within the C-terminal tail. These phosphotyrosine residues serve as docking sites for intracellular substrates and adapter proteins that initiate signaling cascades promoting cell survival, migration and proliferation.

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A universal strategy for stable intracellular antibodies

Cited by 41 publications

References 76 publications

“Inclonals”

“Inclonals”

Targeting antibodies to the cytoplasm

A bispecific transmembrane antibody simultaneously targeting intra‐ and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

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