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Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER.

Böldicke T - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation,Braunschweig, Germany. thomas.boeldicke@helmholtz-hzi.de

ABSTRACT
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface. The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins. The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells. In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing. In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.

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Construction of an scFv ER intrabody. The scheme shows the construction of an scFv ER intrabody starting from a complete hybridoma antibody. Shown are the heavy chain constant domains CH3/1 and CH2, the constant region of the light chain CL, and VH and VL constituting the antigen binding domain.The red square represents the ER retention signal and the yellow rectangle the c-myc tag for detection of the scFv ER intrabody. The 15 amino acid linker assembles the VH and VL domains of the scFv fragment.
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fig02: Construction of an scFv ER intrabody. The scheme shows the construction of an scFv ER intrabody starting from a complete hybridoma antibody. Shown are the heavy chain constant domains CH3/1 and CH2, the constant region of the light chain CL, and VH and VL constituting the antigen binding domain.The red square represents the ER retention signal and the yellow rectangle the c-myc tag for detection of the scFv ER intrabody. The 15 amino acid linker assembles the VH and VL domains of the scFv fragment.

Mentions: The main format of an intrabody is the single-chain variable fragment (scFv) of an antibody, which consists of the H- and L-chain variable antibody domain (VH and VL) held together by a short, flexible linker sequence (Fig.1A and B).The source of the intrabody genes are existing hybridomas. To construct the intrabody, gene VH and VL can be amplified by an immunoglobulin-specific consensus primer, the scFv fragment assembled by overlapping PCR introducing the linker (Gly4Ser)3 and cloned into an ER-targeting vector (Fig. 1B). The ER-targeting vector contains a secretory leader, an ER retention signal and a peptide tag (for detection of the antibody, for example c-myc tag) that are both located at the end of the anti-body gene (Fig. 1B). The ER retention signal (SEKDEL, [23]) retains the antibody inside the lumen of the ER, where its binds the corresponding secretory target molecule and prevents further transport of the antigen to the cell surface (Fig. 1A). In general, expression of the intrabody is performed via the CMV promoter. The other intrabody format used is the Fab fragment. Alternatively, scFv fragments can be selected performed with in vitro display systems, such as phage, bacterial, yeast cell surface or ribosome display. In addition to the most commonly used intrabody formats, the scFv and Fab fragments, other very effective antibody formats have now been constructed: a bispecific intradiabody [44, 45] and single-domain intrabodies [74]. The bispecific intradiabody simultaneously silences two independent signalling pathways and shows high intracellular stability compared to scFv intrabodies [44, 45]. Single-domain antibodies are a new generation of small size, stable antibodies that can be isolated from existing phage display libraries.


Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER.

Böldicke T - J. Cell. Mol. Med. (2007 Jan-Feb)

Construction of an scFv ER intrabody. The scheme shows the construction of an scFv ER intrabody starting from a complete hybridoma antibody. Shown are the heavy chain constant domains CH3/1 and CH2, the constant region of the light chain CL, and VH and VL constituting the antigen binding domain.The red square represents the ER retention signal and the yellow rectangle the c-myc tag for detection of the scFv ER intrabody. The 15 amino acid linker assembles the VH and VL domains of the scFv fragment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401220&req=5

fig02: Construction of an scFv ER intrabody. The scheme shows the construction of an scFv ER intrabody starting from a complete hybridoma antibody. Shown are the heavy chain constant domains CH3/1 and CH2, the constant region of the light chain CL, and VH and VL constituting the antigen binding domain.The red square represents the ER retention signal and the yellow rectangle the c-myc tag for detection of the scFv ER intrabody. The 15 amino acid linker assembles the VH and VL domains of the scFv fragment.
Mentions: The main format of an intrabody is the single-chain variable fragment (scFv) of an antibody, which consists of the H- and L-chain variable antibody domain (VH and VL) held together by a short, flexible linker sequence (Fig.1A and B).The source of the intrabody genes are existing hybridomas. To construct the intrabody, gene VH and VL can be amplified by an immunoglobulin-specific consensus primer, the scFv fragment assembled by overlapping PCR introducing the linker (Gly4Ser)3 and cloned into an ER-targeting vector (Fig. 1B). The ER-targeting vector contains a secretory leader, an ER retention signal and a peptide tag (for detection of the antibody, for example c-myc tag) that are both located at the end of the anti-body gene (Fig. 1B). The ER retention signal (SEKDEL, [23]) retains the antibody inside the lumen of the ER, where its binds the corresponding secretory target molecule and prevents further transport of the antigen to the cell surface (Fig. 1A). In general, expression of the intrabody is performed via the CMV promoter. The other intrabody format used is the Fab fragment. Alternatively, scFv fragments can be selected performed with in vitro display systems, such as phage, bacterial, yeast cell surface or ribosome display. In addition to the most commonly used intrabody formats, the scFv and Fab fragments, other very effective antibody formats have now been constructed: a bispecific intradiabody [44, 45] and single-domain intrabodies [74]. The bispecific intradiabody simultaneously silences two independent signalling pathways and shows high intracellular stability compared to scFv intrabodies [44, 45]. Single-domain antibodies are a new generation of small size, stable antibodies that can be isolated from existing phage display libraries.

Bottom Line: A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation,Braunschweig, Germany. thomas.boeldicke@helmholtz-hzi.de

ABSTRACT
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface. The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins. The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells. In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing. In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.

Show MeSH
Related in: MedlinePlus