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1.
《MABS-AUSTIN》2013,5(6):1010-1035
Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed.  相似文献   

2.
Many therapeutic targets are intracellular proteins and molecules designed to interact with them must effectively bind to their target inside the cell. Intracellular antibodies (intrabodies) recognise and bind to proteins in cells and various methods have been developed to produce such molecules. Intracellular antibody capture (IAC) is based on a genetic screening approach and is a facile methodology with which effective intracellular antibodies can be obtained. During the development of the IAC technology, consensus immunoglobulin variable frameworks were identified which can form the basis of intrabody libraries for direct screening. In this paper, we describe the de novo synthesis of intrabody libraries based on the IAC consensus sequence. The procedure comprises in vitro production of a single antibody gene fragment from oligonucleotides and diversification of CDRs of the immunoglobulin variable domain by mutagenic PCR. Completely de novo intrabody libraries can be rapidly generated in vitro by these approaches. As an example, a single immunoglobulin VH domain intrabody library was screened directly in yeast with an oncogenic BCR-ABL antigen bait and distinct antigen binders were isolated illustrating the functional utility of the library. This second generation IAC approach (IAC2) has many practical advantages, in particular the ability to isolate intrabodies by direct genetic selection, which obviates the need for in vitro production of antigen for pre-selection of antibody fragments.  相似文献   

3.
The specific and high affinity binding properties of intracellular antibodies (intrabodies), combined with their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of applications in the field of functional genomics and gene therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their functions as well as for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. In order to study the biological consequences of knocking down two receptor-tyrosine kinases, we developed a novel intrabody-based strategy. Here we describe the design, engineering, and characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment (scFv) intrabodies demonstrated that the intradiabody is significantly more efficient with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2. In vitro endothelial cell tube formation assays suggest that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and the Tie-2 receptor pathways results in at least additive antiangiogenic effects, which may have implications for future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format for the simultaneous functional knockout of two cell surface receptors.  相似文献   

4.
Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.  相似文献   

5.
There is a major need in target validation and therapeutic applications for molecules that can interfere with protein function inside cells. Intracellular antibodies (intrabodies) can bind to specific targets in cells but isolation of intrabodies is currently difficult. Intrabodies are normally single chain Fv fragments comprising variable domains of the immunoglobulin heavy (VH) and light chains (VL). We now demonstrate that single VH domains have excellent intracellular properties of solubility, stability and expression within the cells of higher organisms and can exhibit specific antigen recognition in vivo. We have used this intracellular single variable domain (IDab) format, based on a previously characterised intrabody consensus scaffold, to generate diverse intrabody libraries for direct in vivo screening. IDabs were isolated using two distinct antigens and affinities of isolated IDabs ranged between 20 nM and 200 nM. Moreover, IDabs selected for binding to the RAS protein could inhibit RAS-dependent oncogenic transformation of NIH3T3 cells. The IDab format is therefore ideal for in vivo intrabody use. This approach to intrabodies obviates the need for phage antibody libraries, avoids the requirement for production of antigen in vitro and allows for direct selection of intrabodies in vivo.  相似文献   

6.

Background  

Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (αT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. αT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence.  相似文献   

7.
Intrabodies are recombinantly expressed intracellular antibody fragments that can be used to specifically bind and inhibit the function of cellular proteins of interest. Intrabodies can be targeted to various cell compartments by attaching an appropriate localization peptide sequence to them. An efficient strategy with a high success rate is to anchor intrabodies in the endoplasmatic reticulum where they can inhibit transitory target proteins by binding and preventing them to reach their site of action. Intrabodies can be assembled from antibody gene fragments from various sources into dedicated expression vectors. Conventionally, antibody cDNA sequences are derived from selected hybridoma cell clones that express antibodies with the desired specificity. Alternatively, appropriate clones can be isolated by affinity selection from an antibody in vitro display library. Here an evaluation of endoplasmatic reticulum targeted intrabodies with respect to other knockdown approaches is given and the characteristics of various intrabody expression vectors are discussed. A step by step protocol is provided that was repeatedly used to construct intrabodies derived from diverse antibody isotypes producing hybridoma cell clones. The inactivation of the cell surface receptor neural cell adhesion molecule (NCAM) by a highly efficacious novel endoplasmatic reticulum-anchored intrabody is demonstrated.  相似文献   

8.
Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.  相似文献   

9.

Background

Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Results

Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.

Conclusion

The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.  相似文献   

10.
The selective knock-down of cellular proteins has proven useful for in vivo studies of protein function and RNAi methods are readily available for this purpose. However, interfering directly at the protein level may have distinct advantages, with the intracellular targeting of antibodies (intrabodies) representing an attractive option, although not a general one. We demonstrate a novel, general strategy named suicide (or silencing) intrabody technology (SIT), based on the inducible degradation of intrabodies, which are equipped with proteasome-targeting sequences and thus converted into suicide intrabodies. We show that suicide intrabodies are able to redirect the target cellular proteins upon stimulus administration to the proteolytic machinery, thus resulting in selective protein knock-down. Remarkably, suicide intrabody acts in a catalytic fashion. SIT is a ligand-inducible strategy, potentially applicable to any protein of interest and does not require the engineering of cellular proteolytic enzymes. SIT represents a general approach to confer “neutralizing” properties to any intrabody, a valuable feature, given the present impossibility to select a priori intrinsically neutralizing antibodies. This knock-down strategy, together with available methods to isolate functional intrabodies, should allow the large-scale investigation of intracellular protein networks.  相似文献   

11.
We have applied in vivo intracellular antibody capture (IAC) technology to isolate human intrabodies which bind to the oncogenic RAS protein. IAC facilitates the capture of antibody fragments, in this case single-chain Fvs (scFvs), which tolerate reducing environments, such as the cytoplasm of cancer cells. Three anti-RAS scFvs with different affinity, solubility and intracellular binding activity were characterized. The anti-RAS scFvs with highest affinity were expressed relatively poorly in mammalian cells, and greater soluble expression was achieved by mutating the antibody framework to canonical consensus scaffolds, previously derived from IAC, without losing antigen specificity. Mutagenesis experiments showed that the consensus scaffolds are functional as intrabody fragments without an intra-domain disulfide bond. Furthermore, we could convert an intrabody which does not bind RAS in mammalian cells into a high-affinity reagent capable of inhibiting RAS-mediated NIH 3T3 transformation by exchanging VH and VL complementarity-determining regions onto its consensus scaffold. These data show that the consensus scaffold is a robust framework by which to improve intrabody function.  相似文献   

12.
The intrabody technology has become a promising therapeutic avenue for a variety of incurable diseases. This technology is an intracellular application of gene-engineered antibodies, aimed at ablating the abnormal function of intracellular molecules. Parkinson's disease (PD) is a common neurodegenerative disease with no cure. Recent studies have explored possible intrabody applications against alpha-synuclein (alpha-syn), whose misfolding is believed to cause a familial form of PD. Here, we review the origin, production, and therapeutic mechanisms of intrabodies and the potential of intrabody protection against alpha-syn toxicity. Furthermore, we propose possible intrabody applications against leucine-rich repeat kinase 2 (LRRK2), whose mutations are the most frequent known cause of familial and sporadic PD.  相似文献   

13.
Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice). iER-VCAM1 mice did not have a lethal phenotype, in contrast to the constitutive knockout of VCAM1, but adult mice exhibited physiological effects in the form of aberrant distribution of immature B-cells in blood and bone marrow. The capability to regulate knock-down strength may spark a new approach for the functional study of membrane and plasma proteins, which may especially be valuable for generating mouse models that more closely resemble disease states than classic knockouts do.  相似文献   

14.
《MABS-AUSTIN》2013,5(6):1394-1401
Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice). iER-VCAM1 mice did not have a lethal phenotype, in contrast to the constitutive knockout of VCAM1, but adult mice exhibited physiological effects in the form of aberrant distribution of immature B-cells in blood and bone marrow. The capability to regulate knock-down strength may spark a new approach for the functional study of membrane and plasma proteins, which may especially be valuable for generating mouse models that more closely resemble disease states than classic knockouts do.  相似文献   

15.
Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.  相似文献   

16.
The expression of intracellular antibodies (intrabodies) in eukaryotic cells has provided a powerful tool to manipulate microbial and cellular signaling pathways in a highly precise manner. However, there have been several technical issues that have restricted their more widespread use. In particular, single-chain antibodies (sFv) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use sFv-phage libraries as a source of intrabodies unless a pre-selection step to identify these rare sFvs from natural libraries or libraries of engineering sFvs that could fold properly in the absence of disulfide bonds were used. Here, we investigated whether target specific sFvs that are isolated from a 15 billion member non-immune human sFv-phage display library could be directly screened in pools as intrabodies without prior knowledge of their individual identity or purity within pools of antigen-specific sFvs. As the target, we used a synthetic transformation effector site 1 (TES1) polypeptide comprising the membrane-most proximal 34 amino acid residues of the carboxy-terminal cytoplasmic tail of the oncogenic latent membrane protein 1 (LMP1) of Epstein Barr virus, which serves as a docking site for adapter proteins of the tumor necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family. Anti-TES1 sFvs, initially identified by phage ELISA screens, were grouped into pools according to the absorbance reading of the antigen-specific phage ELISA assays and then transferred as pools into eukaryotic expression vectors and expressed as cytoplasmic intrabodies. Using the pooling strategy, there was no loss of individual anti-TES1 sFvs in the transfer from prokaryotic to eukaryotic expression vectors. In addition, the initial assignments into sFv pools based on phage ELISA readings allowed the segregation of individual anti-TES1 sFvs into discrete or minimally overlapping intrabody pools. Further assessment of the biological activity of the anti-TES1 intrabody pools demonstrated that they were all able to selectively block F-LMP1-induced NFkappaB activity that was mediated through the TES1-site and to bind LMP1 protein with high efficiency. This direct phage to intrabody screening (DPIS) strategy should allow investigators to bypass much of the in vitro sFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic sFv phage libraries already in existence to identify intrabodies that are active in vivo.  相似文献   

17.
The ability of intracellular antibodies (intrabodies) to block the function of a target protein can be dependent on the stability of the single-chain antibody (sFv) when expressed in the intracellular environment. Low-affinity sFvs capable of reaching high steady-state levels can be more effective modulators of target proteins than high-affinity, unstable sFvs. In an effort to enhance the intracellular stability of sFvs when expressed as intrabodies, we have generated novel sFv-Fc fusion intrabodies. Fusion of the native sFv sequence with the entire heavy chain constant region fragment of IgG results in decreased turnover of the intrabody and enhanced steady-state accumulation of sFv-Fc protein, while maintaining the ability to target intrabody expression to sub-cellular compartments. Here, we describe the rationale and design for this strategy using two anti-cyclin E sFvs constructed for use as intrabodies.  相似文献   

18.
Several approaches have been developed over the past decade to study the complex interactions that occur in biological system. The ability to carry out a comprehensive genetic analysis of an organism becomes more limited and difficult as the complexity of the organism increases because complex organisms are likely to have not only more genes than simple organisms but also more elaborate networks of interactions among those genes. The development of technologies to systematically disrupt protein networks at the genomic scale would greatly accelerate the comprehensive understanding of the cell as molecular machinery. Intracellular antibodies (intrabodies) can be targeted to different intracellular compartments to specifically interfere with function of selected intracellular gene products in mammalian cells. This technique should prove important for studies of mammalian cells, where genetic approaches are more difficult. In the context of large-scale protein interaction mapping projects, intracellular antibodies (ICAbs) promise to be an important tool to knocking out protein function inside the cell. In this context, however, the need for speed and high throughput requires the development of simple and robust methods to derive antibodies which function within cells, without the need for optimization of each individual ICAb. The successful inhibition of biological processes by intrabodies has been demonstrated in a number of different cells. The performance of antibodies that are intracellularly expressed is, however, somewhat unpredictable, because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, we have developed an in vivo selection procedure named Intracellular Antibody Capture Technology (IACT) that allows the isolation of functional intrabodies. The IAC technology has been used for the rapid identification of antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by the selected intracellular antibodies. Several optimizations of the IAC technology for protein knock-out have been developed so far. This system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in different cell lines.  相似文献   

19.
Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.  相似文献   

20.
Although intracellular antibodies (intrabodies) are being explored as putative therapeutic and research reagents, little is known about the principles that dictate the efficacy of these molecules. In our efforts to address this issue, we generated a panel of five intrabodies, directed against catalytically inactive murine caspase-3, by screening single-chain antibody (Fv) phage display libraries. Here we determined criteria that single-chain Fv fragments must fulfill to act as efficient intrabodies. The affinities of these intrabodies, as measured by surface plasmon resonance, varied approximately 5-fold (50-250 nm). Despite their substantial sequence similarity, only two of the five intrabodies were able to significantly accumulate intracellularly. These disparities in intracellular expression levels were reflected by differences in the stability of the purified protein species when analyzed by urea denaturation studies. We observed varied efficiencies in retargeting the antigen murine caspase-3, from the cytosol to the nucleus, mediated by intrabodies tagged with an SV40 nuclear localization signal. Our results demonstrate that the intrinsic stability of the intrabody, rather than its affinity for the antigen, dictates its intracellular efficacy.  相似文献   

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